Comparative physiological, antioxidant and proteomic investigation reveal robust response to cold stress in Digitalis purpurea L.

BACKGROUND OF THE STUDY: Digitalis purpurea (L) is an important medicinal plant growing at Alpine region of Himalayas and withstands low temperatures and harsh climatic conditions existing at high altitude. It serves as an ideal plant system to decipher the tolerance to cold stress (CS) in plants from high altitudes. METHODS AND RESULTS: To understand the complexity of plant response to CS, we performed a comparative physiological and biochemical study complemented with proteomics in one-month-old D. purpurea grown at 25 °C (control) and 4 °C (CS). We observed an enhanced accumulation of different osmo-protectants (glycine betaine, soluble sugar and proline) and higher transcription (mRNA levels) of various antioxidant enzymes with an increased antioxidant enzyme activity in D. purpurea when exposed to CS. Furthermore, higher concentrations of non-enzymatic antioxidants (flavonoids, phenolics) was also associated with the response to CS. Differential proteomic analysis revealed the role of various proteins primarily involved in redox reactions, protein stabilization, quinone and sterol metabolism involved in CS response in D. purpurea.. CONCLUSION: Our results provide a framework for better understanding the physiological and molecular mechanism of CS response in D. purpurea at high altitudes.

Comparative protein analysis of two maize genotypes with contrasting tolerance to low temperature.

BACKGROUND: Low temperature (LT) stress is one of the major environmental stress factors affecting the growth and yield of maize (Zea mays L.). Hence, it is important to unravel the molecular mechanisms behind LT stress tolerance to improve molecular breeding in LT tolerant genotypes. In the present study, two maize genotypes viz. Gurez local from Kashmir Himalaya and tropical grown GM6, were dissected for their LT stress response in terms of accumulation of differentially regulated proteins (DRPs). Leaf proteome analysis at three-leaf stage of maize seedlings subjected to LT stress of 6 °C for a total of 12 h duration was performed using two dimensional gel electrophoresis (2D-PAGE) followed by subsequent identification of the proteins involved. RESULTS: After MALDI-TOF (Matrix-assisted laser desorption/ionization-time of flight) and bioinformatics analysis, 19 proteins were successfully identified in Gurez local, while as 10 proteins were found to get successful identification in GM6. The interesting observations from the present investigation is the identification of three novel proteins viz. threonine dehydratase biosynthetic chloroplastic, thylakoidal processing peptidase 1 chloroplastic, and nodulin-like protein, whose role in abiotic stress tolerance, in general, and LT stress, in particular, has not been reported so far. It is important to highlight here that most of LT responsive proteins including the three novel proteins were identified from Gurez local only, owing to its exceptional LT tolerance. From the protein profiles, obtained in both genotypes immediately after LT stress perception, it was inferred that stress responsive protein accumulation and their expression fashion help the Gurez local in seedling establishment and withstand unfavorable conditions as compared to GM6. This was inferred from the findings of pathway enrichment analysis like regulation of seed growth, timing of floral transition, lipid glycosylation, and aspartate family amino acid catabolic processes, besides other key stress defense mechanisms. However, in GM6, metabolic pathways enriched were found to be involved in more general processes including cell cycle DNA replication and regulation of phenylpropanoid metabolism. Furthermore, majority of the qRT-PCR results of the selected proteins demonstrated positive correlation between protein levels and transcript abundance, thereby strengthening our findings. CONCLUSIONS: In conclusion, our findings reported majority of the identified proteins in Gurez local exhibiting up-regulated pattern under LT stress as compared to GM6. Furthermore, three novel proteins induced by LT stress were found in Gurez local, requiring further functional validation. Therefore, our results offer more insights for elucidating the molecular networks mediating LT stress tolerance in maize.

Proteomics for abiotic stresses in legumes: present status and future directions.

Legumes are the most important crop plants in agriculture, contributing 27% of the world's primary food production. However, productivity and production of Legumes is reduced due to increasing environmental stress. Hence, there is a pressing need to understand the molecular mechanism involved in stress response and legumes adaptation. Proteomics provides an important molecular approach to investigate proteins involved in stress response. Both the gel-based and gel-free-based techniques have significantly contributed to understanding the proteome regulatory network in leguminous plants. In the present review, we have discussed the role of different proteomic approaches (2-DE, 2 D-DIGE, ICAT, iTRAQ, etc.) in the identification of various stress-responsive proteins in important leguminous crops, including soybean, chickpea, cowpea, pigeon pea, groundnut, and common bean under variable abiotic stresses including heat, drought, salinity, waterlogging, frost, chilling and metal toxicity. The proteomic analysis has revealed that most of the identified differentially expressed proteins in legumes are involved in photosynthesis, carbohydrate metabolism, signal transduction, protein metabolism, defense, and stress adaptation. The proteomic approaches provide insights in understanding the molecular mechanism of stress tolerance in legumes and have resulted in the identification of candidate genes used for the genetic improvement of plants against various environmental stresses. Identifying novel proteins and determining their expression under different stress conditions provide the basis for effective engineering strategies to improve stress tolerance in crop plants through marker-assisted breeding.

Environmental stress tolerance in maize (Zea mays): role of polyamine metabolism.

Maize (Zea mays L.), a major multipurpose crop for food, feed and energy is extremely susceptible to environmental perturbations and setting off the major factors for limiting maize yield. Generally, plant yields are reduced and significantly lost to adverse environments and biotic strains. To ensure the safety of living cells under unfavourable circumstances, polyamines (PAs) play an important role in regulating the response under both abiotic and biotic stresses. It is the relative abundance of higher PAs (spermidine, Spd; spermine, Spm) vis-à-vis the diamine putrescine (Put) and PA catabolism that determines the stress tolerance in plants. Climate changes and increasing demands for production of maize have made it pressing to improve the stress tolerance strategies in this plant and it is imperative to understand the role of PAs in response to various environmental perturbations. Here, we critically review and summarise the recent literature on role of PAs in conferring stress tolerance in the golden crop. The responses in terms of PA accumulation, their mechanism of action and all the recent genetic manipulation studies carried out in PA metabolism pathway, ameliorating range of abiotic and biotic stresses have been discussed. As PA metabolism under stress conditions does not operate singly within cells and is always linked to other metabolic pathways in maize, its complex connections and role as a signalling molecule have also been discussed in this review.

Co-over expression of Ascorbate Glutathione pathway enzymes improve mercury tolerance in tomato.

The genetic modification of plants for the removal of inorganic pollutants from contaminated soil and water bodies is an emerging area for addressing environmental concerns. This approach is based on the ability of plants to take up and accumulate heavy metals, with efficiency being dependent on the underlying mechanisms of heavy metal accumulation and tolerance. A robust antioxidant pathway is determinantal for heavy metal uptake and accumulation and, therefore, in this study, we evaluated the transgenic tomato plants installed with Ascorbate Glutathione (ASA-GSH) pathway genes for uptake, accumulation, and response to mercury (Hg). We observed that ASA-GSH overexpressing lines were resilient to Hg stress as they displayed higher photosynthetic activity and increased photosynthetic gas exchange parameters with a concomitant decrease in ion leakage under Hg stress. Additionally, transgenic lines accumulated high osmolytes and showed enhanced activity of antioxidant enzymes. Moreover, the results of SEM and confocal microscopy confirmed least damage to plant tissue in ASA-GSH overexpressing lines compared to wild-type under Hg-stress which was further supported by Atomic absorption study that revealed a significant decline in Hg accumulation in the leaves of transgenic lines compared to wild-type under stress conditions. In conclusion, pyramiding of ASA-GSH pathway genes in tomato plants is an efficient approach for the development of Hg-resistant tomato plants and the reclamation of Hg-contaminated sites.

Nano-pollution: Why it should worry us.

Nanoparticles are immensely diverse both in terms of quality and sources of emission into the environment. Nowadays, nanotechnologies are developing and growing at a rapid pace without specific rules and regulations, leading to a severe effect on environment and affecting the labours in outdoor and indoor workplaces. The continue and enormous use of NPs for industrial and commercial purposes, has put a pressing need to think whether the increasing use of these NPs could overcome the severe environmental effects and unknown human health risks. Only a few studies have been carried out to assess the toxic effect of these NPs resulting from their direct or indirect exposure. There is in an increasing clamour to consider environmental implications of NPs and to monitor the outcome of NP during use in biological testing. There remain many open questions for consideration. An adequate research is required to determine the real toxic effect of these NPs on environment and human health. In this review, we have discussed the negative effects of NPs on environment and biosphere at large and the future research required.

Exogenous brassinosteroid and jasmonic acid improve drought tolerance in Brassica rapa L. genotypes by modulating osmolytes, antioxidants and photosynthetic system.

KEY MESSAGE: Exogenously supplied BR and JA help KS101 and KBS3 genotypes of Brassica rapa to alleviate drought stress by modifying osmolyte concentration, levels of antioxidant enzymes and photosynthetic system. Oilseed plants are susceptible to drought stress and a significant loss in yield has been reported during recent decades. Thus, it is imperative to understand the various underlying drought response mechanisms in Brassica oilseed plants to formulate the sustainable strategies to protect the crop yield under water-limiting conditions. Phytohormones play a key role in fine-tuning various regulatory mechanisms for drought stress adaptation in plants, and the present study explores the response of several physiological stress markers by exogenous supplementation of 24-epibrassinolide (EBL) and jasmonic acid (JA) on two genotypes of Brassica rapa, KS101 and KBS3 under drought stress conditions. The exogenous application of BR and JA, separately or in combination, significantly alleviated the drought stress by improving photosynthetic rate, photosynthetic pigments, stomatal conductance, transpiration rate and antioxidant defence. We observed that concentration of different osmolytes increased and membrane damage significantly reduced by the application of BR and JA. The overall activity of antioxidant enzymes POD, CAT, GR, APX and CAT elevated during all the treatments, be it stress alone or in combination with BR and JA, compared to the control. However, we observed that the BR was much better in mitigating the drought stress compared to JA. Thus, the present study suggests that BR and JA supplementation improves the performance of B. rapa on exposure to drought stress, which hints at the critical role of BR and JA in improving crop productivity in drought-prone areas.

Pyramiding ascorbate-glutathione pathway in Lycopersicum esculentum confers tolerance to drought and salinity stress.

KEY MESSAGE: Stacking Glutathione-Ascorbate pathway genes (PgSOD, PgAPX, PgGR, PgDHAR and PgMDHAR) under stress inducible promoter RD29A imparts significant tolerance to drought and salinity stress in Solanum lycopersicum. Although the exposure of plants to different environmental stresses results in overproduction of reactive oxygen species (ROS), many plants have developed some unique systems to alleviate the ROS production and mitigate its deleterious effect. One of the key pathways that gets activated in plants is ascorbate glutathione (AsA-GSH) pathway. To demonstrate the effect of this pathway in tomato, we developed the AsA-GSH overexpression lines by stacking the genes of the AsA-GSH pathway genes isolated from Pennisetum glaucoma (Pg) including PgSOD, PgAPX, PgGR, PgDHAR and PgMDHAR under stress inducible promoter RD29A. The overexpression lines have an improved germination and seedling growth with concomitant elevation in the survival rate. The exposure of transgenic seedlings to varying stress regiments exhibited escalation in the antioxidant enzyme activity and lesser membrane damage as reflected by decreased electrolytic leakage and little accumulation of malondialdehyde and H2O2. Furthermore, the transgenic lines accumulated high levels of osmoprotectants with increase in the relative water content. The increased photosynthetic activity and enhanced gaseous exchange parameters further confirmed the enhanced tolerance of AsA-GSH overexpression lines. We concluded that pyramiding of AsA-GSH pathway genes is an effective strategy for developing stress resistant crops.

Ectopic expression of a novel cold-resistance protein 1 from Brassica oleracea promotes tolerance to chilling stress in transgenic tomato.

Cold stress is considered as one of the major environmental factors that adversely affects the plant growth and distribution. Therefore, there arises an immediate need to cultivate effective strategies aimed at developing stress-tolerant crops that would boost the production and minimise the risks associated with cold stress. In this study, a novel cold-responsive protein1 (BoCRP1) isolated from Brassica oleracea was ectopically expressed in a cold susceptible tomato genotype Shalimar 1 and its function was investigated in response to chilling stress. BoCRP1 was constitutively expressed in all the tissues of B. oleracea including leaf, root and stem. However, its expression was found to be significantly increased in response to cold stress. Moreover, transgenic tomato plants expressing BoCRP1 exhibited increased tolerance to chilling stress (4 °C) with an overall improved rate of seed germination, increased root length, reduced membrane damage and increased accumulation of osmoprotectants. Furthermore, we observed increased transcript levels of stress responsive genes and enhanced accumulation of reactive oxygen species scavenging enzymes in transgenic plants on exposure to chilling stress. Taken together, these results strongly suggest that BoCRP1 is a promising candidate gene to improve the cold stress tolerance in tomato.

Low temperature elicits differential biochemical and antioxidant responses in maize (Zea mays) genotypes with different susceptibility to low temperature stress.

Maize, a C4 sub-tropical crop, possesses higher temperature optima as compared to the C3 plants. Low temperature (LT) stress confines the growth and productivity of maize. In this context, two maize genotypes, LT tolerant Gurez local and LT susceptible Gujarat-Maize-6 (G-M-6) were analysed in present study for various osmolytes and gene expression of antioxidant enzymes including Ascorbate-glutathione (AsA-GSH) besides trehalose biosynthetic pathways. With the progressive LT treatment, Gurez local showed lesser accumulation of stress markers like hydrogen peroxide (H2O2) and malondialdehyde, a significant increase in osmoprotectants like free proline, total protein, total soluble sugars, trehalose, total phenolics and glycine betaine, and a significant reduction in the plant pigments as compared to the G-M-6. Additionally, Gurez local was found to possess a well-established antioxidant defense system as revealed from the elevated transcripts and enzyme activities of various enzymes of AsA-GSH pathway. Higher gene expression and enzyme activities were exhibited by superoxide dismutase, catalase and peroxidase besides the gene expression of trehalose biosynthetic pathway enzymes. Moreover, through principal component analyses, a positive correlation of all analysed parameters with the LT tolerance was noticed in Gurez local alone demarcating the genotypes on the basis of their extent of LT tolerance. Overall, the present study forms the basis for unravelling of LT tolerance mechanisms and improvement in the performance of the temperate maize. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01020-3.

Combined gas exchange characteristics, chlorophyll fluorescence and response curves as selection traits for temperature tolerance in maize genotypes.

Maize is a low-temperature (LT)-sensitive plant and its physiological responses towards LT of temperate regions developed is an adaptive trait. To further our understanding about the response of maize to LT at the physiological and photosynthesis level, we conducted Infrared Gas Analysis (IRGA using LICOR6400-XT in 45-day-old grown two maize genotypes, one from temperate region (Gurez-Kashmir Himalayas), viz., Gurez local (Gz local), and another from tropics (Gujarat), viz., GM6. This study was carried out to evaluate the underlying physiological mechanisms in the two differentially temperature-tolerant maize genotypes. Net photosynthetic rate (A/PN), 18.253 in Gz local and 25.587 (µmol CO2 m-2 s-1) in GM6; leaf conductance (gs), 0.0102 in Gz local and 0.0566 (mmol H2O m-2 s-1) in GM6; transpiration rate (E), 0.5371 in Gz local and 2.9409 (mmol H2O m-2 s-1) in GM6; and water use efficiency (WUE), 33.9852 in Gz local and 8.7224 (µmol CO2 mmol H2O-1) in GM6, were recorded under ambient conditions. Also, photochemical efficiency of photosystem II (PSII) (Fv/Fm), 0.675 in Gz local and 0.705 in GM6; maximum photochemical efficiency (Fv'/Fm'), 0.310234 in Gz local and 0.401391 in GM6; photochemical quenching (qP), 0.2375 in Gz local and 0.2609 in GM6; non-photochemical quenching (NPQ), 2.0036 in Gz local and 1.1686 in GM6; effective yield of PSII (ФPSII), 0.0789 in Gz local and 0.099 in GM6; and electron transport rate (ETR), 55.3152 in Gz local and 68.112 in GM6, were also evaluated in addition to various response curves, like light intensities and temperature. We observed that light response curves show the saturation light intensity requirement of 1600 µmol for both the genotypes, whereas temperature response curves showed the optimum temperature requirement for Gz local as 20 °C and for GM6 it was found to be 35 °C. The results obtained for each individual parameter and other correlational studies indicate that IRGA forms a promising route for quick and reliable screening of various stress-tolerant valuable genotypes, forming the first study of its kind.

Jasmonic acid ameliorates alkaline stress by improving growth performance, ascorbate glutathione cycle and glyoxylase system in maize seedlings.

Environmental pollution by alkaline salts, such as Na2CO3, is a permanent problem in agriculture. Here, we examined the putative role of jasmonic acid (JA) in improving Na2CO3-stress tolerance in maize seedlings. Pretreatment of maize seedlings with JA was found to significantly mitigate the toxic effects of excessive Na2CO3 on photosynthesis- and plant growth-related parameters. The JA-induced improved tolerance could be attributed to decreased Na uptake and Na2CO3-induced oxidative damage by lowering the accumulation of reactive oxygen species and malondialdehyde. JA counteracted the salt-induced increase in proline and glutathione content, and significantly improved ascorbic acid content and redox status. The major antioxidant enzyme activities were largely stimulated by JA pretreatment in maize plants exposed to excessive alkaline salts. Additionally, increased activities of glyoxalases I and II were correlated with reduced levels of methylglyoxal in JA-pretreated alkaline-stressed maize plants. These results indicated that modifying the endogenous Na+ and K+ contents by JA pretreatment improved alkaline tolerance in maize plants by inhibiting Na uptake and regulating the antioxidant and glyoxalase systems, thereby demonstrating the important role of JA in mitigating heavy metal toxicity. Our findings may be useful in the development of alkali stress tolerant crops by genetic engineering of JA biosynthesis.

Over-expression of Topoisomerase II Enhances Salt Stress Tolerance in Tobacco.

Topoisomerases are unique enzymes having an ability to remove or add DNA supercoils and untangle the snarled DNA. They can cut, shuffle, and religate DNA strands and remove the torsional stress during DNA replication, transcription or recombination events. In the present study, we over-expressed topoisomerase II (TopoII) in tobacco (Nicotiana tabaccum) and examined its role in growth and development as well as salt (NaCl) stress tolerance. Several putative transgenic plants were generated and the transgene integration and expression was confirmed by PCR and Southern blot analyses, and RT-PCR analysis respectively. Percent seed germination, shoot growth, and chlorophyll content revealed that transgenic lines over-expressing the NtTopoIIα-1 gene exhibited enhanced tolerance to salt (150 and 200 mM NaCl) stress. Moreover, over-expression of TopoII lead to the elevation in proline and glycine betaine levels in response to both concentrations of NaCl as compared to wild-type. In response to NaCl stress, TopoII over-expressing lines showed reduced lipid peroxidation derived malondialdehyde (MDA) generation. These results suggest that TopoII plays a pivotal role in salt stress tolerance in plants.

Role of Trichoderma harzianum in mitigating NaCl stress in Indian mustard (Brassica juncea L) through antioxidative defense system.

Salinity stress affected crop production of more than 20% of irrigated land globally. In the present study the effect of different concentrations of NaCl (0, 100, and 200 mM) on growth, physio-biochemical attributes, antioxidant enzymes, oil content, etc. in Brassica juncea and the protective role of Trichoderma harzianum (TH) was investigated. Salinity stress deteriorates growth, physio-biochemical attributes, that ultimately leads to decreased biomass yield in mustard seedlings. Higher concentration of NaCl (200 mM) decreased the plant height by 33.7%, root length by 29.7% and plant dry weight (DW) by 34.5%. On the other hand, supplementation of TH to NaCl treated mustard seedlings showed elevation by 13.8, 11.8, and 16.7% in shoot, root length and plant DW respectively as compared to plants treated with NaCl (200 mM) alone. Oil content was drastically affected by NaCl treatment; however, TH added plants showed enhanced oil percentage from 19.4 to 23.4% in the present study. NaCl also degenerate the pigment content and the maximum drop of 52.0% was recorded in Chl. 'a'. Enhanced pigment content was observed by the application of TH to NaCl treated plants. Proline content showed increase by NaCl stress and maximum accumulation of 59.12% was recorded at 200 mM NaCl. Further enhancement to 70.37% in proline content was recorded by supplementation of TH. NaCl stress (200 mM) affirms the increase in H2O2 by 69.5% and MDA by 36.5%, but reduction in the accumulation is recorded by addition of TH to mustard seedlings. 200 mM NaCl elevated SOD, POD, APX, GR, GST, GPX, GSH, and GSSG in the present study. Further enhancement was observed by the application of TH to the NaCl fed seedlings. NaCl stress suppresses the uptake of important elements in both roots and shoots, however, addition of TH restored the elemental uptake in the present study. Mustard seedlings treated with NaCl and TH showed restricted Na uptake as compared to seedlings treated with NaCl alone. In conclusion, TH proved to be very beneficial in imparting resistance to the mustard plants against NaCl stress through improved uptake of essential elements, modulation of osmolytes and antioxidants.

Cell cycle stage-specific differential expression of topoisomerase I in tobacco BY-2 cells and its ectopic overexpression and knockdown unravels its crucial role in plant morphogenesis and development.

DNA topoisomerases catalyze the inter-conversion of different topological forms of DNA. Cell cycle coupled differential accumulation of topoisomerase I (Topo I) revealed biphasic expression maximum at S-phase and M/G1-phase of cultured synchronized tobacco BY-2 cells. This suggested its active role in resolving topological constrains during DNA replication (S-phase) and chromosome decondensation (M/G1 phase). Immuno-localization revealed high concentrations of Topo I in nucleolus. Propidium iodide staining and Br-UTP incorporation patterns revealed direct correlation between immunofluorescence intensity and rRNA transcription activity within nucleolus. Immuno-stained chromosomes during metaphase and anaphase suggested possible role of Topo I in resolving topological constrains during mitotic chromosome condensation. Inhibitor studies showed that in comparison to Topo I, Topo II was essential in resolving topological constrains during chromosome condensation. Probably, Topo II substituted Topo I functioning to certain extent during chromosome condensation, but not vice-versa. Transgenic Topo I tobacco lines revealed morphological abnormalities and highlighted its crucial role in plant morphogenesis and development.

Plant exomics: concepts, applications and methodologies in crop improvement.

Molecular breeding has a crucial role in improvement of crops. Conventional breeding techniques have failed to ameliorate food production. Next generation sequencing has established new concepts of molecular breeding. Exome sequencing has proven to be a significant tool for assessing natural evolution in plants, studying host pathogen interactions and betterment of crop production as exons assist in interpretation of allelic variation with respect to their phenotype. This review covers the platforms for exome sequencing, next generation sequencing technologies that have revolutionized exome sequencing and led toward development of third generation sequencing. Also discussed in this review are the uses of these sequencing technologies to improve wheat, rice and cotton yield and how these technologies are used in exploring the biodiversity of crops, providing better understanding of plant-host pathogen interaction and assessing the process of natural evolution in crops and it also covers how exome sequencing identifies the gene pool involved in symbiotic and other co-existential systems. Furthermore, we conclude how integration of other methodologies including whole genome sequencing, proteomics, transcriptomics and metabolomics with plant exomics covers the areas which are left untouched with exomics alone and in the end how these integration will transform the future of crops.

Antioxidative response of Lemna polyrrhiza L. to cadmium stress.

Growth, lipid peroxidation, different antioxidative enzymes and metal accumulation were studied in Lemna polyrrhiza treated with different concentrations (1-40 ppm) of CdSO4. The growth of the plant was slightly enhanced with 1 ppm, while higher concentrations retarted growth and multiplication of fronds, the effect being concentration and dose dependant. Increase in malondialdehyde content was insignificant after the first week but a prolonged exposure led to significant (p < 0.05) increase of about 38% and 45% over the control in 20 and 30 ppm, respectively after four weeks. Catalase (EC 1.11.1.6; CAT) activity increased at low concentration, but it declined to 42% and 54% at 40 ppm after 6 and 30 days, respectively Superoxide dismutase (EC 1.15.1.1; SOD), ascorbate peroxidase (EC 1.11.1.11;APx) and glutathione reductase (EC 1.6.4.2) increased at both low as well at high concentrations, but a prolonged exposure to high concentration of Cd (40 ppm) led to significant (p < 0.05) decline in the mean activities of these antioxidant enzymes. Accumulation of Cd in biomass was concentration and time dependant However at high concentration of 40 ppm, Cd accumulation did not increase significantly (p < 0.05) with time. Increased activities of antioxidant enzymes in Cd treated plants suggest that metal tolerance in L. polyrrhiza might be associated to the changes of antioxidant enzymatic activities.