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

Plants sense positional changes relative to the gravity vector. To date, the signaling processes by which the perception of a gravistimulus is linked to the initiation of differential growth are poorly defined. We have investigated the role of inositol 1,4,5-trisphosphate (InsP 3 ) in the gravitropic response of oat (Avena sativa) shoot pulvini. Within 15 s of gravistimulation, InsP 3 levels increased 3-fold over vertical controls in upper and lower pulvinus halves and fluctuated in both pulvinus halves over the first minutes. Between 10 and 30 min of gravistimulation, InsP 3 levels in the lower pulvinus half increased 3-fold over the upper. Changes in InsP 3 were confined to the pulvinus and were not detected in internodal tissue, highlighting the importance of the pulvinus for both graviperception and response. Inhibition of phospholipase C blocked the long-term increase in InsP 3 , and reduced gravitropic bending by 65%. Short-term changes in InsP 3 were unimpaired by the inhibitor. Gravitropic bending of oat plants is inhibited at 4°C; however, the plants retain the information of a positional change and respond at room temperature. Both short-and long-term changes in InsP 3 were present at 4°C. We propose a role for InsP 3 in the establishment of tissue polarity during the gravitropic response of oat pulvini. InsP 3 may be involved in the retention of cold-perceived gravistimulation by providing positional information in the pulvini prior to the redistribution of auxin.

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.

Exogenously applied brassinolide (BL, 10Ϫ9 -10 Ϫ5 m) increased gravitropic curvature in maize (Zea mays) primary roots. The BL-enhanced gravitropic curvature was clearly promoted in the presence of indole-3-acetic acid (IAA, 10 Ϫ10 -10 Ϫ8 m), indicating that BL is interactive with IAA during the gravitropic response. The interactive effect between BL and IAA was completely diminished by treatment of p-chlorophenoxy isobutric acid, an auxin action antagonist. The activation of the gravitropic response by BL in the absence and in the presence of IAA was nullified by application of 2,3,5-triiodobenzoic acid, a polar auxin transport inhibitor. The data indicate that brassinosteroids (BRs) might be involved in auxin-mediated processes for the gravitropic response. Gas chromotography-selected ion-monitoring analysis revealed that maize primary roots contained approximately 0.3 ng g Ϫ1 fresh weight castasterone as an endogenous BR. Exogenously applied castasterone also increased the gravitropic response of maize roots in an IAA-dependent manner. This study provides the first evidence, to our knowledge, for occurrence and gravitropic activity of BRs in plant roots.Since brassinolide (BL) has been identified as a plant growth promoting substance in rape pollen (Grove et al., 1979), over 40 members of related steroids, collectively named as brassinosteroids (BRs), have been characterized in the entire plant kingdom (Kim, 1991; Fujioka, 1999). Early studies investigated possible physiological roles of BRs by exogenous application. The results of those studies suggested that BRs might be involved in the regulation of cell elongation and division, leaf bending, reproductive and vascular development, membrane polarization and proton pump, source/sink definition, and modulation of stress (for review, see Yokota and Takahashi,

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.

In this investigation, two species of Crataegus (hawthorn) were chosen because their polyphenolic constituents have recently received greater attention for the treatment of patients with severe heart disease. One-year-old plants of hawthorn (Crataegus laevigata and C. monogyna) were subjected to water-deficit (continuous water deprivation), cold (4 degrees C), flooding (immersion of roots of plants in water) or herbivory (leaf removal) stress treatments (each of 10 days duration) in order to assess their effects on levels of polyphenolics, namely (-)-epicatechin, catechin, chlorogenic acid, vitexin, vitexin-2"-O-rhamnoside, acetylvitexin-2"-O-rhamnoside, hyperoside, quercetin, and rutin in the leaves. The working hypothesis followed is that one or more of these stress treatment will elicit increases in the levels of these polyphenolics. Cold stress causes increases in levels of vitexin-2"-O-rhamnoside, acetylvitexin-2"-O-rhamnoside, hyperoside, and quercetin in both Crataegus species. Water-deficit stress increased the productivity of chlorogenic acid, catechin, and (-)-epicatechin in both hawthorn species. Flooding and herbivory caused no net increases, and in some cases, decreases in levels of polyphenolics. These studies indicate that either water-deficit stress or cold stress treatments, or a combination of the two, can be used to enhance the levels of desired polyphenolics in the leaves of these two hawthorn species in a photobioreactor system. These results may have significance for hawthorn in adapting to water-deficit or cold stress and are important considerations for the use of hawthorn in the treatment of heart disease in humans.

No abstract

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.