Kazi Md. Kamrul Huda

Global calcium transducer P-type Ca2+-ATPases open new avenues for agriculture by regulating stress signalling

Food security is in danger under the continuous growing threat of various stresses including climate change and global warming, which ultimately leads to a reduction in crop yields. Calcium plays a very important role in many signal transduction pathways including stress signalling. Different extracellular stimuli trigger increases in cytosolic calcium, which is detrimental to plants. To cope with such stresses, plants need to develop efficient efflux mechanisms to maintain ionic homeostasis. The Ca(2+)-ATPases are members of the P-type ATPase superfamily, which perform many fundamental processes in organisms by actively transporting ions across cellular membranes. In recent years, many studies have revealed that, as well as efflux mechanisms, Ca(2+)-ATPases also play critical roles in sensing calcium fluctuations and relaying downstream signals by activating definitive targets, thus modulating corresponding metabolic pathways. As calcium-activated calmodulin (CaM) is reported to play vital roles in stress tolerance, the presence of a unique CaM-binding site in type IIB Ca(2+)-ATPases indicates their potential role in biotic as well as abiotic stress tolerance. The key roles of Ca(2+)-ATPases in transport systems and stress signalling in cellular homeostasis are addressed in this review. A complete understanding of plant defence mechanisms under stress will allow bioengineering of improved crop plants, which will be crucial for food security currently observed worldwide in the context of global climate changes. Overall, this article covers classification, evolution, structural aspects of Ca(2+)-ATPases, and their emerging roles in plant stress signalling.

OsACA6, a P-type IIB Ca2+ATPase promotes salinity and drought stress tolerance in tobacco by ROS scavenging and enhancing the expression of stress-responsive genes

Calcium (Ca²⁺) regulates several signalling pathways involved in growth, development and stress tolerance. Cellular Ca²⁺ homeostasis is achieved by the combined action of channels, pumps and antiporters, but direct evidence for a role of Ca²⁺ATPase pumps in stress tolerance is lacking. Here we report the characterization of a Ca²⁺ ATPase gene (OsACA6) from Oryza sativa, and elucidate its functions in stress tolerance. OsACA6 transcript levels are enhanced in response to salt, drought, abscisic acid and heat. In vivo localization identified plasma membranes as an integration site for the OsACA6-GFP fusion protein. Using transgenic tobacco lines, we demonstrate that over-expression of OsACA6 is triggered during salinity and drought stresses. The enhanced tolerance to these stresses was confirmed by changes in several physiological indices, including water loss rate, photosynthetic efficiency, cell membrane stability, germination, survival rate, malondialdehyde content, electrolyte leakage and increased proline accumulation. Furthermore, over-expressing lines also showed higher leaf chlorophyll and reduced accumulation of H₂O₂ and Na⁺ ions compared to the wild-type. Reduced accumulation of reactive oxygen species (ROS) was observed in transgenic lines. The increased proline accumulation and ROS scavenging enzyme activities in transgenic plants over-expressing OsACA6 efficiently modulate the ROS machinery and proline biosynthesis through an integrative mechanism. Transcriptional profiling of these plants revealed altered expression of genes encoding many transcription factors, stress- and disease-related proteins, as well as signalling components. These results suggest that Ca²⁺ ATPases have diverse roles as regulators of many stress signalling pathways, leading to plant growth, development and stress tolerance.

Pea p68, a DEAD-box helicase, provides salinity stress tolerance in transgenic tobacco by reducing oxidative stress and improving photosynthesis machinery.

Background The DEAD-box helicases are required mostly in all aspects of RNA and DNA metabolism and they play a significant role in various abiotic stresses, including salinity. The p68 is an important member of the DEAD-box proteins family and, in animal system, it is involved in RNA metabolism including pre-RNA processing and splicing. In plant system, it has not been well characterized. Here we report the cloning and characterization of p68 from pea (Pisum sativum) and its novel function in salinity stress tolerance in plant. Results The pea p68 protein self-interacts and is localized in the cytosol as well as the surrounding of cell nucleus. The transcript of pea p68 is upregulated in response to high salinity stress in pea. Overexpression of p68 driven by constitutive cauliflower mosaic virus-35S promoter in tobacco transgenic plants confers enhanced tolerances to salinity stress by improving the growth, photosynthesis and antioxidant machinery. Under stress treatment, pea p68 overexpressing tobacco accumulated higher K+ and lower Na+ level than the wild-type plants. Reactive oxygen species (ROS) accumulation was remarkably regulated by the overexpression of pea p68 under salinity stress conditions, as shown from TBARS content, electrolyte leakage, hydrogen peroxide accumulation and 8-OHdG content and antioxidant enzyme activities. Conclusions To the best of our knowledge this is the first direct report, which provides the novel function of pea p68 helicase in salinity stress tolerance. The results suggest that p68 can also be exploited for engineering abiotic stress tolerance in crop plants of economic importance.

Genome-wide analysis of plant-type II Ca2+ATPases gene family from rice and Arabidopsis: Potential role in abiotic stresses

The Plant Ca(2+)ATPases are members of the P-type ATPase superfamily and play essential roles in pollen tube growth, vegetative development, inflorescence architecture, stomatal opening or closing as well as transport of Ca(2+), Mn(2+) and Zn(2+). Their role in abiotic stress adaptation by activation of different signaling pathways is emerging. In Arabidopsis, the P-type Ca(2+)ATPases can be classified in two distinct groups: type IIA (ECA) and type IIB (ACA). The availability of rice genome sequence allowed performing a genome-wide search for P-type Ca(2+)ATPases proteins, and the comparison of the identified proteins with their homologs in Arabidopsis model plant. In the present study, we identified the P-type II Ca(2+)ATPases from rice by analyzing their phylogenetic relationship, multiple alignment, cis-regulatory elements, protein domains, motifs and homology percentage. The phylogenetic analysis revealed that rice type IIA Ca(2+)ATPases clustered with Arabidopsis type IIA Ca(2+)ATPases and showed high sequence similarity within the group, whereas rice type IIB Ca(2+)ATPases presented variable sequence similarities with Arabidopsis type IIB members. The protein homology modeling, identification of putative transmembrane domains and conserved motifs of rice P-type II Ca(2+)ATPases provided information on their functions and structural architecture. The analysis of P-type II Ca(2+)ATPases promoter regions in rice showed multiple stress-induced cis-acting elements. The expression profile analysis indicated vital roles of P-type II Ca(2+)ATPases in stress signaling, plant development and abiotic stress responses. The comprehensive analysis and expression profiling provided a critical platform for functional characterization of P-type II Ca(2+)ATPase genes that could be applied in engineering crop plants with modified calcium signaling and homeostatic pathways.

Reproductive organ and vascular specific promoter of the rice plasma membrane Ca2+ATPase mediates environmental stress responses in plants.

Background Plasma membrane Ca2+ATPase is a transport protein in the plasma membrane of cells and helps in removal of calcium (Ca2+) from the cell, hence regulating Ca2+ level within cells. Though plant Ca2+ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied. Results The 1478 bp promoter sequence of rice plasma membrane Ca2+ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca2+ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The −1478 to −886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for −1210 and −886 bp flanking region. The −1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The −1210 and −886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the −886 bp and −519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs. Conclusions The rice plasma membrane Ca2+ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the tissue-specificity and inducible nature of this promoter could grant wide applicability in plant biotechnology.

OsACA6, a P-type 2B Ca 2+ ATPase functions in cadmium stress tolerance in tobacco by reducing the oxidative stress load

Main conclusionThe present study demonstrates the first direct evidence of the novel role of OsACA6 in providing Cd2+ stress tolerance in transgenic tobacco by maintaining cellular ion homeostasis and modulating ROS-scavenging pathway.AbstractCadmium, a non-essential toxic heavy metal, interferes with the plant growth and development. It reaches the leaves through xylem and may become part of the food chain, thus causing detrimental effects to human health. Therefore, there is an urgent need to develop strategies for engineering plants for Cd2+ tolerance and less accumulation. The members of P-type ATPases family transport metal ions including Cd2+, and thus play important role an ion homeostasis. The present study elucidates the role of P-type 2B Ca2+ ATPase (OsACA6) in Cd2+ stress tolerance. The transcript levels of OsACA6 were up-regulated upon Cd2+, Zn2+ and Mn2+ exposure. Transgenic tobacco expressing OsACA6 showed tolerance towards Cd2+ stress as demonstrated by several physiological indices including root length, biomass, chlorophyll, malondialdehyde and hydrogen peroxide content. The roots of the transgenic lines accumulated more Cd2+ as compared to shoot. Further, confocal laser scanning microscopy showed that Cd2+ exposure altered Ca2+ uptake in OsACA6 transgenic plants. OsACA6 expression in tobacco also protected the transgenic plants from oxidative stress by enhancing the activity of enzymatic (SOD, CAT, APX, GR) and non-enzymatic (GSH and AsA) antioxidant machinery. Transgenic lines also tolerated Zn2+ and Mn2+ stress; however, tolerance for these ions was not as significant as observed for Cd2+ exposure. Thus, overexpression of OsACA6 confers Cd2+ stress tolerance in transgenic lines by maintaining cellular ion homeostasis and modulating reactive oxygen species (ROS)-scavenging pathway. The results of the present study will help to develop strategies for engineering Cd2+ stress tolerance in economically important crop plants.

Pea p68 Imparts Salinity Stress Tolerance in Rice by Scavenging of ROS-Mediated H2O2 and Interacts with Argonaute

The p68, a prototypic member of DEAD-box protein family, is involved in pre-mRNA splicing, RNA-induced silencing and transcription regulation. However, the role of plant p68 in stress tolerance and molecular targets responsible for this has not been reported. Here, we report the isolation and characterization of salinity-induced pea p68 (Psp68). The expression of Psp68 was more in aerial parts as compared to the roots in response to the abiotic stress. The transgenic-overexpressing Psp68 showed enhanced tolerance to salinity stress by efficiently attenuating ionic adjustment and scavenging reactive oxygen species (ROS). The enhanced tolerance was further confirmed by observing several physiological indices. Psp68-overexpressing rice accumulated higher K+ and Ca2+ and lower Na+ level as compared to the wild-type (WT) plants. Furthermore, Psp68 interacts with pea argonaute (AGO1), a catalytic component of the RNA-induced silencing complex (RISC) responsible for the gene silencing. The microarray analysis showed that Psp68 regulates many transcripts involved in the abiotic and oxidative stress responses as well as gene-silencing mechanisms in rice. Thus, the Psp68 functions as a molecular switch in different signaling pathways leading to stress tolerance. Overall, Psp68 may serve as a useful biotechnological tool for the improvement of stress tolerance crops.

An efficient and rapid regeneration via multiple shoot induction from mature seed derived embryogenic and organogenic callus of Indian maize (Zea mays L.)

An efficient method for in vitro micro propagation and genetic transformation of plants are crucial for both basic and applied research. Maize is one of the most important cereal crops around the world. Regeneration from immature embryo is hampered due to its unavailability round the year. On the contrary mature embryo especially tropical maize is recalcitrant toward tissue culture. Here we report a highly efficient regeneration (90%) system for maize by using 2 different approaches i.e., embryogenic and organogenic callus cultures. Seeds were germinated on MS medium supplemented with 5 mg/l 2,4-D and 3 mg/l BAP. Nodal regions of 2 wks old seedlings were longitudinally split upon isolation and subsequently placed on callus initiation medium. The maximum frequency of embryogenic callus formation (90%) was obtained on MS medium supplemented with 2 mg/l 2,4-D and 1 mg/l BAP in the dark conditions. The compact granular organogenic callus formation (85% frequency) was obtained on MS medium supplemented with 2.5 mg/l 2,4-D and 1.5 mg/l BAP at light conditions. MS medium supplemented with 2 mg/l BAP, 1 mg/l Kinetin and 0.5 mg/l NAA promoted the highest frequency of shoot induction. The highest frequency of root formation was observed when shoots were grown on MS medium. The regenerated plants were successfully hardened in earthen pots after adequate acclimatization. The important advantage of this improved method is shortening of regeneration time by providing an efficient and rapid regeneration tool for obtaining more stable transformants from mature seeds of Indian tropical maize cultivar (HQPM-1).

Salinity and drought tolerant OsACA6 enhances cold tolerance in transgenic tobacco by interacting with stress-inducible proteins

Plant Ca(2+)ATPases regulate many signalling pathways which are important for plant growth, development and abiotic stress responses. Our previous work identified that overexpression of OsACA6 confers salinity and drought tolerance in tobacco. In the present work we report, the function of OsACA6 in cold stress tolerance in transgenic tobacco plants. The expression of OsACA6 was induced by cold stress. The promoter-GUS fusion analyses in the different tissues of transgenic tobacoco confirmed that OsACA6 promoter is cold stress-inducible. Transgenic tobacco plants overexpressing OsACA6 exhibited cold tolerance compared to the wild type (WT) controls. The enhanced tolerance was confirmed by phenotypic analyses as well as by measuring germination, survival rate, chlorophyll content, cell membrane stability, malondialdehyde and proline content. Compared to the WT, the expression of catalase, ascorbate peroxidase and superoxide dismutase increased in the OsACA6 overexpressing plants, which was inversely correlated with the levels of H2O2 in the transgenic lines. We also identified interacting proteins of OsACA6 by using yeast two-hybrid screening assay. Most of the interacting partners of OsACA6 are associated with the widespread biological processes including plant growth, development, signalling and stress adaptation. Furthermore, we also confirmed that OsACA6 is able to self-interact. Overall, these results suggest that OsACA6 plays an important role in cold tolerance at least in part, by regulating antioxidants-mediated removal of reactive oxygen species or by interacting with different calcium signal decoders including calmodulin-like proteins (CaM) calcium/calmodulin dependent protein kinases (CDPKs) and receptor-like protein kinases (RLKs).

Isolation and functional characterization of the promoter of a DEAD-box helicase Psp68 using Agrobacterium-mediated transient assay.

Helicases are molecular motor proteins that perform a variety of cellular functions including transcription, translation, DNA replication and repair, RNA maturation, ribosome synthesis, nuclear export and splicing processes. The p68 is an evolutionarily conserved protein which plays pivotal roles in all aspect RNA metabolism processes. It is well established that helicases provides abiotic stress adaptation in plants but analysis of cis-regulatory elements present in the upstream regions is still infancy. Here we report isolation and functional characterization of the promoter of a DEAD-box helicase Psp68 in response to abiotic stress and hormonal regulation. The promoter of Psp68 was isolated by gene walking PCR from pea genomic DNA library constructed in BD genome walker kit. In silico analysis revealed that promoter of Psp68 contained a TATA, a CAAT motif and also harbors some important stress and hormone associated cis regulatory elements, including E-box, AGAAA, GATA-box, ACGT, GAAAA and GTCTC. Functional analyses were performed by Agrobacterium-mediated transient assay in tobacco leaves. Very high level of GUS activity was observed in agroinfiltrated tobacco leaves by the construct carrying the Psp68 promoter::GUS, subjected to abiotic stress and exogenous hormonal treatments. Stress-inducible nature of Psp68 promoter opens possibility for the study of the gene regulation under stress condition. Therefore, may be useful in the field of agriculture and biotechnology.