iodine starch and water experiment

Why Does Iodine Turn Starch Blue?

Author: Catharina Goedecke

Iodine Test

Using iodine to test for the presence of starch is a common experiment. A solution of iodine (I 2 ) and potassium iodide (KI) in water has a light orange-brown color. If it is added to a sample that contains starch, such as the bread pictured above, the color changes to a deep blue. But how does this color change work?

Starch is a carbohydrate found in plants. It consists of two different types of polysaccharides that are made up of glucose units which are connected in two different ways. One is the linear amylose and the other is the branched amylopectin (pictured below).

Amylose is the compound that is responsible for the blue color. Its chain forms a helix shape, and iodine can be bound inside this helix (pictured below).

Charge-Transfer Complexes

The colors are caused by so-called charge transfer (CT) complexes. Molecular iodine (I 2 ) is not easily soluble in water, which is why potassium iodide is added. Together, they form polyiodide ions of the type I n – , for example,  I 3 – , I 5 – , or I 7 – . The negatively charged iodide in these compounds acts as charge donor, the neutral iodine as a charge acceptor. Electrons in such charge-transfer complexes are easy to excite to a higher energy level by light. The light is absorbed in the process and its complementary color is observed by the human eye.

In the case of the aqueous solution of polyiodides, the absorptions of the different species lead to an overall brownish color. Once amylose is added, it forms another CT complex, Here, the amylose acts as a charge donor and the polyiodide as an acceptor. This complex absorbs light of a different wavelength than polyiodide, and the color turns dark blue.

Polyiodide Chains

The exact structure of the polyiodides inside the amyloid helix is not clear. The amylose-iodine complex is amorphous (i.e., it does not form ordered crystals), which has made it difficult to determine its structure. It has been proposed that the species inside the helix are repeated  I 3 – or I 5 – units.

However, Ram Seshadri, Fred Wudl, and colleagues, University of California, Santa Barbara, USA, have found evidence that infinite polyiodide chains I n x– are contained in the amylose-iodine complex [1]. The team investigated a related system, a pyrroloperylene–iodine complex, to study its properties as an organic electronic conductor. The material is crystalline, and therefore, the team was able to determine its structure using X-ray crystallography. They found nearly linear polyiodide chains in-between stacks of pyrroloperylene. It turned out that the material containing these chains absorbs light at very similar wavelengths to the amylose-iodine complex, which supports the hypothesis that similar polymeric chains form in the iodine test for starch.

[1] Sheri Madhu, Hayden A. Evans, Vicky V. T. Doan-Nguyen, John G. Labram, Guang Wu, Michael L. Chabinyc, Ram Seshadri, Fred Wudl, Infinite Polyiodide Chains in the Pyrroloperylene-Iodine Complex: Insights into the Starch-Iodine and Perylene-Iodine Complexes , Angew. Chem. Int. Ed. 2016 , 55 , 8032–8035. DOI: 10.1002/anie.201601585

• Der Iod-Stärke-Komplex (in German), www.chemieunterricht.de 2006 . (accessed November 24, 2016)
• The structure of the blue starch-iodine complex , Wolfram Saenger, Naturwissenschaften 1984 , 71 , 31–36. DOI: 10.1007/bf00365977

this article is quite helpful .. thank you ✨

Thank you guys

where the h*** is the volume and page number to cite this

Dear Reader, We do not have volumes or page numbers. You can cite the article as: C. Goedecke, Why Does Iodine Turn Starch Blue?, ChemistryViews 2016 . https://doi.org/10.1002/chemv.201600103 Best regards, Your ChemistryViews Team

Interesting light absorbing properties

Nice informative post

Very informative thank you very much

Who is the publisher?

Dear Reader, You can cite the article as: C. Goedecke, Why Does Iodine Turn Starch Blue?, ChemistryViews 2016. https://doi.org/10.1002/chemv.201600103 Best regards, Your ChemistryViews Team

Very interesting and helpful

Very helpful imformation, thank you

Wow that’s awesome

this is awesome

really cool but no words

would iodiDE alone form the same reaction? or is iodiNE specifically needed to react with the glycogen

Thanks for this.. very helpful

What does it mean, when the water become crystal clear after you added iodine into the water?

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Iodine and Starches Visual Science Experiment for Kids

Using iodine to identify starch is a fantastic visual science experiment for kids.

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The kids really enjoyed this simple experiment of using iodine to identify starch. I got to thinking about iodine after writing last week’s water post, because I remembered using iodine tablets to purify water when I went hiking on the Inca trail as a thirteen-year-old. It’s hard to visually explain that experiment, but the starch one is quite striking!

Iodine and Starch Simple Experiment

What You'll Find on This Page

Iodine is normally brown – not very attractive! Add it to starch, however, and you get a beautiful royal purple color! This makes for a truly striking visual science experiment that kids love.

I pulled out some iodine wipes  (warning: iodine STAINS – be careful!), and we set up our experiment. Iodine in liquid form or tablet  form can also be used; simply dissolve the tablets with a little bit of water for this experiment. I put flour, salt, and oatmeal on the plate, and then added baking powder on a whim, just to see what would happen.

How Do Everyday Pantry Items React to Iodine?

Salt doesn’t have any starch, so the iodine stays brown.

Flour has a lot of starch! The iodine turned dark purple! We added a few drops of water with a syringe to help our drop of iodine to mix with the flour.

Oatmeal also has a lot of starch! It turned purple as well, although cooked oatmeal might have allowed the iodine to spread more thoroughly. Or a little more water =)

The baking soda bubbled up and turned purple at the edges. The purple is probably because most baking soda has some starch mixed in, but I’m still not sure why it bubbled. Does anyone know?

Update: It bubbles because baking powder contains baking sodamy IRL friend thanks to my IRL friend Kathy for reminding me of this! Baking soda reacts to acids, so the iodine must be slightly acidic. Here is Kathy’s explanation, if you would like the chemical details (thanks, Kathy!):

I think you created a chemical reaction with the Iodine (I2) baking soda NaHCO3 and water H20 to form a new chemical compound and also CO2 (gas) is a biproduct… which is what the bubbles would be… this is also what makes muffins, cookies, etc rise when baked is the release of the carbon dioxide gas…

Carla added in the comments:

Iodine wipes usually have povidone-iodine, which is a mixture of povidone, hydrogen iodide (which is very soluble in water and would form hydroiodic acid), and elemental iodine (which can act as an acid or base, depending on what you mix it with). Stirring baking soda (a base) into that should give you some excellent fizzing!

The kids thought this was fascinating – and very strange! Johnny asked us to put it in the fridge for a while to see if that changed anything. It didn’t, but I was thrilled to see him taking our experiment one step further!

Have you tried this iodine and starch experiment with your kids? What did they think?

More Science Activities for Kids

Our favorite simple science projects for kids, storybook science: exploring the way things work, science for littles: experimenting with gummy candy, science for littles: weighing, fun science: candy experiments, animal science biology for kids: animal observation log, fun ways to explore the science of flight with kids, growing sugar crystals: delicious science for kids, making pizza with kids: pizza dough science, how to make dry ice ice cream - cool science, growing salt crystals - kids love this visual science experiment, 10 sensational sound activities for preschoolers - explore the science of sound, physics for kids: an easy fluid dynamics experiment, simple experiments for kids: plants and sunlight, diy summer camp.

I’ve teamed up with some fellow bloggers to bring you even more awesome content all month. Click  HERE   for more Chemistry Themed Summer Camp Activities and  HERE  for more DIY Summer Camp Activities!

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MaryAnne Kochenderfer

MaryAnne is a craft loving educator, musician, photographer, and writer who lives in Silicon Valley with her husband Mike and their four children.

46 thoughts on “Iodine and Starches Visual Science Experiment for Kids”

What a fascinating science experiment! Definitely going on my to-do list this year!

I think you are much braver then I am in pulling out the iodine! But it looks like an awesome experiment.

SO COOL!! This would be such a great experiment to do, thanks for sharing!!!

This is such a fun experiment! I remember doing something similar a few years ago with my kiddos.

That is fascinating! I didn’t know iodine would react to starch. I love kitchen science!

This is one of my favorite science experiments – so easy and magical!

This is really interesting – something to do with our own kids this summer. They love experimenting with things around the house. And just scrolling down to your comment section had me seeing lots of other things we can do together this summer.

How fun! My kids would love this!

I love doing experiments with the kids using things/ingredients we have around the house – so much fun!

This is really cool. I think my daughter will love it. Can’t wait to try it with her.

I love this SO much! Very awesome experiment! I want to use this to make a detective chemistry lab! Also…the iodine/baking soda reaction is fantastic…Iodine wipes usually have povidone-iodine, which is a mixture of povidone, hydrogen iodide (which is very soluble in water and would form hydroiodic acid), and elemental iodine (which can act as an acid or base, depending on what you mix it with). Stirring baking soda (a base) into that should give you some excellent fizzing!

Thank you for this additional explanation!

ooooh, I know a little scientist who will LOVE this, thank you!

What a great experiment, so simple, but so interesting! Thanks for linking to Fun Sparks. x

Such an interesting experiment! And great pictures! They show everything so well. Thanks for sharing on weteach.

I loved doing this when I taught Science – it was my first ever lesson and I still remember it after 10+ years.

Thank you also for featuring our snow dough – I’ve linked up again this week with our first exploration of weighing with some snowmen crispie cakes.

I think this is one of the funnest science experiments – so visual and so immediate!

Thanks for linking up again!

It looks like they were having a lot of fun learning! I just read in The Hunger Games where she used iodine drops to purify water. I’m sure I learned that at some point but I think I had forgotten ;)

Apparently if you add vitamin C it gets rid of the icky iodine taste – and protects your from scurvy!

What a fun experiment – my kiddos are very little, but I’m hoping to try a baking soda and vinegar investigation soon!

Baking soda and vinegar was our first – I love that one, because it’s so accessible to even the youngest children. This is my kids’ favorite version: https://www.mamasmiles.com/sensory-play-with-baking-soda-and-vinegar/

This is a great idea, I can’t wait to add this as a lesson in my Little Hands that Cook lessons so they can see why we add all these special ingredients.

What a great addition to the series!

This really looks like fun! JDaniel would love to see the chemical reaction.

What a fun experiment! I remember getting a science kit as a kid and having lots of fun with iodine.

Did you enjoy the Inca Trail as a 13 yo? It’s a dream of my husband and I to hike it but think we may need to do it without kids someday. We have been to Peru, but would love to hike the trail.

It was an amazing experience. We hiked a four-day segment in Bolivia, starting at 14,000 feet the first day, hiking up to 16,000 feet, and then down to 8,000 over the next three days. Parts of the trail were missing, but our guide knew their way. The first day was pretty grueling, but after that it was (literally) all downhill. Definitely something I’ll remember forever!

I love experiments like this! Thanks for sharing at We Teach. My kids and I did an experiment with purple cabbage not too long ago. We soaked a coffee filter with boiled purple cabbage water, dried the filter, and used our filter to test ph levels. I’m sure you’d find the directions for experiments like this online. We’re going to try your experiment with iodine and some of the others you’ve shared. Thanks!

I keep meaning to make ph paper using cabbage – thanks for the reminder! And thank you for visiting from We Teach!

Very fun! There used to be a unit called mystery powders something like this. Doesn’t any liquid and baking soda cause bubbles? Learning like this is so wonderful for your children. Love the photos. Carokyn

Somehow I forgot baking powder has baking soda – thanks for reminding me!

You’re such an amazing, creative mom! I love all the activities you come up with to teach your kids throughout every day!

Really cool! Very curious and had to look into it a little. Looks like baking soda reacts with acids. Iodine must be somewhat acidic…

Thanks, Ann!

Funny, I accidentally did one part of your experiment when I was preparing our traditional gargle solution for Lars (he has a sore throat). Usually it’s soda, salt and iodine, but accidentally I put a baking powder instead. I didn’t see the bubbling, but my gargle turned purple. I was thinking of doing this experiment with Anna, since it’s very cool indeed.

I think Anna would really enjoy this experiment! Another commenter as well as one of my IRL friend reminded me that baking powder has baking soda in it (don’t know how I forgot that!) and that is probably why it is bubbling – the iodine must be slightly acidic? The bubbling was very slight, so you might not see it in a solution.

Just found you site and this stuff is right up my kids alley. My 11 year old is a science nut, she loves experiments.

I bet that was so cool for the kids to see the changes right before their eyes!

They loved it!

How interesting! I had no idea iodine reacts with starch. Love following along with you guys as you learn new things. :)

I did not know about this iodine and starch experiment! Thanks for sharing:)

Great experiment! They look very interested!

Your such a good mom! You kids get experiences in all aspects…experiments…sewing…art..outdoor adventures! Amazing!

We tried the iodine and starch thing when we did the murder mystery and it is so cool. I bet most of the foods I love have starch.

Great experiment! I love Lily’s expression in the first and last pictures.

She was fascinated – and perplexed =)

Comments are closed.

Lab Procedure: Iodine Clock Reaction

Core Concepts

In this lab tutorial, we learn about the iodine clock reaction, including its procedure, underlying chemistry, and data analysis.

Topics Covered in Other Articles

• Lab Safety Rules
• Recrystallization
• Thin Layer Chromatography
• Distillation
• Integrated Rate Laws

What is the Iodine Clock Reaction? 

The Iodine Clock Reaction is a classic chemistry experiment that demonstrates many basic principles of kinetics and redox chemistry . For this, the reaction persists as a staple of general chemistry lab demonstrations.

In this experiment, you prepare two simple, transparent solutions. Once the solutions combine, however, the mixture gradually turns from clear to dark blue to near-black. This color change corresponds to the progress of the reaction, which allows you to visually witness the kinetics in a way that most reactions do not provide.

Interestingly, some chemists colloquially call this reaction the “Egyptian Night” experiment. In Egypt, the darkness of nighttime often arrives rather suddenly, similar to rapid dark color change in this reaction. 

Iodine Clock Procedure

To perform the Iodine Clock Reaction, you need an iodine salt, a reductant, an oxidant, an acid , starch, and water as a solvent. As mentioned before, these components become allocated between three different solutions according to these specifications:

• First Solution: Starch, Water.
• Second Solution: Iodine Salt, Reductant, Water.
• Third Solution: Oxidant, Acid, Water.

Once the solutions mix, the reaction begins.

The most common variant of the Iodine Clock Reaction uses sodium thiosulfate (Na 2 S 2 O 3 ) as the reductant and hydrogen peroxide (H 2 O 2 ) as the oxidant. Potassium iodide (KI) serves as the salt, while sulfuric acid (H 2 SO 4 ) provides the required acidity. Importantly, gloves, safety goggles, and caution should be observed when using sulfuric acid and hydrogen peroxide to prevent chemical burns.

As we’ll find out in a later section, the kinetics of the reaction depends on the concentrations of acid, iodide, and oxidant. Thus, most lab procedures studying reaction kinetics will vary the concentrations of one or more of these species. Aside from that, the reductant concentration tends to be kept low, as very little is required, while the starch tends to be in excess.

The Chemistry of the Iodine Clock

Iodine clock redox and kinetics.

Before the three solutions mix into one, each ionic species dissociates into their respective ions:

KI → K + + I –

Na 2 S 2 O 3 → 2Na + + S 2 O 3 2-

H 2 SO 4 → H + + HSO 4 –

During the reaction, K + , Na + , and HSO 4 – do not participate, remaining as spectator ions. Once the solutions mix, the hydrogen peroxide oxidizes the iodide into diatomic iodine:

2H + + H 2 O 2 + 2I – → I 2 + 2H 2 O

Importantly, as the reaction produces diatomic iodine, the thiosulfate re-reduces the iodine back to iodide:

2S 2 O 3 2- + I 2 → 2I – + S 4 O 6 2-

This back and forth between iodide and iodine continues until all thiosulfate oxidizes away. Afterward, significant quantities of iodide and iodine exist at the same time. They react with one another to form the triiodide ion:

I 2 + I – → I 3 –

This triiodide ion then forms a complex with the starch. This complex is responsible for the increasing dark blue of the reaction vessel. As a side note, due to the striking dark blue of the complex, a mixture of iodine and iodide called Lugol’s iodine is used to test for trace amounts of starch .

Iodine Clock Kinetics

The first reaction, the oxidation, occurs much slower than the reduction, making it the rate-determining step during that first phase of the reaction. Additionally, once the reduction ceases, the oxidation continues to serve as the rate-determining step, as both the formation of the triiodide and the starch complex occur relatively quickly. Thus, for the entirety of the experiment, oxidation determines the progress of the dark blue hue. This is true even though the starch complex is not directly generated from the oxidation.

Aside from the qualitative observation of the increasingly blue reaction vessel, you can periodically measure the starch concentration through spectrophotometry . The resulting data then allows you to quantify the reaction kinetics.

Kinetic Data Analysis

First, you need to do multiple trials of the Iodine Clock with different concentrations of potassium iodide. Then, you quickly place these samples into a spectrophotometer that records concentrations at consistent time intervals. You’d want to set the spectrophotometer to a frequency similar to 600nm to pick up the dark blue of the starch complex.

Next, you graph your data. You should find that the absorbance of each graph increases linearly with time. This makes sense since chemical reactions always initially proceed at linear rates.

Finally, to determine the reaction order with respect to KI, you take the logarithms of the initial concentrations and reaction rates and generate a log/log graph. 

The slope of the resulting slope corresponds to the reaction order in our rate law , due to the properties of logarithms. 

RxnRate = k’[KI] n

log(RxnRate) = log(k’[KI] n ) = nlog([KI]) + log(k’)

k’: Relative rate constant (s -1 )

n: Reaction Order of KI

The graph then generates a trendline of y = x – 1.2883, indicating that the Iodine Clock Reaction is first order with respect to KI (n = 1).

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Iodine Clock Reaction

Try an at home version of this experiment using a few things you may have in your bathroom medicine cabinet. In may ways this experiment feels almost like magic. Two colorless liquids are mixed together and after a few moments the mixture turns a dark blue color. There are actually a couple of simple chemical reactions going on at the same time to make this “clock reaction” occur. This version of the classic “iodine clock reaction” uses safe household chemicals most people have on hand at home.

What you need:

• distilled water (tap water will work OK as well)
• a couple plastic cups
• 1000 mg vitamin C tablets
• tincture of iodine (2%)
• hydrogen peroxide (3%)
• liquid laundry starch

What to do:

• Make a vitamin C solution by crushing a 1000 mg vitamin C tablet and dissolving it in 2 oz of water. Label this as “vitamin C stock solution”.
• Combine 1 tsp of the vitamin C stock solution with 1 tsp of iodine and 2 oz of water. Label this “solution A”.
• Prepare “solution B” by adding 2 oz of water to 3 tsp of hydrogen peroxide and 1/2 tsp of liquid starch solution.
• Pour solution A into solution B, and pour the resulting solution back into the empty cup to mix them thoroughly. Keep pouring the liquid back and fourth between the cups.

What’s going on?

There are actually two chemical reactions going on at the same time when you combine the solutions. During these reactions two forms of iodine created – the elemental form and the ion form.

In Reaction # 1 iodide ions react with hydrogen peroxide to produce iodine element which is blue in the presence of starch. BUT, before that can actually happen, the Vitamin C quickly reacts and consumes the elemental iodine.

The net result, at least for part of the time is that the solution remains colorless with excess of iodide ions being present. Now after a short time as the reactions keep proceeding in this fashion, the Vitamin C gets gradually used up. Once the Vitamin C is used up, the solution turns blue, because now the iodine element and starch are present.

Safety Precautions

Be careful when working with the iodine – it stains, and it stains really well. Be very careful not to spill any of the solution.

Waste Disposal

Dig deeper into the science behind clock reactions in  this paper  from the Journal of Chemical Education.

Middle School Science Blog

Free lesson plans and resources for grades 5-8 by liz belasic (liz larosa), diffusion lab – iodine & cornstarch.

This slideshow requires JavaScript.

Materials and Set Up – this was so easy and inexpensive to do and had the same effect as using dialysis tubing. Great demo/lab as part of our unit on osmosis and diffusion!

For every two students:

• handout from Biology Corner
• large beaker
• inexpensive sandwich bag – non sealing (I used Wegmans 150 ct)
• 1 tbsp corn starch
• 50 mL water
• rubber band
• clothes pin
• graduated cylinder
• 100 mL Iodine dilution

Iodine Preparation

• 20 ml Iodine added to 500 mL of water
• measure out 100 mL of diluted iodine for each group

Prelab Prep:

• Place one bag over each beaker
• Add 1 tbsp of cornstarch to each bag
• Add 50 mL of water to each bag
• Check for leaks
• Use a rubber band on each one to keep closed
• Clip bag to beaker

Observations

• Iodine is able to pass through the plastic bag, the starch is not
• Have students lift the bag out to see the changes that are taking place

Update – I let the set up sit over the weekend, and when I came in today, the water was almost completely clear – looks like just about all of the iodine moved into the bag:

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5 thoughts on “ Diffusion Lab – Iodine & Cornstarch ”

Is there an alternative to iodine or do you know where to find cheap iodine? I don’t have access to a chemistry lab.

I think I bought some from the supermarket or drugstore, sorry I do not recall.

Hi, I’m just finishing up investigations with my 7th graders on osmosis and diffusion. Oddly, I tried this very set up hoping that inexpensive baggies would work. We had tried with a z-loc brand bags and had no luck ( just letters to the company letting them know how non permeable their baggies were) so I used the store brand baggie and we were still disappointed. I guess we all felt better about our sandwiches wrapped in these baggies but not to see the diffusion. We did a great investigation with carrots and salt and then moved onto “naked eggs”. I had been very hopeful the baggie would work. Glad it did for your crew.

Like Liked by 1 person

We used either Wegman’s brand fold over sandwich bags or ShopRite – I threw out the box without writing it down 😦 -Sorry it didn’t work for your class 😦 The carrots sounds like a great idea, I saw one using potato slices that I thought about using as well. I have never tried the egg one, but heard it works well 🙂

Update – it was Wegmans brand, box was still in my room 🙂

Late to the party but I am using ziplock brand locking and it is working but I had to increase the amount of iodine to about 1/2 and 1/2 for it to work in a reasonable timeframe

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Chemistry experiments

Diffusion and Osmosis | Iodine starch experiment with bag | Science Experiments | elearnin

This experiment shows the movement of particles through a membrane For this experiment you will need: • Water • Starch solution • Iodine • Dropper • Zipper plastic bag PROCEDURE: • Mix the starch solution in the water in a beaker. • With the help of the dropper put some iodine solution in the zipper bag. • Zip the plastic bag. • Now turn up side down to check whether there is any leak. • Submerge the plastic bag into the beaker with starch solution. • Leave the arrangement for half an hour. • A layer of deep purple-black color layer is formed on the membrane of the plastic bag and the color slowly diffuses into the starch water. EXPLANATION: Iodine is used to test for the presence of starch. When Iodine reacts with starch, it turns deep purple-black. The iodine molecules are small enough to pass through the membrane of the plastic bag, however starch and water molecules are too big to pass through the membrane. The movement of the iodine through the plastic membrane is functionally the same as movement of molecules through biological membranes, that is, any cell membrane. The molecules will move from higher concentration to lower concentration. Osmosis is the net movement of solvent molecules (in this case, iodine) through a partially permeable membrane (like a plastic bag) into a region of higher solute (water) concentration, in order to equalize the solute concentrations on the two sides. Here, by partially permeable membrane or semi-permeable membrane, we mean a permeable to the solvent, but not the solute. Diffusion is that physical process in which any solvent moves, without input of energy, across a semi permeable membrane separating two solutions of different concentrations. source

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28 Comments

so the iodine diffuses into the semipermeable membrane (the plastic bag) from high concentration to low concentration meaning it doesn’t use any form of energy. when it enters the semipermeable membrane it reacts with the starch and turns a dark purple/blue/black colour. i am currently studying this and although the definition of osmosis was slightly muddled up, it was still a pretty good vid. as osmosis specifically refers to the net movement of water across a semipermeable membrane and on the rare occasion, ethanol too, the movement of the molecules actually refers to diffusion.

The explanation needs to be seriously fixed. How can a wrong statement like this get thousands of views? Its too dangerous for anyone who trusts on everything they see on the internet.

help me I hate this

exiting videos i luv them

What is the ratio of starch to water?

horrible explanation you really need to do your own work and not go on to google or other search engines . common man step up your game.

Thank you alot thiz help me for expo….. again tysm

so what's the starch diffusion or osmosis?

good vid i love it

hi im ms lavender

is it dengrous for our helthly

Osmosis is definitely NOT the net movement of solvent molecules through a partially permeable membrane into a region of higher solute concentration. Osmosis refers ONLY to the movement of WATER across a membrane from an area of higher WATER concentration to an area of lower WATER concentration.

This is so easy as one person Unlike me with a group and I'm little

i really understood this lesson and it was practically the same stuff my teacher taught me thanks

What acts as the cytoplasm and the cell wall?

What mil plastic bags are you using? I tried this and it didn't work, but I was using pretty thick/sturdy bags.

@ Ethiopian boy, why don't appreciate and focus the concept instated finding wrong or correct . Anyway good job bro.

THIS REALLY HELPS! THANK YOU!

Love the visuals but- Your definition of osmosis needs to be corrected- since it is the movement of WATER (not just anything) across a membrane from high to low concentration!

this experiment is too wrong!

This experiment will give a better understanding about osmosis and diffusion

what would be the effect of higher temperature?

Isn't osmosis exclusively the diffusion of water through a membrane and nothing else?

Thank you so much for making this video. It helped me alot with my biology homework. I saw this lab sheet I hadn't done (late/missing work) but this video helped me get the data I needed. 🙂

I'm not quite sure if the explanation is the fact that water molecules are not small enough to make it through the membrane, because they should not be so much bigger than iodine (I would expect them to be smaller, as a matter of fact). A more plausible explanation would be that plastic, being an organic polymer, is mainly non-polar, so is iodine, which allows the latter to pass through the plastic membrane. Starch molecules are indeed too big, but water molecules do not pass through the membrane due to its polarity.

no, starch was placed inside the bag, the iodine reacted with the starch in the bag

I actually had that same question.

So if it turned purple inside the bag, isn't that an indication that starch can pass the membrane too?

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Mystery solved: Why starch turns iodine dark blue

You are free to share this article under the Attribution 4.0 International license.

In the pursuit of a new class of photovoltaic materials, researchers solved a centuries-old mystery of chemistry: Why does an iodine solution turn blue-black when starch is added to the mix?

The exact structural-chemical mechanism that causes the intense deflection of blue light during this transformation has been a subject of active speculation until this point.

University of California, Santa Barbara researchers in the labs of materials professors Fred Wudl and Ram Seshadri report first observation of crystalline infinite iodide polymers, discovered as part of a pyrroloperylene-iodine complex, an organic semiconductor that contains iodine. Their paper appears in the journal Angewandte Chemie .

Candy mistake leads to new molds for silicone

“Every college student taking introductory chemistry learns titration of iodide with thiosulfate solution as part of the curriculum. You add starch as an indicator of iodine to detect the end-point,” explains Seshadri. “When you add iodine to potato starch in solution, it turns a dark blue-black.”

This starch-iodine complex transformation discovered almost exactly 200 years ago, is used in classrooms as a foundational teaching tool in chemistry and biochemistry, such as demonstrating the action of amylose, the enzyme that breaks down starch, in human saliva, or the chemistry behind counterfeit banknote detection pens.

Fast forward two centuries of scientific discovery to the UC Santa Barbara researchers using a technique called Raman spectroscopy, which observes the light-scattering patterns of a molecule that can be a unique fingerprint, to study iodine chains in a semiconducting pyrroloperylene-iodine complex. They initially set out to study this promising organic semiconductor material as part of a new class of solar power-generating materials, a project funded by the US Department of Energy.

Iodine isn’t the best way to prevent C-section infections

“We determined that, when iodide is in the presence of iodine and interspersed between molecules of pyrroloperylene, a polymer chain forms,” Wudl explains. “There is only one other element that can form its own polymeric chain, and that’s sulfur.” Single-element polymeric chains are a rarity, to say the least.

“The problem with sulfur polymer chains is that they’re not crystalline,” Wudl continues. “If there’s no molecule repeating in a precise way you can’t determine where all the atoms are.” The crystalline structure of the polyiodide chain is what allowed the UCSB materials researchers to clearly observe iodine in this form.

What this discovery means to the future of chemistry and materials science, only time will tell, according to Wudl. “If you had told someone in the 1950s there would someday be organic electronic materials they would have laughed you out of the room,” he says. “Discovering new compositions of matter usually leads to new concepts, and these concepts drive technology down the road.”

For now, they agree, the discovery is mainly of academic interest. “If you know where the atoms are, you can use the knowledge to develop things later, such as functional materials for new electronics,” says Seshadri. “At this time, we can say with confidence this is one for the chemistry textbooks.”

Source: UC Santa Barbara

Osmosis and Diffusion

AUSTRALIAN CURRICULUM ALIGNMENT: 

• Movement of materials across membranes occurs via diffusion, osmosis, active transport and/or endocytosis

BACKGROUND: 

The cell membrane maintains the cell a separate entity; it holds the cell contents within, and acts as a barrier to the external environment. It is selectively permeable and has various mechanisms to allow for the exchange of gases and nutrients. These mechanisms allow for the intake of anything that is required and allows for the expulsion of waste and toxins. This membrane does not resemble a sheet or bag; rather, it is many molecules of Phospholipid Bilayers held together by the combined forces of attraction and repulsion. They are comprised of a Phosphate head; which is hydrophilic (water-loving), and a Lipid (fatty acid) tail which is hydrophobic (repelled by water). As the internal and external environments of a cell are aqueous, these molecules arrange themselves into two layers; one with the Phosphate heads oriented out into the external fluid, and the other with the heads oriented inwards into the internal fluid (the Cytoplasm). The Lipid tails are between the two layers of Phosphate heads; thereby, protected from the water, and the strength of this attraction/repulsion mechanism keeps the molecules together as though the membrane were a single entity.

In this practical, dialysis tubing is used as a surrogate cell membrane for a visual demonstration of osmosis and diffusion. A solution containing large molecules (Starch) and small molecules (Glucose) is placed inside the tubing; which is then placed in a solution containing iodine. Students are able to observe as the solution inside the tubing turns dark blue, while the surrounding solution it is submerged in does not. From this, students can use their prior knowledge of the Starch-Iodine complex to surmise that Iodine is able to pass through the membrane while starch is not. The Glucose-testing strips indicate that glucose has been able to pass out of the tubing and into the external fluid. Thus proving the tubing allows movement in both directions. 

This inexpensive and simple experiment provides students with a clear visual result that effectively demonstrates how the size of a molecule can affect its ability to be transported into or out of a cell. It also illustrates the mechanics of diffusion and osmosis by which a cell will attempt to create homeostasis, or equilibrium between its inner and outer environments.

PREPARATION - BY LAB TECHNICIAN

• Cut the dialysis tubing into 15cm lengths and soak for 15 minutes in a beaker filled with room temperature distilled water. Prepare one length of tubing per student or group. However, it is best to prepare extra strips for students, as some strips may tear or leak through handling.  
• To create the Starch solution, dissolve 2g of Starch in 100mL of boiling hot water (2% solution) on a hot plate until the Starch powder has been fully dissolved. Stir as required.  
• To create the Glucose solution, dissolve 30g of Glucose in 100mL water (30% solution) and continue stirring until the glucose has been fully dissolved. 
• Combine the Starch and Glucose solutions in a single beaker. Use a stirring rod to mix well.

METHOD - STUDENT ACTIVITY

Glucose/ Starch Solution

• Measure 5-10 mL of the Glucose/Starch mixture in a small beaker or test tube.
• To determine the initial glucose concentration within the Starch/ Glucose solution, you will first need to dilute a sample of the mixture in water.  To do this, collect 1mL of your mixture using a transfer pipette and add to a test tube filled with 9mL of water. Mix using a clean stirring rod.
• Measure the diluted Starch/Glucose by placing a Glucose-testing strip in the solution, immediately removing it and waiting 60 seconds to observe any colour change. Using the colour guide on the testing strip container, determine the approximate Glucose levels, and record the results.

Iodine Solution

• Fill a large beaker with 100mL water, and add 1mL of Iodine/KI solution. The solution should appear a yellowish colour. 
• Measure the Glucose levels of the Iodine solution with another strip; following the same procedure as before. Ensure you record the results.

  Preparing the "cell" tubing

• Retrieve your soaked piece of dialysis tubing and tie a knot in one end as though you are tying a balloon.
• Using a transfer pipette, half-fill the tubing with your undiluted Starch/Glucose solution and tie the other end to create a “cell”.
• Submerge the “cell” tubing into the Iodine solution.

    Observing changes in the “cell”

• After 15 minutes, observe any colour changes in the tubing and in the beaker solution.
• Measure the Glucose levels in the Iodine solution.
• Carefully open the tubing and pour the contents into a clean beaker.
• To dilute the tubing contents for Glucose testing, collect 1mL of the contents using a pipette and deposit into a test tube filled with 9mL of water.
• Measure the Glucose levels in the diluted contents using a Glucose testing strip following the same procedure as before.
• Record the results of the Glucose testing. 
• Compare the changes in Glucose levels before and after the 15 minute interval.

OBSERVATION AND RESULTS

INVESTIGATION 

• Provide students with the information that you prepared a 100mL solution of 2% starch and a 100mL solution of 30% Glucose. Based on this information, ask your students to calculate the concentration of each in the combined solution. Students should understand that double the volume without extra solute means half the concentration, so what was 2g of Starch in 100mL (2%) is now 2g of Starch in 200mL (1%); and what was 30g of Glucose in 100mL (30%) is now 30g of Starch in 200mL (15%).
• Ask students to identify what occurred the Starch, based on the fact that the blue colour is found inside the cell but not outside of it, students should be able to identify that the Starch has not been able to pass through the tubing, while the Iodine has. Students should understand that the Starch-Iodine complex has therefore been confined to the area where both Starch and Iodine are found, that is, the inside of the cell. 
• Ask students to describe what is suggested by the Glucose results. The appearance of Glucose into the previously Glucose-free solution in the beaker should inform students that Glucose has been able to pass through the membrane.
• To provide students with a deeper understanding surrounding the molecular size of Glucose and Iodine, you may provide students with the information that our dialysis tubing typically allows passage to molecules of up to 12,000 to 14,000 daltons (g/mol). This should provide some guidance of the sizes that Starch molecules can reach. Remind students, however, that the shape of a molecule may affect the passage as a large linear molecule may be able to pass through more easily than a smaller but globular molecule.

TEACHER NOTES

The concentration of Glucose in this practical is quite high to enable shorter waiting times for students. This allows them to more readily measure the glucose which has diffused out of the “cell” using their test strips. However, this also means that the initial concentration is too high to show that the concentration inside the cell has decreased in line with the increase outside the cell. To manage this, students are asked to take a sample of the original combined Glucose/ Starch solution prior to being placed in the “cell” and also a sample of the now-blue solution inside the “cell” at the end of the prac. Both solutions are diluted by a factor of ten to bring the Glucose concentration into the range of the Uriscan strips.

EXTENSION EXERCISE

To observe the process of cell diffusion and osmosis over an extended period of time, make an extra “cell” and keep it in solution until the next class. By the beginning of next class, the Glucose inside and outside the cell should have somewhat equalised. This could be conducted as a class demonstration, or each student may make an extra cell. Once again, dilute both solutions by a factor of ten prior to measuring.

TEACHER TIPS: 

Prepare extra dialysis strips for students, as some strips may tear or leak through handling as students attempt to tie them. 

Time Requirements

• 45 mins  

Material List

Dialysis tubing

• Starch  
• Iodine/KI solution  
• Glucose 
• Glucose-testing strips  
• Test tubes 
• Test tube rack
• Beakers 500mL
• Beakers 100mL
• Transfer pipettes 
• Stirring rod

 Safety Requirements

• Wear appropriate personal protective equipment (PPE); particularly gloves and a lab coat as Iodine will stain clothing and skin on contact. 
• Exercise caution when handling the chemicals used in this prac. 
• Avoid any direct contact with the solution and wash hands thoroughly.   

||Biology||Classroom Practicals||Cells||Year 11&12||

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Detecting starch in food on a microscale

In association with Nuffield Foundation

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Test different foodstuffs for the presence of starch using iodine in this microscale class practical

In this experiment, students conduct qualitative tests to find out whether different foodstuffs contain starch. Working on a microscale, students produce iodine in situ by adding potassium iodide crystals and sodium hypochlorite solution to small samples of various foods. They then note any colour change to blue-black, indicating that starch is present.

A quick and easy class experiment. It should be possible to test a range of foodstuffs in about ten minutes.

• Eye protection
• Clear plastic film (eg acetate sheet as used for an overhead projector)
• Forceps (for handling foodstuffs)
• Paper towels
• Sodium hypochlorite solution, 5% w/v of available chlorine (IRRITANT), 10 cm 3
• Potassium iodide crystals, allow 5–10 small crystals per group
• A range of foodstuffs, broken into small pieces, to include both starchy and non-starch-containing foods

Health, safety and technical notes

• Read our standard health and safety guidance.
• Wear eye protection throughout.
• Sodium chlorate(I) solution (sodium hypochlorite), NaOCl(aq), (IRRITANT at concentration used) – see CLEAPSS Hazcard HC089 . Note this is NOT sodium chlorate(V), NaClO 3 . Sodium chlorate(I) solution can be purchased as such from chemical suppliers. However domestic chlorine-containing bleach solution is quite adequate for this experiment, preferably a cheap brand containing no added detergent or perfumes. Household ‘bleaches’ based on peroxide are becoming more widely available and do not contain chlorine; they should therefore not be used. The sodium chlorate(I) solution should be provided in such a way that students can add a single drop using a plastic dropping pipette. Plastic dropper bottles of capacity 30–60 cm 3 would be suitable for this purpose.
• Potassium iodide crystals, KI(s) –  see CLEAPSS Hazcard HC047b .

It is worth pre-testing the foodstuffs to check that they test correctly – that is, the starchy foods contain enough free starch to give a clear positive test, and the non-starchy foods have not been contaminated by starch-containing material. Note that the amount of free starch present in some uncooked foods may be small, and the test may work more reliably on cooked food.

Suggestions for foodstuffs for testing:

• Place a small piece of each of the foods to be tested on the plastic sheet.
• Place a small potassium iodide crystal on top of the piece of food.
• Add one drop of bleach solution (sodium hypochlorite solution) and allow it to run over both crystal and food.
• If an intense blue-black colour is seen, the food contains starch.
• Clean the plastic sheet with a moistened paper towel.

Teaching notes

The chlorine available from the bleach solution reacts with potassium iodide to form potassium chloride and iodine. The iodine then forms an intense blue-black coloured complex with any starch present. If starch is not present, only the brown colour of iodine in the presence of iodide ions will be seen. The nature of the coloured complex is beyond the level of the students, but note that it is an unstable substance from which the iodine can be easily removed by, for example, sodium thiosulfate.

Each group can be allocated a selection from the range of available foodstuffs, perhaps two starchy foods, and two non-starchy. The class results can then be pooled.

Additional information

This is a resource from the  Practical Chemistry project , developed by the Nuffield Foundation and the Royal Society of Chemistry. This collection of over 200 practical activities demonstrates a wide range of chemical concepts and processes. Each activity contains comprehensive information for teachers and technicians, including full technical notes and step-by-step procedures. Practical Chemistry activities accompany  Practical Physics  and  Practical Biology .

© Nuffield Foundation and the Royal Society of Chemistry

• 11-14 years
• 14-16 years
• 16-18 years
• Practical experiments
• Applications of chemistry

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• Science Fair Project Ideas for Kids, Middle & High School Students

How-to Science Experiments for Kids With Iodine and Cornstarch

How to Make a Vitamin C Indicator

For a handy experiment you can show your young children or let your teens do with your supervision, two well-known experiments exist that demonstrate chemical reactions with iodine and cornstarch. Iodine is a common element found in many medicine cabinets. One of the properties of iodine is that it turns purple in the presence of starch, which is a common staple of most kitchens in the form of cornstarch. You can use this property to look at how starch reacts with different chemical and enzymes. The objective of the first experiment is to show how enzymes in saliva begin to digest the starch in the iodine and starch solution. Hypothesize with your audience how the starch and iodine solution changes when starch is digested. When you add saliva to the iodine and starch solution, the enzyme amylase breaks down starch in saliva to begin digestion, and the solution becomes clear while the control solution that has no saliva remains purple. The objective of the second experiment is to show how much vitamin C is in each juice. Vitamin C buffers the reaction between the iodine and starch and makes the purple color disappear. This experiment hypothesizes that the juice with the highest level of vitamin C will require the fewest drops to clear the purple color from the solution. Orange juice, with the highest vitamin C content will require the fewest drops to stop the reaction while cherry juice will require the most.

Saliva and Starch Digestion

Pour a teaspoon of water into one of the test tubes. Mark this "Tube A" with a piece of masking tape.

Spit into the teaspoon until it is full. Pour the saliva into the second test tube. Mark this "Tube B" with a piece of masking tape.

Measure 1/4 teaspoon of cornstarch and place in each test tube. Shake each tube to dissolve the starch.

Put on the safety glasses. Fill the eye dropper with iodine.

Place four drops of iodine into each test tube. Watch as the fluid in both tubes turns a deep blue color.

Place the tubes in the holder and leave them undisturbed for 30 minutes.

Check the color after 30 minutes. The test tube filled with water and cornstarch will still be purple. But the test tube with saliva will have lightened or even become clear. This is because the enzymes in saliva break down starch. This shows the first steps in digestion.

Exploring Vitamin C Content in Juice

Pour a cup of water into a bowl. Add 2 tablespoons of cornstarch and mix with the fork until the starch is completely dissolved.

Put on the safety glasses. Fill the eyedropper with iodine. Add the iodine to the cornstarch mixture one drop at a time until the entire mixture is a deep blue color. Empty the rest of the eyedropper. Rinse out the dropper with water.

Pour 2 tablespoons of the iodine and cornstarch mixture into four test tubes and place them in the rack. With masking tape and a pen, label each tube for Orange, Lemon, Apple, or Cherry juice.

Fill the eyedropper with orange juice. Put two drops into the first test tube. Swirl the tube to mix the solution. Continue to add juice and swirl until the solution is clear. Record the number of drops needed to make the solution clear.

Repeat with the other three juices, recorded the number of drops for each juice. Because ascorbic acid, or vitamin C, stops the reaction between cornstarch and iodine, the juice with the highest level of vitamin C will require the fewest drops to clear the solution. Juices that contain less vitamin C will require more drops of juice to clear the solution.

Things You'll Need

Tray other juices to figure out which juices have the highest concentration of vitamin C.

Iodine can stain skin, clothing and counter tops. Make sure you perform this for young children, and that teens and older children perform this only under your adult supervision.

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Based in Nashville, Shellie Braeuner has been writing articles since 1986 on topics including child rearing, entertainment, politics and home improvement. Her work has appeared in "The Tennessean" and "Borderlines" as well as a book from Simon & Schuster. Braeuner holds a Master of Education in developmental counseling from Vanderbilt University.

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The Biology Corner

Biology Teaching Resources

Macromolecules Lab – Testing for Starch

Most biology textbooks have a chapter on macromolecules. In chapter 2 of our textbook, students learn about the chemistry of life, which includes the basics of atoms and molecules, properties of water, and carbon compounds. I generally include a food testing lab where students determine the macromolecules found in food. The lesson is engaging, informative, and a lot of fun for everyone involved. It is also a cheap lab, that doesn’t require a lot of materials.

For my AP Biology class, they complete several tests on a blended up “Happy Meal.” The McMush lab is a gross (and popular) activity for my older students, but is quite messy. Though, it is a little much for a freshman biology class that is just getting started with laboratory techniques.

With freshman biology, I simplify this lab into a single test for starches. This is an easy test, where you will only need iodine . Then you provide students with samples to test in well plates, or other containers. Samples can include apples, rice, potatoes, or other common grocery items. If starch is present, the color of the sample with turn dark blue. If no starch is present, the color will remain a yellow-brown.

For many classes, this may be the first lab students complete. It is also a great way to introduce the concept of indicators , which students will see again in a diffusion lab later in the semester.

The Experiment

You can use well plates, petri dishes, or other containers for testing. Basically, whatever you have laying around will probably work. Begin with a short discussion on indicators and a negative and positive control test. Students place a corn starch solution into the container and place a drop of iodine. They should see a dramatic color change, which is the positive test. Then, they compare to a drop placed in plain water, which is the negative test.

Provide students with a variety of foods to test. The handout leaves the data table blank, so students will need to write in the names of foods they are testing. I use apples, bread, rice, potato flakes, milk, and anything else I can find that’s about to expire in the staff refrigerator. Students will get a positive test on anything with starch, like the potato flakes and bread.

In the final part of the lab, students design and conduct an experiment to determine if protein is present in egg whites. This section is open-ended, and students will use what they have learned in the first part to conduct their test using biuret solution , another indicator.

Related Activities

McMush Lab – testing for macromolecules in a Happy Meal

Diffusion Lab – Observing iodine diffuse across a membrane (bag)

Presentation Slides on the Chemistry of Life with student notes

Shannan Muskopf

Impressive experiments with iodine to show your kids

Easy experiments with iodine

The im­por­tance of io­dine in hu­man life

Io­dine is an el­e­ment that holds 53rd place in the pe­ri­od­ic ta­ble. It is a non-ra­dioac­tive non-met­al. It is very im­por­tant in hu­man life. A lack of io­dine in the body caus­es re­tar­da­tion in phys­i­cal and men­tal de­vel­op­ment, and growth de­fi­cien­cies. A deficit of io­dine also caus­es hy­per­thy­roidism. Al­though the io­dine con­tent in the or­gan­ism is low, 25 mg, this does not make it any less im­por­tant for the body. It also takes part in the me­tab­o­lism process. The io­dine in the body is main­ly con­tained in the thy­roid gland. So it is im­por­tant to in­clude ad­di­tion­al io­dine in our diet, es­pe­cial­ly in re­gions where there is a low io­dine con­tent in the wa­ter.

Io­dine is also found in na­ture, for ex­am­ple in al­gae. It is also pro­duced chem­i­cal­ly through cer­tain re­ac­tions.

A lit­tle his­to­ry about the dis­cov­ery of io­dine

In dis­cov­er­ies, ev­ery­thing is al­ways sim­ple and ac­ci­den­tal. The dis­cov­ery of io­dine can be blamed on a cat which knocked over so­lu­tions in flasks. One flask con­tained the re­mains of iod­ic salts treat­ed with salt­peter, and the oth­er con­tained sul­fu­ric acid. The cat’s own­er, the French chemist Bernard Cour­tois, no­ticed a vi­o­lent re­ac­tion when these two com­po­nents mixed, with the re­lease of pur­ple va­por. This was io­dine . An el­e­ment with­out which we could not imag­ine life.

Ex­per­i­ments with io­dine

Io­dine is a very good in­di­ca­tor, so any re­ac­tion with this el­e­ment is very easy to ob­serve. You can con­duct a few ex­per­i­ments with your kids, as these ex­per­i­ments are very sim­ple and ed­u­ca­tion­al. At school they don’t al­ways find the time for such ex­per­i­ments. So you can eas­i­ly car­ry them out at home and show the kids what an in­ter­est­ing sci­ence chem­istry is. Click here to find out how to re­veal fin­ger­prints with the help of io­dine Ex­per­i­ment “Find the starch”

With this ex­per­i­ment we can see what prod­ucts con­tain io­dine , and how much of it. You will need:

• 5% io­dine so­lu­tion;
• dis­pos­able cup;
• prod­ucts with and with­out starch.

First make an io­dine so­lu­tion. Even a child can do this. Take a cup, pour wa­ter into it and add a few drops of io­dine. The so­lu­tion is ready.

Now take dif­fer­ent types of food and place them on a plate: bread, rolled oats, a raw pota­to, a boiled pota­to, a lemon, a radish, a car­rot and a cu­cum­ber. Put sev­er­al drops of io­dine so­lu­tion on them and see how they re­act with io­dine. On the bread, oat meal, cheese and boiled pota­to, a re­ac­tion takes place, and the io­dine turns blue. We draw the con­clu­sion that these prod­ucts con­tain starch. We also con­clude that there is much starch in boiled pota­toes, as the col­or is rich­er. But in radish­es, lemons and cu­cum­bers, we do not ob­serve any starch. So by ex­per­i­ment, we clear­ly see the pres­ence of io­dine in dif­fer­ent types of food.

Ex­per­i­ment “In­ter­ac­tion of starch with io­dine”

To con­duct this ex­per­i­ment, you will need:

• 3 glass­es;

Boil a paste from starch. Take 3 glass­es and pour the paste into the 1st glass, starch with wa­ter into the 2nd, and just wa­ter into the third. Add sev­er­al drops of io­dine to each glass. See the re­sult. In the first cup we ob­serve a so­lu­tion with a rich blue col­or, in the sec­ond a so­lu­tion with a light blue col­or, and in the third a light brown col­or. We may con­clude that the re­ac­tion took place more ac­tive­ly with the paste. Heat-treat­ed starch gave a re­ac­tion more quick­ly.

Ex­per­i­ment “Dis­col­or­ing io­dine”

You can see the re­ac­tion of io­dine and ascor­bic acid For the ex­per­i­ment, take:

• io­dine so­lu­tion;
• 2 glass­es;
• so­lu­tion of ascor­bic acid;

For the so­lu­tion of ascor­bic acid, you will need 20 pills and 60 ml of wa­ter. Pour io­dine into the wa­ter con­tain­ing starch. We get a rich blue col­or. Then mix the so­lu­tion of ascor­bic acid with the io­dine so­lu­tion. The so­lu­tion in­stant­ly be­comes col­or­less. We see some “mag­ic”. The sci­ence of chem­istry does mir­a­cles.

You can car­ry out these ex­per­i­ments to­geth­er with your kids in your spare time. They will find them very in­ter­est­ing and mem­o­rable. Spend time with your chil­dren in a fun and ed­u­ca­tion­al way.

Dozens of experiments you can do at home

One of the most exciting and ambitious home-chemistry educational projects The Royal Society of Chemistry

BIOCHEMINSIDER

Iodine Test: Description, Principle, Procedure And Result Interpretation

What is iodine test.

The iodine test was first described by J.J Colin and H.F Gaultier de Claubry and independently by F. Stromeyer in 1884. The iodine test is used to test for the presence of starch in a given analyte. The test can be qualitative or quantitative.

Objective Of Iodine Test

• To test for the presence of starch in biological molecules.

Principle Of Iodine Test

This test depends upon the property of adsorption possessed by the large polysaccharide molecules. Starch contains alpha-amylose, helical saccharide polymer and amylopectin. Triiodide anion instantly produces an intense blue-black color upon contact with starch. This reaction is as a result of the formation of polyiodide chains from the reaction of starch and iodine. The amylose or straight chain portion of starch, forms helices where iodine molecules assemble, forming a dark-blue/black color. The amylopectic or branched portion of starch forms much shorter helices and iodine molecules are unable to assemble, leading the color to be of an orange/yellow hue.

Generally amylopectin, glycogen and cellulose do not form alpha-helices, they do not complex well with iodine, therefore, they do not show the blue-black color; instead they show a purple or brown color. Monosaccharides on the other hand do not interact with the iodine, therefore no color is produced.

The color obtained depends upon the length of the unbranched or linear chain available for complex formation. Also, the intensity of the color produced decreases with increasing temperature and with the presence of water-miscible organic solvents such as ethanol. Iodine test cannot be performed at very low PH due to the hydrolysis of the starch under these conditions.

Reagent And Material Required

• Iodine Reagent: 0.5 ml iodine diluted in 5 ml distilled water and mixed with 10% potassium iodide to form Iodine solution (Lugol’s iodine)
• Dropper or pipette
• Test sample or solution

Iodine Test Procedure

• Take 1 ml of the test sample in a clean, dry test tube.
• Similarly, take another 1 ml of distilled water in another tube.
• Add about 2-3 drops of Iodine solution to both test tubes and mix thoroughly.
• Observe the appearance of color in the test tubes.
• Heat the test tubes in the water bath until the color disappears.
• Take the test tubes out for cooling.
• Note down the appearance of color seen in the test tubes.

Iodine Test Result Interpretation

• Positive Test : A positive test is indicated by presence of a blue-black or purple color in the test tube. This confirms presence of starch.
• Negative Test: A negative test is indicated by no change in color of iodine solution. This confirms absence of starch.

Limitations Of Iodine Test

• This test cannot be done under acidic conditions as the starch hydrolyses under such circumstances.
• This test is a qualitative test and doesn’t signify the concentration of starch.

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Selective Permeability of Dialysis Tubing Lab: Explained

• Selective Permeability of Dialysis Tubing…

This dialysis tube experiment experiment was conducted to investigate the selective permeability of dialysis tubing. The permeability of the tubing to glucose, starch, and iodine (potassium iodide) was tested. The dialysis tubing was clipped to form a bag so that glucose and starch were fed into the bag through the other end, and was also clipped to avoid the seeping of the solution.

Water with several drops of iodine added to it until it was visibly yellow-amber was added to a 400ml beaker. The bag was then placed in the beaker, which was stirred with a magnetic stirrer. It was left there for 30 minutes. It was seen that the color of the solution in the bag changed to blue-black color, which showed that iodine was able to pass through the membrane into the bag.

The solution in the beaker became pale yellow-amber, showing that starch didn’t pass through the membrane into the beaker. To confirm the presence of glucose in the beaker and the bag, a Benedict test was performed on the solutions including tap water (control) too.

The beaker solution turned light brown after Benedict’s solution was added to it and suspended in a water bath for 10 minutes. The dialysis bag solution also changed to brown color, while tap water remained blue. This experiment showed that dialysis tubing is selective in its permeability to molecules. It was permeable to glucose and iodine but not starch.

INTRODUCTION:

PURPOSE: The purpose of the dialysis bag experiment was to test the permeability of dialysis tubing to glucose, starch, and iodine.

Living cells need to obtain nutrients from their environment and get rid of waste materials to their surroundings. This exchange of materials between the cell and its surroundings is crucial to its existence. Cells have membranes composed of a phospholipid bilayer embedded with proteins.

This cell membrane can distinguish between different substances, slowing or hindering the movement of other substances and allowing others to pass through readily. This property of the cell is known as selective permeability (Ramlingam, 2008).

Selective permeability is a property of a cell membrane that allows it to control which molecules can pass (moving into and out of the cell) through the pores of the membrane. Selectively permeable membranes only allow small molecules such as glucose and amino acids to readily pass through and inhibit larger molecules like protein and starch from passing through it.

The dialysis tubing is a semi-permeable membrane tubing used in separation techniques and demonstration of diffusion, osmosis, and movement of molecules across a restrictive membrane (Todd, 2012). It separates dissolved substances of different molecular sizes in a solution, and some of the substances may readily pass through the pores of the membrane while others are excluded. The dialysis tubing is made up of cellulose fibers shaped in a flat tube.

In this dialysis tubing lab experiment, the selective permeability of dialysis tubing to glucose, starch, and iodine (potassium iodide) will be tested. This experiment consists of two tests: the test for starch and the test for reducing sugar. When iodine (potassium iodide) is added to a solution in which starch is present, the solution turns blue-black or purple; otherwise, it remains yellow-amber.

When Benedict’s reagent is added to a solution in which reducing sugar is present and it is heated in a water bath, the solution turns green, yellow, orange, red, and then brick red or brown (with a high concentration of sugar present). Otherwise, the solution remains blue.

Will glucose, starch, and iodine (potassium iodide) readily pass through the pores of the dialysis tubing?

HYPOTHESIS:

Glucose, starch, and iodine (potassium iodide) will readily pass through the membrane of the dialysis tubing.

Dialysis Lab Report Prediction:

The solution in the bag and the beaker will both turn blue-black due to the presence of iodine and starch; the presence of glucose in the bag and beaker will be investigated using Benedict’s test.

• Dialysis Tubing
• Test Tubes rack
• Benedict’s reagent
• Iodine (Potassium Iodide)

EXPERIMENT PROCEDURE:

1) 250 ml of tap water was added to a beaker. Several droppers of Iodine (Potassium Iodide) solution was added to the water until it was visibly yellow-amber in color. The color was then recorded.

2) The dialysis tubing was soaked in water for a few minutes until it began to open. One end of the bag was folded and clipped in order to secure it so that no solution seeped through.

3) The other end of the tubing was opened so that it forms a bag and 4ml of glucose and 3ml of starch was fed into it. The bag was also closed and its content was mixed. The color of the solution was then recorded.

4) The outside of the bag was rinsed in tap water.

5) The magnetic stirrer and then the bag was placed in the beaker. The other end of the bag was made to hang over the edge of the beaker.

6) The bag was left in the beaker for about 30 minutes, as the beaker was being stirred.

7) After 30 minutes, the bag was carefully removed and made to stand in a dry beaker. The final color of the solutions was recorded.

8) Benedict test was performed to test for the presence of reducing sugar in the solution in the bag, beaker and tap water (serves as control).

• a) 3 test tubes were labelled control, bag and beaker.
• b) 2 ml of water was added to the control test tube. 2 ml of the bag solution was added to the bag test tube and 2 ml of the beaker solution was added to the beaker test tube.
• c) 2 ml of Benedict’s reagent was added to each test tube and was suspended in a boiling water bath for 10 minutes. The color change was recorded.

The solution in the bag turned blue-black in color owing to the movement of molecules of iodine from the beaker to the bag which contains starch. The solution in the beaker turned brown after Benedict’s test, indicating the presence of glucose in the beaker. This means that the dialysis tubing was permeable to both glucose and iodine but not starch. It is known that starch didn’t pass because the solution in the beaker, which contains iodine, didn’t turn blue-black in color but remained yellow-amber.

DISCUSSION:

• How can you explain your results?

From the results of the experiment represented in a tabular form above, the hypothesis suggested before carrying out the experiment turned out to be incorrect. The dialysis tubing was not permeable to all three solutions: glucose, starch, and iodine (potassium iodide). Rather, the tubing was permeable to glucose and iodine but not starch.

This could be known from the color change in the solutions in the beaker and the bag. The dialysis tubing was permeable to iodine, so the content of the bag turned blue-black in color, indicating the presence of starch. Glucose also readily passed through the pores of the membrane. After performing Benedict’s test on the solutions, the bag’s solution as well as the beaker’s solution turned brown in color. This shows the presence of reducing sugar in both solutions, meaning that glucose passed into the beaker from the bag.

• From your results, predict the size of iodine (potassium iodide) relative to starch.

From the results of this experiment, it is obvious that glucose and iodine (potassium iodide) have smaller molecular sizes than starch. Because starch had a larger molecular size, the dialysis tubing was not permeable to it (it didn’t allow it to readily pass through the pores of its membrane).

• What colors would you expect if the experiment started with glucose and iodine (potassium iodide) inside the bag and starch in the beaker? Explain.
• The solution in the bag will remain yellow-amber in color at the end of the experiment.
• The solution in the beaker will turn blue-black in color at the end of the experiment.
• After performing Benedict’s test, both solutions will turn brown in color.

The solution in the bag remained yellow-amber in color at the end of the experiment because the dialysis tubing is not permeable to starch, so starch didn’t pass through from the beaker into the bag. The solution in the beaker turned blue-black in color at the end of the experiment because iodine passed from the bag into the beaker through the membrane. After performing Benedict’s test on the bag and beaker solution, both solutions turned brown in color because the dialysis tubing was permeable to glucose, so glucose readily passed from the bag into the beaker through the membrane.

Precautions

• It was ensured that the right quantity of solutions was used in every part of the experiment.
• It was also ensured that the time required for the successful complement of the experiment was adhered to.
• It was ensured that all apparatus used were handled with caution.
• The dialysis tubing was clipped well on both ends to secure it so that no solution seeped through.

It was concluded that the dialysis tubing investigation doesn’t allow all kinds of substances to pass readily through the pores of its membrane. This means that it is selective in its permeability to substances. The dialysis tubing was permeable to glucose and iodine but not to starch. Starch was excluded because it has a larger molecular size than glucose and iodine.

Ramlingam, S. T. (2008). Modern Biology. Onitsha: African First Publishers.

Todd, I. S. (2012). Dialysis: History, Development and Promise. World Scientific Publishing Co Pte Ltd.

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18 Comments

Could oxygen pass through the dialysis tubing

so what was the chemical formula for this experiment?

After 24 hours of leaving the bag in the iodine solution; -The dialysis bag turned dark blue/purple, Explain. -The fructose test strip turned positive when dipped in the solution, Explain.  

if the dialysis represent the membrane of a root air cell, and the sugar solution inside represent the cells cytoplasm, which is hypotonic, hypertonic or isotonic. is there any movement of iodine molecules?

What is the purpose of the Iodine Solution?

you added starch and glucose to dialysis tubing, a semipermeable membrane that mimics the plasma membrane of cells. The filled tubing which was placed in a beaker of water containing iodine. What is the purpose of the iodine?

Is the iodine entering the dialysis tube an example of diffusion or osmosis? or can osmosis only occur with water?

what was the purpose of placing the dialysis tubing containing starch solution into the beaker of distilled water

What were the limitations of your experiment ?

What about the NaCl? I did this lab but we had a question if NaCl moved out of the tube.

what did not diffuse through the membrane

Starch and Benedict’s solution.

Maybe the starch and its size.

How can you explain the change in weight of the cells?

osmosis of water

Include an analysis maybe? all around good job though!

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Mutations in starch BRANCHING ENZYME 2a suppress the traits caused by the loss of ISOAMYLASE1 in barley

• Original Article
• Open access
• Published: 31 August 2024
• Volume 137 , article number  212 , ( 2024 )

Cite this article

You have full access to this open access article

• Ryo Matsushima   ORCID: orcid.org/0000-0002-8500-7804 1 , 3 ,
• Hiroshi Hisano 1 ,
• June-Sik Kim 1 , 2 ,
• Rose McNelly 3 ,
• Naoko F. Oitome 4 ,
• David Seung 3 ,
• Naoko Fujita 4 &
• Kazuhiro Sato 1  

Key message

The hvbe2a mutations restore the starch-deficient phenotype caused by the hvisa1 and hvflo6 mutations in barley endosperm.

The genetic interactions among starch biosynthesis genes can be exploited to alter starch properties, but they remain poorly understood due to the various combinations of mutations to be tested. Here, we isolated two novel barley mutants defective in starch BRANCHING ENZYME 2a ( hvbe2a-1 and hvbe2a-2 ) based on the starch granule (SG) morphology. Both hvbe2a mutants showed elongated SGs in the endosperm and increased resistant starch content. hvbe2a-1 had a base change in HvBE2a gene, substituting the amino acid essential for its enzyme activity, while hvbe2a-2 is completely missing HvBE2a due to a chromosomal deletion. Further genetic crosses with barley isoamylase1 mutants ( hvisa1) revealed that both hvbe2a mutations could suppress defects in endosperm caused by hvisa1 , such as reduction in starch, increase in phytoglycogen, and changes in the glucan chain length distribution. Remarkably, hvbe2a mutations also transformed the endosperm SG morphology from the compound SG caused by hvisa1 to bimodal simple SGs, resembling that of wild-type barley. The suppressive impact was in competition with floury endosperm 6 mutation ( hvflo6 ), which could enhance the phenotype of hvisa1 in the endosperm. In contrast, the compound SG formation induced by the hvflo6 hvisa1 mutation in pollen was not suppressed by hvbe2a mutations. Our findings provide new insights into genetic interactions in the starch biosynthetic pathway, demonstrating how specific genetic alterations can influence starch properties and SG morphology, with potential applications in cereal breeding for desired starch properties.

Avoid common mistakes on your manuscript.

Introduction

Starch, a glucose-based polymer from plants, is extensively utilized in various food and industrial sectors. Its water-insolubility and lack of osmotic activity make it an ideal substance for long-term storage in seeds, grains, and roots. Within these storage organs, starch forms semi-crystalline starch granules (SGs) produced in specialized plastids called amyloplasts (Gunning and Steer 1996 ). SGs vary in size and shape across different plant species and are broadly categorized as either compound or simple SGs (Tateoka 1962 ; Matsushima et al. 2013 ; Chen et al. 2021 ). Compound SGs consist of assemblies of smaller starch particles, whereas simple SGs are composed of single starch particles. In the case of rice ( Oryza sativa ) endosperm, the compound SGs measure about 10–20 μm in diameter and are made up of polyhedral starch particles, each measuring 3–8 μm (Matsushima et al. 2010 ). Meanwhile, the endosperm of barley ( Hordeum vulgare ) and wheat ( Triticum aestivum ) features two types of simple SGs, the smaller B-type (~ 5 μm diameter) and the larger A-type (10–30 μm diameter), both existing together within a single cell (Jane et al. 1994 ; Matsushima and Hisano 2019 ; Thieme et al. 2023 ). This type is referred to as bimodal simple SGs. The A-type granules begin to form in the early stages of grain development within amyloplasts, followed by the development of B-type granules in the same amyloplasts, which already contain A-type granules (Langeveld et al. 2000 ; Matsushima and Hisano 2019 ; Kamble et al. 2023 ).

The size and shape of SGs are influenced by specific enzymes involved in synthesizing amylopectin, the primary polymer component of SGs, which is made up of α-1,4-linked and α-1,6-branched glucose chains (Smith and Zeeman 2020 ; Tetlow and Bertoft 2020 ). Amylopectin synthesis involves three key reactions: the formation of α-1,4-glycosidic bonds by starch synthases (SSs), which elongate the glucose chains; the creation of α-1,6-glycosidic bonds by branching enzymes (BEs), introducing branches in the structure; and the removal of misplaced glucose branches by hydrolyzing α-1,6 linkages with starch debranching enzymes (DBEs) (Nakamura 2002 ; Pfister and Zeeman 2016 ). In the absence of appropriate trimming by DBEs, improperly positioned branches can prevent the formation of the semi-crystalline structure of amylopectin. Deficiency of one of the DBEs, ISOAMYLASE1 (ISA1), leads to the accumulation of phytoglycogen, a water-soluble α-glucan (Pan and Nelson 1984 ; Nakamura et al. 1997 ; Burton et al. 2002 ). Phytoglycogen is characterized by its extensive branching with short glucan chains and has a molecular weight significantly lower than that of amylopectin. In barley ISA1 mutants ( hvisa1 ), the endosperm forms compound SGs, contrasting with the typical bimodal simple SGs found in the wild type (Burton et al. 2002 ; Matsushima et al. 2023 ). Cereal mutants exhibiting defects in starch biosynthetic enzymes hold considerable interest in the food industry due to their capability to alter the physicochemical and nutritional properties of starch within the grain (Nakamura 2018 ). For example, mutations in the ISA1 gene of maize lead to increased soluble sugars, instead of starch in the grains and have been utilized in breeding sweet corn varieties (Gonzales et al. 1976 ; Pan and Nelson 1984 ; James et al. 1995 ). Furthermore, mutants in the BE genes of rice and maize are noted to enhance the production of resistant starch, known for its resistance to digestion by amylase (Xia et al. 2011 ; Tsuiki et al. 2016 ; Chen et al. 2022 ). The intake of resistant starch changes the composition of gut microbiota in mice, marked by an increase in specific beneficial bacteria (Li et al. 2024 ). This contributes to the improvement in obesity by reducing lipid absorption, decreasing inflammation, and enhancing intestinal barrier strength (Blaak et al. 2020 ).

Recent studies have identified several non-enzymatic proteins that directly interact with starch biosynthetic enzymes and also affect SG shape and size. These proteins include the Arabidopsis thaliana PROTEIN TARGETING TO STARCH (PTST) 1 to 3, the rice PTST2 ortholog, FLOURY ENDOSPERM 6 (FLO6) and its barley and wheat orthologs, HvFLO6 and B-GRANULE CONTENT1 (BGC1), respectively (Peng et al. 2014 ; Seung et al. 2015 , 2017 ; Saito et al. 2018 ; Chia et al. 2020 ). These proteins possess Carbohydrate-Binding Module 48 (CBM48) domains, known for their affinity towards binding starch and maltooligosaccharides (Peng et al. 2014 ; Seung et al. 2015 , 2017 ). CBM48s are also found in the sequences of BEs and DBEs, but not in SSs (Pfister and Zeeman 2016 ). CBM48s probably facilitate the efficient transfer of the substrate to starch biosynthetic enzymes. In the barley hvflo6 mutant (also known as Franubet ), the endosperm develops an unusual mix of simple and compound SGs (DeHaas BW and Goering KJ 1983 ; Chung T-Y 2001 ; Suh et al. 2004 ; Matsushima et al. 2023 ). In wheat, the reduced expression of BGC1 decreases B-type granule content in endosperm (Chia et al. 2017 , 2020 ). In addition, the non-enzymatic protein, LIKE EARLY STARVATION (LESV), was recently reported to be involved in starch biosynthesis in Arabidopsis and rice (Liu et al. 2023 ; Dong et al. 2024 ; Yan et al. 2024 ). In rice, LESV can interact with FLO6 and mediate the localization of starch biosynthetic enzymes to starch (Dong et al. 2024 ; Yan et al. 2024 ).

Studies on the genetic interactions among mutations in starch biosynthesis genes have mainly focused on rice and maize (Ferguson et al. 1979 ; Toyosawa et al. 2016 ; Lee et al. 2017 ; Ida et al. 2021 ; Nagamatsu et al. 2022 ). In barley, the reduction in starch content and the corresponding increase in phytoglycogen in the hvisa1 grains is further enhanced by the hvflo6 mutation (Matsushima et al. 2023 ). The enhancement exceeds just an additive effect, indicating potential genetic interactions between the two mutations. The genetic interaction between hvflo6 and hvisa1 is also observed in the pollen grain, which accumulates starch like the endosperm of cereals. In both wild-type and hvisa1 pollen, simple, rod-shaped SGs predominantly are developed. In the hvflo6 pollen, the number of compound SGs is increased, and this increase is significantly pronounced in the hvflo6 hvisa1 double mutant (Matsushima et al. 2023 ).

In our previous study, we reported on the hvisa1 and hvflo6 mutants in barley, particularly in the elite Japanese malting barley cultivar ‘Haruna Nijo’, using the rapid, simple observation method of SGs (Matsushima et al. 2023 ). Unlike other procedures, this method does not require chemical fixation and resin embedding of samples, making it suitable for dealing with large number of samples (Matsushima et al. 2010 ). In this study, we report newly identified barley mutants, hvbe2a-1 and hvbe2a-2 , which develop elongated SGs in endosperm, that are not present in the wild type. Both mutants had genetic lesions in the HvBE2a gene coding a major BE in barley endosperm. Our analysis using triple and double mutants of hvisa1 , hvflo6 and hvbe2a revealed that hvbe2a mutation has a suppressive effect against hvisa1 mutation in endosperm. hvisa1 mutation altered the SG morphology from a bimodal to a compound type, and the addition of hvbe2a mutation reversed the SG morphology back to the bimodal form. In contrast to the endosperm, the suppressive effect of hvbe2a was not observed in pollen. These findings contribute to our understanding of the genetic network in starch biosynthesis and provide new insights into the determinants of SG morphology.

Materials and methods

Plant material and growth conditions.

Barley cultivar’Haruna Nijo’ was provided from NBRP-Barley ( http://earth.nig.ac.jp/~dclust/cgi-bin/index.cgi?lang=en ).  hvisa1-3 and hvflo6-2 were previously reported (Matsushima et al. 2023 ). hvbe2a-1 and hvbe2a-2 were isolated from the same screening described previously (Matsushima et al. 2023 ). The mutants were crossed with cv Haruna Nijo, and the progeny exhibiting hvbe2a phenotype were used in this study. Double and triple mutants were generated by artificially crossing individual mutants, and multiple homozygous individuals were selected from the F2 progeny using PCR-based genotyping. Barley plants were grown at 22 °C/18 °C in a growth cabinet (NK Systems, LPH-411S) or around 23 °C continuously at 16 h day/8 h night conditions. The shoot weight and number of tillers were measured 28 days after germination.

Observation of SGs by thin-section microscopy with Technovit 7100 Resin

The preparation of Technovit sections to observe SGs in the endosperm is described previously (Matsushima et al. 2014 ).

Purification of starch and quantification of starch granule size distributions

Starch purification was essentially conducted following Kamble et al. ( 2023 ). A grain was ground using the Multi-beads Shocker MB2000 (YASUI KIKAI, Japan), and the sample was resuspended in 2 mL water. The homogenates were filtered through a 100-μm nylon mesh, then centrifuged at 3,000 ×  g for 5 min. The pellet was resuspended in 2 mL water. The starch suspension on a 5-mL cushion of 90% (v/v) Percoll in 50 mM Tris–HCl (pH 8) was centrifuged at 2,500 ×  g for 15 min. The pellet was washed twice in 50 mM Tris–HCl (pH 6.8), 10 mM EDTA, 4% SDS (v/v), 10 mM DTT and then three times in water. Finally, the purified starch was washed with ethanol twice and dried.

To quantify starch granule size distributions, the purified starch was suspended in Isoton II electrolyte solution (Beckman Coulter, Indianapolis), and particle sizes were measured using a Multisizer 4e Coulter Counter (Beckman Coulter) fitted with a 70-μm aperture tube. A minimum of 100,000 particles was measured per sample. These data were used to produce relative percent volume vs. diameter plots. A mixed bimodal distribution (normal and lognormal distributions) was fitted to the relative percent volume vs. diameter plots (Python script available at: https://github.com/DavidSeungLab/Coulter-Counter-Data-Analysis ) to calculate mean diameters of A- and B-type granules by fitting normal curves for A-type and lognormal curves for B-type to the granule size distribution traces (Hawkins et al. 2021 ).

Preparation of antibodies against HvBE2a, HvBE2b and HvBE1

All of the antibodies in this study were raised against synthetic peptides. Anti-HvBE2a antibodies were raised against AAAPGKVLVPDGESDDL (amino acid position from 52 to 68 of HvBE2a) and VDYFTTEHPHDNRPRS (amino acid position from 789 to 804 of HvBE2a). The two antibodies were named anti-HvBE2a-N and anti-HvBE2a-C, respectively. Anti-HvBE2b antibody was raised against AGGPSGEVMI (amino acid position from 58 to 67 of HvBE2b). Anti-HvBE1 antibody was raised against KRGINFVFRSPDKDNK (amino acid position from 810 to 825 of HvBE1). The immunization of rabbits and purification of antibodies were outsourced to Cosmo Bio (Tokyo, Japan) or Eurofins Genomics (Tokyo, Japan).

Immunoblot analysis following SDS-PAGE

Developing grains at 14 days after awn emergence (DAA) were cultivated and stored at − 80 °C before use. The grain was mixed with 150 μL of ice-cold grinding solution (50 mM imidazole–HCl [pH 7.4], 8 mM MgCl 2 , 12.5% [v/v] glycerol), and then crushed using plastic pestles. The homogenates were centrifugated at 16,000 ×  g at 4 °C for 5 min. The supernatant (50 μL) was mixed with 50 μL of SDS-sample buffer (2% [w/v] SDS, 100 mM Tris–HCl [pH 6.8], 2% [v/v] 2-mercaptoethanol, 40% [v/v] glycerol) and incubated at 98 °C for 10 min. 8 μL was subjected to SDS-PAGE, and proteins were transferred electrophoretically to a polyvinylidene difluoride membrane (Millipore). The membrane was then incubated in Phosphate-buffered saline (pH 7.4) plus 0.05% (v/v) Tween–20 with the antibodies for 1 h. Dilutions of the antibodies were 1:3,000—5,000 (v/v). The secondary antibody was an Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey (Cytiva, NA934V), which was diluted (1:5,000). The immunoreactive bands were detected with Immobilon Crescendo Western HRP substrate (Millipore, WBLUR0500).

Quantification of starch and phytoglycogen

Total starch and phytoglycogen quantification in a grain are described previously (Matsushima et al. 2023 ). The Resistant Starch Assay Kit (Megazyme, K-RSTAR) was used according to the manufacturer’s instructions to measure the amount of resistant starch as a percentage of total starch.

Glucan chain length distribution of total α-glucan in grain

The procedures to detect the glucan-chain-length distribution of total α-glucan are the same as in our previous study (Matsushima et al. 2023 ).

Genotyping of mutations

The base change of  hvbe2a-1  mutation was detected by the derived cleaved-amplified polymorphic sequence primers: 5’-TTAGGTGGCGAAGGCTATCTTAATTCCATG-3’ and 5’-GTTCAAATTACAATAAATCGCAACC-3’. The PCR conditions were as follows: 94 °C for 2 min and 35 cycles of 94 °C for 30 s, 53 °C for 45 s, and 68 °C for 1 min. The PCR product was digested with  Nco I, and PCR products were subsequently separated by 15% polyacrylamide gel electrophoresis (PAGE) and detected with ethidium bromide staining. In the case of wild type, a PCR product (109 bp) was digested into 83 and 26 bp. In the case of  hvbe2a-1 , the PCR product was not digested. hvisa1-3 and hvflo6-2  mutation sites were detected according to the previous paper (Matsushima et al. 2023 ).

Genome sequencing of hvbe2a-2

High-molecular-weight DNA was isolated from leaf material of hvbe2a-2 seedlings using NucleoBond HMW DNA (Takara). A total of 0.5 μg of the isolated DNA underwent sequencing library preparation using the MGIEasy FS PCR-Free DNA library Prep Kit (MGI Tech, Shenzhen, China). Whole-genome sequencing was performed on the DNBSEQ-G400RS platform (MGI Tech) by a commercial vendor (Genome-Lead Co., Kagawa, Japan), yielding 375.9 million reads (2 × 150 nucleotides) of the hvbe2a-2 genome. The genomic sequence reads of barley cv Haruna Nijo (1.5 billion reads, 2 × 100 nucleotides) were obtained from a previous report (Sato et al. 2016 ). These sequence reads underwent quality control and were trimmed using Trimmomatic version 0.39 (Bolger et al. 2014 ), then mapped to the reference sequence assembly of Haruna Nijo (Sakkour et al. 2022 ) using bwa-mem version 0.7.17-r1188 (Li and Durbin 2009 ) with the default parameters. Only uniquely mapped reads were retained, then the read depth for genomic regions were assessed and visualized using IGV version 2.16.2 (Robinson et al. 2011 ).

Detection of branching enzyme activity in the developing endosperm following Native-PAGE

After thawing the harvested grains at 14 DAA, the removal of the embryo portion was followed by applying gentle pressure to the opposite side, facilitating the extraction of the endosperm from the grain. The endosperm was homogenized in the ice-cold grinding solution (80 μL), and then crushed using plastic pestles. The homogenates were subjected to centrifugation at 16,000 ×  g at 4 °C for 5 min. Protein concentration of the supernatant was determined using a Bradford Protein Assay Kit (Takara, Japan, T9310A). The supernatant was mixed with sample buffer (400 mM Tris–HCl [pH 7.0], 33% [v/v] glycerol) to adjust the protein concentration to 1 μg/μL. Proteins (7.5 μg) were subjected to Native-PAGE. BE activity staining was assessed using a gel containing 0.0001% oyster glycogen (Yamanouchi and Nakamura 1992 ). The immunoblot analysis following the Native-PAGE is identical to the analysis following SDS-PAGE.

Observation of SGs in pollen grains

To stain SGs in mature pollen, anthers at 3–5 DAA were disrupted with forceps in 120-times diluted Lugol solution on a glass cover slide. The released pollen was squashed by putting gentle pressure on a coverslip to release SGs from pollen vegetative cells. The released SGs were observed with a microscope (Olympus, BX53). For the quantification of compound SGs in pollen, the SGs within the 30 μm × 30 μm field of view were classified into simple and compound forms through visual inspection.

Isolation of pollen grains and protein extraction from pollen

Anthers, at 3 DAA, were collected in a 1.5 mL plastic tube and stored at − 80 °C until use. The anthers were mixed with the ice-cold grinding solution (1 mL) and chopped using a surgical scissor (Hammacher, HSB022-12). The homogenates were then filtered through a 100-μm nylon mesh to remove the debris other than pollen. The flow through was centrifuged at 1,000 ×  g at 4 °C for 1 min. The resulting pellet of pollen grains was resuspended in the ice-cold grinding solution (500 μL) and centrifuged again. The pollen pellet was resuspended once more in the ice-cold grinding solution (50 μL) and disrupted using plastic pestles in the tube. The homogenates were centrifuged at 10,000 ×  g at 4 °C for 2 min. The supernatant was recovered in another tube and protein concentration was measured using the Bradford Protein Assay kit. The supernatant was mixed with grinding solution and SDS-sample buffer to adjust the protein concentration to 0.5 μg/μL and heated immediately at 98 °C for 10 min. The resulting protein extract containing 5 μg of total protein was subjected to SDS-PAGE and immunoblot analysis.

Barley mutants with elongated starch granules in endosperm

In our screen for barley mutants with altered SG morphology, we examined endosperm SGs in a barley sodium-azide-mutagenized population derived from the Japanese elite malting barley cultivar Haruna Nijo (Matsushima et al. 2023 ). We isolated mutants, namely hvbe2a-1 and hvbe2a-2 , which exhibited elongated SGs in the endosperm (Fig.  1 ). Subsequent sequencing analysis identified the causative mutations are in the HvBE2a gene of both mutants (described in a subsequent section); for clarity, we will use the hvbe2a-1 and hvbe2a-2 nomenclature to refer to mutants of interest. Haruna Nijo refers to the wild-type reference in this paper.

Isolation of barley mutants with elongated starch granules. a – c Mature grains of Haruna Nijo a and hvbe2a-1 b and hvbe2a-2 c . Front and side views are shown. Bars = 1 mm. d Single grain weight of Haruna Nijo, hvbe2a-1 and hvbe2a-2 at the mature stage (n = 30, 30, 70, respectively). Statistical comparisons were performed using Welch’s t -test (ns, not significant at p  = 0.05). e – j Iodine-stained thin sections of endosperm cells of Haruna Nijo (e and f), hvbe2a-1 (g and h), and hvbe2a-2 (i and j). Bars = 20 μm. k – m Granule size distributions of Haruna Nijo, hvbe2a-1 and hvbe2a-2 , respectively. The relative percent volume of each diameter was determined using a Coulter Counter (n = 4 each). The graphs are displayed by overlaying the data from four biological replicates with different colors. n – o The average diameter of A- and B-type granules, respectively, extracted from the relative percent volume vs. diameter plots (k-m) by fitting a bimodal mixed normal and lognormal distribution. p Resistant starch amount in total starch of hvbe2a grains (n = 4 each). Statistical comparisons were performed using Wilcoxon rank sum test (*, p  < 0.05). q Glucan chain-length distribution of α-glucans in hvbe2a mutant grains. hvbe2a-1 , hvbe2a-2 and Haruna Nijo are indicated by green, blue and grey lines, respectively. Data are given as means ± SD. All data were obtained from at least three independent grains. r Difference plot corresponding to the glucan-chain-length distribution profile presented in (q). The value for each chain length of Haruna Nijo was subtracted from that of the hvbe2a mutants

The mature grains of these mutants displayed a similar appearance to those of the wild type (Fig.  1 a–c). The grain weight of the hvbe2a-1 and hvbe2a-2 were almost the same as those of Haruna Nijo (Fig.  1 d). The dimensions of grains, including length, width, and thickness, from the hvbe2a-1 mutant were almost the same as those of Haruna Nijo. Certain measurements from the hvbe2a-2 mutant showed small changes compared to Haruna Nijo, but they were not consistently significant for both hvbe2a-1 and hvbe2a-2 (Supplementary Fig. 1a–c). This means that they are not caused by the mutation per se.

Iodine-stained Technovit thin sections of the endosperm revealed that Haruna Nijo developed typical bimodal SGs (Fig.  1 e, f). In contrast, in hvbe2a-1 (Fig.  1 g, h) and hvbe2a-2 (Fig.  1 i, j) mutants, some SGs displayed elongated shapes. Coulter Counter analysis demonstrated that SGs from both Haruna Nijo and the hvbe2a mutants exhibited a similar bimodal distribution (Fig.  1 k–m). The average diameters of A- and B-type SGs were not significantly different between Haruna Nijo and the hvbe2a mutants (Fig.  1 n, o). The minimal changes on the distribution curves detected by the Coulter Counter analysis indicates that the elongated SGs in the hvbe2a mutants were either similar in volume to normal A-type granules, or not the majority. Next, we measured the amount of resistant starch in the hvbe2a mutant grains. Both hvbe2a-1 and hvbe2a-2 grains contained higher amount of resistant starch compared to Haruna Nijo; however, significant variation was observed among the biological replicates (Fig.  1 p). The glucan chain length distribution of α-glucan from hvbe2a grains showed subtle differences compared to Haruna Nijo (Fig.  1 q). The differential plot showed that both hvbe2a mutants had decreased glucose chains at degree of polymerization (DP) 11, while showing an increase in the frequency of chains at DP 17, relative to Haruna Nijo (Fig.  1 r).

Plant appearance at 28 days after germination showed no significant difference in tiller numbers and shoot weights among Haruna Nijo, hvbe2a-1 and hvbe2a-2 mutants (Supplementary Fig. 2a–e). The panicle appearance of hvbe2a mutants was similar to that of Haruna Nijo at 20 DAA and mature stage (Supplementary Fig. 2f–k).

Genetic lesion in BRANCHING ENZYME 2a gene in barley

The F1  plant from the cross between hvbe2a-1 and hvbe2a-2 exhibited elongated SGs just like hvbe2a-1 and hvbe2a-2 (Fig.  2 a–d). This means that hvbe2a-1 and hvbe2a-2 are allelic to each other. The previous study by Regina ( 2010 ) revealed that barley transgenic lines with RNAi-suppressed HvBE2a genes exhibited the occasional alternations of SGs in morphology in endosperm. Furthermore, the RNAi lines showed a decrease in the distribution of glucan chain length around DP10 and an increase around DP15 compared to the parental line (Regina et al. 2010 ). This pattern closely resembled the profile observed in hvbe2a mutants in Fig.  1 r. The phenotypic similarities encouraged us to determine the sequence of the HvBE2a gene in hvbe2a mutants. We amplified the genomic region (chr2H:464,360,851–464,373,337 in Haruna Nijo pseudomolecules v1.) covering the HvBE2a cDNA sequence (AF064560) from hvbe2a - 1 and determined the sequence. In hvbe2a - 1 , the guanine residue located 10,639 bp downstream of the first ATG was replaced by adenine (Fig.  2 e). The predicted HvBE2a protein has a plastidial transit peptide at its N-terminus, a CBM48, and a central (β/α) 8 catalytic module characteristic of the α-amylase family (Catalytic domain). Additionally, it features β-domains typically found in the C-terminus of α-amylase family members (C-domain) (Fig.  2 f). The hvbe2a-1 mutation replaces the glycine residue at position 651with an arginine residue. The glycine residue is conserved across various starch- and glycogen-branching enzymes (Fig.  2 g). We designed derived cleaved-amplified polymorphic sequence (dCAPS) primers to detect the base change in hvbe2a-1 +/– . The dCAPS primers successfully genotyped the wild-type Haruna Nijo, heterozygous HvBE2a/hvbe2a-1 , and homozygous mutation of hvbe2a-1 (Supplementary Fig. 3a). To confirm whether the base change in the hvbe2a-1 mutant co-segregates with the phenotype of the elongated SGs existence in endosperm, we crossed hvbe2a-1 with Haruna Nijo and produced F2 populations. Out of the 70 F2 grains, 19 developed elongated SGs in the endosperm, suggesting that the phenotype segregated in a single recessive manner (χ 2  = 0.17, p = 0.68). Of these 19 grains, fifteen were randomly selected and grown into seedlings for genotyping. The genotyping using the dCAPS primer showed that all the 15 plants were homozygous for the hvbe2a-1 base change (Supplementary Fig. 3b). This result supports the idea that the base change in the HvBEIIa gene of the hvbe2a-1 mutant is responsible for the elongated SG phenotype.

Genetic lesions in hvbe2a-1 and hvbe2a-2. a – b Iodine-stained thin sections of endosperm cells of F 1 grains from a cross between hvbe2a-1 and hvbe2a-2 . Bars = 20 μm. c – d Iodine-stained thin sections of hvbe2a-1 and hvbe2a-1 mutants, respectively. Bars = 20 μm. e The structure of the HvBE2a gene on chr2H:464,360,851..464373337 in Haruna_Nijo_pseudomolecules_v1. The coding and untranslated regions are depicted as blue and white boxes, respectively. Introns are indicated by black lines. The exon–intron structure is based on the reported full-length cDNA (AF064560). The adenine in the translation start codon (ATG) is designated as + 1. hvbe2-1 has a base pair change from G to A at + 10,639, leading to an amino acid substitution of Gly651 by arginine (R). f The protein structure of HvBE2a. The first methionine is designated as + 1. The predicted plastidial transit peptide, carbohydrate-binding module of family 48 (CBM48), the central (β/α) 8 catalytic module of α-amylase family (Catalytic domain) and β-domains typically found in the C terminus of α-amylases family members (C-domain) are depicted according to Pfister and Zeeman ( 2016 ) and Noguchi et al. ( 2011 ). The putative catalytic triad Asp469-Glu524-Asp592 is shown asterisks. g Alignment of sequence around hvbe2-1 mutation with other starch- and glycogen-branching enzymes. HvBE2a, HvBE2b, HvBE1 (HORVU.MOREX.r3.2HG0165780.1, HORVU.MOREX.r3.2HG0170370.1 and HORVU.MOREX.r3.7HG0751660.1), ZmBEIIa, ZmBEIIb and ZmBEI (Zea mays, AAB67316, NP_001105316, and NP_001105370), OsBEIIa, OsBEIIb and OsBEI ( Oryza sativa , AB023498, Os02t0528200-01 and Os06t0726400-01), StBEII and StBEI ( Solanum tuberosum , CAB40748 and CAA49463), ScGLC3 ( Saccharomyces cerevisiae , AAA34632), HsGBEI ( Homo sapiens , NM_000158). Perfectly conserved residues are shown in black. Red arrowheads indicate the residues substituted by hvbe2a-1 mutation. The Clustal W program was used for the alignment. h The deletion on chromosome 2H in hvbe2a-2 including the HvBE2a locus. Gray and blue bars represent the relative mapping depth of the NGS short reads for the Haruna Nijo and hvbe2a-2 genomes, respectively, depicted on a logarithmic scale. The relative positions of three annotated genetic loci, including HvBE2a , are marked within the deletion region spanning from 463,944 to 464,477 kb on chromosome 2H

Next-generation sequencing of the hvbe2a-2 mutant genome, followed by the mapping of the obtained short reads against the Haruna Nijo genome, identified the genomic region with missing reads in hvbe2a-2 (Fig.  2 h). This region, extending from 464,944 to 464,477 kb on chromosome 2H, includes the HvBE2a gene and two other annotated genes. Thus, hvbe2a-2 is missing the HvBE2a gene due to a deletion.

HvBE2a protein accumulation and branching enzyme activity in hvbe2a mutants

Next, we examined the accumulation of the HvBE2a protein and its enzyme activity in hvbe2a mutants. The amino acid sequences of the synthetic peptides used to create the antibodies are shown in Supplemental Fig.  4 . The two anti-HvBE2a antibodies recognize the N-terminus and C-terminus of the mature HvBE2a sequences without the transit peptide, respectively. We refer to the two antibodies as anti-HvBE2a-N and anti-HvBE2a-C. Both antibodies recognized the band around 90 kDa in Haruna Nijo and hvbe2a-1 , but not in hvbe2a-2 mutant in the immunoblot analysis of the developing grains (Fig.  3 a). The band size is consistent with the expected molecular weight of HvBE2a, which is 87.6 kDa, excluding the transit peptide. The absence of the detected band in the hvbe2a-2 is due to the deletion of the HvBE2a gene (Fig.  2 h). In both Haruna Nijo and hvbe2a-1 , anti-HvBE2a-C antibody detected the smaller-sized bands together with the 90-kDa band, which could be degradation products of HvBE2a since they were absent in hvbe2a-2 . Out of the degradation products, the band around 60 kDa, detected with anti-HvBE2a-C antibody, showed significant intensity comparable to that of the full-length band of HvBE2a, implying that it is the most abundant degradation product. This band was not detected with anti-HvBE2a-N antibody, suggesting that the N-terminus is missing in this degradation product. This suggests that HvBE2a is more prone to degradation from the N-terminus.

Starch branching enzyme activity and protein accumulation in hvbe2a mutants. a Immunoblot analysis with anti-HvBE2a-N and anti-HvBE2a-C antibodies after SDS-PAGE of developing endosperm extracts from Haruna Nijo, hvbe2a-1 and hvbe2a-2 . The molecular masses are given on the left in kDa. Membranes are stained with Ponceau-S to verify equal protein loading and transfer. b Native-PAGE activity staining of starch branching enzymes in mutants and immunoblot analysis after Native-PAGE. Proteins from at 14 days after awn emergence (7.5 μg) were loaded on each lane. c – e Immunoblot analysis using anti-HvBE2a-C, anti-HvBE2b and anti-HvBE1, respectively. Closed and open arrowheads indicate the position of HvBE2a and HvBE2b, respectively

Next, we investigated BE activity in the maturing endosperm of hvbe2a mutants. In barley, besides HvBE2a, there are other BE isozymes, namely HvBE2b and HvBE1. These show 76% and 46% amino acid sequence identity with HvBE2a, respectively. To discern their individual activities, we utilized an in-gel Native-PAGE activity assay, which separates these isozymes on the gel. In Haruna Nijo maturing endosperm at 14 DAA, two bands with BE activity were detected with slightly different mobilities on the gel (Fig.  3 b). The upper band was major and the lower band was minor (Fig.  3 b). In the case of hvbe2a-1 and hvbe2a-2 mutants, the upper band was not detected, while the only lower band were detected to have BE activity. To ascertain the precise locations of the bands corresponding to each isozyme in Native-PAGE, we constructed specific antibodies recognizing HvBE2b and HvBE1, respectively in addition to HvBE2a (Supplemental Fig.  4 ). Immunoblot analysis following Native-PAGE revealed that the upper and lower bands with BE activities in Haruna Nijo corresponded to HvBE2a and HvBE2b, respectively (Fig.  3 c, d). This suggests that HvBE2a is the major BE enzyme in the developing endosperm of Haruna Nijo. The protein accumulation level of HvBE2a in hvbe2a-1 was almost the same as in Haruna Nijo. The absence of BE activity from HvBE2a in hvbe2a-1 indicates that the amino acid substitution in hvbe2a-1 does not impact protein accumulation but is crucial for enzymatic activity of HvBE2a. Although HvBE1 was detected as more than two bands with different mobilities on the immunoblotted membrane, no BE activity was observed at these positions under our experimental conditions (Fig.  3 b, e).

Suppressive effect of hvbe2a mutations against starchless phenotype of hvflo6-2 hvisa1-3 grain . a Cross-sections of a mature grain of Haruna Nijo and mutants. Bars = 1 mm. b Single grain weight of triple mutant mature grains ( n  = 6–8). c Starch amount per grain of triple mutant mature grains ( n  = 6–8). Data are given as means ± SD. Statistical comparisons were performed using Tukey’s HSD. The same letters above the bars represent statistically indistinguishable groups, and different letters represent statistically different groups ( p  < 0.05)

hvbe2a suppresses the starchless phenotype of hvflo6 hvisa1

The hvflo6 mutation enhances the hvisa1 phenotype, leading to a significant reduction in starch content in grains and severe grain shrinkage in hvflo6 hvisa1 double mutants (Matsushima et al. 2023 ). In rice, the isa1 phenotype is mitigated by the mutations of be2a and be2b (Lee et al. 2017 ; Nagamatsu et al. 2022 ). We therefore generated the hvflo6 hvisa1 hvbe2a triple mutant to examine the suppressive effect of hvbe2a mutations against the hvflo6 hvisa1 phenotypes. The hvbe2a-1 and hvbe2a-2 mutants did not show any significant differences in grain cross-sections compared to Haruna Nijo (Fig.  4 a). The hvflo6-2 hvisa1-3 grains were shrunken. Interestingly, when hvbe2a-1 and hvbe2a-2 mutations were introduced into the hvflo6-2 hvisa1-3 background, the grain shrinkage phenotype was partially rescued in both triple mutants (Fig.  4 a). The results show that hvbe2a mutations have a suppressive effect on the hvflo6-2 hvisa1-3 phenotype. We also measured the grain weight and starch content in the grains of the triple mutants (Fig.  4 b, c). For the triple mutants, the individual grain weight was higher than that of the hvflo6-2 hvisa1-3 double mutant but lower than that of Haruna Nijo (Fig.  4 b). Similarly, the starch content of the grain in triple mutants was higher than that of the hvflo6-2 hvisa1-3 grain but lower than that of Haruna Nijo (Fig.  4 c). This indicates that loss of HvBE2a function did not completely suppress the double mutant phenotype of hvflo6-2 hvisa1-3 .

To confirm which mutations, hvflo6-2 or hvisa1-3 , are targeted by the suppressive effect of hvbe2a , we generated a series of double mutants, including hvisa1-3 hvbe2a-1, hvisa1-3 hvbe2a-2 , hvflo6-2 hvbe2a-1 and hvflo6-2 hvbe2a-2 . In line with the previous observation (Matsushima et al. 2023 ), the starch content of the single mutants of hvflo6-2 and hvisa1-3 grains was reduced compared to Haruna Nijo (Fig.  5 a). The starch content was higher in the mutants of hvisa1-3 hvbe2a-1 and hvisa1-3 hvbe2a-2 compared to the hvisa1-3 single mutant. Their starch content was restored to the wild-type level (Fig.  5 a). In contrast, there was no significant difference in the starch content of hvflo6-2 hvbe2a-1 and hvflo6-2 hvbe2a-2 grains compared to the hvflo6-2 single mutant (Fig.  5 a). This result means that the suppressive effect of the hvbe2a mutations predominantly targets hvisa1-3 , rather than hvflo6-2 .

Suppressive effect of hvbe2a mutations against hvisa1-3 starch properties. a Starch amount per grain of single and double mutants. Data are given as means ± SD using at least three biological replicates. Statistical comparisons were performed using Welch’s t -test (**, p  < 0.01; and ns, not significant at p  = 0.05). The starch reduction in hvisa1-3 was recovered by adding hvbe2a mutations. In contrast, the starch reduction in hvflo6-2 was not affected by hvbe2a mutations. b Amount of phytoglycogen per mature grain in double and triple mutants. Data are given as means ± SD using at least three biological replicates. Statistical comparisons were performed using Welch’s t -test (**, p  < 0.01). The increases of phytoglycogen in hvisa1-3 and hvflo6-2 hvisa1-3 were suppressed by adding hvbe2a mutations. c Glucan chain-length distribution of α-glucans of mature grains. Haruna Nijo, hvisa1-3 , hvisa1-3 hvbe2a-1, hvisa1-3 hvbe2a-2 are indicated by the black, red, green and blue lines, respectively. The values for hvisa1-3 are identical to the data in Matsushima et al. ( 2023 ). Data are given as means ± SD. All data were obtained from at least three independent grains

We have previously shown that the hvisa1-3 grains accumulate significantly more phytoglycogen compared to the wild type (Matsushima et al. 2023 ). Furthermore, this accumulation was found to be even more pronounced with the addition of the hvflo6-2 mutation. To determine whether the hvbe2a mutations suppress the phytoglycogen accumulation of hvisa1-3 , we measured the phytoglycogen accumulation in hvisa1-3 hvbe2a double mutants. The amount of phytoglycogen in hvisa-3 hvbe2a-1 and hvisa-3 hvbe2a-2 mutants decreased by 88% and 77%, respectively, compared to hvisa-3 (Fig.  5 b). These data demonstrate a noteworthy decrease in phytoglycogen accumulation in both hvisa1-3 hvbe2a-1 and hvisa1-3 hvbe2a-2 mutants, compared to the single hvisa1-3 mutant (Fig.  5 b). This result confirms that the suppressive effect of the hvbe2a mutations primarily targets hvisa1 . We also examined the suppressive effect of hvbe2a mutations on phytoglycogen accumulation in the hvflo6-2 hvisa1-3 background. The amount of phytoglycogen in hvflo6-2 hvisa-3 hvbe2a-1 and hvflo6-2 hvisa-3 hvbe2a-2 triple mutants decreased by 45% and 30%, respectively, compared to hvflo6-2 hvisa-3 double mutants (Fig.  5 b). In the triple mutants, the phytoglycogen were decreased compared to the hvflo6-2 hvisa1-3, but not to the level of hvisa1-3 hvbe2a-1 and hvisa1-3 hvbe2a-2 (Fig.  5 b). This indicates that hvflo6-2 and hvbe2a are in competition to either enhance or suppress the phenotype of hvisa1-3 .

The distribution of glucan chain length in α-glucan from hvisa1-3 hvbe2a-1 and hvisa1-3 hvbe2a-2 grains closely resembles that of Haruna Nijo (Fig.  5 c). While, hvisa1-3 single mutant exhibits a higher abundance of shorter glucose chains (DP < 10) and a lower abundance of longer glucose chains (DP > 10) compared to Haruna Nijo. This also supports the suppressive effect of the hvbe2a mutations against hvisa1-3 .

Transformation of starch granule morphology by hvisa1 and hvbe2a

Next, we investigated the impact of hvbe2a mutations on the SG morphology of the hvisa1-3 mutant. In hvisa1-3 , compound SGs were well developed in the endosperm, replacing the original bimodal simple SGs typical of barley (Fig.  6 a, b). This is consistent with previous observations (Matsushima et al. 2023 ). In the endosperm of hvisa1-3 hvbe2a-1 and hvisa1-3 hvbe2a-2 , typical A- and B-type SGs were developed (Fig.  6 c–f). When SGs of hvisa1-3 were purified and analyzed using a Coulter Counter, the granule size distribution with typical bimodal peaks of A- and B-type SGs was not observed (Fig.  6 g). Instead, a single peak was predominant. The observed peak is most likely attributed to the starch particles that formed the compound SGs of hvisa1-3 and subsequently disintegrated during purification. In the case of hvisa1-3 hvbe2a-1 and hvisa1-3 hvbe2a-2 , the Coulter Counter analysis consistently showed bimodal peaks (Fig.  6 h, i). This suggests that in barley, SGs change from authentic bimodal type to compound type due to the hvisa1 mutation, and the hvbe2a mutation can reverse these compound SGs back to the bimodal type. We have analyzed the average diameters of A- and B-type granules in hvisa1-3 hvbe2a-1 and hvisa1-3 hvbe2a-2 mutants. Our findings reveal that for A-type granules, the diameter in both hvisa1-3 hvbe2a-1 and hvisa1-3 hvbe2a-2 mutants was approximately 80% of that in Haruna Nijo (Fig.  6 j). Similarly, for B-type granules, the diameter in both mutants was around 85% relative to that in Haruna Nijo (Fig.  6 k). The observation of smaller bimodal SGs in the hvisa1-3 hvbe2a mutants, compared to those in Haruna Nijo, indicates that the depletion of HvBE2a does not fully suppress the SG morphological changes induced by hvisa1-3 . We also investigated the impact of the hvbe2a mutations on SG morphology in the hvflo6-2 mutant. In hvflo6-2 , compound SGs were well developed in the endosperm (Fig.  7 a, b), consistent with previous observations (Matsushima et al. 2023 ). In the endosperm of hvflo6-2 hvbe2a-1 and hvflo6-2 hvbe2a-2 , hvbe2a -specific elongated SGs were clearly observed alongside the hvflo6-2 -specific compound SGs (Fig.  7 c–f). This suggests that the effects of the hvflo6-2 and hvbe2a mutations are cumulative with respect to SG morphology. Regarding the α-glucan chain length distribution, hvflo6-2 exhibited a distribution nearly identical to that of Haruna Nijo (Fig.  7 g, h), consistent with previous research (Matsushima et al. 2023 ). When comparing the hvbe2a-1 mutant with the hvflo6-2 hvbe2a-1 double mutant, there was a slight reduction in glucose chains ranging from DP7 to DP15 in the double mutant (Fig.  7 g). Similarly, the hvflo6-2 hvbe2a-2 double mutant showed a slight reduction in glucose chains from DP10 to DP15 compared to the hvbe2a-2 mutant (Fig.  7 h). However, no other significant differences in glucose chain length were observed between the double and single mutants (Fig.  7 g, h). These findings indicate that the genetic interaction between the hvflo6 and hvbe2a mutations differs from the suppressive interaction observed between the hvisa1 and hvbe2a mutations.

Suppressive effect of hvbe2a mutations on hvisa1-3 starch granule morphology in endosperm. a – f Iodine-stained thin sections of endosperm cells of hvisa1-3 (a and b), hvisa1-3 hvbe2a-1 (c and d), and hvisa1-3 hvbe2a-2 (e and f). Bars = 20 μm. g – i Granule size distributions of hvisa1-3 , hvisa1-3 hvbe2a-1 and hvisa1-3 hvbe2a-2 , respectively. The relative percent volume of each diameter was determined using a Coulter Counter. j – k The average diameter of A- and B-type granules, respectively, extracted from the relative percent volume vs. diameter plots of hvisa1-3 hvbe2a-1 and hvisa1-3 hvbe2a-2 (h and i) by fitting a bimodal mixed normal and lognormal distributions (n = 4). The values for Haruna Nijo are identical to the data presented in Fig.  1 (n, o). Data are given as means ± SD. Statistical comparisons were performed using Tukey’s HSD. The same letters above the bars represent statistically indistinguishable groups, and different letters represent statistically different groups ( p  < 0.05)

Starch granule morphology and glucan chain-length distribution of hvflo6 hvbe2a double mutants. a – f Iodine-stained thin sections of endosperm cells of hvflo6-2 (a and b), hvflo6-2 hvbe2a-1 (c and d), and hvflo6-2 hvbe2a-2 (e and f). Bars = 20 μm. g Glucan chain-length distribution of α-glucans of mature grains. Haruna Nijo, hvflo6-2, hvbe2a-1, hvflo6-2 hvbe2a-1 are indicated by the black, red, green and blue lines, respectively. h Glucan chain-length distribution of α-glucans of mature grains. Haruna Nijo, hvflo6-2, hvbe2a-2, hvflo6-2 hvbe2a-2 are indicated by the black, red, green and blue lines, respectively. The values for hvflo6-2 are identical to the data in Matsushima et al. ( 2023 ). Data are given as means ± SD. All data were obtained from at least three independent grains

Absence of suppressive effect of hvbe2a against hvflo6 hvisa1 in pollen

Pollen, as well as endosperm, accumulates storage starch. The rod-shaped SGs were well developed in pollen from Haruna Nijo, hvisa1-3, hvbe2a-1 and hvbe2a-2 (Fig.  8 a). In the hvflo6-2 mutant, a notable increase in compound SGs was observed in addition to the rod-shaped SGs. The proportion of the compound SGs in hvflo6-2 pollen was up to 66.5%, whereas those in Haruna Nijo, hvisa1-3 , hvbe2a-1 , and hvbe2a-2 were less than 9% (Fig.  8 b). The proportion of the compound SGs in the hvisa1-3 hvbe2a-1 and hvisa1-3 hvbe2a-1 pollens were not statistically significant compared to hvisa1-3 pollen (Supplemental Fig.  5 a–b). In contrast, the double mutants, hvflo6-2 hvbe2a-1 and hvflo6-2 hvbe2a-2, exhibited well-developed compound SGs at the same level as hvflo6-2 (Fig.  8 a, b). This indicates that hvbe2a does not suppress hvflo6-2 in terms of compound SG formation in pollen. In contrast, the hvisa1-3 mutation significantly enhanced the formation of compound SGs in hvflo6-2 pollen (Fig.  8 a). In the hvflo6-2 hvisa1-3 pollen, more than 90% of SGs were of the compound type (Fig.  8 b). This implies that HvISA1 is involved in compensating for HvFLO6 function in pollen, especially in the hvflo6-2 background.　We also observed pollen SGs in triple mutants, hvflo6-2 hvisa1-3 hvbe2-1 and hvflo6-2 hvisa1-3 hvbe2-2 . The proportion of compound SGs in these triple mutants did not significantly differ from that in the hvflo6-2 hvisa1-3 mutants (Fig.  8 b). This observation supports the hypothesis that the hvbe2a mutation cannot suppress the formation of compound SGs induced by hvflo6-2 or hvisa1-3 in pollen.

Starch granules in mutant pollen. a Iodine-stained SGs released from squashed pollen grains. Bars = 5 μm. b The percentage of starch granules in compound form, as determined from microscopic images. Data are given as means ± SD. Statistical comparisons were performed using Welch’s t -test (ns, not significant at p  = 0.05). c – e Accumulation of HvBE2a, HvBE2b, and HvBE1 in pollen. Protein extracts from developing endosperm at 14 days after awn emergence (DAA) and mature pollen at 3 DAA from Haruna Nijo and mutants were subjected to SDS-PAGE followed by the immunoblot analysis using anti-HvBE2a-C, anti-HvBE2b, and anti-HvBE1 antibodies. Each lane contains 5 μg of protein. The molecular masses are indicated on the left in kDa. Membranes are stained with CBB to verify equal protein loading and transfer. Arrow and asterisk in d indicate the positions of specific and non-specific bands, respectively

To investigate the protein accumulation of HvBE2a, HvBE2b, and HvBE1 in pollen, pollen protein extracts were subjected to the immunoblot analysis (Fig.  8 c–e). HvBE2a accumulation was not detected in the mutants’ pollen harboring hvbe2a-2 allele ( hvbe2a-2 , hvflo6-2 hvbe2a-2 and hvflo6-2 hvisa1-3 hvbe2a-2 ) because of the deletion of the HvBE2a gene (Fig.  8 c). In pollen, HvBE2a accumulation is lower overall, even in Haruna Nijo, when compared to the endosperm (Fig.  8 c). The HvBE2a band intensity of the Haruna Nijo pollen is slightly stronger compared to that of hvbe2a-1 pollen (Fig.  8 c). This may indicate that the amino acid substitution in hvbe2a-1 affects protein accumulation particularly in pollen. In the hvisa-1-3 and hvflo6-2 mutants, the accumulation of HvBE2a was slightly higher than in Haruna Nijo.

Anti-HvBE2b antibodies recognized the band around 90 kDa in the developing endosperm (Fig.  8 d, arrow). The band size is consistent with the expected molecular weight of HvBE2b (87.9 kDa). The bands with the same size were detected in pollen of hvflo6-2 hvbe2a-1 , hvflo6-2 hvbe2a-2 , hvflo6-2 hvisa1-3 , hvflo6-2 hvisa1-3 hvbe2a-1 , and hvflo6-2 hvisa1-3 hvbe2a-2 . It is noted that these mutants are characterized by the increased proportion of compound SGs in their pollen (Fig.  8 b). Anti-HvBE2b antibodies also detected bands around 100 kDa only in the pollen samples (Fig.  8 d, asterisk). These bands are significantly larger than the expected molecular weight of HvBE2b, and may be non-specific bands unique to the pollen samples. Anti-HvBE1 antibodies recognized the band around 90 kDa in developing endosperm of Haruna Nijo, which is consistent with the expected molecular weight of HvBE1 (86.5 kDa) (Fig.  8 e). In hvisa-1-3 and hvbe2a-2 pollen , HvBE1 was significantly accumulated compared to Haruna Nijo.

Fertility of pollen with compound starch granules

In order to evaluate the fertility of hvflo6-2 pollen with compound SGs, we studied the SGs in mature pollens from heterozygous plant ( HvFLO6 / hvflo6-2 ). We measured the segregation ratio of hvflo6-2 mutant pollen (having compound SGs) to wild-type pollen (containing normal rod-shaped simple SGs) (Table  1 ). The segregation ratio was almost 1:1. This suggests that the hvflo6-2 mutation behaves in a gametophytic manner in pollen and that hvflo6-2 pollen matures successfully without aborting during the developmental stage. We then obtained self-fertilized F2 grains from HvFLO6 / hvflo6-2 heterozygous plants and examined the segregation ratio of hvflo6-2 grains to wild-type grains by observing SG morphology in each grain (Table  2 ). The segregation ratio was 1:3 without any segregation distortion. This indicates that the fertility of hvflo6-2 pollen is not significantly different from that of wild-type pollen. These results suggest that the increase in compound SGs in hvflo6-2 pollen does not significantly affect the pollen fertility.

In our previous paper (Matsushima et al. 2023 ), we presented the co-segregation analysis of shrunken grains caused by the hvflo6-2 hvisa1-3 genotype. This analysis used the selfed population of HvFLO6 / hvflo6-2 hvisa1-3 plants, where hvflo6-2 is heterozygous, and hvisa1-3 is homozygous. In the selfed population, the segregation ratio of shrunken grains to normal grains was approximately 1:3 (Matsushima et al. 2023 ). Genotyping of the grains revealed that all shrunken grains were consistent with the hvflo6-2 hvisa1-3 double homozygous genotype. On the other hand, normal grains were either HvFLO6 / hvflo6-2 hvisa1-3 or HvFLO6 hvisa1-3 . This indicates that the hvflo6-2 mutation segregates as a single recessive allele in the hvisa1-3 background. This undistorted co-segregation experiment suggests that the fertility of hvflo6-2 hvisa1-3 pollen (having compound SGs) is not significantly different from that of hvisa1-3 pollen (containing normal rod-shaped simple SGs).

Isolation of mutants defective in HvBE2a gene

BE is an enzyme that creates branches during amylopectin synthesis (Nakamura 2002 ; Pfister and Zeeman 2016 ). In barley and wheat endosperms, BE2a is the major BE isozyme in endosperm (Regina et al. 2006 , 2010 ). In this study, two barley mutants with genetic lesions in HvBE2a gene were isolated through screening based on the SG morphology (Fig.  1 ). In hvbe2a-1 and hvbe2a-2 mutants, elongated SGs were observed in the endosperm cells (Fig.  1 g–j). Previously, transgenic barley with suppressed HvBE2a expression using RNAi were created, but mutants have not been isolated (Regina et al. 2010 ). The hvbe2a-1 mutant had a nucleotide change causing an amino acid substitution, and the hvbe2a-2 mutant had the complete deletion of the HvBE2a gene (Fig.  2 ). In the hvbe2a-1 mutant, the HvBE2a-1 protein accumulated in similar amounts to the wild-type HvBE2a protein in Haruna Nijo. However, no BE activity was detected from the HvBE2a-1 protein (Fig.  3 b, c). Therefore, the substituted amino acid residue in the HvBE2a-1 protein does not affect protein accumulation but is crucial for the enzymatic activity. This observation aligns with the location of the amino acid substitution near the catalytic triad, and the fact that this residue is conserved among BEs of various plant species as well as glycogen BEs from animals and bacteria (Fig.  2 g).

BEIIb is the primary BE in rice endosperm (Mizuno et al. 1993 ). Rice beIIb mutant EM10 fails to accumulate BEIIb protein, while another beIIb mutant, ssg3 , carries a base substitution leading to an amino acid change in BEIIb (Mizuno et al. 1993 ; Matsushima et al. 2010 ). ssg3 mutation allows the accumulation of BEIIb protein but eliminates its enzymatic activity. EM10 and ssg3 exhibit similar phenotypes, characterized by the reduced polygonal starch particles and altered glucan chain length distributions (Matsushima et al. 2010 ; Nagamatsu et al. 2022 ). However, they differ in starch biosynthetic enzyme complex formation (Crofts et al. 2018 ). This difference is thought to arise from the inactive enzyme still having the ability to contribute to complex formation (Crofts et al. 2018 ). In this study, in the glucan chain length distribution, hvbe2a-1 exhibited a more pronounced reduction around DP11 compared to hvbe2a-2 (Fig.  1 q, r). The difference may be due to the distinct protein complexes of starch biosynthetic enzymes. In the hvbe2a-1 mutant, the HvBE2a protein is present without biochemical activity, which may prevent other isozymes from replacing it. This could lead to a more severe phenotype compared to the complete absence of HvBE2a in hvbe2a-2 . However, we did not observe any morphological differences in SGs between hvbe2a-1 and hvbe2a-2 (Fig.  1 g–j). This is because both mutants have a substantial number of normal SGs along with elongated ones, making it difficult to distinguish differences in SG morphology between hvbe2a mutants.

Suppressive impact of hvbe2a against hvisa1 in endosperm

In hvisa1-3 grains, there was a reduction in total starch and an increase in phytoglycogen. However, these phenotypes were less pronounced in the hvbe2a-1 and hvbe2a-2 background. (Fig.  5 a, b). This suggests that the hvbe2a-1 and hvbe2a-2 mutations are epistatic to the hvisa1-3 mutation. BEs are essential for catalyzing glucose chain branching during amylopectin biosynthesis. However, BEs can sometimes lead to branching at inappropriate positions. In contrast, ISA1 effectively removes glucose chains attached to inappropriate positions on amylopectin to avoid excessive branching caused by BEs (Nakamura 2002 ; Smith and Zeeman 2020 ). The absence of HvBE2a, a major BE in barley endosperm, is expected to reduce the formation of incorrect glucose chains. This may decrease the need for trimming by HvISA1. As a result, the loss of HvBE2a has the potential to alleviate the phenotype of HvISA1.

Previous reports in barley RNAi experiments showed that silencing HvBE2b had less impact on amylopectin chain length distribution than HvBE2a . However, the double silencing of both genes exhibited an enhanced phenotype compared to the HvBE2a single silencing (Regina et al. 2010 ). This suggests a potential functional overlap or compensatory mechanism between HvBE2a and HvBE2b . In the hvbe2a mutant, we detected HvBE2b activity through Native-PAGE activity staining. Thus, HvBE2b is likely to function in the hvbe2a endosperm. It appears that HvBE2b has a minor role in creating inappropriate glucose chains in the endosperm, as there is a significant suppression of phytoglycogen production in hvisa1-3 hvbe2a-1 and hvisa1-3 hvbe2a-2 compared to the hvisa1-3 single mutant (Fig.  5 b). It is also possible that additional DBEs, such as pullulanase, play a crucial role in trimming the inappropriate glucose chains generated by HvBE2b, alongside ISA1. Notably, in rice, pullulanase has been proposed to be involved in debranching inappropriate glucose chains in isa1 　mutant (Fujita et al. 2009 ).

The mutation in BE genes has been shown to alleviate the isa1 phenotype in rice (Lee et al. 2017 ; Nagamatsu et al. 2022 ). Specifically, the beIIb rice mutation reduces the reduction in grain weight of isa1 mutants and changes the glucan chain length distribution of α-glucan from the isa1 -type to the beIIb -type (Nagamatsu et al. 2022 ). This result indicates that beIIb is epistatic to isa1 in rice. Additionally, the beIIa mutation also can mitigate the phenotype caused by isa1 , even though it shows minimal phenotype in rice (Satoh et al. 2003 ; Lee et al. 2017 ). These results indicate that the antagonistic relationship between isa1 and be mutations is common in rice and barley.

The hvisa1 mutant forms compound SGs, while hvisa1-3 hvbe2a double mutants develop bimodal simple SGs (Fig.  6 a–i). This result suggests that in barley, mutations in the HvISA1 and HvBE2a genes enable the conversion between compound and bimodal types of SGs. Soluble phytoglycogen molecules or their degradation products, such as maltooligosaccharides, are proposed to act as substrates for nucleating new granule initiation (Burton et al. 2002 ). The higher level of phytoglycogen in hvisa1 mutants may increase the nucleation events and lead to more starch particles within an amyloplast, resulting in the formation of compound SGs (Burton et al. 2002 ). This idea is supported by the development of simple SGs instead of compound SGs in hvisa1-3 hvbe2a double mutants where the level of phytoglycogen was reduced (Figs. 5 b, 6 a–i). The fact that HvISA1 is not necessary for developing bimodal SGs under conditions with reduced HvBE2a activity suggests that HvISA1 does not play an exclusive function in developing bimodal SGs.

These mutations are unlikely to be involved in the natural variation in SG morphology across different plant species. For example, wild-type rice develops compound SGs, but the rice beIIb mutant increases the number of small spherical starch particles without forming the typical bimodal SGs observed in barley and wheat. The differences in glucan chain length distribution of amylopectin among species are not as pronounced as those caused by mutations in major starch biosynthetic enzymes (Jane et al. 1999 ). This suggests that the variation in SG morphology is influenced by factors other than mutations in starch biosynthetic enzymes.

The minor impact of HvBE2a on starch granule formation in pollen

In cereal pollen, vegetative cells accumulate large amounts of starch (Lee et al. 2022 ). This starch acts as a nutritional source during pollen germination. Some starch-related genes expressed in pollen often function similarly in the endosperm. For instance, waxy mutants of rice, maize and sorghum, possessing mutations in the amylose-synthesizing enzyme, show an absence of amylose in both the endosperm and pollen (Terada et al. 2002 ; Pedersen et al. 2004 ; Talukder et al. 2022 ). The shape of SGs in pollen is commonly consistent among plant species, typically appearing as smaller, rod-shaped, and simple-type (Matsushima and Hisano 2019 ). The consistency of SG shapes in pollen contrasts with the wide range of SG morphologies found in the endosperm. Nevertheless, mutations affecting SG morphologies in the endosperm often lead to alterations in SG shape in pollen (Matsushima et al. 2014 , 2016 , 2023).

In the pollen of Haruna Nijo and the mutants, including hvisa1-3 , hvbe2a-1 , and hvbe2a-2 , the ratio of compound SGs were less than 9%. (Fig.  8 b). In contrast, the hvflo6 pollen exhibited a significantly higher proportion of compound SGs, approximately 66.5% (Fig.  8 b). Furthermore, the hvflo6 phenotype was notably enhanced by the hvisa1 mutation, resulting in over 90% compound SGs in the pollen (Fig.  8 b). This suggests that the HvISA1 gene plays a role in pollen, at least in the hvflo6-2 background. The level of pollen compound SGs in hvflo6-2 hvbe2a-1 and hvflo6-2 hvbe2a-2 were found to be comparable to hvflo6-2 , indicating no significant reduction. Similarly, there were no significant difference observed among hvflo6-2 hvisa1-3 hvbe2a triple mutants and hvflo6-2 hvisa1-3 double mutants (Fig.  8 b). These findings indicate that mutations in hvbe2a did not alleviate the hvflo6-2 hvisa1-3 phenotype in pollen, which contrasts with the traits identified in the endosperm.

The level of HvBE2a accumulation in pollen was lower than in the endosperm (Fig.  8 c). This suggests that HvBE2a has a less significant role in pollen compared to the endosperm. Notably, the accumulation of HvBE2b was increased in hvflo6-2 hvbe2a-1 , hvflo6-2 hvbe2a-2 , hvflo6-2 hvisa1-3 hvbe2a-1 , and hvflo6-2 hvisa1-3 hvbe2a-2 (Fig.  8 d). All of these lines developed compound SGs in pollen despite having the hvbe2a mutations (i.e., the suppressive effect of the hvbe2a mutations is not observed). The HvBE2a depletion leads to the increase of HvBE2b, and the induced HvBE2b may compensate for the HvBE2a function (Fig.  8 d). In the endosperm, due to its high dependency on HvBE2a, HvBE2b may not be able to fully compensate for the depletion of HvBE2a.

Another possible explanation for the different suppressive effects of HvBE2a depletion between pollen and endosperm is that the pathways leading to the increase of compound SGs differ between pollen and endosperm. In the endosperm, the depletion of HvISA1 leads to an increase in compound SGs (Fig.  6 a, b). In contrast, in pollen, the depletion of HvISA1 does not increase compound SGs (Fig.  8 a). This suggests that pollen does not require HvISA1 to maintain normal SG morphology. On the other hand, the absence of HvFLO6 leads to an increase in compound SGs in both pollen and endosperm. This indicates that there are independent pathways involving HvISA1 and HvFLO6 to maintain normal SG morphology in barley. Given that the compound SG increase in hvflo6-2 pollen but not in hvisa1-3 , it is likely that in wild-type pollen, the HvFLO6 pathway primarily ensures the formation of normal simple SGs in pollen. The suppressive effect of HvBE2a depletion on starch accumulation is not significant in hvflo6-2 (Fig.  5 a). This may explain why the suppressive effect of HvBE2a depletion is not observed in pollen, where the HvFLO6 pathway is dominant. However, even considering this possibility, it is difficult to explain why the suppressive effect of HvBE2a depletion is not observed in the hvflo6 hvisa1 double mutant background. In the future, it will be necessary to create additional mutants of HvBE2b and HvBE1 in barley to investigate the contributions of these BEs in pollen.

Mutations in starch-related genes play a crucial role in cereal breeding as they can affect taste, digestibility, and processing qualities. Cereals are versatile crops used for food, feed, malting, and industrial purposes, and their grain starch properties determine their suitability for these different applications. This study demonstrated that genetic interactions among specific mutations can control the amount and characteristics of starch and the SG morphology. Combining mutations to uncover genetic interactions that enhance or suppress traits are crucial for controlling starch properties. Multiple mutants could be used as breeding materials in future breeding efforts.

Data availability

The sequence of HvBE2a, HvBE2b and HvBE1 are available in the Ensemble Plants database ( http://plants.ensembl.org/index.html ) as HORVU.MOREX.r3.2HG0165780.1, HORVU.MOREX.r3.2HG0170370.1 and HORVU.MOREX.r3.7HG0751660.1, respectively. The raw NGS sequence data are available in the DNA Data Bank of Japan Sequence Read Archive (DDBJ DRA) database under specific accession numbers PRJDB17596 for hvbe2a-2 genome and PRJDB4103 for Haruna Nijo genome.

Abbreviations

Days after awn emergence

Degree of polymerization

Hordeum vulgare BRANCHING ENZYME2

Hordeum vulgare ISOAMYLASE1

Hordeum vulgare FLOURY ENDOSPERM 6

Starch granule

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Acknowledgements

The authors would like to thank the National BioResource Project for barley, which is run by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) for distributing the barley grains, and also gratefully thank Brendan Fahy at John Innes Centre for providing technical assistance.

Open Access funding provided by Okayama University. This work was funded by a MEXT Grant-in-Aid for Scientific Research (No. 23K05167 to RM) and grants from G-7 Scholarship Foundation, ASAHI GROUP Foundation, the Foundation for Dietary Scientific Research, and Intensive Support for Young Promising Researchers program by New Energy and Industrial Technology Development Organization (to HH), and the Ohara Foundation.

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Ryo Matsushima, Hiroshi Hisano, June-Sik Kim & Kazuhiro Sato

RIKEN Center for Sustainable Resource Science, Yokohama, 230-0045, Japan

June-Sik Kim

John Innes Centre, Norwich Research Park, Norwich,, NR4 7UH, UK

Ryo Matsushima, Rose McNelly & David Seung

Department of Biological Production, Akita Prefectural University, Akita, 010-0195, Japan

Naoko F. Oitome & Naoko Fujita

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Conceptualization: Ryo Matsushima; Methodology: Ryo Matsushima, Hiroshi Hisano, June-Sik Kim, Rose McNelly, Naoko F. Oitome, David Seung, Naoko Fujita, Kazuhiro Sato; Formal analysis and investigation: Ryo Matsushima; Writing—original draft preparation: Ryo Matsushima; Writing—review and editing: Ryo Matsushima; Funding acquisition: Ryo Matsushima, Hiroshi Hisano; Resources: Hiroshi Hisano, Kazuhiro Sato.

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Matsushima, R., Hisano, H., Kim, JS. et al. Mutations in starch BRANCHING ENZYME 2a suppress the traits caused by the loss of ISOAMYLASE1 in barley. Theor Appl Genet 137 , 212 (2024). https://doi.org/10.1007/s00122-024-04725-7

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Received : 17 April 2024

Accepted : 21 August 2024

Published : 31 August 2024

DOI : https://doi.org/10.1007/s00122-024-04725-7

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