Seminar At IARI

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Published on July 27, 2009

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Slide 1: Indian Agricultural Research Institute, Division of Plant Physiology Slide 2: Indian Agricultural Research Institute, Division of Plant Physiology It is not the strongest of the species that survive, nor the most intelligent, but the one Charls Darwin most responsive to change. Slide 3: Stress’ evoked by climatic change includes: Increased evaporation, Extreme weather, flooding (sea level rise), drought, soil Acidification, Oxygendepletion(anaerobic), Ozone rise in troposphere etc, (UN-IPCC-2008) Developing countries losing about 280 million tons of potential cereal production, valued at $56 billion, as a result of climate change (UN news centre, 26 May 2005) Climate change will cause severe crop losses in Africa and Asia within the next 20 years (Chicago-AFP, 2008) Indian Agricultural Research Institute, Division of Plant Physiology Slide 4: STRESS Stress can be defined as an influence that is outside the normal range of homeostatic control (Lerner, 1999). Any strain or interference that disturbs the functioning of an organism (Encyclopedia Britannica) CLIMATE CHANGE Any change in global temperatures and precipitation over time due to natural variability or to human activity (IPCC)  long-term alteration in global weather patterns, especially increases in temperature and storm activity, regarded as a potential consequence of the greenhouse effect (Encarta) GENETIC ENGINEERING The artificial manupulatin, modification and recombination of DNA or other nucleic acid molecules in order to modify an organism or population of organism (Encyclopaedia Britanica 15 edn.) The branch of biology dealing with the splicing and recombining of specific genetic units from the DNA of living organisms as in order to produce new species or biochemicals (Webster's New World College Dictionary ) Indian Agricultural Research Institute, Division of Plant Physiology Slide 5: Indian Agricultural Research Institute, Division of Plant Physiology Slide 6: Abiotic stresses are the main factors that limit crop productivity. drought, salinity and heavy metals stresses caused yield losses annually to a greater extent (Nafees A. Khan & Sarvajeet Singh, 2008) Abiotic stress reducing average yields of crops by up to 50% (Bray, E. A. 1997) Annually about 42% of the crop productivity is lost owing to various abiotic stress factors (Oerke et.al., 1994) The progressive salinization of soil, estimated at around 20% of irrigated land (Ghassemi et al.,1995) Indian Agricultural Research Institute, Division of Plant Physiology Slide 7: Plant breeders and geneticists have utilized natural variability for stress tolerance within germplasm One special advantage of genetic engineering is the ability to transform plants with genes from other species rather than upregulating an already existing plant stress response Indian Agricultural Research Institute, Division of Plant Physiology Slide 8: Indian Agricultural Research Institute, Division of Plant Physiology Slide 9: Indian Agricultural Research Institute, Division of Plant Physiology Slide 10: Indian Agricultural Research Institute, Division of Plant Physiology Slide 11: Plasmid pPM5 contained an EcoRI-HindIII fragment of 1.75 kb with the reinforced 35S promoter, the HAL1 ORF, and the nos terminator Carmina et al., 2000, plant physiology , 123: 393–402, Indian Agricultural Research Institute, Division of Plant Physiology Slide 12: Saccharomyces genes HAL1 and HAL3, which are involved in theregulation of K1 and Na1 transport respectively introduction of the yeast HAL1 gene (using a modified plasmid with enhancer elements) in tomato(Lycopersicon esculentum cv P-73) TG plant growth was evaluated by measuring rooting capacity, shoot height, number of leaves, and total fresh (FW) and dry (DW) weight of plants after 28 d on culture media with or without NaCl. Carmina et al., 2000, plant physiology , 123: 393–402, Indian Agricultural Research Institute, Division of Plant Physiology Slide 13: Table: Effect of salt stress on shoot growth after 28 d of culture on B1 medium supplemented with NaCl Fig : The relative K1 to Na1 ratio, which was higher in the transgenic families, indicated higher K1 retention under saline conditions Carmina et al., 2000, plant physiology , 123: 393–402, Indian Agricultural Research Institute, Division of Plant Physiology Slide 14: Overexpression of HAL 1 gene in yeast confers a high salt tolerance level by reducing K loss and decreasing intracellular Na from the cells upon salt stress HAL 1 ability to maintain K uptake in the presence of external Na, as shown by the transgenic families (especially TG3), is noteworthy. This ability has been related to salt tolerance in tomato The salt tolerance levels of transgenic tomato plants assayed in this work were higher than that previously observed in melon. This could be due to genetically engineered plasmid used in this work, which had a duplicated 35S promoter. Carmina et al., 2000, plant physiology , 123: 393–402, Indian Agricultural Research Institute, Division of Plant Physiology Slide 15: Plasmid pBY520 with bar gene Xu et al., Plant Physiol. (1996) 11 O: 249-257 Indian Agricultural Research Institute, Division of Plant Physiology Slide 16: HVA7, is a late embryogenesis abundant (LEA) protein gene, from barley (Hordeum vulgare L.) This gene was introduced into rice suspension cells using the Biolistic-mediated transformation method Barley HVA 7 gene regulated by the rice actin 1 gene promoter led to high-level, constitutive accumulation of the HVAl protein in both leaves and roots of transgenic rice plants Xu et al., Plant Physiol. (1996) 11 O: 249-257 Indian Agricultural Research Institute, Division of Plant Physiology Slide 17: Structure of the plasmid pBY520 for expression of HVA7 in transgenic rice. Xu et al., Plant Physiol. (1996) 11 O: 249-257 Indian Agricultural Research Institute, Division of Plant Physiology Slide 18: Table II. fstimated levels of UVA1 protein accumulation in different transgenic lines Using a partially purified HVAl protein preparation as the standard Xu et al., Plant Physiol. (1996) 11 O: 249-257 Indian Agricultural Research Institute, Division of Plant Physiology Slide 19: After 5 d in the stress medium, the germinated seeds (with 0.2- to 0.5-cm-long shoot) were transferred onto MS medium. Both transgenic and control seedlings recovered and resumed normal growth, but transgenic seedlings grew faster during this recovery period, their shoots were significantly longer after 1 week (Table III), Table 111. Seed germination and growth of young seedlings in medium under osmotic stress or salt stress Xu et al., Plant Physiol. (1996) 11 O: 249-257 Indian Agricultural Research Institute, Division of Plant Physiology Slide 20: Appearance and development of stress symptoms occurred much more slowly in transgenic plants than in control plants. Comparison of transgenic plants and NT plants grown under drought and salinity stress conditions. Two NT plants and two R, transgenic plants from each of two lines (numbers 36 and 41) are show here. A, Plants recovered from drought stress. Photograph was taken 21 d after the beginning of initial water stress (three cycles of 5 d of drought stress followed by 2 d of recovery with watering). B, Plants recovered from salt stress. Photograph was taken after 10 d of salt stress in 200 rriM NaCI and 10 d of recovery in tap water Xu et al., Plant Physiol. (1996) 11 O: 249-257 Indian Agricultural Research Institute, Division of Plant Physiology Slide 21: Using a transgenic approach, this study provides direct evidence supporting the hypothesis that LEA proteins play an important role in the protection of plants under wateror salt-stress conditions Transgenic rice plants showed significantly increased tolerance to water deficit and salinity. Transformed Plants maintained higher growth rates than nontransformed control plants under stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by stress and by improved recovery upon the removal of stress conditions. The extent of increased stress tolerance correlated with the leves of the HVAl protein accumulated in the transgenic rice plants Xu et al., Plant Physiol. (1996) 11 O: 249-257 Indian Agricultural Research Institute, Division of Plant Physiology Slide 22: Identification of a leucine-rich repeat RLKgene, Srlk(Salt-induced Receptor-Like Kinase) from the legume Medicago truncatula Srlk Is rapidly induced by salt stress in roots RNAinterference (RNAi) assays specifically targeting Srlk yielded transgenic roots whose growth was less inhibited by the presence of salt in the medium They propose a role for Srlk in the regulation of the adaptation of M. truncatula roots to salt stress Lorenzo et al., 2009, The Plant Cell, 21: 668–680 Indian Agricultural Research Institute, Division of Plant Physiology Slide 23: Analyses of structural properties of the Srlk-predicted protein using Pfam and SMART programs suggest that this protein encodes an LRR-RLK with four domains The previously described LRR-RLK extracellular domains (Shiu and Bleecker, 2001) differ from those of Srlk, suggesting that this receptor recognizes a different signal Lorenzo et al., 2009, The Plant Cell, 21: 668–680 Indian Agricultural Research Institute, Division of Plant Physiology Slide 24: Homology studies have conformed that No function has been assigned to this gene in any species. We determined Srlk expression levels in M. truncatula Jemalong A17 roots submitted to salt (150 mM NaCl), (0, 1 h, 6 h, 1 d, and 4 d, ) Lorenzo et al., 2009, The Plant Cell, 21: 668–680 Indian Agricultural Research Institute, Division of Plant Physiology Slide 25: Mannitol (300 mM), and cold stress treatments (48C) at different time points 6 h and 1 d (quantification of specific PCR amplification products for Srlk gene) Lorenzo et al., 2009, The Plant Cell, 21: 668–680 Indian Agricultural Research Institute, Division of Plant Physiology Slide 26: To investigate the putative role of Srlk in response to salt stress, an RNAi approach was used in M. truncatula RNAi knockdown of candidate genes is an efficient way to suppress gene expression A 203-bp fragment of Srlk with maximal RNAi specificity was used for RNAi constructs Agrobacterium mediated RNAi transfer technique is used to insert Srlk-RNAi Lorenzo et al., 2009, The Plant Cell, 21: 668–680 Indian Agricultural Research Institute, Division of Plant Physiology Slide 27: they compared transgenic Agrobacterium rhizogenes–transformed roots carrying an Srlk-RNAi construct with a gus-RNAi(ß Glucuronidase ) control to rule out any indirect effect induced by the activation of the silencing machinery Two weeks after A. rhizogenes infection in the appropriate medium, plants were transferred to a medium containing 100 mM NaCl and control medium without salt and incubated for six more days Lorenzo et al., 2009, The Plant Cell, 21: 668–680 Indian Agricultural Research Institute, Division of Plant Physiology Slide 28: Root length for each plant was determined from the moment of transfer into these media up to the 6-d period Repression of Srlk expression in M. truncatula plants prevents the inhibition of root growth by salt, suggesting that Srlk-RNAi roots may be less sensitive to this environmental constraint. Lorenzo et al., 2009, The Plant Cell, 21: 668–680 Indian Agricultural Research Institute, Division of Plant Physiology Slide 29: Spatial and temporal expression of Srlk hs been identified using genetic engineering approach By using a 2.2-kb Srlk promoter:GUS fusion DNA construct Two weeks after A. rhizogenes infection, the histological GUS activity of the resulting transgenic root was determined The Srlk promoter was only weakly active in the root epidermis under control conditions Lorenzo et al., 2009, The Plant Cell, 21: 668–680 Indian Agricultural Research Institute, Division of Plant Physiology Slide 30: Srlk expresses in root epidermis and in the root apex, two regions potentially linked to the perception of soil environmental conditions Lorenzo et al., 2009, The Plant Cell, 21: 668–680 Indian Agricultural Research Institute, Division of Plant Physiology Slide 31: Functional analysis of a novel LRR-RLK gene, Srlk, involved in the regulation of early M. truncatula root responses to salt stress Srlk kinase seems to control a salt avoidance response in the model legume M. truncatula Promoter-GUS fusion analyses suggest that the Srlk is expressed in epidermal tissues, as are the HAKtype K+ transporters and the At KC1 and AKT1 (Arabidopsis Shaker K+ channel genes) These results may link the Srlk receptor with perception of high salinity and activation of a signaling pathway leading to plant avoidance of deleterious effects of salt in internal tissues……….. Lorenzo et al., 2009, The Plant Cell, 21: 668–680 Indian Agricultural Research Institute, Division of Plant Physiology Slide 32: Over expression of stress responsive gene SNAC1 (STRESS-RESPONSIVE NAC 1) significantly enhances drought resistance in transgenic rice in the field under severe drought stress conditions at the reproductive stage The transgenic rice also shows significantly improved drought resistance and salt tolerance at the vegetative stage Compared with WT, the transgenic rice are more sensitive to abscisic acid and lose water more slowly by closing more stomatal pores, yet display no significant difference in the rate of photosynthesis Hu et al., 2006, PNAS, 103(35):12987–12992 Indian Agricultural Research Institute, Division of Plant Physiology Slide 33: SNAC1 is induced predominantly in guard cells by drought and encodes a NAM, ATAF, and CUC (NAC) transcription factor with transactivation activity DNA chip analysis revealed that a large number of stress-related genes were up-regulated in the SNAC1-overexpressing rice plants Authors suggest that SNAC1 holds promising utility in improving drought and salinity tolerance in rice Hu et al., 2006, PNAS, 103(35):12987–12992 Indian Agricultural Research Institute, Division of Plant Physiology Slide 34: Hu et al., 2006, PNAS, 103(35):12987–12992 STRESS-INDUCIBLE EXPRESSION OF SNAC1. (a) RNA gel blot analysis of expression of the SNAC1 under drought (DT), salt (200mM), cold (4°C), and ABA treatment (100 M) Indian Agricultural Research Institute, Division of Plant Physiology Slide 35: The temporal and spatial patterns of SNAC1 expression were investigated by transforming a japonica cultivar Nipponbare with a fusion gene of PSNAC1:GFP Hu et al., 2006, PNAS, 103(35):12987–12992 Indian Agricultural Research Institute, Division of Plant Physiology Slide 36: A GFP signal was observed in callus, root, ligule, stamen, and pistil from transgenic plants under normal growth conditions Hu et al., 2006, PNAS, 103(35):12987–12992 Indian Agricultural Research Institute, Division of Plant Physiology Slide 37: When transgenic plants were drought-stressed to the stage of leaf-rolling, strong induction of GFP was detected in leaves and minor induction was observed in roots and nodes, whereas no obvious change of GFP expression level was observed in callus, ligule, stamen, and pistil after drought stress Hu et al., 2006, PNAS, 103(35):12987–12992 Indian Agricultural Research Institute, Division of Plant Physiology Slide 38: Further examination of the GFP signal in the stressed leaves revealed that the signal is localized predominantly in guard cells that constitute the stomata This finding suggests that SNAC1 gene expression was specifically induced in guard cells Hu et al., 2006, PNAS, 103(35):12987–12992 Indian Agricultural Research Institute, Division of Plant Physiology Slide 39: The strongly localized induction of SNAC1 in guard cells of WT plants suggests that increased stomatal closure is a likely target of regulation by SNAC1 It is interesting to note that quite a few rice homologs of genes related to stomatal movement are up regulated in the SNAC1- overexpressing plants Hu et al., 2006, PNAS, 103(35):12987–12992 Indian Agricultural Research Institute, Division of Plant Physiology Slide 40: Overexpression of SNAC1 Can Significantly Improve Drought Resistance severe stress in the field (with soil water content15%), moderate stress in the field (soil water content 28%), and moderate stress in poly(vinyl chloride) (PVC) pipes in which plants were individually stressed Hu et al., 2006, PNAS, 103(35):12987–12992 Indian Agricultural Research Institute, Division of Plant Physiology Slide 41: Cosegregation of SNAC1-overexpressing (RNA gel blot analysis) with the improved drought tolerance in the T1 family of S19. SS(%), seed-setting rate Hu et al., 2006, PNAS, 103(35):12987–12992 Indian Agricultural Research Institute, Division of Plant Physiology Slide 42: All of the SNAC1-overexpressing plants produced significantly (t test, P 0.01) higher spikelet fertility than the negative control under all drought treatments no significant difference was detected between the transgenic plants and the control as evaluated by a number of agronomic traits such as plant height, number of panicles per plant, number of spikelets per panicle, and grain yield per plant, as well as root depth and root volume under unstressed conditions This finding clearly indicates that overexpression of SNAC1 does not affect growth and productivity of the rice plant Besides the improved drought resistance, SNAC1-overexpressing transgenic rice also showed enhanced salt tolerance indicating that there was another potential mechanism of salt tolerance regulated by SNAC1 Hu et al., 2006, PNAS, 103(35):12987–12992 Indian Agricultural Research Institute, Division of Plant Physiology Slide 43: Introduced Arabidopsis thaliana hsp101 (Athsp101) cDNA into the Pusa basmati 1 cultivar of rice (Oryza sativa L.) by Agrobacterium mediated transformation Diagrammatic representation of pUH-Athsp101 construct employed for rice transformation. Katiyar-Agarwal et al., (2003) Pt .Mol. Biol. 51: 677–686 Indian Agricultural Research Institute, Division of Plant Physiology Slide 44: Stable integration and expression of the transgene into the rice genome was demonstrated by Southern, northern and western blot analyses Northern analysis of Athsp101 expression untransformed (C2) and transgenic rice (15, 19 and 43). Katiyar-Agarwal et al., (2003) Pt .Mol. Biol. 51: 677–686 Indian Agricultural Research Institute, Division of Plant Physiology Slide 45: Comparison of survival of transgenic lines after exposure to different levels of high-temperature stress with the untransformed control plants (a)45 ?C for 3 h and then were placed at 28 ?C The optimum temperature for rice growth throughout its life cycle is 25–31 ?C Katiyar-Agarwal et al., (2003) Pt .Mol. Biol. 51: 677–686 Indian Agricultural Research Institute, Division of Plant Physiology Slide 46: Katiyar-Agarwal et al., (2003) Pt .Mol. Biol. 51: 677–686 Indian Agricultural Research Institute, Division of Plant Physiology (B) 47 ?C for 2 h and, after the stress, leaves were excised and the cut plants were placed at 28 ?C (C) 50 ?C for 40 min and, after the stress, leaves were excised and the plants were then placed at 28 ?C for re-growth untransformed (C2) and transgenic lines (15and 43) Slide 47: Katiyar-Agarwal et al., (2003) Pt .Mol. Biol. 51: 677–686 Indian Agricultural Research Institute, Division of Plant Physiology The transgenic rice lines showed significantly better growth performance in the recovery phase following the stress The results showed that all transgenic rice plants survived in the high-temperature range of 45–50 ?C exhibiting vigorous growth during the subsequent recovery at 28 ?C, whereas most of the untransformed plants succumbed. These tests revealed that AtHsp101 imparts basal high temperature tolerance possibly by acting in the post-stress recovery period Slide 48: The chilling sensitivity of plants is closely correlated with the degree of unsaturation of fatty acids Plants with high proportion of cis unsaturated fatty acids such as squash and arabidopsis are resistant to chilling The chloroplast enzyme glycerol-3-phosphate acetyl transferase seems to be important for determining phosphatidyl glycerol fatty acids unsaturation Here the FA unsaturation has been modified by transferring the said enzym DNA from squash and arabidopsis Indian Agricultural Research Institute, Division of Plant Physiology Murata et al., 1992 Slide 49: cDNA for enzyme from Squash and Arabidopsis with CaMV-35S constitute promoter in pBI21 was introduced to Tobacco through Agrobacterium The level of enzym protein was very high (0.1-1%) compare to normal level (0.01%) Indian Agricultural Research Institute, Division of Plant Physiology Murata et al., 1992 Slide 50: Indian Agricultural Research Institute, Division of Plant Physiology Murata et al., 1992 The total FA level was not changed but only the FA composition of Phosphotidyl glycerol was altered Slide 51: a) Transofmant with pBI-121(control) b) Transformant with pARA c) Transformant with pSQ Murata et al., 1992 Indian Agricultural Research Institute, Division of Plant Physiology Slide 52: Leaves of control plant suffered chlorosis Plants with pSQ was severly damaged Plants with pBI-121 show little resistance Plants with pARA resistant to chilling than wild type Indian Agricultural Research Institute, Division of Plant Physiology Murata et al., 1992 Slide 53: It is possible for the alteration of the fatty acids of phosphotidylglycerol by the introduction of an appropriate acytransferase. Indian Agricultural Research Institute, Division of Plant Physiology Murata et al., 1992 Slide 54: Glycine betaine is an osmoprotectant that plays an important role and accumulates rapidly in many plants during salinity or drought stress Choline monooxygenase (CMO) is a major catalyst in the synthesis of glycine betaine. Indian Agricultural Research Institute, Division of Plant Physiology Zhang et al., 2009, Mol Breeding, 23:289–298 Slide 55: CMO gene (AhCMO) cloned from Atriplex hortensis was introduced into cotton (Gossypium hirsutum L.) via Agrobacterium mediation Two transgenic AhCMO cotton lines used to study their salinity tolerance in both greenhouse and field under salinity stress Indian Agricultural Research Institute, Division of Plant Physiology Zhang et al., 2009, Mol Breeding, 23:289–298 Slide 56: Greenhouse study showed that on average, seedlings of the transgenic lines accumulated 26 and 131% more glycine betaine than those of non-transgenic plants under normal and salt-stress (150 mmol l-1 NaCl) conditions respectively Indian Agricultural Research Institute, Division of Plant Physiology Zhang et al., 2009, Mol Breeding, 23:289–298 Slide 57: The osmotic potential, electrolyte leakage and Malondialdehyde (MDA) accumulation were significantly lower in leaves of the transgenic lines after salt stress Indian Agricultural Research Institute, Division of Plant Physiology Zhang et al., 2009, Mol Breeding, 23:289–298 Slide 58: The net photosynthesis rate and Fv/Fm in transgenic cotton leaves were less affected by salinity than in nontransgenic cotton leaves Indian Agricultural Research Institute, Division of Plant Physiology Zhang et al., 2009, Mol Breeding, 23:289–298 Slide 59: Therefore, transgenic cotton over-expressing AhCMO was more tolerant to salt stress due to elevated accumulation of glycine betaine, which provided greater protection of the cell membrane and photosynthetic capacity than in non-transgenic cotton. Indian Agricultural Research Institute, Division of Plant Physiology Zhang et al., 2009, Mol Breeding, 23:289–298 Slide 60: Abiotic stress’ are major cause of concern for the global food security Conventional knowledge has almost saturated in finding the solutions for the sprawling abiotic stress’ resulting due to climatic change and other causes GE has proved its worth in tweaking the plants’ ability to cope with the various abiotic stresses The main advantage of GE is that it can transcend across the species barrier Although much progress has been made through GE in taming stress’ Much is need to be done to realise the fulll potentiality of this technology Indian Agricultural Research Institute, Division of Plant Physiology Slide 61: Understanding the physiological effect…. Brings along undesirable agronomic characters.. In model plants Growth reduction in normal condition…Eg. CBF 1 in tomato Gene transver efficiency for many crops is lower Gene stacking have good future in tackling multiple stress GMO jorgon…………………………….? Indian Agricultural Research Institute, Division of Plant Physiology Slide 62: Indian Agricultural Research Institute, Division of Plant Physiology THANKS Acknowledgement: I extend my sincere thanks to all who helped to prepare this seminar Dr. Promod Kumar Dr. Madan Pal Dr. V.P.Singh Mr. Sudir (Student) Mr. Girish Chakra (Student) Mr. Ashok R.C. (Student) Mr. Samrat Gowda (Student, Genetics)

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