Osmotin and osmotin-like proteins are stress proteins belonging to the plant PR-5 group of proteins induced in several plant species in response to various types of biotic and abiotic stresses. We report here the overexpression of tobacco osmotin in transgenic mulberry plants under the control of a constitutive promoter (CaMV 35S) as well as a stress-inducible rd29A promoter. Southern analysis of the transgenic plants revealed the stable integration of the introduced genes in the transformants. Real-time PCR analysis provided evidence for the expression of osmotin in the transgenic plants under both the constitutive and stress-inducible promoters. Transgenic plants with the stress-inducible promoter were observed to better tolerate salt and drought stress than those with the constitutive promoter. Transgenic plants when subjected to simulated salinity and drought stress conditions showed better cellular membrane stability (CMS) and photosynthetic yield than non-transgenic plants under conditions of both salinity and drought stress. Proline levels were very high in transgenic plants with the constitutive promoter relative to those with the stress-inducible promoter. Fungal challenge undertaken with three fungal species known to cause serious losses to mulberry cultivation, namely, Fusarium pallidoroseum, Colletotrichum gloeosporioides and Colletotrichum dematium, revealed that transgenic plants with osmotin under control of the constitutive promoter had a better resistance than those with osmotin under the control of the stress-inducible promoter. Evaluation in next generation was undertaken by studying bud break in transgenic and non-transgenic plants under simulated drought (2% polyethylene glycol) and salt stress (200 mM NaCl) conditions. The axillary buds of the selected transgenic lines had a better bud break percentage under stressed conditions than buds from non-transgenic mulberry lines. A biotic assay with Bombyx mori indicated that osmotin protein had no undesirable effect on silkworm rearing and feeding. We therefore conclude that 35S transgenic plants are better suited for both abiotic stress also biotic challenges (fungal), while the rd29A transgenic plants are more responsive to drought.

Coping with different kinds of biotic and abiotic stresses is the foundation of sustainable agriculture. Although conventional breeding and marker-assisted selection are being employed in mulberry (Morus indica L.) to develop better varieties, nonetheless the longer time periods required for these approaches necessitates the use of precise biotechnological approaches for sustainable agriculture. In an attempt to improve stress tolerance of mulberry, an important plant of the sericulture industry, an encoding late embryogenesis abundant gene from barley (HVA1) was introduced into mulberry plants by Agrobacterium-mediated transformation. Transgenic mulberry with barley Hva1 under a constitutive promoter actin1 was shown to enhance drought and salinity tolerance. Here, we report that overexpression of barley Hva1 also confers cold tolerance in transgenic mulberry. Further, barley Hva1 gene under control of a stress-inducible promoter rd29A can effectively negate growth retardation under non-stress conditions and confer stress tolerance in transgenic mulberry. Transgenic lines display normal morphology to enhanced growth and an increased tolerance against drought, salt and cold conditions as measured by free proline, membrane stability index and PSII activity. Protein accumulation was detected under stress conditions confirming inductive expression of HVA1 in transgenics. Investigations to assess stress tolerance of these plants under field conditions revealed an overall better performance than the non-transgenic plants. Enhanced expression of stress responsive genes such as Mi dnaJ and Mi 2-cysperoxidin suggests that Hva1 can regulate downstream genes associated with providing abiotic stress tolerance. The investigation of transgenic lines presented here demonstrates the acquisition of tolerance against drought, salt and cold stress in plants overexpressing barley Hva1, indicating that Arabidopsis rd29A promoter can function in mulberry.

Water deficit arises as a result of low temperature, salinity and dehydration, thereby affecting plant growth adversely and making it imperative for plants to surmount such situations by acclimatizing/adapting at various levels. Water deficit stress results in significant changes in gene expression, mediated by interconnected signal transduction pathways that may be triggered by calcium, and regulated via ABA dependent and/or independent pathways. Hence, adaptation of plants to such stresses involves maintaining cellular homeostasis, detoxification of harmful elements and also growth alterations. Stress in general cause excess production of reactive oxygen species (ROS) and the plants overcome the same by either preventing the accumulation of ROS or by eliminating the ROS formed. Ion homeostasis includes processes such as cellular uptake, sequestration and export in conjunction with long distance transport. Requisite amounts of osmolytes are hence synthesized under stress to maintain turgor along with maintaining the macromolecular structures and also for scavenging ROS. Another noteworthy response is the accumulation of novel proteins, including enzymes involved in the biosynthesis of osmoprotectants, heat-shock proteins (HSPs), late embryogenesis abundant (LEA) proteins, antifreeze proteins, chaperones, detoxification enzymes, transcription factors, kinases and phosphatases. The LEAs belong to a redundant protein family and are highly hydrophilic, boiling-soluble, non-globular and therefore have been defined and classified accordingly. The precise function of LEAs is still unknown, but substantial evidence indicates their involvement in dessication tolerance as the expression of LEAs confers increased resistance to stress in heterologous yeast system and also significantly improves water deficit tolerance in transgenic plants. Genetic manipulation of plants towards conferring abiotic stress tolerance is a daunting task, as the abiotic stress tolerance mechanism is highly complex and various strategies have been exploited to address and evaluate the stress tolerance mechanism, and the molecular responses to water deficit via complex signaling networks. Genomic technologies have recently been useful in integrating the multigenicity of the plant stress responses through, transcriptomics, proteomics and metabolite profilling and their interactions. This review deals with the recent developments on genetic approaches for water stress tolerance in plants, with special emphasis on LEAs.

No abstract