植物抗寒及其基因表达研究进展

植物经过逐渐降低的温度从而提高抗寒能力 ,这个过程被人们称为低温驯化。植物低温驯化过程是一个复杂的生理、生化和能量代谢变化过程 ,这些变化主要包括膜系统的稳定性、可溶性蛋白的积累和小分子渗透物质 ,比如脯氨酸、糖等 ,这些变化中的一些是植物抗寒必需的 ,而另外一些变化不是必需的。主要对冷害和低温生理生化变化、低温诱导表达基因的功能和作用、低温驯化的调节机制及其信号转导方面进行了综述。通过差别筛选 c DNA文库的方法已经鉴定了许多低温诱导表达、进而提高植物抗寒能力的基因 ,其中有脱水素、COR基因和 CBF1转录因子等。低温信号的感受、转导和调节表达是低温驯化的关键环节 ,低温信号的转导过程与干旱胁迫之间具有一定的交叉 ,这为利用 ABA等来提高植物抗寒能力成为可能 ,相信不久的将来人们可以通过提高植物抗寒能力从而增加经济产量成为现实。     

AtPP2CG1, a protein phosphatase 2C, positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner

AtPP2CG1 (Arabidopsis thaliana protein phosphatase 2C G Group 1) was predicted as an abiotic stress candidate gene by bioinformatic analysis in our previous study. The gene encodes a putative protein phosphatase 2C that belongs to Group G of PP2C. There is no report of Group G genes involved in abiotic stress so far. Real-time RT-PCR analysis showed that AtPP2CG1 expression was induced by salt, drought, and abscisic acid (ABA) treatment. The expression levels of AtPP2CG1 in the ABA synthesis-deficient mutant abi2-3 were much lower than that in WT plants under salt stress suggesting that the expression of AtPP2CG1 acts in an ABA-dependent manner. Over-expression of AtPP2CG1 led to enhanced salt tolerance, whereas its loss of function caused decreased salt tolerance. These results indicate that AtPP2CG1 positively regulates salt stress in an ABA-dependent manner. Under salt treatment, AtPP2CG1 up-regulated the expression levels of stress-responsive genes, including RD29A, RD29B, DREB2A and KIN1. GUS activity was detected in roots, leaves, stems, flower, and trichomes of AtPP2CG1 promoter-GUS transgenic plants. AtPP2CG1 protein was localized in nucleus and cytoplasm via AtPP2CG1:eGFP and YFP:AtPP2CG1 fusion approaches.     

ABA-hypersensitive germination3 encodes a protein phosphatase 2C (AtPP2CA) that strongly regulates abscisic acid signaling during germination among Arabidopsis protein phosphatase 2Cs

The phytohormone abscisic acid (ABA) regulates physiologically important developmental processes and stress responses. Previously, we reported on Arabidopsis (Arabidopsis thaliana) L. Heynh. ahg mutants, which are hypersensitive to ABA during germination and early growth. Among them, ABA-hypersensitive germination3 (ahg3) showed the strongest ABA hypersensitivity. In this study, we found that the AHG3 gene is identical to AtPP2CA, which encodes a protein phosphatase 2C (PP2C). Although AtPP2CA has been reported to be involved in the ABA response on the basis of results obtained by reverse-genetics approaches, its physiological relevance in the ABA response has not been clarified yet. We demonstrate in vitro and in vivo that the ahg3-1 missense mutation causes the loss of PP2C activity, providing concrete confirmation that this PP2C functions as a negative regulator in ABA signaling. Furthermore, we compared the effects of disruption mutations of eight structurally related PP2C genes of Arabidopsis, including ABI1, ABI2, HAB1, and HAB2, and found that the disruptant mutant of AHG3/AtPP2CA had the strongest ABA hypersensitivity during germination, but it did not display any significant phenotypes in adult plants. Northern-blot analysis clearly showed that AHG3/AtPP2CA is the most active among those PP2C genes in seeds. These results suggest that AHG3/AtPP2CA plays a major role among PP2Cs in the ABA response in seeds and that the functions of those PP2Cs overlap, but their unique tissue- or development-specific expression confers distinct and indispensable physiological functions in the ABA response.     

Plant Cold Acclimation: The Role of Abscisic Acid

The freezing tolerance or cold acclimation of plants is enhanced over a period of time by temperatures below 10°C and by a short photoperiod in certain species of trees and grasses. During this process, freezing tolerance increases 2–8°C in spring annuals, 10–30°C in winter annuals, and 20–200°C in tree species. Gene upregulation and downregulation have been demonstrated to be involved in response to environmental cues such as low temperature. Evidence suggests ABA can substitute for the low temperature stimulus, provided there is also an adequate supply of sugars. Evidence also suggests there may be ABA-dependent and ABA-independent pathways involved in the acclimation process. This review summarizes the role of ABA in cold acclimation from both a historical and recent perspective. It is concluded that it is highly unlikely that ABA regulates all the genes associated with cold acclimation; however, it definitely regulates many of the genes associated with an increase in freezing tolerance.     

Differential expression of wheat genes during cold acclimation

Overwintering crops such as winter wheat display significant increase in freezing tolerance during a period of cold acclimation (CA). To gain better understanding of molecular mechanisms of CA, it is important to unravel functions and regulations of CA-associated genes. Differential screening of a cDNA library constructed from cold acclimated crown tissue of winter wheat identified three novel CA-associated cDNA clones. Nucleotide sequence analysis showed that the clones encode a high mobility globular protein (HMGB1), a glycine-rich RNA-binding protein (TaGRP2), and a LEA D-11 dehydrin (DHN14). Accumulation of the three mRNAs during 14 days of CA was differentially regulated. In response to drought, and ABA, DHN14 mRNA rapidly accumulated while HMGB1 and TaGRP2 mRNA levels remained unchanged. The possible functions of each of these genes in cold acclimation are discussed.     

Suppression of phospholipase Dalpha1 induces freezing tolerance in Arabidopsis: response of cold-responsive genes and osmolyte accumulation

Phospholipase D (PLD; EC 3.1.4.4) plays an important role in membrane lipid hydrolysis and in mediation of plant responses to a wide range of stresses. PLDalpha1 abrogation through antisense suppression in Arabidopsis thaliana resulted in a significant increase in freezing tolerance of both non-acclimated and cold-acclimated plants. Although non-acclimated PLDalpha1-deficient plants did not show the activation of cold-responsive C-repeat/dehydration-responsive element binding factors (CBFs) and their target genes (COR47 and COR78), they did accumulate osmolytes to much higher levels than did the non-acclimated wild-type plants. However, a stronger expression of COR47 and COR78 in response to cold acclimation and to especially freezing was observed in PLDalpha1-deficient plants. Furthermore, a slower activation of CBF1 was observed in response to cold acclimation in these plants compared to the wild-type plants. Typically, cold acclimation resulted in a higher accumulation of osmolytes in PLDalpha1-deficient plants than in wild-type plants. Inhibition of PLD activity by using lysophosphatidylethanolamine (LPE) also increased freezing tolerance of Arabidopsis, albeit to a lesser extent than did the PLD antisense suppression. Exogenous LPE induced expression of COR15a and COR47 in the absence of cold stimulus. These results suggest that PLDalpha1 plays a key role in freezing tolerance of Arabidopsis by modulating the cold-responsive genes and accumulation of osmolytes.     

Differential expression of wheat genes during cold acclimation

Overwintering crops such as winter wheat display a significant increase in freezing tolerance during periods of cold acclimation (CA). To gain a better understanding of the molecular mechanisms of CA, it is important to unravel the functions and regulations of CA-associated genes. Differential screening of a cDNA library constructed from cold acclimated crown tissue of winter wheat identified three novel CA-associated cDNA clones. Nucleotide sequence analysis showed that the clones encode a high mobility globular protein (HMGB1), a glycine-rich RNA-binding protein (TaGRP2), and a LEAD-11 dehydrin (DHN14). Accumulation of the three mRNAs during 14 days of CA was differentially regulated. In response to drought, and ABA, DHN14 mRNA rapidly accumulated while HMGB1 and TaGRP2 mRNA levels remained unchanged. The possible functions of each of these genes in cold acclimation are discussed. The text was submitted by the authors in English.     

Characterization of an 80-kDa dehydrin-like protein in barley responsive to cold acclimation

Barley ( Hordeum vulgare L.) exposed to low temperature increases its freezing tolerance. This increase has been associated with several metabolic changes caused by low temperature, including expression of dehydrins (DHN), a family of proteins induced by dehydration and cold acclimation. DHNs play an undetermined role in dehydration responses during freezing. We have studied the accumulation of an 80-kDa DHN-like protein (P-80) in barley under cold acclimation 6/4°C (day/night), postulating that it is localized in tissues where primary ice nucleation occurs. P-80 was absent in nonacclimated plants and was detectable after 48 h of cold acclimation, reaching a stable level after 6 days. P-80 decreased when plants were returned to 20–25°C. Drought, ABA and high temperature did not increase the levels of P-80, suggesting that its expression could be specifically regulated by cold. Immunolocalization by tissue printing and fresh cross sections of leaves showed the protein to be associated with vascular tissues and epidermis. The localization of P-80 is consistent with our hypothesis because vascular tissue and the epidermis are preferential ice nucleation zones during the onset of freezing. The differential accumulation of P-80 may have an adaptive value by participating in tolerance mechanisms during freeze-induced dehydration.     

Abscisic acid-induced freezing tolerance in the moss Physcomitrella patens is accompanied by increased expression of stress-related genes

Abscisic acid (ABA)-induced genes are implicated in the development of freezing tolerance during cold acclimation in higher plants, but their roles in lower land plants have not been determined. We examined ABA- and cold-induced changes in freezing tolerance and gene expression in the moss Physcomitrella patens. Slow equilibrium freezing to -4 degrees C of P. patens protonemata grown under normal growth conditions killed more than 90% of the cells, indicating that the protonema cells are freezing-sensitive. ABA treatment for 24 h dramatically increased the freezing tolerance of the protonemata, while cold treatment only slightly increased the freezing tolerance within the same period. We examined the expressions of fourteen Physcomitrella patens ABA-responsive genes (PPARs), isolated from ABA-treated protonemata. ABA treatment resulted in a remarkable increase in the expression of all the PPAR genes within 24 h. Several of the PPAR genes (PPAR 1 to 8, and 14) were also responsive to cold, but the response was much slower than that to ABA. Treatment with hyperosmotic concentrations of NaCl and mannitol increased freezing tolerance of protonemata and also increased the expression levels of eleven PPAR genes (PPAR2, 3, 5 to 8, and 10 to 14). These results suggest that ABA and environmental stresses positively affect the expression of common genes that participate in protection of protonema cells leading to the development of freezing tolerance.     

Induced expression of the class II chitinase gene during cold acclimation and dehydration of bermudagrass (Cynodon sp.)

Bermudagrass cultivars vary greatly in their ability to survive freezing temperatures as a result of a differential ability to cold acclimate (CA) at temperatures slightly above 0°C. Little information exists on the genetic and physiological mechanisms associated with the cold acclimation process in bermudagrass. Experiments were conducted to study the changes in chitinase gene expression during cold acclimation of freeze-tolerant bermudagrass cultivars. A chitinase gene (CynCHT1) was isolated from ’Midiron’ bermudagrass. Because the hydrophilic protein putatively encoded by the gene lacked an N-terminal cysteine-rich domain and a hydrophobic C-terminal extension, it was classified a class II chitinase. The expression patterns of this and related chitinase genes in response to CA, drought, and ABA were investigated in freeze-tolerant ’MSU’ (LT₅₀=?11°C), Midiron (LT₅₀=?10°C) and ’Uganda’ (LT₅₀=?8°C) bermudagrasses. Northern-blot analysis indicated expression in the crown tissues induced by CA at 8°C/2°C day/night temperature cycles. Induction of gene expression was evident in tissues sampled at 2 and 28 days after initiating CA. Expression after 2-days de-acclimation at 28°C/24°C was similar to control levels. Significantly higher levels of CA-induced chitinase gene expression were observed in MSU and Midiron, compared to Uganda. Similar expression patterns were observed among the cultivars in responses to drought and ABA. These results suggest that chitinases have important roles in bermudagrass response to low temperature and dehydration stresses.     

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。     

Development of freezing tolerance in different altitudinal ecotypes of <Emphasis Type="Italic">Salix paraplesia</Emphasis>

Salix paraplesia was used as an experimental model to investigate the effect of short day photoperiod (SD) and low temperature (LT) on development of freezing tolerance and on endogenous abscisic acid (ABA) contents. We characterized differences in SD and LT-induced cold acclimation in three ecotypes from different altitudes. The results demonstrated that cold acclimation could be triggered by exposing the plants to SD or LT alone, and that a combination of the different treatments had an additive effect on freezing tolerance in all ecotypes studied. However, the high altitudinal ecotype was more responsive to SD and LT than the low altitudinal ecotype. Development of freezing tolerance induced by SD and LT was accompanied by changes in ABA contents which were ecotype-dependent. Although the stem had higher initial freezing tolerance, the leaves developed freezing tolerance more quickly than the stem and thus leaves may provide an interesting experimental system for physiological and molecular studies of cold acclimation in woody plants.