Seedhabadee Ganeshan

Quantitative expression analysis of selected COR genes reveals their differential expression in leaf and crown tissues of wheat (Triticum aestivum L.) during an extended low temperature acclimation regimen

A number of COR genes (COld-Regulated genes) have been implicated in the acquisition of low temperature (LT) tolerance in wheat (Triticum aestivum L.). This study compared the relative expression patterns of selected COR genes in leaf and crown tissues of wheat near-isogenic lines to increase understanding of the molecular mechanisms underlying LT acclimation. Reciprocal near-isogenic lines were generated such that the dominant Vrn-A1 and recessive vrn-A1 loci were interchanged in a spring cv. Manitou and a winter cv. Norstar. Phenological development, acquisition of LT tolerance, and WCS120 polypeptide accumulation in these genotypes proceeded at rates similar to those previously reported for 6 °C acclimation from 0 to 98 d. However, a differential accumulation of WCS120 polypeptide and expression of the COR genes Wcs120, Wcor410, and Wcor14 was observed in the leaf and crown tissues. COR gene transcript levels peaked at 2 d of the acclimation period in both tissues and differences among genotypes were most evident at this time. COR gene expression was highest for the LT-tolerant and lowest for the tender genotypes. However, expression rates were divergent enough in genotypes with intermediate hardiness that comparisons among tissues and/or times during acclimation often resulted in variable interpretations of the relative expression of the COR genes in the determination of LT tolerance. These observations emphasize the need to pay close attention to experimental conditions, sampling times, and genotype and tissue selection in experiments designed to identify the critical genetic components that interact to determine LT acclimation.

Genome-wide gene expression analysis supports a developmental model of low temperature tolerance gene regulation in wheat (Triticum aestivum L.)

BackgroundTo identify the genes involved in the development of low temperature (LT) tolerance in hexaploid wheat, we examined the global changes in expression in response to cold of the 55,052 potentially unique genes represented in the Affymetrix Wheat Genome microarray. We compared the expression of genes in winter-habit (winter Norstar and winter Manitou) and spring-habit (spring Manitou and spring Norstar)) cultivars, wherein the locus for the vernalization gene Vrn-A1 was swapped between the parental winter Norstar and spring Manitou in the derived near-isogenic lines winter Manitou and spring Norstar. Global expression of genes in the crowns of 3-leaf stage plants cold-acclimated at 6°C for 0, 2, 14, 21, 38, 42, 56 and 70 days was examined.ResultsAnalysis of variance of gene expression separated the samples by genetic background and by the developmental stage before or after vernalization saturation was reached. Using gene-specific ANOVA we identified 12,901 genes (at p < 0.001) that change in expression with respect to both genotype and the duration of cold-treatment. We examined in more detail a subset of these genes (2,771) where expression was highly influenced by the interaction between these two main factors. Functional assignments using GO annotations showed that genes involved in transport, oxidation-reduction, and stress response were highly represented. Clustering based on the pattern of transcript accumulation identified genes that were up or down-regulated by cold-treatment. Our data indicate that the cold-sensitive lines can up-regulate known cold-responsive genes comparable to that of cold-hardy lines. The levels of expression of these genes were highly influenced by the initial rate and the duration of the genes response to cold. We show that the Vrn-A1 locus controls the duration of gene expression but not its initial rate of response to cold treatment. Furthermore, we provide evidence that Ta.Vrn-A1 and Ta.Vrt1 originally hypothesized to encode for the same gene showed different patterns of expression and therefore are distinct.ConclusionThis study provides novel insight into the underlying mechanisms that regulate the expression of cold-responsive genes in wheat. The results support the developmental model of LT tolerance gene regulation and demonstrate the complex genotype by environment interactions that determine LT adaptation in winter annual cereals.

In vitro‐cultured wheat spikes provide a simplified alternative for studies of cadmium uptake in developing grains

BACKGROUND An immature wheat spike culture system was used to monitor cadmium (Cd) accumulation in grains, hulls and awns of bread wheat and durum wheat. Immature spikes were cultured prior to anthesis in a medium containing 50 g L(-1) sucrose and 0.4 g L(-1) L-glutamine, supplemented with 0, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20 or 25 mg L(-1) cadmium chloride (CdCl(2)). Grains were collected at maturity and their Cd accumulation was determined using inductively coupled plasma mass spectrometry (ICP-MS). RESULTS Cd accumulation at CdCl(2) concentrations of 3 mg L(-1) and above was higher in grains of durum wheat compared with bread wheat. In hulls a similar trend was observed at CdCl(2) concentrations above 15 mg L(-1) . Starch concentration in grains increased slightly at 3 and 4 mg L(-1) CdCl(2). Cd accumulation negatively affected grain protein concentration. Expression patterns of Cd-related genes glutathione reductase (TaGR), metallothionein (MT) and phytochelatin synthase (PCS) in spikes cultured in media containing 0, 5, 10, 15 and 25 mg L(-1) CdCl(2) at 5 days post-anthesis showed that TaGR and PCS expression in bread wheat was up-regulated at 5 mg L(-1) CdCl(2) but down-regulated at other CdCl(2) concentrations. However, in durum wheat, expression of all three genes was down-regulated or remained unchanged. CONCLUSION This study demonstrates that immature spike culture can be used to study Cd accumulation in grains and can delineate hyper-accumulating durum wheat from bread wheat at CdCl(2) concentrations of 2 mg L(-1) and above.

Plant Breeding: Antisense ODN Inhibition in in vitro spike cultures as a powerful Diagnostic Tool in Studies on Cereal Grain Development

The study of grain development is perhaps one of the most complex and confounding processes in cereals. In addition to carbon partitioning into the grain, other plant metabolic processes, tissues, organs and environmental factors influence the development of the grain. Due to the importance of cereals as a food/energy source, there has been tremendous effort over the past several decades to understand the grain development process in order to enhance productivity and cater to the food requirements of the ever-growing world population. The significant influence of the environment on grain development has hindered the proper understanding of the factors that play key roles in this process. Therefore, development of a system, wherein the confounding effects of the environment and tissues/organs could be neutralized would be valuable in understanding grain development/filling. Furthermore, with the environment being kept constant, the effects of monitored alterations of physico-chemical factors on grain development can be studied. Field and growth chamber experiments, although valuable, have the disadvantage of being governed by uncontrollable variables. Thus, development of an in vitro approach that would allow for a coordinated, concerted and targeted approach to study grain development would be useful. Even though results from such studies cannot be directly extrapolated to actual field situations, a basic understanding of the grain developmental process can be obtained under various treatments and used as a model on which to build further understanding of grain development under actual growth conditions. However, even with an in vitro approach, every effort should be made to tailor the system to be as close as possible to an in vivo situation. This chapter has the 2-fold purpose of describing the cereal in vitro spike culture as a model system for cereals, and the antisense ODN technology as an emerging means for studying cereal grain development.

Functional Genomics For Crop Improvement

Plant breeding has had a tremendous influence on crop improvement. However, due to dwindling germplasm resources, identification of variability for incorporation into new cultivars is becoming more difficult. Therefore, there has been recourse to alternative approaches including mutagenesis, tissue culture and genetic transformation to aid breeding programs. Furthermore, with the vast repertoire of genome-wide data from different expression profiling techniques such as microarrays, more subtle understanding of gene expression is being obtained and is further helping plant breeders to entertain a different selection approach based on expression quantitative traits to maximize combinations of genes capable of conferring high performance. In this chapter, we review some of the aspects of plant breeding and the influence functional genomics has on breeding programs. Some of the challenges to functional genomics and breeding come from establishment of high-throughput transformation systems to assess gene function, which is limiting functional characterization of numerous genes in their respective crops. Therefore, this chapter also focuses on the need to gain better understanding of the development of gene transfer systems for crop plants to make use of the array of available gene information data.

Gene Transfer Methods

The ability to alter the genetic composition of a plant is fundamental to crop improvement and development of new cultivars with desirable characters. Plant breeders have utilized the naturally occurring genetic variability in existing germ-plasm to develop new lines by sexual hybridization. In the absence of natural variation for a trait, chemical and radiation mutagenesis was used to create genetic variability for use in the development of varieties with desirable traits. In another approach, genes for superior traits in close relatives were identified and recombined by wide hybridization, thereby generating interspecific or intergeneric hybrids between the donor and target species. However, all these chromosome-mediated gene transfers need sexual hybridizations. Sexual compatibility and chromosome pairing are key components for the introgression of a desired trait. To overcome limited sexual compatibility, embryo rescue using in vitro culture techniques was used to induce genetic variability for desirable traits (Raghavan 1986). The development of protoplast culture and somatic cell hybridization was one of the first examples to create genetic variability by asexual means. Furthermore, in vitro culture of plant cells in suboptimal conditions was found to induce genetic variations termed somaclonal variation, subsequently exhibiting an altered pheno-type (Larkin and Scowcroft 1981). The Agrobacterium tumefaciens-mediated integration of foreign DNA into a cells nuclear genome and production of a transgenic plant in which the inserted gene was inherited following Mendelian genetics was the ultimate method to create genetic variation across species, irrespective of genetic proximity or sexual compatibility (Otten et al. 1981).