Evaluation of different methods of protein extraction and identification of differentially expressed proteins upon ethylene-induced early-ripening in banana peels.

BACKGROUND: Banana peels (Musa spp.) are a good example of a plant tissue where protein extraction is challenging due to the abundance of interfering metabolites. Sample preparation is a critical step in proteomic research and is critical for good results. RESULTS: We sought to evaluate three methods of protein extraction: trichloroacetic acid (TCA)-acetone precipitation, phenol extraction, and TCA precipitation. We found that a modified phenol extraction protocol was the most optimal method. SDS-PAGE and two-dimensional gel electrophoresis (2-DE) demonstrated good protein separation and distinct spots of high quality protein. Approximately 300 and 550 protein spots were detected on 2-DE gels at pH values of 3-10 and 4-7, respectively. Several spots were excised from the 2-DE gels and identified by mass spectrometry. CONCLUSIONS: The protein spots identified were found to be involved in glycolysis, the tricarboxylic acid cycle, and the biosynthesis of ethylene. Several of the identified proteins may play important roles in banana ripening.

Involvement of abscisic acid-responsive element-binding factors in cassava (Manihot esculenta) dehydration stress response.

Cassava (Manihot esculenta) is a major staple food, animal feed and energy crop in the tropics and subtropics. It is one of the most drought-tolerant crops, however, the mechanisms of cassava drought tolerance remain unclear. Abscisic acid (ABA)-responsive element (ABRE)-binding factors (ABFs) are transcription factors that regulate expression of target genes involved in plant tolerance to drought, high salinity, and osmotic stress by binding ABRE cis-elements in the promoter regions of these genes. However, there is little information about ABF genes in cassava. A comprehensive analysis of Manihot esculenta ABFs (MeABFs) described the phylogeny, genome location, cis-acting elements, expression profiles, and regulatory relationship between these factors and Manihot esculenta betaine aldehyde dehydrogenase genes (MeBADHs). Here we conducted genome-wide searches and subsequent molecular cloning to identify seven MeABFs that are distributed unevenly across six chromosomes in cassava. These MeABFs can be clustered into three groups according to their phylogenetic relationships to their Arabidopsis (Arabidopsis thaliana) counterparts. Analysis of the 5'-upstream region of MeABFs revealed putative cis-acting elements related to hormone signaling, stress, light, and circadian clock. MeABF expression profiles displayed clear differences among leaf, stem, root, and tuberous root tissues under non-stress and drought, osmotic, or salt stress conditions. Drought stress in cassava leaves and roots, osmotic stress in tuberous roots, and salt stress in stems induced expression of the highest number of MeABFs showing significantly elevated expression. The glycine betaine (GB) content of cassava leaves also was elevated after drought, osmotic, or salt stress treatments. BADH1 is involved in GB synthesis. We show that MeBADH1 promoter sequences contained ABREs and that MeBADH1 expression correlated with MeABF expression profiles in cassava leaves after the three stress treatments. Taken together, these results suggest that in response to various dehydration stresses, MeABFs in cassava may activate transcriptional expression of MeBADH1 by binding the MeBADH1 promoter that in turn promotes GB biosynthesis and accumulation via an increase in MeBADH1 gene expression levels and MeBADH1 enzymatic activity. These responses protect cells against dehydration stresses by preserving an osmotic balance that enhances cassava tolerance to dehydration stresses.

[Parent grouping of 31 elite inbred lines in hot pepper (Capsicum annuum L.)].

Genetic differences were examined among thirty-one elite inbred lines in Capsicum annuum L. Two types of analytic technologies, i.e. SRAP markers and genotypes of traits, were used, and their relative effectiveness was compared. 27 of 30 primer combinations could amplify 310 polymorphic bands among inbred lines, indicating SRAP marker was efficient to detect polymorphism among pepper inbred lines. A dendrogram of 31 inbred lines based on SRAP markers and Yule coefficients could basically separate lines of C. annuum var. grossum and C. annuum var. longum, and reveal the pedigrees of inbred lines. A dendrogram of 31 inbred lines based on genotypes of traits and standardized Euclidean coefficients could separate lines of C. annuum var. grossum and C. annuum var. longum. The SRAP marker genetic distances were correlated with distances based on the genotypes of traits. These results and their application in the development of hot pepper F1 hy-brids were also discussed.

Optimization of fermentation conditions through response surface methodology for enhanced antibacterial metabolite production by Streptomyces sp. 1-14 from cassava rhizosphere.

Streptomyces species 1-14 isolated from cassava rhizosphere soil were evaluated for their antibacterial efficacy against Fusarium oxysporum f.sp. cubense race 4 (FOC4). Of the 63 strains tested, thirteen exhibited potent antibacterial properties and were further screened against eight fungal pathogens. The strain that showed maximum inhibition against all of the test pathogens was identified by 16S rDNA sequencing as Streptomyces sp. 1-14, was selected for further studies. Through the propagation of Streptomyces sp. 1-14 in soil under simulated conditions, we found that FOC4 did not significantly influence the multiplication and survival of Streptomyces sp. 1-14, while indigenous microorganisms in the soil did significantly influence Streptomyces sp. 1-14 populations. To achieve maximum metabolite production, the growth of Streptomyces 1-14 was optimized through response surface methodology employing Plackett-Burman design, path of steepest ascent determinations and Box-Behnken design. The final optimized fermentation conditions (g/L) included: glucose, 38.877; CaCl2•2H2O, 0.161; temperature, 29.97°C; and inoculation amount, 8.93%. This optimization resulted in an antibacterial activity of 56.13% against FOC4, which was 12.33% higher than that before optimization (43.80%). The results obtained using response surface methodology to optimize the fermentation medium had a significant effect on the production of bioactive metabolites by Streptomyces sp. 1-14. Moreover, during fermentation and storage, pH, light, storage temperature, etc., must be closely monitored to reduce the formation of fermentation products with reduced antibacterial activity. This method is useful for further investigations of the production of anti-FOC4 substances, and could be used to develop bio-control agents to suppress or control banana fusarium wilt.

Expression patterns of members of the ethylene signaling-related gene families in response to dehydration stresses in cassava.

Drought is the one of the most important environment stresses that restricts crop yield worldwide. Cassava (Manihot esculenta Crantz) is an important food and energy crop that has many desirable traits such as drought, heat and low nutrients tolerance. However, the mechanisms underlying drought tolerance in cassava are unclear. Ethylene signaling pathway, from the upstream receptors to the downstream transcription factors, plays important roles in environmental stress responses during plant growth and development. In this study, we used bioinformatics approaches to identify and characterize candidate Manihot esculenta ethylene receptor genes and transcription factor genes. Using computational methods, we localized these genes on cassava chromosomes, constructed phylogenetic trees and identified stress-responsive cis-elements within their 5' upstream regions. Additionally, we measured the trehalose and proline contents in cassava fresh leaves after drought, osmotic, and salt stress treatments, and then it was found that the regulation patterns of contents of proline and trehalose in response to various dehydration stresses were differential, or even the opposite, which shows that plant may take different coping strategies to deal with different stresses, when stresses come. Furthermore, expression profiles of these genes in different organs and tissues under non-stress and abiotic stress were investigated through quantitative real-time PCR (qRT-PCR) analyses in cassava. Expression profiles exhibited clear differences among different tissues under non-stress and various dehydration stress conditions. We found that the leaf and tuberous root tissues had the greatest and least responses, respectively, to drought stress through the ethylene signaling pathway in cassava. Moreover, tuber and root tissues had the greatest and least reponses to osmotic and salt stresses through ethylene signaling in cassava, respectively. These results show that these plant tissues had differential expression levels of genes involved in ethylene signaling in response to the stresses tested. Moreover, after several gene duplication events, the spatiotemporally differential expression pattern of homologous genes in response to abiotic and biotic stresses may imply their functional diversity as a mechanism for adapting to the environment. Our data provide a framework for further research on the molecular mechanisms of cassava resistance to drought stress and provide a foundation for breeding drought-resistant new cultivars.