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The resurrection plant Selaginella lepidophylla (Division Lycophyta) belongs to an ancient lineage of vascular plants that can withstand complete desiccation for years and be revived after only a few hours of rehydration. To better understand the molecular basis of dehydration tolerance, a complementary DNA library was constructed from S. lepidophylla microphyll fronds dehydrated for 2.5 h and used to generate an expressed sequence tag (EST) database. ESTs were obtained for 1046 clones representing 874 unique transcripts. Putative functions were assigned to 653 (62.4%) of these clones after comparison with protein databases, whereas 212 (20.2%) sequences having significant similarity to known sequences whose functions are unclear and 181 (17.3%) sequences having no similarity to known sequences. The S. lepidophylla ESTs were compared to the Selaginella moellendorffii EST database using the tBLASTX algorithm. Approximately 36–38% of the S. lepidophylla ESTs exhibited similarity to the S. moellendorffii ESTs, whereas 62–63% were unique to S. lepidophylla and S. moellendorffii , respectively. For those S. lepidophylla ESTs for which functional assignments could be made, the largest functional categories included photosynthesis (17%), followed by primary metabolism (14%) and disease/defense-related functions (11%). S. lepidophylla had a higher relative percentage of ESTs within the transporter, cell structure, secondary metabolism, molecular chaperones (i.e., heat shock proteins), and abiotic stress-related (i.e., late embryogenesis abundance proteins) functional categories than did S. moellendorffii . Thus, S. lepidophylla may serve as a rich genetic resource for the identification of novel genes associated with environmental stress and dehydration tolerance.

We report the analysis and annotation of 146,075 expressed sequence tags from Vitis species. The majority of these sequences were derived from different cultivars of Vitis vinifera, comprising an estimated 25,746 unique contig and singleton sequences that survey transcription in various tissues and developmental stages and during biotic and abiotic stress. Putatively homologous proteins were identified for over 17,752 of the transcripts, with 1,962 transcripts further subdivided into one or more Gene Ontology categories. A simple structured vocabulary, with modules for plant genotype, plant development, and stress, was developed to describe the relationship between individual expressed sequence tags and cDNA libraries; the resulting vocabulary provides query terms to facilitate data mining within the context of a relational database. As a measure of the extent to which characterized metabolic pathways were encompassed by the data set, we searched for homologs of the enzymes leading from glycolysis, through the oxidative/nonoxidative pentose phosphate pathway, and into the general phenylpropanoid pathway. Homologs were identified for 65 of these 77 enzymes, with 86% of enzymatic steps represented by paralogous genes. Differentially expressed transcripts were identified by means of a stringent believability index cutoff of ≥98.4%. Correlation analysis and two-dimensional hierarchical clustering grouped these transcripts according to similarity of expression. In the broadest analysis, 665 differentially expressed transcripts were identified across 29 cDNA libraries, representing a range of developmental and stress conditions. The groupings revealed expected associations between plant developmental stages and tissue types, with the notable exception of abiotic stress treatments. A more focused analysis of flower and berry development identified 87 differentially expressed transcripts and provides the basis for a compendium that relates gene expression and annotation to previously characterized aspects of berry development and physiology. Comparison with published results for select genes, as well as correlation analysis between independent data sets, suggests that the inferred in silico patterns of expression are likely to be an accurate representation of transcript abundance for the conditions surveyed. Thus, the combined data set reveals the in silico expression patterns for hundreds of genes in V. vinifera, the majority of which have not been previously studied within this species.

Plant phosphoenolpyruvate-carboxylase kinase (PEPC-kinase [PpcK]) is the smallest Ser/Thr kinase identified to date, having a molecular mass of approximately 32,000. This novel, monomeric kinase is dedicated to the phosphorylation of plant PEPC, thereby regulating this target enzyme's activity and allosteric properties. Although several recombinant, non-fusion PpcK proteins have been produced recently in Escherichia coli, these are plagued by their high degree of insolubility. Here, we report the use of the native, E. coli NusA protein and a related E. coli expression vector (pET-43a(+) [Novagen]) for enhancing the solubility of this recalcitrant Ser/Thr kinase at least 10-fold by its production as a dual 6xHis-tagged NusA/McPpcK1 fusion protein, which accounts for approximately 10% of the soluble protein fraction from induced cells. Capture of this fusion protein from the centrifuged cell extract by immobilized metal (Ni(2+)) affinity-chromatography, its "on-bead" cleavage by thrombin, and subsequent elution yielded milligram quantities of a "free," approximately 36-kDa form of PpcK for further purification by fast-protein liquid chromatography on blue dextran-agarose or preparative SDS-PAGE. Steady-state kinetic analysis of the former, active preparation revealed that this dedicated kinase discriminates against neither various isoforms of plant PEPC nor certain mutant forms of recombinant C(4) PEPC. Alternatively, the latter, electrophoretically homogeneous sample of the approximately 36-kDa polypeptide was used as antigen for polyclonal-antibody production in rabbits. The antibodies against the recombinant McPpcK1 from Mesembryanthemum crystallinum cross-reacted on Western blots with an enriched preparation of the maize-leaf kinase, but not with the parent crude extract, thus directly documenting this protein's extremely low abundance in vivo. However, these antibodies were effective in immunoprecipitating 32P-based PpcK activity from crude, desalted extracts of maize leaves and soybean root-nodules.

The internal transcribed spacer (ITS) region of 18-26S nuclear ribosomal DNA repeat was sequenced for 31 of the approximately 40 species of Clusia known to occur in Panama. Several species from other genera of the Clusiaceae were used as outgroups in the phylogenetic calculation. High sequence alignment and minimal length variation among ITS-1, 5.8S and ITS-2 sequences facilitated determination of positional homology of nucleotide sizes. Sequence alignment was evaluated with character state (Maximum Parsimony) and distance methods (Neighbour Joining). Phylogenetic trees obtained with the two methods were largely concordant and revealed three main groups that roughly correspond to previous arrangements of species into three large morphological groups, the C. flava group, the C. minor group and the C. multiflora group. Because species of Clusia are either regular C3 plants or exhibit crassulacean acid metabolism (CAM) involving varying proportions of CO2 fixation in the dark versus the light, we mapped photosynthetic pathways onto the cladograms. Photosynthetic pathway classification was based on measurements of 13C/12C ratios of plant carbon and also on information available from the literature. Both the C. flava and C. minor group contained species exhibiting CAM, distributed on distinct branches of the cladograms, whereas the third group (C. multiflora group) was composed of species which are not known to use CAM.

The purpose of this project was to identify large numbers of Arabidopsis genes with essential functions during seed development. More than 120,000 T-DNA insertion lines were generated following Agrobacterium-mediated transformation. Transgenic plants were screened for defective seeds and putative mutants were subjected to detailed analysis in subsequent generations. Plasmid rescue and TAIL-PCR were used to recover plant sequences flanking insertion sites in tagged mutants. More than 4200 mutants with a wide range of seed phenotypes were identified. Over 1700 of these mutants were analyzed in detail. The 350 tagged embryo-defective (emb) mutants identified to date represent a significant advance toward saturation mutagenesis of EMB genes in Arabidopsis. Plant sequences adjacent to T-DNA borders in mutants with confirmed insertion sites were used to map genome locations and establish tentative identities for 167 EMB genes with diverse biological functions. The frequency of duplicate mutant alleles recovered is consistent with a relatively small number of essential (EMB) genes with nonredundant functions during seed development. Other functions critical to seed development in Arabidopsis may be protected from deleterious mutations by extensive genome duplications.

Abiotic stresses reduce plant productivity. We focus on gene expression analysis following exposure of plants to high salinity, using salt-shock experiments to mimic stresses that affect hydration and ion homeostasis. The approach includes parallel molecular and genetic experimentation. Comparative analysis is employed to identify functional isoforms and genetic orthologs of stress-regulated genes common to cyanobacteria, fungi, algae and higher plants. We analyze global gene expression profiles monitored under salt stress conditions through abundance profiles in several species: in the cyanobacterium SynechocystisPCC6803, in unicellular (Saccharomyces cerevisiae) and multicellular (Aspergillus nidulans) fungi, the eukaryotic alga Dunaliella salina, the halophytic land plant Mesembryanthemum crystallinum , the glycophytic Oryza sativa and the genetic model Arabidopsis thaliana. Expanding the gene count, stress brings about a significant increase of transcripts for which no function is known. Also, we generate insertional mutants that affect stress tolerance in several organisms. More than 400 000 T-DNA tagged lines of A. thaliana have been generated, and lines with altered salt stress responses have been obtained. Integration of these approaches defines stress phenotypes, catalogs of transcripts and a global representation of gene expression induced by salt stress. Determining evolutionary relationships among these genes, mutants and transcription profiles will provide categories and gene clusters, which reveal ubiquitous cellular aspects of salinity tolerance and unique solutions in multicellular species. © 2001 Editions scientifiques et medicales Elsevier SAS Arabidopsis T-DNA tag / EST sequencing / functional genomics / gene expression profiles / microarray analysis / phylogenetic profiles of stress tolerance / salinity stress / stress tolerance

A cDNA clone encoding a cysteine protease was isolated from salinity-stressed Mesembryanthemum crystallinum. The deduced sequence of this full-length clone of 1483 bp encodes a pre-proprotein of 367 amino acids with a predicted molecular mass of 41 kDa. Southern blot analysis of genomic DNA indicated that this cDNA is a member of a small multigenic family. Northern and western blot analysis showed that both mRNA and protein expression are strongly induced in leaves in response to salinity stress. mRNA abundance is also enhanced by drought stress and exogenous application of cytokinin and methyl jasmonate, but not ABA. Sequence comparisons showed the encoded gene product shows highest sequence homology (47.7–57.2% identity) to a group of cysteine proteases proposed to have diverse proteolytic functions in higher plants.

Clones coding for a 1100-bp cDNA sequence of phosphoenolpyruvate carboxylase (PEPC) of the constitutive crassulacean acid metabolism (CAM) plant Kalanchoe pinnata (Lam.) Pers., were isolated by reverse transcription-polymerase chain reaction (RT–PCR) and characterised by restriction fragment length polymorphism analysis and DNA sequencing. Seven distinct PEPC isogenes were recovered, four in leaves and three in roots (EMBL accession numbers: AJ344052–AJ344058). Sequence similarity comparisons and distance neighbour-joining calculations separate the seven PEPC isoforms into two clades, one of which contains the three PEPCs found in roots. The second clade contains the four isoforms found in leaves and is divided into two branches, one of which contains two PEPCs most similar with described previously CAM isoforms. Of these two isoforms, however, only one exhibited abundant expression in CAM-performing leaves, but not in very young leaves, which do not exhibit CAM, suggesting this isoform encodes a CAM-specific PEPC. Protein sequence calculations suggest that all isogenes are likely derived from a common ancestor gene, presumably by serial gene duplication events. To our knowledge, this is the most comprehensive identification of a PEPC gene family from a CAM plant, and the greatest number of PEPC isogenes reported for any vascular plant to date.

The common ice plant, Mesembryanthemum crystallinum, is a halophytic (salt-loving) member of the Aizoaceae, which switches from C3 photosynthesis to Crassulacean acid metabolism (CAM) when exposed to salinity or water-deficit stress. CAM is a metabolic adaptation of photosynthetic carbon fixation that improves water use efficiency by shifting net CO2 uptake to the night, thereby reducing transpirational water loss. To improve our understanding of the molecular genetic underpinnings and control mechanisms for Crassulacean acid metabolism (CAM) and other salinity stress response adaptations, a total of 9733 expressed sequence tags (ESTs) from cDNAs derived from leaf tissues of well-watered and salinity-stressed (0.5 M NaCl for 30 and 48 h) were characterized. Clustering and assembly of these ESTs resulted in the identification of a total of 3676 tentative unique gene sequences (1249 tentative consensus sequences and 2427 singleton ESTs) expressed in leaves of ice plant under unstressed and salinity stressed conditions. The same number (2782) of ESTs from each library (total=8346 ESTs) were randomly selected and analyzed to compare expression profiles among the control and salt stressed leaf tissues. EST frequencies for transcripts encoding CAM-related enzymes, pathogenesis-related, senescence-associated, cell death-related, and stress-related proteins such as heat shock proteins (HSPs), chaperones, early light-inducible proteins, ion homeostasis, antioxidative stress, detoxification, and biosynthetic enzymes for osmoprotectants increased 2–12-fold in cDNA libraries constructed from salt stressed plants. In contrast, the frequency of ESTs encoding light-harvesting and photosystem complexes and C3 photosynthetic enzymes decreased 4-fold overall following salinity stress with transcripts for ribulose bisphosphate carboxylase/oxygenase (RuBisCO) subunits decreasing 7-fold. Moreover, stressed plants contained a higher percentage of ESTs encoding novel and/or functionally unknown proteins. The rapid discovery of both known and unknown genes related to stress responses in M. crystallinum demonstrates the great utility of EST analysis in unraveling the complex set of adaptive mechanisms contributing to water use efficiency (CAM) and salinity tolerance.

Repetitive rounds of differential subtraction screening, followed by nucleotide sequence determination and northern-blot analysis, identified 84 salt-regulated (160 mm NaCl for 4 h) genes in Arabidopsis wild-type (Col-0 gl1 ) seedlings. Probes corresponding to these 84 genes and ACP1 , RD22BP1 , MYB2 , STZ , and PAL were included in an analysis of salt responsive gene expression profiles in gl1 and the salt-hypersensitive mutant sos3 . Six of 89 genes were expressed differentially in wild-type and sos3 seedlings; steady-state mRNA abundance of five genes ( AD06C08/ unknown, AD05E05/ vegetative storage protein 2 [ VSP2 ], AD05B11 /S-adenosyl-l-Met:salicylic acid carboxyl methyltransferase [ SAMT ], AD03D05 /cold regulated 6.6/inducible2 [ COR6.6/KIN2 ], and salt tolerance zinc finger [ STZ ]) was induced and the abundance of one gene ( AD05C10 /circadian rhythm-RNA binding1 [ CCR1 ]) was reduced in wild-type plants after salt treatment. The expression of CCR1 , SAMT , COR6.6/KIN2 , and STZ was higher in sos3 than in wild type, and VSP2 and AD06C08/ unknown was lower in the mutant. Salt-induced expression of VSP2 in sos1 was similar to wild type, and AD06C08/ unknown , CCR1 , SAMT , COR6.6/KIN2 , and STZ were similar to sos3. VSP2 is regulated presumably by SOS2/3 independent of SOS1 , whereas the expression of the others is SOS1 dependent. AD06C08/ unknown and VSP2 are postulated to be effectors of salt tolerance whereas CCR1 , SAMT , COR6.6/KIN2 , and STZ are determinants that must be negatively regulated during salt adaptation. The pivotal function of the SOS signal pathway to mediate ion homeostasis and salt tolerance implicates AD06C08 /unknown, VSP2 , SAMT , 6.6/KIN2 , STZ , and CCR1 as determinates that are involved in salt adaptation.