Development of a protocol for the elimination of Cyrtanthus elatus virus-A from Narcissus tazetta by in vitro chemotherapy in combination with electrotherapy.

Narcissus (Narcissus tazetta) is a bulbous ornamental plant propagated vegetatively from bulbs. The Cyrtanthus elatus virus-A (CyEV-A) had been reported to cause a severe mosaic and yellow stripe disease in narcissus. Therefore, this study aimed to develop a protocol for the elimination of CyEV-A from infected bulblets by in vitro chemotherapy (30-50 mg/L ribavirin for 30 days) and electrotherapy (10-30 mA for 20 min), individually and in combination, to produce virus-free plants. The regenerated plants obtained from these treatments were screened for the absence of the CyEV-A by reverse-transcription polymerase chain reaction assays using a set of degenerate primers specific for a potyvirus coat protein gene. The results showed that in vitro chemotherapy (30 mg/L ribavirin for 30 days) alone produced 46.0 % (14/30) of virus-free plants, while electrotherapy (20 mA for 20 min) alone produced 40.0 % (12/30) of virus-free plants. In comparison, a combination of chemotherapy (30 mg/L ribavirin for 30 days) and electrotherapy (20 mA for 20 min) produced 50.0 % (15/30) of virus-free plants. The virus-free plants obtained from this combination treatment exhibited better growth and produced more bulbs compared to the other treatments and control. The protocol may be used for the control of the virus disease in narcissus.

Transcriptome and proteome analyses reveal selenium mediated amelioration of arsenic toxicity in rice (Oryza sativa L.).

Arsenic (As), a chronic poison and non-threshold carcinogen, is a food chain contaminant in rice, posing yield losses as well as serious health risks. Selenium (Se), a trace element, is a known antagonist of As toxicity. In present study, RNA seq. and proteome profiling, along with morphological analyses were performed to explore molecular cross-talk involved in Se mediated As stress amelioration. The repair of As induced structural deformities involving disintegration of cell wall and membranes were observed upon Se supplementation. The expression of As transporter genes viz., NIP1;1, NIP2;1, ABCG5, NRAMP1, NRAMP5, TIP2;2 as well as sulfate transporters, SULTR3;1 and SULTR3;6, were higher in As + Se compared to As alone exposure, which resulted in reduced As accumulation and toxicity. The higher expression of regulatory elements like AUX/IAA, WRKY and MYB TFs during As + Se exposure was also observed. The up-regulation of GST, PRX and GRX during As + Se exposure confirmed the amelioration of As induced oxidative stress. The abundance of proteins involved in photosynthesis, energy metabolism, transport, signaling and ROS homeostasis were found higher in As + Se than in As alone exposure. Overall, present study identified Se responsive pathways, genes and proteins involved to cope-up with As toxicity in rice.

Elimination of Bean yellow mosaic virus from infected cormels of three cultivars of gladiolus using thermo-, electro- and chemotherapy.

Bean yellow mosaic virus (BYMV) is a prevalent virus and major threat to gladiolus cultivation the world over. In the gladiolus repository at CSIR-NBRI, Lucknow, several plants (82-88%) of three economically important cultivars were found infected by BYMV showing severe mosaic and stripe symptoms. Affected plants exhibit diminished quality and quantity of florets and corms, thus reducing their value. Attempts were made to eliminate BYMV from the infected gladiolus cormel explants in vitro through thermotherapy (37 °C for 30 days), chemotherapy (30 mg/L ribavirin for 30 days), and electrotherapy (30 mA for 20 min), either alone and in different combinations. The in vitro regenerated plants were free from BYMV infection when checked by RT-PCR using BYMV-specific primers. The combination of electro- and chemotherapies has given the best response as compared to other treatments. Among the individual therapies, electrotherapy (30 mA/20 min) was found to be the best for and production of BYMV-free gladiolus plants (44-46%) with moderate regeneration efficiency (54-58%) followed by chemotherapy and thermotherapy. However, the cormels obtained from a combination of electro- and chemotherapy treatment (30 mA/20 min + 30 mg/L) has given highest virus free (46-52%) and highest therapy efficiency indices (56%) as compared to other treatments. Further, these cormels showed better developed root systems and produced more cormels which were larger in size as compared to the other treatments and control when grown in tissue culture media.

Ectopic expression of amaranth seed storage albumin modulates photoassimilate transport and nutrient acquisition in sweetpotato.

Storage proteins in plants, because of high nutrient value, have been a subject of intensive investigation. These proteins are synthesized de novo in the cytoplasm and transported to the storage organelles where they serve as reservoir of energy and supplement of nitrogen during rapid growth and development. Sweetpotato is the seventh most important food crop worldwide, and has a significant contribution to the source of nutrition, albeit with low protein content. To determine the behaviour of seed storage proteins in non-native system, a seed albumin, AmA1, was overexpressed in sweetpotato with an additional aim of improving nutritional quality of tuber proteins. Introduction of AmA1 imparted an increase in protein and amino acid contents as well as the phytophenols. The proteometabolomics analysis revealed a rebalancing of the proteome, with no significant effects on the global metabolome profile of the transgenic tubers. Additionally, the slower degradation of starch and cellulose in transgenic tubers, led to increased post-harvest durability. Present study provides a new insight into the role of a seed storage protein in the modulation of photoassimilate movement and nutrient acquisition.

Andrographis paniculata transcriptome provides molecular insights into tissue-specific accumulation of medicinal diterpenes.

BACKGROUND: Kalmegh (Andrographis paniculata) has been widely exploited in traditional medicine for the treatment of infectious diseases and health disorders. Ent-labdane-related diterpene (ent-LRD) specialized (i.e., secondary) metabolites of kalmegh such as andrographolide, neoandrographolide and 14-deoxy-11,12-didehydroandrographolide, are known for variety of pharmacological activities. However, due to the lack of genomic and transcriptomic information, underlying molecular basis of ent-LRDs biosynthesis has remained largely unknown. To identify candidate genes of the ent-LRD biosynthetic pathway, we performed comparative transcriptome analysis using leaf and root tissues that differentially accumulate ent-LRDs. RESULTS: De novo assembly of Illumina HiSeq2000 platform-generated paired-end sequencing reads resulted into 69,011 leaf and 64,244 root transcripts which were assembled into a total of 84,628 unique transcripts. Annotation of these transcripts to the Uniprot, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Carbohydrate-Active Enzymes (CAZy) databases identified candidate transcripts of the ent-LRD biosynthetic pathway. These included transcripts that encode enzymes of the plastidial 2C-methyl-D-erythritol-4-phosphate pathway which provides C5 isoprenoid precursors for the ent-LRDs biosynthesis, geranylgeranyl diphosphate synthase, class II diterpene synthase (diTPS), cytochrome P450 monooxygenase and glycosyltransferase. Three class II diTPSs (ApCPS1, ApCPS2 and ApCPS3) that showed distinct tissue-specific expression profiles and are phylogenetically related to the dicotyledon ent-copalyl diphosphate synthases, are identified. ApCPS1, ApCPS2 and ApCPS3 encode for 832-, 817- and 797- amino acids proteins of 55-63 % identity, respectively. Spatio-temporal patterns of transcripts and ent-LRDs accumulation are consistent with the involvement of ApCPS1 in general (i.e., primary) metabolism for the biosynthesis of phytohormone gibberellin, ApCPS2 in leaf specialized ent-LRDs biosynthesis and ApCPS3 in root diterpene biosynthesis. Moreover, simple sequence repeats (SSRs) that might assist in genotyping and developing specific chemotypes were identified in transcripts of the specialized metabolic pathways, including ent-LRDs. CONCLUSIONS: Comparative analysis of root and leaf transcriptomes disclosed novel genes of the ent-LRD biosynthetic pathway, including three class II diTPSs that showed discrete spatio-temporal expression patterns; thus, suggesting their participation into distinct diterpene metabolic pathways of kalmegh. Overall, these results will be useful in understanding molecular basis of the medicinal ent-LRDs biosynthesis and developing breeding strategies for improving their yields.

Comparative proteomics reveals a role for seed storage protein AmA1 in cellular growth, development, and nutrient accumulation.

Seed storage proteins are known to be utilized as carbon and nitrogen source for growing seedlings and thus are considered as potential candidates for nutritional improvement. However, their precise function remains unknown. We have earlier shown that ectopic expression of a seed storage protein, AmA1, leads to increase in protein besides high tuber yield in potato. To elucidate the AmA1-regulated molecular mechanism affecting increased protein synthesis, reserve accumulation, and enhanced growth, a comparative proteomics approach has been applied to tuber life-cycle between wild-type and AmA1 potato. The differential display of proteomes revealed 150 AmA1-responsive protein spots (ARPs) that change their intensities more than 2.5-fold. The LC-ESI-MS/MS analyses led to the identification of 80 ARPs presumably associated with cell differentiation, regulating diverse functions, viz., protein biogenesis and storage, bioenergy and metabolism, and cell signaling. Metabolome study indicated up-regulation of amino acids paralleling the proteomics analysis. To validate this, we focused our attention on anatomical study that showed differences in cell size in the cortex, premedullary zone and pith of the tuber, coinciding with AmA1 expression and localization. Further, we interrogated the proteome data using one-way analysis of variance, cluster, and partial correlation analysis that identified two significant protein modules and six small correlation groups centered around isoforms of cysteine protease inhibitor, actin, heat shock cognate protein 83 and 14-3-3, pointing toward AmA1-regulated overlapping processes of protein enhancement and cell growth perhaps through a common mechanism of function. A model network was constructed using the protein data sets, which aim to show how target proteins might work in coordinated fashion and attribute to increased protein synthesis and storage reserve accumulation in AmA1 tubers on one hand and organ development on the other.

Next-generation protein-rich potato expressing the seed protein gene AmA1 is a result of proteome rebalancing in transgenic tuber.

Protein deficiency is the most crucial factor that affects physical growth and development and that increases morbidity and mortality especially in developing countries. Efforts have been made to improve protein quality and quantity in crop plants but with limited success. Here, we report the development of transgenic potatoes with enhanced nutritive value by tuber-specific expression of a seed protein, AmA1 (Amaranth Albumin 1), in seven genotypic backgrounds suitable for cultivation in different agro-climatic regions. Analyses of the transgenic tubers revealed up to 60% increase in total protein content. In addition, the concentrations of several essential amino acids were increased significantly in transgenic tubers, which are otherwise limited in potato. Moreover, the transgenics also exhibited enhanced photosynthetic activity with a concomitant increase in total biomass. These results are striking because this genetic manipulation also resulted in a moderate increase in tuber yield. The comparative protein profiling suggests that the proteome rebalancing might cause increased protein content in transgenic tubers. Furthermore, the data on field performance and safety evaluation indicate that the transgenic potatoes are suitable for commercial cultivation. In vitro and in vivo studies on experimental animals demonstrate that the transgenic tubers are also safe for human consumption. Altogether, these results emphasize that the expression of AmA1 is a potential strategy for the nutritional improvement of food crops.

Comparative proteomics of tuber induction, development and maturation reveal the complexity of tuberization process in potato (Solanum tuberosum L.).

Tuberization in potato ( Solanum tuberosum L.) is a developmental process that serves a double function, as a storage organ and as a vegetative propagation system. It is a multistep, complex process and the underlying mechanisms governing these overlapping steps are not fully understood. To understand the molecular basis of tuberization in potato, a comparative proteomic approach has been applied to monitor differentially expressed proteins at different development stages using two-dimensional gel electrophoresis (2-DE). The differentially displayed proteomes revealed 219 protein spots that change their intensities more than 2.5-fold. The LC-ES-MS/MS analyses led to the identification of 97 differentially regulated proteins that include predicted and novel tuber-specific proteins. Nonhierarchical clustering revealed coexpression patterns of functionally similar proteins. The expression of reactive oxygen species catabolizing enzymes, viz., superoxide dismutase, ascorbate peroxidase and catalase, were induced by more than 2-fold indicating their possible role during the developmental transition from stolons into tubers. We demonstrate that nearly 100 proteins, some presumably associated with tuber cell differentiation, regulate diverse functions like protein biogenesis and storage, bioenergy and metabolism, and cell defense and rescue impinge on the complexity of tuber development in potato.