Comparative analysis of expressed sequence tags in resistant and susceptible ecotypes of Arabidopsis thaliana infected with cucumber mosaic virus

Arabidopsis thaliana ecotype Columbia (Col-0) is susceptible to the yellow strain of cucumber mosaic virus [CMV(Y)], whereas ecotype C24 is resistant to CMV(Y). Comprehensive analyses of approximately 9,000 expressed sequence tags in ecotypes Col-0 and C24 infected with CMV(Y) suggested that the gene expression patterns in the two ecotypes differed. At 6, 12, 24 and 48 h after CMV(Y) inoculation, the expression of 6, 30, 85 and 788 genes, respectively, had changed in C24, as opposed to 20, 80, 53 and 150 genes in CMV(Y)-infected Col-0. At 12, 24 and 48 h after CMV(Y) inoculation, the abundance of 3, 10 and 55 mRNAs was altered in both ecotypes. However, at 6 h after CMV(Y) inoculation, no genes were co-induced or co-suppressed in both ecotypes. This differential pattern of gene expression between the two ecotypes at an early stage of CMV(Y) infection indicated that the cellular response for resistance may differ from that resulting in susceptibility at the level detectable by the macroarray. According to the expression pattern at various stages of infection, the expression of many genes could be grouped into clusters using cluster analysis. About 100 genes that encode proteins involved in chloroplast function were categorized into clusters 1 and 4, which had a differentially lower expression in CMV(Y)-inoculated C24. The expression of various genes encoding proteins in the endomembrane system belonged to clusters 2 and 4, which were induced in CMV(Y)-inoculated C24 and Col-0 leaves. Characterization of CMV(Y)-altered gene expression in the two ecotypes will contribute to a better understanding of the molecular basis of compatible and incompatible interactions between virus and host plants.     

Supplementary ultraviolet-B irradiation reveals differences in stress responses between Arabidopsis thaliana ecotypes

Irradiation of Arabidopsis thaliana ecotypes C24, Wassilewskija (Ws) and Columbia-0 (Col-0) with supplementary ultraviolet-A+B (UV-A+B) radiation revealed ecotype-specific differences in expression of the gene for the pathogenesis-related protein PR-5. C24 showed an increased expression level of PR-5 (5- and 20-fold higher compared with Col-0 and Ws, respectively). Expression of other molecular markers such as CHS (encoding chalcone synthase), MEB5.2 [encoding a gene strongly up-regulated by ultraviolet-B (UV-B)] and PYROA [encoding a pyridoxine (Vitamin B6) biosynthesis enzyme] only showed slight differences between ecotypes. Oxidative stress during UVA+B exposure was monitored by staining for H2O2. This analysis also revealed important ecotype-specific differences. 'H2O2 hot spots' were found in C24, whereas an even distribution of H2O2 was found in Ws and Col-0. Necrotic lesions also appeared on C24 leaves after prolonged UV-B exposure. There was a reverse correlation between the H2O2 steady-state concentration and the PR-5 gene expression; Ws showed the highest level of H2O2 accumulation but the lowest expression level of the PR-5 gene. Furthermore, application of paraquat on the rosettes led to similar PR-5 expression and H2O2 accumulation patterns as were found after UV-A+B irradiation. The observed ecotypic differences were also reflected in a statistically significant UV-B-dependent decrease in biomass, rosette size and leaf area for Ws, but not for C24 and Col-0. Our results show that a significant ecotype-specific genetic variability in general UV-B responses in Arabidopsis exists. Moreover, the signal transduction or gene regulation pathway for PR-5 differs from the other molecular markers used in this study.     

Ozone-induced cell death occurs via two distinct mechanisms in Arabidopsis: the role of salicylic acid

Previous studies suggest that salicylic acid (SA) plays an important role in influencing plant resistance to ozone (O3). To further define the role of SA in O3-induced responses, we compared the responses of two Arabidopsis genotypes that accumulate different amounts of SA in response to O3 and a SA-deficient transgenic Col-0 line expressing salicylate hydroxylase (NahG). The differences observed in O3-induced changes in SA levels, the accumulation of active oxygen species, defense gene expression, and the kinetics and severity of lesion formation indicate that SA influences O3 tolerance via two distinct mechanisms. Detailed analyses indicated that features associated with a hypersensitive response (HR) were significantly greater in O3-exposed Cvi-0 than in Col-0, and that NahG plants failed to exhibit these HR-like responses. Furthermore, O3-induced antioxidant defenses, including the redox state of glutathione, were greatly reduced in NahG plants compared to Col-0 and Cvi-0. This suggests that O3-induced cell death in NahG plants is due to the loss of SA-mediated potentiation of antioxidant defenses, while O3-induced cell death in Cvi-0 is due to activation of a HR. This hypothesis is supported by the observation that inhibition of NADPH-oxidases reduced O3-induced H2O2 levels and the O3-induced cell death in Cvi-0, while no major changes were observed in NahG plants. We conclude that although SA is required to maintain the cellular redox state and potentiate defense responses in O3 exposed plants, high levels of SA also potentiate activation of an oxidative burst and a cell death pathway that results in apparent O3 sensitivity.     

Ozone-induced ethylene production is dependent on salicylic acid,and both salicylic acid and ethylene act in concert to regulate ozone-induced cell death

Ethylene is known to influence plant defense responses including cell death in response to both biotic and abiotic stress factors. However, whether ethylene acts alone or in conjunction with other signaling pathways is not clearly understood. Ethylene overproducer mutants, eto1 and eto3, produced high levels of ethylene and developed necrotic lesions in response to an acute O3 exposure that does not induce lesions in O3-tolerant wild-type Col-0 plants. Treatment of plants with ethylene inhibitors completely blocked O3-induced ethylene production and partially attenuated O3-induced cell death. Analyses of the responses of molecular markers of specific signaling pathways indicated a relationship between salicylic acid (SA)- and ethylene-signaling pathways and O3 sensitivity. Both eto1 and eto3 plants constitutively accumulated threefold higher levels of total SA and exhibited a rapid increase in free SA and ethylene levels prior to lesion formation in response to O3 exposure. SA pre-treatments increased O3 sensitivity of Col-0, suggesting that constitutive high SA levels prime leaf tissue to exhibit increased magnitude of O3-induced cell death. NahG and npr1 plants compromised in SA signaling failed to produce ethylene in response to O3 and other stress factors suggesting that SA is required for stress-induced ethylene production. Furthermore, NahG expression in the dominant eto3 mutant attenuated ethylene-dependent PR4 expression and rescued the O3-induced HR (hypersensitive response) cell death phenotype exhibited by eto3 plants. Our results suggest that both SA and ethylene act in concert to influence cell death in O3-sensitive genotypes, and that O3-induced ethylene production is dependent on SA.     

Differential ozone sensitivity among <Emphasis Type="Italic">Arabidopsis</Emphasis> accessions and its relevance to ethylene synthesis

We compared the physiological and molecular responses of two Arabidopsis accessions, Col-0 and Ws-2, to ozone (O(3)) exposure. Observation of visible injury as well as ion-leakage analysis demonstrated clear differences between the O(3)-tolerant accession Col and the O(3)-sensitive accession Ws. RNA-blot analysis showed that O(3)-induced increases in mRNA levels of several ethylene-inducible genes and a salicylic acid-inducible gene were substantially higher in Ws than in Col. The time-course of induction of various mRNA levels shows that the expression of ethylene-inducible genes was rapidly, and more strongly, induced by O(3) in Ws than in Col, suggesting that Ws exhibits higher ethylene-signaling. Both the level of mRNA for an O(3)-inducible 1-aminocyclopropane-1-carboxylate synthase and the level of ethylene generation after 3 h of O(3)-exposure were higher in Ws than in Col. O(3)-induced leaf damage was attenuated by pretreatment with ethylene biosynthesis- and signaling-inhibitors, indicating that ethylene signaling is required for O(3)-induced leaf injury in Ws. On the other hand, an ethylene-overproducing mutant of Col, eto1-1, displayed significantly increased O(3)-induced leaf injury compared to wild type plants. These results indicate that the difference in O(3) sensitivity is dependent on the difference in ethylene production rate between these two accessions. Finally, we investigated the relationship between the degree of leaf damage and the level of ethylene evolution in 20 different Arabidopsis accessions. Based on the result, the accessions were classified into four types. However, most of them showed significant correlation between the ethylene production level and the degree of leaf injury, suggesting that ethylene signaling is an important factor in the natural variety of O(3) sensitivity among Arabidopsis accessions.     

Sugars and circadian regulation make major contributions to the global regulation of diurnal gene expression in Arabidopsis

The diurnal cycle strongly influences many plant metabolic and physiological processes. Arabidopsis thaliana rosettes were harvested six times during 12-h-light/12-h-dark treatments to investigate changes in gene expression using ATH1 arrays. Diagnostic gene sets were identified from published or in-house expression profiles of the response to light, sugar, nitrogen, and water deficit in seedlings and 4 h of darkness or illumination at ambient or compensation point [CO(2)]. Many sugar-responsive genes showed large diurnal expression changes, whose timing matched that of the diurnal changes of sugars. A set of circadian-regulated genes also showed large diurnal changes in expression. Comparison of published results from a free-running cycle with the diurnal changes in Columbia-0 (Col-0) and the starchless phosphoglucomutase (pgm) mutant indicated that sugars modify the expression of up to half of the clock-regulated genes. Principle component analysis identified genes that make large contributions to diurnal changes and confirmed that sugar and circadian regulation are the major inputs in Col-0 but that sugars dominate the response in pgm. Most of the changes in pgm are triggered by low sugar levels during the night rather than high levels in the light, highlighting the importance of responses to low sugar in diurnal gene regulation. We identified a set of candidate regulatory genes that show robust responses to alterations in sugar levels and change markedly during the diurnal cycle.     

二氧化硫胁迫导致拟南芥防护基因表达改变

研究SO2熏气对拟南芥细胞中mRNA和蛋白质表达的影响,分析植株对逆境胁迫的响应机制.结果表明,30 mg·m-3 SO2 熏气72 h后拟南芥地上组织中差异表达1倍以上的基因有494个,其中抗氧化酶、谷胱甘肽硫转移酶(GST)、硫氧还蛋白等多种与逆境生理关系密切的基因表达上调;2.5～30 mg·m-3 SO2 熏气可导致超氧化物岐化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GPX)和GST的活性诱导性增高,SOD、CAT同工酶谱带特征改变.研究结果表明,SO2 胁迫能够诱导拟南芥中防护基因在mRNA和蛋白质表达水平的改变,这些基因的差异性表达可能对逆境生理过程有益.     

Mutants of Arabidopsis thaliana with pleiotropic effects on the expression of the gene for β-amylase and on the accumulation of anthocyanin that are inducible by sugars

We identified a mutant of Arabidopsis thaliana ecotype Col-0 in which significantly reduced levels of expression of the gene for β-amylase ( ATβ-Amy ) were detected in leaves in response to high concentrations of sucrose, glucose or fructose. Genetic studies, including a cross with transgenic plants that harbored the ATβ-Amy:GUS transgene with the promoter of ATβ-Amy , indicated that this phenotype was caused by a recessive mutation, Iba1 , that affected expression of ATβ-Amy in trans . We also found a reduced level of sugar-induced expression of ATβ-Amy in the Landsberg erecta (L er ) ecotype compared with other ecotypes. This phenotype seemed to be due to a recessive trait, provisionally designated Iba2 , that was linked to neither erecta nor Iba1 . The Iba2 mutation also affected expression of ATβ-Amy:GUS transgene. Accumulation of starch and sugars after treatment of leaves with sucrose was not affected in the Iba1 mutant and L er plants. However, both Iba1 mutant and L er plants accumulated low levels of anthocyanin in response to sucrose, results that suggested the existence of some genetic linkage between regulation of the expression of ATβ-Amy and regulation of the accumulation of anthocyanin. Although the Iba1 and Iba2 mutations did not affect sugar-inducible gene expression in general, the expression of sugar-regulated genes other than the gene for β-amylase was differentially affected in the Iba1 mutant and L er plants. These results suggest that the sugar-regulated expression of many genes in plants might be mediated by multiple signal-transduction pathways.     

ERECTA, an LRR receptor-like kinase protein controlling development pleiotropically affects resistance to bacterial wilt

Bacterial wilt, one of the most devastating bacterial diseases of plants worldwide, is caused by Ralstonia solanacearum and affects many important crop species. We show that several strains isolated from solanaceous crops in Europe are pathogenic in different accessions of Arabidopsis thaliana. One of these strains, 14.25, causes wilting symptoms in A. thaliana accession Landsberg erecta (Ler) and no apparent symptoms in accession Columbia (Col-0). Disease development and bacterial multiplication in the susceptible Ler accession depend on functional hypersensitive response and pathogenicity (hrp) genes, key elements for bacterial pathogenicity. Genetic analysis using Ler x Col-0 recombinant inbred lines showed that resistance is governed by at least three loci: QRS1 (Quantitative Resistance to R. solanacearum) and QRS2 on chromosome 2, and QRS3 on chromosome 5. These loci explain about 90% of the resistance carried by the Col-0 accession. The ERECTA gene, which encodes a leucine-rich repeat receptor-like kinase (LRR-RLK) and affects development of aerial organs, is dimorphic in our population and lies close to QRS1. Susceptible Ler plants transformed with a wild-type ERECTA gene, and the LER line showed increased disease resistance to R. solanacearum as indicated by reduced wilt symptoms and impaired bacterial growth, suggesting unexpected cross-talk between resistance and developmental pathways.     

Arabidopsis sos1 mutant in a salt-tolerant accession revealed an importance of salt acclimation ability in plant salt tolerance

An analysis of the salinity tolerance of 354 Arabidopsis thaliana accessions showed that some accessions were more tolerant to salt shock than the reference accession, Col-0, when transferred from 0 to 225 mM NaCl. In addition, several accessions, including Zu-0, showed marked acquired salt tolerance after exposure to moderate salt stress. It is likely therefore that Arabidopsis plants have at least two types of tolerance, salt shock tolerance and acquired salt tolerance. To evaluate a role of well-known salt shock tolerant gene SOS1 in acquired salt tolerance, we isolated a sos1 mutant from ion-beam-mutagenized Zu-0 seedlings. The mutant showed severe growth inhibition under salt shock stress owing to a single base deletion in the SOS1 gene and was even more salt sensitive than Col-0. Nevertheless, it was able to survive after acclimation on 100 mM NaCl for 7 d followed by 750 mM sorbitol for 20 d, whereas Col-0 became chlorotic under the same conditions. We propose that genes for salt acclimation ability are different from genes for salt shock tolerance and play an important role in the acquisition of salt or osmotic tolerance.