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...

Histone deacetylase (HDAC) is a chromatin-remodeling factor that contributes to transcriptional repression in eukaryotes. In Arabidopsis (Arabidopsis thaliana), the transcription factors LEAFY COTYLEDON1 (LEC1), FUSCA3 (FUS3), and ABSCISIC ACID INSENSITIVE3 (ABI3) play key roles in embryogenesis. Although the repression of embryogenesis-related genes during germination has been proposed to occur, the role of HDAC in this process has not been elucidated. To address this question, the effects of an HDAC inhibitor and suppression of the Arabidopsis HDAC genes on this process were investigated. Here, we show that treatment of an HDA6 repression line with the HDAC inhibitor trichostatin A resulted in growth arrest and elevated transcription of LEC1, FUS3, and ABI3 during germination. The growth-arrest phenotype of the repression line was suppressed by lec1, fus3, and abi3. An HDA6/HDA19 double-repression line displayed arrested growth after germination and the formation of embryo-like structures on the true leaves of 6-week-old plants even without trichostatin A. The growth-arrest phenotype of this line was rescued by lec1. These results suggest that during germination in Arabidopsis, HDA6 and HDA19 redundantly regulate the repression of embryonic properties directly or indirectly via repression of embryo-specific gene function.

Background and Purpose-Hyperglycemia is linked to a worse outcome after ischemic stroke. Among the manifestations of brain damage caused by ischemia are blood-brain barrier (BBB) disruption and edema formation. Oxidative stress and matrix metalloproteinase-9 (MMP-9) activation are implicated in BBB dysfunction after ischemia/reperfusion injury. Our present study was designed to clarify the relation among hyperglycemia, oxidative stress, and MMP-9 activation associated with BBB dysfunction after transient focal cerebral ischemia (tFCI). Methods-We used a model of 60 minutes of middle cerebral artery occlusion on the following animals: normoglycemic wild-type rats, wild-type rats with hyperglycemia induced by streptozotocin, and human copper/zinc superoxide dismutase (SOD1) transgenic rats with streptozotocin-induced hyperglycemia. We evaluated edema volume, Evans blue leakage, and oxidative stress, such as the carbonyl groups and oxidized hydroethidine (HEt), SOD activity, and gelatinolytic activity, including MMP-9. Results-Hyperglycemia significantly increased edema volume and Evans blue leakage. Moreover, it enhanced the levels of the carbonyl groups, the oxidized HEt signals, and MMP-9 activity after tFCI without alteration in SOD activity. Gelatinolytic activity and oxidized HEt signals had a clear spatial relation in the hyperglycemic rats. SOD1 overexpression reduced the hyperglycemia-enhanced Evans blue leakage and MMP-9 activation after tFCI. Conclusions-Hyperglycemia increases oxidative stress and MMP-9 activity, exacerbating BBB dysfunction after ischemia/reperfusion injury. Superoxide overproduction may be a causal link among hyperglycemia, MMP-9 activation, and BBB dysfunction.

When grafting or wounding disconnects stem tissues, new tissues are generated to restore the lost connection. In this study, the molecular mechanism of such healing was elucidated in injured stems of Arabidopsis. Soon after the inflorescence stems were incised, the pith cells started to divide. This process was strongly inhibited by the elimination of cauline leaves, shoot apices, or lateral buds that reduced the indole-3-acetic acid supply. Microarray and quantitative RT-PCR analyses revealed that genes related to cell division, phytohormones, and transcription factors were expressed because of incision. Among them, two plant-specific transcription factor genes, ANAC071 and RAP2.6L, were abundantly expressed. ANAC071 was expressed at 1-3 d after cutting exclusively in the upper region of the cut gap, with concomitant accumulation of indole-3-acetic acid. In contrast, RAP2.6L was expressed at 1 d after cutting exclusively in the lower region, with concomitant deprivation of indole-3-acetic acid. The expression of ANAC071 and RAP2.6L were also promoted by ethylene and jasmonic acid, respectively. In transformants suppressing the function of RAP2.6L or ANAC071, the division of pith cells was inhibited. Furthermore, the ethylene signaling-defective ein2 mutant showed incomplete healing. Hence, plant-specific transcription factors differentially expressed around the cut position were essential for tissue reunion of Arabidopsis wounded flowering stems and were under opposite control by polar-transported auxin, with modification by the ethylene and jasmonic acid wound-inducible hormones.regeneration | meristem | stress

SummaryTwo cDNA clones, cATMPK1 and cATMPK2, encoding MAP kinases (mitogen-activated protein kinases) have been cloned from Arabidopsis thaliana and their nucleotide sequences have been determined. Putative proteins encoded by ATMPK1 and ATMPK2 genes, designated ATMPK1 and ATMPK2, contain 370 and 376 amino acid residues, respectively, and are 88.7% identical at the amino acid sequence level. ATMPK1 and ATMPK2 exhibit significant similarity to rat ERK2 (49%) and Xenopus MAP kinase (50%). The amino acid residues corresponding to the sites of phosphorylation (Thr-Glu-Tyr) that are involved in the activation of MAP kinases are conserved in ATMPK1 and ATMPK2. Northern blot analysis indicates that the ATMPK1 and ATMPK2 mRNAs are significantly present in all the organs except seeds. Genomic Southern blot analysis suggests that there are a few additional genes that are related to ATMPK1 and ATMPK2 in the Arabidopsis genome. Purified Xenopus MAP kinase kinase (MAPK kinase) phosphorylates ATMPK1 and ATMPK2 proteins that have been expressed in Escherichia coil, activating these enzymes. A rapid and transient activation of 46-kDa protein kinase activity that phosphorylated myelin basic protein (MBP) was detected when auxinstarved tobacco BY-2 cells were treated with synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D).

SUMMARYTranspiration and gas exchange occur through stomata. Thus, the control of stomatal aperture is important for the efficiency and regulation of water use, and for the response to drought. Here, we demonstrate that SIZ1-mediated endogenous salicylic acid (SA) accumulation plays an important role in stomatal closure and drought tolerance. siz1 reduced stomatal apertures. The reduced stomatal apertures of siz1 were inhibited by the application of peroxidase inhibitors, salicylhydroxamic acid and azide, which inhibits SA-dependent reactive oxygen species (ROS) production, but not by an NADPH oxidase inhibitor, diphenyl iodonium chloride, which inhibits ABA-dependent ROS production. Furthermore, the introduction of nahG into siz1, which reduces SA accumulation, restored stomatal opening. Stomatal closure is generally induced by water deficit. The siz1 mutation caused drought tolerance, whereas nahG siz1 suppressed the tolerant phenotype. Drought stresses also induced expression of SA-responsive genes, such as PR1 and PR2. Furthermore, other SA-accumulating mutants, cpr5 and acd6, exhibited stomatal closure and drought tolerance, and nahG suppressed the phenotype of cpr5 and acd6, as did siz1 and nahG siz1. Together, these results suggest that SIZ1 negatively affects stomatal closure and drought tolerance through the accumulation of SA.

SummarySomatic embryogenesis is an obvious experimental evidence of totipotency, and is used as a model system for studying the mechanisms of de-differentiation and re-differentiation of plant cells. Although Arabidopsis is widely used as a model plant for genetic and molecular biological studies, there is no available tissue culture system for inducing somatic embryogenesis from somatic cells in this plant. We established a new tissue culture system using stress treatment to induce somatic embryogenesis in Arabidopsis. In this system, stress treatment induced formation of somatic embryos from shoot-apical-tip and¯oral-bud explants. The somatic embryos grew into young plantlets with normal morphology, including cotyledons, hypocotyls, and roots, and some embryo-speci®c genes (ABI3 and FUS3) were expressed in these embryos. Several stresses (osmotic, heavy metal ion, and dehydration stress) induced somatic embryogenesis, but the optimum stress treatment differed between different stressors. When we used mannitol to cause osmotic stress, the optimal conditions for somatic embryogenesis were 6±9 h of culture on solid B5 medium containing 0.7 M mannitol, after which the explants were transferred to B5 medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 4.5 lM), but no mannitol. Using this tissue culture system, we induced somatic embryogenesis in three major ecotypes of Arabidopsis thaliana ± Ws, Col, and Ler.

Intercellular spaces are often the first sites invaded by pathogens. In the spaces of tobacco mosaic virus (TMV)-infected and necrotic lesion-forming tobacco (Nicotiana tabacum L.) leaves, we found that an inducer for acidic pathogenesis-related (PR) proteins was accumulated. The induction activity was recovered in gel-filtrated fractions of low molecular mass with a basic nature, into which authentic spermine (Spm) was eluted. We quantified polyamines in the intercellular spaces of the necrotic lesion-forming leaves and found 20-fold higher levels of free Spm than in healthy leaves. Among several polyamines tested, exogenously supplied Spm induced acidic PR-1 gene expression. Immunoblot analysis showed that Spm treatment increased not only acidic PR-1 but also acidic PR-2, PR-3, and PR-5 protein accumulation. Treatment of healthy tobacco leaves with salicylic acid (SA) caused no significant increase in the level of endogenous Spm, and Spm did not increase the level of endogenous SA, suggesting that induction of acidic PR proteins by Spm is independent of SA. The size of TMV-induced local lesions was reduced by Spm treatment. These results indicate that Spm accumulates outside of cells after lesion formation and induces both acidic PR proteins and resistance against TMV via a SA-independent signaling pathway.