Drought continues to be a primary driver of famine and crop loss around the world, with the accompanied water shortages not only affecting food production, but also drinking water availability. It is predicted that, by 2025, over two and a half billion people will be facing those sort of water shortages, especially as the effects of climate change worsen.

For agriculture, there are several crops that have been found to be specially sensitive to drought due to their high liquid content and growing conditions, such as soybeans and rice. Since these foods make up major staples for dozens of countries around the world, the ability to combat the impact of drought is of high importance. It is estimated that food production in rice-primary countries will need to increase by 40% in the next ten years to not only meet demand, but to also offset the crop losses from a changing environment. Food security has never been more important.

Thanks to modern biotechnology, however, new genetic improvement techniques have allowed for the development and testing of crops with far more in-depth changes than ever before. The Omics fields have reached a level where we can move beyond just transferring traits and instead focus on altering regulatory pathways that control traits overall. Transcription factors, signaling molecules, cytokinin metabolism and more are steadily being unveiled as options for cascading effects beyond single changes, allowing for far greater improvements.

A Breakdown of Terminology

Before we start on the research, there are some terms that should be explained. They won’t be used very often below, but understanding how they are grouped is crucial to analyzing drought tolerance studies. It’s pretty simple though. There are four main categories of experimentation that can be done with crops in regards to this topic or, more specifically, four mechanisms that can be altered.

The first is drought avoidance, which is the method of broadening water supplies in a plant to avoid drought becoming an issue at all. This is done by either increasing the speed of closing gas exchange systems so water loss is minimized, multiplying the speed and amount of water uptake through the roots, or by augmenting the rate of the plant moving from vegetative growth to the reproductive stage. The latter is relevant because any severe drought that occurs in the middle of this conversion can completely abort the reproductive cycle as a whole, thus preventing the formation of seeds to harvest.

The second mechanism is drought tolerance itself, otherwise known as the ability of the plant to continue growing and producing necessary nutrients even under drought conditions. Rephrased, it is the plant’s capability to tolerate drought. This system is where all of the regulatory pathways come into play and the thousands of genes to manipulate ratchet up the complexity of genome modification.

Third is drought escape, which holds several similarities to the final option in drought avoidance. The mechanisms to escape drought are largely those that rapidly speed up the move from one growth stage to another before the effects of drought cause a slowdown in plant activity.

Lastly and more straightforwardly is drought recovery or genes that help the plant recover its photosynthesis efficiency and nutritional and water uptake systems back to normal levels after experiencing drought conditions. These types of genes are often combined with drought tolerance modification since their combination can greatly improve the ability of a plant to grow through drought and get back to maximum efficiency afterwards.

These categories make up the genetic groupings that researchers study in order to improve crop yields and the ability to resist the abiotic stress of drought. In many cases, genes from different categories may be mixed and matched and one experiment may not focus on just one set of genes, but several to see if combinatorial effects can improve the benefits from each.

With that explanation out of the way, let’s get into the research.

The Rice Rosetta Stone

Out of all the crops that have been deemed a focus for drought tolerance research, there are none that have proven more significant than rice itself. Due to its submerged growing nature, the evolution of rice as a crop has included the necessity of developing genes that can control for both flood and drought resistance. This has proven to be both its blessing and its curse.

Modern rice crops require a truly astronomical amount of water. Even those farmers that rely on merely rainfed plots find that their rice crops do not grow to their full capability. There is just not enough water from rain to give them what they need. Irrigation has become one of the only options left and also long-term water saving methods. This is why many current pictures of rice farming in places like China show a form of terraced farming that allows for easier irrigation and also the flow of water is kept in each terrace, while also allowing for it to flow to lower terraces.

In scientific terms, rice finds itself having very low Water Use Efficiency (WUE). And it remains extremely sensitive to drought conditions, even with the high amounts of water it is practically drowned in. The amount of water required is expected to climb due to higher evaporation from climate change heat. This will also lead to gradual draining of fresh water supplies in river basins and this may make rice farming impossible on the large scale by as early as 2025.

Unless we are able to develop a drought tolerant variety by that point.

Transcription Factor Families

The results of drought on rice include conditions such as a delayed flowering time, a lower number of spikelets forming, and the grain filling rate being reduced. For farmers, this usually entails harvesting less than a ton of actual rice grains per hectare, a very low collection amount for contemporary crops.

The traits that plants have developed to deal with this include better water uptake and retention and the ability to modulate their growth and time of development to keep their interior resources high. Almost all of these have been set up to respond to several groups of transcription factor families that are often shared between plant species.

Among the largest of these is the NAC family, with over 150 transcription factors in rice alone. The name of this superfamily is obtained from the first letter of three individual protein families (NAM, ATAF, and CUC) that were placed into a single group due to their very similar DNA-binding domains and how this causes them to be mutually activated under the same conditions.

Previous studies have already shown that specific transcription factors in the main rice species, Oryza sativa, are responsible for regulating a variety of abiotic stress responses. For drought tolerance, these have been seen to largely affect root growth and the strengthening of water uptake pathways such as the xylem.

Connecting Roots With Drought

The OsNAC6 gene has been recognized as a key regulator for salinity response, blast disease resistance, and drought tolerance. It does so indirectly though by upregulating other direct genes that are considered stress-inducible genes responding to the environment. Since the gene has also been found to control root growth during early life stages of the rice plant, it has been suggested that there might be a connection between this and its drought-related capabilities.

In a study in June of this year, researchers investigated this possibility by making transgenic rice lines that overexpressed the gene, with one group using the root-specific promoter version and another using the constitutive (entire plant) promoter. An OsNAC6 mutant with the gene knocked out was also used as a control.

As expected, the first two groups saw drought tolerance and the latter control saw drought susceptibility. Though in actual multi-year field testing, the root-specific group far outstripped the resistance of the rest, showcasing the connection the gene has between root growth and resisting drought. Further genetic testing helped identify the particular genes that OsNAC6 upregulates, giving options for future experimentation.

Avoiding Growth Retardation

A main issue in searching for and expressing drought tolerance genes is that they often have the side effect of retarding plant growth over time. This is likely due to the plant tradeoff system between stress response and growth. Improving the ability of plants to respond to abiotic stresses in turn has them focus less on cell growth and duplication.

It has been one of the primary concerns in transgenic plants for this field thanks to how unavoidable it seems. The long-term activation period of overexpressed genes exacerbates the problem. Choosing less optimal expression genes that are not as effective and turn off faster would just reduce their response rate to the relevant stresses, which would also retard their growth because of that.

A group of researchers in April set out on a different tactic. Pyramiding, that is stacking on top of, of genes is a common occurrence in current biotech research. It seems like basic sense that stacking multiple different genes of the same type, such as for Bt toxin production, would increase the overall output and impact. And that is certainly the case.

But stacking of unrelated genes for different and, possibly, cross-purposes is not common at all. It’s practically unheard of, actually. But the researchers decided to see what would happen if they combined overexpression of a stress response gene against dehydration (DREB1A) with overexpression of a cell elongation for growth gene (OsPIL1). Normally, the latter is down-regulated under drought conditions as the plant puts more energy toward dealing with the water shortage and not toward plant growth.

What they discovered was a welcome surprise. The two genes appear to work independently of each other and do not affect the expression of their opposite. So, while one led the rice plant to respond to drought better, the other importuned the plant to both grow more and to flower earlier.

The acceleration of the latter did not happen at full efficiency, it should be noted. The amount of growth was only partially what would have been expected for just overexpression of the gene by itself, but the fact that there was any improvement at all compared to the lines with only DREB1A expression was a good sign.

The scientists hope that this revelation on stacking of opposite traits will lead to further studies that can improve the available drought resistant lines to match the yield and output of their non-drought tolerant counterparts.

Extending The Roots

One of the key responses to drought conditions in plants is the closing of the stomata on the leaves to reduce water evaporation and loss through them. However, since these are the dominant method of gas exchange and respiration for plants, this causes the predictable consequence of reducing growth.

Since the roots are the first identifiers and responders to drought, along with being the primary water intake systems, an improvement in the roots can lead to a longer time before drought response is triggered, giving plants more time to grow before that occurrence. But the root systems have a fairly huge array of genetic pathways and transcription factors controlling them, making it difficult to untangle which genes affect which others and how.

Another large protein family in plants is called the AP2/ERF superfamily, with ten different subgroupings. Yet, due to this vastness, the amount these groups have been studied is limited, with only Group VIII and Group IX having any meaningful characterization. Individual genes may have been studied among the rest, but the multiple groups’ motifs (their physical protein structure) and activities (the other genes they may alter and regulate) have yet to be conglomerated into a working description.

Some ERF transcription factors have been found to influence drought tolerance by raising the number of osmoprotectants (soluble sugars and some amino acids) that protect against osmotic shock. That is, the change in concentration of certain molecules in the environment around the cell, which a sudden decrease in water would modify.

Among these osmoprotectant molecules, calcium ions (Ca2+) are a highly important signaling molecule and help send signals related to environmental stress. Entire pathways are built around controlling calcium signaling. But the specific manner in how certain complex proteins called CaM-like proteins (CMLs) interact with calcium ions and regulate these stress responses isn’t well understood.

A study from just a few months ago was testing rice plant response to overexpression of a particular ERF pathway gene, OsERF48, with another root-specific vs entire plant comparison. The root-specific group, as usual, saw an expansion of root growth that in turn increased drought tolerance.

When running a transcriptomic analysis, they saw that OsERF48 actually regulated another gene called OsCML16 from the CML family and it is this latter gene and protein that causes the root growth. Though they also appear to together adjust the regulation of many other genes down the line, with the ERF gene being the master regulator of how much expression is enacted.

With this, yet another gene and pathway is further explained and opens up more regulatory options for genome modification that can give rice plants (and other plants besides) drought tolerance.

The Stomata Hormone

In a final look into the genes that rice provide, we go back to a previously mentioned subject involving stomata and the ongoing theme of water upkeep. This takes us to another gene family, but a much smaller one in comparison. The ASR family, which stands for abscisic acid, stress, and ripening all focus around the production of the mentioned acid which will be abbreviated as ABA from hereon out.

ABA acts as a core plant hormone that controls far too many regulatory systems to count, but is highly known for its use in regulating stress genes and triggering the closing of stomata. Past research has found that ABA is able to cause the production of hydrogen peroxide (H2O2) to act as signaling molecules for mediating stomatal closure.

The issue is that the genes that actually manage this two-part pathway are yet to be understood and while many ASR genes have been identified and relate to various stress responses, the exact response genes for drought tolerance haven’t been deciphered. Even comparing homologous ASR proteins between different plants (such as ASR1 from each) hasn’t helped, as they don’t always involve the same stress response, despite being physically similar. So sequence homology alone won’t be enough.

Researchers in a February study chose to feature upland rice, a group of cultivars known for their drought tolerance traits, and the specific genes OsASR3, OsASR5, and OsASR6. Out of the three identified options, OsASR5 was chosen for extended testing with an overexpressed transgenic line compared to a line with the same gene knocked out.

The latter obviously showed heightened drought sensitivity and a reduced amount of water content in the plant. The overexpressed line meanwhile had a greater amount of ABA produced and was very sensitive to the effects of ABA on the germination and just after germination stages. H2O2 was also produced at a higher level, leading to more stomatal closure and less water loss from evaporation.

A last thing found was that the OsASR5 protein itself acts as a chaperone protein and is believed to keep several other stress genes from inactivating, helping protect the plant more from other stress conditions like heat shock, which often accompanies environmental drought.

Other Drought Tolerance Research

With the immensity that is rice covered fairly thoroughly, we will now go through a hodgepodge list of other recent research with several crops and the genetic insights they have revealed. There will be no real attempt to create a connecting narrative back to the previous sections, though some of the studies below will indeed discuss protein families and transcription factors related to those already explained. Just not the genes from rice themselves.

Field Testing of Soybeans

Another major crop commodity with a heavy reliance on water sources is the soybean. This has been especially so for Brazil, one of the highest producers of soybeans in the world, over the past several decades and the issues they have dealt with involving drought. Research has shown that the same DREB and ABA genes in other species play just as much of an important role as they do in rice. Commonly in studies, the version of the gene found in the model organism Arabidopsis thaliana has been transferred into soybeans to increase drought tolerance.

However, a lot of this research was done under controlled greenhouse conditions, which don’t always translate the same when tested in the field proper. They can’t properly account for the random variance of water and environmental exposure in a true field test. But, since field tests are usually a requirement before commercialization and release, they have to be done at some point for any trait that scientists wish to bring to farmers.

Thus, this study in April took three genes from Arabidopsis named AtDREB1A, AtDREB2CA, and AtAREB1FL and created transgenic soybean lines under both irrigated and non-irrigated conditions to see how well these modifications took to the field. The best lines to cultivate could also be found and later chosen for true cultivar breeding.

What was discovered was that the individual molecular components worked on multiple expression levels to regulate several pathways. ABA-independent and -dependent transcription factors nonetheless utilized similar mechanisms to respond to drought. Comparatively, however, the DREB transgenic soybeans did not perform significantly differently than the wild-type control group, whereas the AREB transgenic line showed a higher performance than the wild-type and all the other GM lines.

This difference under field conditions requires further study to understand why one set of genetic components can better modulate and respond to drought conditions when overexpressed as compared to the others.

Controlling Negative Feedback

Not all genes play positive roles in trait development. Negative feedback loops and pathways exist to downregulate and dampen the effects of other regulatory systems. When it comes to ABA production and signaling, there is a group of molecules with the long name of Clade A PP2C phosphatases.

When there is an absence of ABA in the plant, these phosphatases dephosphorylate other proteins and work to encourage germination and root growth. In short, they actively oppose the drought stress response that tries to conserve water and shore up a stock of nutrients. This can be beneficial in some cases relating to desired growth, but in many others it leads to drought sensitivity and possible death of the plant if not properly controlled.

Therefore, Chinese scientists in March published a study where they used genomic modeling to identify and characterize 13 of these PP2C genes in maize and narrowed their focus onto one named ZmPP2C-A10 that held a strong negative correlation with drought tolerance during the seedling stage, where it instead pushes for more growth and makes the seedling more sensitive to harm from drought.

A genomic database search helped them to identify even more of these genes and even some similar ones in rice, allowing for them to be appropriately named. Then they created an overexpressed transgenic maize (and Arabidopsis) line for the A10 gene. These lines had higher germination rates and growth than their wild-type counterparts, but when exposed to drought conditions lost water easily and less survived.

Prior sequencing of varieties of the gene from multiple maize crops earlier in the study had identified a variant with an upstream part of the gene being deleted. This upstream element controlled activation of the gene when the cell’s endoplasmic reticulum (ER) stress response is triggered. Since this segment was deleted in this allelic variant of the gene, this activation does not occur.

Testing with a transgenic line using this precise variant of the A10 gene showed enhanced drought tolerance as compared to the other transgenic lines and more so than the wild-type controls. This evidence provides a method to help control the negative feedback effects of at least this particular PP2C gene and also indicates the involvement of the ER stress signaling system with drought tolerance pathways. More research is clearly needed to investigate this connection.

Slowing Stomata Development

Stomata have been a continual reference point in this article thus far due to how relevant they are to water loss in plants. Transpiration, the movement of water through and out of plants, is the primary method of water loss for plants and it happens through the stomata. So they deserve all the attention they have received from scientists. But, thus far, we’ve only looked at them in regards to regulating the opening and closing of these pores and what reaction this has on the plant’s growth.

Now, let’s look at what happens when you control the original development of the stomata themselves. The density and number of stomata that form on plant leaves has been found, at least in regards to negative feedback, to be regulated by a peptide family called epidermal patterning factors (EPFs). Studies in the last 2000’s were able to characterize in greater detail the first two members of this family.

An interesting fact about these peptides is that they work extracellularly, moving through the epidermal cell layer to activate signaling pathways that suppress stomata development. EPF2 has been found to work earlier in the plant cycle to prevent cells from turning into stomatal differentiation types, thereby preventing stomata from being created. EPF1, meanwhile, works later in the cycle to stop division of existing stomata cells and thereafter increase the space between each stomata.

The only existing issue in regards to the EPF family is that almost all of our knowledge about it has come from just Arabidopsis testing and not testing in crops themselves. This is an even greater problem since the two classes of flowering plants, the monocots vs the dicots, have significant structural differences in their stomata and patterning. Luckily, the genetic components appear to have similar ortholog genes that did not differentiate much over evolutionary time. Even so, EPF mechanisms in the grasses has yet to be studied in much depth.

To test whether they function identically, in June, scientists from the United Kingdom created an overexpressed transgenic barley for the gene HvEPF1. What this caused was, as just mentioned, a reduction in stomata density, showing that the gene is indeed identical across the species. This reduction also helped to limit leaf gas exchange and water loss, but the researchers were happily surprised to find that this had no significant effect on the barley’s grain yield.

With that exposed, it opens up the ability to use these negative stomata feedback loops to heavily increase water use efficiency while not harming the fitness of the crops.

Autophagy and Recycling

On to something completely different. One of the repercussions of drought stress in plants is the formation of reactive oxygen species (ROS) that accumulate within the cell. These can cause damage to the cellular membrane and to enzymes and proteins in the cell itself. To prevent this, there are scavenging systems in place to collect and detoxify the cell.

In a related vein, the autophagy system works to recycle any nutrients and other components in the cell that aren’t functioning or are no longer needed. The autophagosomes work similar to lysosomes, in that they engulf their target components and degrade them. This autophagy system is often activated during abiotic stress due to the common buildup of unwanted compounds in the cell at those times. Past research shows that autophagy is also involved in removing ROS themselves and other oxidized materials.

The autophagy-related protein (ATG) family therefore is connected to and improves the tolerance of plants to abiotic stress by recycling the harmful components such stress causes. For apples, very little is known about their autophagy system, with the MdATG18a gene being the first one in apples to be cloned for use in transgenic studies.

Researchers in China used this cloned component to make overexpressed transgenic tomato and apple plants for the gene with the Cauliflower mosaic virus promoter, one of the strongest plant-based promoters out there, being attached to it. They saw that this overexpression enhanced drought tolerance for both, with the apple plant in particular having a higher production of ATGs and other recycling systems as compared to the wild-type.

The amplified autophagy allowed for greater antioxidant capability and the removal of oxidized proteins more rapidly, minimizing damage to the plant cells and having them respond more favorably to drought conditions. Thus, another point of attack to deal with drought tolerance in plants.

Regulatory Research On The Rise

Even with the extensive breakdown of multiple regulatory systems in the article above, this is but a taste of what is out there and what scientists have been working on. The use of biotechnology techniques and applications has continued to boom in recent years and it has reached the point where it would be impossible to keep up with all of it, even just within a specific topic like drought tolerance in plant crops.

This was more or less a random selection of contemporary research, chosen to purposefully have an assortment of different options to discuss. I hope you were able to learn at least a little about how plants work and how we might manipulate their genomes and physical traits to suit our needs.

Thank you for your time, dear reader.

References

1. Pandey, V. & Shukla, A. (July 2015) Acclimation and Tolerance Strategies of Rice under Drought Stress. Rice Science 22 (4), 147–161. doi:10.1016/j.rsci.2015.04.001

2. Belavadi, V. V., Karaba, N. N., & Gangadharappa, N. R. (2017). Chapter: “Genetic Engineering of Component Traits of Drought Tolerance in Rice”. Agriculture under Climate Change: Threats, Strategies and Policies. Allied Publishers. Retrieved from https://books.google.com/books?hl=en&lr=&id=4fyGDgAAQBAJ&oi=fnd&pg=PA139#v=onepage&q&f=false

3. Nakashima, K. & Suenaga, K. (2017) Toward the Genetic Improvement of Drought Tolerance in Crops. Japan Agricultural Research Quarterly: JARQ 51, 1–10. Retrieved from https://www.jstage.jst.go.jp/article/jarq/51/1/51_1/_pdf

4. Lee, D.-K. et al. (June 2017) The rice OsNAC6 transcription factor orchestrates multiple molecular mechanisms involving root structural adaptions and nicotianamine biosynthesis for drought tolerance. Plant Biotechnology Journal 15, 754–764. doi:10.1111/pbi.12673

5. Kudo, M. et al. (Apr 2017) Double overexpression of DREB and PIF transcription factors improves drought stress tolerance and cell elongation in transgenic plants. Plant Biotechnology Journal 15, 458–471.

6. Jung, H. et al. (Oct 2017) Overexpression of OsERF48 causes regulation of OsCML16, a calmodulin-like protein gene that enhances root growth and drought tolerance. Plant Biotechnology Journal 15 (10), 1295–1308.

7. Li, J. et al. (Feb 2017) OsASR5 enhances drought tolerance through a stomatal closure pathway associated with ABA and H2O2 signalling in rice. Plant Biotechnology Journal 15 (2), 183–196.

8. Fuganti-Pagliarini, R. et al. (2017) Characterization of Soybean Genetically Modified for Drought Tolerance in Field Conditions. Frontiers in Plant Science 8, 448. doi:10.3389/fpls.2017.00448

9. Xiang, Y., Sun, X., Gao, S., Qin, F. & Dai, M. (Mar 2017) Deletion of an Endoplasmic Reticulum Stress Response Element in a ZmPP2C-A Gene Facilitates Drought Tolerance of Maize Seedlings. Molecular Plant 10, 456–469. Retrieved from http://www.cell.com/molecular-plant/pdf/S1674-2052(16)30227-1.pdf

10. Hughes, J. et al. (June 2017) Reducing Stomatal Density in Barley Improves Drought Tolerance without Impacting on Yield. Plant Physiology 174, 776–787. doi:10.1104/pp.16.01844. Retrieved from http://www.plantphysiol.org/content/plantphysiol/174/2/776.full.pdf

11. Sun, X. et al. (Aug 2017) Improvement of drought tolerance by overexpressing MdATG18a is mediated by modified antioxidant system and activated autophagy in transgenic apple. Plant Biotechnology Journal. doi:10.1111/pbi.12794

Photo CCs: Heat affected crop during a green drought from Wikimedia Commons

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