Abdullah A. Al-Doss

Engineering Melon Plants with Improved Fruit Shelf Life Using the TILLING Approach

Background Fruit ripening and softening are key traits that have an effect on food supply, fruit nutritional value and consequently, human health. Since ethylene induces ripening of climacteric fruit, it is one of the main targets to control fruit over ripening that leads to fruit softening and deterioration. The characterization of the ethylene pathway in Arabidopsis and tomato identified key genes that control fruit ripening. Methodology/Principal Findings To engineer melon fruit with improved shelf-life, we conducted a translational research experiment. We set up a TILLING platform in a monoecious and climacteric melon line, cloned genes that control ethylene production and screened for induced mutations that lead to fruits with enhanced shelf life. Two missense mutations, L124F and G194D, of the ethylene biosynthetic enzyme, ACC oxidase 1, were identified and the mutant plants were characterized with respect to fruit maturation. The L124F mutation is a conservative mutation occurring away from the enzyme active site and thus was predicted to not affect ethylene production and thus fruit ripening. In contrast, G194D modification occurs in a highly conserved amino acid position predicted, by crystallographic analysis, to affect the enzymatic activity. Phenotypic analysis of the G194D mutant fruit showed complete delayed ripening and yellowing with improved shelf life and, as predicted, the L124F mutation did not have an effect. Conclusions/Significance We constructed a mutant collection of 4023 melon M2 families. Based on the TILLING of 11 genes, we calculated the overall mutation rate of one mutation every 573 kb and identified 8 alleles per tilled kilobase. We also identified a TILLING mutant with enhanced fruit shelf life. This work demonstrates the effectiveness of TILLING as a reverse genetics tool to improve crop species. As cucurbits are model species in different areas of plant biology, we anticipate that the developed tool will be widely exploited by the scientific community.

Expression of the Aeluropus littoralis AlSAP gene in rice confers broad tolerance to abiotic stresses through maintenance of photosynthesis

The expression of AlSAP, in rice cv. Nipponbare, enhances plant tolerance to cold, drought and salt stresses. AlSAP lines showed 100% survival rate and set seeds while control plants did not recover from the cold treatment. Under a severe drought stress treatment (fraction of transpirable soil water down to 0.1), AlSAP lines exhibited enhanced Transpiration Efficiency (TE) and maintained a high A (Assimilation rate) value (22 µmol·m(-2)  s(-1) ) while these values dramatically decreased (A = 4 µmol·m(-2)  s(-1) ) in control plants which were subsequently unable to recover from the stress. Of noteworthy is that AlSAP rice plants yielded a similar and a 60% seed set under control and stress conditions respectively, with regard to wild-type (WT) plants grown under control conditions. This indicates that AlSAP expression imposes no yield penalty and allows seed production even following a severe drought stress at the vegetative stage. Furthermore, AlSAP rice was shown to accumulate transcripts of a pilot set of eight stress-related genes at a significantly higher level than WT plants, both under control and stressed conditions. The results suggest that AlSAP expression generates stress tolerance in plants through maintenance of the photosynthetic apparatus integrity and by stimulating an endogenous adaptive potential which is not effectively accomplished in WT plants.

Characterisation of alleles of tomato light signalling genes generated by TILLING

Targeting Induced Local Lesions IN Genomes (TILLING) combines chemical mutagenesis with high throughput screening to allow the generation of alleles of selected genes. In this study, TILLING has been applied to produce a series of mutations in genes encoding essential components of the tomato light signal transduction pathway in an attempt to enhance fruit nutritional quality. Point mutations to DEETIOLATED1 (DET1), which is responsible for the high pigment2 (hp2) tomato mutant, resulted in elevated levels of both carotenoid and phenylpropanoid phytonutrients in ripe fruit, whilst immature fruit showed increased chlorophyll content, photosynthetic capacity and altered fruit morphology. Furthermore, genotypes with mutations to the UV-DAMAGED DNA BINDING PROTEIN 1 (DDB1), COP1 and COP1like were also characterised. These genotypes largely did not display phenotypes characteristic of mutation to light signalling components but their characterisation has enabled interrogation of structure function relationships of the mutated genes.

Identification of new TRAP markers linked to chlorophyll content, leaf senescence, and cell membrane stability in water-stressed wheat

In order to identify target region amplification polymorphism (TRAP) markers linked to three physiological traits in wheat (Triticum aestivum L.), the segregating F4 population from the cross between drought-sensitive (Yecora Rojo) and drought-tolerant (Pavon 76) genotypes was made. The parents and 150 F4 families were evaluated phenotypically for drought tolerance using two irrigation treatments [2.5 and 7.5 m3(H2O) m−2(soil)]. Using 40 different TRAP primer combinations tested for polymorphism in parental and F4 family genotypes, the results revealed that quantitative trait locus (QTL) for chlorophyll content was associated with TRAP 5, TRAP 14, and TRAP 20 and explained 18, 16, and 23 % phenotypic variation, respectively. The genetic distance between chlorophyll content QTL and TRAP 5, TRAP 14, and TRAP 20 were 12.3, 19.8, and 13.6 cM, respectively. QTL for flag leaf senescence was associated with TRAP 2, TRAP 3, TRAP 15, and TRAP 16 and explained 33, 27, 28, and 23 % phenotypic variations, respectively. The genetic distance between flag leaf senescence QTL and TRAP 2, TRAP 3, TRAP 15, and TRAP 16 were 9.4, 14.7, 18.1, and 17.3 cM, respectively. QTL for cell membrane stability was associated with TRAP 8, TRAP 9, and TRAP 37 and explained 27, 30, and 24 % phenotypic variation, respectively. The markers TRAP 8, TRAP 9, and TRAP 37 had genetic distances of 17.0, 10.0, and 9.0 cM, respectively. Therefore, these TRAP markers can be used in breeding for drought tolerance in wheat.

Soil Seed Bank of a Desert Range Site Infested with Rhazya stricta in Raudhat al-Khafs, Saudi Arabia

Knowledge of the dynamics of soil seed bank in rangelands is important in planning for range management and improvement. A three-year study of soil seed bank was conducted in an area infested with Rhazya stricta Decne. The object of the study was to evaluate annual and seasonal variations in the soil seed bank over three degrees of infestation by R. stricta (low, moderate, and heavy). Soil samples were placed in germination trays in a growth chamber and irrigated. Emerging seedlings were identified and counted by species. Species were grouped into shrubs, legumes, grasses, and forbs. Results revealed the presence of seeds of 44 species, mostly annuals. Low similarity was observed between above-ground vegetation and soil seed components. Significant annual and seasonal variations were observed among groups of species. Generally, seed bank size and species richness were higher under the lower level of R. stricta infestation. It is concluded that most of the desirable species that have the potential of growing in the area are disappearing.

Mapping of QTLs associated with abscisic acid and water stress in wheat

A segregating F4 population from the cross between drought sensitive (Yecora Rojo) and drought tolerant (Pavon 76) genotypes was made to identify molecular markers linked to a wheat (Triticum aestivum L.) abscisic acid (ABA) content at two water regimes. The parents and 150 F4 lines were evaluated phenotypically for drought tolerance using two irrigation treatments [0.25 and 0.75 m3(H2O) m−2(soil)]. Forty different target region amplification polymorphism (TRAP) primer combinations, 98 different sequence-related amplified polymorphism (SRAP) primer combinations, and 400 simple sequence repeat (SSR) primers were tested for polymorphism among the parental genotypes and the F4 lines. Seven loci in the F4 lines treated with the drought stress were identified. Single quantitative trait loci (QTLs) were located on chromosomes 1B, 2A, 3A, 5D, and 7B and each of them explained from 15 to 31 % of phenotypic variance with a LOD value of 7.2 to 15.7. Five QTLs were located on chromosome 4A and six QTLs on chromosome 5A. In control (well-watered) F4 lines, two QTLs were mapped on chromosome 3B and one QTL on each chromosome 5B and 5D. Statistically the most significant groups of QTLs for the ABA content were identified in the regions of chromosomes 3B, 4A, and 5A mostly near to Barc164, Wmc96, and Trap9 markers. Therefore, these markers linked to QTLs for the drought-induced ABA content can be further used in breeding for drought tolerance in wheat.

The promoter of the AlSAP gene from the halophyte grass Aeluropus littoralis directs a stress-inducible expression pattern in transgenic rice plants

Key messageWhen fused to “PrAlSAP” promoter, transcripts ofgusAexhibited similar accumulation patterns in transgenic rice asAlSAPtranscripts inA. littoralis. PrAlSAPcan be used for engineering abiotic stress tolerance.AbstractWe previously showed that ectopic expression of a stress-associated protein gene from Aeluropus littoralis (AlSAP) enhances tolerance to multiple abiotic stresses in tobacco, wheat and rice. The ortholog of AlSAP in rice is OsSAP9. Here, we demonstrate that AlSAP transcripts accumulate in Aeleuropus in response to multiple abiotic stresses and at a higher level in roots, while those of OsSAP9 are preferentially induced by cold and heat treatments and accumulate preferentially in leaves of rice. In silico analysis of the AlSAP promoter “PrAlSAP” predicted several cis-acting elements responsible for gene regulation by dehydration, salt, heat, ABA, SA, wounding and tissue-specific expression. The PrAlSAP promoter was fused to the gusA gene and used to produce transgenic rice plants. Transcripts of gusA exhibited similar accumulation patterns in transgenic rice as AlSAP transcripts in A. littoralis. Indeed, accumulation of gusA transcripts was higher in roots than in leaves and induced by salt, drought, cold and heat treatments. GUS activity was confirmed in roots, coleoptiles, leaves and glumes, but absent in the root cell elongation zone and in dry seeds. A wound treatment strongly induced GUS accumulation in leaves and imbibed seeds. Altogether, these results indicate that the regulatory regions of two ortholog genes “AlSAP” and “OsSAP9” have diverged in the specificity of the signals promoting their induction, but that the trans-acting elements allowing the correct spatiotemporal regulation and stress induction of PrAlSAP exist in rice. Therefore, the AlSAP promoter appears to be an interesting candidate for engineering abiotic stress tolerance in cereals.

Rapid detection and characterization of Salmonella enterica serovars by multiplex polymerase chain reaction assay

Multiplex polymerase chain reaction (PCR) was used for molecular typing of Salmonella enterica serovars in Egypt. During the summer of 2010, a total of 1075 samples were collected from cattle, sheep and poultry farms to be subjected for isolation of Salmonella (290 rectal swabs from cattle, 335 rectal swabs from sheep and 450 cloacal swabs from poultry). Bacteriological examination revealed the isolation of 68 Salmonella belonging to 13 different Salmonella serovars. The most common serovars were Salmonella Typhimurium (16 isolates), Salmonella Enteritidis (13 isolates), Salmonella Kentucky (eight isolates) and Salmonella Arizona (seven isolates). Other serovars typed were Salmonella Heidelberg (four), Salmonella Cerro (four), Salmonella Gallinarum (three), Salmonella Virginia (three), Salmonella Paratyphi-A (three), Salmonella Dublin (two), Salmonella Agona (two), Salmonella Hadar (two) and Salmonella Bardo (one). The results of molecular techniques highlight the usefulness of the multiplex PCR for the rapid detection of the two serotypes of Salmonella from field samples especially after pre-enrichment on Rappaport-Vassiliadis (RV) media. Moreover, detecting S. Typhimurium and S. Enteritidis by this assay was carried out within two days as opposed to five to six days by the bacteriological and serological methods. Key words: Multiplex polymerase chain reaction (PCR), Salmonella Enteritidis, Salmonella Typhimurium , Egypt.

Ectopic Expression of Aeluropus littoralis Plasma Membrane Protein Gene AlTMP1 Confers Abiotic Stress Tolerance in Transgenic Tobacco by Improving Water Status and Cation Homeostasis

We report here the isolation and functional analysis of AlTMP1 gene encoding a member of the PMP3 protein family. In Aeluropus littoralis, AlTMP1 is highly induced by abscisic acid (ABA), cold, salt, and osmotic stresses. Transgenic tobacco expressing AlTMP1 exhibited enhanced tolerance to salt, osmotic, H2O2, heat and freezing stresses at the seedling stage. Under greenhouse conditions, the transgenic plants showed a higher level of tolerance to drought than to salinity. Noteworthy, AlTMP1 plants yielded two- and five-fold more seeds than non-transgenic plants (NT) under salt and drought stresses, respectively. The leaves of AlTMP1 plants accumulated lower Na+ but higher K+ and Ca2+ than those of NT plants. Tolerance to osmotic and salt stresses was associated with higher membrane stability, low electrolyte leakage, and improved water status. Finally, accumulation of AlTMP1 in tobacco altered the regulation of some stress-related genes in either a positive (NHX1, CAT1, APX1, and DREB1A) or negative (HKT1 and KT1) manner that could be related to the observed tolerance. These results suggest that AlTMP1 confers stress tolerance in tobacco through maintenance of ion homeostasis, increased membrane integrity, and water status. The observed tolerance may be due to a direct or indirect effect of AlTMP1 on the expression of stress-related genes which could stimulate an adaptive potential not present in NT plants.

Anther culture response in wheat (Triticum aestivum L.) genotypes with HMW alleles

The objective of this study was to develop a simple anther culture protocol for a range of Saudi wheat genotypes. Seven wheat genotypes were evaluated in anther culture on five different medium protocols for their ability to initiate callus and green plants. The estimates of significance for the effects of genotypes, and medium protocols used, and their interactions on callus induction, callus weight and shoot formation derived from anther explants indicated that the in vitro traits were significantly influenced by the genotypes, medium protocols, and their interactions. The percentage of explants that developed calli ranged from 0.41% (Lang) to 15.39% (Irena) averaged across the five medium protocols with an average 4.45%. The genotype Irena produced the highest average means of shoot formation (69.65%) across medium protocols. The genotype Yecora Rojo (13.73%) was significantly inferior to all other tested genotypes for shoot formation.