Deepwater rice (Oryza sativa) is adapted to survive conditions of severe flooding over extended periods of time. During such periods adventitious roots develop to provide water, nutrients, and anchorage. In the present study the growth of adventitious roots was induced by treatment with ethylene but not auxin, cytokinin, or gibberellin. Root elongation was enhanced between 8 and 10 h after submergence. The population of cells in the S phase and expression of the S-phase-specific histone H3 gene increased within 4 to 6 h. Within 6 to 8 h the G2-phase population increased. Cell-cycle activation was accompanied by sequential induction of a cdc2-activating kinase homolog, R2, of two cdc2 genes, cdc2Os-1 and cdc2Os-2, and of three cyclin genes, cycA1;3, cycB2;1, and cycB2;2, but only induction of the R2 gene expression preceded the induction of the S phase, possibly contributing to cell-cycle regulation in the G1 phase. Both cdc2 genes were expressed at slightly higher levels during DNA replication. Transcripts of the A-type cyclin accumulated during the S and G2 phases, and transcripts of the B-type cyclins accumulated during the G2 phase. Cyclin expression was induced at all nodes with a similar time course, suggesting that ethylene acts systemically and that root primordia respond to ethylene at an early developmental stage.

SummaryMethylthioadenosine (MTA) is formed as a by-product of ethylene biosynthesis from S-adenosyl-L-methionine (AdoMet). The methionine cycle regenerates AdoMet from MTA. In two independent differential screens for submergence-induced genes and for 1-aminocyclopropane-1-carboxylic acid (ACC)-induced genes from deepwater rice (Oryza sativa L.) we identified an acireductone dioxygenase (ARD). OsARD1 is a metal-binding protein that belongs to the cupin superfamily. Acireductone dioxygenases are unique proteins that can acquire two different activities depending on the metal ion bound. Ectopically expressed apo-OsARD1 preferentially binds Fe 2þ and reconstituted Fe-OsARD1 catalyzed the formation of 2-keto-pentanoate and formate from the model substrate 1,2-dihydroxy-3-ketopent-1-ene and dioxygen, indicating that OsARD1 is capable of catalyzing the penultimate step in the methionine cycle. Two highly homologous ARD genes were identified in rice.OsARD1 mRNA levels showed a rapid, early and transient increase upon submergence and after treatment with ethylene-releasing compounds. The second gene from rice, OsARD2, is constitutively expressed. Accumulation of OsARD1 transcript was observed in the same internodal tissues, i.e. the meristem and elongation zone, which were previously shown to synthesize ethylene. OsARD1 transcripts accumulated in the presence of cycloheximide, an inhibitor of protein synthesis, indicating that OsARD1 is a primary ethylene response gene. Promoter analysis suggests that immediate-early regulation of OsARD1 by ethylene may involve an EIN3-like transcription factor. OsARD1 is induced by low levels of ethylene. We propose that early feedback activation of the methionine cycle by low levels of ethylene ensures the high and continuous rates of ethylene synthesis required for longterm ethylene-mediated submergence adaptation without depleting the tissue of AdoMet.

Using subtractive hybridization a submergence-induced gene was identified from deepwater rice, OsUsp1, that encodes a homologue of the bacterial universal stress protein family. Sequence analysis revealed that OsUSP1 is most closely related to the bacterial MJ0577-type of ATP-binding USP proteins which have been suggested to act as a molecular switch. USP protein homologues appear to be ubiquitous in plants and are encoded by gene families, but are absent in animal species. In the youngest internode of deepwater rice plants, OsUsp1 expression was very strongly induced within 1 h of submergence. Elevated transcript levels were observed in dividing cells, in expanding cells and in differentiated tissue indicating that USP1 mediates a general process. Gene induction was shown to be regulated by ethylene with a highly similar expression pattern to that observed with submergence treatment. Based on sequence information and on expression data it is hypothesized that OsUSP1 plays a role in ethylene-mediated stress adaptation in rice.

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In deepwater rice (Oryza sativa L.), the youngest internode can be induced to grow rapidly with submergence. This growth response is mediated by gibberellic acid (GA). It is based on both increased cell growth and increased cell division activity. In order to understand whether acceleration of the cell division cycle is a result of increased cell size, we have analyzed various growth and division parameters at the cellular and molecular levels. Flow-cytometric analysis of cells from the intercalary meristem showed that the S phase population increased within 4±6 h after submergence and the G2 phase population increased within 6±8 h, indicating activation of the cell division cycle at the G1®S phase transition. Expression of the G1 and S phase-speci®c cdc2Os-2 and histone H3 genes was induced prior to S phase induction and also prior to induction of growth of meristematic cells which started with a lag phase of 4 h, indicating that transcript levels increased in response to submergence directly and not as a result of the submergence-induced accelerated growth of cells. Transcripts of the two mitotic cyclins cycOs1 and cycOs2 accumulated to signi®cant levels in G2 phase. In parallel, activity of the histone H1 kinase which represents the G2/M phase-speci®c cyclin-dependent cdc2 kinase complex increased, supporting earlier ®ndings that the activity of this kinase complex may, at least in part, be regulated at the level of cyclin expression. The molecular evidence presented therefore indicates that induction of cell division activity at G1®S occurs independently of cell growth. It also indicates that control of cell division through cell size is exerted at a level other than cdc2 gene expression.Abbreviations: ABA = cis-abscisic acid; CDK = cyclin-dependent cdc2 kinase; GA = gibberellic acid Correspondence to: M. Sauter;