Khaled Sassi

Identification and characterization of drought stress responsive genes in faba bean (Vicia faba L.) by suppression subtractive hybridization

Faba bean (Vicia faba L.) ranks fourth in food legume crop production in the world. However, drought is a potential major constraint to faba bean production and improved faba bean cultivars and development of drought-resistant varieties play a key role in enhancing faba bean crop production. In this study, suppression subtractive hybridization (SSH) technique was used to study differential expression in response to water stress and to identify genes involved in molecular mechanism of drought tolerance. A forward subtractive cDNA library induced by water deficit conditions was constructed used Hara faba bean cultivar grown in pots and treated with either well-watered (WW) or water-stressed (WS). A total of 28 clones were identified as drought stress induced. After sequencing, ten unique expressed sequence tags (ESTs) were obtained by clustering and blast analysis which showed homology to known drought responsive genes including heat shock protein (HSP), late embryogenic abundant (LEA), zinc finger protein transcription factors (ZFP), lipid transfer protein (LTP), chlorophyll a/b-binding protein (ChlBP), thioredoxin h (Trx h), and ATP synthase as well as some functionally unknown transcripts. Their expression was characterized in Leaf, root, flower, cotyledon, and stem tissue. Quantitative RT-PCR analysis revealed that eight genes were consistently up-regulated in Hara compared to Giza3 cultivar, known as drought-tolerant and sensitive respectively under water deficit treatment. The expression of six genes was differentially expressed in different stages of water stress faba bean plant. Drought responsive genes showed changed expression patterns, indicating that they may play important roles in faba bean water stress response. Furthermore, these results indicate that drought-induced genes are related to metabolic pathways and genetic regulation of stress and development and can serve as a foundation for future studies to elucidate drought stress mechanisms of faba bean.

Identification and Analysis of Differentially Expressed Genes During Seed Development Using Suppression Subtractive Hybridization (SSH) in Phaseolus vulgaris

Interspecific hybridization in the genus Phaseolus, conducted to introgress desired traits into common bean (Phaseolus vulgaris L.), leads to the abortion of immature embryos, usually at early developmental stages. Little is known about the physiological responses of embryo dysfunction in P. vulgaris during the early stages of embryogenesis and the genes that are involved in these responses. Identification of the genes involved in Phaseolus embryogenesis may provide information that will help to understand the molecular basis of Phaseolus embryo dysfunction. To investigate the genes expressed during Phaseolus seed development, we constructed a suppression subtractive hybridization (SSH) library using cDNA from abortive seed development as the driver and those from normal seed development as tester. The differentially expressed cDNA fragments were identified by differential screening. We identified 72 unique ESTs of which we selected 12 candidates on the basis of their redundancy. These candidates were subjected to a validation procedure based on the study of their expression level by real-time PCR. Sequence analysis revealed that most of the differentially expressed genes are related to metabolism and regulation such as protein synthesis. Some genes also encoded transcription factors. These genes showed high mRNA transcript levels in seed tissues and little or no expression in other tissues (root, stem, flower, leaf, and cotyledon). Seven genes were chosen and their expression profile during seed development in P. vulgaris was analyzed by real-time PCR using RNA preparations originating from different seed development stages. This study revealed hitherto unknown genes putatively involved in dicotyledonous embryogenesis, which serve as a starting point for understanding Phaseolus embryogenesis.

Effect of drought stress on chlorophyll fluorescence, antioxidant enzyme activities and gene expression patterns in faba bean (Vicia faba L.)

ABSTRACT Drought is a major environmental stress that limits faba bean growth, leading to considerable reduction in their productivity. Two genotypes (Giza 3 and Hara) were evaluated under three water treatments (90%, 60% and 30% field capacity). The results showed that water stress affects negatively chlorophyll fluorescence parameters at different levels of water deficit and the variations of Fv/Fm, ΦPSII and electron transport rate in Giza 3 were largely higher than those in Hara. Drought preferentially enhanced the activities of superoxide dismutase, catalase and guaiacol peroxidase whereas it did not affect ascorbate peroxidase activity. Real-time reverse transcriptase-polymerase chain reaction results demonstrated that the expression of aquaporin (PIP), myeloblastosis (MYB), nine-cis-epoxycarotenoid dioxygenase, late embryogenesis abundant, dehydrins (Dhn), heat shock protein (HSP) and APETALA2/ethylene-responsive factor was modified in response to drought conditions and that all genes were differentially expressed in Hara and Giza 3. The observed expression patterns of these genes might be due to water deficit intensity according to cultivar’s tolerance to drought. Hara with the highest level of antioxidant enzyme activities, the accumulation of proline content and the expression level of Dhn, HSP and MYB gene throughout all conditions tested is reported more drought tolerant than Giza 3. This study should contribute to explaining the physiological, biochemical and molecular mechanism tolerance drought stress and improving breeding program in faba bean.

In silico identification and characterization of putative differentially expressed genes involved in common bean (Phaseolus vulgaris L.) seed development

Two genotypes of common bean (Phaseolus vulgaris L.) were studied to determine the structural cause of seed abortion in this species. In the non-abortive control (wild-type, cultivar BAT93), the histological analysis revealed a classical pattern of seed development and showed coordinated differentiation of the embryo proper, suspensor, endosperm tissue and seed coat. In contrast, the ethyl methanesulfonate (EMS) mutant (cultivar BAT93) showed disruption in the normal seed development leading to embryo abortion. Aborted embryos from these degenerate seeds showed abnormalities in suspensor and cotyledons at the globular, heart, torpedo and cotyledon stages. Exploring the feasibility of incorporating the available online bioinformatics databases, we identified 22 genes revealing high homology with genes involved in Arabidopsisthaliana embryo development and expressed in common bean immature seeds. The expression patterns of these genes were confirmed by RT–PCR. All genes were highly expressed in seed tissues. To study the expression profiles of isolated genes during Phaseolus embryogenesis, six selected genes were examined by quantitative RT–PCR analysis on the developing embryos of wild-type and EMS mutant plants. All selected genes were expressed differentially at different stages of embryo development. These results could help to improve understanding of the mechanism of common bean embryogenesis.

Genetic variabilitly in Tunisian populations of faba bean (Vicia faba L. var. major) assessed by morphological and SSR markers

The genetic diversity of 21 faba bean populations was examined using morphological and molecular markers. DNA was extracted from 189 individuals and 8 microsatellite markers were genotyped individually in these 21 populations. A total of 53 alleles were obtained in all populations, with an average of 6.62 alleles per locus. The expected and observed heterozygosity was 0.38 and 0.62 respectively. The average polymorphism index content of SSR markers was 0.61, ranging from 0.31 to 0.81. The unweighted pair group method with arithmetic mean dendrogram clustered all the populations into two groups, each for them subdivided into 3 sub-groups according to geographical origin. Morphological variation showed that the populations were not grouped according to their geographical origin. Therefore, patterns of differentiation of morphological traits did not coincide with molecular differentiation, indicating that morphological variation does not reflect genetic subdivision in studied faba bean populations. Analysis of molecular variance revealed high levels of genetic variation (83%) within population and provides a good base for designing genetic improvement programs. The result of Principal Component Analysis (PCA) revealed that three dimensional principal components (PC1, PC2 and PC3) contributed 40.56% of the total variability and accounted with values of 20.64, 11.22 and 8.70%, respectively. Cluster analysis based on PCA indicated three separate groups of populations. The genetic relationships found between the 21 populations samples were the same in both the PCA and STRUCTURE analysis which support the results observed. These data may serve as a foundation for the development of faba bean breeding programs.

FOOD WASTAGE BY TUNISIAN HOUSEHOLDS

Food waste (FW) is seen as an obstacle to achieving food and nutrition security and food systems sustainability. It is known in literature that households are significant contributors to the total amount of FW. This paper reports on results of an online survey that was conducted from February to April 2015 with a random sample of 281 Tunisian adults. The aim of the survey is to assess the knowledge and relative importance of FW; attitudes towards FW; impacts of behaviors regarding food and food management; quantity and value of FW; as well as barriers and willingness to behavioral change. The sample was not gender-balanced (71.2% female and 28.8% male). The majority of the respondents was young (70.8% aged between 18 and 34 years) and has high education level (95.4% having university and PhD degrees). Food waste is prevalent in Tunisia as about the half of respondents declare that they throw food. The most wasted food products are fruits, vegetables, and cereals and bakery products. Only 42.7% of respondents declared that the economic value of food waste generated each month is more than 6US

Changes in DNA-methylation during zygotic embryogenesis in interspecific hybrids of beans (Phaseolus ssp.)

. Most of Tunisian respondents have a good understanding of food labels that is probably due to the high education level of the sample. About 37% of respondents throw weekly at least 250 g of still consumable food. To reduce FW in Tunisia it is important to set a strategy at all food chain levels. There is also an urgent need to raise people’s and organizations awareness towards this problem. This article provides a basis for the development of other more context specific investigations and interventions for the prevention of household FW in Tunisia.