Systems biology of seeds: decoding the secret of biochemical seed factories for nutritional security.

Seeds serve as biochemical factories of nutrition, processing, bio-energy and storage related important bio-molecules and act as a delivery system to transmit the genetic information to the next generation. The research pertaining towards delineating the complex system of regulation of genes and pathways related to seed biology and nutrient partitioning is still under infancy. To understand these, it is important to know the genes and pathway(s) involved in the homeostasis of bio-molecules. In recent past with the advent and advancement of modern tools of genomics and genetic engineering, multi-layered 'omics' approaches and high-throughput platforms are being used to discern the genes and proteins involved in various metabolic, and signaling pathways and their regulations for understanding the molecular genetics of biosynthesis and homeostasis of bio-molecules. This can be possible by exploring systems biology approaches via the integration of omics data for understanding the intricacy of seed development and nutrient partitioning. These information can be exploited for the improvement of biologically important chemicals for large-scale production of nutrients and nutraceuticals through pathway engineering and biotechnology. This review article thus describes different omics tools and other branches that are merged to build the most attractive area of research towards establishing the seeds as biochemical factories for human health and nutrition.

Containment evaluation, cold tolerance and toxicity analysis in Osmotin transgenic tomato (Solanum lycopersicum L. cv. Pusa Ruby).

The present study reports engineered cold tolerance and toxicity analysis in genetically modified tomato (Solanum lycopersicum L. cv. Pusa Ruby) developed through constitutive over expression of Nicotiana tabacum Osmotin gene. Rate of seed germination, seedling establishment and growth remained unaffected in the transgenic tomato in response to a low temperature (15 °C) treatment, but were significantly (P ≤ 0.05) reduced in the wild type. At reproductive stage, the wild type plants failed to recover at the low temperature (4.0 °C) treatment for 10 days but the transgenic plants survived successfully without any leaf senescence or other visible chilling injury symptoms. The quantitative transcript expression analysis confirmed up regulation of the transgene by 55% in the transgenic plants on cold treatment for 2 h whereas, the transcripts were not detected in the wild type. Containment evaluation under normal environmental conditions revealed similar morphology in both the transgenic and wild type tomato plants however an average fruit yield was higher in the transgenic plants (725.91 ± 39.27 g) than the wild type (679.84 ± 28.80 g). The composition of mature fruits in terms of element content was at par in both the transgenic and wild type except significantly higher Ca and Mg contents in the transgenic fruits than that of the wild type. Further, acute and sub-acute toxicity tests conducted in the adult female Wister rats revealed no mortality or significant changes in general and psychological behaviour, at par food intake and body weight and, normal biochemical, and hematological parameters for animals fed with the wild type or transgenic tomato fruits as compared to the control group, confirming its safety for animal consumption.

Bioactive potential of Indian stinging plants leaf extract against pathogenic fungi.

Background & methods We investigated the in vitro antioxidant and antifungal activity by agar disc diffusion assay of leaf extract of some stinging plants namely, Urtica dioica L., Tragia involucrate L., Carduus nutans L. and Mucuna pruriens (L.) DC., against pathogenic fungi causing infections/diseases. Results M. pruriens (Disc 4), T. involucrate (Disc 2), U. dioica (Disc 1) showed significant antifungal activity against all tested pathogens, while C. nutans (Disc 3) showed intermediate activity against only Chaetomium globosum (Cg). The leaf extract of M. Pruriens showed maximum total phenol content (~1004 µg g-1 dry wt) followed by T. involucrate, C. nutans and U. dioica. However, the ascorbate was observed highest in T. involucrate (~10.3 µg g-1 dry wt) followed by M. pruriens (~9.2 µg g-1 dry wt) but the difference was not significant (p ≤ 0.05). Likewise, M. pruriens showed maximum anthocyanin content (~0.3 µg g-1 dry wt). The activity of antioxidant enzymes revealed that M. Pruriens showed maximum ascorbate peroxidase (APX) activity, while the highest guaiacol peroxidase (GPX) and catalase (CAT) activities were observed in C. nutans and U. dioica, respectively. Conclusions M. Pruriens showed potential in vitro antioxidant and antifungal activity against studied pathogens that may be used for ethno-pharmacological uses.

Identification and molecular characterization of Dof transcription factor gene family preferentially expressed in developing spikes of Eleusine coracana L.

We report 48 putative DNA binding with one finger (Dof) TF genes from genome and transcriptome data of finger millet (Eleusine coracana L.; FM), involved in plant developmental process. To characterize seed-specific Dof genes, transcript profiles of 32 EcDof identified from transcriptome data of developing spikes of FM genotypes were further analyzed in different tissues (root, stem, and leaf) and developmental stages of spikes (S1, S2, S3, and S4) in two FM genotypes [GE1437 (low protein genotype; LPG) and GE3885 (high protein genotype; HPG)]. More than 50% of identified EcDof genes showed expression during seed development processes. Among these, seven genes (EcDof 3, EcDof 5, EcDof 15, EcDof 18, EcDof 22, EcDof 23, and EcDof 31) expressed maximally at specific stages of seed development. Fourteen EcDof genes showed that differential transcript accumulation in vegetative tissue as well as in developing spikes suggests involvement during seed filling and also throughout the plant development. In addition, three EcDof genes (EcDof 9, EcDof 25, and EcDof 28) expressed preferentially at root and stem tissue. The 3D structural prediction of EcDof proteins showed variability in structural attributes. Molecular docking results showed strong binding affinity for seed-specific EcDof-EcO2 with α-prolamine promoters. The identified and characterized EcDof genes will help to dissect the roles of FM seed-specific Dof genes.

Finger Millet: A "Certain" Crop for an "Uncertain" Future and a Solution to Food Insecurity and Hidden Hunger under Stressful Environments.

Crop growth and productivity has largely been vulnerable to various abiotic and biotic stresses that are only set to be compounded due to global climate change. Therefore developing improved varieties and designing newer approaches for crop improvement against stress tolerance have become a priority now-a-days. However, most of the crop improvement strategies are directed toward staple cereals such as rice, wheat, maize etc., whereas attention on minor cereals such as finger millet [Eleusine coracana (L.) Gaertn.] lags far behind. It is an important staple in several semi-arid and tropical regions of the world with excellent nutraceutical properties as well as ensuring food security in these areas even during harsh environment. This review highlights the importance of finger millet as a model nutraceutical crop. Progress and prospects in genetic manipulation for the development of abiotic and biotic stress tolerant varieties is also discussed. Although limited studies have been conducted for genetic improvement of finger millets, its nutritional significance in providing minerals, calories and protein makes it an ideal model for nutrition-agriculture research. Therefore, improved genetic manipulation of finger millets for resistance to both abiotic and biotic stresses, as well as for enhancing nutrient content will be very effective in millet improvement. Key message: Apart from the excellent nutraceutical value of finger millet, its ability to tolerate various abiotic stresses and resist pathogens make it an excellent model for exploring vast genetic and genomic potential of this crop, which provide us a wide choice for developing strategies for making climate resilient staple crops.

Stinging plants: as future bio-weapon.

In the present opinion paper, we have been introducing for the first time the stinging plants and/or their biological toxins as novel bio-threat agents that may be used for the development of bio-weapons for self-defence purpose. The selected studied stinging plants are having dual role as nutraceutical and ethno-pharmacological uses apart from their less explored stinging property. However, future detailed work is required for identification and characterization of the precise stinging chemical components that will be used for the formulation of novel bio-warfare agents for self-defence purpose.

Systems Biology for Smart Crops and Agricultural Innovation: Filling the Gaps between Genotype and Phenotype for Complex Traits Linked with Robust Agricultural Productivity and Sustainability.

In recent years, rapid developments in several omics platforms and next generation sequencing technology have generated a huge amount of biological data about plants. Systems biology aims to develop and use well-organized and efficient algorithms, data structure, visualization, and communication tools for the integration of these biological data with the goal of computational modeling and simulation. It studies crop plant systems by systematically perturbing them, checking the gene, protein, and informational pathway responses; integrating these data; and finally, formulating mathematical models that describe the structure of system and its response to individual perturbations. Consequently, systems biology approaches, such as integrative and predictive ones, hold immense potential in understanding of molecular mechanism of agriculturally important complex traits linked to agricultural productivity. This has led to identification of some key genes and proteins involved in networks of pathways involved in input use efficiency, biotic and abiotic stress resistance, photosynthesis efficiency, root, stem and leaf architecture, and nutrient mobilization. The developments in the above fields have made it possible to design smart crops with superior agronomic traits through genetic manipulation of key candidate genes.

Overexpression of NAC gene from Lepidium latifolium L. enhances biomass, shortens life cycle and induces cold stress tolerance in tobacco: potential for engineering fourth generation biofuel crops.

We report elevated biomass and altered growth characteristics of tobacco plants up on transformation with a NAC (NAM, ATAF1/2,CUC2) gene (GenBank Accession FJ754254) isolated from Lepidium latifolium L. (LlaNAC). Transgenic plants showed significant differences in fresh weight, midrib length of longest leaf, leaf area, height of the plant, root and shoot weights, etc. during vegetative phase. On 100th day after sowing (DAS), plants of transgenic lines were 2-3 times taller than the wild type plants, though no significant difference was recorded in moisture contents of any of the plant tissues. Over-expression of NAC gene up to 2,000 fold was recorded in leaves of transgenic plants on 100th DAS. Interestingly, transgenic plants showed significantly shortened (P(t) = 0.02-0.04) life cycle, as they showed a completely altered growth behaviour. Transgenic plants entered reproductive phase earlier by 60 days, with lines NC2 and NC7b entering first, followed by line NC10. However, the time period spent in the reproductive phase by the plant was nearly twice in case of transgenic lines NC2, NC7b and NC10, as compared to the wild type plants. Despite that, these lines completed their life cycle in 45-60 days lesser than the time taken by wild-type tobacco plants. No difference was recorded in fruit and seed yield of transgenic or wild type plants. To the best of our knowledge, this is the first report on over-expression of NAC gene causing altered growth and biomass patterns. We expect this study to become an important reference towards future engineering of plants for fuel and fodder purposes.

Fluctuation of Dof1/Dof2 expression ratio under the influence of varying nitrogen and light conditions: involvement in differential regulation of nitrogen metabolism in two genotypes of finger millet (Eleusine coracana L.).

In order to gain insights into the mechanism of high nitrogen use efficiency (NUE) of finger millet (FM) the role of Dof2 transcription factor (TF), which is a repressor of genes involved in C/N metabolism was investigated. The partial cDNA fragment of EcDof2 (912-bp; GenBank acc. no. KF261117) was isolated and characterized from finger millet (FM) that showed 63% and 58% homology with Dof2 of Zea mays at nucleotide and protein level, respectively. Its expression studies were carried out along with the activator EcDof1 in two genotypes (GE3885, high protein genotype (HPG); GE1437, low protein genotype (LPG)) of FM differing in grain protein contents (13.8% and 6.2%) showed that EcDof2 is expressed in both shoot and root tissues with significantly (p≤0.05) higher expression in the roots. The diurnal expression of both EcDof1 and EcDof2 in shoots was differential having different time of peak expression indicating a differential response to diurnal condition. Under continuous dark conditions, expression of EcDof1 and EcDof2 oscillated in both the genotypes whereas on illumination, the fold expression of EcDof1 was higher as compared to EcDof2. Under increasing nitrate concentration, EcDof2 expression increases in roots and shoots of LPG while it remains unchanged in HPG. However, the EcDof1 expression was found to increase in both genotypes. Further, time kinetics studies under single nitrate concentration revealed that EcDof2 was repressed in the roots of both genotypes whereas EcDof1 oscillated with time. The EcDof1/EcDof2 ratio measured showed differential response under different light and nitrogen conditions. It was higher in the roots of HPG indicating higher activation of genes involved in N uptake and assimilation resulting in high grain protein accumulation. The results indicate that both light and nitrogen concentration influence Dof1 and Dof2 expression and suggests a complex pattern of regulation of genes influenced by these plant specific TFs. In nutshell, the Dof1/Dof2 ratio can serve as an index for measuring the N responsiveness and NUE of crops and can be further validated by Dof2 knock down approach.

RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.).

Curcin, a type I ribosomal inhibiting protein-RIP, encoded by curcin precursor gene, is a phytotoxin present in Jatropha (Jatropha curcas L.). Here, we report designing of RNAi construct for the curcin precursor gene and further its genetic transformation of Jatropha to reduce its transcript expression. Curcin precursor gene was first cloned from Jatropha strain DARL-2 and part of the gene sequence was cloned in sense and antisense orientation separated by an intron sequence in plant expression binary vector pRI101 AN. The construction of the RNAi vector was confirmed by double digestion and nucleotide sequencing. The vector was then mobilized into Agrobacterium tumefaciens strain GV 3101 and used for tissue culture independent in planta transformation protocol optimized for Jatropha. Germinating seeds were injured with a needle before infection with Agrobacterium and then transferred to sterilized sand medium. The seedlings were grown for 90 days and genomic DNA was isolated from leaves for transgenic confirmation based on real time PCR with NPT II specific dual labeled probe. Result of the transgenic confirmation analysis revealed presence of the gene silencing construct in ten out of 30 tested seedlings. Further, quantitative transcript expression analysis of the curcin precursor gene revealed reduction in the transcript abundance by more than 98% to undetectable level. The transgenic plants are being grown in containment for further studies on reduction in curcin protein content in Jatropha seeds.

Analysis of Jatropha curcas transcriptome for oil enhancement and genic markers.

Oil-rich seeds of Jatropha curcas are being focussed as a source of bio-diesel. However, prior to its industrial use, a lot of crop improvement efforts are required in Jatropha. Availability of a large number of EST sequences of Jatropha in public domain allow identification of candidate genes for several agronomic characters including oil content in seeds. Here, we have analysed 42,477 ESTs of Jatropha spanning 22.9 Mbp for microsatellites and fatty acid metabolism related sequences. Unigene sequences were built using CAP 3 programme resulted in 12,358 contigs and 5,730 singlets. Nearly, 8 % unigenes showed presence of microsatellites, slightly over-represented compared to their occurrence in ESTs. Most of the microsatellites were either di- or tri-nucleotide repeats, while other categories of tetra-, penta- and hexa-nucleotide repeats together constituted ~4 % of total microsatellites. Assessment of functional relevance of unigenes was carried out using Blast2GO using its default settings. The overall sequence similarity level against sequences in 'nr' database was >80 %. A total of 931 sequences that participated in any of the pathways related to fatty acid or lipid metabolism were found at GO level 6. Among these, GO terms "Fatty acid metabolic process" and "Fatty acid biosynthetic process" were most over-represented. Overall, our work has due relevance in identifying molecular markers for the candidate genes for oil content in Jatropha seeds, and will prove to be an important reference for further studies for identification of trait specific markers in Jatropha.

Semi-quantitative analysis of transcript accumulation in response to drought stress by Lepidium latifolium seedlings.

Cross-amplification of five Arabidopsis abiotic stress-responsive genes (AtPAP, ZFAN, Vn, LC4 and SNS) in Lepidium has been documented in plants raised out of seeds pre-treated with potassium nitrate (KNO 3) for assessment of enhanced drought stress tolerance. cDNA was synthesized from Lepidium plants pre-treated with KNO 3 (0.1% and 0.3%) and exposed to drought conditions (5% and 15% PEG) at seedling stage for 30 d. Transcript accumulation of all the five genes were found suppressed in set of seedlings, which were pre-treated with 0.1% KNO 3 and were exposed to 15% PEG for 30 d. The present study establishes that different pre-treatments may further enhance the survivability of Lepidium plants under conditions of drought stress to different degrees.

Cloning and characterization of GPAT gene from Lepidium latifolium L.: a step towards translational research in agri-genomics for food and fuel.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes first and the rate limiting step in glycerolipid synthesis pathway, which in turn contribute to stabilization of plasma membrane structure and oil lipid synthesis in plant cells. Here, we report cloning and characterization of GPAT gene from Lepidium latifolium (LlaGPAT). The cDNA sequence (1,615 bp) of LlaGPAT gene consisted of 1,113 bp ORF encoding a protein of 370 aa residues, with deduced mass of 41.2 kDa and four acyltransferase (AT) motifs having role in catalysis and in glycerol-3-phosphate binding. Southern blot analysis suggested presence of a single copy of the gene in the genome. Tissue specific expression of the gene was seen more abundantly in aerial parts, compared to the roots. Quantitative real-time PCR indicated down-regulation of the gene by cold (4 °C), drought (PEG6000), salt (300 mM NaCl) and ABA (100 µM) treatments. Considering the vitality of the function of encoded enzyme, LlaGPAT can be considered a potential candidate gene for genetic engineering of oil yields and abiotic stress management in food as well as fuel crops.

DRE-binding transcription factor gene (LlaDREB1b) is regulated by various abiotic stresses in Lepidium latifolium L.

We have isolated and in silico characterized a cold-induced LlaDREB1b encoding a putative DRE-binding transcription factor from Lepidium latifolium. Its cDNA (JN214345) sequence (998 bp) consisted of a 642 bp ORF, 168 and 188 bp of 5' and 3' UTR regions, respectively, encoding a protein of 213 aa with deduced molecular mass 23.85 kDa and pI of 4.63. In silico and phylogenetic analysis further suggested that the protein showed features of a typical member of the AP2/EREBP family of DNA-binding proteins. Southern blot analysis indicated that multiple copies of the gene could be present in the genome. Its transcripts were abundant in leaves, petiole and stem, but scarce in roots and could strongly be induced by cold treatment (4 °C), weakly by drought and salt stress, and did not respond to ABA treatment. Thus, LlaDREB1b is a potential candidate for abiotic stress-tolerance engineering in crop plants upon its further functional validation.

Isolation and characterization of cold responsive NAC gene from Lepidium latifolium.

Cold stress is one of the major limiting factor in crop productivity. Plants growing in colder regions acclimatize to severe conditions owing to the presence of 'cold stress tolerant genes'. Isolation and functional characterization of these genes are important before their exploitation in modern agricultural practices. Here, we have cloned full length NAC gene (1,388 bp) from Lepidium latifolium (LlaNAC). This gene belongs to NAP sub-group which also includes ANAC056 of Arabidopsis thaliana, nearest relative of LlaNAC. Upstream analysis and microarray data analysis of ANAC056 suggested that LlaNAC might also be ABA-regulated. However, quantitative transcript expression analysis revealed that LlaNAC transcript upregulated by cold stress and downregulated in response to varying concentrations of abscisic acid, salicylic acid, calcium chloride and ethylene. There is also a possibility that the gene may be getting regulated by a pathway whose components are still unknown. Any further investigations to understand the mechanism of regulation of LlaNAC gene expression are likely to find immense importance in plant biotechnology and crop improvement.

Chlorophyllase in Piper betle L. has a role in chlorophyll homeostasis and senescence dependent chlorophyll breakdown.

Total chlorophyll content and chlorophyllase (chlorophyll-chlorophyllido hydrolase EC 3.1.1.14) activity in fresh leaves of Piper betle L. landrace KS was, respectively, twofold higher and eight fold lower than KV, showing negative correlation between chlorophyll and chlorophyllase activity. Specific chlorophyllase activity was nearly eightfold more in KV than KS. ORF of 918 nt was found in cloned putative chlorophyllase cDNAs from KV and KS. The gene was present as single copy in both the landraces. The encoded polypeptide of 306 amino acids differed only at two positions between the KV and KS; 203 (cysteine to tyrosine) and 301 (glutamine to glycine). Difference in chlorophyllase gene expression between KV and KS was evident in fresh and excised leaves. Up regulation of chlorophyllase gene by ABA and down regulation by BAP was observed in both the landraces; however, there was quantitative difference between KV and KS. Data suggests that chlorophyllase in P. betle is involved in chlorophyll homeostasis and chlorophyll loss during post harvest senescence.

Female plants of Hippophae salicifolia D. Don are more responsive to cold stress than male plants.

Hippophae salicifolia D. Don (Seabuckthorn) grows in stressful environment of high altitude under conditions of low temperature and low availability of water. We have studied gender based differences in physiochemical response to cold stress in male and female plants of Seabuckthorn. After 24 h of cold stress about 32 and 66 % higher electrolyte leakage (EL) was recorded in male and female plants respectively. Relative water content (RWC) at the end of 24 h stress was higher in male plants (~64 %) compared to female plants (~60 %). Proline content in leaf samples of cold stressed male and female plants also increased upon cold stress. After 24 h about 2.7 fold higher amount of proline was assessed in male and female in comparison to control plants. Similarly, about two fold increase in the specific activities of superoxide dismutase (SOD) and catalase was also observed upon cold stress in male and female plants. These findings have important inferences for community of molecular biologists exploring seabuckthorn genome for agronomically important genes.

Isolation and characterization of ripening related pectin methylesterase inhibitor gene from banana fruit.

Identification of ethylene-regulated and ripening-related genes from banana (Musa acuminata Var. Harichaal) fruits using DDRT-PCR led to the isolation of differentially expressed partial cDNA of pectin methylesterase inhibitor (MaPMEI) gene. Its full-length cDNA sequence consisted of a 567 bp ORF, encoding a protein of 189 aa with deduced molecular mass 19.6 kDa. Expression pattern of MaPMEI gene revealed that upon ethylene treatment, this gene is up-regulated initially giving maximum expression in post-climacteric stage then decreases slightly in later stages of ripening. 1-MCP, a known ethylene perception inhibitor, inhibits both fruit ripening as well as the transcript level of this gene. Also, the transcripts of MaPMEI gene were not detected during the short time ethylene treatment suggesting this gene appears to be not directly induced by ethylene. Interestingly, MaPMEI gene showed fruit specific expression that indicates its possible role in the regulations of PMEs in fruits. In silico analysis revealed a predicted signal peptide sequence necessary for localization of MaPMEI in the cell wall. Furthermore, the four Cys residues involved in disulfide bridges are conserved in MaPMEI similar to other PMEIs and invertase inhibitors. Phylogenetic analysis further suggests that the MaPMEI identified in this study is more closely related to PMEIs than to invertase inhibitors.

Role of chlorophyllase in chlorophyll homeostasis and post-harvest breakdown in Piper betle L. leaf.

Piper betle L., a dioecious shade-loving perennial climber is one of the important Pan-Asiatic plants. More than hundred landraces having marked variation in leaf chlorophyll (Chl) content are in cultivation in India. In this study, role of chlorophyllase (Chlase) in Chl homeostasis and post-harvest breakdown was investigated in two contrasting P. betle landraces Kapoori Vellaikodi (KV) with light green and Khasi Shillong (KS) with dark green leaves. The two landraces showed negative correlation between Chl content and Chlase activity in fresh as well as stored leaves. Accumulation of chlorophyllide a (Chlid a) was correlated with the level of Chlase activity, which was higher in KV than KS. The overall response of abscisic acid (ABA) and benzylaminopurine (BAP) was similar in KV and KS, however, the time-course was different. ABA-induced Chl loss was accompanied by rise in Chlase activity in KV and KS and the delay in Chl loss by BAP was accompanied by reduction in Chlase activity. While there were significant differences in Chlase activity in KV and KS, only minor differences were observed in the enzyme properties like pH and temperature optima, Km and Vmax. No landrace-related differences were observed on the effect of metal ions and functional group reagents/amino acid effectors on Chlase activity. These results showed that despite significant differences in Chl content and Chlase activity between landraces KV and KS, the properties of Chlase were similar. The findings show that in P. betle Chlase is involved in Chl homeostasis and also in Chl degradation during post-harvest storage and responds to hormonal regulations. These findings might be useful in predicting the stability of Chl during post-harvest storage and also the shelf-life in other P. betle landraces.

Breaking seed dormancy in Hippophae salicifolia, a high value medicinal plant.

Seed dormancy is an important limiting factor in exploitation of an economically important species to its fullest. Hippophae salicifolia D. Don (seabuckthorn), a rich source of medicinal metabolites shows both exogenous and endogenous dormancy. Evidently, we recorded a high seed viability (94 %) but poor germination (22 %) of untreated seeds. We applied different pre-sowing seed priming treatments including NaCl (50, 100, 200, 500 mM), KNO3 (0.1, 0.2, 0.3 %), Thiourea (1, 2, 3 %), GA3 (100, 250, 500 mg/L), Sulphuric acid (98 %) and cold (4 °C) and warm water (65 °C) stratifications to explore improvements in its germination percentage, if any. We found KNO3 (0.1 %) and Thiourea (1 %) treatments to be superior to other methods for enhancement of mean seed germination percentage of H. salicifolia. Considering the practical applicability and cost effectiveness, these treatments can be applied to overcome seed dormancy and recommended for mass multiplication through seeds of H. salicifolia.