Agro-morphological and genetic diversity studies in Rice (Oryza sativa L.) germplasm using microsatellite markers.

BACKGROUND: Knowledge of the genetic diversity and population structure of germplasm collections is an important foundation for crop improvement. Rice production across a broad range of rice-growing environments results in a diverse array of local rice varieties. Many rice varieties have been lost as a result of biodiversity loss and are now grown in Pakistan. METHODS AND RESULTS: To protect the biodiversity of rice varieties, an experiment was carried out to check the genetic and morphological variations between 8 exotic and 7 local rice genotypes, using 5 different SSR markers, i.e., RM3, RM259, RM341, RM520, and RM11943. The analysis of morphological and quality traits of rice observed significant variation across genotypes. The results revealed that genotype Irri-Pak attained the highest plant height and primary branch plant-1, while genotype Mushkan produced a higher number of productive tillers and obtained a higher fertility factor (%). Similarly, the highest value for panicle length was observed for genotype Faker-e-Malakand, 1000-grains weight in genotype Calmochi, and maximum days to maturity was noticed in genotype Swati-2014. Moreover, the genotype Brio attained the highest value of stem diameter, while maximum seed length was noted in the genotype Sug Dasi. The highest number of primary branches plant-1 in genotype Ibge-I and secondary branches plant-1 in genotype Calmochi were noticed. A higher concentration of sodium and potassium was observed for the genotype Marte, while the genotype Muskan attained the maximum content of copper. Moreover, the highest concentration of iron in genotype Originario, zinc in genotype JP-5, and cadmium content were noticed in genotype Ibge. Similarly, the dendrogram analysis for quantitative parameters showed three clusters at 74.13% similarities. Whereas all the genotypes of European origin formed a separate cluster. A set of 5 simple sequence repeat primers, covering four chromosomes, amplified a total of 14 alleles and showed 100% polymorphism with an average PIC value ranging from 0.39 to 0.91. The UPGMA cluster analysis separated the 15 rice genotypes into 3 main groups based on 32.5% similarities and the highest genetic distance (45.1%) was observed between two genotypes (Fakher-e-malakand and Musa), having different geographical origins. There was no genetic distance between the genotypes Marte and Brio, irrespective of having the same origin. CONCLUSIONS: The maximum genetic distances were noted for genotype, Fakhre-e-Malakand and Musa having a different origin, while the minimum genetic distance was shown by genotypes, Marte and Onice, from the same origin.

Quantitative proteomics reveals that peroxidases play key roles in post-flooding recovery in soybean roots.

Soybean is an important legume crop that exhibits markedly reduced growth and yields under flooding conditions. To unravel the mechanisms involved in recovery after flooding in soybean root, gel-free proteomic analysis was performed. Morphological analysis revealed that growth suppression was more severe with increased flooding duration. Out of a total of 1645 and 1707 identified proteins, 73 and 21 proteins were changed significantly during the recovery stage following 2 and 4 days flooding, respectively. Based on the proteomic, clustering, and in silico protein-protein interaction analyses, six key enzymes were analyzed at the mRNA level. Lipoxygenase 1, which was increased at the protein level during the recovery period, was steadily down-regulated at the mRNA level. The peroxidase superfamily protein continuously increased in abundance during the course of recovery and was up-regulated at the mRNA level. HAD acid phosphatase was decreased at the protein level and down-regulated at the transcript level, while isoflavone reductase and an unknown protein were increased at both the protein and mRNA levels. Consistent with these findings, the enzymatic activity of peroxidase was decreased under flooding stress but increased significantly during the recovery sage. These results suggest that peroxidases might play key roles in post-flooding recovery in soybean roots through the scavenging of toxic radicals.

Proteomic insight into soybean response to flooding stress reveals changes in energy metabolism and cell wall modifications.

Soybean is a legume crop enriched with proteins and oil. It is frequently exposed to anthropogenic and natural flooding that limits its growth and yield. Current study applied gel-free proteomic techniques to unravel soybean response mechanism to flooding stress. Two-days-old soybeans were flooded for 4 days continuously and root samples were collected at days 2 to 6 for proteomic and enzymatic analyses. Age-matched untreated soybeans were collected as control. After protein extraction, purification and tryptic digestion, the peptides were analyzed on nano-liquid chromatography-mass spectrometry. A total of 539 and 472 proteins with matched peptides 2 or more were identified in control and flooded seedlings, respectively. Among these 364 proteins were commonly identified in both control and flooded soybeans. Fourty-two protein's abundances were changed 4-fold after 2-days of flooding stress as compared to starting point. The cluster analysis showed that highly increased proteins included cupin family proteins, enolase, pectin methylesterase inhibitor, glyoxalase II, alcohol dehydrogenase and aldolase. The enzyme assay of enolase and pectin methylesterase inhibitor confirmed protein abundance changes. These findings suggest that soybean adopts the less energy consuming strategies and brings biochemical and structural changes in the cell wall to effectively respond to flooding stress and for the survival.

Targeted metabolomics reveals fatty acid abundance adjustments as playing a crucial role in drought-stress response and post-drought recovery in wheat.

Drought stress is one of the abiotic stresses restricting plant development, reproductive growth, and survival. In the present study, the effect of drought stress and post-drought recovery for the selected local wheat cultivar, Atta Habib, was studied. Wheat was grown for 16 days followed by drought stress for 7 days and allowed to recover for 7 days after the removal of the drought stress. Same-aged untreated plants were also grown as a control. The effect of drought stress and post-drought recovery on morphology (root length, shoot length, root weight, and shoot weight), enzymatic activity, and fatty acid profile were analyzed. The results showed that shoot weight (93.1 mg), root weight (85.2 mg), and shoot length (11.1 cm) decreased in the stressed plants but increased steadily in the recovered plants compared to the same-aged control plants, while root length showed a higher increase (14.0 cm) during drought stress and tended to normalize during the recovery phase (13.4 cm). The ascorbate peroxidase activity increased in the stressed plants (5.44 unit/mg protein) compared to the control, while gradually normalizing in the recovery phase (5.41 unit/mg protein). Gas chromatography coupled mass spectrometric analysis revealed abundance changes in important fatty acids, such as palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid. Palmitic acid (39.1%) and oleic acid (2.11%) increased in the drought-stressed plants, while a reduction in linoleic acid (6.85%) and linolenic acid (51.18%) was observed compared to the same-aged control plants, i.e., palmitic (33.71%), oleic (0.95%), linoleic (7.52%), and linolenic acid (55.23%). The results suggest that wheat tries to recover in the post-drought stage by repairing oxidative damage through ascorbate peroxidase, and by adjusting fatty acid abundances under drought stress and during the post-drought phase in an effort to maintain membranes' integrity and a suitable fat metabolism route, thus helping recovery. Targeted metabolomics may be further used to explore the role of other metabolites in the drought-stress response mechanism in wheat. Furthermore, this relatively little explored avenue of post-drought recovery needs more detailed studies involving multiple stress durations.

Pyramiding of Four Broad Spectrum Bacterial Blight Resistance Genes in Cross Breeds of Basmati Rice.

Pyramiding of major resistance (R) genes through marker-assisted selection (MAS) is a useful way to attain durable and broad-spectrum resistance against Xanthomonas oryzae pv. oryzae pathogen, the causal agent of bacterial blight (BB) disease in rice (Oryza sativa L.). The present study was designed to pyramid four broad spectrum BB-R genes (Xa4, xa5, xa13 and Xa21) in the background of Basmati-385, an indica rice cultivar with much sought-after qualitative and quantitative grain traits. The cultivar, however, is susceptible to BB and was therefore, crossed with IRBB59 which possesses R genes xa5, xa13 and Xa21, to attain broad and durable resistance. A total of 19 F1 plants were obtained, some of which were backcrossed with Basmati-385 and large number of BC1F1 plants were obtained. In BC1F2 generation, 31 phenotypically superior genotypes having morphological features of Basmati-385, were selected and advanced up to BC1F6 population. Sequence-tagged site (STS)-based MAS was carried out and phenotypic selection was made in each successive generation. In BC1F6 population, potentially homozygous recombinant inbred lines (RILs) from each line were selected and evaluated on the bases of STS evaluation and resistance to local Xanthomonas oryzae pv. oryzae (Xoo) isolates. Line 23 was found pyramided with all four BB-R genes i.e., Xa4, xa5, xa13 and Xa21. Five genotypes including line 8, line 16, line 21, line 27 and line 28 were identified as pyramided with three R genes, Xa4, xa5 and xa13. Pathological study showed that rice lines pyramided with quadruplet or triplet R genes showed the highest level of resistance compared to doublet or singlet R genes. Thus, line 23 with quadruplet, and lines 8, 16, 21, 27, and 28 with triplet R genes, are recommended for replicated yield and resistance trials before release as new rice varieties. Further, traditional breeding coupled with MAS, is a solid way to attain highly effective BB-resistant rice lines with no yield cost.

Proteomics, physiological, and biochemical analysis of cross tolerance mechanisms in response to heat and water stresses in soybean.

Water stress (WS) and heat stress (HS) have a negative effect on soybean plant growth and crop productivity. Changes in the physiological characteristics, proteome, and specific metabolites investigated on molecular and cellular functions were studied in two soybean cultivars exposed to different heat and water stress conditions independently and in combination. Leaf protein composition was studied using 2-DE and complemented with MALDI TOF mass spectrometry. While the two cultivars displayed genetic variation in response to water and heat stress, thirty-nine proteins were significantly altered in their relative abundance in response to WS, HS and combined WS+HS in both cultivars. A majority of these proteins were involved in metabolism, response to heat and photosynthesis showing significant cross-tolerance mechanisms. This study revealed that MED37C, a probable mediator of RNA polymerase transcription II protein, has potential interacting partners in Arabidopsis and signified the marked impact of this on the PI-471938 cultivar. Elevated activities in antioxidant enzymes indicate that the PI-471938 cultivar can restore the oxidation levels and sustain the plant during the stress. The discovery of this plant's development of cross-stress tolerance could be used as a guide to foster ongoing genetic modifications in stress tolerance.

Organ-Specific Proteomic Analysis of Early Vegetative Stage Soybean.

BACKGROUND: Soybean is a nutritionally important source of proteins and oils. Organspecific analyses of the plants provided valuable information about physiological changes occurring in particular organ. However, studies on the vegetative stage soybean during the growth and development are lacking. Analyzing the growth stages of soybean at proteomic level is particularly important for understanding the growth dynamics. OBJECTIVES: The main objective of this study was to investigate the organ-specific growth pattern of proteins during early growth stage. METHODS: Soybean (Glycine max L. cv Enrei) seeds were sown in each seedling case for 10 days and roots, hypocotyls, and leaves were collected. Proteins were extracted and analyzed by nano-liquid chromatography mass spectrometry. Mole percent abundance was calculated using emPAI values. To determine the functional role of the proteins identified in the MS analysis, functional categorization was performed using MapMan bin codes. Visualization of protein abundance ratio was performed using MapMan software. Enzyme activity and quantitative reverse transcription-polymerase chain reaction analyses were performed. RESULTS: A total of 357, 360, and 392 proteins were identified in root, hypocotyl, and leaf of vegetative stage soybean, respectively. Proteins related to stress, cell organization, transport, signaling, and mitochondrial electron transport chain decreased in root, hypocotyl, and leaf. Proteins related to protein metabolism, glycolysis, and cell wall were comparable in root and hypocotyl; however, in leaf, glycolysis and cell wall related proteins were decreased. Aldehyde dehydrogenase was decreased in abundance and activity in hypocotyl and leaf as compared to root. Major latex proteins 43 and 423 changed in abundance in an organ-specific manner. The mRNA expression level of major latex proteins exhibited a differential expression in the hypocotyl of soybean during flooding stress and recovery. CONCLUSION: Our results suggest that aldehyde dehydrogenase and major latex proteins play key roles in growth of soybean in an organ-specific way.

Proteomic analysis of soybean root including hypocotyl during recovery from drought stress.

UNLABELLED: Soybean is a nutritionally important crop that exhibits reductions in growth and yield under drought stress. To investigate soybean responses during post-drought recovery, a gel-free proteomic technique was used. Two-day-old soybeans were treated with drought stress for 4days and recovered for 4days. Root including hypocotyl was collected during the drought treatment and recovery stage. Seedling growth was suppressed by drought stress, but recovered following stress removal. The malondialdehyde content increased under drought stress, but decreased during the recovery stage. A total of 792 and 888 proteins were identified from the control and recovering seedlings, respectively. The identified proteins were related to functional categories of stress, hormone metabolism, cell wall, secondary metabolism, and fermentation. Cluster analysis indicated that abundances of peroxidase and aldehyde dehydrogenase were highly changed in the seedlings during the post-drought recovery. The activity of peroxidase decreased under drought conditions, but increased during recovery. In contrast, the activity of aldehyde dehydrogenase was increased in response to drought stress, but decreased during the recovery stage. These results suggest that peroxidase and aldehyde dehydrogenase play key roles in post-drought recovery in soybean by scavenging toxic reactive oxygen species and reducing the load of harmful aldehydes. BIOLOGICAL SIGNIFICANCE: Post-drought recovery response mechanisms in soybean root including hypocotyl were analyzed using gel-free proteomic technique. A total of 643 common proteins between control and drought-stressed soybeans changed significantly in abundance over time. The proteins that changed during post-drought recovery were assigned to protein, stress, hormone metabolism, secondary metabolism, cell wall, redox, and glycolysis categories. The analysis revealed that peroxidase and aldehyde dehydrogenase were increased in protein abundance under drought stress. The enzyme activity of peroxidase decreased under drought but increased during recovery. The activity of aldehyde dehydrogenase was increased under drought stress but decreased during recovery stage. Peroxidase and aldehyde dehydrogenase reduce the toxic reactive oxygen species and aldehydes from the plant, respectively, and help to recover from drought stress. The study provides information about post-drought recovery mechanism in soybean.

Proteomic analysis of soybean hypocotyl during recovery after flooding stress.

Soybean is a nutritionally important crop, but exhibits reduced growth and yields under flooding stress. To investigate soybean responses during post-flooding recovery, a gel-free proteomic technique was used to examine the protein profile in the hypocotyl. Two-day-old soybeans were flooded for 2 days and hypocotyl was collected under flooding and during the post-flooding recovery period. A total of 498 and 70 proteins were significantly changed in control and post-flooding recovering soybeans, respectively. Based on proteomic and clustering analyses, three proteins were selected for mRNA expression and enzyme activity assays. Pyruvate kinase was increased under flooding, but gradually decreased during post-flooding recovery period at protein abundance, mRNA, and enzyme activity levels. Nucleotidylyl transferase was decreased under flooding and increased during post-flooding recovery at both mRNA expression and enzyme activity levels. Beta-ketoacyl reductase 1 was increased under flooding and decreased during recovery at protein abundance and mRNA expression levels, but its enzyme activity gradually increased during the post-flooding recovery period. These results suggest that pyruvate kinase, nucleotidylyl transferase, and beta-ketoacyl reductase play key roles in post-flooding recovery in soybean hypocotyl by promoting glycolysis for the generation of ATP and regulation of secondary metabolic pathways. BIOLOGICAL SIGNIFICANCE: This study analyzed post-flooding recovery response mechanisms in soybean hypocotyl, which is a model organ for studying secondary growth, using a gel-free proteomic technique. Mass spectrometry analysis of proteins extracted from soybean hypocotyls identified 20 common proteins between control and flooding-stressed soybeans that changed significantly in abundance over time. The hypocotyl proteins that changed during post-flooding recovery were assigned to protein, development, secondary metabolism, and glycolysis categories. The analysis revealed that three proteins, pyruvate kinase, nucleotidylyl transferase, and beta-ketoacyl reductase, were increased in hypocotyl under flooding conditions and during post-flooding recovery. The proteins are involved in glycolysis, nucleotide synthesis and amino acid activation, and complex fatty acid biosynthesis.