Full-length cDNA corresponding to Arabidopsis (Arabidopsis thaliana) gene At2g31690, which has been annotated in GenBank as a putative triacylglycerol (TAG) lipase, was obtained by reverse transcription-polymerase chain reaction using RNA from senescing rosette leaves of Arabidopsis as a template. The cognate protein was found to contain the lipase active site sequence, and corresponding recombinant protein proved capable of deesterifying TAG. In vitro chloroplast import assays indicated that the lipase is targeted to chloroplasts. This was confirmed by confocal microscopy of rosette leaf tissue treated with fluorescein isocyanate-labeled, lipase-specific antibody, which revealed that lipase protein colocalizes with plastoglobular neutral lipids. Western-blot analysis indicated that the lipase is expressed in roots, inflorescence stems, flowers, siliques, and leaves and that it is strongly up-regulated in senescing rosette leaf tissue. Transgenic plants with suppressed lipase protein levels were obtained by expressing At2g31690 cDNA in antisense orientation under the regulation of a constitutive promoter. Transgenic plants bolted and flowered at the same time as wild-type plants, but were severely stunted and exhibited delayed rosette senescence. Moreover, the stunted growth phenotype correlated with irregular chloroplast morphology. The chloroplasts of transgenic plants were structurally deformed, had reduced abundance of thylakoids that were abnormally stacked, and contained more plastoglobular neutral lipids than chloroplasts of wild-type plants. These observations collectively indicate that this TAG lipase plays a role in maintaining the structural integrity of chloroplasts, possibly by mobilizing the fatty acids of plastoglobular TAG.

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Eukaryotic translation initiation factor 5A (eIF5A) is thought to facilitate protein synthesis by participating in the nuclear export of specific mRNAs. In Arabidopsis, there are three isoforms of eIF5A. One of them, AteIF5A1, has been shown to be expressed in vascular tissue, specifically developing vessel members, using GUS as a reporter. In order to determine whether AteIF5A1 plays a role in xylem formation, its full-length cDNA was constitutively over-expressed in transgenic Arabidopsis plants. Microscopic analysis revealed that the cross-sectional area of the xylem in the main inflorescence stems of transgenic plants was 1.9-fold higher than those of corresponding inflorescence stems of wild-type plants. In wild-type stems, the primary xylem typically comprised six cell layers and was approximately 105 mum thick, but increased to 9-11 cell layers, 140-155 mum thick, in transgenic stems. Similarly, the secondary xylem increased from six cell layers, approximately 70 mum thick, in control stems to approximately 9 cell layers, 95-105 mum thick, in transgenic stems. Moreover, constitutive down-regulation of AteIF5A1 using antisense technology resulted in the major suppression of xylem formation compared with control plants, and the antisense transgenic plants were also stunted. These data collectively indicate that eIF5A1 plays a role in xylogenesis.

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