Reduced Expression of PRX2/ATPRX1, PRX8, PRX35, and PRX73 Affects Cell Elongation, Vegetative Growth, and Vasculature Structures in Arabidopsis thaliana.

Class III peroxidases (PRXs) are involved in a broad spectrum of physiological and developmental processes throughout the life cycle of plants. However, the specific function of each PRX member in the family remains largely unknown. In this study, we selected four class III peroxidase genes (PRX2/ATPRX1, PRX8, PRX35, and PRX73) from a previous genome-wide transcriptome analysis, and performed phenotypic and morphological analyses, including histochemical staining, in PRX2RNAi, PRX8RNAi, PRX35RNAi, and PRX73RNAi plants. The reduced mRNA levels of corresponding PRX genes in PRX2RNAi, PRX8RNAi, PRX35RNAi, and PRX73RNAi seedlings resulted in elongated hypocotyls and roots, and slightly faster vegetative growth. To investigate internal structural changes in the vasculature, we performed histochemical staining, which revealed alterations in cell wall structures in the main vasculature of hypocotyls, stems, and roots of each PRXRNAi plant compared to wild-type (Col-0) plants. Furthermore, we found that PRX35RNAi plants displayed the decrease in the cell wall in vascular regions, which are involved in downregulation of lignin biosynthesis and biosynthesis-regulated genes' expression. Taken together, these results indicated that the reduced expression levels of PRX2/ATPRX1, PRX8, PRX35, and PRX73 affected hypocotyl and root elongation, vegetative growth, and the vasculature structures in hypocotyl, stem, and root tissues, suggesting that the four class III PRX genes play roles in plant developmental processes.

BAK7 displays unequal genetic redundancy with BAK1 in brassinosteroid signaling and early senescence in Arabidopsis.

BRI1-Associated kinase 1 (BAK1), a five leucine-rich-repeat containing receptor-like serine/threonine kinase, has been shown to have dual functions: mediating brassinosteroid (BR) signaling and acting in the BR-independent plant defense response. Sequence analysis has revealed that BAK1 has two homologs, BAK7 and BAK8. Because BAK8 deviates from the canonical RD kinase motif, we focused on the functional analysis of BAK7. The expression pattern and tissues in which BAK7 appeared partially overlapped with those observed for BAK1. Expression levels of BAK7 increased in the bak1 mutant. Overexpression of BAK7 rescued the bri1 mutant phenotype, indicating that BAK7 can compensate for BAK1 in BR-mediated processes, especially in the absence of BAK1. However, root and hypocotyl elongation patterns of transgenic plants overexpressing BAK1 or BAK7 appeared to be different from the patterns observed in a BRI1 overexpressor. Furthermore, the sensitivity of transgenic plants overexpressing BAK7 to brassinazole, a biosynthetic inhibitor of brassinolide (BL), did not change compared to that of wild-type plants. In addition, we generated transgenic plants expressing BAK7 RNA interference constructs and found severe growth retardation and early senescence in these lines. Taken together, these results suggest that BAK7 is a component of the BR signaling pathway, with varying degrees of genetic redundancy with BAK1, and that it affects plant growth via BL-independent pathways in vivo.

Modulations of AtGSTF10 expression induce stress tolerance and BAK1-mediated cell death.

Glutathione-S-transferases are essential proteins involved in cellular detoxification. The expression of GSTs has been studied extensively under various environmental stressors including xenobiotics. Here, we have isolated AtGST10, one of the phi classes of AtGSTs on the basis of its interaction with BAK1 in a yeast two-hybrid screen. BAK1 is an LRR-RLK, acting in both brassinosteroid signaling and plant defense responses. We found that AtGSTF10 binds to BAK1 through its N-terminal domain. AtGSTF10 is expressed ubiquitously in plant tissues, and the endogenous transcript level of AtGSTF10 was not induced by plant growth regulators or abiotic stressors, except drought, unlike other GSTs. Overexpression of AtGSTF10 conferred higher tolerance to salt and disturbed redox status of transgenic plants. The down-regulation of AtGSTF10 produced by RNA interference caused reduced tolerance to abiotic stress and an accelerated senescence of transformants, indicating that AtGSTF10 is involved in stress tolerance and the BAK1-mediated spontaneous cell death signaling pathway in Arabidopsis.

Hormone- and light-regulated nucleocytoplasmic transport in plants: current status.

The gene regulation mechanisms underlying hormone- and light-induced signal transduction in plants rely not only on post-translational modification and protein degradation, but also on selective inclusion and exclusion of proteins from the nucleus. For example, plant cells treated with light or hormones actively transport many signalling regulatory proteins, transcription factors, and even photoreceptors and hormone receptors into the nucleus, while actively excluding other proteins. The nuclear envelope (NE) is the physical and functional barrier that mediates this selective partitioning, and nuclear transport regulators transduce hormone- or light-initiated signalling pathways across the membrane to mediate nuclear activities. Recent reports revealed that mutating the proteins regulating nuclear transport through the pores, such as nucleoporins, alters the plant's response to a stimulus. In this review, recent works are introduced that have revealed the importance of regulated nucleocytoplasmic partitioning. These important findings deepen our understanding about how co-ordinated plant hormone and light signal transduction pathways facilitate communication between the cytoplasm and the nucleus. The roles of nucleoporin components within the nuclear pore complex (NPC) are also emphasized, as well as nuclear transport cargo, such as Ran/TC4 and its binding proteins (RanBPs), in this process. Recent findings concerning these proteins may provide a possible direction by which to characterize the regulatory potential of hormone- or light-triggered nuclear transport.

Ethylene-induced opposite redistributions of calcium and auxin are essential components in the development of tomato petiolar epinastic curvature.

Calcium has been suggested as an important mediator of gravity signaling transduction within the root cap statocyte. In a horizontally-placed root, it is redistributed in the direction of the gravity vector (i.e. it moves downward) and its redistribution is closely correlated with auxin downward movement. However, the involvement of calcium in the regulation of ethylene-induced epinasty and auxin movement is not known. In this report, we examined the involvement of calcium in lateral auxin transport during ethylene-induced epinasty in an effort to understand the relationship among calcium, auxin, and ethylene. Ethylene-induced epinasty was further stimulated by exogenously applied Ca2+, the calcium effect being the strongest among divalent cations tested. Pretreatment with NPA, an auxin transport inhibitor, negated the promotive effect of calcium ions on the petiolar epinasty. Ethylene caused redistribution/differential accumulation of 45Ca2+ toward the morphologically lower (abaxial) side of the leaf petioles, an effect opposite to that of 14C-IAA redistribution. Verapamil, a Ca2+ channel blocker, inhibited ethylene-induced epinasty, as well as the redistribution of 14C-IAA and 45Ca2+. When the petiole was inverted in the presence or absence of ethylene, the direction of 45Ca2+ differential accumulation was still toward the morphologically abaxial side of the petiole during epinastic movement regardless of gravitational direction. These results suggest that gravity-insensitive, ethylene-induced Ca2+ redistribution and accumulation toward the abaxial side are closely coupled to the adaxial auxin redistribution/accumulation and, in turn, to the petiolar epinasty.

The tobacco gene Ntcyc07 confers arsenite tolerance in Saccharomyces cerevisiae by reducing the steady state levels of intracellular arsenic.

We cloned a plant gene, Ntcyc07, conferring arsenite tolerance by expressing a tobacco expression library in WT yeast (Y800). Expression of Ntcyc07 increased the tolerance to As(III) and decreased its accumulation, suggesting that the enhanced As(III) tolerance resulted from a reduction of the intracellular arsenic level. Interestingly, expression of Ntcyc07 increased the expression of the As(III) export carrier ACR3, but repressed that of As(III) uptake channel FPS1. Ntcyc07p interacted with Acr1p, which is the transcriptional activator of ACR3, but not with the ACR3 promoter. Taken together, the data indicated that Ntcyc07p promoted As(III) tolerance by decreasing the intracellular level of As(III) via increasing the expression of ACR3 and reducing that of FPS1.

Elongation and gravitropic responses of Arabidopsis roots are regulated by brassinolide and IAA.

Exogenously applied brassinolide (BL) increased both gravitropic curvature and length of primary roots of Arabidopsis at low concentration (10(-10) M), whereas at higher concentration, BL further increased gravitropic curvature while it inhibited primary root growth. BRI1-GFP plants possessing a high steady-state expression level of a brassinosteroid (BR) receptor kinase rendered the plant's responses to gravity and root growth more sensitive, while BR-insensitive mutants, bri1-301 and bak1, delayed root growth and reduced their response to the gravitropic stimulus. The stimulatory effect of BL on the root gravitropic curvature was also enhanced in auxin transport mutants, aux1-7 and pin2, relative to wild-type plants, and increasing concentration of auxin attenuated BL-induced root sensitivity to gravity. Interestingly, IAA treatment to the roots of bri1-301 and bak1 plants or of plants pretreated with a BL biosynthetic inhibitor, brassinazole, increased their sensitivity to gravity, while these treatments for the BL-hypersensitive transgenic plants, BRI1-GFP and 35S-BAK1, were less effective. Expression of a CYP79B2 gene, encoding an IAA biosynthetic enzyme, was suppressed in BL-hypersensitive plant types and enhanced in BL-insensitive or -deficient plants. In conclusion, our results indicate that BL interacts negatively with IAA in the regulation of plant gravitropic response and root growth, and its regulation is achieved partly by modulating biosynthetic pathways of the counterpart hormone.

Arabidopsis CYP85A2, a cytochrome P450, mediates the Baeyer-Villiger oxidation of castasterone to brassinolide in brassinosteroid biosynthesis.

The conversion of castasterone (CS) to brassinolide (BL), a Baeyer-Villiger oxidation, represents the final and rate-limiting step in the biosynthesis of BL in plants. Heterologously expressed Arabidopsis thaliana CYP85A2 in yeast mediated the conversion of CS to BL as well as the C-6 oxidation of brassinosteroids (BRs). This indicated that CYP85A2 is a bifunctional enzyme that possesses BR C-6 oxidase and BL synthase activity. CYP85A2 is thus a cytochrome P450 that mediates Baeyer-Villiger oxidation in plants. Biochemical, physiological, and molecular genetic analyses of Arabidopsis CYP85A2 loss-of-function and overexpression lines demonstrated that CS has to be a bioactive BR that controls the overall growth and development of Arabidopsis plants. Mutant studies also revealed that BL may not always be necessary for normal growth and development but that Arabidopsis plants acquire great benefit in terms of growth and development in the presence of BL.

Auxin-induced reactive oxygen species production requires the activation of phosphatidylinositol 3-kinase.

We recently reported that production of reactive oxygen species (ROS) is essential for auxin-induced gravitropic signaling. Here, we investigated the role of phosphatidylinositol 3-kinase and its product, PtdIns(3)P, in auxin-mediated ROS production and the root gravitropic response. Pretreatment with LY294002, an inhibitor of PtdIns 3-kinase activity, blocked auxin-mediated ROS generation, and reduced the sensitivity of root tissue to gravistimulation. The amount of PtdIns(3)P increased in response to auxin, and this effect was abolished by pretreatment with LY294002. In addition, sequestration of PtdIns(3)P by transient expression of the endosome binding domain in protoplasts abrogated IAA-induced ROS accumulation. These results indicate that activation of PtdIns 3-kinase and its product PtdIns(3)P are required for auxin-induced production of ROS and root gravitropism.

Novel biosynthetic pathway of castasterone from cholesterol in tomato.

Endogenous brassinosteroids (BRs) in tomato (Lycopersicon esculentum) seedlings are known to be composed of C27- and C28-BRs. The biosynthetic pathways of C27-BRs were examined using a cell-free enzyme solution prepared from tomato seedlings that yielded the biosynthetic sequences cholesterol --> cholestanol and 6-deoxo-28-norteasterone <--> 6-deoxo-28-nor-3-dehydroteasterone <--> 6-deoxo-28-nortyphasterol --> 6-deoxo-28-norcastasterone --> 28-norcastasterone (28-norCS). Arabidopsis CYP85A1 that was heterologously expressed in yeast mediated the conversion of 6-deoxo-28-norCS to 28-norCS. The same reaction was catalyzed by an enzyme solution from wild-type tomato but not by an extract derived from a tomato dwarf mutant with a defect in CYP85. Furthermore, exogenously applied 28-norCS restored the abnormal growth of the dwarf mutant. These findings indicate that the C-6 oxidation of 6-deoxo-28-norCS to 28-norCS in tomato seedlings is catalyzed by CYP85, just as in the conversion of 6-deoxoCS to CS. Additionally, the cell-free solution also catalyzed the C-24 methylation of 28-norCS to CS in the presence of NADPH and S-adenosylmethionine (SAM), a reaction that was clearly retarded in the absence of NADPH and SAM. Thus it seems that C27-BRs, in addition to C28-BRs, are important in the production of more active C28-BRs and CS, where a SAM-dependent sterol methyltransferase appears to biosynthetically connect C27-BRs to C28-BRs. Moreover, the tomato cell-free solution converted CS to 26-norCS and [2H6]CS to [2H3]28-norCS, suggesting that C-28 demethylation is an artifact due to an isotope effect. Although previous feeding experiments employing [2H6]CS suggested that 28-norCS was synthesized from CS in certain plant species, this is not supported in planta. Altogether, this study demonstrated for the first time, to our knowledge, that 28-norCS is not synthesized from CS but from cholesterol. In addition, CS and [2H6]CS were not converted into BL and [2H6]BL, respectively, confirming an earlier finding that the active BR in tomato seedlings is not BL but CS. In conclusion, the biosynthesis of 28-norBRs appears to play a physiologically important role in maintaining homeostatic levels of CS in tomato seedlings.

Cytochrome P450-catalyzed brassinosteroid pathway activation through synthesis of castasterone and brassinolide in Phaseolus vulgaris.

The last reaction in the biosynthesis of brassinolide has been examined enzymatically. A microsomal enzyme preparation from cultured cells of Phaseolus vulgaris catalyzed a conversion from castasterone to brassinolide, indicating that castasterone 6-oxidase (brassinolide synthase) is membrane associated. This enzyme preparation also catalyzed the conversions of 6-deoxocastasterone and typhasterol to castasterone which have been reported to be catalyzed by cytochrome P450s, CYP85A1 of tomato and CYP92A6 of pea, respectively. The activities of these enzymes require molecular oxygen as well as NADPH as a cofactor. The enzyme activities were strongly inhibited by carbon monoxide, an inhibitor of cytochrome P450, and this inhibition was recovered by blue light irradiation in the presence of oxygen. Commercial cytochrome P450 inhibitors including cytochrome c, SKF 525A, 1-aminobenzotriazole and ketoconazole also inhibited the enzyme activities. The present work presents unanimous enzymological evidence that cytochrome P450s are responsible for the synthesis of brassinolide from castasterone as well as of castasterone from typhasterol and 6-deoxocastasterone, which have been deemed activation steps of BRs.

Changes in phosphorylation of 50 and 53 kDa soluble proteins in graviresponding oat (Avena sativa) shoots.

The present work indicates that phosphorylation of a 50 kDa soluble protein is involved in the gravitropic response in graviresponsive pulvini of oat (Avena sativa) stems. This 50 kDa protein shows a differential pattern of phosphorylation between lower and upper halves of pulvini both in vivo and in vitro. The differential phosphorylation of this protein is detected only when stem segments are gravistimulated for short and long time periods. The differential phosphorylation of the 50 kDa protein occurs as early as 5 min after the initiation of gravistimulation. This corresponds closely to the presentation time of 5.2 min. This differential phosphorylation pattern was changed by treatments with cycloheximide, implying that a newly-synthesized protein is involved in the differential phosphorylation during the gravitropic response. An autophosphorylation experiment shows that the 50 kDa protein has kinase activity. The phosphorylation patterns of a 53 kDa protein were similar to those of the 50 kDa protein, but were only expressed in vitro. These findings indicate that the differential phosphorylation of the 50 (and 53 kDa) soluble proteins in graviresponding oat shoots may be an important component of the gravity signal transduction pathway.

The putative transcriptional activator MSN1 promotes chromium accumulation in Saccharomyces cerevisiae.

Yeast is a good system for studying molecular mechanisms of metal tolerance. Using a mini-Tn mutagenized yeast pool, we isolated a chromate-tolerant mutant, CrT9, that displayed metal-specific tolerance since it was only tolerant to Cr(VI), not to Cr(III), Cd, As, or Fe. The Cr-tolerance of CrT9 appeared to be due to reduced Cr accumulation as it accumulated only 56% as much as WT (Y800). Using IPCR (inverse PCR), we found that the mini-Tn had been inserted at nt 741 of the transcriptional activator, MSN1. MSN1 is a multifunctional protein involved in invertase activity, iron uptake, starch degradation, pseudohyphal growth, and osmotic gene expression. We found that there was only one mini-Tn insertion in CrT9 since MSN1 and mini-Tn probes hybridized to the same DNA fragment, and the MSN1 probe detected an enlarged MSN1 mRNA. When we over-expressed MSN1 in CrT9 and WT, both accumulated larger amounts of Cr. We conclude that Cr accumulation in S. cerevisiae is promoted by the transcriptional activator MSN1.

ABA and polyamines act independently in primary leaves of cold-stressed tomato (Lycopersicon esculentum).

The effects of ABA and putrescine, a polyamine, on cold-induced membrane leakage were investigated using primary leaves of wild-type and an ABA-deficient mutant, flacca, of tomato (Lycopersicon esculentum Mill.). The amount of chilling-induced electrolyte leakage from flacca leaves was much higher than that from the wild-type leaves. When applied exogenously ABA reduced cold-induced electrolyte leakage from leaves of both wild-type and the flacca mutant. However, the cold-induced electrolyte leakage from flacca leaves was not as pronounced as in the wild-type indicating that ABA is an important mediator in response to cold stress in the leaves. Putrescine reduced cold-induced electrolyte leakage from both wild-type and flacca leaves. Synthesis of putrescine in the leaves was increased by cold treatment. DFMO, a biosynthetic inhibitor of the polyamine, increased electrolyte leakage from cold-treated leaves, and exogenously applied putrescine decreased the enhanced leakage to the control level. Therefore, this polyamine is thought also to be involved in the response to cold stress of tomato leaves. Both ABA and putrescine were protective against cold stress, but exogenously applied ABA decreased the endogenous level of putrescine in the leaves. Furthermore, the DMFO-increased electrolyte leakage in cold-stressed leaves was completely abolished by the application of ABA. These results suggest that ABA is a major regulator in the response to cold stress in tomato leaves and that it does not exert its role via putrescine in the response to cold stress.