De novo genetic variation revealed in somatic sectors of single Arabidopsis plants.

Concern over the tremendous loss of genetic diversity among many of our most important crops has prompted major efforts to preserve seed stocks derived from cultivated species and their wild relatives.  Arabidopsis thaliana propagates mainly by self-fertilizing, and therefore, like many crop plants, theoretically has a limited potential for producing genetically diverse offspring. Despite this, inbreeding has persisted in Arabidopsis for over a million years suggesting that some underlying adaptive mechanism buffers the deleterious consequences of this reproductive strategy. Using presence-absence molecular markers we demonstrate that single Arabidopsis plants can have multiple genotypes. Sequence analyses reveal single nucleotide changes, loss of sequences and, surprisingly, acquisition of unique genomic insertions. Estimates based on quantitative analyses suggest that these genetically discordant sectors are very small but can have a complex genetic makeup. In ruling out more trivial explanations for these data, our findings raise the possibility that intrinsic drivers of genetic variation are responsible for the targeted sequence changes we detect. Given the evolutionary advantage afforded to populations with greater genetic diversity, we hypothesize that organisms that primarily self-fertilize or propagate clonally counteract the genetic cost of such reproductive strategies by leveraging a cryptic reserve of extra-genomic information.

Arabidopsis eIF5A3 influences growth and the response to osmotic and nutrient stress.

AteIF5A3, one of three genes encoding eukaryotic translation initiation factor 5A (eIF5A) in Arabidopsis thaliana, and corresponding genes PdeIF5A3 from Populus deltoides (eastern cottonwood) and SleIF5A4 from Solanum lycopersicum (tomato) were constitutively over-expressed in A. thaliana. The resultant transgenic plants exhibited enhanced vegetative and reproductive growth. Indeed, the increase in seed yield relative to empty vector controls for the PdeIF5A3 over-expressing plants ranged from 50% to 300% depending on the line. The PdeIF5A3 over-expressing plants also exhibited enhanced fitness when exposed to osmotic and nutrient (N, P and K) stress. The spatial localization of AteIF5A3 was visualized by confocal microscopy using transgenic plants expressing P(AteIF5A3) :GFP-AteIF5A3. GFP fluorescence reflecting expression of AteIF5A3 was detectable in the phloem, particularly companion cells, of roots, stems and leaves, in the epidermal cells of the root tip, in the columella cells of the root cap and in the chalazal tissue of fertilized ovules, which all play a pivotal role in nutrient or hormone translocation. Thus, AteIF5A3 appears to be involved in supporting growth and to play a regulatory role in the response of plants to sub-lethal osmotic and nutrient stress.

Eukaryotic translation initiation factor 5A is involved in pathogen-induced cell death and development of disease symptoms in Arabidopsis.

Eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein found in all eukaryotic kingdoms. This study demonstrates that plant eIF5A is involved in the development of disease symptoms induced by a common necrotrophic bacterial phytopathogen. Specifically, AteIF5A-2, one of the three eIF5A genes in Arabidopsis (Arabidopsis thaliana), is shown to regulate programmed cell death caused by infection with virulent Pseudomonas syringae pv tomato DC3000 (Pst DC3000). Transgenic Arabidopsis plants with constitutively suppressed AteIF5A-2 exhibited marked resistance to programmed cell death induced by virulent Pst DC3000, and there was a corresponding reduction in pathogen growth and development of disease symptoms in the plant tissue. Constitutive overexpression of AteIF5A-2 circumvented the apparent posttranscriptional regulation of AteIF5A-2 protein expression characteristic of wild-type plants but did not increase susceptibility to virulent Pst DC3000 ingression. The transgenic plants with constitutive AteIF5A-2 overexpression did, however, display phenotypes consistent with precocious cell death. The results indicate that AteIF5A-2 is a key element of the signal transduction pathway resulting in plant programmed cell death.

Modulation of eIF5A1 expression alters xylem abundance in Arabidopsis thaliana.

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.

Characterization of a plastid triacylglycerol lipase from Arabidopsis.

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.

Eukaryotic translation initiation factor 5A induces apoptosis in colon cancer cells and associates with the nucleus in response to tumour necrosis factor alpha signalling.

Eukaryotic translation initiation factor 5A (eIF5A) is thought to function as a nucleocytoplasmic shuttle protein. There are reports of its involvement in cell proliferation, and more recently it has also been implicated in the regulation of apoptosis. In the present study, we examined the effects of eIF5A over-expression on apoptosis and of siRNA-mediated suppression of eIF5A on expression of the tumour suppressor protein, p53. Over-expression of either eIF5A or a mutant of eIF5A incapable of being hypusinated was found to induce apoptosis in colon carcinoma cells. Our results also indicate that eIF5A is required for expression of p53 following the induction of apoptosis by treatment with Actinomycin D. Depiction of eIF5A localization by indirect immunofluorescence has indicated, for the first time, that the protein is rapidly translocated from the cytoplasm to the nucleus by death receptor activation or following treatment with Actinomycin D. These findings collectively indicate that unhypusinated eIF5A may have pro-apoptotic functions and that eIF5A is rapidly translocated to the nucleus following the induction of apoptotic cell death.

Regulation of senescence by eukaryotic translation initiation factor 5A: implications for plant growth and development.

Regulation of protein synthesis is increasingly being recognized as an important determinant of cell proliferation and senescence. In particular, recent evidence indicates that eukaryotic translation initiation factor 5A (eIF-A) plays a pivotal role in this determination. Separate isoforms of eIF-5A appear to facilitate the translation of mRNAs required for cell division and cell death. This raises the possibility that eIF-5A isoforms are elements of a biological switch that is in one position in dividing cells and in another position in dying cells. Changes in the position of this putative switch in response to physiological and environmental cues are likely to have a significant impact on plant growth and development.