Characterization of the Arabidopsis thermosensitive mutant atts02 reveals an important role for galactolipids in thermotolerance

Plants are constantly challenged with various abiotic stresses in their natural environment. Elevated temperatures have a detrimental impact on overall plant growth and productivity. Many plants increase their tolerance to high temperatures through an adaptation response known as acquired thermotolerance. To identify the various mechanisms that plants have evolved to cope with high temperature stress, we have isolated a series of Arabidopsis mutants that are defective in the acquisition of thermotolerance after an exposure to 38 degrees C, a treatment that induces acquired thermotolerance in wild-type plants. One of these mutants, atts02, was not only defective in acquiring thermotolerance after the treatment, but also displayed a reduced level of basal thermotolerance in a 30 degrees C growth assay. The affected gene in atts02 was identified by positional cloning and encodes digalactosyldiacylglycerol synthase 1 (DGD1) (the atts02 mutant was, at that point, renamed dgd1-2). An additional dgd1 allele, dgd1-3, was identified in two other mutant lines displaying altered acquired thermotolerance, atts100 and atts104. Expression patterns of several heat shock proteins (HSPs) in heat-treated dgd1-2 homozygous plants were similar to those from identically treated wild-type plants, suggesting that the thermosensitivity in the dgd1-2 mutant was not caused by a defect in HSP induction. Lipid analysis of wild-type and mutant plants indicated a close correlation between the ability to acquire thermotolerance and the increases in digalactosyldiacylglycerol (DGDG) level and in the ratio of DGDG to monogalactosyldiacylglycerol (MGDG). Thermosensitivity in dgd1-2 and dgd1-3 was associated with (1) a decreased DGDG level and (2) an inability to increase the ratio of DGDG to MGDG upon exposure to a 38 degrees C sublethal temperature treatment. Our results suggest that the DGDG level and/or the ratio of DGDG to MGDG may play an important role in basal as well as acquired thermotolerance in Arabidopsis.     

Enhanced Thermostability of Arabidopsis Rubisco activase improves photosynthesis and growth rates under moderate heat stress

Plant photosynthesis declines when the temperature exceeds its optimum range. Recent evidence indicates that the reduction in photosynthesis is linked to ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) deactivation due to the inhibition of Rubisco activase (RCA) under moderately elevated temperatures. To test the hypothesis that thermostable RCA can improve photosynthesis under elevated temperatures, we used gene shuffling technology to generate several Arabidopsis thaliana RCA1 (short isoform) variants exhibiting improved thermostability. Wild-type RCA1 and selected thermostable RCA1 variants were introduced into an Arabidopsis RCA deletion (Deltarca) line. In a long-term growth test at either constant 26 degrees C or daily 4-h 30 degrees C exposure, the transgenic lines with the thermostable RCA1 variants exhibited higher photosynthetic rates, improved development patterns, higher biomass, and increased seed yields compared with the lines expressing wild-type RCA1 and a slight improvement compared with untransformed Arabidopsis plants. These results provide clear evidence that RCA is a major limiting factor in plant photosynthesis under moderately elevated temperatures and a potential target for genetic manipulation to improve crop plants productivity under heat stress conditions.     

The thylakoid lumen protease Deg1 is involved in the repair of photosystem II from photoinhibition in Arabidopsis

Deg1 is a Ser protease peripherally attached to the lumenal side of the thylakoid membrane. Its physiological function is unknown, but its localization makes it a suitable candidate for participation in photoinhibition repair by degradation of the photosystem II reaction center protein D1. We transformed Arabidopsis thaliana with an RNA interference construct and obtained plants with reduced levels of Deg1. These plants were smaller than wild-type plants, flowered earlier, were more sensitive to photoinhibition, and accumulated more of the D1 protein, probably in an inactive form. Two C-terminal degradation products of the D1 protein, of 16 and 5.2 kD, accumulated at lower levels compared with the wild type. Moreover, addition of recombinant Deg1 to inside-out thylakoid membranes isolated from the mutant could induce the formation of the 5.2-kD D1 C-terminal fragment, whereas the unrelated proteases trypsin and thermolysin could not. Immunoblot analysis revealed that mutants containing less Deg1 also contain less FtsH protease, and FtsH mutants contain less Deg1. These results suggest that Deg1 cooperates with the stroma-exposed proteases FtsH and Deg2 in degrading D1 protein during repair from photoinhibition by cleaving lumen-exposed regions of the protein. In addition, they suggest that accumulation of Deg1 and FtsH proteases may be coordinated.     

Isolation of Arabidopsis mutants lacking components of acquired thermotolerance

Acquired thermotolerance is a complex physiological phenomenon that enables plants to survive normally lethal temperatures. This study characterizes the temperature sensitivity of Arabidopsis using a chlorophyll accumulation bioassay, describes a procedure for selection of acquired thermotolerance mutants, and provides the physiological characterization of one mutant (AtTS02) isolated by this procedure. Exposure of etiolated Arabidopsis seedlings to 48 degrees C or 50 degrees C for 30 min blocks subsequent chlorophyll accumulation and is eventually lethal. Arabidopsis seedlings can be protected against the effects of a 50 degrees C, 30-min challenge by a 4-h pre-incubation at 38 degrees C. By the use of the milder challenge, 44 degrees C for 30 min, and protective pretreatment, mutants lacking components of the acquired thermotolerance system were isolated. Putative mutants isolated by this procedure exhibited chlorophyll accumulation levels (our measure of acquired thermotolerance) ranging from 10% to 98% of control seedling levels following pre-incubation at 38 degrees C and challenge at 50 degrees C. The induction temperatures for maximum acquired thermotolerance prior to a high temperature challenge were the same in AtTS02 and RLD seedlings, although the absolute level of chlorophyll accumulation was reduced in the mutant. Genetic analysis showed that the loss of acquired thermotolerance in AtTS02 was a recessive trait. The pattern of proteins synthesized at 25 degrees C and 38 degrees C in the RLD and AtTS02 revealed the reduction in the level of a 27-kD heat shock protein in AtTS02. Genetic analysis showed that the reduction of this protein level was correlated with the acquired thermotolerance phenotype.     

FtsH proteases in chloroplasts and cyanobacteria

FtsH is a membrane-bound ATP-dependent metalloprotease complex found in prokaryotes and organelles of eukaryotic cells. It consists of one or two trans -membrane helices at its amino-terminus, a highly conserved ATPase domain, which relates it to the AAA protein family, and a zinc-binding domain towards its carboxy-terminus that serves as the proteolytic site. Most bacteria contain a single FtsH gene, but the cyanobacterium Synechocystis has four. The Arabidopsis thaliana genome contains 12 genes encoding FtsH proteins, nine of them can be targeted to chloroplasts, whereas the other three are mitochondrial. Chloroplast FtsH protease is located in the thylakoid membrane, where it forms complexes, most likely hexamers, whose ATPase and proteolytic domains are exposed to the stroma. It is involved in the degradation of the D1 protein of photosystem II reaction centre during its repair from photoinhibition, as well as in the degradation of unassembled proteins in the thylakoid and the stroma. In Arabidopsis , FtsH2 is the most abundant isomer, followed by FtsH5, 8 and 1. This hierarchy is well reflected in the severity of the variegated phenotype of mutants in these genes.     

ATP-dependent FtsH and Lon chloroplast proteases

Arabidopsis thaliana proteome contains 667 proteases; some tens of them are chloroplast-targeted proteins, encoded by genes orthologous to the ones coding for bacterial proteolytic enzymes. It is thought that chloroplast proteases are involved in chloroplasts' proteins turnover and quality control (maturation of nucleus-encoded proteins and removal of nonfunctional ones). Some ATP-dependent chloroplast proteases belonging to FtsH family (especially FtsH2 and FtsH5) are considered to be involved in numerous aspects of chloroplast and whole plant maintenance under non-stressing as well as stressing conditions. This notion is supported by severe phenotype appearance of mutants deficient in these proteases. In contrast to seemingly high physiological importance of chloroplast members of FtsH protease family, only a few individual proteins have been identified so far as their physiological targets (i.e. Lhcb1, Lhcb3, PsbA and Rieske protein). Our knowledge regarding structure and molecular mechanisms of these enzymes' action is limited when compared with what is known about FtsHs of bacterial origin. Equally limited is the knowledge about ATP-dependent Lon4 protease being the single known chloroplast-targeted ortholog of Lon protease of Escherichia coli.     

Identification of genetic diversity and mutations in higher plant acquired thermotolerance

Plants experience high air and soil temperatures during periods of drought and when fields receive limited irrigation. Elevated plant temperatures that occur under these conditions negatively impact plant health and productivity. Plants, like all organisms, respond to an elevation in temperature by the synthesis of heat shock proteins (HSP). The appearance of plant HSP is strongly correlated to the development of a condition termed 'acquired thermotolerance'. Acquired thermotolerance is induced by pre-exposure to elevated but non-lethal temperatures and leads to enhanced protection of plant cells from subsequent heat induced injury. Although the correlation between the development of acquired thermotolerance and the appearance of HSP is strong, a cause-and-effect relationship between the two has been difficult to demonstrate. To understand the relationship between HSP and acquired thermotolerance, mutations would be required that result in a coordinate change in the expressions of HSP. This paper describes research efforts leading to the development of a screening procedure for the isolation and characterization of acquired thermotolerance mutants. This method for identifying mutants is based on the inhibition of chlorophyll accumulation in etiolated tissue following challenges at lethal temperatures and the prevention of this inhibition by pre-incubation at a non-lethal elevated temperature; i.e. acquired thermotolerance. Arabidopsis thaliana mutants deficient in varying levels of acquired thermotolerance have been identified from both the RLD and Columbia ecotypes and these mutants are currently undergoing a detailed characterization at both the protein and molecular levels.     

Genetic analysis reveals domain interactions of Arabidopsis Hsp100/ClpB and cooperation with the small heat shock protein chaperone system

We have defined amino acids important for function of the Arabidopsis thaliana Hsp100/ClpB chaperone (AtHsp101) in acquired thermotolerance by isolating recessive, loss-of-function mutations and a novel semidominant, gain-of-function allele [hot1-4 (A499T)]. The hot1-4 allele is unusual in that it not only fails to develop thermotolerance to 45 degrees C after acclimation at 38 degrees C, but also is sensitive to 38 degrees C, which is a permissive temperature for wild-type and loss-of-function mutants. hot1-4 lies between nucleotide binding domain 1 (NBD1) and NBD2 in a coiled-coil domain that is characteristic of the Hsp100/ClpB proteins. We then isolated two classes of intragenic suppressor mutations of hot1-4: loss-of-function mutations (Class 1) that eliminated the 38 degrees C sensitivity, but did not restore thermotolerance function to hot1-4, and Class 2 suppressors that restored acquired thermotolerance function to hot1-4. Location of the hot1-4 Class 2 suppressors supports a functional link between the coiled-coil domain and both NBD1 and the axial channel of the Hsp100/ClpB hexamer. In addition, the strongest Class 2 suppressors restored solubility of aggregated small heat shock proteins (sHsps) after heat stress, revealing genetic interaction of the Hsp100/ClpB and sHsp chaperone systems. These results also demonstrate that quantitative phenotypes can be used for in vivo genetic dissection of protein mechanism in Arabidopsis.     

Trehalose synthesis is important for the acquisition of thermotolerance in Schizosaccharomyces pombe

Yeast cells show an adaptive response to a mild heat shock, resulting in thermotolerance acquisition. This is accompanied by induction of heat-shock protein (hsp) synthesis and rapid accumulation of trehalose. Genetic approaches to determine the specific role of trehalose in heat-induced thermotolerance in Saccharomyces cerevisiae have been hampered by the finding that deletion of TPS1 , the gene encoding trehalose-6-phosphate synthase, causes a variety of pleiotropic effects, including inability to grow on glucose-containing media. Here, we have studied a tps1 mutant of the yeast Schizosaccharomyces pombe that reportedly has no such growth defects. We show that tps1 mutants have a serious defect in heat shock-induced acquisition of thermotolerance if conditioned at highly elevated temperatures (40–42.5°C), which, in wild-type cells, prevent hsp but not trehalose synthesis. In contrast, hsp synthesis appears to become particularly important under conditions in which trehalose synthesis is either absent (in tps1 mutant strains) or not fully induced (conditioning at moderately elevated temperatures, i.e. 35°C). In addition, pka1 mutants deficient in cAMP-dependent protein kinase were examined. Unconditioned pka1 cells had low levels of trehalose but a high basal level of thermotolerance. It was found that pka1 mutant cells, contrary to wild-type cells, accumulated large amounts of trehalose, even during a 50°C treatment. pka1 tps1 double mutants lacked this ability and showed reduced intrinsic thermotolerance, indicating a particularly important role for trehalose synthesis, which takes place during the challenging heat shock.     

Arabidopsis mutants reveal that short- and long-term thermotolerance have different requirements for trienoic fatty acids

The photosynthetic thylakoid has the highest level of lipid unsaturation of any membrane. In Arabidopsis thaliana plants grown at 22°C, approximately 70% of the thylakoid fatty acids are trienoic - they have three double bonds. In Arabidopsis, and other species, the levels of trienoic fatty acids decline substantially at higher temperatures. Several genetic studies indicate that reduced unsaturation improves photosynthetic function and plant survival at high temperatures. Here, these studies are extended using the Arabidopsis triple mutant, fad3-2 fad7-2 fad8 that contains no detectable trienoic fatty acids. In the short-term, fluorescence analyses and electron-transport assays indicated that photosynthetic functions in this mutant are more thermotolerant than the wild type. However, long-term photosynthesis, growth, and survival of plants were all compromised in the triple mutant at high temperature. The fad3-2 fad7-2 fad8 mutant is deficient in jasmonate synthesis and this hormone has been shown to mediate some aspects of thermotolerance; however, additional experiments demonstrated that a lack of jasmonate was not a major factor in the death of triple-mutant plants at high temperature. The results indicate that long-term thermotolerance requires a basal level of trienoic fatty acids. Thus, the success of genetic and molecular approaches to increase thermotolerance by reducing membrane unsaturation will be limited by countervailing effects that compromise essential plant functions at elevated temperatures.     

Modulation of nitrosative stress by S-nitrosoglutathione reductase is critical for thermotolerance and plant growth in Arabidopsis

Nitric oxide (NO) is a key signaling molecule in plants. This analysis of Arabidopsis thaliana HOT5 (sensitive to hot temperatures), which is required for thermotolerance, uncovers a role of NO in thermotolerance and plant development. HOT5 encodes S-nitrosoglutathione reductase (GSNOR), which metabolizes the NO adduct S-nitrosoglutathione. Two hot5 missense alleles and two T-DNA insertion, protein null alleles were characterized. The missense alleles cannot acclimate to heat as dark-grown seedlings but grow normally and can heat-acclimate in the light. The null alleles cannot heat-acclimate as light-grown plants and have other phenotypes, including failure to grow on nutrient plates, increased reproductive shoots, and reduced fertility. The fertility defect of hot5 is due to both reduced stamen elongation and male and female fertilization defects. The hot5 null alleles show increased nitrate and nitroso species levels, and the heat sensitivity of both missense and null alleles is associated with increased NO species. Heat sensitivity is enhanced in wild-type and mutant plants by NO donors, and the heat sensitivity of hot5 mutants can be rescued by an NO scavenger. An NO-overproducing mutant is also defective in thermotolerance. Together, our results expand the importance of GSNOR-regulated NO homeostasis to abiotic stress and plant development.     

Attachment to roots and virulence of a chvB mutant of Agrobacterium tumefaciens are temperature sensitive

Agrobacterium tumefaciens chvB mutants are unable to produce beta-1,2 glucan. They are nonattaching and avirulent and show reduced motility at room temperature. At lower temperatures (16 degrees C), chvB mutants became virulent on Bryophyllum daigremontiana and Lycopersicon esculentum and were able to attach to L. esculentum, Arabidopsis thaliana, Daucus carota, and Tagetes erecta roots. The mutant bacteria also recovered wild-type motility at lower temperatures. Two other nonattaching mutants of A. tumefaciens, AttR and AtrA, were unaffected by the lowered temperature, remaining nonattaching and avirulent.     

Nuclear-mitochondrial cross-talk during heat shock in Arabidopsis cell culture

Apart from energy generation, mitochondria perform a signalling function determining the life and death of a cell under stress exposure. In the present study we have explored patterns of heat-induced synthesis of Hsp101, Hsp70, Hsp17.6 (class I), Hsp17.6 (class II) and Hsp60, and the development of induced thermotolerance in Arabidopsis thaliana cell culture under conditions of mitochondrial dysfunction. It was shown that treatment by mitochondrial inhibitors and uncouplers at the time of mild heat shock downregulates HSP synthesis, which is important for induced thermotolerance in plants. The exposure to elevated temperature induced an increase in cell oxygen consumption and hyperpolarization of the inner mitochondrial membrane. Taken together, these facts suggest that mitochondrial functions are essential for heat-induced HSP synthesis and development of induced thermotolerance in A. thaliana cell culture, suggesting that mitochondrial-nuclear cross-talk is activated under stress conditions. Treatment of Arabidopsis cell culture at 50 degrees C initiates a programmed cell death determined by the time course of viability decrease, DNA fragmentation and cytochrome c release from mitochondria. As treatment at 37 degrees C protected Arabidopsis cells from heat-induced cell death, it may be suggested that Hsp101, Hsp70 and small heat-shock proteins, the synthesis of which is induced under these conditions, are playing an anti-apoptotic role in the plant cell. On the other hand, drastic heat shock upregulated mitochondrial Hsp60 synthesis and induced its release from mitochondria to the cytosol, indicating a pro-apoptotic role of plant Hsp60.