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Biochemical basis of micronutrient deficiency tolerance in triticale x wheat derivative lines
(Punjab Agricultural University, Ludhiana, 2019) Ravneet Kaur; Bhatia, Surekha
The present study was aimed to investigate the biochemical basis and molecular marker profile of genes associated with micronutrient uptake in parental lines viz rye, triticale (TL 2908, TL 2942), bread wheat lines (PBW 343, PBW550, HD 2967, HD 3086) and durum wheat lines (PDW 233, PDW 291, WHD 943). Five days old seedlings of these parental lines were transferred to three set of treatments (T1-control, T2- micronutrient deficient (stress), T3-with micronutrient supply (alkaline stress pH-9) for 30 days. Rye and triticale lines have longer, hairy and thick roots with high number as compared to bread wheat and durum wheat lines. Roots and shoots of rye and triticale lines accumulated higher fresh/dry weight than bread wheat and durum wheat lines irrespective of treatments. Both stressed conditions caused reduction in roots/shoots fresh weight and dry weight of all parental lines however, rye and triticale lines accumulated greater biomass under alkaline stress conditions on 21 DAT. Rye and triticale lines had higher phytosiderophore content (118 to 498 µmole of Cu equivalents/gFW) than bread wheat (19 to 112 µmole of Cu equivalents/gFW) and durum wheat (6 to 68 µmole of Cu equivalents/gFW) under different treatments during whole period of experiment. In rye and triticale, it was increased from 7 DAT to 21 DAT and decreased afterward on 30 DAT. However, in bread wheat and durum wheat lines, this increase was found only up to 14 DAT. Increase or decrease in release of PSs from roots of parental lines was positively correlated with the higher micronutrient content in roots (r=>0.9) and shoots (r=>0.8) of these lines. Highest micronutrient efficiency was found in rye (73.62-86.08%) followed by triticale lines (70.61-81.06%), bread wheat lines (36.82-66.14%) and durum wheat lines (29.42-62.38%). Further higher activities of SOD, CAT and CA enzymes in rye and triticale lines than bread wheat and durum wheat lines were found which was probably linked with higher micronutrient efficiency corresponding to efficient utilization of micronutrients in rye and triticale lines. Rye and triticale also exhibited higher activities of NAAT and MAS enzymes. Parental lines and triticale X wheat derivative lines were tested for presence of 5R chromosomes using seven 5R chromosome specific markers. Only three markers (SCM 138, KU 140, KU 640) showed expected polymorphism in parental lines were further used to evaluate triticale X wheat derivative lines. Fifteen rye specific primers of Ids3 gene were designed after in-silico sequence analysis. Five primers showed polymorphism in parental lines as well as triticale X wheat derivative lines. Higher release of PSs was estimated from the roots of triticale X wheat derivative lines which were positive for Ids3 and 5R chromosome specific markers. Fourteen triticale X wheat derivative lines with higher PSs under micronutrient deficient conditions, were selected for their further utilization in wheat breeding program for improvement and development of efficient varieties with higher nutritional value.
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Evaluation of different okra (Abelmoschus esculentus L. moench) genotypes for nutritional and antinutritional components
(Punjab Agricultural University, Ludhiana, 2018) Parminder Kumar; Sangha, Manjeet Kaur
The present study evaluated green pods and mature seeds of 166 okra genotypes for phenotypic trait, nutritional and antinutritional component variation. Phenotypic variation was observed for 32 qualitative and quatitative traits. Pearson product correlation correlated fruits/plant positively and significantly with number of internodes and plant height. Fruit total carbohydrates varied from 39.11-82.1% (DW basis). Total soluble sugars (TSS), reducing sugars, starch, sucrose and mucilage ranged from 3.39-20.98%, 0.67-1.86%, 30.26-65.6%, 11.34-16.9% and 3.01-8.14% respectively in pods. Total carbohydrates, TSS and reducing sugars in seeds varied from 15.79- 38.99%, 3.67-9.78% and 1.39-4.35% respectively. Crude protein, TSP and free amino acids in pods varied from 13.3%- 33.8%, 8.17- 21.0% and 0.62- 5.07% respectively. In seeds they ranged from 14.49- 37.83%, 6.61- 25.41%, and 0.44- 1.39% respectively. Lysine, methionine and tryptophan varied from 0.54- 1.29%, 0.06- 1.44 (mg/g) and 0.62- 2.23% respectively. Crude fiber, ADF and NDF in pods ranged from 5.21-8.98%, 8.29-14.92 and 12.73-24.35%. The seed oil content varied from 2.33-18.01%. 16:0, 18:0, 18:1 and 18:2 registered variations of 24.52- 45.64%, 0.01- 6.81%, 21.62- 39.63% and 23.95- 48.78% respectively. In pods total phenols, odihydroxy phenols and flavonols ranged from 385.71-939.421mg/100g, 20.09-79.451mg/100g and 11.43-98.1mg/100g DW. Whereas phenols and flavonols in seeds ranged from 3.18-7.32 mg/100g and 12.17-39.35 mg/100g. Chlorophyll a, b and carotenoids in fruits varied from 1.38- 11.15, 0.42-5.57 and 0.24-1.86mg/g respectively. Vitamin C and E ranged from 18.7-33.90.59 mg/g and 0.16-0.59 mg/g respectively. FRAP (2.28 mg/g AAE) and DPPH (89.89% inhibition) activity was maximum in Punjab Padmini and IC0506229 respectively. The carotenoids and tocopherols in seeds varied from 0.36-1.43mg/100g and 298.8-985.3ppm respectively. Phytate, saponins, tannins and oxalate in pods ranged from 1.7- 2.94%, 0.41- 1.32%, 10.36- 99.76% and 0.41- 0.95% respectively. In seeds the range was 0.21-0.75%, 1.72-3.44%, 11.67-38.47% and 0.27-1.62% respectively. Principal components analysis showed total variation of 77.17% and 82.49% in pods and mature seeds respectively.Genotypes (IC0043741, IC0506056, IC0506096, IC0510679, POL-311, POL-325) with high total protein content were subjected to SDS-PAGE. Globulins constituted maximum portion (30.53%) of seed protein followed by albumins (17.87%), glutelins (7.86%) and prolamines (4.43%). SDS pattern of the glutelins showed maximum variability. Fifteen elite genotypes (with high levels of quality parameters and low levels of antinutritionals) were selected out which can be exploited for okra improvement programmes and industrial and pharmaceutical applications.
Open Access
Exploring potential of plant ribosome inactivating proteins (RIPs) in combating leaf curl and fungal foliar diseases of cotton
(Punjab Agricultural University, Ludhiana, 2017) Brar, Gundeep Kaur; Sangha, Manjeet Kaur
Ribosome inactivating proteins (RIPs) are N-glycosydases, which catalyse the depurination of conserved single adenine (A4034) in yeast ribosomal RNA, leading to cell death. They are endowed with antimicrobial activities against bacteria, fungi and viruses in plants and animals. Cotton leaf curl disease (CLCuD) transmitted by whitefly (Bemisia tabaci) affects cotton production in northern India. In the present study, RIPs were purified by ion exchange chromatography (SP and CM sepharose) from the seeds of seven crops viz. Momordica charantia, Momordica balsmina, Spinacia oleracea, Phaseolus vulgaris, Cucurbita moschata, Zea mays and Hordeum vulgare. RIPs purified from M. balsmina and M. charantia showed maximum per cent recovery of 12.8 and 11.06 respectively. They also depicted maximum RIP activity on yeast ribosomal RNA by qualitative depurination assay. Depurination assay was carried out on ribosomes isolated from yeast. Both purified RIPs gave a single homogenous band on PAGE and SDS-PAGE of 28kDa. They registered a pI value of 10 on isoelectric focusing, showing them to be highly basic. Secondary structure determination of balsmina and charantia RIPs by CD spectroscopy revealed the presence of both alpha helices (23.5% and 32.8%) and beta sheets (24.6% and 43.6%). The RIPs also exhibited superoxide dismutase and chitinase activity. The purified balsmin RIP @0.250 mg/ml and charantia RIP @0.250 mg/ml and @0.500 mg/ml were sprayed on two cotton cultivars LH2076 and F1378, before and after whitefly infestation. Disease incidence was recorded after 28 and 40 days of RIP treatment. The disease incidence was found to be lower (20%-60%) in plants sprayed with RIPs as compared to their respective controls (100%). The latency or absence of virus was verified by PCR analysis using coat protein and beta DNA primers. The results correlated with disease incidence data. Charantia and balsmina RIPs also exhibited antifungal activity against Alternaria alternata and Myrothecium roridum. Inhibition of fungal growth by balsmin RIP @0.250 mg/ml on both fungi varied from 60.09% and 55.10% respectively, where as with charantia RIP 78.8% and 56.2% inhibition respectively was observed. Charantia RIP at 0.500 mg/ml inhibited the mycelial growth to 87.41% and 75.39% respectively. The spore germination inhibition studies revealed an EC50 value of 0.2 mg/ml for both the RIPs. Catalase, peroxidase, total sugars, reducing sugars, proteins, proline and total antioxidant activity increased in RIPs treated plants, highest increase was recorded in plants treated with charantia @0.500 mg/ml, thus depicting the role of RIPs in systemic acquired resistance (SAR). Therefore, the study depicts the antiviral and antifungal activity of RIPs purified from Momordica charantia and Momordica balsmina along with their role in SAR.
Open Access
Re-established desiccation tolerance as a mechanism of stress tolerance in wheat
(Punjab Agricultural University, Ludhiana, 2017) Satinder Kour; Zhawar, Vikramjit Kaur
Re-established desiccation tolerance (Re-DT) is a mechanism of extreme level of tolerance for almost complete water loss. It was found higher in wheat cultivar PBW 644 (drought tolerant) than PBW 343 (drought susceptible). Higher level of Re-DT in PBW 644 was related to higher reduction of water level so to arrest metabolic activity to avoid increases of ROS and oxidative toxicity, induction of special antioxidants like aldehyde dehydrogenase for detoxification and effective recycling of ascorbate under desiccation. This type of response in PBW 644 helped this plant to revive with higher survival upon rehydration. Contrary to it, PBW 343 showed higher water content, metabolic activity, ROS and oxidative toxicity under desiccation, though it induced antioxidant enzymes but failed to induce effective detoxification system (aldehyde dehydrogenase) and ascorbate recycling, therefore produced poor survival upon rehydration. Re-DT was ABA-regulated mechanism of cross tolerance. It involved ABA/ROS/NO-pathways for induction of antioxidant activity under desiccation and subsequent rehydration. It was improved by ABA and stress factors like water deficit stress, salt stress and heat stress. Ten wheat cultivars were screened for Re-DT and other related features. Four cultivars (HD 3086, PBW 725, PBW 658, PBW 677) showed a clear difference from six cultivars (gladius, kirchauff, HD 2967, BWL 5200, BWL 5202, PBW 723) for having higher level of Re-DT. Seeds of these four cultivars also showed higher dormancy, ABA sensitivity, less level of ROS/nitric oxides in embryos at 0 and 90 days of after-ripening. Laboratory testing of abiotic (osmotic, salt and heat) stress tolerance at seedlings stage found higher increase of root length and root to shoot length ratios by these four cultivars compared to other six. Therefore, this study indicated the presence of relatively higher level of vegetative stress tolerance in HD 3086, PBW 725, PBW 658, PBW 677compared to other six cultivars.
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BIOCHEMICAL CHARACTERIZATION OF WHEAT GENOTYPES WITH RESPECT TO NITROGEN USE EFFICIECNY
(PAU Ludhiana, 2012) Gurpreet Kaur; Bavita, Asthir
Increased use of nitrogen (N) fertilizer has raised concerns because N surplus causes environmental contamination and also high cost associated with its production. Thus, improving nitrogen use efficiency (NUE) through identification of efficient genotypes is becoming a necessity. In this two years study (2009-10 and 2010-11), 18 wheat genotypes (PBW 621, PBW 636, PBW 590, DBW 17, HD 2967, PBW 509, BW 9178, BW 9183, BW 8989, BW 9022, PBW 343, PBW 550, GLU 1101, GLU 1356, GLU 2001, GLU 700, PH132-4836, PH132-4840) selected on the basis of their commercial relevance or distinct genetic background were used for studying N metabolism at four N doses including the presently recommended N dose (RDN) (120 Kg N/ha), suboptimal N doses [RDN-50% (60 Kg N/ha) and RDN-25% (90 Kg N/ha)] and supraoptimal N dose [RDN+25% (150 Kg N/ha)]. Enzymes involved in N assimilation [nitrate reductase (NR), nitrite reductase (NIR), glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH)] in relation to deposition of amino acids and proteins were studied at tillering (30 days after sowing; DAS), anthesis (about 90-100 DAS) and post-anthesis (15 days post anthesis) stages. PBW 621 showed higher activities of NR, NIR and GS at RDN-25%. At RDN-50%, PBW 636 and GLU 1356 showed highest GS activity. HD 2967 and PBW 590 revealed maximum activities of these enzymes at RDN and RDN+25%. Widely grown cultivar PBW 343 and advanced breeding lines BW 9178, BW 9183, BW 8989 and BW 9022 showed low efficiency for N assimilating enzymes. GLU 1356 showed high grain protein content indicating higher translocation of assimilates from flag leaf to sink. Sugar and starch content was higher in the PBW 343, BW 9178, BW 8989 and BW 9022 genotypes in which amino acid and protein content was less. PBW 621 showed higher NUE and yield compared to other genotypes. NR and GS enzymes were positively correlated with NUE and yield indicating that these might be the rate limiting steps in N metabolism. Biochemical similarity between PBW 621, PBW 636 and GLU 1356 was authenticated from cluster analysis. Tiller culture technique did not reveal much difference with respect to PBW 621 and PBW 343 in N metabolism. However, hydroponically raised seedlings showed complementary results with field studies in identifying genotypes with maximum NUE. Due to stable performance of PBW 621, PBW 636 and GLU 356 at suboptimal doses over two years, these genotypes hold future potential for developing new cultivars with improved NUE.
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Comparative studies on heat and drought tolerance mechanisms in wheat using biochemical traits
(PAU, 2014) Shashi Bala; Asthir, Bavita
In semi-tropical regions, particularly in the Punjab (India), drought and high-temperature stress are erious constraints for wheat production. Six wheat cultivars PBW 343, PBW 550, PBW 621, PBW 175, C 306 and HD 2967 were extensively characterized for biochemical and physiological parameters under heat and drought conditions. High temperature (HT) elevated activities of syringaldazine peroxidase (SPX), guaiacol peroxidase (GPX) and coniferyl alcohol peroxidase (CPX) in C 306 and HD 2967 cultivars while, drought stress accentuated arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) activities in PBW 621 and PBW 175. Both heat and drought stress enhanced activities of diamine oxidase (DAO) polyamine oxidase (PAO) along with contents of H2O2. Amongst studied POXs, SPX activity was relatively high and coincided well with lignin deposition in leaves during vegetative stage while at 15 days post anthesis stage, CPX was the main peroxidase in the grain. Activity of ADC/ODC paralleled with putrescine and spermidine contents under drought stress condition. Higher build up of thiobarbituric acid reactive substances in PBW 343 and PBW 550 indicated higher membrane disruption during stress. Grain carbon metabolism was also altered under heat and drought stress condition as indicated by significant reduction in enzymes activities of ADP-Glucose pyrophosphorylase (AGPase), alkaline inorganic pyrophosphatase and phosphofructokinase (PFK) in conjunction with reduced starch content. However, contents of reducing sugars and fructans were increased while amylose and amylopectin content decreased. Correlation analysis revealed positive correlation of amylose with alkaline inorganic pyrophosphatase and starch with AGPase and PFK. Role of PFK was evident from correlation between AGPase and PFK indicating its predominant role in providing substrate for AGPase activity. Thousand grain weights decreased more under heat stress as compared to drought stress indicating higher severity of the former stress. Our results reveal variable response of cell wall peroxidases, amine oxidase and starch synthesising enzymes under two stress conditions. Increased activities peroxidases and contents of lignin, H2O2 and TBARS antioxidants, and other metabolites viz. Sugar and fructans alleviate the effect of heat and drought stress. Based on the results, we infer that cell wall peroxidase under heat stress and polyamines under drought led to enhanced abiotic stress tolerance for plant survival.
Open Access
Study of grain carbon metabolism in relation to nitrogen use efficiency in wheat
(PAU, 2015) Kaur, Balraj; Asthir, Bavita
The present study was conducted to evaluate the effect of application of nitrogen (N) under field conditions on six wheat genotypes (GLU 1356, HD 2967, PBW 621, PBW 636, PBW 550 and PBW 343) at three levels of N optimal N dose (120 Kg N/ha), sub-optimal N dose (90 Kg N/ha) and supra-optimal N dose (150 Kg N/ha). Enzymes involved in N metabolism (viz. nitrate reductase , glutamine synthetase, glutamate synthase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase); and carbon metabolism [viz. sucrose synthase (synthesis), sucrose phosphate synthase, Sucrose synthase cleavage, acid invertase, neutral invertase, phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICDH)] along with various metabolites were studied in roots, flag leaf and grains at different developmental stages. Results indicated that supra-optimal dose upregulated N and carbon metabolizing enzymes, whereas sub-optimal N dose indicated that aminotransferases enhanced the protein content while increased activities of invertases provide carbon skeleton for amino acid. Apparently, PEPC, ICDH and MDH resulted in assimilating ammonium ions thereby reducing its toxicity. Interestingly at sub-optimal N dose, higher activity of polyamine catabolizing enzymes results in speeding up oxidation of various polyamines to cope up with N deficiency in plant. Putrescine was found to be higher at early stage whereas spermidine and spermine were found to be higher towards mature stages of grain indicating the role of putrescine in grain filling process. Using, hydroponic culture technique in lab providing N sources viz. 4mM KNO3, NH4Cl and NH4NO3 for 12 days, the size of shoot and root system decreased significantly when plants were supplied with NH4Cl as exclusive N source. NH4Cl speed up amino acid content by elevated glutamate dehydrogenase, aminotransferases and PEPC activities and thereby decreasing NH4+ toxicity. Whereas under NH4NO3 treatment, N and carbon metabolism were elevated as compared to NH4Cl and control conditions but less than KNO3 source indicating inhibition of NH4+ toxicity by NO3- uptake. Tiller culturing technique also supported the above findings of grain filling processes, soluble sugars/starch were strongly correlated to invertase activities whereas proteins to aminotransferases. GLU 1356, HD 2967 showed highest value of nitrogen use efficiency followed by PBW 621, PBW 636, PBW 550 and PBW 343, therefore, categorized as N efficient genotypes.
Unknown
Effect of sulphur and nitrogen supply on their metabolism and protein quality of soybean (Glycine max (L.) Merrill)
(PAU, 2014) Sharma, Anju; Sharma, Sucheta
Field experiments were conducted to evaluate the effect of nitrogen (urea @ 31.25 kg ha-1) and sulphur (gypsum @ 20 kg ha-1) either alone or in combination on nodulation, nitrogen and sulphur assimilation in roots, nodules, leaves and developing seeds and protein quality in soybean (Glycine max (L.) Merrill). Treatment of nitrogen or sulphur significantly increased nodulation, leghaemoglobin content and vegetative growth in soybean. Glutamate dehydrogenase activity in cytosol and bacteroid fractions of nodules increased significantly in all the treatments from 70 to 90 days after sowing. Fertilizer treatments resulted in higher activities of nitrate reductase, glutamine synthetase, glutamate synthase, glutamate dehydrogenase, ATP sulphurylase, serine acetyl transferase, O-acetyl serine (thiol) lyase and increased nitrate content in leaves and developing seeds as compared to control. Sulphur alone or in combination with nitrogen maximally increased free amino acids, sulphur-containing amino acids and decreased N:S ratio in leaves and developing seeds. The contents of albumin and globulin fractions in developing and mature seeds increased under the effect of nitrogen and sulphur alone or in combination as compared to control. Gypsum alone or combined with nitrogen increased glycinin fraction of globulin whereas decreased β-conglycinin content thus improving 11S:7S ratio. Total seed yield, nutrient uptake, total soluble protein, free amino acid, methionine, cysteine, glutathione, palmitic and linoleic acid contents in seeds increased significantly under treatments of sulphur and nitrogen. Results suggest that gypsum @ 20 kg ha-1 alongwith recommended dose of urea @ 31.25 kg ha-1 can be used as optimum recommended source for improving soybean quality under local agroclimatic conditions of Punjab.
Open Access
Biochemical evaluation of irrigated flooded transplanted and aerobic rice (Oryza sativa L.)
(PAU, 2014) Manisha Kumari; Asthir, Bavita
Aerobic rice offers an attractive alternative approach over transplanting system as it uses less water with low labour expenses. Ten rice cultivars viz. PR 120, PR 115, PR 116, Feng Ai Zaan, Vandana, IR 71701-28-1-4, PAU 201, Punjab Mehak 1, MAS 946 and UPLRi 7 were extensively characterised for biochemical and physiological parameters under lab and field conditions. In flag leaf and grains, activities of antioxidative enzymes viz. ascorbate peroxidase (APx), guaiacol peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD) along with polyamine oxidase (PAO) and contents of ascorbate, α-tocopherol and polyamines increased gradually till mid milky stage and thereafter declined towards maturity under both planting conditions. Transplanted conditions led to higher activities of antioxidative enzymes (APx, GPx, CAT) alongwith contents of ascorbate, α-tocopherol in PR 116, PAU 201 and Punjab Mehak 1 whereas aerobic condition had elevated levels of the PAO, SOD activities and contents of polyamines, lipid peroxide and hydrogen peroxide in Feng Ai Zaan, PR 115 and PR 120 cultivars which indicated differential response of cultivars and their superior tolerance mechanism. Contents of total sugars, reducing sugars and sucrose alongwith activities of sucrose synthase (cleavage) and invertase (acid and neutral) first increased gradually till anthesis in leaf and 14 days post anthesis in grains and thereafter declined towards maturity under both planting conditions. Similar to antioxidant defense and polyamine metabolism, aerobic conditions significantly decreased activities of invertases (acid and neutral) and sucrose synthase cleavage along with decrease in yield and starch content in all cultivars. In contrast, activities of sucrose synthase/phosphate synthase and soluble protein content increased in conjunction with an increase in the amino acids content under aerobic conditions. Our results reveal variable response of antioxidant, amine oxidase and sucrose metabolising enzymes within genotypes under two planting conditions. Diminished activities of antioxidant enzymes and contents of antioxidants, and other metabolites viz. sugar, starch and protein were exhausted to combat water stress. Based on the results, we infer that antioxidative defense mechanism in transplanted rice and polyamine metabolism in direct-seeded ones led to enhanced tolerance for their survival. This differential response of genotypes under two planting conditions give a clear indication that metabolic pathway differ under two planting conditions to counteract reactive oxygen species generated during stress conditions in aerobic rice.

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