Physiological responses induced by phospholipase C isoform 5 upon heat stress in Arabidopsis thaliana.

Plant's perception of heat stress involves several pathways and signaling molecules, such as phosphoinositide, which is derived from structural membrane lipids phosphatidylinositol. Phospholipase C (PLC) is a well-known signaling enzyme containing many isoforms in different organisms. In the present study, Phospholipase C Isoform 5 (PLC5) was investigated for its role in thermotolerance in Arabidopsis thaliana. Two over-expressing lines and one knock-down mutant of PLC5 were first treated at a moderate temperature (37 °C) and left for recovery. Then again exposed to a high temperature (45 °C) to check the seedling viability and chlorophyll contents. Root behavior and changes in 32Pi labeled phospholipids were investigated after their exposure to high temperatures. Over-expression of PLC5 (PLC5 OE) exhibited quick and better phenotypic recovery with bigger and greener leaves followed by chlorophyll contents as compared to wild-type (Col-0) and PLC5 knock-down mutant in which seedling recovery was compromised. PLC5 knock-down mutant illustrated well-developed root architecture under controlled conditions but stunted secondary roots under heat stress as compared to over-expressing PLC5 lines. Around 2.3-fold increase in phosphatidylinositol 4,5-bisphosphate level was observed in PLC5 OE lines upon heat stress compared to wild-type and PLC5 knock-down mutant lines. A significant increase in phosphatidylglycerol was also observed in PLC5 OE lines as compared to Col-0 and PLC5 knock-down mutant lines. The results of the present study demonstrated that PLC5 over-expression contributes to heat stress tolerance while maintaining its photosynthetic activity and is also observed to be associated with primary and secondary root growth in Arabidopsis thaliana.

AtCIPK16, a CBL-interacting protein kinase gene, confers salinity tolerance in transgenic wheat.

Globally, wheat is the major source of staple food, protein, and basic calories for most of the human population. Strategies must be adopted for sustainable wheat crop production to fill the ever-increasing food demand. Salinity is one of the major abiotic stresses involved in plant growth retardation and grain yield reduction. In plants, calcineurin-B-like proteins form a complicated network with the target kinase CBL-interacting protein kinases (CIPKs) in response to intracellular calcium signaling as a consequence of abiotic stresses. The AtCIPK16 gene has been identified in Arabidopsis thaliana and found to be significantly upregulated under salinity stress. In this study, the AtCIPK16 gene was cloned in two different plant expression vectors, i.e., pTOOL37 having a UBI1 promoter and pMDC32 having a 2XCaMV35S constitutive promoter transformed through the Agrobacterium-mediated transformation protocol, in the local wheat cultivar Faisalabad-2008. Based on their ability to tolerate different levels of salt stress (0, 50, 100, and 200 mM), the transgenic wheat lines OE1, OE2, and OE3 expressing AtCIPK16 under the UBI1 promoter and OE5, OE6, and OE7 expressing the same gene under the 2XCaMV35S promoter performed better at 100 mM of salinity stress as compared with the wild type. The AtCIPK16 overexpressing transgenic wheat lines were further investigated for their K+ retention ability in root tissues by utilizing the microelectrode ion flux estimation technique. It has been demonstrated that after 10 min of 100 mM NaCl application, more K+ ions were retained in the AtCIPK16 overexpressing transgenic wheat lines than in the wild type. Moreover, it could be concluded that AtCIPK16 functions as a positive elicitor in sequestering Na+ ions into the cell vacuole and retaining more cellular K+ under salt stress to maintain ionic homeostasis.

32Pi Labeled Transgenic Wheat Shows the Accumulation of Phosphatidylinositol 4,5-bisphosphate and Phosphatidic Acid Under Heat and Osmotic Stress.

The ensuing heat stress drastically affects wheat plant growth and development, consequently compromising its grain yield. There are many thermoregulatory processes/mechanisms mediated by ion channels, lipids, and lipid-modifying enzymes that occur in the plasma membrane and the chloroplast. With the onset of abiotic or biotic stresses, phosphoinositide-specific phospholipase C (PI-PLC), as a signaling enzyme, hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to generate inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) which is further phosphorylated into phosphatidic acid (PA) as a secondary messenger and is involved in multiple processes. In the current study, a phospholipase C (PLC) signaling pathway was investigated in spring wheat (Triticum aestivum L.) and evaluated its four AtPLC5 overexpressed (OE)/transgenic lines under heat and osmotic stresses through 32Pi radioactive labeling. Naturally, the wheat harbors only a small amount of PIP2. However, with the sudden increase in temperature (40°C), PIP2 levels start to rise within 7.5 min in a time-dependent manner in wild-type (Wt) wheat. While the Phosphatidic acid (PA) level also elevated up to 1.6-fold upon exposing wild-type wheat to heat stress (40°C). However, at the anthesis stage, a significant increase of ∼4.5-folds in PIP2 level was observed within 30 min at 40°C in AtPLC5 over-expressed wheat lines. Significant differences in PIP2 level were observed in Wt and AtPLC5-OE lines when treated with 1200 mM sorbitol solution. It is assumed that the phenomenon might be a result of the activation of PLC/DGK pathways. Together, these results indicate that heat stress and osmotic stress activate several lipid responses in wild-type and transgenic wheat and can explain heat and osmotic stress tolerance in the wheat plant.

Spider toxin (Hvt) gene cloned under phloem specific RSs1 and RolC promoters provides resistance against American bollworm (Heliothis armigera).

Spider venoms are neurotoxin proteins that can kill insects. Spider toxin Hvt gene was cloned under two phloem specific RSs1 and RolC promoters, transformed into tobacco plants through Agrobacterium-mediated transformation and tested against Heliothis armigera larvae. Transgenic plants were confirmed through PCR. First instar larvae of H. armigera were released on detached leaves of transformed and non-transformed plants. Insect bioassays showed 93-100% mortality of H. armigera larvae within 72 h on the leaves of transgenic plants while all larvae survived and continued feeding on detached leaves from non-transformed control plants. The Hvt gene expressing under phloem specific RSs1 and RolC promoters could therefore be used for developing H. armigera-resistant, genetically-modified crops.

Hazards of nitrogen fertilizers and ways to reduce nitrate accumulation in crop plants.

In modern agriculture, farm produce accumulates a lot of nitrates that can reach toxic levels owing to the unfair use of nitrogen fertilizers, cultural methods, farming policies in multiple areas of the world, thereby increasing concerns about the availability of hygienic food supply and environmental hazards. Over the past few decades, global interest in achieving greater output through intensive fertilization has been a growing trend. The fertilizer based on urea or ammonium mainly yields ammonium, which is then transformed to nitrate through the oxidation process that is biologically mediated. Nitrate tends to accumulate differently in distinct crop plants and distinct components of agricultural commodities based on species, crop variety, genetic history, environmental circumstances, harvest phase, post-harvest storage conditions, agronomic variables, nature, and fertilizer application rate. The current article highlights various factors that could directly or indirectly contribute to the accumulation of nitrates in different parts of crop plants and discusses strategies to minimize the accumulation of nitrates in farm produce, thus ensuring healthy food supply and protecting the environment from the accumulation of nitrates.

Excessive use of nitrogenous fertilizers: an unawareness causing serious threats to environment and human health.

Farmers occasionally need to add nitrogen fertilizer to their farms and gardens to make available just the precise nutrients for their plants' growth. The applications of inorganic nitrogen fertilizers to various crops have been continuously increasing since last many decades globally. Although nitrogen fertilizer contributes substantially to yield enhancement, but excessive use of this manure has posed serious threats to environment and human health. Rate of nitrogen fertilizers application has a close relationship with nitrate accumulation in surrounding environment, groundwater, as well as leafy and root vegetables. Consumption of diets having high nitrate contents has contributed to endogenous nitrosation, which could lead to thyroid condition, various kinds of human cancers, neural tube defects (during fetus development), and diabetes. In this short review, the authors have tried to create awareness among general public, farming community, health practitioners, and agricultural scientists for the risk involved with excessive use of nitrogen fertilizers to human health. Carcinogenic activity and other adverse effects of N-nitroso compounds might be prevented by consuming vitamin C and antioxidants containing fruits and vegetables.

Identification of differentially expressed genes in developing cotton fibers (Gossypium hirsutum L) through differential display

Cotton fibers are differentiated, non-dividing cells that originate from the epidermal layer of developing ovules. To identify genes involved in cotton fiber development, we performed non-radioactive differential display reverse transcriptase PCR (DDRT-PCR) on the purified mRNA. This technique was tested on mRNA isolated from five different developmental stages of cotton fiber including 0, 5, 10, 15 and 20 DPA (days after pollination). The mRNA purified from total RNA was reversibly transcribed using three anchored oligo-dT primers. Polymerase chain reaction (PCR) amplification of each cDNA preparation was carried out in combination with seven arbitrary primers. The amplified products were resolved on 1 percent agarose gel containing ethidium bromide. DNA was extracted from seventeen differentially expressed bands and cloned in pTZ57R/T vector. The sequencing and BLAST search analysis indicated that 12 of the differentially expressed genes matched the previously characterized genes, while 3 of them matched the uncharacterized sequences of cotton fiber expressed sequence tags (ESTs) reported previously to be associated with cotton fiber and 2 of the clones had homology with putative proteins. The technique can be used to efficiently identify differentially expressed genes and can be expanded to large scale studies by increasing the number of random decamers.