PhD (Plant Production) / Department of Plant Production / Planting chickpea genotypes at different dates within the same season may expose the crop to different environmental factors (temperature and moisture) during their vegetative and reproduction stages. Thus, knowledge of optimum planting date that minimises extreme temperature and water stress conditions during crital stages of chickpea plant development may increase biomass and grain yield. The objective of the study was to determine the effect of planting date and genotype on aboveground biomass and grain yield of chickpea under climate change scenario in North Eastern Region of South Africa. The hypothesis tested was that planting date and genotype have an effect on biomass and grain yield of chickpea under climate change scenario. Thus, a study design incorporating a combination of field and modelling experiments was set to run in 2014 and 2015 winter planting seasons at the University of Venda, South Africa. Field experiments determined the effect of planting date and genotype on chickpea flower retention and pod abortion, aboveground biomass and grain yield, water use and radiation use efficiency, whilst modelling experiments calibrated and validated the FAO AquaCrop model to simulate chickpea aboveground biomass and grain yield using climate datasets (1950 - 2100), simulated from 15 global circulation models (GCMs) under the representative carbon dioxide concentration pathways (RCP) 4.5 and 8.5. Field experiments results showed significant effect of planting date and genotype on biomass and grain yield of chickpea. Planting early, particularly under well-watered conditions appeared to be the most suitable sowing period for chickpea in this region. In contrast, late planting had lowest biomass and grain yield. The high grain yield in early planting (1.99 t ha-1) was supported by greater yield components (seed weight (13.8 gm-2) and pod weight 23 gm-2), number of pods per plant (75 pods plant-1) and harvest index (43 %)). Moreover, plant phenological factors such as plant height (46 cm) and number of branches per plant (16 branches) were also greater in early planting, with late planting recording lowest values in all the measured parameters. In addition, the greater biomass and grain yield in early planting compared with the normal and late sowings was caused by greater intercepted radiation (91%), improved flower retention (45.2%) and minimised water use (174 mm) and pod abortion (13.6%). Late maturing genotypes (Range 4 & 5) showed greater water use efficiency of grain yield (7.3 & 7.1 kg ha-1 mm-1) and had the highest radiation use efficiency of grain yield, which was on average 7.2% (0.07 g MJ-1) greater than ICCV9901, and 15.6% (0.13 g MJ-1) greater than Range 1 & 3, but this depended on soil moisture availability. vi The simulation results, indicated a significant increase in temperature (by 4.2 to 5.5 oC) over a period from 1950 to 2100. This increase lead to a concomitant increase in chickpea evapotranspiration and accumulated growing degree days. Moreover, optimal planting date for chickpea shifted from mid-month of April during 1950 to end of May in 2100 and reduced growing season length from 140 days in 1950 to 85 days in 2100. Aboveground biomass increased from 2.0 & 2.05 t ha-1 in 1950 to 4.3 & 4.57 t ha-1 in 2100, respectively in RCP 4.5 and 8.5, whilst grain yield increased from 1.07 & 1.08 t ha-1 in 1950 to 1.68 & 2.21 t ha-1 in 2100, respectively under RCP 4.5 and 8.5. Planting dates that were recommended by AquaCrop model recorded the highest increase in aboveground biomass and grain yield compared with early, normal and late planting dates. Late maturing genotypes (Range 4 & 5) showed greater grain yield and biomass, whilst early and medium maturing genotypes had low biomass and grain yield. The study recommend early planting date together with late maturing chickpea genotypes (Range 4 and 5) in the region so as to improve water use efficiency, radiation use efficiency, heat use efficiency and aboveground biomass and grain yield of the crop under the present time and under climate change scenario. The early maturing genotype (Range 1) and medium maturing genotypes (Range 3 and ICCV9901) may only be recommended under normal planting date, although there will not be any significant yield advantages compared with late maturing genotypes. The study also recommend the use of planting dates generated by AquaCrop model so as to improve biomass and grain yield when chickpea is sown under climate change scenario in Southern Africa. The yield improvement using AquaCrop recommended planting dates was partly caused by greater water use efficiency, heat use efficiency and corbon dioxide productivity. Given the potential importance of planting dates in improving current and future productivity of chickpea shown in the study, there is need to work on development of a sowing (planting date) criteria for chickpea in the / NRF

In plants, calcium is a ubiquitous signaling molecule and changes in cytosolic calcium levels reported in response to various abiotic and biotic stresses like salt stress, drought, pathogen attack and phytohormone signaling. Any calcium- mediated signal transduction process involves the establishment of a signal-specific change in the cytosolic calcium concentration termed as ‗calcium signature‘ which is decoded by the specific group of proteins called ‗calcium sensors‘ (eg: Calmodulin (CaM) and Ca2+ - regulated kinases). Plants have a novel group of kinases designated as Ca2+- dependent protein kinases (CDPK; EC 2.7.1.37). CDPKs are biochemically distinct from other Ca2+- dependent kinases, such as Ca2+- and phospholipid- dependent protein kinases, as they are activated directly by Ca2+-and are independent of CaM. They exist as monomeric serine/threonine protein kinases and consist of four domains namely an amino-terminal variable domain, a kinase domain, an autoinhibitory domain and a calmodulin-like domain (CaM-LD). CDPKs represent a unique class of Ca2+ sensors, having protein kinase as well as CaM-LD in a single polypeptide chain, enabling them to couple the calcium sensor directly to its responder (kinase). In the absence of calcium signature, CDPKs activity is inhibited by the autoinhibitory domain, which acts as a pseudo-substrate of kinase domain and thus blocks the active site of the enzyme. In the presence of calcium signature, CDPKs undergo conformational changes leading to removal of the inhibition. Besides plants, CDPKs are also reported in few protozoans viz Plasmodium falciparam, Paramecium and Taxoplasma. However, CDPKs are not found in the eukaryotic genome of yeast, nematodes, fruitflies and humans. In the current study, we have cloned CDPK1 gene from Cicer arietinum (CaCDPK1) in pRSET-A expression vector and expressed it in Escherichia coli BL21pLysS strain. However, while expressing the recombinant CaCDPK1 in E.coli, most of the recombinant CaCDPK1 protein was expressed as insoluble form. Therefore, we focused our efforts on optimizing the culture conditions for achieving the maximum yield of soluble recombinant CaCDPK1. Expression of the soluble CaCDPK1 was achieved by optimizing the different conditions like IPTG concentrations, temperature and growth time after induction. Maximum amount of soluble expression of recombinant CaCDPK1 was achieved by inducing the bacterial culture with 0.1 mM IPTG at 0.6 OD and growing it further for 4 h at 25°C. As with several other CDPKs, CaCDPK1 was found to get autophosphorylated in a calcium-dependent manner. To find the significance of autophosphorylation, we measured the substrate phosphorylation activity of the native and autophosphorylated CaCDPK1, which revealed that the autophosphorylation enhances the kinase activity of CaCDPK1 by 2-fold. Autophosphorylation was linearly dependant on concentrations of the enzyme suggesting that the autophosphorylation in CaCDPK1 occurs via an intra-molecular mechanism. Further analysis of autophosphorylation shows that autophosphorylation happens before substrate phosphorylation and provides calcium -independent substrate phosphorylation property. It also reduces the lag phase for activation of the enzyme and utilizes both ATP and GTP as phosphor-donor, but ATP is preferred over GTP. Autophosphorylation was found to occur at serine and threonine residues. The MALDI MS/MS analysis of the cold ATP autophosphorylated CaCDPK1 showed Thr- 339, Ser- 357, and Ser- 367 residues could be the potential autophosphorylation sites in CaCDPK1. Phospholipids, the major structural components of membranes, can also have functions in regulating signaling pathways in plants under biotic and abiotic stress conditions. The effects of adding phospholipids on the activity of stress-induced calcium dependent protein kinase (CaCDPK1) from chickpea are reported in this study. Both autophosphorylation as well as phosphorylation of the added substrate were enhanced specifically by phosphatidylcholine and to a lesser extent by phosphatidic acid, but not by phosphatidylethanolamine. Diacylgylerol, the neutral lipid known to activate mammalian PKC, stimulated CaCDPK1 but at higher concentrations. Increase in Vmax of the enzyme activity by these phospholipids significantly decreased the Km indicating that phospholipids enhance the affinity towards its substrate. In the absence of calcium, addition of phospholipids had no effect on the negligible activity of the enzyme. Intrinsic fluorescence intensity of the CaCDPK1 protein was quenched on adding PA and PC. Higher binding affinity was found with PC (K½ = 1.3 nM) when compared to PA (K½ = 56 nM). We also found that the concentration of PA increased in chickpea plants under salt stress. The stimulation by PA and PC suggests regulation of CaCDPK1 by these phospholipids during stress response. In the current study we also investigated CaCDPK1 interactions with calcium ions to address the Ca2+ -induced conformational changes in CaCDPK1 by using circular dichroism (CD), fluorescence spectroscopy and isothermal titration (ITC). Isothermal calorimetric analysis of calcium binding to CaCDPK1 shows a biphasic curve with two Kd of 27 nM and 1.72 µM respectively. The fluorescence measurements showed quenching in fluorescence intensity with a 5 nm red shift. The plot of changes in intensity against calcium concentrations again showed a biphasic curve, indicating that there may be more than one kind of Ca2+ binding sites. 8-anilinonaphthalene-1-sulfonic acid (ANS) binding showed that calcium bound form of CaCDPK1 exposes hydrophobic surfaces which may act as binding sites for other proteins. CD analysis of CaCDPK1 showed that it‘s an alpha helical rich protein and its helical content increases after binding to calcium. Taken all together this study describes the successful heterologous expression of Cicer arietinum CDPK isoform 1 in E.coli. and demonstrates that the autophoshorylation happens via an intra-molecular mechanism and it increases the kinase activity of CaCDPK1 at least by 2-fold. We also report here that CaCDPK1 prefers ATP as phosphodonor over GTP. The present study also shows the activation of CaCDPK1 by PC and PA, but not by PE or diacylglycerol. Both phospholipids were able to bind to CaCDPK1 and increased its Vmax and affinity towards the exogenous substrate, histone III-S. The current study also shows that calicum binding induces conformational changes in CaCDPK1 and the all four EF hand motifs of CaCDPK1 do not function in an equivalent manner.

Chickpea (Cicer arietinum L.) production on the semi-arid Canadian Prairies is challenging due to a short growing season and low and variable moisture. The current recommended chickpea population density of 44 plants m-2 is based on preliminary studies and a narrow range of 20 to 50 plants m-2. The aims of this study were to i) determine optimum population density of varying chickpea canopy types, i.e., leaf type and growth habit, by investigating seed yield responses at 30 to 85 plants m-2 and ii) identify desirable parental traits for breeding programs by assessing growth and yield parameter responses to varying leaf types and growth habits at a range of population densities. Field experiments were conducted from 2002 to 2005. Canopy measurements and calculated variables included light interception, biomass, growth rate, seed yield, harvest index, ascochyta blight severity and radiation- and water use efficiencies. The plant density which produced the highest seed yield when averaged over years for each location for each treatment revealed that a plant density of at least 55 plants m-2 produced a 23% to 49% seed yield increase above that of the currently recommended plant density. This indicates that a higher seed yield average over the long term in spite of periodic low seed yield episodes will be more profitable to producers. Increasing plant density increased lowest pod height significantly in all except one location-year but did not explicitly increase ascochyta blight severity or decrease individual seed size. This suggests that increasing the recommended chickpea plant density on the Canadian Prairies will increase seed yield but would neither negatively impact individual seed size nor ascochyta blight severity, especially, when combined with good agronomic practices. Fern-leaved cultivars had significantly higher maximum intercepted light (62 to 91%), seed yield (136 to 369 g m-2), harvest index (0.33 to 0.53), yield-based water use efficiency (0.56 to 1.06 g m-2 mm-1) and lower ascochyta blight severity (3 to 27%) than the unifoliate cultivars in all location-years. The fern-leaved cultivars also tended to show significantly higher cumulative intercepted radiation (221 to 419 MJ m-2) and biomass (306 to 824 g m-2) but leaf type showed no consistent effect on radiation use efficiency. Cultivars with bushy growth habit generally performed better regarding maximum intercepted light (62 to 90%), cumulative intercepted radiation (233 to 421 MJ m-2), biomass (314 to 854 MJ m-2), seed yield (120 to 370 g m-2), harvest index (0.37 to 0.50), yield-based water use efficiency (0.56 to 1.06 g m-2 mm-1) and ascochyta blight severity (7 to 36%) than the erect cultivars. The overall performance of the spreading cultivar was generally intermediate between the bushy and erect cultivars except for ascochyta blight severity where the spreading cultivar exhibited significantly lower disease severity (3 to 36%). Radiation use efficiency was generally not influenced by growth habit. Increasing plant population density generally increased intercepted light, biomass and cumulative intercepted radiation on each sampling day after seeding resulting in a general increase in seed yield. Harvest index, however, remained constant and ascochyta blight severity was generally stable but radiation use efficiency decreased with increasing population density. Chickpea cultivars with fern leaves and bushy growth habit at higher than currently recommended population densities would best utilize the limited resources of the short-season Canadian prairie environment to maximize and stabilize seed yield.

Differences in pea (Pisum sativum L.) and chickpea (Cicer arietinum L.) microbial compatibility and/ or their associated farming practices may influence root fungi of the following crop and affect the yield. The main objective of this research was to explain the difference in durum wheat (Triticum turgidum L.) yield the year after pea and chickpea crops through changes in the functional diversity of wheat root fungi. The effect of fungicides used on chickpea on the root fungi of a following durum wheat crop was studied using plate culture and pyrosequencing. Pyrosequencing detected more Fusarium spp. in the roots of durum wheat after fungicide-treated chickpea than in non-fungicide treated chickpea. Plate culture revealed that the functional groups of fungi responded differently to fungicide use in the field but the effect on total community was non-significant. Highly virulent pathogens were not affected, but antagonists were suppressed. More fungal antagonists were detected after the chickpea CDC Luna than CDC Vanguard. Fungal species responded differently to the use of fungicides in vitro, but the aggregate inhibition effect on antagonists and highly virulent pathogens was similar. The effect of chickpea vs. pea previous crop and different chickpea termination times on root fungi of a following durum wheat crop was studied. The abundance of Fusarium spp. increased after cultivation of both cultivars of chickpea as compared to pea according to pyrosequencing and was negatively correlated with durum yield. Plate culture analysis revealed that fungal antagonists were more prevalent after pea than both cultivars of chickpea and chickpea CDC Vanguard increased the abundance of highly virulent pathogens. The abundance of highly virulent pathogens in durum wheat roots was negatively correlated to durum yield. Early termination of chickpea did not change the community of culturable fungi in the roots of a following durum crop. It is noteworthy that Fusarium redolens was identified for the first time in Saskatchewan and its pathogenicity was confirmed on durum wheat, pea and chickpea. The classical method of root disease diagnostics in cereals is based on the examination of the subcrown internode. I evaluated the method by comparing the fungal communities associated with different subterranean organs of durum wheat. The fungal community of the subcrown internode was different from that of roots and crown, suggesting cautious use of this method.

Chickpea (Cicer arietinum L.) production on the semi-arid Canadian Prairies is challenging due to a short growing season and low and variable moisture. The current recommended chickpea population density of 44 plants m-2 is based on preliminary studies and a narrow range of 20 to 50 plants m-2. The aims of this study were to i) determine optimum population density of varying chickpea canopy types, i.e., leaf type and growth habit, by investigating seed yield responses at 30 to 85 plants m-2 and ii) identify desirable parental traits for breeding programs by assessing growth and yield parameter responses to varying leaf types and growth habits at a range of population densities. Field experiments were conducted from 2002 to 2005. Canopy measurements and calculated variables included light interception, biomass, growth rate, seed yield, harvest index, ascochyta blight severity and radiation- and water use efficiencies. The plant density which produced the highest seed yield when averaged over years for each location for each treatment revealed that a plant density of at least 55 plants m-2 produced a 23% to 49% seed yield increase above that of the currently recommended plant density. This indicates that a higher seed yield average over the long term in spite of periodic low seed yield episodes will be more profitable to producers. Increasing plant density increased lowest pod height significantly in all except one location-year but did not explicitly increase ascochyta blight severity or decrease individual seed size. This suggests that increasing the recommended chickpea plant density on the Canadian Prairies will increase seed yield but would neither negatively impact individual seed size nor ascochyta blight severity, especially, when combined with good agronomic practices. Fern-leaved cultivars had significantly higher maximum intercepted light (62 to 91%), seed yield (136 to 369 g m-2), harvest index (0.33 to 0.53), yield-based water use efficiency (0.56 to 1.06 g m-2 mm-1) and lower ascochyta blight severity (3 to 27%) than the unifoliate cultivars in all location-years. The fern-leaved cultivars also tended to show significantly higher cumulative intercepted radiation (221 to 419 MJ m-2) and biomass (306 to 824 g m-2) but leaf type showed no consistent effect on radiation use efficiency. Cultivars with bushy growth habit generally performed better regarding maximum intercepted light (62 to 90%), cumulative intercepted radiation (233 to 421 MJ m-2), biomass (314 to 854 MJ m-2), seed yield (120 to 370 g m-2), harvest index (0.37 to 0.50), yield-based water use efficiency (0.56 to 1.06 g m-2 mm-1) and ascochyta blight severity (7 to 36%) than the erect cultivars. The overall performance of the spreading cultivar was generally intermediate between the bushy and erect cultivars except for ascochyta blight severity where the spreading cultivar exhibited significantly lower disease severity (3 to 36%). Radiation use efficiency was generally not influenced by growth habit. Increasing plant population density generally increased intercepted light, biomass and cumulative intercepted radiation on each sampling day after seeding resulting in a general increase in seed yield. Harvest index, however, remained constant and ascochyta blight severity was generally stable but radiation use efficiency decreased with increasing population density. Chickpea cultivars with fern leaves and bushy growth habit at higher than currently recommended population densities would best utilize the limited resources of the short-season Canadian prairie environment to maximize and stabilize seed yield.