Zohreh Tabaeizadeh

Drought-Induced Responses in Plant Cells

Plants subjected to water stress undergo numerous physiological and metabolic changes. A general decrease in photosynthetic rate is among the most common responses. This is due to a programmed process involving the closure of stomata and reduction in the activity of photosynthetic enzymes. The plant hormone abscisic acid plays an important role in this process. Accumulation of compatible solutes, during water stress, is thought to be an adaptive response which has been developed by some plant species. Engineering the genes involved in the synthesis of these compounds, into nonaccumulating plants, has demonstrated promising results for genetic improvement of drought tolerance. Drought stress induces alteration of gene expression. A large number of genes which are upregulated by water stress have been isolated and characterized. Proteins encoded by some of these genes share several characteristics. The biochemical role of most of these gene products is unknown, but potential adaptive functions have been suggested. Abscisic acid is involved in the regulation of some of these genes.

Isolation of an osmotic stress- and abscisic acid-induced gene encoding an acidic endochitinase from Lycopersicon chilense.

We have identified one osmotic stress- and abscisic acid-responsive member of the endochitinase (EC 3.2.1.14) gene family from leaves of drought-stressed Lycopersicon chilense plants, a natural inhabitant of extremely arid regions in South America. The 966-bp full-length cDNA (designated pcht28) encodes an acidic chitinase precursor with an amino-terminal signal peptide. The mature protein is predicted to have 229 amino acid residues with a relative molecular mass of 24 943 and pI value of 6.2. Sequence analysis revealed that pcht28 has a high degree of homology with class II chitinases (EC 3.2.1.14) from tomato and tobacco. Expression of the pcht28 protein in Escherichia coli verified that it is indeed a chitinase. Northern blot analysis indicated that this gene has evolved a different pattern of expression from that of other family members reported thus far. It is highly induced by both osmotic stress and the plant hormone abscisic acid. Southern blot analysis of genomic DNA suggested that the pcht28-related genes may form a small multigene family in this species. The efficiency of induction of the gene by drought stress, in leaves and stems, is significantly higher in L. chilense than in the cultivated tomato. It is speculated that, besides its general defensive function, the pcht28-encoded chitinase may play a particular role in plant development or in protecting plants from pathogen attack during water stress.

Transgenic tomato plants expressing a Lycopersicon chilense chitinase gene demonstrate improved resistance to Verticillium dahliae race 2

Abstract An acidic endochitinase gene (pcht28) isolated from Lycopersicon chilense was introduced into tomato (L. esculentum) through Agrobacterium-mediated transformation, using the CAMV 35S promoter. Transgenic plants demonstrated a high level of constitutive expression of pcht28 and chitinase enzyme activity. Kanamycin-resistant R1 plants (resulting from self-pollination of transgenic plants) as well as R2 plants were evaluated for their tolerance to Verticillium dahliae (race 1 and 2 for R1 plants and race 2 for R2 plants) in the greenhouse. They demonstrated a significantly (P<0.05) higher level of tolerance to the fungi compared to the nontransgenic plants, as measured by foliar disease symptoms, vascular discoloration, and vascular discoloration index. The transgenic plants produced in this study represent a source of genetic resistance to Verticillium dahliae.

Chitinase: Differential induction of gene expression and enzyme activity by drought stress in the wild (Lycopersicon chilense Dun.) and cultivated (L. esculentum Mill.) tomatoes

Summary We have previously reported the isolation of a drought and abscisic acid (ABA)-induced chitinase gene from Lycopersicon chilense , a drought tolerant wild tomato (Chen et al., 1994. Mol. Gen. Genet. 245: 195–202). We have therefore carried out a comparative study in order to examine chitinase gene expression and enzyme activity in Lycopersicon chilense (LA2747 and LA1930) and L. esculentum (cultivar Starfire) during water stress. Both enzyme assay and northern blot analysis revealed that chitinase expression was differentially induced by drought in the two species. Higher induction of chitinase occurred in tolerant species compared to the sensitive one. Among the genotypes examined, L. chilense LA2747 which is the most drought tolerant, presented the highest level of chitinase induction, while the lowest level was found in L. esculentum , Starfire. Similar results were obtained using cell suspensions treated with ABA and mannitol. Both ABA treatment and osmotic stress induced chitinase gene expression in the cell suspensions and this induction was more pronounced in the wild tomato compared to the cultivated one. Drought induced accumulation of chitinase mRNA was investigated in L. chilense , LA2747 leaves, stems and roots. The results revealed that the chitinase gene was organ specifically expressed during drought stress. Leaves of drought stressed plants showed the highest expression and roots showed the lowest. The accumulation of chitinase during drought stress was also confirmed by in situ hybridization. Leaf water potentials (LWP) were determined in the three genotypes during drought stress. A correlation was found between the chitinase gene expression and leaf water potential during drought stress. The highest level of leaf water potential was maintained in L. chilense LA2747 which also demonstrates the highest level of chitinase expression among the three genotypes. Our results suggest that the L. chilense chitinase gene might be involved in drought tolerance of this species.

Identification and immunolocalization of a 65 kDa drought induced protein in cultivated tomatoLycopersicon esculentum

SummaryA 65 kDa protein with a pI value of 5.2 accumulated gradually in tomato leaves during water stress. Protein levels returned to those of the control upon rehydration of the plants. Antiserum raised against the protein, purified from two dimensional electrophoresis gels, was obtained and used as a probe to localize the protein in tomato leaves by immunofluorescence and immunogold labeling. The protein was found to be mainly localized in different areas of nuclei (peripheral chromatin masses, nucleoli and nucleoplasm), chloroplasts, and some leaf cell cytoplasmic regions. Quantification of the gold labeling clearly demonstrated that the amount of the protein increased significantly in nuclei and chloroplasts of cells in drought-stressed plants. Cytological changes occurring in leaf tissues during water stress are also reported.

Transformation of the wild tomatoLycopersicon chilense Dun. byAgrobacterium tumefaciens.

SummaryLeaf disc transformation-regeneration technique was applied to the drought tolerant wild relative of cultivated tomato,Lycopersicon chilense, using a plasmid construct which contained the coding sequences of neomycin phosphotransferase (NPTII) and chloramphenicol acetyltransferase (CAT) genes. The two genotypes used, LA2747 and LA1930, showed a distinct difference in their aptitude to transformation; a higher success rate was obtained for the first genotype in every stage of the process. Shoots were formed on the regeneration medium containing 100 μg/ml kanamycin through direct or indirect organogenesis. Root formation became only possible when the concentration of kanamycin was reduced to 50 μg/ml. Expression of chloramphenicol acetyltransferase gene was observed in all of the kanamycin-screened plants after they matured; the activity of the gene was absent or low in some of the young plants. The presence of the CAT gene in transgenic plants was further confirmed by Southern blot analysis. Although transgenic plants grew to maturity, they did not produce fruit, owing to the self incompatibility ofL. chilense.

Somatic embryogenesis and plant regeneration in Triticum aestivum x Leymus angustus F1 hybrids and the parental lines

Somatic embryos and plants were produced from cultured inflorescence and leaf segments of Triticum aestivum X Leymus anaustus F1 hybrids and the parental lines. Inflorescences showed a better capacity for somatic embryogenesis and plant regeneration than leaves. Leymus anaustus produced the highest number of embryogenic calli, while the hybrids were intermediate between this species and Triticum aestivum. Presence of 2,4-D was shown to be essential for induction and maintenance of somatic embryogenesis. Addition of five amino acids (glutamine, proline, asparagine, aspartic acid and glutamic acid) did not have any marked effect when they were used in the callus induction medium. The regenerated plants had the same morphology as the original plants. No cytological modification was observed in the examined plants.

Genetic transformation of a pasture legume,Lotuscorniculatus, L. (birdsfoot trefoil)

SummaryA rapid procedure for introducing foreign genes inLotuscorniculatus based on the induction of hairy roots byAgrobacteriumrhizogenes was developed. Expression of chloramphenicol acetyltransferase and neomycin phosphotransferase II was revealed in transgenic plants. Southern blot hybridization was used to confirm the genetic transformation. The transgenic plants looked normal and did not show any morphological modification compared to the seed grown plants.

Embryogenic cell suspensions of Triticum aestivum X Leymus angustus F1 hybrids : characterization and plant regeneration

SummaryEmbryogenic cell suspension cultures were established from Triticum aestivum X Leymus angustus F1 hybrids, using compact nodular calli derived from inflorescence segments. Calli originating from leaf segments did not give rise to stable cell suspensions. Growth measurements of the cell suspensions revealed that they continued rapid growth up to 10 days after subculturing. Flow cytometric studies of the cell cycle over a 7 day culture period showed that the majority of cells were in G1 phase while the rest were either in S or G2. During the 7 days of culture, no significant differences in DNA distribution patterns were observed. The cells from suspension cultures produced somatic embryos when they were transferred to different solid media. The embryos germinated and gave rise to plantlets which were successfully rooted and transferred to soil.