Several genes are known to regulate circadian rhythms in Arabidopsis, but the identity of the central oscillator has not been established. LHY and CCA1 are related MYB-like transcription factors proposed to be closely involved. Here we demonstrate that, as shown previously for CCA1, inactivation of LHY shortens the period of circadian rhythms in gene expression and leaf movements. By constructing lhy cca1-1 double mutants, we show that LHY and CCA1 are partially redundant and essential for the maintenance of circadian rhythms in constant light. Under light/dark cycles the lhy cca1-1 plants show dramatically earlier phases of expression of GI and TOC1, genes associated with the generation of circadian rhythms and the promotion of LHY and CCA1 expression. We conclude that LHY and CCA1 appear to be negative regulatory elements required for central oscillator function.

;Protein phosphorylation has pivotal roles in ABA and osmotic stress signaling in higher plants. Two protein phosphatase genes, ABI1 and ABI2, are known to regulate these signaling pathways in Arabidopsis. The identity of ABAactivated protein kinases required for the ABA signaling, however, remains to be elucidated. Here we demonstrate that two protein kinases, p44 and p42, were activated by ABA in Arabidopsis T87 cultured cells, and at least one protein kinase, p44, was activated not only by ABA but also by low humidity in Arabidopsis plants. Analysis of T-DNA knockout mutants and biochemical analysis using a specific antibody revealed that the p44 is encoded by a SnRK2-type protein kinase gene, SRK2E. The srk2e mutation resulted in a wilty phenotype mainly due to loss of stomatal closure in response to a rapid humidity decrease. ABAinducible gene expression of rd22 and rd29B was suppressed in srk2e. These results show that SRK2E plays an important role in ABA signaling in response to water stress.

The circadian clock acts as the timekeeping mechanism in photoperiodism. In Arabidopsis thaliana, a circadian clockcontrolled flowering pathway comprising the genes GIGANTEA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) promotes flowering specifically under long days. Within this pathway, GI regulates circadian rhythms and flowering and acts earlier in the hierarchy than CO and FT, suggesting that GI might regulate flowering indirectly by affecting the control of circadian rhythms. We studied the relationship between the roles of GI in flowering and the circadian clock using late elongated hypocotyl circadian clock associated1 double mutants, which are impaired in circadian clock function, plants overexpressing GI (35S:GI), and gi mutants. These experiments demonstrated that GI acts between the circadian oscillator and CO to promote flowering by increasing CO and FT mRNA abundance. In addition, circadian rhythms in expression of genes that do not control flowering are altered in 35S:GI and gi mutant plants under continuous light and continuous darkness, and the phase of expression of these genes is changed under diurnal cycles. Therefore, GI plays a general role in controlling circadian rhythms, and this is different from its effect on the amplitude of expression of CO and FT. Functional GI:green fluorescent protein is localized to the nucleus in transgenic Arabidopsis plants, supporting the idea that GI regulates flowering in the nucleus. We propose that the effect of GI on flowering is not an indirect effect of its role in circadian clock regulation, but rather that GI also acts in the nucleus to more directly promote the expression of flowering-time genes. INTRODUCTIONInduction of flowering in response to daylength synchronizes flowering to the changing seasons and is believed to be important in adaptation of plants to growth at different latitudes (Ray and Alexander, 1966). Physiological experiments implicated the circadian clock as the timekeeping mechanism that enables the measurement of daylength Yanovsky and Kay, 2003). Forward genetics in Arabidopsis thaliana identified a genetic pathway that promotes flowering specifically on exposure to long days (LDs) (Searle and Coupland, 2004), and the role of the circadian clock in photoperiodic time measurement was confirmed by demonstrating that transcription of the genes that act in this pathway is circadian clock controlled. Mutations in one of these genes, GIGANTEA (GI), both impair circadian rhythms and delay flowering. Here, we use moleculargenetic approaches to compare the role of GI in the circadian system with its function in controlling flowering. GI, CONSTANS (CO), and FLOWERING LOCUS T (FT)were placed in the Arabidopsis photoperiod pathway based on genetic analysis (Redei, 1962;Koornneef et al., 1991Koornneef et al., , 1998. Loss-offunction mutations in each of these genes delay flowering under LDs but have little or no effect under short days (SDs). Genetic epistasis and analysis of expression of these three genes in mutant and wild-type background...

ABI1 and ABI2 encode PP2C-type protein phosphatases and are thought to negatively regulate many aspects of abscisic acid (ABA) signaling, including stomatal closure in Arabidopsis. In contrast, SRK2E/OST1/SnRK2.6 encodes an Arabidopsis SnRK2 protein kinase and acts as a positive regulator in the ABA-induced stomatal closure. SRK2E/OST1 is activated by osmotic stress as well as by ABA, but the independence of the two activation processes has not yet been determined. Additionally, interaction between SRK2E/OST1 and PP2C-type phosphatases (ABI1 and ABI2) is not understood. In the present study, we demonstrated that the abi1-1 mutation, but not the abi2-1 mutation, strongly inhibited ABA-dependent SRK2E/OST1 activation. In contrast, osmotic stress activated SRK2E/OST1 even in abi1-1 and aba2-1 plants. The C-terminal regulatory domain of SRK2E/OST1 was required for its activation by both ABA and osmotic stress in Arabidopsis. The C-terminal domain was functionally divided into Domains I and II. Domain II was required only for the ABA-dependent activation of SRK2E/OST1, whereas Domain I was responsible for the ABA-independent activation. Full-length SRK2E/OST1 completely complemented the wilty phenotype of the srk2e mutant, but SRK2E/OST1 lacking Domain II did not. Domain II interacted with the ABI1 protein in a yeast two-hybrid assay. Our results suggested that the direct interaction between SRK2E/OST1 and ABI1 through Domain II plays a critical role in the control of stomatal closure.

SummaryMitogen-activated protein kinase (MAP kinase, MAPK) cascades play pivotal roles in signal transduction of extracellular stimuli, such as environmental stresses and growth regulators, in various organisms. Arabidopsis thaliana MAP kinases constitute a gene family, but stimulatory signals for each MAP kinase have not been elucidated. Here we show that environmental stresses such as low temperature, low humidity, hyper-osmolarity, touch and wounding induce rapid and transient activation of the Arabidopsis MAP kinases ATMPK4 and ATMPK6. Activation of ATMPK4 and ATMPK6 was associated with tyrosine phosphorylation but not with the amounts of mRNA or protein. Kinetics during activation differ between these two MAP kinases. These results suggest that ATMPK4 and ATMPK6 are involved in distinct signal transduction pathways responding to these environmental stresses.

We describe here the cloning and characterization of a cDNA encoding a protein kinase that has high sequence homology to members of the mitogen-activated protein kinase (MAPK)

The plant hormone jasmonic acid (JA) plays a key role in the environmental stress responses and developmental processes of plants. Although ATMYC2/JASMONATE-INSENSITIVE1 (JIN1) is a major positive regulator of JA-inducible gene expression and essential for JA-dependent developmental processes in Arabidopsis thaliana, molecular mechanisms underlying the control of ATMYC2/JIN1 expression remain largely unknown. Here, we identify a mitogen-activated protein kinase (MAPK) cascade, MAPK KINASE 3 (MKK3)-MAPK 6 (MPK6), which is activated by JA in Arabidopsis. We also show that JA negatively controls ATMYC2/JIN1 expression, based on quantitative RT-PCR and genetic analyses using gain-of-function and loss-offunction mutants of the MKK3-MPK6 cascade. These results indicate that this kinase unit plays a key role in JA-dependent negative regulation of ATMYC2/JIN1 expression. Both positive and negative regulation by JA may be used to fine-tune ATMYC2/JIN1 expression to control JA signaling. Moreover, JA-regulated root growth inhibition is affected by mutations in the MKK3-MPK6 cascade, which indicates important roles in JA signaling. We provide a model explaining how MPK6 can convert three distinct signals-JA, pathogen, and cold/salt stress-into three different sets of responses in Arabidopsis.

The isolation and characterization is reported of a cDNA for delta 1-pyrroline-5-carboxylate (P5C) synthetase (cAtP5CS), an enzyme involved in the biosynthesis of proline, from a cDNA library prepared from a dehydrated rosette plant of Arabidopsis thaliana. Southern blot analysis suggested that only one copy of the corresponding gene (AtP5CS) is present in A. thaliana. The deduced amino acid sequence of the P5CS protein (AtP5CS) from A. thaliana exhibited 74% homology to that of the P5CS from Vigna aconitifolia. Northern blot analysis revealed that the gene for P5CS was induced by dehydration, high salt and treatment with ABA, while it was not induced by heat or cold treatment. Moreover, the simultaneous accumulation of proline was observed as a result of the former treatments in A. thaliana. A cDNA for P5C reductase (cAtP5CR) was also isolated from A. thaliana and Northern blot analysis was performed. The AtP5CR gene was not induced to a significant extent by dehydration or high-salt stress. These observations suggest that the AtP5CS gene plays a principal role in the biosynthesis of proline in A. thaliana under osmotic stress.