磷脂酶Dδ参与植物的低温驯化过程

对经低温驯化和未经低温驯化的磷脂酶Dδ（PLDδ）基因敲除突变体与野生型植株进行冻害胁迫处理后,比较2种基因型植株的抗冻性。结果发现,经低温驯化的PLDδ敲除突变体的抗冻性明显低于野生型,而未经低温驯化的PLD礅除突变体与野生型的抗冻性没有显著差异,表明PLDδ参与植物的低温驯化过程。对PLDδ的作用途径进行分析,发现PLDδ在低温驯化过程中不参与抗氧化酶活性的调节,对脯氨酸和可溶性糖的积累起负调节作用,但是参与低温信号转导物质ABA诱导抗冻性的过程。     

磷脂酶Dδ参与植物的低温驯化过程

对经低温驯化和未经低温驯化的磷脂酶Dδ (PLDδ)基因敲除突变体与野生型植株进行冻害胁迫处理后, 比较2种基因型植株的抗冻性。结果发现, 经低温驯化的PLDδ敲除突变体的抗冻性明显低于野生型, 而未经低温驯化的PLDδ敲除突变体与野生型的抗冻性没有显著差异, 表明PLDδ参与植物的低温驯化过程。对PLDδ的作用途径进行分析, 发现PLDδ在低温驯化过程中不参与抗氧化酶活性的调节, 对脯氨酸和可溶性糖的积累起负调节作用, 但是参与低温信号转导物质ABA诱导抗冻性的过程。     

Heterosis in the freezing tolerance of crosses between two Arabidopsis thaliana accessions (Columbia-0 and C24) that show differences in non-acclimated and acclimated freezing tolerance

Heterosis is broadly defined as the increased vigour of hybrids in comparison to their parents. In the model plant Arabidopsis thaliana, a significant heterosis effect on leaf-freezing tolerance was observed in the F(1) generation of a cross between the accessions Columbia-0 (Col) and C24. Parental Col plants were significantly more freezing-tolerant than C24 plants in both the acclimated and non-acclimated (NA) states. Mid-parent heterosis was observed in the F(1) plants, both in the basic tolerance of non-adapted plants and in freezing tolerance after cold acclimation. Best-parent heterosis, on the other hand, was only found after cold acclimation. The heterosis effect was reduced in the F(2) populations such that only mid-parent heterosis was evident. The leaf content of soluble sugars (fructose (Fru), glucose (Glc), sucrose (Suc) and raffinose (Raf)) increased dramatically in the F(1) plants after cold acclimation as compared to the parental lines. The content of proline (Pro), however, was only moderately increased in the F(1) plants under the same conditions. Correlation analyses showed that only Raf content was consistently related to leaf-freezing tolerance in both the acclimated and NA states. A quantification of mRNA levels in leaves of parental and F(1) lines using quantitative real-time RT-PCR showed no clear indication for an involvement of the investigated genes (CBF (C-repeat binding factor)1, CBF2, (cold-regulated protein (COR) 6.6, COR15a, COR15b, COR47 and COR78) in the heterosis effect.     

HOS1, a genetic locus involved in cold-responsive gene expression in arabidopsis.

Low-temperature stress induces the expression of a variety of genes in plants. However, the signal transduction pathway(s) that activates gene expression under cold stress is poorly understood. Mutants defective in cold signaling should facilitate molecular analysis of plant responses to low temperature and eventually lead to the identification and cloning of a cold stress receptor(s) and intracellular signaling components. In this study, we characterize a plant mutant affected in its response to low temperatures. The Arabidopsis hos1-1 mutation identified by luciferase imaging causes superinduction of cold-responsive genes, such as RD29A, COR47, COR15A, KIN1, and ADH. Although these genes are also induced by abscisic acid, high salt, or polyethylene glycol in addition to cold, the hos1-1 mutation only enhances their expression under cold stress. Genetic analysis revealed that hos1-1 is a single recessive mutation in a nuclear gene. Our studies using the firefly luciferase reporter gene under the control of the cold-responsive RD29A promoter have indicated that cold-responsive genes can be induced by temperatures as high as 19 degrees C in hos1-1 plants. In contrast, wild-type plants do not express the luciferase reporter at 10 degrees C or higher. Compared with the wild type, hos1-1 plants are l ess cold hardy. Nonetheless, after 2 days of cold acclimation, hos1-1 plants acquired the same degree of freezing tolerance as did the wild type. The hos1-1 plants flowered earlier than did the wild-type plants and appeared constitutively vernalized. Taken together, our findings show that the HOS1 locus is an important negative regulator of cold signal transduction in plant cells and that it plays critical roles in controlling gene expression under cold stress, freezing tolerance, and flowering time.     

Clinal variation in the non-acclimated and cold-acclimated freezing tolerance of Arabidopsis thaliana accessions

Arabidopsis thaliana is a geographically widely spread species consisting of local accessions differing both genetically and phenotypically. These differences may constitute environmental adaptations and a latitudinal cline in freezing tolerance has been shown previously. Many plants, including Arabidopsis, exhibit increased freezing tolerance after cold exposure (cold acclimation). Here we present evidence for geographical clines (both latitudinal and longitudinal) in acclimated (ACC) and non-acclimated (NA) freezing tolerance, estimated from electrolyte leakage measurements on 54 accessions. Leaf Pro contents were not correlated with freezing tolerance, while sugar contents (Glc, Fru, Suc, Raf) were in the ACC, but not the NA state. Expression levels of 14 cold-induced genes were investigated before and after 2 weeks of cold acclimation by quantitative RT-PCR. Expression of the CBF1, 2 and 3 genes was not correlated with freezing tolerance. The expression of some CBF-regulated (COR) genes, however, was correlated specifically with ACC freezing tolerance. A tight correlation between CBF and COR gene expression was only observed under non-acclimating conditions, where CBF and COR expression were also correlated with the expression of PRR5, a component of the circadian clock. Collectively, this study sheds new light on the molecular determinants of plant-freezing tolerance and cold acclimation and their geographical dependence.     

Differential degradation of extraplastidic and plastidic lipids during freezing and post-freezing recovery in Arabidopsis thaliana

Changes in membrane lipid composition play important roles in plant adaptation to and survival after freezing. Plant response to cold and freezing involves three distinct phases: cold acclimation, freezing, and post-freezing recovery. Considerable progress has been made toward understanding lipid changes during cold acclimation and freezing, but little is known about lipid alteration during post-freezing recovery. We previously showed that phospholipase D (PLD) is involved in lipid hydrolysis and Arabidopsis thaliana freezing tolerance. This study was undertaken to determine how lipid species change during post-freezing recovery and to determine the effect of two PLDs, PLDalpha1 and PLDdelta, on lipid changes during post-freezing recovery. During post-freezing recovery, hydrolysis of plastidic lipids, monogalactosyldiacylglycerol and plastidic phosphatidylglycerol, is the most prominent change. In contrast, during freezing, hydrolysis of extraplastidic phospholipids, phosphatidylcholine and phosphatidylethanolamine, occurs. Suppression of PLDalpha1 decreased phospholipid hydrolysis and phosphatidic acid production in both the freezing and post-freezing phases, whereas ablation of PLDdelta increased lipid hydrolysis and phosphatidic acid production during post-freezing recovery. Thus, distinctly different changes in lipid hydrolysis occur in freezing and post-freezing recovery. The presence of PLDalpha1 correlates with phospholipid hydrolysis in both freezing and post-freezing phases, whereas the presence of PLDdelta correlates with reduced lipid hydrolysis during post-freezing recovery. These data suggest a negative role for PLDalpha1 and a positive role for PLDdelta in freezing tolerance.     

The plasma membrane-bound phospholipase Ddelta enhances freezing tolerance in Arabidopsis thaliana

Freezing injury is a major environmental limitation on the productivity and geographical distribution of plants. Here we show that freezing tolerance can be manipulated in Arabidopsis thaliana by genetic alteration of the gene encoding phospholipase Ddelta (PLDdelta), which is involved in membrane lipid hydrolysis and cell signaling. Genetic knockout of the plasma membrane-associated PLDdelta rendered A. thaliana plants more sensitive to freezing, whereas overexpression of PLDdelta increased freezing tolerance. Lipid profiling revealed that PLDdelta contributed approximately 20% of the phosphatidic acid produced in wild-type plants during freezing, and overexpression of PLDdelta increased the production of phosphatidic acid species. The PLDdelta alterations did not affect the expression of the cold-regulated genes COR47 or COR78 or alter cold-induced increases in proline or soluble sugars, suggesting that the PLD pathway is a unique determinant of the response to freezing and may present opportunities for improving plant freezing tolerance.     

The role of raffinose in the cold acclimation response of Arabidopsis thaliana

In many plants raffinose family oligosaccharides are accumulated during cold acclimation. The contribution of raffinose accumulation to freezing tolerance is not clear. Here, we investigated whether synthesis of raffinose is an essential component for acquiring frost tolerance. We created transgenic lines of Arabidopsis thaliana accessions Columbia-0 and Cape Verde Islands constitutively overexpressing a galactinol synthase (GS) gene from cucumber. GS overexpressing lines contained up to 20 times as much raffinose as the respective wild-type under non-acclimated conditions and up to 2.3 times more after 14 days of cold acclimation at 4 degrees C. Furthermore, we used a mutant carrying a knockout of the endogenous raffinose synthase (RS) gene. Raffinose was completely absent in this mutant. However, neither the freezing tolerance of non-acclimated leaves, nor their ability to cold acclimate were influenced in the RS mutant or in the GS overexpressing lines. We conclude that raffinose is not essential for basic freezing tolerance or for cold acclimation of A. thaliana.     

Heterosis in the freezing tolerance, and sugar and flavonoid contents of crosses between Arabidopsis thaliana accessions of widely varying freezing tolerance

Heterosis is defined as the increased vigour of hybrids in comparison to their parents. We investigated 24 F(1) hybrid lines of Arabidopsis thaliana generated by reciprocally crossing either C24 or Col with six other parental accessions (Can, Co, Cvi, Ler, Rsch, Te) that differ widely in their freezing tolerance. The crosses differed in the degree of heterosis for freezing tolerance, both in the non-acclimated state and after a 14 d cold acclimation period. Crosses with C24 showed more heterosis than crosses with Col, and heterosis was stronger in acclimated than in non-acclimated plants. Leaf content of soluble sugars and proline showed more deviation from mid-parent values in crosses involving C24 than in those involving Col, and deviations were larger in acclimated than in non-acclimated plants. There were significant correlations between the content of different sugars and leaf freezing tolerance, as well as between heterosis effects in freezing tolerance and sugar content. Flavonoid content and composition varied between accessions, and between non-acclimated and acclimated plants. In the crosses, large deviations from the mid-parent values in the contents of different flavonols occurred, and there were strikingly strong correlations between both flavonol content and freezing tolerance, and between heterosis effects in freezing tolerance and flavonol content.     

拟南芥CBF1与植物对低温和干旱的抗性

对冷驯化过程中基因表达差异的认识,使抗冻基因(COR)的克隆及其功能的分析成为研究冷驯化过程的主要目标。在拟南芥和其他抗冻植物中,分离了许多COR基因,这些基因对植物抗冻起着非常重要的作用。在拟南芥COR调控的研究中,发现了CBF转录因子的基因家族,其中CBF1能调控一组COR基因的表达。近年来,在冷敏植物如番茄和玉米中也发现了CBF类似基因,拟南芥CBF1基因在转基因番茄中的过量表达提高了植株的抗寒和抗旱性。这一研究结果展示了拟南芥CBF1类似基因的应用可能为冷敏植物抗寒和抗旱性的品种改良提供一条新的途径。     

Overexpression of Multiple Dehydrin Genes Enhances Tolerance to Freezing Stress in Arabidopsis

To elucidate the contribution of dehydrins (DHNs) to freezing stress tolerance in Arabidopsis, transgenic plants overexpressing multiple DHN genes were generated. Chimeric double constructs for expression of RAB18 and COR47 (pTP9) or LTI29 and LTI30 (pTP10) were made by fusing the coding sequences of the respective DHN genes to the cauliflower mosaic virus 35S promoter. Overexpression of the chimeric genes in Arabidopsis resulted in accumulation of the corresponding dehydrins to levels similar or higher than in cold-acclimated wild-type plants. Transgenic plants exhibited lower LT50 values and improved survival when exposed to freezing stress compared to the control plants. Post-embedding immuno electron microscopy of high-pressure frozen, freeze-substituted samples revealed partial intracellular translocation from cytosol to the vicinity of the membranes of the acidic dehydrin LTI29 during cold acclimation in transgenic plants. This study provides evidence that dehydrins contribute to freezing stress tolerance in plants and suggests that this could be partly due to their protective effect on membranes.