Cloning and characterisation of heat shock and wound-induced genes in pea (pisum sativum L.)

Plant productivity in many regions of the world is limited primarily as a result of environmental stresses. High temperature and wounding caused by pest and pathogen infection are among the main factors accounting for unpredictable and often severe yield losses worldwide. These stresses, force the plants to alter then gene expression in order to adapt to the changed environment. Attempts were made in the study to isolate and characterise the differentially expressed heat shocked and wound-induced genes to understand the underlying molecular mechanism of heat shock and wounding response. The isolation of the promoters and their use to derive the tissue-specific and high expression of the linked coding sequences will be proved practically more significant. A cDNA clone designated LP 19 was isolated from a differential screening of a cDNA library prepared from lignifying pods of pea line L59. Sequence homology analysis showed that LP19 belongs to the hsp70 gene family. Northern analysis of RNA from pods from pea lines of different genotypes, showing the presence or absence of pod lignification, showed that LP 19 expression was specifically associated with lignification. Several cDNA species derived from transcripts of the LP 19 gene were subsequently isolated, which showed varying positions of poly (A) addition to the 3' untranslated region. Southern blotting of genomic DNA indicated the presence of single gene corresponding to LP 19.The pea hsp70 gene corresponding to LP 19 was isolated from a pea genomic library using LP 19 as a probe. The pea hsp70(LP19) gene predicts an open reading frame encoding a polypeptide of 648 amino acid residues. This sequence is similar to other plant hsp70 proteins. However, unlike most other plant hsp70 genes, the pea hsp70(LP19) gene lacks an intron. 1.8 kb of 5' flanking sequence of hsp70(LP19) gene was also sequenced. The promoter region contains 6 putative consensus heat shock elements (HSEs) as well as 4 A-T rich sites upstream from TATA box. Induction of gene expression of the pea hsp70(LP19) was observed in all organs of the plant after heat shock; the highest level of expression was observed in root, followed by stem and least in leaves. A similar expression pattern for a corresponding gene was observed in chickpea (Cicer arietinum L.). Other stress conditions such as salt stress and wounding failed to induce the expression of hsp70LP(19) gene both in pea and chickpea. The pea hsp70(LP19) promoter region, including 1.8 kb 5'-flanking sequence, and the first 18 amino acids of the coding region, was fused with coding sequence for P- glucuronidase (GUS). Tobacco plants were transformed with this chimaeric gene in order to study tissue specific and developmental expression of the hsp70(LP19) promoter. Histological staining of GUS activity in transgenic tobacco plants showed that protein was present predominantly in the phloem tissue in stem, root and petioles In addition, developmental expression of the hsp70(LP19) gene promoter, without heat shock, was observed in petals, pollen grains, developing seeds as well as in germinating seeds and seedlings at different stages of growth. Quantitative assay of GUS activity by fluorometric assay was used to follow the time course of protein accumulation. Activity was detected within few minutes of the start of heat shock and increased to a maximum after 6 hrs. A high level of GUS activity was observed only in the heat shocked parts of the plant; no endogenous signal that spread systemically from the heat shocked areas to the rest of the plant was observed.Pea and chickpea plants showed a transient increase of polyphenol oxidase (PPO) with maximum level at 48 hrs after wounding. No systemic induction of PPO was observed in unwounded parts in response to both wounding and MeJA treatment. In order to isolate transcripts expressed differentially in response to wounding, a pea subtractive cDNA library was made. 21 subtracted cDNA clones were partially sequenced. Most of the subtracted cDNA clones showed homology with wound or pathogen induced sequences. Northern analysis of the genes corresponding to the subtracted cDNA clones (SC3, SC7, SC12, SC33, SC57 and SC58), indicated differential expression in response to wounding. Full length or nearly full length cDNAs corresponding to 4 subtracted cDNA clones, designated SC10, SC15, SC57 and SC58, were isolated and sequenced. These cDNA clones will be further studied and efforts will be made to isolate their promoters. The tissue-specific expression will be carried out by using promoter-reporter system. These isolated cDNA clones were partially characterised and will be available for further studies to isolate their respective promoters. The tissue specific expression will be carried out by using promoter-reporter system

Expression profiling of Crambe abyssinica under arsenate stress identifies genes and gene networks involved in arsenic metabolism and detoxification

Background Arsenic contamination is widespread throughout the world and this toxic metalloid is known to cause cancers of organs such as liver, kidney, skin, and lung in human. In spite of a recent surge in arsenic related studies, we are still far from a comprehensive understanding of arsenic uptake, detoxification, and sequestration in plants. Crambe abyssinica, commonly known as \u27abyssinian mustard\u27, is a non-food, high biomass oil seed crop that is naturally tolerant to heavy metals. Moreover, it accumulates significantly higher levels of arsenic as compared to other species of the Brassicaceae family. Thus, C. abyssinica has great potential to be utilized as an ideal inedible crop for phytoremediation of heavy metals and metalloids. However, the mechanism of arsenic metabolism in higher plants, including C. abyssinica, remains elusive. Results To identify the differentially expressed transcripts and the pathways involved in arsenic metabolism and detoxification, C. abyssinica plants were subjected to arsenate stress and a PCR-Select Suppression Subtraction Hybridization (SSH) approach was employed. A total of 105 differentially expressed subtracted cDNAs were sequenced which were found to represent 38 genes. Those genes encode proteins functioning as antioxidants, metal transporters, reductases, enzymes involved in the protein degradation pathway, and several novel uncharacterized proteins. The transcripts corresponding to the subtracted cDNAs showed strong upregulation by arsenate stress as confirmed by the semi-quantitative RT-PCR. Conclusions Our study revealed novel insights into the plant defense mechanisms and the regulation of genes and gene networks in response to arsenate toxicity. The differential expression of transcripts encoding glutathione-S-transferases, antioxidants, sulfur metabolism, heat-shock proteins, metal transporters, and enzymes in the ubiquitination pathway of protein degradation as well as several unknown novel proteins serve as molecular evidence for the physiological responses to arsenate stress in plants. Additionally, many of these cDNA clones showing strong upregulation due to arsenate stress could be used as valuable markers. Further characterization of these differentially expressed genes would be useful to develop novel strategies for efficient phytoremediation as well as for engineering arsenic tolerant crops with reduced arsenic translocation to the edible parts of plants

Elucidating the Response of Crop Plants towards Individual, Combined and Sequentially Occurring Abiotic Stresses

In nature, plants are exposed to an ever-changing environment with increasing frequencies of multiple abiotic stresses. These abiotic stresses act either in combination or sequentially, thereby driving vegetation dynamics and limiting plant growth and productivity worldwide. Plants’ responses against these combined and sequential stresses clearly differ from that triggered by an individual stress. Until now, experimental studies were mainly focused on plant responses to individual stress, but have overlooked the complex stress response generated in plants against combined or sequential abiotic stresses, as well as their interaction with each other. However, recent studies have demonstrated that the combined and sequential abiotic stresses overlap with respect to the central nodes of their interacting signaling pathways, and their impact cannot be modelled by swimming in an individual extreme event. Taken together, deciphering the regulatory networks operative between various abiotic stresses in agronomically important crops will contribute towards designing strategies for the development of plants with tolerance to multiple stress combinations. This review provides a brief overview of the recent developments in the interactive effects of combined and sequentially occurring stresses on crop plants. We believe that this study may improve our understanding of the molecular and physiological mechanisms in untangling the combined stress tolerance in plants, and may also provide a promising venue for agronomists, physiologists, as well as molecular biologists

Discovery of miRNAs and Development of Heat-Responsive miRNA-SSR Markers for Characterization of Wheat Germplasm for Terminal Heat Tolerance Breeding

A large proportion of the Asian population fulfills their energy requirements from wheat (Triticum aestivum L.). Wheat quality and yield are critically affected by the terminal heat stress across the globe. It affects approximately 40% of the wheat-cultivating regions of the world. Therefore, there is a critical need to develop improved terminal heat-tolerant wheat varieties. Marker-assisted breeding with genic simple sequence repeats (SSR) markers have been used for developing terminal heat-tolerant wheat varieties; however, only few studies involved the use of microRNA (miRNA)-based SSR markers (miRNASSRs) in wheat, which were found as key players in various abiotic stresses. In the present study, we identified 104 heat-stress-responsive miRNAs reported in various crops. Out of these, 70 miRNA-SSR markers have been validated on a set of 20 terminal heat-tolerant and heat-susceptible wheat genotypes. Among these, only 19 miRNA-SSR markers were found to be polymorphic, which were further used to study the genetic diversity and population structure. The polymorphic miRNA-SSRs amplified 61 SSR loci with an average of 2.9 alleles per locus. The polymorphic information content (PIC) value of polymorphic miRNA-SSRs ranged from 0.10 to 0.87 with a mean value of 0.48. The dendrogram constructed using unweighted neighbor-joining method and population structure analysis clustered these 20 wheat genotypes into 3 clusters. The target genes of these miRNAs are involved either directly or indirectly in providing tolerance to heat stress. Furthermore, two polymorphic markers miR159c and miR165b were declared as very promising diagnostic markers, since these markers showed specific alleles and discriminated terminal heat-tolerant genotypes from the susceptible genotypes. Thus, these identified miRNA-SSR markers will prove useful in the characterization of wheat germplasm through the study of genetic diversity and population structural analysis and in wheat molecular breeding programs aimed at terminal heat tolerance of wheat varieties

Increased Cuticle Waxes by Overexpression of WSD1 Improves Osmotic Stress Tolerance in Arabidopsis thaliana and Camelina sativa

To ensure global food security under the changing climate, there is a strong need for developing ‘climate resilient crops’ that can thrive and produce better yields under extreme environmental conditions such as drought, salinity, and high temperature. To enhance plant productivity under the adverse conditions, we constitutively overexpressed a bifunctional wax synthase/acyl-CoA:diacylglycerol acyltransferase (WSD1) gene, which plays a critical role in wax ester synthesis in Arabidopsis stem and leaf tissues. The qRT-PCR analysis showed a strong upregulation of WSD1 transcripts by mannitol, NaCl, and abscisic acid (ABA) treatments, particularly in Arabidopsis thaliana shoots. Gas chromatography and electron microscopy analyses of Arabidopsis seedlings overexpressing WSD1 showed higher deposition of epicuticular wax crystals and increased leaf and stem wax loading in WSD1 transgenics compared to wildtype (WT) plants. WSD1 transgenics exhibited enhanced tolerance to ABA, mannitol, drought and salinity, which suggested new physiological roles for WSD1 in stress response aside from its wax synthase activity. Transgenic plants were able to recover from drought and salinity better than the WT plants. Furthermore, transgenics showed reduced cuticular transpirational rates and cuticle permeability, as well as less chlorophyll leaching than the WT. The knowledge from Arabidopsis was translated to the oilseed crop Camelina sativa (L.) Crantz. Similar to Arabidopsis, transgenic Camelina lines overexpressing WSD1 also showed enhanced tolerance to drought stress. Our results clearly show that the manipulation of cuticular waxes will be advantageous for enhancing plant productivity under a changing climate

Graphitic Carbon Nitride (C3N4) Reduces Cadmium and Arsenic Phytotoxicity and Accumulation in Rice (Oryza sativa L.)

The present study investigated the role of graphitic carbon nitride (C3N4) in alleviating cadmium (Cd)- and arsenic (As)-induced phytotoxicity to rice (Oryza sativa L.). A high-temperature pyrolysis was used to synthesize the C3N4, which was characterized by transmission electron microscopy, Fourier-transform infrared spectroscopy, and dynamic light scattering. Rice seedlings were exposed to C3N4 at 50 and 250 mg/L in half-strength Hoagland’s solution amended with or without 10 mg/L Cd or As for 14 days. Both Cd and As alone resulted in 26–38% and 49–56% decreases in rice root and shoot biomass, respectively. Exposure to 250 mg/L C3N4 alone increased the root and shoot fresh biomass by 17.5% and 25.9%, respectively. Upon coexposure, Cd + C3N4 and As + C3N4 alleviated the heavy metal-induced phytotoxicity and increased the fresh weight by 26–38% and 49–56%, respectively. Further, the addition of C3N4 decreased Cd and As accumulation in the roots by 32% and 25%, respectively, whereas the metal contents in the shoots were 30% lower in the presence of C3N4. Both As and Cd also significantly altered the macronutrient (K, P, Ca, S, and Mg) and micronutrient (Cu, Fe, Zn, and Mn) contents in rice, but these alterations were not evident in plants coexposed to C3N4. Random amplified polymorphic DNA analysis suggests that Cd significantly altered the genomic DNA of rice roots, while no difference was found in shoots. The presence of C3N4 controlled Cd and As uptake in rice by regulating transport-related genes. For example, the relative expression of the Cd transporter OsIRT1 in roots was upregulated by approximately threefold with metal exposure, but C3N4 coamendment lowered the expression. Similar results were evident in the expression of the As transporter OsNIP1;1 in roots. Overall, these findings facilitate the understanding of the underlying mechanisms by which carbon-based nanomaterials alleviate contaminant-induced phyto- and genotoxicity and may provide a new strategy for the reduction of heavy metal contamination in agriculture

Unlocking the potential of nanoscale sulfur in sustainable agriculture

The global population is growing rapidly, which poses a significant challenge to food security. Innovation in agricultural technologies is necessary to achieve sustainable development in agriculture and combat food insecurity. Nanotechnology has emerged as a promising tool in agriculture; compared to conventional agricultural chemicals, demonstrated benefits include increased efficiency of delivery and utilization of both nutrients and pesticides, as well as nanoscale-specific stimulation of stress tolerance pathways. Among the many studied nanomaterials, nano-sulfur has demonstrated superior effects at enhancing plant resilience to pathogens and abiotic stresses, as well as improving plant growth and nutritional quality of edible tissues. A number of published studies have investigated the physiological effects (growth promotion, disease resistance) of single or several sulfur and sulfide compounds on crop species. However, there is no systematic analysis of this literature, including the effects and specific mechanisms of various sulfur forms in agricultural applications. In this review, we will discuss the effects of sulfur (including nano-sulfur) on crop species, the underlying mechanisms of action for their transport and transformation in the soil-plant system, and evaluate their suitability in sustainable agricultural development. Additionally, we discuss the current challenges and knowledge gaps for nanoscale sulfur use in agriculture, and describe future research directions to advance our understanding of the sustainable use of this material at the scale of individual fields

Unlocking the potential of nanoscale sulfur in sustainable agriculture

The global population is growing rapidly, which poses a significant challenge to food security. Innovation in agricultural technologies is necessary to achieve sustainable development in agriculture and combat food insecurity. Nanotechnology has emerged as a promising tool in agriculture; compared to conventional agricultural chemicals, demonstrated benefits include increased efficiency of delivery and utilization of both nutrients and pesticides, as well as nanoscale-specific stimulation of stress tolerance pathways. Among the many studied nanomaterials, nano-sulfur has demonstrated superior effects at enhancing plant resilience to pathogens and abiotic stresses, as well as improving plant growth and nutritional quality of edible tissues. A number of published studies have investigated the physiological effects (growth promotion, disease resistance) of single or several sulfur and sulfide compounds on crop species. However, there is no systematic analysis of this literature, including the effects and specific mechanisms of various sulfur forms in agricultural applications. In this review, we will discuss the effects of sulfur (including nano-sulfur) on crop species, the underlying mechanisms of action for their transport and transformation in the soil-plant system, and evaluate their suitability in sustainable agricultural development. Additionally, we discuss the current challenges and knowledge gaps for nanoscale sulfur use in agriculture, and describe future research directions to advance our understanding of the sustainable use of this material at the scale of individual fields

Effect of stem structural characteristics and cell wall components related to stem lodging resistance in a newly identified mutant of hexaploid wheat (Triticum aestivum L.)

In wheat, lodging is affected by anatomical and chemical characteristics of the stem cell wall. Plant characteristics determining the stem strength were measured in lodging tolerant mutant (PMW-2016-1) developed through mutation breeding utilizing hexaploid wheat cultivar, DPW-621-50. Various anatomical features, chemical composition, and mechanical strength of the culms of newly developed lodging-tolerant mutant (PMW-2016-1) and parent (DPW-621-50), were examined by light microscopy, the Klason method, prostate tester coupled with a Universal Tensile Machine, and Fourier Transform Infrared Spectroscopy. Significant changes in the anatomical features, including the outer radius of the stem, stem wall thickness, and the proportions of various tissues, and vascular bundles were noticed. Chemical analysis revealed that the lignin level in the PMW-2016-1 mutant was higher and exhibited superiority in stem strength compared to the DPW-621-50 parent line. The force (N) required to break the internodes of mutant PMW 2016-1 was higher than that of DPW-621-50. The results suggested that the outer stem radius, stem wall thickness, the proportion of sclerenchyma tissues, the number of large vascular bundles, and lignin content are important factors that affect the mechanical strength of wheat stems, which can be the key parameters for the selection of varieties having higher lodging tolerance. Preliminary studies on the newly identified mutant PMW-2016-1 suggested that this mutant may possess higher lodging tolerance because it has a higher stem strength than DPW-621-50 and can be used as a donor parent for the development of lodging-tolerant wheat varieties

A novel stress-associated protein 'AtSAP10' from Arabidopsis thaliana confers tolerance to nickel, manganese, zinc, and high temperature stress.

We describe here the functional characterization of a novel AtSAP10, a member of the Stress Associated Protein (SAP) gene family, from Arabidopsis thaliana ecotype Columbia. AtSAP10 contains an A20 and AN1 zinc-finger domain at the N- and C-terminal, respectively. Arabidopsis SAP10 showed differential regulation by various abiotic stresses such as heavy metals and metalloids (Ni, Cd, Mn, Zn, and As), high and low temperatures, cold, and ABA. Overexpression of AtSAP10 in Arabidopsis conferred strong tolerance to heavy metals such as Ni, Mn, and Zn and to high temperature stress. AtSAP10 transgenic plants under these stress conditions grew green and healthy, attained several-fold more biomass, and had longer roots as compared to wild type plants. Further, while these transgenic plants accumulated significantly greater amounts of Ni and Mn in both shoots and root tissues, there was no significant difference in the accumulation of Zn. AtSAP10 promoter-GUS fusion studies revealed a root and floral organ-specific expression of AtSAP10. Overexpression of AtSAP10-GFP fusion protein showed the localization in both nucleus and cytoplasm. Taken together, these results showed that AtSAP10 is a potentially useful candidate gene for engineering tolerance to heavy metals and to abiotic stress in cultivated plants