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Item Saccharomyces cerevisiae polo-like kinase, Cdc5 exhibits ATP-dependent Mg2+ -enhanced kinase activity in vitro(Elsevier B.V., 2019) Chauhan, Sujata; Samanta, Subhasis; Sharma, Nitin; Thakur, Jitendra K.; Dev, Kamal; Sourirajan, AnuradhaPhosphorylation of proteins on serine/threonine residues represents an important biochemical mechanism to regulate several cellular processes. Polo-like kinases (PLKs) are a family of serine-threonine kinases that play an imminent role in cell cycle regulation in yeast to humans, and thus an important therapeutic target for cancers. The present study provides insights into the enzymatic features of Saccharomyces cerevisiae PLK, Cdc5 using in vitro casein phosphorylation assays. The recombinant yeast PLK, GST-Cdc5 showed maximum casein phosphorylation activity at 30 °C, pH 9 and 45 min of incubation period. GST-Cdc5 exhibited a KM of 1.35 μM for casein, and high affinity for ATP, since addition of non-radioactive ATP chased out casein phosphorylation by radiolabeled ATP. The recombinant enzyme showed maximum kinase activity at 2.7 μM of GST-Cdc5. Casein was found to be the best in vitro substrate of GST-Cdc5 followed by BSA (Bovine Serum Albumin) and MBP (Myelin Basic Protein). Of the metal ions tested, Mg2+ (at 20 mM) was found to enhance GST-Cdc5 kinase activity, while Ca2+ (at 5 mM) and Mn2+ (at 10 mM) inhibited the same. The presence of EDTA, SDS and PMSF inhibited phosphorylation by GST-Cdc5, while DTT had no effect. The recombinant GST-Cdc5 can be used as a tool for deciphering PLKs’ structure and functions, which are still at infancy.Item Production and cytological characterization of a synthetic amphiploid derived from a cross between Oryza sativa and Oryza punctata(NRC Research Press, 2019) Kumar, Kishor; Neelam, Kumari; Singh, Gurpreet; Mathan, Jyotirmaya; Ranjan, Aashish; Brar, Darshan Singh; Singh, KuldeepOryza punctata Kotschy ex Steud. (BB, 2n = 24) is a wild species of rice that has many useful agronomic traits. An interspecific hybrid (AB, 2n = 24) was produced by crossing O. punctata and Oryza sativa variety Punjab Rice 122 (PR122, AA, 2n = 24) to broaden the narrow genetic base of cultivated rice. Cytological analysis of the pollen mother cells (PMCs) of the interspecific hybrids confirmed that they have 24 chromosomes. The F1 hybrids showed the presence of 19–20 univalents and 1–3 bivalents. The interspecific hybrid was treated with colchicine to produce a synthetic amphiploid (AABB, 2n = 48). Pollen fertility of the synthetic amphiploid was found to be greater than 50% and partial seed set was observed. Chromosome numbers in the PMCs of the synthetic amphiploid were 24II, showing normal pairing. Flow cytometric analysis also confirmed doubled genomic content in the synthetic amphiploid. Leaf morphological and anatomical studies of the synthetic amphiploid showed higher chlorophyll content and enlarged bundle sheath cells as compared with both of its parents. The synthetic amphiploid was backcrossed with PR122 to develop a series of addition and substitution lines for the transfer of useful genes from O. punctata with least linkage drag.Item Overexpression of OsPUB41, a rice E3 ubiquitin ligase induced by cell wall degrading enzymes, enhances immune responses in rice and Arabidopsis(BioMed Central Ltd, 2019) Kachewar, Neha Rajendra; Gupta, Vishal; Ranjan, Ashish; Patel, Hitendra Kumar; Sonti, Ramesh V.Background: Cell wall degrading enzymes (CWDEs) induce plant immune responses and E3 ubiquitin ligases are known to play important roles in regulating plant defenses. Expression of the rice E3 ubiquitin ligase, OsPUB41, is enhanced upon treatment of leaves with Xanthomonas oryzae pv. oryzae (Xoo) secreted CWDEs such as Cellulase and Lipase/Esterase. However, it is not reported to have a role in elicitation of immune responses. Results: Expression of the rice E3 ubiquitin ligase, OsPUB41, is induced when rice leaves are treated with either CWDEs, pathogen associated molecular patterns (PAMPs), damage associated molecular patterns (DAMPs) or pathogens. Overexpression of OsPUB41 leads to induction of callose deposition, enhanced tolerance to Xoo and Rhizoctonia solani infection in rice and Arabidopsis respectively. In rice, transient overexpression of OsPUB41 leads to enhanced expression of PR genes and SA as well as JA biosynthetic and response genes. However, in Arabidopsis, ectopic expression of OsPUB41 results in upregulation of only JA biosynthetic and response genes. Transient overexpression of either of the two biochemically inactive mutants (OsPUB41C40A and OsPUB41V51R) of OsPUB41 in rice and stable transgenics in Arabidopsis ectopically expressing OsPUB41C40A failed to elicit immune responses. This indicates that the E3 ligase activity of OsPUB41 protein is essential for induction of plant defense responses. Conclusion: The results presented here suggest that OsPUB41 is possibly involved in elicitation of CWDE triggered immune responses in rice.Item Physiological plasticity to high temperature stress in chickpea: Adaptive responses and variable tolerance(Elsevier B.V., 2019) Pareek, Akanksha; Rathi, Divya; Mishra, Divya; Chakraborty, Subhra; Chakraborty, NiranjanHigh temperature stress (HTS) is one of the most crucial factors that limits plant growth and development, and reduces crop yields worldwide. Cool-season crops, particularly the legumes, are severely affected by increasing ambient temperature associated with global climate change. We characterized the HTS-induced modulations of morpho-physicochemical traits and gene expression of several chickpea genotypes and the metabolic profile of the tolerant cultivar. Higher water use efficiency and photosynthetic capacity, minimal membrane lipid peroxidation in conjunction with increased abundance of osmolytes and secondary metabolites depicted thermotolerance of ICC 1205. The adaptive responses were accompanied by high transcript abundance of heat shock proteins and antioxidant enzymes. To integrate stress-responsive signalling and metabolic networks, the HTS-induced physicochemical analysis was further extended to metabolite profiling of the thermotolerant cultivar. The screening of the metabolome landscape led to the identification of 49 HTS-responsive metabolites that include polycarboxylic acid, sugar acids, sugar alcohols and amino acids which might confer thermotolerance in chickpea. The present study, to our knowledge, is the most comprehensive of its kind in dissecting cultivar-specific differential adaptive responses to HTS in chickpea, which might potentiate the identification of genetic traits extendible to improvement of thermotolerance of crops.Item The PHYTOGLOBIN-NO cycle regulates plant mycorrhizal symbiosis(Elsevier B.V., 2019) Kumari, Aprajita; Pathak, Pradeep Kumar; Loake, Gary J.; Gupta, Kapuganti JagadisThe production of the redox-active signaling molecule, NO, has long been associated with interactions between microbes and their host plants. Emerging evidence now suggests that specific NO signatures and cognate patterns of PHYTOGLOBIN1 (PHYTOGB1) expression, a key regulator of cellular NO homeostasis, may help determine either symbiosis or pathogenicity.Item Alternative oxidase (AOX) senses stress levels to coordinate auxin-induced reprogramming from seed germination to somatic embryogenesis- A role relevant for seed vigor prediction and plant robustness(Frontiers Media S.A., 2019) Mohanapriya, Gunasekaran; Bharadwaj, Revuru; Noceda, Carlos; Costa, José Hélio; Kumar, Sarma Rajeev; Sathishkumar, Ramalingam; Thiers, Karine Leitão Lima; Macedo, Elisete Santos; Silva, Sofia; Annicchiarico, Paolo; Groot, Steven P.C.; Kodde, Jan; Kumari, Aprajita; Gupta, Kapuganti Jagadis; Arnholdt-Schmitt, BirgitSomatic embryogenesis (SE) is the most striking and prominent example of plant plasticity upon severe stress. Inducing immature carrot seeds perform SE as substitute to germination by auxin treatment can be seen as switch between stress levels associated to morphophysiological plasticity. This experimental system is highly powerful to explore stress response factors that mediate the metabolic switch between cell and tissue identities. Developmental plasticity per se is an emerging trait for in vitro systems and crop improvement. It is supposed to underlie multi-stress tolerance. High plasticity can protect plants throughout life cycles against variable abiotic and biotic conditions. We provide proof of concepts for the existing hypothesis that alternative oxidase (AOX) can be relevant for developmental plasticity and be associated to yield stability. Our perspective on AOX as relevant coordinator of cell reprogramming is supported by real-time polymerase chain reaction (PCR) analyses and gross metabolism data from calorespirometry complemented by SHAM-inhibitor studies on primed, elevated partial pressure of oxygen (EPPO)–stressed, and endophyte-treated seeds. In silico studies on public experimental data from diverse species strengthen generality of our insights. Finally, we highlight ready-to-use concepts for plant selection and optimizing in vivo and in vitro propagation that do not require further details on molecular physiology and metabolism. This is demonstrated by applying our research & technology concepts to pea genotypes with differential yield performance in multilocation fields and chickpea types known for differential robustness in the field. By using these concepts and tools appropriately, also other marker candidates than AOX and complex genomics data can be efficiently validated for prebreeding and seed vigor prediction.Item Biological mechanisms of plant interactions with a combination of biotic and abiotic stresses(Frontiers Media S.A., 2019) Morel, Jean-benoit; Senthil-Kumar, Muthappa; Ballini, ElsaThis Research Topic addresses the way plants respond to a combination of different types of stresses, for instance, how a plant deals with simultaneous attacks by pathogens (biotic stress) and environmental stresses (abiotic stress). While most plant research has been focused on the understanding of individual biotic or abiotic stress responses, we propose to encourage submissions on a broader approach that better represents stress conditions encountered in the field. Indeed, in nature or under field conditions plants are not just dealing with one environmental stress: they are often coping with several simultaneously occurring biotic and abiotic stresses. Recent findings indicate that predicting the plant response to a combination of stresses cannot be made from the knowledge of an individual stress, and therefore remains a major challenge. It remains to be determined what biological systems respond to combined stresses. Hormones, with their complex regulation and cross-talk, as well as molecules like calcium or reactive oxygen species, are likely to play a key role in combined stresses as potential integrators of multiple signaling pathways. Moreover, given the importance of epigenetic changes in response to a given stress, the role of epigenetic modifications during combined stresses needs further attention. Combined stresses may not only be concomitant but be successive, either during a plant’s life or across generations. In that respect, trans-generational, epigenetic changes and more generally stress memory mechanisms could also be important for our understanding of combinational stresses. Interestingly, dual RNA-Seq and other approaches also indicate that besides the measured changes in plants, pathogen transcriptional programs are also severely and indirectly affected by stresses applied to their host plants. This Research Topic welcomes the submission of all article types, with a preference for Original Research, Reviews, and Opinions, focusing on the following: (1) analysis at the molecular and physiological level of combined stresses, and if possible also describing the behavior of the pathogen, (2) genetic and epigenetic studies of combined biotic and abiotic stresses, (3) studies describing at the plant level or within how a well-known mechanism involved in one type of stress is affected by another type of stress (e.g., how Resistance gene function is altered by heat), (4) studies using bioinformatics/computational tools to analyze various publicly available transcriptomic/proteomic/metabolomic datasets, provided that the genomic studies have adequate biological/functional validation, All types of common pathogens (viruses, bacteria, fungi, nematodes) and insects are welcome, which exclude probiotic or beneficial organisms; abiotic stresses may also include agronomical constraints, like nutrient fertilization. In this Research Topic, ‘combined stress’ is understood as a combination of one biotic stress with at least one abiotic stress. Studies need to provide a mechanisms or a molecular level understanding of stress interaction, and not just an evaluation. Manuscripts dealing with each type of stress separately (e.g., “gene X confers both drought tolerance and pathogen resistance”) will not be considered.Item Chilli leaf curl virus infection downregulates the expression of the genes encoding chloroplast proteins and stress-related proteins(Springer Nature Publishing AG, 2019) Kushwaha, Nirbhay Kumar; Mansi; Sahu, Pranav Pankaj; Prasad, Manoj; Chakrabroty, SupriyaVirus infection alters the expression of several host genes involved in various cellular and biological processes in plants. Most of the studies performed till now have mainly focused on genes which are up-regulated and later projected them as probable stress tolerant/susceptible genes. Nevertheless, genes which are down-regulated during plant-virus interaction could also play a critical role on disease development as well as in combating the virus infection. Hence, to identify such down-regulated genes and pathway, we performed reverse suppression subtractive hybridization in Capsicum annuum var. Punjab Lal following Chilli leaf curl virus (ChiLCV) infection. The screening and further processing suggested that majority of the genes (approximately 35% ESTs) showed homology with the genes encoding chloroplast proteins and 16% genes involved in the biotic and abiotic stress response. Additionally, we identified several genes, functionally known to be involved in metabolic processes, protein synthesis and degradation, ribosomal proteins, energy production, DNA replication and transcription, and transporters. We also found 3% transcripts which did not show homology with any known genes. The redundancy analysis revealed the maximum percentage of chlorophyll a-b binding protein (15/96) and auxin-binding proteins (13/96).Item Differential RNA editing of mitochondrial genes in WA-cytoplasmic based male sterile line pusa 6A, and its maintainer and restorer lines(Elsevier B.V., 2019) Ngangkham, Umakanta; Parida, Swarup K.; Singh, Ashok Kumar; Mohapatra, TrilochanRNA editing changes the nucleotides at the transcript level of mitochondrial genes which results in synthesis of functional proteins. This study was designed to find the editing sites which could be implicated in male fertility restoration and to develop editing based markers for differentiation of cytoplasmic male sterility and maintainer lines from each other. DNA and RNA from young panicles were isolated from three-line system of hybrid rice PRH10, wild abortive (WA) cytoplasm based male sterile (A line Pusa 6A), maintainer (B line Pusa 6B) and restorer (R line PRR78) lines. Pusa 6A and PRR78 having the same WA cytoplasm are allo-nuclear and iso-cytpolasmic lines. The genomic and cDNA amplicons for eight mitochondrial genes (18SrRNA, atp6, atp9, cobII, coxI, coxIII, nadI and rps3) were sequenced and compared. Differences in genomic and cDNA sequences were considered as editing. Two hundred and thirty editing sites having base substitution or insertion/deletion were identified with the highest in 18SrRNA (5.74%) and the lowest in coxI (0.60%). The highest editing sites were observed in fertile maintainer Pusa 6B followed by PRR78 and Pusa 6A, of which random five editing sites in five different rice mitochondrial transcripts namely atp9, cobII, coxIII, rps3 and 18SrRNA were chosen and validated through cleaved amplified polymorphism sequence (CAPS) analysis and found to be partially edited in four genes. The identical editing sites of different mitochondrial genes from maintainer and restorer lines might reflect their possible contribution to fertility restoration of sterile WA cytoplasm.Item Tomato roots exhibit in vivo glutamate dehydrogenase aminating capacity in response to excess ammonium supply(Elsevier B.V., 2019) Vega-Mas, I.; Rossi, M.T.; Gupta, Kapuganti Jagadis; González-Murua, C.; Ratcliffe, R.G.; Estavillo, J.M.; González-Moro, M.B.In higher plants ammonium (NH4+) assimilation occurs mainly through the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway. Nevertheless, when plants are exposed to stress conditions, such as excess of ammonium, the contribution of alternative routes of ammonium assimilation such as glutamate dehydrogenase (GDH) and asparagine synthetase (AS) activities might serve as detoxification mechanisms. In this work, the in vivo functions of these pathways were studied after supplying an excess of ammonium to tomato (Solanum lycopersicum L. cv. Agora Hybrid F1) roots previously adapted to grow under either nitrate or ammonium nutrition. The short-term incorporation of labelled ammonium (15NH4+) into the main amino acids was determined by GC–MS in the presence or absence of methionine sulphoximine (MSX) and azaserine (AZA), inhibitors of GS and GOGAT activities, respectively. Tomato roots were able to respond rapidly to excess ammonium by enhancing ammonium assimilation regardless of the previous nutritional regime to which the plant was adapted to grow. The assimilation of 15NH4+ could take place through pathways other than GS/GOGAT, since the inhibition of GS and GOGAT did not completely impede the incorporation of the labelled nitrogen into major amino acids. The in vivo formation of Asn by AS was shown to be exclusively Gln-dependent since the root was unable to incorporate 15NH4+ directly into Asn. On the other hand, an in vivo aminating capacity was revealed for GDH, since newly labelled Glu synthesis occurred even when GS and/or GOGAT activities were inhibited. The aminating GDH activity in tomato roots responded to an excess ammonium supply independently of the previous nutritional regime to which the plant had been subjected.