Should Hydrogen Peroxide Be Used in the Garden?
If you spend any time on social media or reading popular gardening blogs you already know that hydrogen peroxide does all kinds of useful things in the garden. You will see blog headings such as, “11 Mega Reasons why Hydrogen Peroxide for Plants is a Must” and “10 Amazing Uses of Hydrogen peroxide for Plants in the Garden.”
This stuff must be fantastic! Or not.
Not every claim is a complete myth, but many of these claims are just wishful thinking. Time to look at some science and get down to the reality of using hydrogen peroxide in the garden.
Should Hydrogen Peroxide Be Used in the Garden?
What is Hydrogen Peroxide?
Hydrogen peroxide, or peroxide for short, is a simple chemical with the formula H2O2. It is water with an extra oxygen atom attached.
Peroxide is sold in most pharmacies as a disinfectant in either a 3% or 6% solution, but is also available at higher concentrations. When applied to bacteria or fungi, it will kill them. You might remember using it to sterilize a cut, although this is no longer recommended since it also damages tissue in the cut, making it more difficult to heal.
It is very reactive and easily loses the extra oxygen when it comes into contact with all kinds of other chemicals. Spraying it into the air, on soil or even adding it to water will cause it to degrade rapidly producing oxygen and water. Light will also degrade it, explaining why it is kept in brown plastic containers. Any mixtures for plant use need to be used right away.
Soil Organic Matter Can Be Measured Using Hydrogen Peroxide
When peroxide is mixed with soil it will react with living microbes as well as dead organic matter. In fact, an older method for measuring the amount of organic matter in soil used peroxide as the main reagent. During this reaction oxygen is produced and can be observed as bubbles.
What Are the Claims for Hydrogen Peroxide and Plants?
“It makes plants think that plain water is actually rainwater and you know how good that is for plants!”. Rainwater does contain very low levels of peroxide. Other claims include the following.
- Aerates the soil
- Disinfects pots, tools, benches and greenhouses
- Cures root rot caused by waterlogged soil
- Fights fungal diseases
- Disinfects growing media
- Sanitizes seeds
- Speeds up seed germination
- Fertilizes plants
- Boosts root growth
- Repels insects
- Kills Weeds
- Treats water
Peroxide Aerates the Soil
Plants don’t grow well in compacted soil and it is claimed that pouring peroxide onto it will reduce compaction. The peroxide releases oxygen and somehow this opens up the pores in soil.
This is nonsense. Peroxide will release oxygen, but it will not build up enough pressure to open up new pores in soil. The peroxide is degraded quickly as it reacts with organic matter and in a few minutes you are left with soil that is just as compacted as before.
Disinfects Pots, Tools, Benches and Greenhouses
Hydrogen peroxide is a disinfectant, so this will work, however commercial greenhouses don’t use it. They prefer to use products that combine hydrogen peroxide and peroxyacetic acid because they work better.
Comments like, “If you find a plant disease in your backyard, use a hydrogen peroxide solution to disinfect everything that might have come into contact with your troubled plants” are misleading. Firstly, most diseases in the garden don’t pose a problem and don’t need to be treated. Secondly, it is next to impossible to disinfect the soil in a home garden.
Soil and plant material will deactivate the peroxide, so material being disinfected should be washed first.
Peroxide Cures Root Rot
The claim is that root rot is caused by waterlogged soil due to overwatering. The water fills the air spaces, resulting in low oxygen levels which makes it easier for root rot fungus to take hold. Adding peroxide to the soil adds oxygen thereby improving the waterlogged soil.
This problem is real and it can be caused by overwatering. “Most plants can be affected by some form of root rot, usually caused by species of water molds: Phytophthora and Pythium, or by species of fungi: Rhizoctonia, Fusarium, and Thielaviopsis. These opportunistic, soil-borne plant pathogens infect plant root systems, where they thrive under low oxygen.”
“For plants with root rot or fungal infections, use 1 tablespoon hydrogen peroxide per cup of water”. Using the 3% standard solution this results in 0.2% solution which is now so dilute it won’t add much oxygen to the soil. The other thing to consider is that this is 99.8% water. The problem being solved is, too much water in the soil. Does adding more water really help?
Trying to solve root rot on plants in the ground is difficult. The best approach is to let the soil dry out to give plants a chance to fight the infection. Then solve the real soil problems, such as compaction, high water table, poor drainage etc. Follow that with proper watering.
Peroxide Cures Fungal Diseases
“You can use hydrogen peroxide to combat every kind of fungal infection on your plants.” One site recommends using a 0.75% solution for spraying plants, which is a 200 mM solution.
I am always suspicious when a product cures all kinds of fungi infections. Even commercial fungicides are only effective on some fungi.
A study looked at preventing powdery mildew from infecting greenhouse-grown cucumbers by using a hydrogen peroxide spray. They found that peroxide solutions of 15 and 20 mM concentration reduced PM from 90% to 12%. However:
- the plants were sprayed after transplanting and before the disease started.
- 50 mM solutions damaged the plants.
Powdery mildew seems to be a difficult fungus to cure once plants have it. Most treatments need to be started early to prevent an initial infection. It is also important to recognize that the suggested concentration of 200 mM would harm the plants. Every plant has a different sensitivity to chemicals but what you find on gardening blogs is a standard concentration for “all plants” and “all diseases”.
Peroxide is a fungicide and will kill fungal organisms, so there is no doubt it does work in some cases. The problem for the gardener is to know which cases work, when to spray and what concentration to use. This kind of information is almost nonexistent.
Disinfect Growing Media
Soak your media in peroxide and it will kill some of the microbes – but is this a good thing?
While some people are talking about disinfecting media, others are adding microbes to make plants grow better. A lot of the commercial potting soil I see has mycorrhizal fungi added. Peroxide will kill these organisms.
Even if you disinfect the soil completely, as soon as you add plants and set it in a window or on a patio outside, microbes from the air or plant will contaminate it. Trying to sterilize soil and keep it sterile is impossible without special lab equipment. Gardeners should not even bother to try.
Use Peroxide to Sanitize Seeds
“Let the seeds soak in 3% hydrogen peroxide for five minutes. Then thoroughly wash off the chemical by running water over the seeds for a minute.”
This recommendation has the same problem as some of the other claimed benefits, namely a one-size-fits-all approach. This advice assumes no seed is affected adversely by the soak and it assumes all seed will be totally sanitized – that is not likely to be the case. “A seed treatment method that works for one type of seed may not be as effective for another, because surface structure varies for different seed types.” The treatment can also adversely affect the germination rate.
I have grown several thousand different species of plants from seed and have never sterilized them. I do use the baggy method for germination (I have a video showing you how) which lends itself to fungal infection and yet I have had few issues. Sanitizing seeds might have helped in some cases, but in general, seeds do not need to be sanitized.
One concern with seedlings is damping off disease. The best way to prevent this is by keeping soil drier and running a fan 24/7. In wet conditions even sterilized seed will get damping off.
There is one case were seed sanitation may be a good idea and that is for growing sprouts (i.e. microgreens). “Raw or lightly cooked sprouts are a common source of foodborne illness.” Unlike the above recommendation, UC Davis suggests to, “treat the seed by heating on the stovetop for five minutes in a solution of 3% hydrogen peroxide preheated to 140°F (60°C).”
Speed Up Seed Germination
The claim goes something like this. High levels of oxygen are needed by sprouting seeds and peroxide provides a simple way to provide the extra oxygen. Or, some claim that the peroxide breaks down the seed coat and makes it easier for the seed to germinate. It is suggested to use a 3% soak for 30 minutes.
A peroxide soak has been used to speed up the germination of some seeds.
A four hour soak in 3% peroxide increased the germination rate and increased the number of seeds that germinated for Ribes cereum. However, an 8 hour soak had the opposite effect.
A hydrogen peroxide concentration of over 1% reduced the number of lettuce seed that germinated, but even at 0.1%, the length of the radical (root) was reduced in size.
Peroxide plays a critical role in seed germination. As soon as water is absorbed, natural peroxide levels influence several of the key processes that need to happen before seeds germinate and higher levels can speed up the process. It is also important to note that excess peroxide results in seed deterioration and a loss of seed vigor. The seed produces this needed peroxide on its own and does not need gardeners to supply it. Adding it in the right concentration and using the right soak duration will likely speed up germination, but doing it wrong, can kill the seed.
Peroxide soaks can help with germination but the actual mechanism is complex and still being elicited by scientists. It is not as simple as providing oxygen or softening the seed coat. It does not work for all seeds and too much can harm them. The blanket statements on the internet about speeding up all seed germination is false since most types of seed have not even been tested. If you are working with seeds that germinate slowly, you will most likely need to run some tests to see what works.
Some say that peroxide makes a good fertilizer. Hydrogen peroxide is hydrogen and oxygen – how can anyone suggest this as a good fertilizer?
Boost Root Growth with Peroxide
It is claimed that watering plants with hydrogen peroxide solution will add more oxygen into your soil which increases nutrient uptake by roots, thereby increasing growth. Use about two teaspoons of 35% hydrogen peroxide to around one gallon of water and then use it on your garden every other time you go out to water your plants.
It is unlikely that the excess oxygen increases root growth, except in cases where the plant is stressed by conditions like compaction. Peroxide in plant cells do play a role is things like potassium absorption by roots. It is possible that extra peroxide in soil has some impact on nutrient absorption by roots, but I can’t find scientific support for pouring peroxide onto the soil to enhance root growth. Remember that the oxygen provided by a peroxide soak will only last a few minutes.
Current science does not support this practice.
Peroxide Repels Insects
“A 1% hydrogen peroxide solution is safe to use and will keep away insects and kill any eggs. Aphids will be deterred from sitting on the leaves of your plants with just a spritz of this solution.”
Do aphids sit on leaves? I see them mostly on the stems and buds!
If you spray an insect or eggs it is quite likely it will be harmed and might even be killed.
Some claim peroxide kills larvae and eggs of fungus gnats, but I could not find a reliable source that confirms this. There is also no evidence it kills aphids.
The claim says that spraying plants with peroxide repels insects. How can this be? When peroxide is sprayed on plants it will degrade quickly as it reacts with the microbes that cover the leaves and sunlight hits it. After that, it’s just water. Last time I checked water is not very effective at repelling insects.
Peroxide Kills Weeds
The claim is that a 10% spray will kill off unwanted plants. Given the above mentioned phytotoxicity levels, this is quite possible but it would also kill non-weed plants.
If this works, it is surprising that it is never mentioned in government weed control procedures. Also note this requires a higher concentration of peroxide than the normal consumer product and that inhaling this higher concentration is harmful to lungs.
“Mix some peroxide into your watering can before you take it out into the garden. As hydrogen peroxide has strong oxidation, it will remove any harmful chemicals or pesticides found in ordinary tap water. This includes getting rid of chlorine which is added to water at treatment plants.”
Hydrogen peroxide is a strong oxidizer and it does react with some chemicals. It certainly does not “remove all harmful chemicals”. It is also possible that the reaction produces a chemical that is even more harmful. Each chemical of concern should be researched and evaluated on its own merits.
Peroxide will react with chlorine in drinking water, provided the pH is above 7. The reaction converts chlorine into hydrochloric acid – is that better for plants?
The truth is that the levels of chlorine in drinking water are not harmful to plants, so there is no problem to solve.
Natural Hydrogen Peroxide in Plants
Hydrogen peroxide is made by plants and used to control a number of internal hormonal systems. In low amounts it can trigger a plant to initiate the production of natural pesticides which in turn protect the plant from insects and diseases. For example, in pepper plants a peroxide spray can induce a plant to protect itself from a virus attack.
In high amounts peroxide is very toxic to plants.
Toxicity to Plants
When microgreen and lettuce seedlings were treated, some damage to leaves was seen at 0.0025%. This study recommends an upper limit of 0.01%, but this does vary by species. Mature plants are probably less sensitive since their leaves are tougher, and coated with microbes. The peroxide will react with the microbes and decompose before entering the leaf.
One popular site suggests using a spray of 0.75% to control diseases which is almost 100 times above the safe limit for seedlings.
Should Gardeners Use Hydrogen Peroxide in the Garden?
Most of the claims made on gardening blogs and social media are not supported by science. Some are true in select cases, but the typical claim is usually stated in an all encompassing way – kills all insects – making the claim untrue.
Popular suggestions also ignore the potential harm these treatments can do. Peroxide can be toxic to plants at some of the suggested doses.
Peroxide is certainly a good way to sanitize solid surfaces. It will kill microbes, but it affects both the good and bad guys.
I would not use it except for special cases that really need a solution and where there is some scientific evidence that it works. If you have a problem it might be worth your while to do some experimentation by applying different concentrations and seeing if there is an improvement in plants.
Most people use seed that germinates fairly quickly so there is no problem to solve. I know that 10 days for tomato seed seems like a long time, but it isn’t compared to slow germinating seeds, such as clematis. Experimentation with seeds would be a good place for some citizen science work. The key here is to use controls. Some of the seed needs to be treated in a normal way to compare it to seed treated with peroxide. You should also count germinated seeds – don’t just use a gut feeling that tells you it worked. If you do such work, post it on our Facebook Group.
23 Responses to ‘Should Hydrogen Peroxide Be Used in the Garden?’
Thank you for providing a truly scientific blog. I am relatively uneducated. I too read about the benefits of H2O2. Fortunately, despite the lack of a scientific education, I am a diehard researcher. When I start reading about something that will cure everything, I get very nervous. Consequently, I look for papers from mainstream State universities, Cornell, etc. Your blog opened my eyes and led me to further research. Ultimately I conclude that H202 probably does more harm than anything else.
I suggest that gardeners research Trichoderma harzianum, strain T-22 (https://biocontrol.entomology.cornell.edu/pathogens/trichoderma.php).
My original problem was that I didn’t know enough about planting anything so I planted roses into a former rose garden, sans fumigating the soil. Now my roses practically have everything a rose can get, e.g. black spot, brown spot, etc. plus every bug you can imagine because they are so weakened by the fungi they have no resistance.
I am trying to go all-biological controls as I don’t see any future with chemicals because every bug or fungi eventually become resistant.
Again, thank you for your rational, scientifically substantiated report. I deeply appreciate any advice you may wish to send me.
iNDEED, APHIDS DO LIVE ON LEAVES! MY YOUNG PRUNE PLUM ( SUMMERLAND, B.C.) WAS BEING DEVESTATED BY APHIDS. CATNIP GROWING UNDERNEAH THE TREE HELPED CONFUSE THE APHIDS. i TRIED 3%h2o2 ON SOME OF THE WORST EFFECTED NEW SHOOTS AND IT KILLED THE APHIDS, AND SEEMED TO DETER THE ANTS FROM RETURNING TO THE SHOOTS. BUT THE GROWING TIPS OF THE TREATED SHOOTS ARE NOT AS HEALTHY AS THE SHOOTS THAT i JUST USED A STROMG SPRAY OF WATER ON TO DISLODGE THE APHIDS. PERHAPS FOR SOME APPLICATIONS H2O2 MAY HAVE SOME USES, BUT IT CERTAINLY IS NOT THE PANACEA SOME CLAIM IT TO BE.
I planted a red oak tree (1 & 1/2” ) In a heavily watered lawn area. In the spring it put on nice foliage but then the leaves got brown edges then died. New leaves would grow and do the same thing. The internet suggested h202 with water to kill fungus & enhance oxygen. I tried the recommended tablespoon per gallon and had positive results – new leaves did not die. However, the ultimate solution has been to move the tree to a more dry location – waiting to see if it works. So far the tree has continued the same cycle of losing leaves.
How do you know that the tree did not regrow on its own, without the peroxide adding any benefit?
Thanks so much for clarifying this for me.
Oh, sure, that figures. After reading SEVERAL blogs/opinions on the internet extolling its virtues, I had bought a litre of 29% food-grade hydrogen peroxide to use, diluted, of course, on my indoor plants. I have been battling against tiny black flies, which I’m pretty sure is a fungus gnat, but then I’m not an entomologist, so my assessment is not conclusive. I have tried a product called Nema Globe Pot Popper with little success, and I’ve been using yellow sticky tape catchers, but the problem persists.
Would you kindly share any recommendations either for a solution to the problem or for an internet resource? Any direction would be greatly appreciated. Thank you.
Very well written. As a very serious plant enthusiast AND a third year biotechnology student I cringe when people say to use peroxide for gnats or bacterial and fungal infections!
Thank you for putting this together so cohesively and understandably.
According to published literatures in journals, it stated h2O2 plays a part in plant growth.
I have posted 4 citations below for reference. Please use your institution to access the papers. and you can access further evidence from the numerous citation found in the published papers below.
Jamaludin, R., Mat, N., Suryati Mohd, K., Afiza Badaluddin, N., Mahmud, K., Hailmi Sajili, M. and Khandaker, M.M., 2020. Influence of Exogenous Hydrogen Peroxide on Plant Physiology, Leaf Anatomy and Rubisco Gene Expression of the Ficus deltoidea Jack var. Deltoidea. Agronomy, 10(4), p.497.
Černý, M., Habánová, H., Berka, M., Luklová, M. and Brzobohatý, B., 2018. Hydrogen peroxide: its role in plant biology and crosstalk with signalling networks. International journal of molecular sciences, 19(9), p.2812.
Nasir, N.N.N.M., Khandaker, M.M., Mohd, K.S., Badaluddin, N.A., Osman, N. and Mat, N., 2020. Effect of Hydrogen Peroxide on Plant Growth, Photosynthesis, Leaf Histology and Rubisco Gene Expression of the Ficus deltoidea Jack Var. deltoidea Jack. Journal of Plant Growth Regulation, pp.1-22.
Barba‐Espin, G., Diaz‐Vivancos, P., Clemente‐Moreno, M.J., Albacete, A., Faize, L., Faize, M., Pérez‐Alfocea, F. and Hernández, J.A., 2010. Interaction between hydrogen peroxide and plant hormones during germination and the early growth of pea seedlings. Plant, Cell & Environment, 33(6), pp.981-994.
May i also know your credentials? Are you a plant biologist or just a hobbyist.
It will be great to back your statements above with papers published in academic journals.
Otherwise your blog is pretty much just another opinionate piece rebuking H2O2 uses, no difference from other bloggers who claim H2O2 is useful.
And some of these opinionate writers on the positive effect of H2O2 are also Master Gardeners, a fancy title with no recognition by academic institution.
Please note, i am only sharing evidence and facts backed by academics with papers published in reputable journals. I am not bothered who is right or wrong. But hobbyist should be bothered if they are reading and following blindly.
I am a biochemist.
I have no idea what point you are trying to make. Why not state your point, and then provide one reference that supports your point.
I have 70 rose bushes of a variety of types. From the time I bought and planted them, I used Neem and potassium soap to ward off fungus. They bloomed gloriously, but by the time the blooms faded, leaves had black spots, turned yellow and dropped. I had 70 stalks with no leaves. I used H2O2, 30% with water, in the ground and sprayed then with governor (a natural product available in Mexico, where I live). Now, about 2.5 months later, all of my roses have leaves and buds. Daily I pick off spotted and yellow leaves. It worked for me when nothing else did.
You claim that peroxide worked, but lets look at your facts.
1) You used both peroxide and governor – so you don’t know which if either made a difference.
2) You pick off infected leaves daily – if peroxide worked, then there would not no leaves with black spot to pick off.
Clearly the peroxide is not working.
You erred because you reacted to incomplete information. We sprayed the governor on leaves for weeks without stopping the black spots and yellowing. When picking off the affected leaves failed to correct the problem, I stopped doing it. I then read about H2O2, applied it as the processor indicated, and started to see improvement. Only when new leaves appeared did I resume picking off the handful of black-spotted leaves. I have 70 roses. All are now healthy, although we watch then carefully. I, too, am a scientist, a researcher. And I strive to be civil instead of a know-it-all.
I can only comment on the information you provide.
But you say “when new leaves appeared did I resume picking off the handful of black-spotted leaves”. So the plants are still getting black spot.
As a scientist you know that you need controls to reach any conclusion. How did you set up the controls?
We’re is your scientific proof that some of these h202 methods don’t work. You stated leave it to science but I see non of that just your personal 2 cents. We’re are the web links and proof, actual scientific proof
The references to scientific data I found are in the article. If you have additional ones to prove the conclusion wrong – post them here.
Robert, thank you for your posting your clarification on H2O2. The fad is taking the place by storm and in my opinion, has little effect on anything except to leave a wake of damage. Granted it is wonderful for some things, but no where near a panacea.
“10 amazing uses….” Yep, amazing – I’d be amazed too if they worked.
Robert I started a garden this year and your blog has been invaluable. I just bought your book on soil and can’t wait to learn more about how the two Orchid Lights Azaleas I purchased stand no chance in my (lab tested) 7.6 PH soil (I’ve got you beat!).
But really, thanks for your work it’s been very helpful and also enjoyable to read !
Thank you Robert!
Thanks for helping clear the confusion. The one thing I will add on aphids, they do get on apple and cherry leaves, at least here in Oregon. I had a horrible infestation this year. However, I don’t use peroxide on them.
“Trying to sterilize soil and keep it sterile is impossible without special lab equipment. Gardeners should not even bother to try.”
I don’t think anyone seriously talks sterilising soil in a strict sense. More common I think is heat-treated soil, which is sufficient to destroy weed seeds, and presumably some amount of microbes/fungi to reduce the chance of damping off fungus being present.
I assume the intent with the peroxide is a quicker route to this level of ‘treated’ soil.
“Some claim peroxide kills larvae and eggs of fungus gnats, but I could not find a reliable source that confirms this”
I appreciate all efforts at trying to find supporting papers, as I know it can be quite time consuming, but on the note of treatment of fungus gnats, a cursory google suggests 125 ppm hydrogen peroxide in a hydroponic setting was effective , but they do also note negative effects on germination. If you have the time, a list of concentrations and what they were effective at would be an excellent resource for gardeners to know which cases work, and what concentration to use.
“Do aphids sit on leaves?”
Perhaps you’ve only had minor infestations so they are mostly at the buds, but both my Jalepeno and tomato plants have had significant numbers on the leaves, and that is usually the point at which I spot them!
The study you linked.
I have a problem with it. They treated the seed starting trays with peroxide (of unknown concentration) and then left the seed to germinate and grow. After 15 days they counted larvae. Presumably there were no larvae at the beginning of the experiment. Peroxide diluted in water, sitting in a warm greenhouse with lots of light decomposes quickly. It makes no sense that it was still controlling larvae two weeks later.
The number of larvae were reduced compared to water, but there were still quite a few. How effective was the peroxide? Why did it not kill them all?
They also report that the concentration used affected lettuce seed and seedlings as well as cucumber seedlings.
Comparative transcriptome analysis of wheat embryo and endosperm responses to ABA and H2O2 stresses during seed germination
Wheat embryo and endosperm play important roles in seed germination, seedling survival, and subsequent vegetative growth. ABA can positively regulate dormancy induction and negatively regulates seed germination at low concentrations, while low H2O2 concentrations promote seed germination of cereal plants. In this report, we performed the first integrative transcriptome analysis of wheat embryo and endosperm responses to ABA and H2O2 stresses.
We used the GeneChip® Wheat Genome Array to conduct a comparative transcriptome microarray analysis of the embryo and endosperm of elite Chinese bread wheat cultivar Zhengmai 9023 in response to ABA and H2O2 treatments during seed germination. Transcriptome profiling showed that after H2O2 and ABA treatments, the 64 differentially expressed genes in the embryo were closely related to DNA synthesis, CHO metabolism, hormone metabolism, and protein degradation, while 121 in the endosperm were involved mainly in storage reserves, transport, biotic and abiotic stresses, hormone metabolism, cell wall metabolism, signaling, and development. Scatter plot analysis showed that ABA treatment increased the similarity of regulated patterns between the two tissues, whereas H2O2 treatment decreased the global expression similarity. MapMan analysis provided a global view of changes in several important metabolism pathways (e.g., energy reserves mobilization, cell wall metabolism, and photosynthesis), as well as related functional groups (e.g., cellular processes, hormones, and signaling and transport) in the embryo and endosperm following exposure of seeds to ABA and H2O2 treatments during germination. Quantitative RT-PCR analysis was used to validate the expression patterns of nine differentially expressed genes.
Wheat seed germination involves regulation of a large number of genes involved in many functional groups. ABA/H2O2 can repress/promote seed germination by coordinately regulating related gene expression. Our results provide novel insights into the transcriptional regulation mechanisms of embryo and endosperm in response to ABA and H2O2 treatments during seed germination.
Cereals are important to humankind, with over 2000 million tonnes harvested annually and used for food, livestock feed, and industrial raw materials. Wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD), an allohexaploid species, is one of the most important and widely cultivated cereal crops and is a main food source for more than 40 % of the global population . Wheat grains include mainly embryo and endosperm, and both play important roles in seed germination, seedling survival, and subsequent vegetative growth. The embryo forms radicle, plumule, and new plants, while the endosperm, which contains reserve substances, supplies nutrients for subsequent plant growth, which in turn affects wheat yield and quality.
Similar to most flowering plants, development and germination of wheat seeds are separated by a period of quiescence, which in many cases is also a dormancy phase. Only after breaking dormancy can the quiescent embryo germinate after imbibition. These processes have been investigated intensively at the physiological and molecular levels [2, 3]. Seed germination commences with imbibition, the uptake of water by the quiescent dry seed, and terminates with elongation of the embryonic axis . Wheat seed germination undergoes a three-phase process of physiological and morphological changes, including a rapid initial uptake phase, a plateau phase, and a further water-uptake phase, corresponding to switches from the degradation of small-molecule sucrose to the metabolism of three major nutrients and photosynthesis . These metabolic processes play key roles in seed germination by providing the required energy.
Abscisic acid (ABA) is a major hormone during seed germination . The interactions among ABA, gibberellin (GA), ethylene, and brassinosteroids (BR) control the interconnected molecular processes of dormancy release and germination in eudicot seeds, such as Arabidopsis and tobacco [7, 8]. ABA is an important plant hormone that at low concentrations positively regulates dormancy induction and negatively regulates seed germination. ABA not only inhibits water uptake by preventing cell wall loosening of the embryo  but also specifically inhibits endosperm rupture rather than testa (i.e., seed coat) rupture . Seeds undergo changes in both ABA content and sensitivity during germination in response to internal and external changes. Several studies to date have explored the roles of ABA during seed germination in both model plants and crop species such as Arabidopsis [11, 12], barley [13, 14], rice , lettuce , tomato , and coffee . Particularly, most studies on the functions of ABA involved in seed germination have focused on the model plant Arabidopsis, including on the regulation of, and the protein kinases required for, ABA signaling during seed germination [19, 20] and transcriptional regulation of ABA-responsive genes in germinating seeds .
Treatment with hydrogen peroxide (H2O2) at low concentrations promotes seed germination of cereal plants, but a high H2O2 concentration limits germination of seeds, such as those of barley, wheat, and rice , Arabidopsis , pea [23, 24], maize [25, 26], Zinnia elegans , Jatropha curcas , and oat . Endogenous H2O2 is generated in chloroplasts, mitochondria, and peroxisomes following exposure to a wide variety of abiotic and biotic stimuli . Besides the important signaling function in response to environmental stimuli, H2O2 has toxic effects . Catalase was proposed to be the most important H2O2-consuming enzyme in the presence of physiological concentrations of H2O2 . The exogenous application of H2O2 can increase endogenous seed H2O2 content and cause carbonylation of storage proteins and several metabolic enzymes, thus enhancing seed germination . Exogenously applied H2O2 ameliorates seed germination: one explanation is that the scavenging activity for H2O2 is high, resulting in the production of O2 for mitochondrial respiration [33, 34]. Another explanation is that H2O2 facilitates cracking of hard seeds, allowing them to interact with water .
In recent years, along with considerable progress in plant genomics, various transcriptomics and proteomics approaches have been used to investigate the mechanisms of seed germination and responses to various abiotic stresses in several plant species, such as Arabidopsis [35, 36], barley [37, 38], maize [39, 40], and rice [41, 42]. However, the majority of these studies focused on changes in the transcriptome of only one organ (embryo or endosperm) under one stress treatment, such as ABA or H2O2. An integrative transcriptome analysis of the responses of wheat embryo and endosperm to ABA and H2O2 stresses has not been reported to date.
In this study, we used the elite Chinese bread wheat cultivar Zhenmai 9023 and performed the first comparative transcriptome microarray analysis of the responses of embryo and endosperm to ABA and H2O2 treatments during seed germination using the GeneChip® Wheat Genome Array (Affymetrix, Santa Clara, CA). Numerous differentially expressed genes in embryo and endosperm responsive to ABA and H2O2 stresses involved in seed germination were identified. Our results provide novel insights into the molecular mechanisms of wheat seed germination and responses to abiotic stresses.
Transcriptome expression profiling of the response to ABA and H2O2 stress during seed germination
The global transcriptome changes of embryo and endosperm in response to ABA and H2O2 treatments during wheat seed germination were investigated using the Affymetrix GeneChip® Wheat Genome Array. We grew wheat seeds in pure water as a contrast check (CK_embryo and CK_endosperm), while seeds in the experimental groups were separately treated with 100 mg/L ABA and 100 μmol H2O2 = (ABA_embryo and ABA_endosperm, H2O2_embryo and H2O2_endosperm). When seeds were imbibed in water for 16 h and the radicle emerged from episperm, we harvested the embryo and endosperm tissues separately in triplicate (18 individual samples). All microarray data from three biological replicates obtained in this study have been deposited in the NCBI GEO database. The normalized expression values obtained from three biological replicates based on independently treated plant materials are shown in Additional file 1.
The degree of reproducibility was evaluated based on the square of the Pearson correlation coefficient, represented by the R 2 value, between each biological replicate, of which all were larger than 0.99, indicating relatively good repeatability. All samples were substantiated by one unsupervised hierarchical clustering-based classification procedure, and the replicates were clustered as neighboring clades (Fig. 1).
Hierarchical cluster dendrogram of normalized transcript abundances from 18 experiments including three biological replicates based on complete distance linkage. Two tissue fractions (E/A, red; Em, blue) were analyzed under different treatment (ABA, H2O2 and CK) during wheat germination
The dendrogram showed major differences among all samples between embryo and endosperm during ABA and H2O2 treatment processes based on variations in transcribed gene sets (Fig. 1). The clustering of embryo and endosperm tissues suggests a smooth transition between different treatments in the same tissue. The two tissues showed distinct expression profiles, and tissue expression variability was greater than in biological treatments, showing clear tissue-specific expression. For example, the difference in gene expression patterns between ABA-embryo and ABA-endosperm was greater than that between ABA-embryo and CK-embryo, similar to the comparison of H2O2 and CK. Interestingly, excluding the relatively large expression differences between the two tissues, there was a marked difference between the ABA-treated group and the other two groups (H2O2-treated group and CK group; these were grouped under the second stratum of hierarchical clustering) in both embryo and endosperm samples. One possible explanation for the considerable changes is that H2O2 treatment more accurately mimics natural biological processes than ABA treatment.
To increase our understanding of the expression differences between embryo and endosperm under ABA and H2O2 treatments, we performed a heat map analysis of significant differentially expressed genes (Additional file 2). As shown in Additional file 3, the whole cluster was divided into three groups and marked with three colors (blue, yellow, and red) on the right side. In the blue group, endosperm genes were mostly downregulated, while those in embryo were upregulated. In the yellow group, most genes in both embryo and endosperm were upregulated under H2O2 treatment. However, in ABA-treated tissues, approximately 20 % of genes in embryo and 60 % in endosperm were upregulated. In the red group the genes in the ABA-treated tissues were downregulated, while those under H2O2 treatment were upregulated. In our results, we have found several genes which showing that the expression of the H2O2 samples is not significantly different but expression for these genes in the ABA samples is significantly different, such as BJ293360 (hormone metabolism), BJ296527 (Biodegradation of Xenobiotics), BJ299669 (storage proteins), CA719001 (starch cleavage), BQ169398 (starch cleavage) (Additional file 1 and Additional file 2). It verified the above results again on the other hand that H2O2 treatment was more accurately mimics natural biological processes than ABA treatment.
Differential gene expression in embryo and endosperm in response to ABA and H2O2 treatments during seed germination
Significance analysis of microarrays (SAM) with a stringent 5 % false discovery rate (FDR) was applied to compare gene expression changes in embryo and endosperm under ABA and H2O2 treatments. The numbers of differentially expressed genes (DEGs) in embryo and endosperm in response to the treatments are shown in Fig. 2a and b. There are 6106 differentially expressed genes (DEGs) are regulated under the ABA treatment in the upregulated direction and 6521 DEGs in the downregulated direction compared to control groups. While in the H2O2 treated groups, there are 2064 DEGs in the upregulated direction and 1710 in the downregulated direction compared to control groups. In total, 4007 genes were downregulated while 2783 genes were upregulated in the ABA_embryo group, which showed that the sum total number (6790) of expression changes was similar to the ABA_endosperm (5837) containing 3323 upregulated and 2514 downregulated genes. Interestingly, the number of upregulated and downregulated genes under H2O2 treatment in embryo contrasted with those in endosperm (Fig. 2a). This sharp contrast suggested that the sensitivity of embryo and endosperm to H2O2 was different, and the embryo showed a different sensitivity in terms of response to ABA or H2O2 treatment. A greater number of genes were repressed than induced in embryo and there were more induced genes in endosperm exposed to ABA treatment (Fig. 2a). These results suggested that ABA repressed seed germination mainly by repressing embryo germination, while H2O2 induced seed germination mainly by activating endosperm genes.
Differential gene expression in embryo and endosperm responsive to ABA and H2O2 treatments during seed germination. a Histogram of differentially expressed genes; b Venn diagram analysis of the differentially expressed genes under the ABA, H2O2 treatment compared to the control group. ABA treated means DEGs in the ABA_embryo and ABA_endosperm; H2O2 treated means H2O2_embryo and H2O2_endosperm. Figure 2B (left) is all the DEGs (upregulated or downregulated) that identified in four groups: ABA_embryo group, ABA_endosperm group, H2O2_embryo group and H2O2_ endosperm group shared with each other. Figure 2B (right) is 10,185 DEGs in the ABA-treated groups and 3756 in the H2O2-treated groups. 3746 DEGs were shared in ABA-treated groups and H2O2-treated groups
Figure 2b (left panel) showed all the DEGs (upregulated or downregulated) that identified in four groups: ABA_embryo group, ABA_endosperm group, H2O2_embryo group and H2O2_ endosperm group shared with each other. We identified 10,185 DEGs in the ABA-treated groups and 3756 in the H2O2-treated groups (Fig. 2b right panel). There were 2442 DEGs not only in endosperm but also in embryo under ABA treatment, and 18 DEGs were identified in the above two tissues under H2O2 treatment. In total, 3746 DEGs were expressed in the above two treated tissues, of which 17 genes were differentially expressed in both embryo and endosperm under ABA and H2O2 treatments (Fig. 2b).
The key DEGs expressed under both ABA and H2O2 treatments in the embryo and endosperm are listed in Tables 1 and 2, respectively. In the embryo, 64 DEGs were associated mainly with DNA synthesis, CHO metabolism, hormone metabolism, and protein degradation. These functional groups belong to cellular processes and metabolic pathways. Half (50 %) of the 64 genes were assigned to cellular processes, 34 % to metabolic pathways, and only a few genes to development protein and hormone/signaling (Table 1). In the endosperm, 121 DEGs were closely related to the metabolism of storage reserves, transport, biotic and abiotic stresses, hormone metabolism, cell wall metabolism, signaling, and development. These functional classes are mostly involved in metabolic pathways, with about half of the assigned genes. The numbers of other assigned genes related to development proteins, transport, hormone/signaling, and cellular processes were similar (Table 2), all of which are important for seed germination and metabolic pathways. In the two gene lists, almost all of the genes were expressed only in embryo or endosperm, excluding CA602902 (related to stress defense) (Tables 1 and 2), which was expressed in both embryo and endosperm, suggesting that stress defense is important in both tissues during seed germination.
Embryo and endosperm transcript levels, determined based on Z-score transformation, are shown in scatter plots (Fig. 3). This transformation was used to compare the expression levels between two tissues. With ABA treatment, a greater number of genes were expressed in both embryo and endosperm compared to under CK and H2O2 treatments. ABA treatment increased the similarity of the gene expression patterns between the two tissues: R 2 = 0.868 in the ABA treatment compared to 0.843 in the CK. The H2O2-regulated group showed decreased global expression similarity compared to ABA treatment.
Comparison of the embryo and endosperm transcriptome of the control and treatment group. (Left list) Plot of Z-score transformed embryo versus endosperm expression data shows similar expression of most genes in both tissues. (Right list) The number of detected expressed genes in embryo, endosperm, or both tissues
To further investigate the transcriptome changes of embryo and endosperm in response to ABA and H2O2 treatments during seed germination, we compared the transcriptome data using MapMan software, which is a user-driven tool for mapping transcriptome data, define functional categories, and identify significantly overrepresented functional groups.
During seed maturation, reserve materials such as starch, sucrose, lipids, and storage proteins are gradually accumulated. Metabolic activities within the seed are significantly downregulated during dormancy and then reactivated during germination. Many hormones—including ABA, GA, H2O2, BR, ethylene, auxin (IAA), and jasmonate (JA)—are involved in seed germination. As shown in Fig. 4, the gene expression profiles during seed germination under ABA and H2O2 treatments, respectively, were determined. H2O2 treatment resulted in the upregulation of genes involved in seed germination in both embryo and endosperm. In contrast, a greater number of genes were expressed under ABA treatment and showed downregulated expression pattern. This indicated that H2O2 could promote seed germination, while ABA represses seed germination. According to our results, H2O2 treatment affected the expression of only a small number of genes in the embryo (Fig. 2a), but these genes caused marked effects and changes (Fig. 4). These effects and changes were mainly in glycolysis, sucrose degradation, cell wall, lipid metabolism, and photosynthesis (Fig. 4a and b). Lipids are found in the embryo, and genes associated with FA synthesis and beta-oxidation were largely upregulated in H2O2-treated embryos. Indeed, genes involved in photosynthesis, such as light reactions and photorespiration, were strongly upregulated. Another interesting result is that genes related to ascorbate and glutathione were upregulated in H2O2-treated embryo compared to the endosperm. This suggested that embryo is more sensitive to oxidative stress than endosperm.
MapMan metabolism overview maps showing differences in transcript levels between ABA/H2O2 treatment and CK during seed germination. a ABA vs CK and H2O2 vs CK in the embryo. b ABA vs CK and H2O2 vs CK in the endosperm. Log2 ratios for average transcript abundance were based on three replicates of AffymetrixGeneChip ® Wheat Genome Array. The resulting file was loaded into the MapMan Image Annotator module to generate the metabolism overview map. On the logarithmic color scale, blue represents downregulated transcripts, and red represents upregulated transcripts
The accumulated reserve materials of wheat seeds are stored mainly in the seed endosperm. During seed germination, they begin to metabolize along with imbibition. Sucrose, one type of deposit, is a micromolecule substance that is easily mobilized. Our results showed that the inhibition of ABA and the ability of H2O2 to promote sucrose mobilization were most distinct in embryo and endosperm (Fig. 4). The majority of genes related to sucrose mobilization were downregulated under ABA treatment, while several were upregulated under H2O2 treatment. In the process of sucrose degradation, sucrose synthase (SUSY) is the most important enzyme, and the related key gene to SUSY is CA623473 (Additional file 1). Other enzymes, such as hexokinase and fructokinase, also play a role. Besides energy provision, sucrose acts as transmembrane transporter in the cell membrane.
The main reserve deposit of wheat seeds is in the form of starch, which is mainly present in endosperm. Different treatments result in distinct changes in starch cleavage and synthesis in endosperm. Two important enzymes, amylase for wheat starch cleavage and UDP-glucose pyrophosphorylase (UGPase) for starch synthesis, are required during metabolic processes. The majority of genes related to starch cleavage were downregulated, while those for starch synthesis were upregulated under ABA treatment. However, H2O2 treatment resulted in the opposite changes (Fig. 4). Glycolysis and the TCA cycle are very important metabolic pathways during seed germination. Based on our results, the expression levels of genes related to both glycolysis and the TCA cycle in endosperm were higher than those in embryo, indicating that the primary pathways occurred in endosperm to provide energy for wheat seed germination.
During seed germination, rapid water absorption leads to seed expansion and penetration of the embryonic axis. According to our gene expression data, genes related to major constituents of the cell wall (cellulose, pectin, hemi-cellulose, and expansins) were activated during imbibition. This suggests that the cell wall was undergoing continuous modification and synthesis; however, it was also experiencing continuous degradation. Several important enzymes related to degradation such as cellulases and pectatelyases are activated early during seed germination. Hence, genes associated with cell wall synthesis and degradation were activated during the process of imbibition. We speculated that the majority of the fractured cell wall might be degraded into small molecules to provide raw materials for cell wall synthesis during seed germination. However, our gene expression data showed that most cell wall-related genes were upregulated under H2O2 treatment and downregulated under ABA treatment. Furthermore, these related genes were more activated in H2O2-treated embryos than other treatments (Fig. 4).
As shown in Fig. 4a, genes related to photosynthetic processes, such as the light reactions and the Calvin cycle, became activated in the embryo. However, in the endosperm, the photosynthesis-related genes remained inactivated (Fig. 4b). This suggested that photosynthesis genes were activated first in the embryo, and that their expression was promoted by H2O2.
Verification of gene expression patterns using qRT-PCR
Quantitative real-time polymerase chain reaction (qRT-PCR) with specific primers was used to confirm the expression of nine representative genes (Additional file 4). These genes are involved in starch synthesis, fermentation, RNA regulation of transcription, cell wall modification and precursor synthesis, abiotic stress, transport, and hormone metabolism, and play pivotal roles in seed germination. Optimization experiments showed higher amplification efficiency and specificity of nine targeted genes (Additional file 5). As shown in Fig. 5, the expression patterns of five genes (CD491559, CA498269, AY543540.1, Y09916.1, and CK198230) were consistent with those determined by transcriptome microarray analysis. The expression patterns of the other four genes (BJ249131, AY485121.1, BQ170546, and CA645154) generally followed the transcriptional expression models.
Verification of 9 key gene expression patterns by qRT-PCR. The horizontal axis is the different treatment in the different tissues (6 groups) during seed germination, and the vertical axis is the expression of each group after normalized fold
Tissue differential expression and significant functional classes in wheat embryo and endosperm in response to ABA and H2O2 treatments
Our results showed that embryo and endosperm exhibit different responses when exposed to ABA and H2O2 treatments. Treatment with a low concentration of H2O2 facilitates seed germination, while low concentrations of ABA repress seed germination. Thus, there is tissue differential expression during seed germination in response to ABA and H2O2 treatments, similar to in germinating Arabidopsis seeds . Heat map analysis of differential genes showed that genes are expressed differentially not only in number, but also in classification, between embryo and endosperm (Additional file 3). During seed germination, the key functional class in embryo is cellular processes, while that in endosperm is metabolic pathways. These results suggest that genes in embryo and endosperm show tissue-differential expression.
According to our results, lipid degradation is repressed by ABA in the embryo, but not obvious in endosperm tissues. In endosperm, ABA inhibited mainly storage reserve metabolism (Fig. 4b). Apparently, a tissue-specific response to ABA sensitivity exists between embryo and endosperm. This differential sensitivity of lipid mobilization to ABA in the embryo and endosperm was confirmed in Arabidopsis and tobacco seeds [12, 44], suggestive of wide conservation and functional differentiation of embryo and endosperm among seed plants.
In this study, we detected several significant functional classes in embryo and endosperm during seed germination based on analysis of differentially expressed genes. We hypothesize that the cellular processes are more important than other functional classes in embryo during seed germination, as suggested by the assigned gene numbers, similar to the metabolic pathways in endosperm. Transport, a functional class important to metabolism, was present only in endosperm based on the classification of differentially expressed genes (Table 2). This suggested that endosperm, the main tissue for energy supply, could provide the energy for seed germination through various metabolic pathways. Small molecules generated during the germination process were transported to the embryo and other locations to support germination processes, such as DNA synthesis, bud germination, and development. Another difference between embryo and endosperm is that the cell wall-related genes in embryo were involved in degradation and modification, while those in endosperm were involved in synthesis and modification. This may be because penetration of the embryonic axis occurs in embryo during seed germination, and nutrients are required. According to our results, only CA602902 was expressed in embryo and endosperm (Tables 1 and 2). The CA602902 gene was involved in biotic stress responses, and similar genes (prp4, At3g19690, Os07g0129200) have been found in maize , Arabidopsis , and rice .
The endosperm in mature cereal seeds of comparatively large sizes is important to understanding the regulation of seed germination. In wheat, endosperm accounts for about 90 % of the whole seed and plays a vital role in seed germination. According to this study, an important role of endosperm in wheat seeds during imbibition is to provide energy for seed germination and the post-germination period. In the seeds, hydrolytic enzymes are secreted from the aleurone layer into the free endosperm to mobilize starch, protein, and lipid reserves. Carbon in the form of sucrose from endospermic reserves is transported to the embryo to fuel post-germinative growth . Similar results have been reported in other angiosperm seeds, such as Arabidopsis  and barley . Our results demonstrated that ABA repressed seed germination by inhibiting the activity of hydrolytic enzymes such as amylase, hexokinase, PPFK, and PK during reserve mobilization. A previous study also showed that an ABA-induced protein kinase could mediate ABA suppression of amylase expression .
Our results demonstrated that activation of cell wall genes was associated with seed expansion and penetration of the embryonic axis during seed imbibition. ABA repressed the expression of genes related to the cell wall, while H2O2 induced the expression of these genes in both embryo and endosperm. Endosperm is considered a barrier to radicle protrusion in many angiosperm seeds. Furthermore, endosperm weakening can mediate control of radicle protrusion during Brassicaceae seed germination . The other functions of endosperm were to control germination by secreting cell wall-loosening enzymes such as β-1, 4-glucanase, polygalacturonase, and expansins to degrade cell walls of the endosperm and seed coat, thus removing mechanical barriers to radicle emergence [12, 43]. According to our transcriptome data, β-1,4-glucanase and polygalacturonase are two important cell wall-degradative enzymes, named cellulase and pectinase, respectively (Additional file 1). Expansins facilitate cell wall extensions, possibly by disrupting hydrogen bonding between hemicellulosic wall components and cellulose microfibrils . Activation of the genes related to these three types of enzymes results in cell wall degradation, modification, and synthesis. Thus, we hypothesize that ABA/H2O2 repressed/induced seed germination by inhibiting/facilitating gene expression of these enzymes.
In tobacco, the micropylar endosperm region could function as a water reservoir for the embryo . Wheat, similar to tobacco, contains comparatively large endosperms in mature seeds. It is likely that wheat endosperm plays a unique role in preserving water.
Signaling function and regulation of H2O2
Our results showed that exogenous application of H2O2 could promote seed germination, as has been reported previously . H2O2 has two important roles: serving as a signal in response to environmental stimuli and regulating hormonal metabolism, with effects on accelerating seed germination [11, 52].
Our recent work showed that when placed in water, wheat seeds activate a series of mechanisms that respond to biotic and abiotic stresses during germination due to changes in the external environment . Mitogen-activated protein kinase (MAPK) is believed to play a key role in these biotic and abiotic responses. MAPKs receive hormonal and other signals, and mediate transcription factors through the MAPK cascade reaction. These transcription factors then regulate defensive genes encoding stress-related proteins that function in the responses to external biotic and abiotic stresses . The regulation of defensive genes can protect germinating seeds against damage from biotic and abiotic stresses. Similar results have been reported in Arabidopsis , rice , and tobacco . H2O2 serves as a second messenger in cellular signal transduction pathways, and can lead to the activation of MAPKs [24, 56, 57]. Therefore, we propose that H2O2 promotes seed germination by regulating the activation of MAPKs. AtMPK6, an Arabidopsis MAPK, is involved in signal transduction pathways responding to these biotic and abiotic stresses for reactive oxygen species (ROS) [58, 59]. In the present study, we identified a gene (AY173962.1) that is similar to At2g43790, which encodes AtMPK6; the encoded protein may have the same function as AtMPK6.
Two major plant hormones, ABA and GA, play an important role in controlling wheat seed germination. Both ABA and GA are under the regulation of H2O2 in seed dormancy and germination . H2O2 upregulates ABA catabolism, resulting in a decreased ABA content, and promotes GA biosynthesis during imbibition, while ABA plays an important role in enhancing seed dormancy and delaying germination. Hence, the decreased ABA content could benefit seed germination, which indirectly shows that H2O2 promotes seed germination. Exogenous H2O2 can increase ABA catabolism by enhancing the expression of CYP707A genes in Arabidopsis . CYP707A1 encoded by At4g19230, a member of the CYP707A gene family, may play an important role in determining ABA levels. In our study, we identified an important gene, BJ291883 (Table 1), which may have the same function. GA has an antagonistic role with ABA in seed germination. For example, our results showed that ABA suppressed the expression of amylase, while GA induces transcription of amylase in cereal seeds [50, 60, 61]. GA was found to promote seed germination in many species, such as Arabidopsis [62, 63] and maize .
Reactive oxygen species (ROS) in seed germination
As byproducts of aerobic metabolism, reactive oxygen species (ROS) such as H2O2, O2 − , hydroxyl radicals, and superoxide radicals are produced during seed germination. The accumulation of ROS not only leads to cell injury and disturbances in seed germination but also functions as a signaling molecule and is involved in a wide range of responses to various stimuli . The balance between ROS production and scavenging regulates their accumulation, and antioxidative mechanisms are important for the scavenging of ROS. Levels of antioxidant compounds, such as ascorbate and glutathione, increase during wheat and Pinuspinea seed germination [66, 67]. Similar results were found in our study: two differentially expressed genes (CA607898 and BE426829) related to ascorbate and glutathione were detected in endosperm (Table 2). The expression levels of CA607898 and BE426829 increased by about two to three-fold under H2O2 treatment, indicating that detoxifying enzymes and antioxidant compounds were strongly expressed, possibly due to increased H2O2 toxicity. ABA-treated endosperm showed upregulation of CA607898 and downregulation of BE426829, possibly because of the different results of ABA signal transduction affected by H2O2.
In this study, we performed a global transcriptome profiling analysis using the Affymetrix GeneChip® Wheat Genome Array to characterize gene expression changes in embryo and endosperm in response to ABA and H2O2 treatments during wheat seed germination. Microarray analysis enabled detection of a large number of genes in germinating seeds related to ABA and H2O2 responses. The dendrogram analysis was suggestive of major differences between embryo and endosperm under ABA and H2O2 treatment during seed germination. The differential expression analysis between CK-treated and ABA/H2O2-treated tissues identified a number of differentially expressed genes in the two tissues under different treatments. The differentially expressed genes in embryo under ABA and H2O2 treatments were closely related to DNA synthesis, CHO metabolism, hormone metabolism, and protein degradation, while those in endosperm under ABA and H2O2 treatments were related mainly to the metabolism of storage reserves, transport, biotic and abiotic stresses, hormone metabolism, cell wall metabolism, signaling, and development. Scatter plot analysis showed that regulation patterns in the ABA-treated group were similar between the two tissues, while the H2O2-treated group showed greater expression differences. MapMan analysis provided a global view of the changes in several important metabolic processes (e.g., energy reserve mobilization, cell wall metabolism, and photosynthesis) and functional groups (e.g., cellular processes, hormones and signaling and transport) in embryo and endosperm following exposure to ABA and H2O2 treatment during germination. qRT-PCR analysis was used to validate the expression patterns of nine genes. Our results provide novel insights into the mechanisms of transcriptional regulation in embryo and endosperm in response to ABA and H2O2 treatments during seed germination.
Plant material and treatments
Arrays were performed on isolated embryo and endosperm tissues from Zhengmai 9023, an elite Chinese bread wheat cultivar (Triticum aestivum L.) with high yield performance and superior quality . Seeds were germinated on wet filter paper in Petri dishes with three biological replicates, and incubated at 25 °C in a growth chamber in the dark. Tissues were harvested from seeds under 100 μmol H2O2 and 100 mg/L ABA treatment, respectively until radicles just break through the sporniodem. Embryo and endosperm samples were collected by manual dissection as described, and stored in RNAlater solution prior to RNA extraction (Qiagen). Three biological replicates for two tissues under ABA and H2O2 treatments were used for microarray hybridization.
RNA isolation and microarray hybridization
Total RNA was extracted from materials using the Trizol® Plus RNA Purification Kit (Invitrogen, Carlsbad, CA) with an on-column DNase treatment. Purified total RNA samples were quantified with Agilent 2100Bioanalyzer (Agilent Technologies, Palo Alto, CA), and satisfactory purity was indicated by A260:280 ratios about 2.0 in 10 mM Tris–HCl (pH 7.5). Integrity of total RNA samples was assessed by denaturing formaldehyde gel electrophoresis, where the presence of sharp 28S and 18S ribosomal RNA bands at an intensity ratio of
2:1 (28S:18S) indicated good integrity. After that, high quality RNAs can be used to the subsequent high-throughput experiments. Total RNAs were incubated with OligodT/T7 primers and reverse-transcribed into double-stranded cDNA. The amplified RNAs were purified and labeled by biotin with Affymetrix’s IVT labelingkit. The biotinylated cDNAs were fragmented and hybridized to the Affymetrix GeneChip ® Wheat Genome Array (Affymetrix, Inc., Santa Clara, CA) for 16 h. The wheat genome array includes 61,127 probe sets representing 55,052 transcripts for all 21 wheat chromosomes in the genome. 59,356 probes sets represent modern hexaploid (A, B and D genomes) bread wheat (T. aestivum) and are derived from the public content of the T. aestivum UniGene Build #38. 1215 probe sets are derived from ESTs of a diploid near relative of the A genome (T. monococcum), a further 539 represent ESTs of the tetraploid (A and B genomes) macaroni wheat species T. turgidum, and five are from ESTs of a diploid near relative of the D genome known as Aegilops tauschii. After washing and staining, the results were scanned and recorded.
Data treatments and significant differential gene analysis
The microarray imaging data were analyzed with Microarray suite version 5.0 (Affymetrix Inc.), followed by Spotfire (Spotfire, Somerville, MA). Three biological replicates per treatment were hybridized independently to the Affymetrix ATH1 array, washed, stained, and scanned following the procedures described in the Affymetrix technical manual. The expression levels of genes were measured by detection calls and signal intensities using the Micro Array Suite 5.0 software with a target signal of 100. Sixty four Affymetrix controls and 5623 wheat genes that are detected as absent in all 18 chips were removed from the 22,810 probe sets. All microarray data from three biological replicates obtained in this study have been deposited in the NCBI GEO database, which are accessible through GEO Series accession number GSE64030 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE64030). All pairwise differentially expressed genes were identified using SAM software using the data of all the remaining 17,123 wheat probe sets. A false discovery rate parameter of 1 % was used for the SAM analysis. Following SAM analyses, genes that were called absent more than twice among three replicas in both control and treatment arrays were regarded as not expressed in both conditions and then removed from the above list. Z-score transformation was performed as described . This transformation normalizes the data according to the distance of each log10 value from the mean log10 value, expressed in terms of number of standard deviations.
For the MapMan analysis, input files were created by calculating the natural log ratio of the mean detection of the three control samples to the mean detection in the treatment samples. Genes called absent in two out of the three replicates were regarded as not expressed under that particular experimental condition. Final analyses were performed with MapMan version 1.6.1, including automatic application of the Wilcoxon rank sum test . Comparison with public domain Affymetrix ATH1 data sets was achieved by downloading entire data sets from NascArrays and from Nakabayashi et al. . Probe sets were identified that exhibited two fold or greater changes in expression in response to H2O2 and ABA treatments.
We did the hierarchical clustering to analyze the gene expression profile based on methods described by Eisen et al. . A software named cluster3 was used to do the clustering analysis. The parameters are following: % present is set to > =80, sd is 2, all ratio values are log transformed (base 2 for simplicity), we also selected the median and normalize, then the Euclidean distance similarity metric was used to define the similarity and the hierarchical clusters were assembled using the complete linkage clustering method, the k-means was default.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Representative differentially expressed genes were verified by qRT-PCR. After RNA isolation, first-strand cDNA was synthesized in a 20-μl volume containing 0.5 μl AMV reverse transcriptase (Promega), 0.5 μl RNase inhibitor (Promega), 1 μl oligodT primer, 2 μl dNTP mixture, 4 μl MgCl2 (25 mM), 2 μl 10 × reverse transcriptase buffer and 4 μl RNA sample. The reaction mixture was incubated at 42 °C for 60 min.
Double standard curve method was used to detect the gene expression levels. ADP-ribosylation factor was used as the internal control, which was identified as one of the most stably expressed genes . Gene-specific primers were designed using Primer 5.0, and their specificities were checked by the melting curves of the RT-PCR products. Each qRT-PCR reaction was performed in 20-μl volumes containing 10 μl 2 × SYBR Premix Ex Taq (TaKaRa), 2 μl 50-fold diluted cDNA, 0.4 μl of each gene-specific primer, and 7.2 μl ddH2O. PCR conditions were as follows: 95 °C for 3 min, 45 cycles of 15 s at 95 °C, 57 °C for 15 s and 72 °C for 20 s. Three replicates were used for each sample. Reactions were conducted in a CFX96 Real-Time PCR Detection System (Bio-Rad). All data were analyzed with CFX Manager Software (Bio-Rad).