Ghassen Abid

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.

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.

Effect of genotype and growing season on nitrate accumulation and expression patterns of nitrate transporter genes in potato (Solanum tuberosum L.)

ABSTRACT Nitrate (NO3‒) is the most important source of mineral nitrogen (N) affecting the vegetative growth, the tuber yield and the quality of potato (Solanum tuberosum L.). The screening of 16 potato cultivars for low nitrate content was reported and the effects of genotype and experimental factors on nitrate accumulation were studied. Both genotype and growing season had a highly significant effect on dry weight, number of tubers/plant, yield and nitrate accumulation in potato which varied significantly from one genotype to another. Furthermore, nitrate accumulation varies with the season, being higher in the first season (autumn) than in the second season (spring), for all the genotypes. The cultivars Safrane and Universa followed by Actrice and Alaska produced the highest tuber yield/plant during the first and second season, respectively, accumulating higher quantities of nitrate compared to other cultivars. Ponomera cultivar was of interest from a breeding perspective due to both combining best tuber yield/plant and low nitrate accumulation for both growing season. Expression pattern of nitrate transporter genes (StNRT1.2, StNRT1.5 and StNRT2.1) has been reported. Thus, these genes were differentially expressed which could be explained in part by the difference in nitrate accumulation rate between the studied cultivars. The results showed that high accumulator genotype got significant higher expression level of StNRT1.2, StNRT1.5 or StNRT2.1 which lead to the higher nitrate uptake rate and consequently higher nitrate accumulation than low accumulator genotype.

Nitrate Content and Expression of Putative Nitrate Transporter Genes in Lettuce Fertilized with Nitrogen Fertilizers

ABSTRACT Nitrate accumulation in plants is influenced by environmental conditions and genetic variability. It is important to develop approaches that could be used to reduce nitrate levels in lettuce (Lactuca sativa L.) because nitrate accumulated in leaves can cause harmful health effects in humans. A greenhouse experiment was conducted to determine effects of ammonium nitrate or urea nitrogen on lettuce fresh weight, nitrate content, and characterization of nitrate transporter genes in lettuce. Head fresh weight in cultivars grown with the nitrogen fertilizers differed. All cultivars accumulated more nitrates with urea than with ammonium nitrate fertilizer. ‘Great Lakes’ had lower nitrate accumulation than other cultivars due to urea or ammonium nitrate treatment. Lettuce nitrate transporter genes LsNRT1 and LsNRT2 were detected and their expression level varied due to cultivar and leaf position. ‘Great Lakes’ had low mRNA levels of LsNRT1 and LsNRT2 in leaves compared to other cultivars. ‘Great Lakes’ could be used for lettuce production due to high yield and lower nitrate content.

Identification of Differentially Expressed Putative Nitrate Transporter Genes in Lettuce

ABSTRACT Nitrogen is important to plant growth and development, with nitrate being the major form absorbed by crops. Lettuce (Lactuca sativa L.) accumulates high levels of nitrate, which can cause harmful health effects in humans. Information is needed regarding accumulation of nitrate among lettuce genotypes. The lettuce cvs. Vitalia (type Butterhead); Great Lakes (type Iceberg); L01 (type Iceberg), and L02 (type Romaine) were used to determine expression patterns of nitrate transporter gene LsNRT in the cultivars and in nitrate accumulation. A computer modeling analysis indicated that genes LsNRT1 and LsNRT2 encode for low- and high-affinity nitrate transport, respectively, in lettuce. To better characterize nitrate transport in lettuce, the relative expression of these genes encoding nitrate transport was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). Higher nitrate accumulator cultivars posses higher nitrate transporter transcripts than low accumulators, indicating stronger ability to assimilate absorbed nitrate in the higher than the low accumulator. Increased knowledge of gene systems and identification of genotypes with low nitrate concentrations can be used in breeding programs.

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.

Identification and characterization of two faba bean ( Vicia faba L.) WRKY transcription factors and their expression analysis during salt and drought stress

Drought and salinity are two major environmental factors limiting faba bean growth, leading to considerable reduction in their productivity. The WRKY gene family act as major transcription factors that might play an important role in abiotic stress tolerance. In the present study, two partial sequences sharing significant homology with known WRKY genes were isolated from faba bean by polymerase chain reaction (PCR) amplification using degenerate primers targeting the well-conserved WRKY domain. The isolated WRKY gene fragments were designated as VfWRKY1 and VfWRKY2 showing 62% similarity between them. Sequence and phylogenetic analyses revealed that VfWRKY1 and VfWRKY2 belong to WRKY group I and could be grouped with their orthologues from other plant species. The gene expression profile of VfWRKY1 and VfWRKY 2 in faba bean showed that they are significantly accumulated in various plant organs. Further, quantitative real-time PCR analysis showed that both transcripts were responsive to drought and salt stress, and also they are genotype dependent, meaning that different faba bean cultivars respond in a different way to drought and salt challenge. The expression patterns obtained suggest the important roles of VfWRKY1 and VfWRKY2 in drought and salt stress response and tolerance. This knowledge might be helpful in the identification of drought-tolerant cultivars and provide potential candidate markers for faba bean breeding in order to develop osmotic-stress-tolerant cultivars.