Development of a citrus genome-wide EST collection and cDNA microarray as resources for genomic studies.

A functional genomics project has been initiated to approach the molecular characterization of the main biological and agronomical traits of citrus. As a key part of this project, a citrus EST collection has been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in the citrus genome. Functional annotation of unigenes which have Arabidopsis orthologues (68% of all unigenes) revealed gene representation in every major functional category, suggesting that a genome-wide EST collection was obtained. A Citrus clementina Hort. ex Tan. cv. Clemenules genomic library, that will contribute to further characterization of relevant genes, has also been constructed. To initiate the analysis of citrus transcriptome, we have developed a cDNA microarray containing 12,672 probes corresponding to 6875 putative unigenes of the collection. Technical characterization of the microarray showed high intra- and inter-array reproducibility, as well as a good range of sensitivity. We have also validated gene expression data achieved with this microarray through an independent technique such as RNA gel blot analysis.

Hormonal regulation of a cysteine proteinase gene, EPB-1, in barley aleurone layers: cis- and trans-acting elements involved in the co-ordinated gene expression regulated by gibberellins and abscisic acid.

The synthesis of EPB, a cysteine proteinase responsible for the degradation of seed endosperm storage proteins in barley (Hordeum vulgare), is induced by gibberellins (GA) and repressed by abscisic acid (ABA). The EPB gene family consists of two very similar members, EPB-1 and EPB-2, with the former being more highly induced by GA. We have functionally characterized the cis-acting elements in the EPB-1 promoter and determined that a gibberellin response element (GARE), a pyrimidine box and an upstream element are necessary for GA induction. By comparison with the promoters of alpha-amylase genes, which are also induced by GA, we suggest that GARE is coupled with the upstream element and the pyrimidine box to form a GA response complex. In addition, we have shown that the 3'-untranslated/untranscribed region of the EPB-1 gene is required for a low background expression in the absence of GA. Constitutive expression of a transcription factor, GAMyb, in the absence of GA leads to the transactivation of EPB-1 expression in a dosage dependent manner with the highest level comparable to that in fully GA-induced tissue. Co-expression of a truncated version of GAMyb containing only the DNA binding domain blocks the GA-induction of EPB-1, further supporting the role of GAMyb in the regulation of gene expression. Although ABA is very effective in blocking the GA induction of EPB-1, it has no effect on the GAMyb-mediated expression of EPB-1. We suggest that ABA acts upstream of the formation of functional GAMyb which co-ordinates the hormonal regulation of a diverse group of genes in cereal aleurone layers, including those encoding EPB and alpha-amylases.

Cloning and characterization of TPE4A, a thiol-protease gene induced during ovary senescence and seed germination in pea.

A cDNA clone encoding a thiol-protease (TPE4A) was isolated from senescent ovaries of pea (Pisum sativum) by reverse transcriptase-polymerase chain reaction. The deduced amino acid sequence of TPE4A has the conserved catalytic amino acids of papain. It is very similar to VSCYSPROA, a thiol-protease induced during seed germination in common vetch. TPE4A mRNA levels increase during the senescence of unpollinated pea ovaries and are totally suppressed by treatment with gibberellic acid. In situ hybridization indicated that TPE4A mRNA distribution in senescent pea ovaries is different from that of previously reported thiol-proteases induced during senescence, suggesting the involvement of different proteases in the mobilization of proteins from senescent pea ovaries. TPE4A is also induced during the germination of pea seeds, indicating that a single protease gene can be induced during two different physiological processes, senescence and germination, both of which require protein mobilization.

A major cysteine proteinase, EPB, in germinating barley seeds: structure of two intronless genes and regulation of expression.

The barley cysteine proteinase B (EPB) is the main protease responsible for the degradation of endosperm storage proteins providing nitrogenous nutrients to support the growth of young seedlings. The expression of this enzyme is induced in the germinating seeds by the phytohormone, gibberellin, and suppressed by another phytohormone, abscisic acid. In situ hybridization experiments indicate that EPB is expressed in the scutellar epithelium within 24 h of seed germination, but the aleurone tissue surrounding the starchy endosperm eventually becomes the main tissue expressing this enzyme. The EPB gene family of barley consists of two very similar genes, EPB1 and EPB2, both of which have been mapped to chromosome 3. The sequences of EPB1 and EPB2 match with the two previously published cDNA clones indicating that both genes are expressed. Interestingly, neither of these genes contain any introns, a rare phenomenon in which all members of a small gene family are active intronless genes. Sequence comparison indicates that the barley EPB family can be classified as cathepsin L-like endopeptidases and is most closely related to two legume cysteine proteinases (Phaseolus vulgaris EP-C1 and Vigna mungo SHEP) which are also involved in seed storage protein degradation. The promoters of EPB1 and EPB2 have been linked to the coding sequence of a reporter gene, GUS, encoding beta-glucuronidase, and introduced into barley aleurone cells using the particle bombardment method. Transient expression studies indicate that EPB promoters are sufficient to confer the hormonal regulation of these genes.