Cloning of new members of heat shock protein HSP101 gene family in wheat (Triticum aestivum (L.) Moench) inducible by heat, dehydration, and ABA(1).

We have cloned two cDNAs, TaHSP101B and TaHSP101C, encoding two heat stress-inducible members of HSP101/ClpB family in bread wheat (Triticum aestivum (L.) Moench.). Proteins encoded by these cDNAs are highly similar at the primary sequence level and diverged from the previously reported TaHSP101 (designated TaHSP101A) both in the consensus ATP/GTP-binding region II and in the carboxy terminal region. The HSP101 gene was determined to be a single copy gene or a member of a small gene family in hexaploid wheat. Messages encoding HSP101 proteins were inducible by heat stress treatments in both wheat leaves and roots. Accumulation of the TaHSP101C mRNA was less abundant than that of TaHSP101B mRNA. We are showing for the first time that in addition to heat stress, expression of HSP101 mRNAs in wheat leaves was induced by a 2-h dehydration and a treatment with 5x10(-5)M ABA, but not affected by chilling or wounding, indicating that HSP101 proteins may be involved in both heat and drought responses in wheat.

Characterization of a maize heat-shock protein 101 gene, HSP101, encoding a ClpB/Hsp100 protein homologue.

Heat shock protein 101 (HSP101) cDNA and genomic clones from maize have been isolated. The structure of maize HSP101 reveals the presence of six exons interrupted by five introns. Maize HSP101 contains a predicted open reading frame that translates into a 912-aa sequence with a mass of 101kDa. Initiation of transcription was mapped 146 bases upstream of the AUG codon. Five heat shock element (HSE) boxes were found within the proximal 289 bases of the promoter region. Southern blot analysis of genomic DNA indicates that the maize genome contains only one copy of HSP101. A protein sequence comparison showed that maize Hsp101 belongs to the heat shock 100kDa and caseino-lytic protease B protein family (Hsp100/ClpB) that plays important roles in bacteria and yeast in the survival to extremely high temperatures and the control of proteolysis. Accumulation of HSP101 mRNA was strong under heat shock conditions, but not detectable after cold or osmotic stress treatments or by exogenous application of ABA. The analysis of the predicted supersecondary structure of maize Hsp101 showed that a coiled-coil located in the middle region of the protein is evolutionarily conserved in all members of the Clp A, B and C subfamilies. It is proposed that these supersecondary structures may have important roles in Clp function.

Cloning and characterization of cDNAs encoding oat PF1: a protein that binds to the PE1 region in the oat phytochrome A3 gene promoter.

In monocotyledons, the expression of the oat phytochrome A gene (PHYA) is down-regulated by phytochrome itself. This autoregulatory repression is the most rapid light-induced effect on gene expression reported in plants to date. A functional analysis of the oat PHYA3 gene minimal promoter in a rice transient expression assay has identified two promoter elements, PE1 and PE3, that interact synergistically in positive regulation. We have isolated an oat cDNA clone (pO2) that encodes a DNA-binding protein that binds to the PE1 region of the oat PHYA3 gene promoter. The in vitro binding properties of the pO2-encoded protein, towards DNA probes containing either the PE1 sequence or linker-substitution mutations in PE1, correlate with the activity of these DNA elements in the rice transient expression assay. These mutations are known to abolish expression of a reporter gene in vivo. Binding of these linker-substitution mutants to the pO2-encoded protein in vitro was lower by one to two orders of magnitude than the binding of the native PE1 region. We suggest, therefore, that the pO2 clone may encode the putative nuclear factor, oat PF1, that is involved in positive regulation of PHYA3 by binding to PE1 in vivo. pO2 encodes a 170-amino-acid-long protein that contains three repeats of the 'AT-hook' DNA-binding motif found in high mobility group I-Y (HMGI-Y) proteins. Oat PF1 is highly similar to rice PF1 and to the protein encoded by soybean cDNA SB16. They all have a strong similarity in their N-terminus to the pea H1 histone, and the presence of several AT-hook DNA-binding motifs in their C-terminal halves.

Absence of heat shock protein synthesis in isolated mitochondria and plastids from maize.

Examination of the proteins synthesized by isolated mitochondria, chloroplasts, or proplastids from maize tissues showed that a heat treatment at 40 degrees C does not induce or enhance the synthesis of any protein when compared to preparations treated at the control temperature of 28 degrees C. These observations are consistent with the results obtained by labeling proteins in vivo under sterile conditions. In vivo labeling in the presence of cycloheximide during heat shock showed no heat shock protein synthesis. Labeling in the presence of chloramphenicol during heat shock showed a similar heat shock protein pattern as in the absence of the inhibitor. It is concluded that maize organelles do not synthesize heat shock proteins and that, if present, they may be due to bacterial contamination.

Effect of heat shock on the metabolism of glutathione in maize roots.

High performance liquid chromatography analyses revealed that glutathione (GSH) and cysteine are two of the major low molecular weight thiol compounds in maize root extracts. Treatment of maize roots to heat shock temperatures of 40 degrees C resulted in a decrease of cysteine levels and an increase of GSH levels. Pulse labeling of maize roots with [(35)S]cysteine showed that the rate of incorporation of (35)S into GSH or glutathione disulfide (GSSG) in heat shocked tissues was twice that in nonheat shocked tissues. In addition, extracts from heat shocked maize, barley, and soybean tissues contained an unidentified low molecular weight compound that increased from 1.2- to 8-fold within 2 hours of heat shock treatment depending on the tissue and plant involved. Our results indicate that during heat shock there is an increase in the activity of the GSH synthetizing capacity in maize root cells. The elevated synthesis of GSH may be related to the cells capacity to cope with heat stress conditions.

PF1: an A-T hook-containing DNA binding protein from rice that interacts with a functionally defined d(AT)-rich element in the oat phytochrome A3 gene promoter.

Phytochrome-imposed down-regulation of the expression of its own phytochrome A gene (PHYA) is one of the fastest light-induced effects on transcription reported in plants to date. Functional analysis of the oat PHYA3 promoter in a transfection assay has revealed two positive elements, PE1 and PE3, that function synergistically to support high levels of transcription in the absence of light. We have isolated a rice cDNA clone (pR4) encoding a DNA binding protein that binds to the AT-rich PE1 element. We tested the selectivity of the pR4-encoded DNA binding activity using linker substitution mutations of PE1 that are known to disrupt positive expression supported by the PHYA3 promoter in vivo. Binding to these linker substitution mutants was one to two orders of magnitude less than to the native PE1 element. Because this is the behavior expected of positive factor 1 (PF1), the presumptive nuclear transcription factor that acts in trans at the PE1 element in vivo, the data support the conclusion that the protein encoded by pR4 is in fact rice PF1. The PF1 polypeptide encoded by pR4 is 213 amino acids long and contains four repeats of the A-T hook DNA binding motif found in high-mobility group I-Y (HMGI-Y) proteins. In addition, PF1 contains an 11-amino acid-long hydrophobic region characteristic of HMG I proteins, its N-terminal region shows strong similarities to a pea H1 histone sequence and a short peptide sequence from wheat HMGa, and it shows a high degree of similarity along its entire length to the HMG Y-like protein encoded by a soybean cDNA, SB16. In vitro footprinting and quantitative gel shift analyses showed that PF1 binds preferentially to the PE1 element but also at lower affinity to two other AT-rich regions upstream of PE1. This feature is consistent with the binding characteristics of HMG I-Y proteins that are known to bind to most runs of six or more AT base pairs. Taken together, the properties of PF1 suggest that it belongs to a newly described family of nuclear proteins containing both histone H1 domains and A-T hook DNA binding domains.

Cloning, sequence analysis, and expression of a cDNA encoding a plastid-localized heat shock protein in maize.

We have cloned and characterized a cDNA encoding a maize (Zea mays L.) heat shock protein (HSP), HSP26. The mRNA of HSP26 is present as a single mRNA species of 1.1 kilobase pairs in size and is detectable when maize seedlings are treated at 40 degrees C but not at 28 degrees C. Accumulation of HSP26 mRNA was detected after 10 minutes of incubation at 40 degrees C, reaching the maximum level after 1 hour. Comparison of the deduced amino acid sequence of maize HSP26 to other HSPs indicated a strong homology to the sequences of two nuclear encoded HSPs that are transported into the chloroplasts during heat shock: pea HSP21 and soybean HSP22. Maize HSP26 was also found to cross-react with anti-pea chloroplast HSP21 antibodies. Because of the sequence homology between maize HSP26, soybean HSP22, and pea HSP21, in vitro chloroplast protein import experiments were conducted. The in vitro synthesized maize HSP26 is specifically imported to the soluble fraction of the chloroplast and processed to a smaller polypeptide. The sequence homology and antibody cross-reactivity between maize HSP26 and pea HSP21 have allowed us to conclude that maize HSP26 is a nuclear-encoded, plastid-localized protein in maize.

COVASIAM: an image analysis method that allows detection of confluent microbial colonies and colonies of various sizes for automated counting.

In this work we introduce the confluent and various sizes image analysis method (COVASIAM), an automated colony count technique that uses digital imaging technology for detection and separation of confluent microbial colonies and colonies of various sizes growing on petri dishes. The proposed method takes advantage of the optical properties of the surfaces of most microbial colonies. Colonies in the petri dish are epi-illuminated in order to direct the reflection of concentrated light coming from a halogen lamp towards an image-sensing device. In conjunction, a multilevel threshold algorithm is proposed for colony separation and counting. These procedures improved the quantification of colonies showing confluence or differences in size. We tested COVASIAM with a sample set of microorganisms that form colonies with contrasting physical properties: Saccharomyces cerevisiae, Aspergillus nidulans, Escherichia coli, Azotobacter vinelandii, Pseudomonas aeruginosa, and Rhizobium etli. These physical properties range from smooth to hairy, from bright to opaque, and from high to low convexities. COVASIAM estimated an average of 95.47% (sigma = 8.55%) of the manually counted colonies, while an automated method based on a single-threshold segmentation procedure estimated an average of 76% (sigma = 16.27) of the manually counted colonies. This method can be easily transposed to almost every image-processing analyzer since the procedures to compile it are generically standard.

The yeast heat shock transcription factor contains a transcriptional activation domain whose activity is repressed under nonshock conditions.

Transcription of heat shock genes is induced by exposure of cells to elevated temperatures or other stress conditions. In yeast, it is thought that induction of transcription is mediated by conversion of a DNA-bound transcriptionally inactive form of the heat shock transcription factor (HSTF) to a DNA-bound transcriptionally active form. We have identified domains in HSTF involved in transcriptional activation and in repression of transcriptional activation at non-shock temperatures. We present evidence that a temperature-regulated transcriptional activation domain exists in HSTF and that this domain is essential for survival of yeast cells at heat shock temperatures. We propose a model for temperature-regulated transcriptional activation by a derepression mechanism.

Three maize root-specific genes are not correctly expressed in regenerated caps in the absence of the quiescent center.

The quiescent center is viewed as an architectural template in the root apical meristem of all angiosperm and gymnosperm root tips. In roots of Arabidopsis thaliana (L.) Heynh., the quiescent center inhibits differentiation of contacting initial cells and maintains the surrounding initial cells as stem cells. Here, the role of the quiescent center in the development of the maize (Zeca mays L.) root cap has been further explored. Three maize root-specific genes were identified. Two of these were exclusively expressed in the root cap and one of them encoded a GDP-mannose-4,6-dehydratase. Most likely these two genes are structural, tissue-specific markers of the cap. The third gene, a putative glycine-rich cell wall protein, was expressed in the cap and in the root epidermis and, conceivably is a positional marker of the cap. Microsurgical and molecular data indicate that the quiescent center and cap initials may regulate the positional and structural expression of these genes in the cap and thereby control root cap development.

A developmentally regulated hydroxyproline-rich glycoprotein from the cell walls of soybean seed coats.

In soybean seeds the level of hydroxyproline is regulated in a developmental and tissue-specific manner. The seed coat contains approximately 77% of the total hydroxyproline in the seed at all stages of development. We determined the ratio of hydroxyproline to dry weight in a number of tissues within the seed; however, only the seed coat shows an increase in this ratio during development. Within the many cell layers of the seed coat, hydroxyproline is most abundant in the external layer. The hydroxyproline is present as an hydroxyproline-rich cell wall glycoprotein. The protein is rich in hydroxyproline (36%), lysine (11%), proline (10%), histidine (9%), tyrosine (9%), and serine (8%). The carbohydrate portion is 90 mole% arabinose and 10 mole% galactose. The arabinose residues are attached to hydroxyproline mostly in the form of trisaccharides. The apparent molecular weight of this glycoprotein is 100,000 daltons.

A Selaginella lepidophylla trehalose-6-phosphate synthase complements growth and stress-tolerance defects in a yeast tps1 mutant.

The accumulation of the disaccharide trehalose in anhydrobiotic organisms allows them to survive severe environmental stress. A plant cDNA, SlTPS1, encoding a 109-kD protein, was isolated from the resurrection plant Selaginella lepidophylla, which accumulates high levels of trehalose. Protein-sequence comparison showed that SlTPS1 shares high similarity to trehalose-6-phosphate synthase genes from prokaryotes and eukaryotes. SlTPS1 mRNA was constitutively expressed in S. lepidophylla. DNA gel-blot analysis indicated that SlTPS1 is present as a single-copy gene. Transformation of a Saccharomyces cerevisiae tps1Delta mutant disrupted in the ScTPS1 gene with S. lepidophylla SlTPS1 restored growth on fermentable sugars and the synthesis of trehalose at high levels. Moreover, the SlTPS1 gene introduced into the tps1Delta mutant was able to complement both deficiencies: sensitivity to sublethal heat treatment at 39 degrees C and induced thermotolerance at 50 degrees C. The osmosensitive phenotype of the yeast tps1Delta mutant grown in NaCl and sorbitol was also restored by the SlTPS1 gene. Thus, SlTPS1 protein is a functional plant homolog capable of sustaining trehalose biosynthesis and could play a major role in stress tolerance in S. lepidophylla.