Afif Hassairi

Overexpression of wheat dehydrin DHN-5 enhances tolerance to salt and osmotic stress in Arabidopsis thaliana

Late Embryogenesis Abundant (LEA) proteins are associated with tolerance to water-related stress. A wheat (Triticum durum) group 2 LEA proteins, known also as dehydrin (DHN-5), has been previously shown to be induced by salt and abscisic acid (ABA). In this report, we analyze the effect of ectopic expression of Dhn-5 cDNA in Arabidopsis thaliana plants and their response to salt and osmotic stress. When compared to wild type plants, the Dhn-5 transgenic plants exhibited stronger growth under high concentrations of NaCl or under water deprivation, and showed a faster recovery from mannitol treatment. Leaf area and seed germination rate decreased much more in wild type than in transgenic plants subjected to salt stress. Moreover, the water potential was more negative in transgenic than in wild type plants. In addition, the transgenic plants have higher proline contents and lower water loss rate under water stress. Also, Na+ and K+ accumulate to higher contents in the leaves of the transgenic plants. Our data strongly support the hypothesis that Dhn-5, by its protective role, contributes to an improved tolerance to salt and drought stress through osmotic adjustment.

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.

Transformation of two potato cultivars ‘Spunta’ and ‘Claustar’ (Solanum tuberosum) with lettuce mosaic virus coat protein gene and heterologous immunity to potato virus Y

Abstract The coat protein (CP) gene of the lettuce mosaic potyvirus (LMV) was introduced into two potato cultivars—Spunta and Claustar—via Agrobacterium tumefaciens -mediated transformation. Gene expression was verified by Southern. Northern and Western blot. The regenerated plants have been tested for resistance against potato virus Y (PVY-0) in greenhouse. We observed an immune phenotype in four transgenic lines expressing the CP-LMV protein at different detectable levels. However, in two other transgenic plant lines, symptoms were developed at a frequency of 25 and 60% with a delay in their appearance. Transgene transcripts of the CP-LMV gene were detected by Northern blot in these two lines, but the protein could not be detected by Western blot. The correlation between the presence of the CP protein at a detectable level and the expression of an immune phenotype is discussed.

Marker-free transgenic durum wheat cv. Karim expressing the AlSAP gene exhibits a high level of tolerance to salinity and dehydration stresses

We have recently isolated the AlSAP (stress-associated protein) gene from the halophyte grass Aeluropus littoralis and demonstrated that AlSAP expression improves tolerance to continuous salt and drought stresses in transgenic tobacco. To extend these findings to an important crop, we generated marker-free transgenic durum wheat plants of the commercial cv. Karim expressing the AlSAP gene. The integration and expression of AlSAP in T3 homozygous plants were ascertained by Southern, Northern and Western blotting respectively. AlSAP wheat lines exhibited improved germination rates and biomass production under severe salinity and osmotic stress conditions. Following a long-term salt or drought stress greenhouse trial, AlSAP lines produced normally filled grains whereas wild-type (WT) plants either died at the vegetative stage under salt stress or showed markedly reduced grain filling under drought stress. Measurements of the RWC (relative water content) and endogenous Na+ and K+ levels in leaves of AlSAP plants, showed a lower water loss rate and a higher Na+ accumulation in senescent-basal leaves, respectively, compared to those of WT plants. Taken together, these results extend to cereals the high potential of the AlSAP gene for engineering effective drought and salt tolerance.

Adventitious shoot production from strips of stem in the Dutch elm hybrid 'Commelin' : Plantlet regeneration and neomycin sensitivity

The regenerative ability of small strips of stem of the Dutch elm hybrid ‘Commelin’ was tested as well as its sensitivity to neomycins. Cambium explants (1 mm thick), were excised from woody stems collected in the field. Up to 20 buds/explant were induced within 2–3 weeks giving 2–5 rootable shoots/explant after 5–6 weeks. Shoot excision every week from week three improved the yield up to 7 shoots per explant. Fourteen and 2.9 μM GA3 promoted shoot growth. Cytokinins (1 μM zeatin or 5 μM BA or 0.05 μM TDZ) completely inhibited shoot production and promoted callus formation. Kanamycin and paromomycin at between 240 and 360 μM inhibited shoot formation as did geneticin at 80 μM. The shoot-forming ability of the explants was high from leaf fall in the autumn to the spring flush, but could be maintained up to September by using cold storage (5–7 °C). Ninety-six percent of the shoots rooted with 0.5 μM IBA and were successfully acclimatized despite having a large basal callus.

Promoter of the AlSAP gene from the halophyte grass Aeluropus littoralis directs developmental-regulated, stress-inducible, and organ-specific gene expression in transgenic tobacco

In our recent published work it has been demonstrated that AlSAP, a gene encoding an A20/AN1 zinc-finger protein (stress-associated protein) of the C4 halophyte grass Aeluropuslittoralis, is inducible by various abiotic stresses and by hormonal stimuli. To further investigate the regulation of the gene, a 586-bp genomic fragment upstream of the AlSAP translated sequence has been isolated, cloned, and designated as the “PrAlSAP” promoter. Sequence analysis of “PrAlSAP” revealed the presence of cis-regulatory elements which could be required for abiotic stress, abscisic acid (ABA), and salicylic acid (SA) responsiveness and for tissue-specific and vascular expression. The PrAlSAP promoter was fused to the β-glucuronidase (gusA) gene and the resulting construct transferred into tobacco. Histochemical assays of stably transformed tobacco plants showed that PrAlSAP is active in this heterologous C3 system. While full-length gusA transcripts accumulated in whole 15, 30, and 45-day-old plants, GUS histochemical staining was only observed in leaves and stems of 45-day-old, or older, transgenic seedlings. Histological sections prepared at this stage revealed activity localized in leaf veins (phloem and bundle sheath) and stems (phloem and cortex) but not in roots. Furthermore, gusA transcripts accumulated in an age-dependent manner with a basipetal pattern in leaf and stem tissues throughout the plant. In flowers, GUS expression was detected in sepals only. The accumulation of gusA transcripts was up-regulated by salt, dehydration, ABA, and SA treatment. Altogether, these results show that, when used in a heterologous dicot system, PrAlSAP is an age-dependent, abiotic-stress-inducible, organ-specific and tissue-specific promoter.

In vitro budding ability of woody internode and Agrobacterium susceptibility as prerequisites for elm genetic transformation

Summary In order to improve elm resistance to dutch elm disease by genetic engineering, woody internodes of field grown elms Commelin were tested for regeneration capacity and susceptibility to Agrobacterium tumefaciens . After a drastic surface disinfection (especially burning), the explants were introduced in vitro on a medium containing cef otaxime (500 mg - L ∔1 ). Budding took place within 2 weeks on the cambial part of the explants. After 1-2 months, the number of rootable shoots per explant was 3 -6, increasing up to 13 by shoot excision. Shoot rooting (70 %) and acclimatization (survival 80 %) were easy. Agrobacterium tumefaciens wild strains (Ach5, T37, C58, 82-139) were either inoculated on shoots or cocultivated on internodes. Octopine/agropine type Ach5 induced tumors (> 50 %) where agropine was often detected. Efficiency of 82-139 was lower. However, it was very effective in improving budding ability of internodes up to 11. A strategy to obtain transgenic elm based on these results is now in progress.

Tobacco plants transformed with an untranslatable form of the coat protein gene of the Potato virus Y are resistant to viral infection

The coat protein (CP) gene of the Potato virus Y common strain (PVYo), rendered untranslatable by introduction of a stop codon immediately after the initiation codon, was cloned into an Agrobacterium tumefaciens transformation vector. The regenerated transgenic tobacco plants were analyzed for the presence of the CP gene. Although transgene transcripts were observed in all transgenic lines tested, three different phenotypes of resistance were observed, including high resistance, partial resistance, and susceptibility. An inverse correlation between the transgene transcript steady-state levels and resistance of transgenic plants to PVYo was observed. The results obtained suggest that the resistance observed in the transgenic plants is essentially due to the presence of multiple PVY transgenes that accumulate low levels of the transgene transcript. A post-transcriptional RNA degradation process appears to support the transgene transcript reduction within the RNA-mediated resistance mechanism.

Identification of TIFY/JAZ family genes in Solanum lycopersicum and their regulation in response to abiotic stresses

Plant phenotypic plasticity determines plant adaptation to changing environments and agricultural productivity. Phytohormones are essential plant signalling molecules regulating this plasticity through complex signalling networks. Jasmonates (JAs) are key phytohormones regulating many aspects of growth, development and defence responses. An important role of JAs in tolerance to abiotic stresses is also emerging. The expression of JAZ (JASMONATE-ZIM-DOMAIN PROTEIN) genes, encoding for the key repressors in the JA-pathway, is regulated by multiple abiotic stresses, suggesting a role for the JAZ proteins in response to these stresses. The JAZ proteins belong to the TIFY family, well described in many plant species. However, only the role of few tomato JAZ proteins in response to microbial infection has been analysed so far. Here, we identify the members of the tomato TIFY family, and characterize them phylogenetically. In addition, we analyse the transcriptional regulation of several SlJAZ in response to abiotic stresses and hormone treatments both in root and leaves to assess their specific expression in response to stresses. Most SlJAZ are JA-induced and responsive to one or more abiotic stresses, providing clues for functional analysis of JAZ genes in abiotic responses in tomato.

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.