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The 70-kDa heat shock proteins (HSP70s) are a conserved class of chaperones that play critical roles during the normal life cycle of plants. HSP70s are particularly involved in the regulation of biotic and abiotic stress responses. In this paper, the potential roles of this protein were investigated. A reverse genetic approach was employed for transient silencing of hsp70 gene in tomato (Solanum lycopersicum L.) to evaluate different growth and physiological parameters under normal conditions and during the response to drought stress. A combined ANOVA (analysis of variance) and HCA (hierarchical clustering analysis) showed that hsp70 silencing led to severe growth retardation and mortality, significant membrane damage and leakage, decline in relative water content, low rate of pigment accumulation, and reduced antioxidant enzyme activity under normal and drought stress conditions. Among the different parameters, proline was the only trait that was unaffected by gene silencing and accumulated by similar amounts to that of nonsilent plants. In conclusion, HSP70 played critical roles in maintaining the cellular homeostasis of plants during adaptation to drought and under normal plant life conditions. It was speculated that proline was, to some extent, involved in improving the loss of protein folding or function resulting from HSP70 deficiency, and played a crucial role in the adaptation of plants on exposure to stress.

The natural variation of drought tolerance among eight Iranian bermudagrass (Cynodon dactylon L.) accessions was evaluated and compared with a commonly used variety by measuring various morphological, growth, physiological and molecular responses during mild and severe drought stress conditions. A combined multivariable analysis using principal component analysis, hierarchical clustering analysis, as well as drought-tolerance ranking measurement showed that the accessions were clustered into three groups in terms of drought tolerance. This method evaluated three accessions (Maragh, Abyaneh, and Badrood) as superior drought-tolerant, three (Karshahi, Josheghan, and Kashan) as relatively drought-tolerant, and three (Ardeha, SWI-7, and Khonb) as drought-sanative genotypes. Drought-tolerant accessions generally showed improved growth and relative water content (RWC), minor electrolyte leakage (EL) and lipid peroxidation (MDA), higher content of pigments (Chlorophyll and carotenoid) and dark green color index (DGCI), more significant up-regulation of two stress-related genes (ECERIFERUM1 and Delta 1-pyrroline-5-carboxylate synthetase), as well as higher accumulation of proline content compared to sensitive accessions under drought stress. On the other hand, drought-sensitive accessions typically had more activity of antioxidant enzymes (catalase and ascorbate peroxidase), and higher amounts of soluble sugars (glucose, mannose, and rhamnose), compared to tolerant accessions under drought stress. In conclusion, different defensive strategies may be recruited by tolerant or sensitive bermudagrass genotypes during encountering to drought stress.

Calcineurin-B-like proteins (CBLs) are essential components of the calcium signaling network and act during plant's response to stress and normal conditions. A combined research strategy of in-silico analysis and gene silencing experiment was employed to investigate the role of different classes of CBLs in tomato (Solanum lycopersicum L.) during the response to drought stress. Two different classes of CBL genes, including SlCBL3-1, and SlCBL3-2, with the minimum and a maximum number of drought-responsive cis-elements, were selected and were targeted for transient gene silencing in tomato followed by the drought treatment. The effect of silencing events was evaluated by determining of further growth and physiological traits in plants under both control and drought stress conditions. The results showed that silencing of SlCBL3-1 significantly reduced shoot and root growth, relative water content (RWC), and the concentration of pigments while increased free radical accumulation, lipid peroxidation, and leakage from the cells. On the other hand, no antioxidant enzyme activity or proline induction was triggered in plants after SlCBL3-1 silencing. Some of these adverse events were more significantly enhanced when the silenced plants were exposed to drought stress. Overall, a significant role for SlCBL3-1 in the life cycle of plant suggested under both normal and stress conditions. The SlCBL3-2 silencing showed more efficient plants recovery from silencing or drought stress conditions. Therefore, SlCBL3-2 gene may act as a negative regulator under stress conditions. The results might provide new theoretical insight and genetic resources for developing resistant crops against environmental stresses.

Heat shock proteins (HSPs) have vital roles during plant adaptation to biotic and abiotic stresses, as well as stress-free conditions. In the present study, we used a heterologous strategy of virus induced gene silencing to investigate the role of different classes of HSPs in Nicotiana benthamiana. Different growth and physiological parameters in silenced plants were evaluated under both control and salt stress conditions. Among the treatments, silencing of HSP70, especially under salinity regime, was found to have stronger impacts on growth, protein concentration, the accumulation of photosynthetic pigments, proline and total soluble carbohydrates content, malondialdehyde, the activity of antioxidant enzymes, performance index, relative water content and the ratio of K+/Na+, suggesting a more prominent role for HSP70 in both stressful and stress-free life cycle of the plants. Principal component analysis and hierarchical clustering indicated that HSP70 gene was silenced, the plants might effectively respond to stress by a higher accumulation of compatible solutes, like proline to protect the cell membranes and proteins from damage.

The growth and physiological parameters of fourteen tomato cultivars were measured during both mild and severe drought. The tomato cultivars showed decrease in growth traits, relative water content and pigments content under both drought treatments compared with the control. By contrast, proline accumulation, malondialdehyde and electrolyte leakage increased in the cultivars. The activity of superoxide dismutase was more correlated to drought stress than those of ascorbate peroxidase or catalase. The combined approach of principal component analysis, hierarchical clustering analysis and calculating mean tolerance value showed relative differences in drought tolerance among the cultivars. The studied cultivars were clustered into four groups. Four cultivars were placed in cluster I as the drought-tolerant group. Moderately tolerant cultivars were grouped into Cluster II. The clusters III and IV represented the sensitive and highly sensitive cultivars, respectively. Early orbana, Roma and Cal-j cultivars were indicated as the highly sensitive tomato cultivars. It was concluded that such an approach could be used as a useful tool to screen the other abiotic or even biotic stress tolerance in tomato.

Eleven genes encoding Calcineurin B-Like proteins with a high degree of sequence conservation were identified using bioinformatics approaches in tomato. These proteins classified into five clusters including SlCBL1, SlCBL3, SlCBL4, SlCBL8 and SlCBL10 using orthology-based method of nomenclature. Sequence analysis showed that all five members of SlCBL1 and SlCBL4 contained a myristoylation conserved motif (MGXXXS/T) at their N-terminals. Semi-quantitative RT-PCR showed that among the SlCBL1 members, SlCBL1-3 up-regulated under both drought and virus stresses, as well as the combined treatment. Although, both SlCBL3-1 and SlCBL3-2 up-regulated under both drought and virus stresses in both susceptive and resistant cultivars, the combined stress did not have any additional effect on the expression. Among SlCBL4 members, only SlCBL4-1 up-regulated under drought or virus attack. There was a diverse pattern of expression between the two SlCBL8 members under different stresses in both cultivars. SlCBL10 showed no change in expression pattern under drought or virus stresses in susceptive cultivar and this gene showed to be up-regulated under drought in resistant cultivar. Overall, it was concluded that changes in the expression pattern of CBL genes under biotic and abiotic stresses seemingly induced various CBL/CIPK patways in suseptive or resistant plants.

In this work, we investigated the importance of phytoene desaturase (PDS) during salt stress in plants. For this aim, we transiently suppressed the corresponding gene of Nicotiana benthamiana using a Tobacco Rattle Virus silencing system. After salinity stress, the silenced plants were assayed for different growth and physiological parameters. The silenced plants were found to be induced more stunted and feeble shoots and roots than the negative controls. The results indicated that the concurrent incidence of salinity stress and viral infection did not have an additive influences on the plant growth and physiological parameters. Importantly, our results showed a more detrimental influence of salinity on plant growth and physiology after PDS silencing. Compared to the control, salt-stressed plants contained more proline, and malondialdehyde, less amount of photosynthetic pigments, higher Na+/K+ ratios in shoots and roots and lower relative water content after PDS silencing. The activity of antioxidant enzymes were also decreased in the plants coped with salinity or Tobacco Rattle Virus. It was also represented that simultaneous silencing of the PDS gene and salinity treatment resulted in a significant decrease of the performance index (PIABS) and an increase of dissipation per active reaction center (DIo/RC) reflecting severe injuries in photosynthetic machinery under such a condition. Overall results suggested that PDS silencing could increase the sensitivity of plants to salinity.

VIGS (virus induced gene silencing) is considered as a powerful genomics tool for characterizing the function of genes in a few closely related plant species. The investigations have been carried out mainly in order to test if a pre-existing VIGS vector can serve as an efficient tool for gene silencing in a diverse array of plant species. Another route of investigation has been the constructing of new viral vectors to act in their hosts. Our approach was the creation of a heterologous system in which silencing of endogenous genes was achieved by sequences isolated from evolutionary remote species. In this study, we showed that a TRV-based vector cloned with sequences from a gymnosperm, Taxus baccata L. silenced the endogenous phytoene desaturase in an angiosperm, N. benthamiana. Our results showed that inserts of between 390 and 724 bp isolated from a conserved fragment of the Taxus PDS led to silencing of its homolog in tobacco. The real time analysis indicated that the expression of PDS was reduced 2.1- to 4.0-fold in pTRV-TbPDS infected plants compared with buffer treated plants. Once the best insert is identified and the conditions are optimized for heterologous silencing by pTRV-TbPDS in tobacco, then we can test if TRV can serve as an efficient silencing vector in Taxus. This strategy could also be used to silence a diverse array of genes from a wide range of species which have no VIGS protocol. The results also showed that plants silenced heterologously by the VIGS system a minimally affected with respect to plant growth which may be ideal for studying the genes that their complete loss of function may lead to decrease of plant growth or plant death.

A highly promising procedure to obtain seedlings of Taxus baccata L. has been developed, which involves a combination of in vitro embryo culture and growth under hydroponic conditions. Embryos isolated from freshly collected seeds were 100% sterile, even though the seeds were not treated with acid or soaked in water prior to culturing. The embryo germination level of non-leached seeds was slightly lower (85%) than those leached in running water for 7 d (100%). The leached embryos germinated with extended roots while the non-leached embryos had abnormal shapes. The embryos cultured on media supplemented with an absorbent (PVP or activated charcoal) had extended roots and shoots and were a larger size without any browning, as compared to those grown without the supplement; activated charcoal gave better results. There were no significant differences in germination rates of T. baccata embryos between the media with differing strengths of macronutrients; however, for further development of the shoot, it was necessary to sub-culture the seedlings in MS in the light. To obtain seedlings with longer roots, they had to be maintained in one-half strength MS in darkness. Approximately 90% of the plants survived when grown hydroponically for 2 mo. The surviving plants showed well-extended roots and were a good starting material for genomic, proteomic, and conservational studies as well as Taxol permeabilization investigations.

Background and Aims: Virus-Induced Gene Silencing (VIGS) is a virus vector technology that exploits antiviral defense mechanism. By infecting plants with recombinant viruses containing host genes inserted in the viral genome, VIGS achieves the RNA silencing process. The purpose of this study was to investigate the possibility of tomato (Lycopersicon esculentum Mill.) and tobacco (Nicotiana benthamiana) phytoene desaturase (PDS) gene silencing, using VIGS technique by VIGS vector containing tomato PDS. Methods: PDS gene encodes one of the important enzymes in the carotenoid biosynthesis pathway. In this method Tobacco rattle tobravirus (TRV) vectors including pTRV1 ،pTRV2 and pTRV2PDS were used. The pTRV2PDS vector carried inserts derived from tomato PDS gene. Vectors were transformed into Escherichia coli strain DH5α. Agrobacterium tumefaciens strain GV3101 cells were transformed by the vectors containing specific genes. Results: Colony PCR with specific primers proved the presence of the PDS and RNAdependent RNA polymerase genes in selected agrobacterium random colonies. Agrobactrium containing pTRV1 ،pTRV2 and pTRV2PDS was inoculated into induction media culture. Finally, acetosyringone was added to pTRV1 culture and then pTRV1 and pTRV2PDS cultures mixed in a 1:1 ratio. Mixed culture was infiltrated into the test plant seedlings using 1ml needless syringe. Conclusion: Results showed the gene silencing in the form of photobleaching and mosaic occurred in tomato and tobacco plant leaves, respectively. However, plant infiltration with pTRV1 and pTRV2 (without PDS gene as control) mixed culture did not show any photobleaching.