The methionine-rich low-molecular-weight chloroplast heat-shock protein: evolutionary conservation and accumulation in relation to thermotolerance

The evolutionary conservation of the low-molecular-weight chloroplast-localized heat-shock protein (LMW chlpHsp) in vascular plants was examined using immunological methods. An antibody (Abmet) specific to the LMW chlpHsp was produced using a synthetic 28-residue peptide containing the most conserved elements of its unique "methionine-rich domain" as an antigen. This antibody detected a heat-inducible low-molecular-weight chloroplast protein in plants of six divergent Anthophyta species, including C3, C4, CAM, monocot, and dicot species. Abmet also detected a LMW chlpHsp in species from the Divisions Psilotophyta, Equisetophyta, Polypodiophyta, and Ginkgophyta. A preliminary examination of the relationship between accumulation of the LMW chlpHsp and habitat was also conducted. Seven Anthophyta species originating from both warm- and cool-temperature habitats were grown at 28C and then heat stressed at 40C. A positive qualitative relationship between the accumulation of the LMW chlpHsp and organismal thermotolerance in these species was observed; similar results were obtained separately with four nonAnthophyta species. The strong evolutionary conservation of this LMW Hsp and its localization to the chloroplast, and the correlation between production of this protein and plant thermotolerance, suggest that the LMW chlpHsp plays an important role in adaptation to heat stress.     

Heat sensitivity in a bentgrass variant. Failure to accumulate a chloroplast heat shock protein isoform implicated in heat tolerance

Two variants of creeping bentgrass (Agrostis stolonifera cv palustris), developed using tissue culture, have been used to determine the roles of chloroplast-localized small heat shock proteins (CP-sHSPs) in heat tolerance. Results from previous research indicate that the heat-tolerant variant expressed two additional CP-sHSP isoforms not expressed in the heat-sensitive variant, that accumulation of the additional CP-sHSP isoforms was genetically linked to thermotolerance, and that the presence of the additional isoforms in the heat-tolerant variant provided greater protection to photosystem II during heat stress. To determine the basis of the differential expression, we isolated the genes encoding the CP-sHSPs from both variants and characterized their structure and expression. Two genes, ApHsp26.2 and ApHsp26.7a, were isolated from the heat-tolerant variant, and three genes, ApHsp26.2m, ApHsp26.8, and ApHsp26.7b, were isolated from the heat-sensitive variant. The sequence of ApHsp26.2m from the heat-sensitive variant was identical to ApHsp26.2, except for a point mutation that generated a premature stop codon. Therefore, the protein product of ApHsp26.2m did not accumulate in the heat-sensitive line. Mass spectrometry analysis confirmed that ApHsp26.2 encoded for the CP-sHSP isoforms unique to the heat-tolerant variant. An identical mutation was detected in one of the three parental lines used to develop the creeping bentgrass variants. This suggests that ApHsp26.2m was inherited from this parent and did not arise from a mutation that occurred during tissue culture. The presence of two isoforms encoded by the same gene might be due to differential processing of the N-terminal amino acids during or after import into the chloroplast.     

Effects of temperature stress on bean-nodulating Rhizobium strains

High soil temperatures in tropical areas limit nodulation and dinitrogen fixation by strains of Rhizobium. Several heat-tolerant bean-nodulating Rhizobium strains have been isolated previously. However, the basis of their resistance to heat remains unknown. In this study, we compared the effects of heat on symbiotic nitrogen fixation, cell survival, amino acid uptake, and protein synthesis in a heat-tolerant (CIAT899) and a heat-sensitive (CNPAF512) bean-nodulating Rhizobium strain. Acetylene reduction activity of nodulated roots excised from unstressed plants was strongly diminished at 35 or 40 degrees C when plants were nodulated either by CIAT899 or by CNPAF512. When these strains were tested under free-living conditions, survival at 40 degrees C as well as the kinetics of l-[S]methionine uptake and protein synthesis at 35 and 40 degrees C indicated the higher tolerance of CIAT899 than of CNPAF512 to thermal stress. The synthesis of heat shock proteins was detected in both strains, although at different temperatures. Increased synthesis of 14 heat shock proteins in CNPAF512 and of 6 heat shock proteins in CIAT899 was observed at 40 and 45 degrees C, respectively. A heat shock protein of approximately 21 kDa, of which the synthesis was strongest in both Rhizobium strains upon a temperature shift up, was also conserved in several other bean-nodulating rhizobia. Acquired thermotolerance in CIAT899 was shown to depend on protein synthesis.     

The Distribution of Stomata and Photosynthetic Pathway in Leaves

The stoma freguency and distribution on both epidermis of leaves of 65 species of C3 plants, 49 species of C4 plants and 16 species of CAM plants were studied by impres- sion method with colorless nail oil. The results indicated that distribution of stoma on both epidermis of leaves was in relation to the photosynthetic pathway and living type. The stoma ratio of upper/lower of C3 plants was 0.45 (sun herb) and 0.07 (shade herb), no stoma was found on upper epidermis of leaves of ferns, shrubs and trees. Plants with C4 photosynthetic pathway, except a few species, showed a higher stoma ratio of upper/lower 0.64 (Cyperaceae), 0.82 (dicot) and 0.94 (Oramineae). Much less stoma frequency was found in CAM plants than in C3 and C4 plant, but the stoma ratio of upper/lower was equal. In C3 crop plants, the species with shorter growth period and higher yield, such as sunflower, peanut and some leafy vegetables had higher stoma ratio of upper/lower. It is supposed that the stoma ratio of upper/lower might be used as an helpful index for identifing the C4 plants in monocot grass. It was also considered that the photosynthetic rate of amphistomatous leaves of C3 plants with same living type was related to the stoma ratio of upper/lower.     

High correlation between thermotolerance and photosystem II activity in tall fescue

Heat stress affects a broad spectrum of cellular components and metabolism. The objectives of this study were to investigate the behavior of Photosystem II (PSII) in tall fescue (Festuca arundinacea Schreb) with various thermotolerance capacities and to broaden our comprehension about the relationship between thermotolerance and PSII function. Heat-tolerant and heat-sensitive accessions were incubated at 24 °C (control) and 46 °C (heat stress) for 5 h. The fluorescence transient curves (OJIP curves), slow Chl fluorescence kinetic, and light response curve were employed to study the behavior of PSII subjected to heat stress. After heat stress, performance index for energy conservation from photons absorbed by PSII antenna until the reduction of PSI acceptors (PI_Total), the value of electrons produced per photon (a), and the maximal rate of electron transport (ETR_max) of heat-tolerant accessions were lower than those of heat-sensitive accessions. Relatively lower reactive oxygen species (ROS) contents were detected in heat-tolerant accessions. Simultaneously, there was a significant decline in the quantum yield of photochemical energy conversion in PS II (Y(II)), probability that a PSII Chl molecule functions as reaction center (γ_RC), and the increase of quantum yield for non-regulated non-photochemical energy loss (Y(NO)) in heat-tolerant accessions. Moreover, a significant inverse correlation between heat tolerance indexes (HTI) and Y(II) was observed. Therefore, maintaining a lower photochemical activity in heat-tolerant accessions could be a crucial strategy to improve their thermotolerance. This finding could be attributed to the structural difference in the reaction center, and for heat-tolerant accessions, it could simultaneously limit energy input into linear electron transport, and dissipate more energy through non-regulated non-photochemical energy loss processes.     

The Presence of GSI-Like Genes in Higher Plants: Support for the Paralogous Evolution of GSI and GSII Genes

Glutamine synthetase type I (GSI) genes have previously been described only in prokaryotes except that the fungus Emericella nidulans contains a gene (fluG) which encodes a protein with a large N-terminal domain linked to a C-terminal GSI-like domain. Eukaryotes generally contain the type II (GSII) genes which have been shown to occur also in some prokaryotes. The question of whether GSI and GSII genes are orthologues or paralogues remains a point of controversy. In this article we show that GSI-like genes are widespread in higher plants and have characterized one of the genes from the legume Medicago truncatula. This gene is part of a small gene family and is expressed in many organs of the plant. It encodes a protein similar in size and with between 36 and 46% amino acid sequence similarity to prokaryotic GS proteins used in the analyses, whereas it is larger and with less than 25% similarity to GSII proteins, including those from the same plant species. Phylogenetic analyses suggest that this protein is most similar to putative proteins encoded by expressed sequence tags of other higher plant species (including dicots and a monocot) and forms a cluster with FluG as the most divergent of the GSI sequences. The discovery of GSI-like genes in higher plants supports the paralogous evolution of GSI and GSII genes, which has implications for the use of GS in molecular studies on evolution. Received: 4 May 1999 / Accepted: 17 September 1999     

Interactive effects of high temperature and elevated carbon dioxide concentration on cowpea [Vigna unguiculata (L.) Walp.]

Limitations in carbohydrate supplies have been implicated as a factor responsible for reproductive failure under heat stress. Heat stress affects two stages of reproductive development in cowpea [Vigna unguiculata (L.) Walp.], and genotypes are available with tolerance and sensitivity to heat during these different stages. The objectives of this study were to determine the responses of these cowpea lines to ambient and elevated [CO₂], under heat stress and optimal temperature, and test whether differences in carbohydrate supplies due to genotypes, CO₂ enrichment and heat stress are associated with differences in sensitivity to heat during reproductive development. Plants were grown in reach-in growth chambers and subjected to day/night temperatures of either 33/20 or 33/30°C, and [CO₂] levels of either 350 or 700 μmol mol^-1. Under intermediate night temperature (33/20°C), all lines set substantial numbers of pods. Under high night temperature (33/30°C) with either ambient or elevated [CO₂], one heat-sensitive line produced no flowers and the other set no pods, whereas the heat-tolerant line abundantly set pods. High night temperature reduced the overall carbohydrate content of the plants, especially peduncle sugars, and caused decreases in photosynthetic rates. The high pod set of the heat-tolerant line, under high night temperature, was associated with higher levels of sugars in peduncles compared with the heat-sensitive lines. The heat-tolerant line accumulated substantial shoot biomass, exhibited less accumulation of starch in leaves, and possibly had less down-regulation of photosynthesis in response to CO₂ enrichment and heat stress than the heat-sensitive lines. Elevated [CO₂] resulted in higher overall carbohydrate levels in heat-sensitive lines (starch in leaves, stems and peduncles), but it did not increase their heat tolerance with respect to flower production or pod set. Heat-induced damage to floral buds and anthers in the sensitive lines was associated with low sugars levels in peduncles, indicating that heat had greater effects on assimilate demand than on leaf assimilate supply. The heat-tolerant line was the most responsive genotype to elevated [CO₂] with respect to pod production under either high or intermediate temperatures.     

利用全测序基因组数据对被子植物纤维素合成酶基因的进化分析

利用已经分离的植物纤维素合成酶基因的Cellulose_synt结构域为检索序列,从NCBI和其他数据库中调取已完成测序的物种的纤维素合成酶的氨基酸序列,共涉及10个物种的171个基因,基于以上氨基酸序列,应用MEGA4.0生成系统进化树。结果表明:CesA基因和Csl基因直向的相似度远大于平行的相似度,且它们的分化可能在单子叶和真双子叶植物分化之前,单子叶和真双子叶植物的最近共同祖先中至少存在7个CesA基因,综合已知的模式植物CesA基因的功能(初生壁或次生壁形成特异性),可为推测其他物种中该基因的功能提供帮助。     

The Relationship Between Yield and the Antioxidant Defense System in Tomatoes Grown Under Heat Stress

Four putative heat-tolerant tomato (Lycopersicum esculentum) cultivars (Tamasabro, Heat Wave, LHT-24, and Solar Set) and one putative heat-sensitive tomato culti-var (Floradade) were grown in the field under non-stress (average daily temperature of 26°C) and heat-stress (average daily temperature of 34°C) conditions. At anthesis, approximately five weeks after being transplanted to the field, leaf samples were collected for antioxidant analyses. Yield was determined by harvesting ripe fruit seven weeks after the collection of leaf samples. Heat stress resulted in a 79.1% decrease in yield for the heat-sensitive Floradade, while the fruit yield in the heat-tolerant cultivars Heat Wave, LHT-24, Solar Set, and Tamasabro was reduced 51.5%, 22.1%, 43.8%, and 34.8% respectively. When grown under heat stress, antioxidant activities were also greater in the heat-tolerant cultivars. Superoxide dismutase (SOD) activity increased up to 9-fold in the heat-tolerant cultivars but decreased 83.1% in the heat-sensitive Floradade. Catalase, peroxidase, and ascorbate peroxidase activity increased significantly in all cultivars. Only Heat Wave showed a significant increase in glutathione reductase in response to heat stress but all heat-tolerant cultivars exhibited significantly lower oxidized ascorbate/reduced ascorbate ratios, greater reduced glutathione/oxidized glutathione ratios, and greater a-tocopherol concentrations compared to the heat-sensitive cultivar Floridade. These data indicate that the more heat-tolerant cultivars had an enhanced capacity for scavenging active oxygen species and a more active ascorbate-glutathione cycle and suggest a strong correlation between the ability to up-regulate the antioxidant defense system and the ability of tomatoes to produce greater yields when grown under heat stress.     

Evolutionary physiology: the extent of C4 and CAM photosynthesis in the genera Anacampseros and Grahamia of the Portulacaceae

The Portulacaceae is one of the few terrestrial plant families known to have both C(4) and Crassulacean acid metabolism (CAM) species. There may be multiple origins of the evolution of CAM within the Portulacaceae but the only clear evidence of C(4) photosynthesis is found in members of the genus Portulaca. In the Portulaca, CAM succulent tissue is overlaid with the C(4) tissue in a unique fashion where both pathways are operating simultaneously. Earlier reports have shown that the clade containing the genera Anacampseros and Grahamia may also contain C(4) photosynthetic species similar to the Portulaca, which would indicate multiple origins of C(4) photosynthesis within the family. The aim of the present study was to ascertain the true photosynthetic nature of these genera. An initial survey of the carbon isotope composition of the Anacampseros ranged from -12.6 per thousand to -24.0 per thousand, indicating very little CAM activity in some species, with other values close to the C(4) range. Anacampseros (=Grahamia) australiana which had been previously identified as a C(4) species had a carbon isotope composition value of -24.0 per thousand, which is more indicative of a C(3) species with a slight contribution of CAM activity. Other Anacampseros species with C(4)-like values have been shown to be CAM plants. The initial isotope analysis of the Grahamia species gave values in the range of -27.1 per thousand to -23.6 per thousand, placing the Grahamia species well towards the C(3) photosynthetic range. Further physiological studies indicated increased night-time CO(2) uptake with imposition of water stress, associated with a large diurnal acid fluctuation and a marked increased phosphoenolpyruvate carboxylase activity. This showed that the Grahamia species are actually facultative CAM plants despite their C(3)-like carbon isotope values. The results indicate that the Grahamia and Anacampseros species do not utilize the C(4) photosynthetic pathway. This is the first to identify that the Grahamia species are facultative CAM plants where CAM can be induced by water stress. This work supports earlier physiological work that indicates that this clade containing Anacampseros and Grahamia species comprises predominantly facultative CAM plants. This report suggests there may be only one clade which contains C(4) photosynthetic members with CAM-like characteristics.