PROPOSAL

Candidate Proteins and Genes for Drought Resistance in Kazakhstan Wheat

Tamara E. Lee, Ph.D., Senior Scientist, Institute of Plant Physiology, Genetics and Bioengineering, Department of Photosynthesis, Almaty, Kazakhstan. E-mail: aksai252@ yahoo.com; tamlee@mail.ru

Hans J. Bohnert, Ph.D., Professor, Department of Plant Biology, University of Illinois, Urbana, IL 61801, USA

Abstract.

We target drought stress responses of cereal crops using molecular genetic, tissue culture, biochemical and physiological techniques. Our goal is to study and understand mechanisms of stress tolerance in wheat breeding lines adapted to growing conditions in Kazakhstan. Included is a genomics approach for the identification of "candidate genes" that will help in molecular breeding. We emphasize (three objectives) transcripts and proteins essential to carbon and nitrogen allocation under stress, based on preliminary data, and on the genetic trait of leaf rolling. Specifically:

· Analysis of transcript expression profiles in tissues of wheat subjected to stress that mimics conditions found in the field, utilizing microarrays of up to 10,000 genes (ESTs) and small arrays, which contain the transcripts for enzymes in the nitrogen reduction, carbon assimilation and amino acid transport pathways.

· Analysis of protein amounts for the targeted pathways and analysis of enzyme activities.

· Manipulation of external or internal conditions to observe and pinpoint the effectors that affect gene expression and/or protein activity in targeted pathways.

The two laboratories utilize complementary techniques. At the institute in Kazakhstan, tissue culture and regeneration facilities, growth rooms and greenhouses are used to focus mainly on protein isolation and the characterization of enzyme activities with plant materials adapted to local conditions. At the university of Illinois, transcript profiles and transcript regulation will be studied with RNA from plant material grown in Kazakhstan. The exchange of people will disseminate information and knowledge. The expected results will augment the wheat breeding programs in Kazakhstan by providing a biochemical understanding of characters that distinguish tolerant and sensitive lines. The results on biochemical and transcript behavior under stress will provide candidate genes that can then be incorporated into breeding programs.

Drought resistance is desirable trait for grain production in Kazakhstan, where low precipitation in spring often arrests seedling development, affecting the physiology of the subsequent developmental phases: vegetative growth, earring, flowering and grain maturation. Selection of stress-tolerant genotypes of wheat and other cereal crops will contribute to improved yield and grain quality under sub-optimal environmental conditions.

Project Narrative

a. Approach, Objectives, Milestones and Measurements.

Our hypothesis states that by a combination of biochemical analyses and microarray analyses of transcripts, we will be able to pinpoint genes, proteins and enzyme activities that indicate superior performance under drought stress conditions in cereals. Modern crop breeding incorporates new approaches and techniques, supplied by plant physiology, biochemistry, biotechnology, molecular biology and genetics. Future crop improvement for enhanced stress resistance is possible through the application of modern genetic manipulation of primary metabolic processes such as C- and N-assimilation to support the production potential of crops. We list three objectives that can reasonably be tackled during a two-year work period.

· Analysis of transcript expression profiles in different tissues of rice, corn, barley and wheat subjected to drought stress regimes that mimic those found in field situations. We will utilize arrays that include transcripts for enzymes in nitrogen and carbon utilization and allocation pathways.

· Analysis of proteins for the targeted pathways and analysis of their activities.

· Manipulation of external or internal conditions to observe and pinpoint the effectors (e.g., plant hormones: ABA, cytokinin; metabolite levels; phosphorylation state of enzymes such as PEPc, PPDK, NR, etc.) that affect gene expression and/or protein activity of the targeted pathways.

Through classical plant breeding crop varieties have been adapted to produce in different environments. The most persistent problem, however, has not resulted in significant improvement - how to maintain high yield, or at least tolerable yield, under drought conditions (no water) and in soils with high sodium salinity (difficulty to extract the water). Clearly, plants cannot grow without water but such an unrealistic goal is not our objective. What can be accomplished, we think, is that periods of drought can be tolerated longer without totally compromising growth if existing tolerance genes can be accumulated through breeding. Molecular genetics can be used to identify candidate genes and their biochemical analysis can provide mechanisms through which they act. Drought periods are especially damaging during two phases of the life of a (wheat) plant - early growth and anthesis when flowers become fertilization-competent.

Our research will target these two periods of highest sensitivity to yield reduction to monitor which genes are differently expressed in drought-tolerant and -sensitive varieties. Also, the research is based on correlations established in local breeding programs, which have established phenotypes with superior preformance under stress. The results of these studies indicated that drought-based yield reduction and yield reduction due to nitrogen deficiency are tightly correlated.

The research will result in data that can be used in breeding programs of cereals, even though some of our work will be done with rice and barley for which we have many more available genes than for wheat. We base this conviction on the co-linearity of cereal genomes, which indicates that gene positions along chromosomes of all grass crops are largely conserved. Thus, finding the position of a character on the rice genome provides, with high probability, the position of this character on the wheat chromosome map.

b. Laboratory Competence - Results documenting Feasibility of the Approach.

The laboratory in Kazakhstan, in particular Dr. Lee, is working on stress-specific projects. Substantial changes in the levels of GS and aminating GDH activities were noticed in RL-genotypes under osmotic shock and salinity (Lee & Lips, 2001). In addition, the team of Prof. Gilmanov, Head of Laboratory Enzymes Structure and Regulation, has experience in studying key nitrogen metabolism enzymes under stress conditions for over 25 years. Opposite effects of drought and salinity on GS, NADH-GDH, AO were observed in wheat roots and leaves. Plants with increased GS and NADH-GDH (amination) activities were more tolerant to drought. The induction of PPDK and the activation/induction of PEPc under stress conditions was higher in stress-tolerant genotypes. They have described the enzyme complex MDH-GOT catalyzing the irreversible transfer of ammonia from glutamate to oxaloacetate (Gilmanov et al., 1989). This complex consists of malate dehydrogenase (MDH) and glutamate-oxaloacetate transaminase (GOT) and is responsible for transamination between glutamate and oxaloacetate, generated by malate oxidation, resulting in 2-oxoglutarate and aspartate. The activity of this complex in cereals under rust infection was 100-fold higher than the activity of glutamate dehydrogenase (GDH) deamination reaction (Koldasova et al., 1997). Cereals with high activity of MDH-GOT do not accumulate ammonia, because most of the glutamate resulting from protein hydrolysis turns into aspartate and formate in addition to NADH and 2-oxoglutarate. The levels of activity of GDH, MDH-GOT seem to play an important role in detoxification and protein turnover under abiotic stress. Prof.Gilmanov and coworkers discovered a cytokinin-based signal transduction mechanism. Cytokinin lead to the formation of a mediator, which is transported to other organs and binds to fusicoccins receptors leading to increases in cytosolic Ca²⁺. This activates proteinkinase-C which in turn phosphorylates and activates the anabolic enzymes (Gilmanov et.al., 1993; Gilmanov, 2001)

Figure 1: Pathway schematic for nitrogen assimilation in plants. Under stress conditions, an important element increasing performance is the continued activity of these enzymes.

The Illinois team has been working on plant stress responses for over 10 years. Projects target salinity and drought stress responses of plants using molecular genetic, biochemical and physiological techniques (Vernon & Bohnert, 1992; Tarzynski et al., 1993; Shen et al., 1997; Nelson et al., 1998; Bohnert & Bressan, 2001; Hasegawa et al., 2000).

A multi-investigator project (NSF, Plant genome program, "Functional Genomics of Plant Stress Tolerance", PI: HJ Bohnert) includes work with corn, barley and rice lines with variable salinity and drought stress tolerance characteristics. We generated cDNA libraries from stressed plants and carried out EST-sequencing and microarray analysis with probes from stressed and unstressed plants (Kawasaki et al., 2001) and includes an evolutionary treatment of the salinity stress response pathways (Yale and Bohnert, 2001). Also, we collaborate with the International Rice Research Institute (IRRI, Philippines) on drought-related projects. Responses to drought at seedling emergence, vegetative growth and during anthesis are monitored by microarray analysis with a segregating population of 217 lines from a cross between the drought-tolerant rice variety "Azucena" and drought-sensitive "IR64" (Wade et al., 2000; Edmeades et al., 2000). Results from these studies can be transferred to other cereal crops based on the co-linearity of the grass genomes.

The lab works with microarrays from three cereal crops: corn, rice and barley. Some results (with rice and salinity stress) have been published (Kawasaki et al., 2001). The comparison between salt-tolerant and salt-sensitive rice lines indicated that these lines are distinguished by how they induce, or fail to induce, pathways that lead to tolerance. We have repeated the experiments with corn, with very similar results (H Wang, HJ Bohnert, to be submitted), indicating that we may be able to generalize between cereal crops. At present, microarrays for corn and rice include ~8000 transcripts. We are comparing drought, salinity, low temperature and radical oxygen challenge. For barley ~2,500 transcripts (ESTs) have been obtained from drought-stressed plants and printed on microarrays. We are comparing varieties distinguished by different drought tolerance levels.

One result is particularly relevant for our work. Microarrays that contain corn, rice and barley genes on one slide were used to monitor cross-hybridization with the rationale of studying whether one cereal crop gene array ("Grass-Array") might be used for more than one cereal grass species. This seems to be the case (Ozturk, Talamé, Wang, Kawasaki and Bohnert; unpublished). In most cases where we compare nucleotide sequences, the different grass species show close to 90% or higher identity. Tests indicated that we can reliably monitor transcript behavior in corn on a rice array when the nucleotide homology is higher than ~87%. One note of caution is that absolute amounts of transcripts for any given gene can be dramatically different in the different species. Thus, in order to compare across grasses, one must compare the control and experimental conditions on the same slide (after differential fluorescent labeling the RNAs from control and stressed states).

Specific Objectives and Experimental Plan

The Kazakh team will study the mechanism regulating expression of genes of some key enzymes of nitrogen and carbon assimilation, such as GDH, MDH-GOT; PEPc, PPDK and molybdate-containing enzymes, such as NR, AO, XDH. They will determine their critical involvement in tolerance acquisition and the factors that lead to the induction of protein activity of these key-enzymes in plant nitrogen and carbon metabolism. The Kazakhstan scientists will investigate the extent to which hormone (ABA) levels in drought-stressed tissues constitute a determining factor in controlling the activity of enzymes that are at the basis of C/N balance and allocation. Changes in nitrogen (nitrate) uptake by roots and its assimilation are of central importance since they determine the supply of organic nitrogen to the sustained development of the shoot. Equally, the provision of carbon skeletons is essential, and, finally, the transport vehicles that lead to distribution of (amino acids) nitrogen are essential biochemical functions that must be maintained under stress conditions.

Biochemical and physiological Studies. The major pathway for ammonium assimilation by plants (Lea and Miflin, 1974; Stewart et al.,1980; Lea et al.,1990; Oaks, 1994) involves the glutamate synthase cycle including the combined actions of glutamine synthetase (GS: EC 6.3.1.2) and glutamate synthase (GOGAT: EC 1.4.1.13) (Figure 1). The products of this cycle are usually glutamate (GLU) and glutamine (GLN) which are loaded onto the xylem sap by xylem parenchyma cells and transported to the shoot of the plant. This pathway, however, is considerably altered under stress conditions (Cramer and Lips, 1995).

(1) We will detect and measure changes of enzymes involved in nitrogen assimilation that are activated and/or induced by abiotic stresses - glutamate dehydrogenase (GDH), pyruvate-phosphate dikinase (PPDK), asparagine synthetase (AS), phosphoenol pyruvate carboxylase (PEPc) and the enzyme complex MDH-GOT.

(2) We will study the development of “emergency” pathways for the supply of carbon skeletons that replace or supplement the provision of mitochondrial oxo-glutarate for ammonium assimilation under stress conditions. Studies of the activation/induction under stress of the enzymes involved in the alternative ammonium acceptors carbon compounds: PEPc, PPDK, AS and MDH-GOT (malate dehydrogenase; glutamate-oxaloacetate aminotransferase). In part, specific abtibodies can be used (provided by colleagues in the US and Germany).

(3) Studies of the roles of stress activated/induced Molybdenum (Mo)-enzymes – nitrate reductase (NR), aldehyde oxidase (AO) and xanthine dehydrogenase (XDH) on the mechanism to maintain nitrogen assimilation in roots under stress conditions.

(4) Studies of xylem sap composition in stress tolerant and sensitive cultivars. Osmotic stress increase root amino acid pools and, in sensitive varieties, transport to the shoot is blocked at the xylem level. We will determine changes in the transport of nitrogen compounds, especially amino acids and hormones (ABA, cytokinin), through the xylem from root to shoot. Samples will be collected in Kazakhstan and analyzed in Illinois. Enzymatically, the key enzymes of nitrogen assimilation will be investigated, in particular the phosphorylated form of NADP-GDH, whose function is controlled by cytokinin. We will study the NADP-GDH activity and cytokinin signal transduction (and related transcripts) under stress.

Alternative Pathways of Carbon Skeletons for Ammonium Assimilation under Stress. PEPc activity increased significantly in tomato plants exposed to salinity (Cramer and Lips, 1995). PEPc activity was higher in roots than in shoots, while organic acid concentration was higher in shoots. These result suggest that a significant part of the organic acids produced in the roots were used as carbon skeleton for amination and transamination reactions. The increased activities of NR, PEPc and GS with salinity and N concentration in the medium explain the increased production of organic nitrogen observed in annual ryegrass plant under saline conditions (Sagi et al., 1998).

Roots of rice under stress induced PPDK (pyruvate-phosphate dikinase), an enzyme which regenerates PEP from pyruvate, one of the substrates of PEPc (Moons et al., 1998), presumably as part of the plant efforts to provide alternative carbon skeletons (OAA - oxaloacetate) to sustain ammonium assimilation in roots of stressed plants. The increase of PPDK activity parallel to enhanced activity of PEPc and the production of aspartate and asparagine, suggest an “emergency” supply system of carbon skeletons for ammonium assimilation in stressed plants. This “emergency” path is activated when mitochondria has to focus its production on ATP for osmoregulation through enhance uptake of ions from the medium at the expense of isocitrate export to provide 2-oxoglutarate to GOGAT. Drought and salinity greatly affect nitrogen metabolism in plants, and it has been recently observed that the enhanced activities of PEPc and PPDK in roots of stress plants contribute to the supply of oxaloacetate as an alternative carbon skeleton for ammonium assimilation when the availability of oxoglutarate is curtailed. Drought and salt tolerant crops capable of activating alternative mechanisms of nitrogen assimilation in roots are also capable of maintaining productivity under salinity and drought conditions.

Focus on an important Trait. "Rolling leaf" (RL) wheat genotypes show remarkable drought resistance. They were obtained in Kazakhstan by traditional breeding based on the crossing of a variety of wheat genotypes carrying the RL1 and RL2 genes. These genes were genetically identified and have been located on chromosomes 6A and 4D (Bogdanova & Shulembaeva, 1988). They contribute to the expression of traits such as rolling leaves, which improve water balance and contribute to water conservation at high temperature and/or drought affected soils. Wheat (Triticum aestivum L.) doubled haploid RL-lines (DHL) were obtained by modern haploid biotechnology through the culture of isolated anthers and microspores in vitro (Anapiyaev, 1999). The haploid technology permits production of stable lines of hybrid populations and new high yielding lines (De Buyser et.al., 1987). Two shoot-related mechanisms of drought avoidance, stomatal closure and leaf rolling, are evident in rice and both reduce water loss by transpiration (Price et al., 1997). Rolling also reduces the absorption of heat and light. A similar relationship between leaf water potential, leaf rolling and stomatal closure in different varieties were described (O’Toole & Cruz, 1980). Leaf rolling and stomatal closure contribute to increased water use efficiency in water stressed rice (Dingkuhn et al., 1989). We will compare these elite germplasm developed in Kazakhstan with less tolerant lines both at the protein and at the transcript level.

The Illinois team will provide and utilize cloned transcripts and provide microarray slides for this project. Our plan is to have these arrayed DNA elements ready for colleagues from Kazakhstan to work in the laboratory at Urbana for four months of each of the two years.

(1) ESTs (cloned cDNAs, partially sequenced) and microarray slides are available from several grass species. We list the DNAs that are available, and indicate that our ongoing work will add more ESTs during the coming year.

Corn (B73) root and leaf clones from young plants ~8,200 ESTs

Rice (Pokkali, IR29, Nipponbare) root, leaf ~8,500 ESTs

Barley (Tokak - drought tolerant Turkish variety) root, leaf ~2,700 ESTs

In addition, the lab utilizes ESTs from Arabidopsis (>9,000), Mesembryanthemum (>15,000), tobacco (~2,200) and yeast (whole genome array). For rice, corn and barley microarrays that contain these ESTs are available. In addition, we have printed and available approximately 70 slides that contain 4,000 ESTs each from the three grass species on one slide. Funds in this project will be used to generate microarray elements for the enzymes targeted in this study and for a number of control genes (see below) from a Kazakh wheat line.

(2) A "grass array", already available (see above), will utilize the high sequence identity among cereal crop transcript sequences.

(3) We will obtain additional sequences that will be assembled into a project-specific microarray slide. Most of the target gene transcripts for enzymes in C and N assimilation are available in corn and rice (http://www.stress-genomics.org). Missing transcripts will be obtained before colleagues from Kazakhstan come to Illinois (year one). We will assemble microarray slides for the transcripts encoding enzymes in nitrogen and carbon assimilation - based on a list of enzymes that has been provided by Dr. Lee. We will obtain these relevant transcripts from wheat by PCR amplification utilizing degenerate (for genes that have not yet been sequenced in wheat) and precise primers (for genes that have already been sequenced in wheat). Based on our experience in other projects, it is advantageous to design primers such that we obtain coding region and 3'UTR probes separately. This requires that we sequence any cDNA from both the 5' and 3' end and then design new primers that are specific for the 3'UTR. In addition, we will sequence a sufficient number of cDNAs to obtain with high probability the most abundant and, hopefully, all isoforms for the enzymes that are in the center of the project. Several of these genes must exist in multiple forms, based on the ploidy of bread wheat, and also based on the fact that isoforms are present for different compartments. In addition, we will add DNA elements to the microarrays that represent whatever transaminases we can find in our EST collections, the entire photosynthesis machinery (already available), and transcripts for both carbon and amino acid transporters (Rentsch et al., 1995).

We expect that we will have approximately 150 (plus 3'UTR) transcripts for these pathways. In addition to those transcripts, we will also print ESTs for functions that indicate cell growth and maintenance, cell cycle, stomatal behavior, ion and water homeostasis, and transcript for selected developmental stages, such as tillering, root growth, and flowering and seed and embryo development. These will be obtained by PCR amplification, cloning and sequencing of the DNA.

c. Anticipated Results

Drought-based and nitrogen deficiency-based yield reduction are tightly correlated. Our research is based on correlations established in breeding programs to which we will add biochemical behavior of essential proteins and novel molecular genetic markers. The results of these studies are of mutual benefit to both countries. As in Kazakhstan, drought stress is a growing concern in US agriculture because of dwindling fresh water reserves. Our project will result in data that can be used in a Kazakh wheat breeding program, even though some of our work will utilize rice and barley. The work will be significant based on the co-linearity of cereal genomes. Based on the exchange of collaborators, our project generates opportunities for interaction, education and training of young scientists and leads to a sharing of resources.

We expect applicable results from this work - a collection (electronic and as cDNA clones) of candidate genes for molecular breeding, metabolic understanding of essential drought defense mechanisms and recommendations for breeding projects that implement the knowledge acquired. Also, the results can become the basis for transgenic approaches to generate plants with enhanced abiotic stress tolerance.

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