Glycosyl hydrolases of cell wall are induced by sugar starvation in Arabidopsis

Three Arabidopsis genes encoding a putative beta-galactosidase (At5g56870), beta-xylosidase (At5g49360) and beta-glucosidase (At3g60140) are induced by sugar starvation. The deduced proteins belong to the glycosyl hydrolase families 35, 3 and 1, respectively. They are predicted to be secretory proteins that play roles in modification of cell wall polysaccharides based on amino acid similarity. The beta-galactosidase encoded by At5g56870 was identified as a secretory protein in culture medium of suspension cells by mass spectrometry analysis. This protein was specifically detected under sugar-starved conditions with a specific antibody. Induction of these genes was repressed in suspension cells grown with galactose, xylose and glucose, as well as with sucrose. In planta, expression of the genes and protein accumulation were detected when photosynthesis was inhibited. Glycosyl hydrolase activity against galactan also increased during sugar starvation. The amount of monosaccharide in pectin and hemicellulose in detached leaves decreased in response to sugar starvation. These findings suggest that the cell wall may function as a storage reserve of carbon in addition to providing physical support for the plant body.     

Enzymatic activity and motility of recombinant Arabidopsis myosin XI, MYA1

We expressed recombinant Arabidopsis myosin XI (MYA1), in which the motor domain of MYA1 was connected to an artificial lever arm composed of triple helical repeats of Dictyostelium alpha-actinin, in order to understand its motor activity and intracellular function. The V(max) and K(actin) of the actin-activated Mg(2+) ATPase activity of the recombinant MYA1 were 50.7 Pi head(-1) s(-1) and 30.2 microM, respectively, at 25 degrees C. The recombinant MYA1 could translocate actin filament at the maximum velocity of 1.8 microm s(-1) at 25 degrees C in the in vitro motility assay. The value corresponded to a motility of 3.2 microm s(-1) for native MYA1 if we consider the difference in the lever arm length, and this value was very close to the velocity of cytoplasmic streaming in Arabidopsis hypocotyl epidermal cells. The extent of inhibition by ADP of the motility of MYA1 was similar to that of the well-known processive motor, myosin V, suggesting that MYA1 is a processive motor. The dissociation rate of the actin-MYA1-ADP complex induced by ATP (73.5 s(-1)) and the V(max) value of the actin-activated Mg(2+) ATPase activity revealed that MYA1 stays in the actin-bound state for about 70% of its mechanochemical cycle time. This high ratio of actin-bound states is also a characteristic of processive motors. Our results strongly suggest that MYA1 is a processive motor and involved in vesicle transport and/or cytoplasmic streaming.     

Identification of the gravitropism-related rice gene LAZY1 and elucidation of LAZY1-dependent and -independent gravity signaling pathways

We identified the gene responsible for three allelic lazy1 mutations of Japonica rice (Oryza sativa L.) by map-based cloning, complementation and RNA interference. Sequence analysis and database searches indicated that the wild-type gene (LAZY1) encodes a novel and unique protein (LAZY1) and that rice has no homologous gene. Two lazy1 mutants were LAZY1 null. Confirming and advancing the previously reported results on lazy1 mutants, we found the following. (i) Gravitropism is impaired, but only partially, in lazy1 coleoptiles. (ii) Circumnutation, observed in dark-grown coleoptiles, is totally absent from lazy1 coleoptiles. (iii) Primary roots of lazy1 mutants show normal gravitropism and circumnutation. (iv) LAZY1 is expressed in a tissue-specific manner in gravity-sensitive shoot tissues (i.e. coleoptiles, leaf sheath pulvini and lamina joints) and is little expressed in roots. (v) The gravitropic response of lazy1 coleoptiles is kinetically separable from that absent from lazy1 coleoptiles. (vi) Gravity-induced lateral translocation of auxin, found in wild-type coleoptiles, does not occur in lazy1 coleoptiles. Based on the genetic and physiological evidence obtained, it is concluded that LAZY1 is specifically involved in shoot gravitropism and that LAZY1-dependent and -independent signaling pathways occur in coleoptiles. It is further concluded that, in coleoptiles, only the LAZY1-dependent gravity signaling involves asymmetric distribution of auxin between the two lateral halves and is required for circumnutation.     

Involvement of Polypyrimidine Tract-Binding Protein (PTB)-Related Proteins in Pollen Germination in Arabidopsis

The pollen grains of most angiosperms contain stores of RNAsand their translation products required for pollen germinationand subsequent early elongation of pollen tubes. Polypyrimidinetract-binding protein (PTB), which is involved in the regulationof pre-mRNA alternative splicing, internal ribosomal entry site(IRES)-mediated translation and mRNA localization/sorting, isknown to act as a bridging molecule between RNAs and a varietyof cellular factors to fulfill cellular functions in both thenucleus and cytoplasm. Moreover, it has been reported that PTBplays roles in the differentiation and development of animalcells and tissues. In the Arabidopsis genome, there are twoPTB-related genes, tentatively termed AtPTB1 and AtPTB2. Inthe present study, the physiological functions of AtPTBs wereinvestigated using genetic and cytological approaches. The AtPTBpromoter was highly active in vegetative cells of mature pollengrains, and AtPTB was localized in the nucleus and cytoplasmof these vegetative cells. Mutations in the AtPTB genes resultedin decreased germination efficiency, and this effect was rescuedby introduction of the AtPTB2 promoter::AtPTB2–GFP. Takentogether, these findings suggest that AtPTB is involved in pollengermination through possible RNA metabolism processes in late-maturingand mature pollen grains.     

AHK5 histidine kinase regulates root elongation through an ETR1-dependent abscisic acid and ethylene signaling pathway in Arabidopsis thaliana

The Arabidopsis thaliana genome encodes a small family of histidine (His) protein kinases, some of which have redundant functions as ethylene receptors, whereas others serve as cytokinin receptors. The most poorly characterized of these is authentic histidine kinase 5 (AHK5; also known as cytokinin-independent 2, CKI2). Here we characterize three independent ahk5 mutants, and show that they have a common phenotype. Our results suggest that AHK5 His-kinase acts as a negative regulator in the signaling pathway in which ethylene and ABA inhibit the root elongation through ETR1 (an ethylene receptor).     

Requirement for the gravity-controlled transport of auxin for a negative gravitropic response of epicotyls in the early growth stage of etiolated pea seedlings

Gravity-controlled transport of auxin was studied for a negative gravitropic response in the early growth stage of etiolated pea (Pisum sativum L. cv. Alaska) seedlings, in which epicotyl bending was observed near the cotyledon nodes of the seedlings grown continuously from seeds germinated in a horizontal or an inclined position. Increased expression of an auxin-inducible gene, PsIAA4/5, was observed in the elongated side of epicotyls grown in a horizontal or an inclined position. Regardless of the conditions of seed germination, polar auxin transport in the proximal side of the first internodes of the seedlings was significantly higher than in the distal side. Polar auxin transport in the proximal side of epicotyls grown in an inclined position was significantly lower than in those grown in a horizontal position. In contrast, lateral auxin distribution from the proximal to distal sides in epicotyls grown in an inclined position was significantly higher than in epicotyls grown in a horizontal position. Accumulation of PsPIN1 mRNA encoding a putative auxin efflux facilitator, which was observed in vascular tissue, cortex and epidermis in the proximal and distal sides of epicotyls, was markedly influenced by gravistimulation. These results strongly suggest that gravistimulation induces changeable polar auxin transport and one-way lateral auxin distribution in epicotyls as well as asymmetric auxin accumulation in the proximal and distal sides of epicotyls, resulting in a negative gravitropic response of epicotyls in the early growth stage of pea seedlings.     

Genetic linkages between circadian clock-associated components and phytochrome-dependent red light signal transduction in Arabidopsis thaliana

The current best candidates for Arabidopsis thaliana clock components are CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) and its homolog LHY (LATE ELONGATED HYPOCOTYL). In addition, five members of a small family, PSEUDO-RESPONSE REGULATORS (including PRR1, PRR3, PRR5, PRR7 and PRR9), are believed to be another type of clock component. The originally described member of PRRs is TOC1 (or PRR1) (TIMING OF CAB EXPRESSION 1). Interestingly, seedlings of A. thaliana carrying a certain lesion (i.e. loss-of-function or misexpression) of a given clock-associated gene commonly display a characteristic phenotype of light response during early photomorphogenesis. For instance, cca1 lhy double mutant seedlings show a shorter hypocotyl length than the wild type under a given fluence rate of red light (i.e. hypersensitivity to red light). In contrast, both toc1 single and prr7 prr5 double mutant seedlings with longer hypocotyls are hyposensitive under the same conditions. These phenotypes are indicative of linkage between the circadian clock and red light signal transduction mechanisms. Here this issue was addressed by conducting combinatorial genetic and epistasis analyses with a large number of mutants and transgenic lines carrying lesions in clock-associated genes, including a cca1 lhy toc1 triple mutant and a cca1 lhy prr7 prr5 quadruple mutant. Taking these results together, we propose a genetic model for clock-associated red light signaling, in which CCA1 and LHY function upstream of TOC1 (PRR1) in a negative manner, in turn, TOC1 (PRR1) serves as a positive regulator. PRR7 and PRR5 also act as positive regulators, but independently from TOC1 (PRR1). It is further suggested that these signaling pathways are coordinately integrated into the phytochrome-mediated red light signal transduction pathway, in which PIF3 (PHYTOCHROME-INTERACTING FACTOR 3) functions as a negative regulator immediately downstream of phyB.     

Arabidopsis cytokinin-resistant mutant, cnr1, displays altered auxin responses and sugar sensitivity

Based upon the phenotype of young, dark-grown seedlings, a cytokinin-resistant mutant, cnr1, has been isolated, which displays altered cytokinin- and auxin-induced responses. The mutant seedlings possess short hypocotyls and open apical hooks (in dark), and display agravitropism, hyponastic cotyledons, reduced shoot growth, compact rosettes and short roots with increased adventitious branching and reduced number of root hairs. A number of these features invariably depend upon auxin/cytokinin ratio but the cnr1 mutant retains normal sensitivity towards auxin as well as auxin polar transport inhibitor, TIBA, although upregulation of primary auxin-responsive Aux/IAA genes is reduced. The mutant shows resistance towards cytokinin in hypocotyl/root growth inhibition assays, displays reduced regeneration in tissue cultures (cytokinin response) and decreased sensitivity to cytokinin for anthocyanin accumulation. It is thus conceivable that due to reduced sensitivity to cytokinin, the cnr1 mutant also shows altered auxin response. Surprisingly, the mutant retains normal sensitivity to cytokinin for induction of primary response genes, the type-A Arabidopsis response regulators, although the basal level of their expression was considerably reduced as compared to the wild-type. The zeatin and zeatin riboside levels, as estimated by HPLC, and the cytokinin oxidase activity were comparable in the cnr1 mutant and the wild-type. The hypersensitivity to red light (in hypocotyl growth inhibition assay), partial photomorphogenesis in dark, and hypersensitivity to sugars, are some other features displayed by the cnr1 mutant. The lesion in the cnr1 mutant has been mapped to the top of chromosome 1 where no other previously known cytokinin-resistant mutant has been mapped, indicating that the cnr1 mutant defines a novel locus involved in hormone, light and sugar signalling.     

Differential Expression Control and Polarized Distribution of Plasma Membrane-Resident SYP1 SNAREs in Arabidopsis thaliana

Membrane trafficking to the plasma membrane (PM) is a highlyorganized process which enables plant cells to build up theirbodies. SNARE (soluble N-ethylmaleimide-sensitive factor attachmentprotein receptor) genes, which encode the proteins involvedin membrane trafficking, are much more abundant in the Arabidopsisgenome than in that of any other eukaryote. We have previouslyshown that a large number of SNARE molecules in the Arabidopsiscell are localized predominantly on the PM. In the present study,in order to elucidate the physiological function of each PM-localizedSNARE, we analyzed the spatiotemporal expression profiling ofnine SYP1s that are resident in the PM of Arabidopsis, and usedthe information thus acquired to generate transgenic Arabidopsisplants expressing green fluorescent protein-fused Qa-SNAREsunder control of their authentic promoters. Among the nine SYP1s,only SYP132 is expressed ubiquitously in all tissues throughoutplant development. The expression patterns of the other SYP1s,in contrast, are tissue specific, and all different from oneanother. A particularly noteworthy example is SYP123, whichis predominantly expressed in root hair cells during root development,and shows a focal accumulation pattern at the tip region ofroot hairs. These results suggest that SYP132 is involved inconstitutive membrane trafficking to the PM throughout plantdevelopment, while the other SYP1s are involved in membranetrafficking events such as root formation or tip growth of roothair, with some redundancy.     

The CCCH-type zinc finger proteins AtSZF1 and AtSZF2 regulate salt stress responses in Arabidopsis

The molecular mechanism governing the response of plants to salinity stress, one of the most significant limiting factors for agriculture worldwide, has just started to be revealed. Here, we report AtSZF1 and AtSZF2, two closely related CCCH-type zinc finger proteins, involved in salt stress responses in Arabidopsis. The expression of AtSZF1 and AtSZF2 is quickly and transiently induced by NaCl treatment. Mutants disrupted in the expression of AtSZF1 or AtSZF2 exhibit increased expression of a group of salt stress-responsive genes in response to high salt. Significantly, the atszf1-1/atszf2-1 double mutant displays more sensitive responses to salt stress than the atszf1-1 or atszf2-1 single mutants and wild-type plants. On the other hand, transgenic plants overexpressing AtSZF1 show reduced induction of salt stress-responsive genes and are more tolerant to salt stress. We also showed that AtSZF1 is localized in the nucleus. Taken together, these results demonstrated that AtSZF1 and AtSZF2 negatively regulate the expression of salt-responsive genes and play important roles in modulating the tolerance of Arabidopsis plants to salt stress.     

Identification of genes expressed in vascular tissues using NPA-induced vascular overgrowth in Arabidopsis

The genetic basis of vascular differentiation and function isrelatively poorly understood, partly due to the difficulty ofscreening for mutants defective in internal vascular tissues.Here we present an approach based on a predicted increase invascular-related gene expression in response to an auxin transportinhibitor-induced vascular overgrowth. We used microarray analysesto identify 336 genes that were up-regulated 2-fold in shoottissues of Arabidopsis thaliana showing vascular overgrowth.Promoter–marker gene fusions revealed that 38 out of 40genes with 4-fold up-regulation in vascular overgrowth tissueshad vascular-related expression in transgenic Arabidopsis plants.Obtained expression patterns included cambial tissues and differentiatingxylem, phloem and fibers. A total of 15 genes were found tohave vascular-specific expression patterns in the leaves and/orinflorescence stems. This study provides empirical evidenceof the efficiency of the approach and describes for the firsttime the in situ expression patterns of the majority of theassessed genes.     

Genetic linkages of the circadian clock-associated genes, TOC1, CCA1 and LHY, in the photoperiodic control of flowering time in Arabidopsis thaliana

In Arabidopsis thaliana, the flowering time is regulated through the circadian clock that measures day-length and modulates the photoperiodic CO-FT output pathway in accordance with the external coincidence model. Nevertheless, the genetic linkages between the major clock-associated TOC1, CCA1 and LHY genes and the canonical CO-FT flowering pathway are less clear. By employing a set of mutants including an extremely early flowering toc1 cca1 lhy triple mutant, here we showed that CCA1 and LHY act redundantly as negative regulators of the photoperiodic flowering pathway. The partly redundant CCA1/LHY functions are largely, but not absolutely, dependent on the upstream TOC1 gene that serves as an activator. The results of examination with reference to the expression profiles of CO and FT in the mutants indicated that this clock circuitry is indeed linked to the CO-FT output pathway, if not exclusively. For this linkage, the phase control of certain flowering-associated genes, GI, CDF1 and FKF1, appears to be crucial. Furthermore, the genetic linkage between TOC1 and CCA1/LHY is compatible with the negative and positive feedback loop, which is currently believed to be a core of the circadian clock. The results of this study suggested that the circadian clock might open an exit for a photoperiodic output pathway during the daytime. In the context of the current clock model, these results will be discussed in connection with the previous finding that the same clock might open an exit for the early photomorphogenic output pathway during the night-time.     

Characterization of OsPID, the rice ortholog of PINOID, and its possible involvement in the control of polar auxin transport

PINOID, a serine threonine protein kinase in Arabidopsis, controls auxin distribution through a positive control of subcellular localization of PIN auxin efflux carriers. Compared with the rapid progress in understanding mechanisms of auxin action in dicot species, little is known about auxin action in monocot species. Here, we describe the identification and characterization of OsPID, the PINOID ortholog of rice. Phylogenetic analysis showed that the rice genome contains a single PID ortholog, OsPID. Constitutive overexpression of OsPID caused a variety of abnormalities, such as delay of adventitious root development, curled growth of shoots and agravitropism. Abnormalities observed in the plants that overexpress OsPID could be phenocopied by treatment with an inhibitor of active polar transport of auxin, indicating that OsPID could be involved in the control of polar auxin transport in rice. Analysis of OsPID mRNA distribution showed a complex pattern in shoot meristems, indicating that it probably plays a role in the pattern formation and organogenesis in the rice shoot.     

Protophloem differentiation in early Arabidopsis thaliana development

During Arabidopsis embryogenesis, procambial cells undergo coordinated, asymmetric cell divisions, giving rise to vascular precursor cells (protophloem and protoxylem precursors). After germination, these cells terminally differentiate into specialized conducting cells, referred to as protophloem and protoxylem cells. Few readily identifiable markers of the onset of specification and differentiation are available, hampering the molecular genetic analysis of protophloem development. Confocal microscopy was used to investigate the patterning and differentiation of phloem cells during early plant development. Longitudinal divisions of phloem initials allowed the identification of protophloem precursor cells and adjacent metaphloem initials along the length of the plant. During germination, protophloem differentiation was observed at two independent locations, in the cotyledons and the hypocotyl. In both locations, differentiation was concomitant with cell elongation. We identified five gene-trap lines (PD1-PD5) with marker gene expression in immature protophloem elements. The spatio-temporal marker expression pattern of the lines divides them into two groups. The early specification markers PD4 and PD5 were expressed in developing organs before procambium formation and then became restricted to phloem initial cells. The protophloem precursor markers PD1-PD3 were expressed in differentiating protophloem cells at different stages of their development. All markers were expressed transiently and iteratively during the differentiation of protophloem in newly formed organs. Flanking genes were identified for four out of five gene-trap insertion lines. The possible function of these genes with respect to phloem differentiation is discussed.