A proteomics study of the mung bean epicotyl regulated by brassinosteroids under conditions of chilling stress.

Mung bean CYP90A2 is a putative brassinosteroid (BR) synthetic gene that shares 77% identity with the Arabidopsis CPD gene. It was strongly suppressed by chilling stress. This implies that exogenous treatment with BR could allow the plant to recover from the inhibited growth caused by chilling. In this study, we used proteomics to investigate whether the mung bean epicotyl can be regulated by brassinosteroids under conditions of chilling stress. Mung bean epicotyls whose growth was initially suppressed by chilling partly recovered their ability to elongate after treatment with 24-epibrassinolde; 17 proteins down-regulated by this chilling were re-up-regulated. These up-regulated proteins are involved in methionine assimilation, ATP synthesis, cell wall construction and the stress response. This is consistent with the re-up-regulation of methionine synthase and S-adenosyl-L-methionine synthetase, since chilling-inhibited mung bean epicotyl elongation could be partially recovered by exogenous treatment with DL-methionine. This is the first proteome established for the mung bean species. The regulatory relationship between brassinosteroids and chilling conditions was investigated, and possible mechanisms are discussed herein.

Chilling stress suppresses chloroplast development and nuclear gene expression in leaves of mung bean seedlings.

Etiolated leaves of 28 degrees C-dark-grown mung bean (Vigna radiata L. cv. 2937) seedlings fail to turn green after being shifted to a light and cold environment. At the visible phenotypic level, incapability of leaf greening is the only failure event for the de-etiolation of mung bean seedlings at low temperature. Ultrastructural studies revealed that chloroplast development was completely suppressed by chilling treatment. A cDNA library originating from 28 degrees C-light-grown seedling leaves was constructed for screening cold-suppressed (cos) genes. Thirteen full-length cDNA clones were obtained, with 12 clones encoding chloroplast proteins, which, according to their known physiological functions, were important for chloroplast development and photosynthesis. Another cos cDNA encodes CYP90A2, which is a cytochrome P450 protein involved in the biosynthesis of brassinosteroid hormones. All cos genes are light-regulated at normal temperature. The influence of chilling stress on cos expression was examined in 10 degrees C-light- and 10 degrees C-dark-grown etiolated seedlings, and in 10 degrees C-light-grown green plants. The data show that cos expression in these three treatments is severely suppressed. This suppression is controlled at the transcriptional level, as demonstrated by nuclear runoff experiments, and is reversible because cos mRNAs accumulate again after the cold-treated plants have been transferred to 28 degrees C.

Azetidine-induced accumulation of class I small heat shock proteins in the soluble fraction provides thermotolerance in soybean seedlings.

Accumulation of class I small heat shock proteins (sHSPs) is induced by the proline analog, azetidine-2-carboxylic acid (Aze) in soybean seedlings to a level similar to that induced by exposure to 40 degrees C. However, only the treatment with 10 mM Aze for 6 h and subsequently with 10 mM proline for 24 h protected the seedlings from damage during subsequent exposure to 45 degrees C as assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. A chaperone activity assay showed that the purified class I sHSPs induced by Aze were functional in vitro and protected proteins from thermal denaturation. Amino acid composition analysis indicated that Aze was not incorporated into de novo synthesized class I sHSPs. Accumulation of class I sHSPs in the soluble post-ribosomal supernatant fraction was found to be important for acquisition of thermotolerance. We suggest that both the accumulation of class I sHSPs and their presence in the soluble fraction are important for establishment of thermotolerance.

Characterization of the genomic structures and selective expression profiles of nine class I small heat shock protein genes clustered on two chromosomes in rice (Oryza sativa L.).

The cytosolic class I small heat shock proteins (sHSP-CI) represent the most abundant sHSP in plants. Here, we report the characterization and the expression profile of nine members of the sHSP-CI gene family in rice (Oryza sativa Tainung No.67), of which Oshsp16.9A, Oshsp16.9B, Oshsp16.9C, Oshsp16.9D and Oshsp17.9B are clustered on chromosome 1, and Oshsp17.3, Oshsp17.7, Oshsp17.9A and Oshsp18.0 are clustered on chromosome 3. Oshsp17.3 and Oshsp18.0 are linked by a 356-bp putative bi-directional promoter. Individual gene products were identified from the protein subunits of a heat shock complex (HSC) and from in vitro transcription/ translation products by two-dimensional gel electrophoreses (2-DE). All sHSP-CI genes except Oshsp17.9B were induced strongly after a 2-h heat shock treatment. The genes on chromosome 3 were induced rapidly at 32 and 41 degrees C, whereas those on chromosome 1 were induced slowly by similar conditions. Seven of these genes, except Oshsp16.9D and Oshsp17.9B, were induced by arsenite (As), but only genes on chromosome 3 were strongly induced by azetidine-2-carboxylic acid (Aze, a proline analog) and cadmium (Cd). A similar expression profile of all sHSP-CI genes at a lower level was evoked by ethanol, H2O2 and CuCl2 treatments. Transient expression assays of the promoter activity by linking to GUS reporter gene also supported the in vivo selective expression of the sHSP-CI genes by Aze treatment indicating the differential induction of rice sHSP-CI genes is most likely regulated at the transcriptional level. Only Oshsp16.9A abundantly accumulated in mature dry seed also suggested additionally prominent roles played by this HSP in development.

Allelopathic potential of Macaranga tanarius (L.) muell.-arg.

Macaranga tanarius is widely distributed in the abandoned lowlands of Taiwan where substantial amounts of leaves accumulate on the ground. A unique pattern of weed exclusion underneath trees is often found and thought to result from allelopathic interactions. Density-dependent phytotoxicity analysis of Lactuca sativa L. (lettuce) growing in soil mixed with the powder of M. tanarius leaves showed a significant deviation from the expected yield-density relationship. Lettuce growth was most suppressed in the low seed density experiment suggesting that the phytotoxins produced during leaf decomposition inhibit the growth of lettuce seedlings. Bidens pilosa and Leucaena leucocephala, growing in soil mixed with the leaf powder of M. tanarius were also suppressed. Aqueous leaf extracts were bioassayed against lettuce and B. pilosa, and exhibited a significant suppression in radicle growth. Compounds identified from leaves included nymphaeol-A (1), nymphaeol-B (2), nymphaeol-C (3), quercetin (4), abscisic acid (ABA) (5), blumenol A (6), blumenol B (7), roseoside II (8), tanariflavanone A (9), and tanariflavanone B (10), ABA was the major growth inhibitor. At concentrations of 20 ppm, ABA suppressed lettuce germination, while at 120 ppm it inhibited the growth of Miscanthus floridulus, Chloris barbata, and Bidens pilosa. At 600 ppm, quercetin, blumenol A, and blumenol B, caused 20-25% inhibition of radicle and shoot growth of M. floridulus. The amount of ABA in M. tanarius leaves was approximately 3-5 microg g(-1) dry weight, significantly higher than previously reported. We conclude that the pattern of weed exclusion underneath stands of M. tanarius and its invasion into its adjacent grassland vegetation results from allelopathic interactions.

Wound-response regulation of the sweet potato sporamin gene promoter region.

Sporamin, a tuberous storage protein of sweet potato, was systemically expressed in leaves and stems by wound stimulation. In an effort to demonstrate the regulatory mechanism of wound response on the sporamin gene, a 1.25 kb sporamin promoter was isolated for studying the wound-induced signal transduction. Two wound response-like elements, a G box-like element and a GCC core-like sequence were found in this promoter. A construct containing the sporamin promoter fused to a beta-glucuronidase (GUS) gene was transferred into tobacco plants by Agrobacterium-mediated transformation. The wound-induced high level of GUS activity was observed in stems and leaves of transgenic tobacco, but not in roots. This expression pattern was similar to that of the sporamin gene in sweet potatoes. Exogenous application of methyl jasmonate (MeJA) activated the sporamin promoter in leaves and stems of sweet potato and transgenic tobacco plants. A competitive inhibitor of ethylene (2,5-norbornadiene; NBD) down-regulated the effect of MeJA on sporamin gene expression. In contrast, salicylic acid (SA), an inhibitor of the octadecanoid pathway, strongly suppressed the sporamin promoter function that was stimulated by wound and MeJA treatments. In conclusion, wound-response expression of the sporamin gene in aerial parts of plants is regulated by the octadecanoid signal pathway.

Functional regions of rice heat shock protein, Oshsp16.9, required for conferring thermotolerance in Escherichia coli.

Rice (Oryza sativa) class I low-molecular mass (LMM) heat shock protein (HSP), Oshsp16.9, has been shown to be able to confer thermotolerance in Escherichia coli. To define the regions for this intriguing property, deletion mutants of this hsp have been constructed and overexpressed in E. coli XL1-blue cells after isopropyl beta-D-thioglactopyranoside induction. The deletion of amino acid residues 30 through 36 (PATSDND) in the N-terminal domain or 73 through 78 (EEGNVL) in the consensus II domain of Oshsp16.9 led to the loss of chaperone activities and also rendered the E. coli incapable of surviving at 47.5 degrees C. To further investigate the function of these two domains, we determined the light scattering changes of Oshsp16.9 mutant proteins at 320 nm under heat treatment either by themselves or in the presence of a thermosensitive enzyme, citrate synthase. It was observed that regions of amino acid residues 30 through 36 and 73 through 78 were responsible for stability of Oshsp16.9 and its interactions with other unfolded protein substrates, such as citrate synthase. Studies of two-point mutants of Oshsp16.9, GST-N74E73K and GST-N74E74K, indicate that amino acid residues 73 and 74 are an important part of the substrate-binding site of Oshsp16.9. Non-denaturing gel analysis of purified Oshsp16.9 revealed that oligomerization of Oshsp16.9 was necessary but not sufficient for its chaperone activity.