Catherine A. Whitelaw

Temporal and spatial expression of a polygalacturonase during leaf and flower abscission in oilseed rape and Arabidopsis.

During leaf abscission in oilseed rape (Brassica napus), cell wall degradation is brought about by the action of several hydrolytic enzymes. One of these is thought to be polygalacturonase (PG). Degenerate primers were used to isolate a PG cDNA fragment by reverse transcriptase-polymerase chain reaction from RNA extracted from ethylene-promoted leaf abscission zones (AZs), and in turn a full-length clone (CAW471) from an oilseed rape AZ cDNA library. The highest homology of this cDNA (82%) was to an Arabidopsis sequence that was predicted to encode a PG protein. Analysis of expression revealed that CAW471 mRNA accumulated in the AZ of leaves and reached a peak 24 h after ethylene treatment. Ethylene-promoted leaf abscission in oilseed rape was not apparent until 42 h after exposure to the gas, reaching 50% at 48 h and 100% by 56 h. In floral organ abscission, expression of CAW471 correlated with cell separation. Genomic libraries from oilseed rape and Arabidopsis were screened with CAW471 and the respective genomic clones PGAZBRAN and PGAZATisolated. Characterization of these PG genes revealed that they had substantial homology within both the coding regions and in the 5′-upstream sequences. Fusion of a 1,476-bp 5′-upstream sequence ofPGAZAT to β-glucuronidase or green fluorescent protein and transformation of Arabidopsis revealed that this fragment was sufficient to drive expression of these reporter genes in the AZs at the base of the anther filaments, petals, and sepals.

Cloning and sequencing of cDNAs for hypothetical genes from chromosome 2 of Arabidopsis.

About 25% of the genes in the fully sequenced and annotated Arabidopsis genome have structures that are predicted solely by computer algorithms with no support from either nucleic acid or protein homologs from other species or expressed sequence matches from Arabidopsis. These are referred to as “hypothetical genes.” On chromosome 2, sequenced by The Institute for Genomic Research, there are approximately 800 hypothetical genes among a total of approximately 4,100 genes. To test their expression under various growth conditions and in specific tissues, we used six cDNA populations prepared from cold-treated, heat-treated, and pathogen (Xanthomonas campestris pv campestris)-infected plants, callus, roots, and young seedlings. To date, 169 hypothetical genes were tested, and 138 of them are found to be expressed in one or more of the six cDNA populations. By sequencing multiple clones from each 5′- and 3′-rapid amplification of cDNA ends (RACE) product and assembling the sequences, we generated full-length sequences for 16 of these genes. For 14 genes, there was one full-length assembly that precisely supported the intron-exon boundaries of their gene predictions, adding only 5′- and 3′-untranslated region sequences. However, for three of these genes, the other assemblies represent additional exons and alternatively spliced or unspliced introns. For the remaining two genes, the cDNA sequences reveal major differences with predicted gene structures. In addition, a total of six genes displayed more than one polyadenylation site. These data will be used to update gene models in The Institute for Genomic Research annotation database ATH1.

Spatial and Temporal Expression of Abscission-Related Genes During Ethylene-Promoted Organ Shedding

During the course of development a spectrum of organs are shed from a plant [1]. Although the cellular and molecular mechanisms by which the abscission process is brought about have yet to be ascertained, the final event is the dissolution of the cell wall at the site where shedding takes place. A major difficulty encountered when studying abscission is that only a few rows of cells may undergo separation. In bean, there is convincing evidence that only one or two rows of cells may contribute to the loss of the primary leaf [2] while in tomato flowers there may be 5-10 rows of cells involved in the process [3]. In an attempt to examine the molecular changes which lead to abscission we have chosen to study the shedding of leaflets in Sambucus nigra [4,5,6]. This is a well characterised system and as the leaflet abscission zone is a composite of three sites where cell separation takes place, as many as 50 rows of cells may be involved in the process [7]. Moreover, leaflet abscission can be induced in approximately 24 hours by exposure to ethylene (10 μ1 1-1) and the use of explants leads to a close degree of synchronisation of the process [5].

Differential display: analysis of gene expression during plant cell separation processes.

An essential component in the study of cell growth and development in any organism is the analysis of differential gene expression. There are numerous techniques available for comparison of two or more systems at the molecular level, including subtractive hybridization, reverse transcriptase (RT), polymerase chain reaction (PCR), differential screening of cDNA libraries, and, more recently, cDNA microarrays. Differential display has advantages in that it is relatively less time-consuming and can result in the identification of rare cDNA, which may be missed by conventional cDNA library screening. In addition, cDNA microarrays are a valuable asset to the analysis of regulated gene expression but the technique is expensive to employ. Although we successfully applied differential display to isolate novel mRNAs that are up- and downregulated during cell separation processes in plants, the technique can be applied to any system where two or more mRNA sets are to be compared.