Scientists share keys to drought tolerance

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Scientists share keys to drought tolerance
Some of the world's leading crop researchers met in Adelaide recently to discuss breeding strategies for increasing plants' water-use efficiency
By Gio Braidotti

With global grain stocks at their lowest levels in decades and water stress accounting for a glaring proportion of global shortfalls, the 2007 Genomics Symposium in Adelaide allowed some of the world's best drought-tolerance pre-breeders to put their heads together on new ways to test, select and dissect traits that can maintain yields under drought.

[Photo (left) by Melissa Marino: CSIRO's Dr Richard Richards (left) with an Indian researcher during a recent workshop (co-sponsored by the Australian Centre for International Agricultural Research) in New Delhi that brought together leading researchers from around the world seeking to improve wheat's water-use efficiency.]

Convened by Professor Peter Langridge of the Australian Centre for Plant Functional Genomics (ACPFG), the Genomics of Drought Symposium played host to more than 130 scientists from Australia, the US, Europe, Mexico, China, India, South Africa, Syria, Canada, Israel, Azerbaijan and Japan.

Among them were Dr Richard Richards and his CSIRO Plant Industry team, which ranks among the world's most successful at devising ways to select for improved yields during drought.  

Also present was Dr Steve Jefferies from AGT, the company responsible for Gladius pbrlogo and Axe pbrlogo, the southern wheat varieties with an enhanced ability to stay green during intermittent and terminal droughts.

Despite the conventional breeders' successes to date, the consensus is that drought tolerance remains a tough trait to study, with the difficulty due to three basic issues:
the variable nature of drought, further confounded by climate change;
the gene-by-environment (GxE) describes how genes respond to different environments; and
pleiotropy, the ability of a single genetic change to ripple unintended physiological effects throughout a plant.
These conundrums made Dr Jefferies, for one, sceptical that a panacea can be delivered in the form of a single 'drought resistance' gene. Nonetheless, he said gene biotechnologies - primarily genomics and transgenics (genetic modifications/GM) - had an important role to play.

Overall, genomics is currently playing a more peripheral role in the work of conventional pre-breeders, with scientists attempting to dissect the genetic basis of drought tolerance and map traits to discrete chromosomal subregions (called QTLs or quantitative trait loci).

However, these studies are coming up against GxE (environmental) effects in that QTLs are often proving specific to particular trial sites. Similarly, observations made in greenhouses under artificial drought conditions have been known to produce misleading results.
In view of these hurdles, ACPFG's own efforts were notable because of efforts to circumvent these challenges. ACPFG has teamed up with CSIRO and AGT to map QTLs for two wheat varieties, Drysdale pbrlogo and Gladius pbrlogo, which have tried-and-tested paddock records of maintaining yields in dry years.

For Gladius pbrlogo, mapping efforts are targeting the genes that underlie both the drought and heat-tolerance traits. At stake is the possibility of identifying a wheat equivalent of sorghum's stay-green trait. A similar approach is being taken with the transpiration efficiency of Drysdale pbrlogo in conjunction with the NSW Department of Primary Industries.

In addition, ACPFG is also testing crosses between Gladius pbrlogo and Drysdale pbrlogo to determine whether fundamentally different mechanisms are at work in the two varieties. If so, the opportunity arises to combine them in a bid for either greater water-use efficiency or protection against a broader range of drought conditions.

Numerous other QTL-mapping projects are taking place around the world and the hope is that molecular markers will soon be developed that can track inheritance of some proportion of a drought-tolerance trait. The markers are especially important as they can facilitate combining several drought-tolerance mechanisms from different breeding programs into single cultivars.
Meanwhile, the conventional pre-breeders are pushing on, content to continue to invent ways to measure and select for physiological adaptations that help plants cope when paddocks dry up and temperatures start climbing. On that front, Dr Richards said that Australian grain production was nowhere near its upper biological limit and that many opportunities exist to not only maintain, but also increase production. At CSIRO, getting 'more crop per drop' of rain is implicitly seen as part of the production challenge.

"Current district average yields are typically falling 50 to 60 per cent short of the yield potential," Dr Richards said. "That provides an opportunity for breeders, agronomists and growers to bring about a doubling in crop production, to an average four tonnes per hectare."

Confident that the environment can cope with that doubling, he proceeded to map his 'best breeding bets' for achieving yield increases, although he stressed these are needed alongside improved rotations, better fertiliser efficiency, and even the inclusion of native perennials into farm production systems.

At CSIRO's Canberra and Brisbane laboratories, Dr Greg Rebetzke, Dr Linda Tabe, Dr Fernanda Dreccer and other scientists are tackling these 'best bets', which include:

making use of the full growing season with sowing and flowering time modifications;
increasing the proportion of carbon allocated to grain;
selecting for longer coleoptiles (an emerging shoot's protective sheath);
more vigorous early growth;
maximising transpiration efficiency;
reducing the number of wasteful tillers; and
increasing storage of carbohydrates in the stem for use during grain filling.
Additionally, a Mediterranean-based genomic project discussed by Professor Roberto Tuberosa of the University of Bologna in Italy, further identified canopy height and the length of the peduncle (the stem that supports the flower cluster) as traits worth pursuing in durum and bread wheat.

Across Australia similar mapping efforts are under way. Dr Mehmet Çakir of Murdoch University in Perth outlined a project to map QTLs for drought adaptations in the western growing region; Dr Glenn McDonald of the Molecular Plant Breeding CRC and Ali Izanloo and James Edwards of ACPFG presented work on the southern region; and Dr Lynne McIntyre of the Brisbane arm of CSIRO Plant Industry reported on adaptations to the northern region.

Looking to the future, Dr Richards thinks pre-breeding efforts will increasingly focus on ways to increase grain number and improve root characteristics to overcome limitations below ground. However, the challenge remains the same: using the sum of available ingenuity to invent appropriate ways to measure and select for paddock-based biological activities that promote yield gains under both dry and favourable conditions.

More information: Professor Peter Langridge, ACPFG, 08 8303 7182 

Drought tolerance: the GM route
In the race to produce drought-resistant crop varieties, conventional breeders have proven they can meet the challenge, releasing wheat varieties such as Drysdale pbrlogo, Rees pbrlogo, Gladius pbrlogo and Axe pbrlogo that perform well under both water sufficient and deficient conditions.

In the meantime, the transgenic, or GM, front must first go through a gene-discovery phase, sifting through entire genomes for those sequences that can nudge plant physiology to make better use of available water. Only then can the genes be fed into variety-development programs.

Worldwide, some 50 genes have been reported to confer drought tolerance when over-expressed in transgenic plants. As a result, the number of GM field-trials is on the rise.

However, to date no transgenic drought-tolerant crop has been commercialised. Instead, several noteworthy programs are under way that could see a GM variety available, perhaps within a decade:

undergoing bio-safety testing in Egypt in preparation for commercialisation is a variety of wheat containing a gene from barley, which requires less water than its conventional counterpart;
Monsanto's drought-tolerant corn is reported to have a yield advantage over non-transgenic varieties under water-deficient field-trials; while development of drought-tolerant soybean and cotton are also in the pipeline; and
Bayer, Syngenta, Dow, BASF and Dupont all have extensive research programs in the area of drought tolerance.
However, the complexity of a plant's response to water stress tends to cause drought-tolerance transgenes to have additional, usually unpredictable, and often unwanted effects on other traits, including yield and quality. Scientists call a gene's ability to ripple consequences in this way the 'pleiotropic effect'.

The most notable and problematic is the tendency of drought-tolerant GM lines to not perform as well under favourable conditions. This appears to be the case for CIMMYT's GM wheat and Monsanto's GM corn. The main reason, according to the CEO of the Australian Centre for Plant Functional Genomics Professor Peter Langridge, is that the transgene slows down plant development, resulting in smaller plants and delayed flowering.

The flaw is a profound one. It amounts to shifting the yield losses experienced in dry seasons onto the good years.

Fortunately, the possibility of a 'pleiotropic' yield penalty was anticipated by scientists, who believe they can fine-tune a transgene's performance. However, this fine-tuning has meant an extra R&D phase to engineer a switch that turns the drought-tolerance gene 'on' only under stress conditions.

The switch is itself a piece of DNA, but one that does not function as a gene. Instead, the DNA is normally found adjacent to genes where it promotes that gene's expression. Collectively called 'promoters', specific examples are known that make gene expression responsive to dehydration.

It is this type of promoter that is expected to bail out the transgenes and unleash a second round of field-trials. At that stage, the GM lines move into real-world paddocks ... and go up against the 'gene-by-environment effect' and the variability of drought events.

For researchers, it is still a hard road ahead, and so too for graingrowers who need, with increasing urgency, more robust crop varieties 

The enduring difficulty of breeding for drought tolerance
The pleiotropic effect
The ability of single genetic changes to ripple unintended effects throughout a plant is possible because as the building blocks of traits, genes are so basic and versatile they can be re-used in many different plant processes. By analogy, a piano can be resolved into strings, hammers and foot pedals when attempting to improve its performance, or into more basic units such as wood, nails and nylon. The more versatile the building block, the more likely it is to have pleiotropic effects throughout the piano if changed in some way.

In fact, it is very difficult mentally to conceptualise the causal level in which genes are operating. That makes it difficult for scientists to predict the effects of genetic modifications, or conversely it allows those scientists with a feel for a gene's pleiotropic nature to make improbable progress. Drought tolerance is not the only trait causing breeders pleiotropic problems. For instance, botanists at Oregon State University in the US have discovered that a single plant gene that causes resistance to one disease can also cause changes that make the plant more susceptible to a different disease.

The gene-by-environment (GxE) effect
While non-scientists easily deal with GxE - being familiar with the way nutrition, stress or exercise affects biology - scientists struggle with it as the result of a historical quirk. Originally, science had to deliberately silence the environment's impact on genetics in order to resolve chromosomes into individual genes. An ingenious gene-detecting method was devised that involved making one random mutation to a genome and then comparing the mutant with a normal counterpart that was identical in every other way - including nutrition, daylight exposure, and environment.

Any trait differences between them could then be attributed to the mutated bit of DNA, which acquired a name that reflected the altered characteristic. For example, there is a stretch of fruit fly genome called 'wingless'. While the method proved astute for isolating genes under lab conditions, the approach tended to shield molecular biology from the role environment plays in determining the organism.

That deficit inspired pre-breeders such as CSIRO's Dr John Passioura to head out in search of paddock-based methods that are alive to the two-way interplay between genetics and environment. In the process, he laid the foundations for the development of drought-tolerant wheat varieties such as Drysdale pbrlogo and Rees pbrlogo.

The variability of drought events
To help deal with the variability of drought events, Dr Richard Trethowan of the University of Sydney reported on international efforts led by CIMMYT to define and divide the world's growing regions into 'agro-ecological zones' relevant for drought-tolerance-breeding efforts. Trial sites have been established and categorised according to crop adaptation requirements, disease spectrums and crop management systems. This resolved into 12 mega-environments: six for spring wheat and six for winter varieties that are hosting international variety trials, including wheat lines from Australian projects. The power of computer modelling is also being applied, with CSIRO's Dr Scott Chapman applying cropping simulation models to more than 100 years of weather records in the northern growing region. In both sorghum and wheat he has found that drought patterns around flowering can be shown to account for GxE effects in breeding trials. 

Australia joins the transgene hunt
A small stretch of DNA, just six or so bases long, is sufficient to provide a gene with the means to sense the occurrence of a stress. One specific example of such a sequence - the dehydration response element (DRE) - serves to provide genome-wide docking sites for proteins that re-orchestrate gene expression to deal with water stress.

[Photo (left) by Gio Braidotti: Two of Australia's promising young grains researchers, Alex Smart (left)and Sarah Morran of the ACPFG, at the Genomics of Drought Symposium in Adelaide.]

That fact has caught the interest of transgenic pre-breeders around the world, with speakers at the Genomics of Drought Symposium proposing that DRE binding (DREB) factors are worth testing as 'drought-resistance' transgenes in GM crop varieties.

Speaking on the last day of the symposium, Sarah Morran, a postgraduate researcher at the Australian Centre for Plant Functional Genomics (ACPFG), has systematically tackled that proposition. She reported using the DRE as bait to fish among wheat molecules and pull out those of interest.

Her work has resulted in the discovery of two novel genes (TaDREB2 and TaDREB3), which have been introduced and over-expressed in barley (Golden Promise) and wheat (Bobwhite). For both transgenes, Ms Morran found that GM lines performed better than the parent varieties under water-restricted conditions in glasshouse trials.

However, Ms Morran also detected disadvantageous pleiotropic effects. Efforts are now under way to reduce these unwanted traits. Like transgenic pre-breeders worldwide, the scientists have come full circle, relying on DRE-like DNA elements to restrict transgene activity to the occurrence of water stress.

More information: Sarah Morran, ACPFG, email 

Monsanto's transgene
News broke during the Genomics of Drought Symposium that Monsanto has developed transgenic corn lines reported to deliver improved corn yields on water-limited fields. The news arrived via Dr Robert Sharp of the University of Missouri in the US whose work on maintaining root growth under water deficit is partly supported by Monsanto.

According to the report published in the Proceedings of the National Academy of Science, Monsanto took a functional genomics approach to the gene discovery problem. Like Australia's Sarah Morran from the Australian Centre for Plant Functional Genomics ACPFG, Monsanto screened those proteins that bind adjacent to genes and mediate changes in gene expression in response to water stress.

The search resulted in the identification of a corn gene (ZmNF-YB2) that, when over-expressed in GM lines, results in tolerance to drought in greenhouse tests and field-trials as measured by chlorophyll content, stomatal conductance, leaf temperature, reduced wilting, and maintenance of photosynthesis. However, under adequate water conditions, the GM lines suffer a yield penalty.

As such, the authors conclude their lines will have "the most significant impact on severely water-limited corn production systems", and that more work is needed to understand the effects of the technology over a greater range of environments.

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