Feature: Designing drought tolerant crops

In the past decade, the zero-till revolution has rolled across wheat farms on the margins of the arid zone in south-eastern Australia, helping wheat growers survive the longest and deepest drought since European settlement.

By planting directly into undisturbed soil and stubble, zero-till farmers have been able to conserve moisture and organic matter, to obtain modest but economic yields on as little as 100 millimetres of rain during the growing season.

But agronomic advances alone will not ensure the wheat industry’s continued viability as global warming increases average temperatures across the wheat belt by as much as two degrees Celsius by mid-century.

The industry’s future rests on the ability of molecular plant breeders to develop new wheat varieties with greater tolerance of abiotic stresses: heat, water deficits and salinity.

Professor Julian Schroeder, Novartis Torrey Mesa Institute Chair in Plant Science and Distinguished Professor in the Division of Biological Sciences at the University of California San Diego, will describe his group’s findings on CO₂-sensing mechanisms in his Annals of Botany Lecture at the OzBio2010 Conference in Melbourne in September.

Holding breath

Schroeder says researchers have known for decades that increased concentrations of atmospheric carbon dioxide induce partial closing of the stomata, the tiny leaf pores through which plants lose water to the atmosphere during photosynthesis. This contraction of the stomata enhances a plant’s water-use efficiency during photosynthesis, improving its drought tolerance.

The mechanism means there’s an upside to rapidly rising concentrations of CO₂ in the atmosphere. Greater water-use efficiency associated with the increase in CO₂ concentrations from 270 ppm at the beginning of the industrial revolution 200-odd years ago, to 390 ppm today, has probably been a significant factor in increasing yields from today’s grain crops.

The question, says Schroeder, is why plants have evolved such a dynamic response to increased CO₂, when CO₂ levels in the atmosphere typically change over timescales of thousands to millions of years? When night falls, most plants – except for cacti and other succulents with Crassulacean Acid Metabolism (CAM) – cease photosynthesis, and stop drawing down CO₂ from the atmosphere. Oxygen use increases, and the concentration of CO₂ inside the leaves rises because respiration (consumption of CO₂) prevails in the absence of photosynthesis.

The stomatal guard cells react to the rising internal concentration of CO₂ by closing the stomata, and reducing water loss through dark respiration. “The question is, what are the implications if atmospheric CO₂ concentrations continue to rise,” asks Schroeder. “What happens during the daytime?”

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He suggests plant water-use efficiency is likely to increase – but only up to a point. “If the stomatal pores narrow too much it will conserve water, but it could inhibit CO₂ absorption. Until a few years ago, nothing was known about the genetic mechanisms that mediate CO₂ signal transduction in response to changes in CO₂ concentrations.”

As well as causing stomatal apertures to narrow, elevated CO₂ also increases terrestrial temperatures by acting as a blanket that reduces the re-radiation of long-wave infra-red radiation from the Earth’s surface back into space – the well-known greenhouse effect.

“Narrowing of the stomatal aperture could add insult to injury, because transpiration cools plant leaves,” he said. “Reducing the stomatal apertures at elevated CO₂ reduces the cooling effect and could increase heat stress on the plant. That could become a significant issue for Australia,” he says.

“Globally, many studies have predicted the importance of the response. However, until a few years ago, we knew nothing of the genes and mechanisms that mediate CO₂-induced stomatal closing.”

Thirsty work

In his Annals of Botany Lecture, Schroeder will describe the results of his group’s search for genes involved in the CO₂-sensing pathway that regulate the stomatal aperture. In the past four years his team has published a number of papers – some in collaboration with other groups – describing their discoveries.

After sifting through many candidate genes, they identified two homologous CO₂-binding proteins, carbonic anhydrases, that mediate the CO₂-response, while ruling out leaf photosynthesis as mechanism for the stomatal pore CO₂ response.

Disrupting both genes in Arabidopsis resulted in a much slower and weaker CO₂-induced stomatal closing response, compared to the wild-type control plants. In his talk, Schroeder will describe how the genes signal to the ion channels in the stomatal guard cells.

His team also over-expressed the carbonic anhydrases in guard cells, using a strong guard-cell promoter his laboratory had isolated. Over-expression of the CO₂-binding proteins increased the instantaneous water-use efficiency of the plants by over 40 per cent.

Schroeder says earlier research by Dr Susanne von Caemmerer, of the Australian National University School of Biological Sciences in Canberra, suggested CO₂ control of stomatal aperture operates independently of photosynthesis. His own research group’s work supports these findings with genetic evidence for the mechanisms involved.

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Last December, Professor Sean Cutler’s research group at University of California Riverside, in collaboration with Schroeder’s team and several others, published a paper in Science describing how the phytohormone abscisic acid (ABA) regulates the early signalling pathways by which plants sense and respond to environmental stress.

This research, conducted by several laboratories in the US, Germany and other countries, has signposted the route for molecular biologists to dissect and manipulate the mechanisms of drought tolerance. In doing so, they have solved another long-standing puzzle of great relevance to Australia and many other regions: how do plants turn on a drought-tolerance response?

In the greenhouse

In 2009, Cutler’s team at UC Riverside and Professor Erwin Grill’s team at the Technical University of Munich independently showed how abscisic acid mediates the early signalling mechanisms that control the guard cells surrounding stomata. Schroeder’s laboratory independently identified the ABA receptors using a proteomics approach.

They found that plant roots synthesise ABA in response to water deficits. The hormone is translocated to the leaves, where it initiates signalling cascades that reduce water loss. “Plants lose over 90 per cent of their water by evaporation through their stomatal breathing pores. Abscisic acid inhibits water loss by signalling the two guard cells surrounding each breathing pore to swell and close,” says Schroeder.

“We set out to dissect signal transduction in the two guard cells by identifying the major classes of ion channels that coordinate the drought response, including anion, potassium and calcium channels. It turns out that signal transduction via these guard-cell ion channels is important for many other responses in plants.”

Several years ago, while working to identify the early upstream signals that regulate ion-channel activity, Schroeder’s team found that a well-known phosphatase protein, ABI1, functions the furthest upstream of all known early ABA signalling mechanisms. This prompted his laboratory to isolate the protein complexes attached to the ABI1 phosphatase to determine whether other, unknown ABA-signalling or receptor proteins interact with ABI1.

Noriyuki Nishimura in Schroeder’s laboratory employed a proteomics approach to search for the ABI1 phosphatase target proteins. Using the ABI1 phosphatase as ‘bait’, he searched guard-cell protein extracts to see which proteins it hooked. The fishing expedition yielded a new sub-family of abscisic acid receptor proteins.

In Munich, Grill’s team used yeast two-hybrid screening to search for interactors of the related ABI2 protein phosphatase techniques to identify the same family of receptors. “The family contains 14 genes, which is probably why classical genetic screens failed to identify them,” says Schroeder.

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Another of Schroeder’s collaborators, X-ray crystallographer Professor Elizabeth Getzoff, of the Scripps Research Institute in California, together with Schroeder, led one of five groups that resolved the protein’s structure. It revealed that abscisic acid binds within a shared structural motif – a water-filled cavity – common to all proteins in the newly identified family of ABA receptors.

PYR/RCAR ABA receptors act as positive regulators of abscisic acid-mediated pathways, but Schroeder says research is only in its early stages, and it remains to be determined how the different pathways interact to coordinate the drought response.

The simplest hypothesis, he says, is that it involves differential expression of members of the PYR/RCAR receptor family in target tissues. “We have found that knocking out these receptors results in plants that do not close their stomatal pores in response to drought. Thus the receptors play a central role in ABA-induced closing of stomata to conserve water.”

Grains of salt

Schroeder’s lab has also identified a sodium membrane transporter protein in Arabidopsis that regulates sodium accumulation in leaf tissues.

Research published by Schroeder’s lab over the past 10 years showed that the AtHKT1;1 transporter protein protects Arabidopsis against stress from over-accumulation of sodium in its leaves. “The photosynthetic and metabolic machinery of leaves is most sensitive to over-accumulation of sodium ions from salty soils,” says Schroeder. “We found that the AtHKT1;1 sodium transporter functions as a sodium-excluder in leaves, by removing sodium ions from the xylem sap of plants.”

Australian wheat researcher, Rana Munns, at the CSIRO Plant Industry in Canberra, has intensively studied salinity tolerance. She discovered three major quantitative trait loci (QTLs) associated with high tolerance in bread wheat, but it has proven difficult to identify the genes involved at these loci because wheat has such a large polyploidy genome, says Schroeder.

“Rana explored the mapping regions for these QTLs to see if any of the loci harbour AtHKT1;1-like genes, and has shown that all three carry polymorphisms in wheat homologs of the AtHKT1;1 gene.

“Rana’s research has demonstrated that some primitive wild diploid wheat varieties have superior resistance to salinity, so one can now use HKT genes as molecular markers to track introgression of the gene in breeding programs to improve the salinity tolerance of tetraploid durum wheat, and hexaploid bread wheats.”