Plant acclimation to freezing temperatures is very complex. Many temperate plants increase in freezing tolerance upon exposure to a period of low but non-freezing temperatures, an adaptive process known as cold acclimation. This acclimation phenomenon has encouraged investigations of physiological, biochemical, and molecular changes that are associated with the development of freezing tolerance. Although many biochemical and gene-expression changes occur during cold acclimation, few have been unequivocally demonstrated to contribute to the development of freezing tolerance. However, in the last few years, exciting new progress has been made through the use of mutational analysis and molecular genetic approaches. We now recognize that several interacting signal pathways are activated to bring about cold acclimation and ensure the winter survival of plants. The challenge for the future is to understand these pathways at a mechanistic level. Facile map-based cloning in Arabidopsis and techniques (such as DNA micro-arrays) for transcript profiling will provide the tools needed for this task.

The polyunsaturated fatty acids (PUFAs) linoleic acid (18:2) and ␣-linolenic acid (18:3) in triacylglycerols (TAG) are major factors affecting the quality of plant oils for human health, as well as for biofuels and other renewable applications. These PUFAs are essential fatty acids for animals and plants, but also are the source of unhealthy trans fats during the processing of many foodstuffs. PUFAs 18:2 and 18:3 are synthesized in developing seeds by the desaturation of oleic acid (18:1) esterified on the membrane lipid phosphatidylcholine (PC) on the endoplasmic reticulum. The reactions and fluxes involved in this metabolism are incompletely understood, however. Here we show that a previously unrecognized enzyme, phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), encoded by the Arabidopsis ROD1 gene, is a major reaction for the transfer of 18:1 into PC for desaturation and also for the reverse transfer of 18:2 and 18:3 into the TAG synthesis pathway. The PDCT enzyme catalyzes transfer of the phosphocholine headgroup from PC to diacylglycerol, and mutation of rod1 reduces 18:2 and 18:3 accumulation in seed TAG by 40%. Our discovery of PDCT is important for understanding glycerolipid metabolism in plants and other organisms, and provides tools to modify the fatty acid compositions of plant oils for improved nutrition, biofuel, and other purposes.Arabidopsis ͉ lipid metabolism ͉ oilseeds

Temperate plants develop a greater ability to withstand freezing in response to a period of low but nonfreezing temperatures through a complex, adaptive process of cold acclimation. Very little is known about the signaling processes by which plants perceive the low temperature stimulus and transduce it into the nucleus to activate genes needed for increased freezing tolerance. To help understand the signaling processes, we have isolated mutants of Arabidopsis that are constitutively freezing-tolerant in the absence of cold acclimation. Freezing tolerance of wild-type Arabidopsis was increased from ؊5.5°C to ؊12.6°C by cold acclimation whereas the freezing tolerance of 26 mutant lines ranged from ؊6.8°C to ؊10.6°C in the absence of acclimation. Plants with mutations at the eskimo1 (esk1) locus accumulated high levels of proline, a compatible osmolyte, but did not exhibit constitutively increased expression of several cold-regulated genes involved in freezing tolerance. RNA gel blot analysis suggested that proline accumulation in esk1 plants was mediated by regulation of transcript levels of genes involved in proline synthesis and degradation. The characterization of esk1 mutants and results from other mutants suggest that distinct signaling pathways activate different aspects of cold acclimation and that activation of one pathway can result in considerable freezing tolerance without activation of other pathways.

Background: Sorghum [Sorghum bicolor (L.) Moench] is ranked as the fifth most important grain crop and serves as a major food staple and fodder resource for much of the world, especially in arid and semi-arid regions. The recent surge in sorghum research is driven by its tolerance to drought/heat stresses and its strong potential as a bioenergy feedstock. Completion of the sorghum genome sequence has opened new avenues for sorghum functional genomics. However, the availability of genetic resources, specifically mutant lines, is limited. Chemical mutagenesis of sorghum germplasm, followed by screening for mutants altered in important agronomic traits, represents a rapid and effective means of addressing this limitation. Induced mutations in novel genes of interest can be efficiently assessed using the technique known as Targeting Induced Local Lesion IN Genomes (TILLING).

PCR has become one of the most popular techniques in functional genomics. Projects in both forward and reverse genetics routinely require PCR amplification of thousands of samples. Processing samples to extract DNA of sufficient purity for PCR is often a limiting step. We have developed a simple 96-well plate-based high-throughput DNA extraction method that is applicable to many plant species. The method involves a simple incubation of plant tissue samples in a DNA extraction buffer followed by a neutralization step. With the addition of a modified PCR buffer, the extracted DNA enabled the robust amplification of genomic fragments from samples of Arabidopsis, tobacco, sorghum, cotton, moss, and even pine needles. Several thousand DNA samples can be economically processed in a single day by one person without the use of robotics. This procedure will facilitate many technologies including high-throughput genotyping, map-based cloning, and identification of T-DNA or transposontagged mutants for known gene sequences.

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Sorghum (Sorghum bicolor) is a versatile C4 crop and a model for research in family Poaceae. High-quality genome sequence is available for the elite inbred line BTx623, but functional validation of genes remains challenging due to the limited genomic and germplasm resources available for comprehensive analysis of induced mutations. In this study, we generated 6400 pedigreed M4 mutant pools from EMS-mutagenized BTx623 seeds through single-seed descent. Whole-genome sequencing of 256 phenotyped mutant lines revealed >1.8 million canonical EMS-induced mutations, affecting >95% of genes in the sorghum genome. The vast majority (97.5%) of the induced mutations were distinct from natural variations. To demonstrate the utility of the sequenced sorghum mutant resource, we performed reverse genetics to identify eight genes potentially affecting drought tolerance, three of which had allelic mutations and two of which exhibited exact cosegregation with the phenotype of interest. Our results establish that a large-scale resource of sequenced pedigreed mutants provides an efficient platform for functional validation of genes in sorghum, thereby accelerating sorghum breeding. Moreover, findings made in sorghum could be readily translated to other members of the Poaceae via integrated genomics approaches.