Soo Chul Chang

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.

Use of plant growth-promoting rhizobacteria to control stress responses of plant roots

AbstractsEthylene is a key gaseous hormone that controls various physiological processes in plants including growth, senescence, fruit ripening, and responses to abiotic and biotic stresses. In spite of some of these positive effects, the gas usually inhibits plant growth. While chemical fertilizers help plants grow better by providing soil-limited nutrients such as nitrogen and phosphate, over-usage often results in growth inhibition by soil contamination and subsequent stress responses in plants. Therefore, controlling ethylene production in plants becomes one of the attractive challenges to increase crop yields. Some soil bacteria among plant growth-promoting rhizobacteria (PGPRs) can stimulate plant growth even under stressful conditions by reducing ethylene levels in plants, hence the term “stress controllers” for these bacteria. Thus, manipulation of relevant genes or gene products might not only help clear polluted soil of contaminants but contribute to elevating the crop productivity. In this article, the beneficial soil bacteria and the mechanisms of reduced ethylene production in plants by stress controllers are discussed.

Quantification of major isoflavonoids and l-canavanine in several organs of kudzu vine (Pueraria montana) and in starch samples derived from kudzu roots

Abstract Isoflavonoids (daidzein, genistein, daidzin, genistin and puerarin) were extracted and analyzed quantitatively by high pressure liquid chromatography from different organs of kudzu vine ( Pueraria montana ) and starch samples derived from kudzu roots. Aerial parts of kudzu plants (young shoots, leaf blades and leaf petioles) contained relatively low levels of all isoflavonoids examined, whereas seeds and seedlings were intermediate in isoflavonoid levels and roots consistently had the highest levels, particularly puerarin and the glucosyl conjugates of genistein and daidzein, namely, genistin and daidzin. Interestingly, commercially available kudzu root starch from Japan does not contain the isoflavonoids of interest, whereas homemade kudzu root starch contains all studied isoflavonoids in various amounts, and especially, high levels of puerarin. Shoots of light-grown kudzu seedlings, when compared with shoots of dark-grown seedlings had higher levels of all isoflavonoids with the exception of daidzin. In contrast, for seedling roots, such differences were not greatly different between light-grown and dark-grown plants. Light-grown intact kudzu seedlings had significantly higher levels of soluble proteins than dark-grown seedlings. Mass spectrometer analyzes of all kudzu samples for the toxic non-protein amino acid, l -canavanine, indicated it to be absent using this method of detection and level of sensitivity.

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.

Brassinosteroids affect ethylene production in the primary roots of maize (Zea mays L.)

To better understand the physiological roles of brassinosteroids (BRs) in the primary roots of maize, we examined their effect on ethylene production. Exogenously applied brassinolide (BL; 10-9 to 10-7 M) incrementally increased the level ethylene in a dose-dependent manner. This BL-induced production was enhanced in the presence of IAA, thereby implying a synergistic effect between BR and IAA. At 10-7 M BL, the level of free ACC was increased, but that of conjugated ACC was diminished. Moreover, greater concentrations of BL proportionally increased ACC oxidase activity. In contrast, higher levels of IAA increased the endogenous content of conjugated ACC as well as ACC synthase activity. Based on these results, we conclude that BR activates ethylene production mainly via ACC oxidase, and interacts with IAA to produce ethylene. However, the functional site for ethylene production is different for each hormone.

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.

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.

Risks and Benefits Associated with Genetically Modified (GM) Plants

Genetically modified (GM) plants are those whose genomes have been modified by the introduction of foreign DNA constructs derived from bacteria, fungi, viruses, or animals. The most common genetically modified plants include soybeans, maize/corn, rapeseed mustard, potatoes, cotton, sugarcane, tomato, rice, and aspen/Populus.

Overexpression of Brassica rapa NGATHA1 Gene Confers De-Etiolation Phenotype and Cytokinin Resistance on Arabidopsis thaliana

Brassica rapa NGATHA1 (BrNGA1) encodes a B3-type transcription factor. By analyzing Arabidopsis overexpressors of BrNGA1 (BrNGA1ox), we have previously demonstrated that BrNGA1 may be involved in negative regulation of cell proliferation during lateral organ and root growth. In the present study, we have found that BrNGA1ox seedlings grown in the dark display de-etiolation phenotypes, such as short hypocotyls, open and elongated cotyledons, and developing true leaves. BrNGA1ox seedlings as well as adult plants and calli are also resistant specifically to exogenous cytokinins. These data raise the possibility that the de-etiolation phenotypes of BrNGA1ox seedlings may result from an alteration in cytokinin response. We set out to test whether the de-etiolation phenotype is due to cytokinin overproduction or constitutively activated cytokinin response. First, BrNGA1ox was crossed to the CKX2ox plant, an overexpression line of CYTOKIN OXIDASE 2, which is responsible for degradation of active cytokinins. We found, however, no difference in the de-etiolation and shoot growth phenotypes between BrNGA1ox and BrNGA1ox CKX2ox plants. Next, we measured the transcripts level of ARR5 and ARR7, frequently employed as molecular markers for cytokinin signaling and yet found no difference in their transcripts levels of the wild-type and BrNGA1ox seedlings and shoots. These data indicate that biological role of BrNGA1 involved in de-etiolation seems to be associated with neither cytokinin overproduction nor its altered signaling. Possible molecular mechanisms by which BrNGA1 may interfere with cytokinin responses and etiolation are discussed.