The peri-cell-cycle in Arabidopsis.

The root systems of plants proliferate via de novo formed meristems originating from differentiated pericycle cells. The identity of putative signals responsible for triggering some of the pericycle cells to re-enter the cell cycle remains unknown. Here, the cell cycle regulation in the pericycle of seedling roots of Arabidopsis thaliana (L.) HEYNH: is studied shortly after germination using various strategies. Based on the detailed analysis of the promoter-beta-glucuronidase activity of four key cell cycle regulatory genes, combined with cell length measurements, microdensitometry of DNA content, and experiments with a cell cycle-blocking agent, a model is proposed for cell cycle regulation in the pericycle at the onset of lateral root initiation. The results clearly show that before the first lateral root is initiated, the pericycle consists of dissimilar cell files in respect of their cell division history. Depending on the distance behind the root tip and on position in relation to the vascular tissue, particular pericycle cells remain in the G(2) phase of the cell cycle and are apparently more susceptible to lateral root initiation than others.

Developmental expression of the Arabidopsis thaliana CycA2;1 gene.

The associations of cyclins with highly conserved cyclin-dependent kinases are key events in the regulation of cell cycle progression. The spatio-temporal expression of an Arabidopsis thaliana (L.) Heynh. mitotic cyclin, Arath;CycA2;1, was studied by histochemical beta-glucuronidase (GUS) analysis and in-situ hybridizations. The CycA2,1] promoter was active in the egg apparatus before fertilization. During embryogenesis, CycA2;1:gus expression was found in the embryo and the developing endosperm. Throughout plant development, CycA2;1 transcripts were found in both dividing and non-dividing cells, indicating that the expression of this cyclin is not a limiting factor for cell division. In the pericycle and stelar parenchyma, CycA2;1 transcripts were located at the xylem poles, a position that can be correlated with competence for lateral root formation. In addition, CycA2;1:gus expression was upregulated in roots by auxins and in the shoot apex by cytokinins. Transcription of CycA2;1 was shown by reverse transcription-polymerase chain reaction to be strongly induced by sucrose in A. thaliana cell suspensions.

Expression of cell cycle regulatory genes and morphological alterations in response to salt stress in Arabidopsis thaliana.

Hyperosmotic stress severely affects plant growth and development. To examine the effect of salt stress on cell cycle activity in Arabidopsis thaliana (L.) Heynh., the transcriptional regulation of a cyclin-dependent kinase, CDC2aAt, and two mitotic cyclins, Arath;CycB1;1 and Arath;CycA2;1, was studied by using the beta-glucuronidase (gus) reporter gene. Moreover, the mRNA abundance of these cell cycle genes as well as CDC2bAt were monitored during salt stress. Upon NaCl treatment, the promoter activities and transcript levels of all cell cycle genes diminished initially in the shoot apex and were subsequently induced during salt-stress adaptation. Additionally, the promoter activities of CDC2aAt and CycA2;1 decreased in the vascular cylinder of the root in correlation with reduced lateral root formation. In the root tips, a regression of CDC2aAt, CycA2;1, and CycB1;1:gus expression was observed, concomitant with a shrinkage of the root meristem and inhibition of root growth. Our data indicate that salt stress interferes with cell cycle regulation at the transcriptional level, resulting in an adaptive growth response.

The Rhodococcus fascians-plant interaction: morphological traits and biotechnological applications.

Rhodococcus fascians is a Gram-positive bacterium that infects dicotyledonous and monocotyledonous plants, leading to an alteration in the normal growth process of the host. The disease results from the modulation of the plant hormone balances, and cytokinins are thought to play an important role in the induction of symptoms. Generally, on the aerial parts of the plants, existing meristems were found to be most sensitive to the action of R. fascians, but, depending on the infection procedure, differentiated tissues as well gave rise to shoots. Similarly, in roots not only actively dividing cells, but also cells with a high competence to divide were strongly affected by R. fascians. The observed symptoms, together with the determined hormone levels in infected plant tissue, suggest that auxins and molecules of bacterial origin are also involved in leafy gall formation. The complexity of symptom development is furthermore illustrated by the necessary and continuous presence of the bacteria for symptom persistence. Indeed, elimination of the bacteria from a leafy gall results in the further development of the multiple embryonic buds of which it consists. This interesting characteristic offers novel biotechnological applications: a leafy gall can be used for germplasm storage and for plant propagation. The presented procedure proves to be routinely applicable to a very wide range of plants, encompassing several recalcitrant species.

A new D-type cyclin of Arabidopsis thaliana expressed during lateral root primordia formation.

D-type cyclins are believed to regulate the onset of cell division upon mitogenic signaling. Here, the isolation is reported of a new D-type cyclin gene (CYCD4;1) of Arabidopsis thaliana (L.) Heynh. during a two-hybrid screen using the cyclin-dependent kinase CDC2aAt as bait. Transcription of CYCD4;1 can be induced by sucrose. The co-regulated expression of CYCD4;1 and CDC2aAt in starved suspension cultures upon mitogenic stimulation indicates that the formation of a complex between these two partners is important for the resumption of cell division activity. By in-situ hybridizations CYCD4;1 was shown to be expressed during vascular tissue development, embryogenesis, and formation of lateral root primordia. Expression during the latter process suggests that the induced expression of D-type cyclins by mitogenic stimuli might be one of the rate-limiting events for the initiation of lateral roots.

Molecular markers and cell cycle inhibitors show the importance of cell cycle progression in nematode-induced galls and syncytia.

Root knot and cyst nematodes induce large multinucleated cells, designated giant cells and syncytia, respectively, in plant roots. We have used molecular markers to study cell cycle progression in these specialized feeding cells. In situ hybridization with two cyclin-dependent kinases and two cyclins showed that these genes were induced very early in galls and syncytia and that the feeding cells progressed through the G2 phase. By using cell cycle blockers, DNA synthesis and progression through the G2 phase, or mitosis, were shown to be essential for gall and syncytium establishment. When mitosis was blocked, further gall development was arrested. This result demonstrates that cycles of endoreduplication or other methods of DNA amplification are insufficient to drive giant cell expansion. On the other hand, syncytium development was much less affected by a mitotic block; however, syncytium expansion was inhibited.

Two Arabidopsis cyclin promoters mediate distinctive transcriptional oscillation in synchronized tobacco BY-2 cells.

Cyclins are cell cycle regulators whose proteins oscillate dramatically during the cell cycle. Cyclin steady-state mRNA levels also fluctuate, and there are indications that both their rate of transcription and mRNA stability are under cell cycle control. Here, we demonstrate the transcriptional regulation of higher eukaryote cyclins throughout the whole cell cycle with a high temporal resolution. The promoters of two Arabidopsis cyclins, cyc3aAt and cyc1At, mediated transcriptional oscillation of the beta-glucuronidase (gus) reporter gene in stably transformed tobacco BY-2 cell lines. The rate of transcription driven by the cyc3aAt promoter was very low during G1, slowly increased during the S phase, peaked at the G2 phase and G2-to-M transition, and was down-regulated before early metaphase. In contrast, the rate of the cyc1At-related transcription increased upon exit of the S phase, peaked at the G2-to-M transition and during mitosis, and decreased upon exit from the M phase. This study indicates that transcription mechanisms that seem to be conserved among species play a significant role in regulating the mRNA abundance of the plant cyclins. Furthermore, the transcription patterns of cyc3aAt and cyc1At were coherent with their slightly higher sequence similarity to the A and B groups of animal cyclins, respectively, suggesting that they may fulfill comparable roles during the cell cycle.

Three discrete classes of Arabidopsis cyclins are expressed during different intervals of the cell cycle.

We have isolated cDNAs encoding four additional mitotic-like cyclins from Arabidopsis: cyc2aAt, cyc2bAt, cyc3aAt, and cyc3bAt. Examination of amino acid sequences deduced from plant cyclin cDNAs isolated so far showed that they can be grouped into three distinct classes. The members of each plant cyclin family are more related to each other than to any animal or yeast cyclin. Reverse transcription-PCR analysis demonstrated that cyc2aAt was expressed in all plant organs, whereas cyc2bAt mRNAs were found only in roots; cyc3aAt was not expressed in leaves and was barely expressed in flowers. On the other hand, cyc3bAt transcripts were observed in all organs. Whole-mount in situ hybridizations on roots showed that the cyclin mRNAs were confined to parts of the roots with mitotic activity. Furthermore, results of whole-mount in situ hybridizations on roots treated with either oryzalin or hydroxyurea suggest that the different cyclin classes have distinct functions in the cell cycle.