Programmed cell death (PCD) is a physiological cell death process involved in the selective elimination of unwanted cells (Ellis et al., 1991). In animals, these unwanted cells include those that have served temporary functions, such as tadpole tail cells at metamorphosis; cells that are overproduced, such as vertebrate neurons; cells that are unwanted or present in inappropriate positions, such as cells between the developing digits and Mullerian duct cells required in females but not males; and cells that die during the process of cell specialization, such as keratinocytes at the surface of the skin (Jacobsen et al., 1997). PCD in specific cell types can also give rise to disease. These cell types include helper T cells, which undergo PCD in AIDS, and selected brain neurons, which die during Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and Lou Gehrig’s disease (Duke et al., 1996). Thus, PCD plays an important role in cell and tissue homeostasis and specialization, tissue sculpting, and disease. PCD in Caenorhabditis elegans and other animals depends on the induction and action of specific genes that bring about the controlled disassembly of a cell (Wadewitz and Lockshin, 1988; Ellis et al., 1991). This disassembly involves the condensation, shrinkage, and fragmentation of the cytoplasm and nucleus and the fragmentation of the nuclear DNA into -50-kb and, in some cases, -0.14-kb pieces that represent oligonucleosomes (Cohen, 1993; Eastman et al., 1994). These DNA fragments can be detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) of DNA 3’-OH groups in sections of cells (Gavrieli et al., 1992). When PCD features marginalization of the chromatin in the nucleus, breakage of the cytoplasm and nucleus into small, sealed packets, and processing and fragmentation of the DNA at nucleosome linker sites, the process is termed apoptosis (Cohen, 1993). However, not all PCD involves all of these changes (Schwartz et al., 1993). Many of the proteins responsible for PCD in animal cells have now been identified. Proteases of the interleukin-1 pconverting enzyme (ICE) family bring about cell condensation and shrinkage (Martin and Green, 1995), and endonucleases cause the fragmentation of nuclear DNA (Eastman et al., 1994). The basic leucine zipper transcription factor ces-2 (for

Programmed Cell Death in Plants.

The role of ABA and MAPK signaling pathways in plant abiotic stress responses.

Antioxidant enzymes play an important role in conferring abiotic stress tolerance. Superoxide dismutase (SOD) is the first enzyme in the enzymatic antioxidative pathway. Halophytic plants like mangroves have been reported to have a high level of SOD activity, which plays a major role in defending the mangrove species against severe abiotic stresses. We had previously reported the isolation of Sod1, a cDNA encoding a cytosolic copper zinc superoxide dismutase from the mangrove plant Avicennia marina and its mRNA expression pattern during various oxidative and abiotic stresses. The present study is an extension of the previous study in further characterizing the Sod1 cDNA by transforming it into rice and analysing the transgenic plants for abiotic stress tolerance. Southern hybridization of A. marina genomic DNA using Sod1, revealed that this gene in A. marina genome is present as a single copy. The cDNA was cloned into a binary vector (pCAMBIA 1300) and transformed into indica rice var Pusa Basmati-1. Southern hybridization analysis of transgenic rice plants revealed stable integration of the Sod1 transgene in the rice genome. The mRNA transcript of Sod1 was detected by Northern hybridisation in the transgenic rice plants. SOD isozyme assay of the transgenic rice plants revealed the stable expression of the transgenic Sod1 protein. The transgenic plants were more tolerant to methyl viologen mediated oxidative stress in comparison to the untransformed control plants. The transgenic plants also withstood salinity stress of 150 mM of NaCl for a period of eight days while the untransformed control plants wilted at the end of the stress treatment in hydroponics. Pot grown transgenic plants could also tolerate salinity stress better than the untransformed control plants, when irrigated with saline water. The transgenic plants also revealed better tolerance to drought stress in comparison to untransformed control plants.

Over expression of cytosolic copper/zinc superoxide dismutase from a mangrove plant Avicennia marina in indica Rice var Pusa Basmati-1 confers abiotic stress tolerance

Salinity is one of the major environmental factors limiting growth and productivity of rice plants. In this study, the effect of salt stress on phospholipid signaling responses in rice leaves was investigated. Leaf cuts were radiolabeled with 32P-orthophosphate and the lipids extracted and analyzed by thin-layer chromatography, autoradiography and phosphoimaging. Phospholipids were identified by co-migration of known standards. Results showed that 32P(i) was rapidly incorporated into the minor lipids, phosphatidylinositol bisphosphate (PIP2) and phosphatidic acid (PA) and, interestingly, also into the structural lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), which normally label relatively slowly, like phosphatidylcholine (PC) and phosphatidylinositol (PI). Only very small amounts of PIP2 were found. However, in response to salt stress (NaCl), PIP2 levels rapidly (<30 min) increased up to 4-fold, in a time- and dose-dependent manner. PA and its phosphorylated product, diacylglycerolpyrophosphate (DGPP), also increased upon NaCl stress, while cardiolipin (CL) levels decreased. All other phospholipid levels remained unchanged. PA signaling can be generated via the combined action of phospholipase C (PLC) and diacylglycerol kinase (DGK) or directly via phospholipase D (PLD). The latter can be measured in vivo, using a transphosphatidylation assay. Interestingly, these measurements revealed that salt stress inhibited PLD activity, indicating that the salt stress-induced PA response was not due to PLD activity. Comparison of the 32P-lipid responses in salt-tolerant and salt-sensitive cultivars revealed no significant differences. Together these results show that salt stress rapidly activates several lipid responses in rice leaves but that these responses do not explain the difference in salt tolerance between sensitive and tolerant cultivars.

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Phospholipid Signaling Responses in Salt-Stressed Rice Leaves

The green plants and insects represent the two dominant groups of living organisms on Earth. The green plants occupy the most capacious segment among all biological organisms, whereas the insects are the most specious group. These two ‘empires’ are interconnected as well as interdependent. Green plants are the primary producers of food, and all animals being heterotrophs depend directly or indirectly on plant-produced food. In turn, nearly three fourths of all angiosperms require the services of insect pollinators. The entomophilic flowering plants and their insect pollinators thus represent the most evident and widely applicable example of mutualism among living organisms. But a wide variety of phytophagous insects also flourishes, diversifies and sustains on these plants. Consequently, the plants have evolved a dizzying array of morphological and biochemical (constitutive as well as induced) barriers for protection against insects and other herbivores. Evolutionary interactions between plants and insects may have contributed to the increased biodiversity and success of both these groups. The study of these interrelationships, as outlined in this chapter, is of great practical significance for the future agricultural production. The development of pest-resistant cultivars of crop plants and progress in integrated pest management both require an intricate understanding of insect-plant relationships.

Harcharan S. Dhariwal

Papers

Genetic Engineering for Abiotic Stress Tolerance in Plants