Summary• Phytosulfokine-α (PSK-α) is a disulfated pentapeptide described to act as a growth factor in suspension cells. In this study, the involvement of PSK signaling through the PSK receptor gene AtPSKR1 in Arabidopsis root growth was assessed.• Expression studies of PSK precursor genes and of AtPSKR1 were performed in roots with RT-PCR and P:GUS analyses. Root elongation, lateral root formation, cell production and root cell elongation were analyzed in wild-type (wt) and in the receptor knockout mutant Atpskr1-T treated with or without synthetic PSK-α.• Phytosulfokine and AtPSKR1 genes are differentially expressed in roots. PSK-α induced root growth in a dose-dependent manner without affecting lateral root density. Kinematic analysis established that enhancement of root growth by PSK-α was mainly caused by an increase in cell size. In Atpskr1-T, the primary roots were shorter as a result of reduced mature cell size and a smaller root apical meristem composed of fewer cells than in wt.• The results indicate that PSK-α signaling through AtPSKR1 affects root elongation primarily via control of mature cell size. Root organogenesis, on the other hand, is not controlled by PSK-α.

SummaryThe methionine (Met) cycle contributes to sulfur metabolism through the conversion of methylthioadenosine (MTA) to Met at the expense of ATP. MTA is released as a by-product of ethylene synthesis from Sadenosylmethionine (AdoMet). Disruption of the Met cycle in the Arabidopsis mtk mutant resulted in an imbalance of AdoMet homeostasis at sulfur-limiting conditions, irrespective of the sulfur source supplied to the plants. At a low concentration of 100 lM sulfate, the mtk mutant had reduced AdoMet levels and growth was retarded as compared with wild type. An elevated production of ethylene was measured in seedlings of the ethylene-overproducing eto3 mutant. When Met cycle knockout and ethylene overproduction were combined in the mtk/eto3 double mutant, a reduced capacity for ethylene synthesis was observed in seedlings. Even though mature eto3 plants did not produce elevated ethylene levels, and AdoMet homeostasis in eto3 plants did not differ from that in wild type, shoot growth was severely retarded. The mtk/eto3 double mutant displayed a metabolic plant phenotype that was similar to mtk with reduced AdoMet levels at sulfurlimiting conditions. We conclude from our data that the Met cycle contributes to the maintenance of AdoMet homeostasis, especially when de novo AdoMet synthesis is limited. Our data further showed that the Met cycle is required to sustain high rates of ethylene synthesis. Expression of the Met cycle genes AtMTN1, AtMTN2, AtMTK, AtARD1, AtARD2, AtARD3 and AtARD4 was not regulated by ethylene. This result is in contrast to that found in rice where OsARD1 and OsMTK are induced in response to ethylene. We hypothesize that the regulation of the Met cycle by ethylene may be restricted to plants that naturally produce high quantities of ethylene for a prolonged period of time.

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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.