WEREWOLF and ENHANCER of GLABRA3 are interdependent regulators of the spatial expression pattern of GLABRA2 in Arabidopsis.

In multicellular organisms, cell fates are specified through differential regulation of transcription. Epidermal cell fates in the Arabidopsis thaliana root are precisely specified by several transcription factors, with the GLABRA2 (GL2) homeodomain protein acting at the farthest downstream in this process. To better understand the regulation of GL2 expression, we ectopically expressed WEREWOLF (WER) and ENHANCER OF GLABRA3 (EGL3) in various tissues and examined GL2 expression. Here we show that WER expressed ubiquitously in the root induced GL2 expression only in the root epidermis, whereas co-expression of WER and EGL3 induced GL2 expression in the corresponding tissues. We also found that GL3 accumulated in the nucleus at the early meristematic region and EGL3 accumulated later in the nucleus of epidermal cells. We further found that ectopic expression of WER and EGL3 in ground tissues inhibited GL2 expression in the epidermis. Our results suggest that the co-expression of WER and EGL3 is sufficient for driving GL2 and CPC expression.

Ethylene negatively regulates EXPA5 expression in Arabidopsis thaliana.

We examined the effects of ethylene on the expression of Arabidopsis expansins (AtEXPs). Among the AtEXPs tested, transcription of the AtEXPA5 gene was reduced most by exogenous ethylene. 2-Aminoethoxyvinylglycine, an ethylene biosynthesis inhibitor, increased AtEXPA5 transcription. Ethylene insensitive (ein7) and constitutive (ctr1) mutants resulted in increased and decreased transcription, respectively, thereby suggesting that ethylene endogenously downregulates AtEXPA5 expression. Hypocotyl elongation followed the same trend as AtEXPA5 expression, implying that changes in hypocotyl elongation reflect changes in AtEXPA5 expression. A transgenic plant line that overexpresses AtEXPA5, 35S-EXPA5, showed a reduced response to exogenous ethylene in terms of hypocotyl lengths when compared to wild-type and expA5-1, a knockout mutant. These results and the dose-dependent effect of aminocyclopropane-1-carboxyl acid on hypocotyl elongation implicate AtEXPA5 overexpression in making tissues more sensitive to high doses of ethylene. In summary, AtEXPA5 appears to respond to ethylene and play a role in ethylene regulating hypocotyl elongation in Arabidopsis thaliana.

The effect of ascorbic acid and dehydroascorbic acid on the root gravitropic response in Arabidopsis thaliana.

The effects of ascorbic acid (AA) and dehydroascorbic acid (DHA), one of products of the disproportionation of monodehydroascorbate (MDHA) by AA oxidase (AAO, EC 1.10.3.3), on the gravitropic curvature of Arabidopsis roots were characterized by biochemical and genetic approaches. Exogenously applied AA and DHA both stimulated root gravitropic responses in a concentration-dependent fashion. AA also changed the Indole-3-acetic acid (IAA) distribution in the roots after gravistimulation. In an effort to determine the relationship between AA and DHA in the gravitropic response, changes in the amount of reduced AA were evaluated in Arabidopsis under a variety of conditions. The expression level of an AAO gene (AAO1) was increased upon gravistimulation. Brassinolide (BL), indole-3-acetic acid (IAA), and AA also increased the transcript levels of this gene. Root elongation and the gravitropic response were both suppressed in the AA biosynthesis mutant, vtc1, which has a greatly reduced level of total AA. Furthermore, the line of AAO double mutants (aao1-1 X aao3-1, 41-21) showed a reduced gravitropic response and reduced root elongation. Taken together, the results of this study imply that both AA and DHA help to determine the redox environment for the root gravitropic response, but DHA, rather than AA, is a major player in the regulation of the gravitropic response mediated by AA in the roots of Arabidopsis thaliana.

Brassinosteroids control AtEXPA5 gene expression in Arabidopsis thaliana.

To elucidate the spatial and temporal roles of EXPANSIN A5 (AtEXPA5) in growth and development of Arabidopsis thaliana, phenotypic alterations in loss-of-function mutants were observed. Seedlings of the null mutant, expA5-1, had shorter roots and hypocotyls than those of wild-type plants under both light and dark conditions. Compared to wild-type plants, the mutants had smaller rosette leaves. AtEXPA5 was dominantly expressed in aerial parts of A. thaliana, especially in the inflorescence stems and flowers. Expression of AtEXPA5 was enhanced by exogenously applied brassinosteroids. AtEXPA5 expression was reduced in a brassinosteroid-deficient mutant (det2) and a signaling mutant (bri1-301), while it was increased in bzr1-1D, a dominant mutant of a brassinosteroid transcription factor. A double mutant, bzr1-1DXexpA5-1, showed reduced growth compared to the bzr1-1D mutant. In addition, the brassinazole resistance of bzr1-1D was impaired in the double mutant. These findings indicate that AtEXPA5 is a growth-regulating gene whose expression is controlled by brassinosteroid signaling downstream of BZR1 in A. thaliana.

Analysis of phosphorylation of the BRI1/BAK1 complex in arabidopsis reveals amino acid residues critical for receptor formation and activation of BR signaling.

The plasma membrane-localized BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED KINASE1 (BAK1) are a well-known receptor pair involved in brassinosteroids (BR) signaling in Arabidposis. The formation of a receptor complex in response to BRs and the subsequent activation of cytoplasmic domain kinase activity share mechanistic characteristics with animal receptor kinases. Here, we demonstrate that BRI1 and BAK1 are BR-dependently phosphorylated, and that phosphorylated forms of the two proteins persist for different lengths of time. Mutations of either protein abolished phosphorylation of the counterpart protein, implying transphosphorylation of the receptor kinases. To investigate the specific amino acids critical for formation of the receptor complex and activation of BAK1 kinase activity, we expressed several versions of BAK1 in yeast and plants. L32E and L46E substitutions resulted in a loss of binding of BAK1 to BRI1, and threonine T455 was essential for the kinase activity of BAK1 in yeast. Transgenic bri1 mutant plants overexpressing BAK1(L46E) displayed reduced apical dominance and seed development. In addition, transgenic wild type plants overexpressing BAK1(T455A) lost the phosphorylation activity normally exhibited in response to BL, leading to semi-dwarfism. These results suggest that BAK1 is a critical component regulating the duration of BR efficacy, even though it cannot directly bind BRs in plants.

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.

The production of hypericins in two selected Hypericum perforatum shoot cultures is related to differences in black gland structure.

In vitro shoot cultures of Hypericum perforatum derived from wild populations grown in Armenia have a wide variation of hypericin and pseudohypericin metabolite content. We found that a germ line denoted as HP3 produces six times more hypericin and fourteen times more pseudohypericin than a second line labeled HP1. We undertook a structural comparison of the two lines (HP1 and HP3) in order to see if there are any anatomical or morphological differences that could explain the differences in production of these economically important metabolites. Analysis by LM (light microscopy), SEM (scanning electron microscopy), and TEM (transmission electron microscopy) reveals that the hypericin/pseudohypericin-containing black glands located along the margins of the leaves consist of a peripheral sheath of flattened cells surrounding a core of interior cells that are typically dead at maturity. The peripheral cells of the HP3 glands appear less flattened than those of the HP1 glands. This may indicate that the peripheral cells are involved in hypericin/pseudohypericin production. Furthermore, we find that these peripheral cells undergo a developmental transition into the gland's interior cells. The fact that the size of the peripheral cells may correlate with metabolite production adds a new hypothesis for the actual site of hypericin synthesis.

Changes in starch and inositol 1,4,5-trisphosphate levels and auxin transport are interrelated in graviresponding oat (Avena sativa) shoots.

This study was conducted to unravel a mechanism for the gravitropic curvature response in oat (Avena sativa) shoot pulvini. For this purpose, we examined the downward movement of starch-filled chloroplast gravisensors, differential changes in inositol 1,4,5-trisphosphate (IP(3)) levels, transport of indole-3-acetic acid (IAA) and gravitropic curvature. Upon gravistimulation, the ratio for IAA levels in lower halves versus those in upper halves (L/U) increased from 1.0 at 0 h and reached a maximum value of 1.45 at 8 h. When shoots were grown in the dark for 10 d, to deplete starch in the chloroplast, the gravity-induced L/U of IAA was reduced to 1.0. N-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), both auxin transport inhibitors, significantly reduced the amount of gravitropic curvature and gravity-induced lateral IAA transport, but did not reduce the gravity-induced late change in the L/U ratio of IP(3) levels. U73122, a specific phospholipase C (PLC) inhibitor, decreased gravity-induced curvature. Because U73122 reduced the ratio of L/U of IAA imposed by gravistimulation, it is clear that IAA transport is correlated with changes in IP(3) levels upon gravistimulation. These results indicate that gravistimulation-induced differential lateral IAA transport may result from the onset of graviperception in the chloroplast gravisensors coupled with gravity-induced asymmetric changes in IP(3) levels in oat shoot pulvini.

Regulation of isoflavone production in hydroponically grown Pueraria montana (kudzu) by cork pieces, XAD-4, and methyl jasmonate.

A mini-hydroponic growing system was employed for seedlings of kudzu vine (Pueraria montana) and contents of isoflavones (daidzein, genistein, daidzin, genistin, and puerarin) from shoot and root parts of seedlings were analyzed quantitatively. In addition, exogenous cork pieces, polymeric adsorbent, XAD-4, and universal elicitor, methyl jasmonate (MeJA), were used to regulate the production of these isoflavones. It was shown that cork pieces up-regulate the production of daidzein and genistein up to seven- and eight-fold greater than the levels obtained for control roots. In contrast, levels of glucosyl conjugates, daidzin and genistin, decrease up to five- and eight-fold, respectively. Cork treatment also induces the excretion of the root isoflavone constituents into the growth medium. Minimal levels of isoflavones are absorbed by the cork pieces. XAD-4 stimulates the production of glucosyl conjugates, daidzin and genistin, in root parts about 1.5-fold greater than that obtained in control roots. These are the highest amounts of daidzin and genistin that are observed (5.101 and 6.759 mg g(-1) dry weight, respectively). In contrast to these two adsorbents, MeJA increases the accumulation of isoflavones in shoot rather than in root parts of seedlings, about three- to four-fold over control levels, with the exception of genistein. These studies reveal new observations on the regulation of isoflavone production in hydroponically grown Pueraria montana plants by two adsorbents (cork pieces and XAD-4) and MeJA elicitor.

Isoflavone levels in five soybean (Glycine max) genotypes are altered by phytochrome-mediated light treatments.

The objective of the present study was to determine whether concentrations of different isoflavones (puerarin, genistein, genistin, daidzein, and daidzin) in shoots and roots of five selected soybean genotypes would respond the same or differently to red (650 nm peak transmittance) and far-red (750 nm peak transmittance) light treatments given under controlled environments. Levels of isoflavones (mg g(-1) dry weight biomass) present in seeds, control roots, and shoots and 10 day light-treated seedlings (light, dark, red, and far-red wavelengths) of soybean (Glycine max) were determined by high-performance liquid chromatography analysis in comparison with known isoflavone standards. Seeds of the five soybean genotypes studied consistently stored most of their isoflavones as glucosyl conjugates (e.g., daidzin, genistin, and puerarin). For the five soybean genotypes, isoflavone levels were lower in the seeds as compared with roots plus shoots of control, time zero (first true leaf stage) seedlings. Following 10 days of the respective light treatments, we found that (i) isoflavone levels were enhanced in dark-grown plants over light-grown plants for three of the five genotypes (a new finding) and the reverse occurred for a single genotype (a typical response of legumes) and (ii) generally, far-red end of day (EOD) light treatment enhanced total isoflavone levels in roots plus shoots over red EOD light treatment. Results from the present study show that phytochrome does appear to play a role in regulating isoflavone levels in developing soybean seedlings and that this influence by red/far-red-mediated phytochrome reactions is strongly dependent on the genotypes selected for study.

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.

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.

Identification and characterization of an auxin-inducible protein kinase, VrCRK1, from mungbean.

An auxin-inducible protein kinase, VrCRK1, was isolated by a differential reverse transcriptase-polymerase chain reaction, using mRNAs extracted from auxin-treated mungbean hypocotyls. VrCRK1 exhibits high homology with plant CDPKs over catalytic domains, however, it does not have any calcium-binding EF-hand which is typically shown in plant CDPKs. Auxin treatment increased the expression level of VrCRK1. However, the increased level was reduced to basal level by treatment with PCIB, an auxin inhibitor. When extracts of mungbean hypocotyls were immunoprecipitated and the resultant immunoprecipitates were used as the enzyme source, kinase activity of VrCRK1 was found, and such activity was also increased by auxin treatment. In transgenic tobacco plants that express VrCRK1, the transcript levels of some auxin-dependent genes were elevated as much as those in wild type plants treated with auxin. These results indicate that gene expression of VrCRK1 is specifically induced by auxin, and that VrCRK1 may play a role in auxin signaling via protein phosphorylation.

Antioxidant capacity of polyphenolic extracts from leaves of Crataegus laevigata and Crataegus monogyna (Hawthorn) subjected to drought and cold stress.

Crataegus laevigata and Crataegus monogyna (hawthorn) were subjected to drought and cold stress treatments, and polyphenolic extracts from control and stress-treated plants were assayed for antioxidant capacities using a modified version of the Total Antioxidant Status Assay (Randox, San Francisco, CA). In addition, these plants were analyzed for levels of flavanol-type substance [(-)-epicatechin] and flavonoid (vitexin 2' '-O-rhamnoside, acetylvitexin 2' '-O-rhamnoside, and hyperoside) constituents that are important metabolites in hawthorn herbal preparations used to treat patients with heart disease. Drought and cold stress treatments caused increases in levels of (-)-epicatechin and hyperoside in both Crataegus species. Such treatments also enhanced the antioxidant capacity of the extracts. The results from this study thus indicate that these kinds of stress treatments can enhance the levels of important secondary metabolites and their total antioxidant capacities in leaves of Crataegus.

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.

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.