Differential regulation of small heat-shock genes in plants: analysis of a water-stress-inducible and developmentally activated sunflower promoter

We have isolated two sunflower genes, Ha hsp 18.6 G2 and Ha hsp 17.7 G4, that encode small heat shock proteins (sHSPs). RNAse A protection experiments, carried out with RNA probes transcribed from each gene and hybridized to sunflower total RNA, allowed us to distinguish their mRNA accumulation patterns. In sunflower, Ha hsp 17.7 G4 mRNAs accumulated during zygotic embryogenesis at 25°C. In vegetative tissues, these mRNAs accumulated in response to either heat shock (42°C), abscisic acid (ABA), or mild water stress treatments. In all cases, the mRNAs were transcribed from the same initiation site. In contrast, Ha hsp 18.6 G2 mRNAs accumulated only in response to heat-shock. This result demonstrates differential regulation of these two sHSP genes. The complex regulation depicted by the Ha hsp 17.7 G4 promoter has been further analyzed in transgenic tobacco, using G4::GUS translational fusions. Developmental induction of Ha hsp 17.7 G4 during zygotic embryogenesis was faithfully reproduced in the transgenic plants. 5-distal sequences (between -1132 and -395) were required to confer a preferential spatial expression of GUS activity in the cotyledons. More proximal sequences (from -83 to +163) conferred to the chimeric genes most of the developmental regulation, and the responses to ABA and heat shock characteristic of the Ha hsp 17.7 G4 promoter. The water stress response of this gene was not reproduced in transgenic tobacco and, thus, could be uncoupled from its regulation during embryogenesis.     

Reduction of photosynthetic apparatus plays a key role in survival of the microalga Haematococcus pluvialis (Chlorophyceae) at freezing temperatures

The microalga Haematococcus pluvialis is a biotechnologically important microorganism producing a ketocarotenoid astaxanthin. Haematococcus exists either as metabolically active vegetative cells with a high chlorophyll content or astaxanthin-rich haematocysts (aplanospores). This microalga featuring outstanding tolerance to a wide range of adverse conditions is a highly suitable model for studies of freezing tolerance in phototrophs. The retention of H. pluvialis cell viability after freezing–thawing is ascribed to elevated antioxidant enzyme activity and high ketocarotenoid content. However, we report that only haematocysts characterized by a lower photosynthetic activity were resistant to freezing–thawing even without cryoprotectant addition. The key factors of haematocyst freezing tolerance were assumed to be a low water content, rigid cell walls, reduction of the membranous structures, photosynthesis downregulation, and low chlorophyll content. Collectively, viability of Haematoccus after freezing–thawing can be improved by forcing the transition of vegetative cells to freeze-tolerant haematocysts before freezing.

     

Molecular cloning, characterization, and expression analysis of a cytosolic HSP90 gene from Haematococcus pluvialis

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone that plays key roles in the folding, maintenance of structural integrity, and regulation of a subset of cytosolic proteins. In this study, the cDNA of Haematococcus pluvialis HSP90 (designated HpHSP90) was cloned by the combination of homology cloning and rapid amplification of cDNA ends approaches. The full-length cDNA of HpHSP90 was of 2,606 bp, including an open reading frame of 2,109 bp encoding a polypeptide of 702 amino acids with predicted molecular weight of 80.14 kDa and theoretical isoelectric point of 5.07. BLAST analysis revealed that HpHSP90 shared high similarity with other known HSP90s, and the five conserved amino acid blocks defined as HSP90 protein family signatures were also identified in HpHSP90, which indicated that HpHSP90 should be a cytosolic member of the HSP90 family. Under different stress conditions, messenger RNA (mRNA) expression levels of HpHSP90 were quantified by quantitative RT-PCR. To H. pluvialis kept at different temperatures for 1 h, maximum HpHSP90 expression was observed in the range 5 to 10°C and 35 to 40°C and the expression level of HpHSP90 at 40°C was the highest (threefold compared with that at 25°C). In H. pluvialis kept at 35°C for different times, the mRNA expression level of HpHSP90 reached a maximum level after 7 h and then dropped progressively. The results indicate that HpHSP90 responded to cold and heat stresses with a temperature-dependent expression pattern as well as exposure time effect and could be used as a molecular biomarker in adverse stress environment.     

Differential proteomic analysis during the vegetative phase change and the floral transition in Malus domestica

In order to identify the proteomic changes of apple (Malus domestica Borkh.) during the vegetative phase change and the floral transition, leaf protein of juvenile, adult vegetative and reproductive phase in a seedling ('Jonathan' × 'Golden Delicious') was extracted and analyzed by 2-D electrophoresis and Matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Seventy two gel spots with significant expression differences between ontogenetic phases were obtained. Five protein spots were only detected in leaves of juvenile phase and 11 were not; 17 spots were found exclusively in adult vegetative leaves; and only one spot solely appeared in reproductive leaves while 12 did not. Twenty six of the differentially expressed proteins identified were involved in photosynthesis. Seven enzymes were related to respiration and carbohydrate metabolism. Fifteen other proteins also presented qualitative or quantitative differences among developmental phases. The spatial distribution of one differentially expressed protein, serine hydroxymethyltransferase, was confirmed by enzyme linked immunosorbent assay and immunohistochemistry. These results strongly support the idea that the vegetative phase change and the floral transition are regulated independently during developmental process.