The expression of heat shock proteins (Hsps) induced by nonlethal heat treatment confers acquired thermotolerance (AT) to organisms against subsequent challenges of otherwise lethal temperature. After the stress signal is removed, AT gradually decays, with decreased Hsps during recovery. AT of sufficient duration is critical for sessile organisms such as plants to survive repeated heat stress in their environment, but little is known regarding its regulation. To identify potential regulatory components, we took a reverse genetics approach by screening for Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutants that show decreased thermotolerance after a long recovery (2 d) under nonstress conditions following an acclimation heat treatment. Among the tested mutants corresponding to 48 heat-induced genes, only the heat shock transcription factor HsfA2 knockout mutant showed an obvious phenotype. Following pretreatment at 37°C, the mutant line was more sensitive to severe heat stress than the wild type after long but not short recovery periods, and this could be complemented by the introduction of a wild-type copy of the HsfA2 gene. Quantitative hypocotyl elongation assay also revealed that AT decayed faster in the absence of HsfA2. Significant reduction in the transcript levels of several highly heat-inducible genes was observed in HsfA2 knockout plants after 4 h recovery or 2 h prolonged heat stress. Immunoblot analysis showed that Hsa32 and class I small Hsp were less abundant in the mutant than in the wild type after long recovery. Our results suggest that HsfA2 as a heat-inducible transactivator sustains the expression of Hsp genes and extends the duration of AT in Arabidopsis.

Plants and animals share similar mechanisms in the heat shock (HS) response, such as synthesis of the conserved HS proteins (Hsps). However, because plants are confined to a growing environment, in general they require unique features to cope with heat stress. Here, we report on the analysis of the function of a novel Hsp, heat-stress-associated 32-kD protein (Hsa32), which is highly conserved in land plants but absent in most other organisms. The gene responds to HS at the transcriptional level in moss (Physcomitrella patens), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa). Like other Hsps, Hsa32 protein accumulates greatly in Arabidopsis seedlings after HS treatment. Disruption of Hsa32 by T-DNA insertion does not affect growth and development under normal conditions. However, the acquired thermotolerance in the knockout line was compromised following a long recovery period (.24 h) after acclimation HS treatment, when a severe HS challenge killed the mutant but not the wild-type plants, but no significant difference was observed if they were challenged within a short recovery period. Quantitative hypocotyl elongation assay also revealed that thermotolerance decayed faster in the absence of Hsa32 after a long recovery. Similar results were obtained in Arabidopsis transgenic plants with Hsa32 expression suppressed by RNA interference. Microarray analysis of the knockout mutant indicates that only the expression of Hsa32 was significantly altered in HS response. Taken together, our results suggest that Hsa32 is required not for induction but rather maintenance of acquired thermotolerance, a feature that could be important to plants.

A DNA cassette containing an Arabidopsis C repeat/dehydration-responsive element binding factor 1 (CBF1) cDNA and a nos terminator, driven by a cauliflower mosaic virus 35S promoter, was transformed into the tomato (Lycopersicon esculentum) genome. These transgenic tomato plants were more resistant to water deficit stress than the wild-type plants. The transgenic plants exhibited growth retardation by showing dwarf phenotype, and the fruit and seed numbers and fresh weight of the transgenic tomato plants were apparently less than those of the wild-type plants. Exogenous gibberellic acid treatment reversed the growth retardation and enhanced growth of transgenic tomato plants, but did not affect the level of water deficit resistance. The stomata of the transgenic CBF1 tomato plants closed more rapidly than the wild type after water deficit treatment with or without gibberellic acid pretreatment. The transgenic tomato plants contained higher levels of Pro than those of the wild-type plants under normal or water deficit conditions. Subtractive hybridization was used to isolate the responsive genes to heterologous CBF1 in transgenic tomato plants and the CAT1 (CATALASE1) was characterized. Catalase activity increased, and hydrogen peroxide concentration decreased in transgenic tomato plants compared with the wild-type plants with or without water deficit stress. These results indicated that the heterologous Arabidopsis CBF1 can confer water deficit resistance in transgenic tomato plants.Many environmental stresses, such as heat, salinity, low temperature, and drought, and developmental processes, such as seed maturation, cause water deficit in plants (Ingram and Bartels, 1996). To understand water deficit stress at the molecular level, many genes have been isolated, such as rd (responsive to dehydration), erd (early responsive to dehydration), and Lea (late embryogenesis abundant; Shinozaki and Yamaguchi-Shinozaki, 2000). The accumulation of LEA protein occurs during seed maturation, desiccation, and increases in vegetative tissue when plants are exposed to water deficit (Ingram and Bartels, 1996). Overexpression of a barley (Hordeum vulgare) group 3 LEA protein gene, HVA1, enhances tolerance of water deficit and salt stress in transgenic rice (Oryza sativa; Xu et al., 1996). Arabidopsis RD29A (COR78) responds to water deficit and low-temperature stresses (Horvath et al., 1993; Yamaguchi-Shinozaki and Shinozaki, 1993). Study of the promoter RD29A has lead to the characterization of a 9-bp element, TACCGACAT, referred to as dehydration-responsive element (DRE), that is also found in the promoter regions of many water deficit and cold responsive genes, such as RD17, ERD10, KIN1, COR15a, and COR6.6 (Yamaguchi-Shinozaki and Shinozaki, 1994; Wang et al., 1995; Thomashow, 1999). The DRE element contains a 5-bp core sequence of CCGAC, also known as C repeat (CRT), that plays an important role in regulating gene expression in response to low temperature, water deficit, and high salinity (Baker et al., 1994; YamaguchiShin...

To survive adverse and ever-changing environmental conditions, an organism must be able to adapt. It has long been established that the cellular reaction to stress includes the upregulation of genes coding for specific stress-responsive factors. In the present study, we demonstrate that during the early steps of the heat stress response, 25% of the Arabidopsis seedling transcriptome is targeted for rapid degradation. Our findings demonstrate that this process is catalyzed from 5' to 3' by the cytoplasmic exoribonuclease XRN4, whose function is seemingly reprogrammed by the heat-sensing pathway. The bulk of mRNAs subject to heat-dependent degradation are likely to include both the ribosome-released and polysome associated polyadenylated pools. The cotranslational decay process is facilitated at least in part by LARP1, a heat-specific cofactor of XRN4 required for its targeting to polysomes. Commensurate with their respective involvement at the molecular level, LARP1 and XRN4 are necessary for the thermotolerance of plants to long exposure to moderately high temperature, with xrn4 null mutants being almost unable to survive. These findings provide mechanistic insights regarding a massive stress-induced posttranscriptional downregulation and outline a potentially crucial pathway for plant survival and acclimation to heat stress.

The posttranslational addition of small ubiquitin-like modifier (SUMO) is an essential protein modification in plants that provides protection against numerous environmental challenges. Ligation is accomplished by a small set of SUMO ligases, with the SAP-MIZ domain-containing SIZ1 and METHYL METHANESULFONATE-SENSITIVE21 (MMS21) ligases having critical roles in stress protection and DNA endoreduplication/repair, respectively. To help identify their corresponding targets in , we used and mutants for proteomic analyses of SUMOylated proteins enriched via an engineered SUMO1 isoform suitable for mass spectrometric studies. Through multiple data sets from seedlings grown at normal temperatures or exposed to heat stress, we identified over 1000 SUMO targets, most of which are nuclear localized. Whereas no targets could be assigned to MMS21, suggesting that it modifies only a few low abundance proteins, numerous targets could be assigned to SIZ1, including major transcription factors, coactivators/repressors, and chromatin modifiers connected to abiotic and biotic stress defense, some of which associate into multisubunit regulatory complexes. SIZ1 itself is also a target, but studies with mutants protected from SUMOylation failed to uncover a regulatory role. The catalog of SIZ1 substrates indicates that SUMOylation by this ligase provides stress protection by modifying a large array of key nuclear regulators.

SummaryAbiotic stresses impact negatively on plant growth, profoundly affecting yield and quality of crops. Although much is known about plant responses, very little is understood at the molecular level about the initial sensing of environmental stress. In plants, hypoxia (low oxygen, which occurs during flooding) is directly sensed by the Cys-Arg/N-end rule pathway of ubiquitin-mediated proteolysis, through oxygen-dependent degradation of group VII Ethylene Response Factor transcription factors (ERFVIIs) via amino-terminal (Nt-) cysteine [1, 2]. Using Arabidopsis (Arabidopsis thaliana) and barley (Hordeum vulgare), we show that the pathway regulates plant responses to multiple abiotic stresses. In Arabidopsis, genetic analyses revealed that response to these stresses is controlled by N-end rule regulation of ERFVII function. Oxygen sensing via the Cys-Arg/N-end rule in higher eukaryotes is linked through a single mechanism to nitric oxide (NO) sensing [3, 4]. In plants, the major mechanism of NO synthesis is via NITRATE REDUCTASE (NR), an enzyme of nitrogen assimilation [5]. Here, we identify a negative relationship between NR activity and NO levels and stabilization of an artificial Nt-Cys substrate and ERFVII function in response to environmental changes. Furthermore, we show that ERFVIIs enhance abiotic stress responses via physical and genetic interactions with the chromatin-remodeling ATPase BRAHMA. We propose that plants sense multiple abiotic stresses through the Cys-Arg/N-end rule pathway either directly (via oxygen sensing) or indirectly (via NO sensing downstream of NR activity). This single mechanism can therefore integrate environment and response to enhance plant survival.

SUMMARYInositol hexakisphosphate (IP 6 ) provides a phosphorous reservoir in plant seeds; in addition, along with its biosynthesis intermediates and derivatives, IP 6 also plays important roles in diverse developmental and physiological processes. Disruption of the Arabidopsis inositol pentakisphosphate 2-kinase coding gene AtIPK1 was previously shown to reduce IP 6 content in vegetative tissues and affect phosphate (Pi) sensing. Here we show that AtIPK1 is required for sustaining plant growth, as null mutants are non-viable. An incomplete loss-of-function mutant, atipk1-1, exhibited disturbed Pi homeostasis and overaccumulated Pi as a consequence of increased Pi uptake activity and root-to-shoot Pi translocation. The atipk1-1 mutants also showed a Pi deficiency-like root system architecture with reduced primary root and enhanced lateral root growth. Transcriptome analysis indicated that a subset of Pi starvation-responsive genes was transcriptionally perturbed in the atipk1-1 mutants and the expression of multiple genes involved in Pi uptake, allocation, and remobilization was increased. Genetic and transcriptional analyses suggest that disturbance of Pi homeostasis caused by atipk1 mutation involved components in addition to PHR1(-like) transcription factors. Notably, the transcriptional increase of a number of Pi starvation-responsive genes in the atipk1-1 mutants is correlated with the reduction of histone variant H2A.Z occupation in chromatin. The myo-inositol-1-phosphate synthase mutants, atmips1 and atmips2 with comparable reduction in vegetative IP 6 to that in the atipk1-1 mutants did not overaccumulate Pi, suggesting that Pi homeostasis modulated by AtIPK1 is not solely attributable to IP 6 level. This study reveals that AtIPK1 has important roles in growth and Pi homeostasis.

The reprogramming of gene expression in heat stress is a key determinant to organism survival. Gene expression is downregulated through translation initiation inhibition and release of free mRNPs that are rapidly degraded or stored. In mammals, heat also triggers 5′-ribosome pausing preferentially on transcripts coding for HSC/HSP70 chaperone targets, but the impact of such phenomenon on mRNA fate remains unknown. Here, we provide evidence that, in Arabidopsis thaliana, heat provokes 5′-ribosome pausing leading to the XRN4-mediated 5′-directed decay of translating mRNAs. We also show that hindering HSC/HSP70 activity at 20°C recapitulates heat effects by inducing ribosome pausing and co-translational mRNA turnover. Strikingly, co-translational decay targets encode proteins with high HSC/HSP70 binding scores and hydrophobic N-termini, two characteristics that were previously observed for transcripts most prone to pausing in animals. This work suggests for the first time that stress-induced variation of translation elongation rate is an evolutionarily conserved process leading to the polysomal degradation of thousands of ‘non-aberrant’ mRNAs.