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1.
Plant Physiol Biochem ; 211: 108613, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38696868

ABSTRACT

Ionic and metal toxicity in plants is still a global problem for the environment, agricultural productivity and ultimately poses human health threats when these metal ions accumulate in edible organs of plants. Metal and ion transport from cytosol to the vacuole is considered an important component of metal and ion tolerance and a plant's potential utility in phytoremediation. Finger millet (Eleusine coracana) is an orphan crop but has prominent nutritional value in comparison to other cereals. Previous transcriptomic studies suggested that one of the calcium/proton exchanger (EcCAX3) is strongly upregulated during different developmental stages of spikes development in plant. This finding led us to speculate that high calcium accumulation in the grain might be because of CAX3 function. Moreover, phylogenetic analysis shows that EcCAX3 is more closely related to foxtail millet, sorghum and rice CAX3 protein. To decipher the functional role of EcCAX3, we have adopted complementation of yeast triple mutant K677 (Δpmc1Δvcx1Δcnb1), which has defective calcium transport machinery. Furthermore, metal tolerance assay shows that EcCAX3 expression conferred tolerance to different metal stresses in yeast. The gain-of-function study suggests that EcCAX3 overexpressing Arabidopsis plants shows better tolerance to higher concentration of different metal ions as compared to wild type Col-0 plants. EcCAX3-overexpression transgenic lines exhibits abundance of metal transporters and cation exchanger transporter transcripts under metal stress conditions. Furthermore, EcCAX3-overexpression lines have higher accumulation of macro- and micro-elements under different metal stress. Overall, this finding highlights the functional role of EcCAX3 in the regulation of metal and ion homeostasis and this could be potentially utilized to engineer metal fortification and generation of stress tolerant crops in near future.


Subject(s)
Arabidopsis , Eleusine , Plants, Genetically Modified , Stress, Physiological , Eleusine/genetics , Eleusine/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Stress, Physiological/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Gene Expression Regulation, Plant/drug effects , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/drug effects , Phylogeny , Antiporters/metabolism , Antiporters/genetics , Metals/metabolism , Calcium/metabolism , Cation Transport Proteins , Arabidopsis Proteins
2.
Trends Plant Sci ; 2024 May 22.
Article in English | MEDLINE | ID: mdl-38782620

ABSTRACT

Potassium (K) is an essential macronutrient for plant development. Although the low-K+-responsive calcium (Ca2+) signaling pathway is known, its regulator remained elusive. Li et al. recently demonstrated that the target of rapamycin complex (TORC) and Ca2+ signaling pathways show reciprocal regulation of K+-responsive growth in plants.

3.
Int J Biol Macromol ; 257(Pt 2): 128769, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38096937

ABSTRACT

WRKY as the name suggests, are the transcription factors (TFs) that contain the signature WRKY domains, hence named after it. Since their discovery in 1994, they have been well studied in plants with exploration of approximately 74 WRKY genes in the model plant, Arabidopsis alone. However, the study of these transcription factors (TFs) is not just limited to model plant now. They have been studied widely in crop plants as well, because of their tremendous contribution in stress as well as in growth and development. Here, in this review, we describe the story of WRKY TFs from their identification to their origin, the binding mechanisms, structure and their contribution in regulating plant development and stress physiology. High throughput transcriptomics-based data also opened a doorway to understand the comprehensive and detailed functioning of WRKY TFs in plants. Indeed, the detailed functional role of each and every WRKY member in regulating the gene expression is required to pave the path to develop holistic understanding of their role in stress physiology and developmental processes in plants.


Subject(s)
Arabidopsis , Transcription Factors , Transcription Factors/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Plants/genetics , Plants/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Plant Development , Phylogeny , Stress, Physiological/genetics , Gene Expression Regulation, Plant
4.
Plant Physiol Biochem ; 206: 108237, 2024 Jan.
Article in English | MEDLINE | ID: mdl-38109831

ABSTRACT

Pathogen severely affects plant mitochondrial processes including respiration, however, the roles and mechanism of mitochondrial protein during the immune response remain largely unexplored. The interplay of plant hormone signaling during defense is an outcome of plant pathogen interaction. We recently discovered that the Arabidopsis calcineurin B-like interacting protein kinase 9 (AtCIPK9) interacts with the voltage-dependent anion channel 3 (AtVDAC3) and inhibits MV-induced oxidative damage. Here we report the characterization of AtVDAC3 in an antagonistic interaction pathway between abscisic acid (ABA) and salicylic acid (SA) signaling in Pseudomonas syringae -Arabidopsis interaction. In this study, we observed that mutants of AtVDAC3 were highly susceptible to Pseudomonas syringae infection as compared to the wild type (WT) Arabidopsis plants. Transcripts of VDAC3 and CIPK9 were inducible upon ABA application. Following pathogen exposure, expression analyses of ABA and SA biosynthesis genes indicated that the function of VDAC3 is required for isochorisimate synthase 1 (ICS1) expression but not for Nine-cis-epoxycaotenoid dioxygenase 3 (NCED3) expression. Despite the fact that vdac3 mutants had increased NCED3 expression in response to pathogen challenge, transcripts of ABA sensitive genes such as AtRD22 and AtRAB18 were downregulated even after exogenous ABA application. VDAC3 is required for ABA responsive genes expression upon exogenous ABA application. We also found that Pseudomonas syringae-induced SA signaling is downregulated in vdac3 mutants since overexpression of VDAC3 resulted in hyperaccumulation of Pathogenesis related gene1 (PR1) transcript. Interestingly, ABA application prior to P. syringae inoculation resulted in the upregulation of ABA responsive genes like Responsive to ABA18 (RAB18) and Responsive to dehydration 22 (RD22). Intriguingly, in the absence of AtVDAC3, Pst challenge can dramatically increase ABA-induced RD22 and RAB18 expression. Altogether our results reveal a novel Pathogen-SA-ABA interaction pathway in plants. Our findings show that ABA plays a significant role in modifying plant-pathogen interactions, owing to cross-talk with the biotic stress signaling pathways of ABA and SA.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Dioxygenases , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Salicylic Acid/pharmacology , Salicylic Acid/metabolism , Plant Growth Regulators/metabolism , Abscisic Acid/metabolism , Dioxygenases/genetics , Voltage-Dependent Anion Channels/genetics , Voltage-Dependent Anion Channels/metabolism , Pseudomonas syringae/physiology , Plant Diseases/genetics , Gene Expression Regulation, Plant , Protein Serine-Threonine Kinases/metabolism
5.
Biochem J ; 480(13): 1015-1034, 2023 07 12.
Article in English | MEDLINE | ID: mdl-37418287

ABSTRACT

Plants require the major macronutrients, nitrogen (N), phosphorus (P) and potassium (K) for normal growth and development. Their deficiency in soil directly affects vital cellular processes, particularly root growth and architecture. Their perception, uptake and assimilation are regulated by complex signalling pathways. To overcome nutrient deficiencies, plants have developed certain response mechanisms that determine developmental and physiological adaptations. The signal transduction pathways underlying these responses involve a complex interplay of components such as nutrient transporters, transcription factors and others. In addition to their involvement in cross-talk with intracellular calcium signalling pathways, these components are also engaged in NPK sensing and homeostasis. The NPK sensing and homeostatic mechanisms hold the key to identify and understand the crucial players in nutrient regulatory networks in plants under both abiotic and biotic stresses. In this review, we discuss calcium signalling components/pathways underlying plant responses to NPK sensing, with a focus on the sensors, transporters and transcription factors involved in their respective signalling and homeostasis.


Subject(s)
Calcium , Plants , Calcium/metabolism , Plants/metabolism , Soil , Homeostasis , Transcription Factors/genetics , Transcription Factors/metabolism
6.
Int J Biol Macromol ; 245: 125492, 2023 Aug 01.
Article in English | MEDLINE | ID: mdl-37343610

ABSTRACT

Calcium (Ca2+) signaling plays a major role in regulating multiple processes in living cells. The photoreceptor potential in Chlamydomonas triggers the generation of all or no flagellar Ca2+ currents that cause membrane depolarization across the eyespot and flagella. Modulation in membrane potential causes changes in the flagellar waveform, and hence, alters the beating patterns of Chlamydomonas flagella. The rhodopsin-mediated eyespot membrane potential is generated by the photoreceptor Ca2+ current or P-current however, the flagellar Ca2+ currents are mediated by unidentified voltage-gated calcium (VGCC or CaV) and potassium channels (VGKC). The voltage-gated ion channel that associates with ChRs to generate Ca2+ influx across the flagella and its cellular distribution has not yet been identified. Here, we identified putative VGCCs from algae and predicted their novel properties through insilico analysis. We further present experimental evidence on Chlamydomonas reinhardtii VGCCs to predict their novel physiological roles. Our experimental evidences showed that CrVGCC4 localizes to the eyespot and flagella of Chlamydomonas and associates with channelrhodopsins (ChRs). Further in silico interactome analysis of CrVGCCs suggested that they putatively interact with photoreceptor proteins, calcium signaling, and intraflagellar transport components. Expression analysis indicated that these VGCCs and their putative interactors can be perturbed by light stimuli. Collectively, our data suggest that VGCCs in general, and VGCC4 in particular, might be involved in the regulation of the photobehavioral response of Chlamydomonas.


Subject(s)
Chlamydomonas reinhardtii , Chlamydomonas , Chlamydomonas/metabolism , Calcium/metabolism , Calcium Channels/metabolism , Chlamydomonas reinhardtii/metabolism , Calcium Signaling
7.
Int J Biol Macromol ; 243: 125135, 2023 Jul 15.
Article in English | MEDLINE | ID: mdl-37247713

ABSTRACT

Translocation of channelrhodopsins (ChRs) is mediated by the intraflagellar transport (IFT) machinery. However, the functional role of the network involving photoreceptors, IFT and other proteins in controlling algal ciliary motility is still not fully delineated. In the current study, we have identified two important motifs at the C-terminus of ChR1, VXPX and LKNE. VXPX is a known ciliary targeting sequence in animals, and LKNE is a well-known SUMOylation motif. To the best of our knowledge, this study gives prima facie insight into the role of SUMOylation in Chlamydomonas. We prove that VMPS of ChR1 is important for interaction with GTPase CrARL11. We show that SUMO motifs are present in the C-terminus of putative ChR1s from green algae. Performing experiments with n-Ethylmaleimide (NEM) and Ubiquitin-like protease 1 (ULP-1), we show that SUMOylation may modulate ChR1 protein in Chlamydomonas. Experiments with 2D08, a known sumoylation blocker, increased the concentration of ChR1 protein. Finally, we show the endogenous SUMOylated proteins (SUMOylome) of C. reinhardtii, identified by using immunoprecipitation followed by nano-LC-MS/MS detection. This report establishes a link between evolutionarily conserved SUMOylation and ciliary machinery for the maintenance and functioning of cilia across the eukaryotes. Our enriched SUMOylome of C. reinhardtii comprehends the proteins related to ciliary development and photo-signaling, along with the orthologue(s) associated to human ciliopathies as SUMO targets.


Subject(s)
Chlamydomonas reinhardtii , Animals , Humans , Chlamydomonas reinhardtii/metabolism , Channelrhodopsins/metabolism , Tandem Mass Spectrometry , Biological Transport , Signal Transduction
8.
Plant Cell Environ ; 46(7): 1985-2006, 2023 07.
Article in English | MEDLINE | ID: mdl-37132157

ABSTRACT

Reactive oxygen species (ROS) and calcium (Ca2+ ) signalling are interconnected in the perception and transmission of environmental signals that control plant growth, development and defence. The concept that systemically propagating Ca2+ and ROS waves function together with electric signals in directional cell-to-cell systemic signalling and even plant-to-plant communication, is now firmly imbedded in the literature. However, relatively few mechanistic details are available regarding the management of ROS and Ca2+ signals at the molecular level, or how synchronous and independent signalling might be achieved in different cellular compartments. This review discusses the proteins that may serve as nodes or connecting bridges between the different pathways during abiotic stress responses, highlighting the crosstalk between ROS and Ca2+ pathways in cell signalling. We consider putative molecular switches that connect these signalling pathways and the molecular machinery that achieves the synergistic operation of ROS and Ca2+ signals.


Subject(s)
Calcium , Plants , Reactive Oxygen Species/metabolism , Calcium/metabolism , Plants/metabolism , Stress, Physiological , Signal Transduction
9.
Adv Protein Chem Struct Biol ; 134: 307-370, 2023.
Article in English | MEDLINE | ID: mdl-36858740

ABSTRACT

Protein phosphorylation is a vital reversible post-translational modification. This process is established by two classes of enzymes: protein kinases and protein phosphatases. Protein kinases phosphorylate proteins while protein phosphatases dephosphorylate phosphorylated proteins, thus, functioning as 'critical regulators' in signaling pathways. The eukaryotic protein phosphatases are classified as phosphoprotein phosphatases (PPP), metallo-dependent protein phosphatases (PPM), protein tyrosine (Tyr) phosphatases (PTP), and aspartate (Asp)-dependent phosphatases. The PPP and PPM families are serine (Ser)/threonine (Thr) specific phosphatases (STPs) that dephosphorylate Ser and Thr residues. The PTP family dephosphorylates Tyr residues while dual-specificity phosphatases (DsPTPs/DSPs) dephosphorylate Ser, Thr, and Tyr residues. The composition of these enzymes as well as their substrate specificity are important determinants of their functional significance in a number of cellular processes and stress responses. Their role in animal systems is well-understood and characterized. The functional characterization of protein phosphatases has been extensively covered in plants, although the comprehension of their mechanistic basis is an ongoing pursuit. The nature of their interactions with other key players in the signaling process is vital to our understanding. The substrates or targets determine their potential as well as magnitude of the impact they have on signaling pathways. In this article, we exclusively overview the various substrates of protein phosphatases in plant signaling pathways, which are a critical determinant of the outcome of various developmental and stress stimuli.


Subject(s)
Phosphoprotein Phosphatases , Protein Processing, Post-Translational , Animals , Phosphorylation , Protein Kinases , Signal Transduction
10.
Adv Protein Chem Struct Biol ; 134: 371-439, 2023.
Article in English | MEDLINE | ID: mdl-36858741

ABSTRACT

Calcium (Ca2+) signaling is versatile communication network in the cell. Stimuli perceived by cells are transposed through Ca2+-signature, and are decoded by plethora of Ca2+ sensors present in the cell. Calmodulin, calmodulin-like proteins, Ca2+-dependent protein kinases and calcineurin B-like proteins are major classes of proteins that decode the Ca2+ signature and serve in the propagation of signals to different parts of cells by targeting downstream proteins. These decoders and their targets work together to elicit responses against diverse stress stimuli. Over a period of time, significant attempts have been made to characterize as well as summarize elements of this signaling machinery. We begin with a structural overview and amalgamate the newly identified Ca2+ sensor protein in plants. Their ability to bind Ca2+, undergo conformational changes, and how it facilitates binding to a wide variety of targets is further embedded. Subsequently, we summarize the recent progress made on the functional characterization of Ca2+ sensing machinery and in particular their target proteins in stress signaling. We have focused on the physiological role of Ca2+, the Ca2+ sensing machinery, and the mode of regulation on their target proteins during plant stress adaptation. Additionally, we also discuss the role of these decoders and their mode of regulation on the target proteins during abiotic, hormone signaling and biotic stress responses in plants. Finally, here, we have enumerated the limitations and challenges in the Ca2+ signaling. This article will greatly enable in understanding the current picture of plant response and adaptation during diverse stimuli through the lens of Ca2+ signaling.


Subject(s)
Calcium , Calmodulin , Signal Transduction
11.
Int J Biol Macromol ; 237: 124163, 2023 May 15.
Article in English | MEDLINE | ID: mdl-36965564

ABSTRACT

Ca2+ signaling is an important biological process that enable to perceive and communicate information in the cell. Our current understanding of the signaling system suggests that plants and animals have certain differences in signal-sensing mechanisms. The Ca2+-mediated CBL-CIPK module has emerged as a major sensor responder network for Ca2+ signaling and has been speculated to be involved in plant terrestrial life adaptation. This module has previously been reported in Archaeplastids, Chromalveolates, and Excavates. In our experimental analysis of Chlamydomonas reinhardtii CBLs, we proved that the CrCBL1 protein interacts with Phototropin and Channelrhodopsin, and the expression of CrCBLs is modulated by light. Further analysis using chlorophyte and streptophyte algal sequences allowed us to identify the differences that have evolved in CBL and CIPK proteins since plants have progressed from aquatic to terrestrial habitats. Moreover, an investigation of Klebsormidium CBL and CIPK genes led us to know that they are abiotic stress stimuli-responsive, indicating that their role was defined very early during terrestrial adaptations. Structure-based prediction and Ca2+-binding assays indicated that the KnCBL1 protein in Klebsormidium showed a typical Ca2+-binding pocket. In summary, the results of this study suggest that these stress-responsive proteins enable crosstalk between Ca2+ and light signaling pathways very early during plant adaptation from aquatic to terrestrial habitats.


Subject(s)
Arabidopsis , Chlorophyta , Protein Serine-Threonine Kinases/metabolism , Arabidopsis/genetics , Calcium/metabolism , Calcium-Binding Proteins/metabolism , Plant Proteins/genetics , Plants/metabolism , Stress, Physiological , Calcium Signaling
12.
Biochem J ; 480(9): 555-571, 2023 05 15.
Article in English | MEDLINE | ID: mdl-36916393

ABSTRACT

Plant growth and development are governed by selective protein synthesis and degradation. Ubiquitination mediated protein degradation is governed by activating enzyme E1 followed by conjugating enzyme E2 and E3 ligase. Plant Armadillo (ARM) repeat/U-box (PUB) protein family is one of the important classes of E3 ligase. We studied the function of AtPUB2 by loss-of-function (knockout and knock down mutants) and gain-of-function (CaMV 35S promoter driven overexpression lines) approach in Arabidopsis. Under normal growth condition, we observed that loss-of-function mutant plants did not show any significant difference in growth when compared with wild-type possibly due to functional redundancy between PUB2 and PUB4. However, AtPUB2-OE lines exhibit early flowering and improved vegetative growth. Also, AtPUB2-OE seedlings showed sensitive phenotype in the presence of exogenous cytokinin. We found that AtPUB2 expression is induced under oxidative stress. Subcellular localization analysis shows that AtPUB2 is predominantly localized in the nucleus. We performed the phenotypic analysis under oxidative stress condition induced by methyl viologen (MV) and observed that overexpression lines display tolerance to oxidative stress in light and dark conditions. Furthermore, we found less amount of ROS accumulation, enhanced proline accumulation and decreased levels of MDA after MV treatment in AtPUB2-OE lines. PUB2-OE lines showed enhanced oxidative stress marker genes expression. By in vitro auto-ubiquitination assay, we also show that it possesses the E3 ligase activity. Overall, our findings suggest the possible role of AtPUB2 in plants ability to tolerate oxidative stress by enhancing the activity of antioxidant enzymes, which in turn improves ROS scavenging activity and homeostasis.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Reactive Oxygen Species/metabolism , Oxidative Stress , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Plants, Genetically Modified , Gene Expression Regulation, Plant , Stress, Physiological/genetics , Plant Proteins/metabolism
13.
Int J Biol Macromol ; 234: 123522, 2023 Apr 15.
Article in English | MEDLINE | ID: mdl-36758765

ABSTRACT

Glutamate receptors like channels (GLRs) are ligand gated non-selective cation channels and are multigenic in nature. They are homologs of mammalian ionic glutamate receptors (iGLRs) that play an important role in neurotransmission. It has been more than 25 years of discovery of plant GLRs, since then, significant progress has been made to unravel their structure and function in plants. Recently, the first crystal structure of plant GLR has been resolved that suggests that, though, plant GLRs contain the conserved signature domains of iGLRs, their unique features enable agonist/antagonist-dependent change in their activity. GLRs exhibit diverse subcellular localization and undergo dynamic expression variation in response to developmental and environmental stress conditions in plants. The combined use of genetic, electrophysiology and calcium imaging using different genetically encoded calcium indicators has revealed that GLRs are involved in generating calcium (Ca2+) influx across the plasma membrane and are involved in shaping the Ca2+ signature in response to different developmental and environmental stimuli. These findings indicate that GLRs influence cytosolic Ca2+ dynamics, thus, highlighting "GLR-Ca2+-crosstalk (GCC)" in developmental and stress-responsive signaling pathways. With this background, the present review summarises the recent developments pertaining to GLR function, in the broader context of regulation of stress tolerance in plants.


Subject(s)
Calcium , Animals , Calcium/metabolism , Calcium Signaling , Mammals/metabolism , Plants/genetics , Plants/metabolism , Receptors, Glutamate/genetics , Receptors, Glutamate/metabolism
14.
Physiol Mol Biol Plants ; 29(12): 1777-1811, 2023 Dec.
Article in English | MEDLINE | ID: mdl-38222278

ABSTRACT

Calcium ion (Ca2+) is a multifaceted signaling molecule that acts as an important second messenger. During the course of evolution, plants and animals have developed Ca2+ signaling in order to respond against diverse stimuli, to regulate a large number of physiological and developmental pathways. Our understanding of Ca2+ signaling and its components in physiological phenomena ranging from lower to higher organisms, and from single cell to multiple tissues has grown exponentially. The generation of Ca2+ transients or signatures for various stress factor is a well-known mechanism adopted in plant and animal systems. However, the decoding of such remarkable signatures is an uphill task and is always an interesting goal for the scientific community. In the past few decades, studies on the concentration and dynamics of intracellular Ca2+ are significantly increasing and have become a trend in modern biology. The advancement in approaches from Ca2+ binding dyes to in vivo Ca2+ imaging through the use of Ca2+ biosensors to achieve spatio-temporal resolution in micro and milliseconds range, provide us phenomenal opportunities to study live cell Ca2+ imaging or dynamics. Here, we describe the usage, improvement and advancement of Ca2+ based dyes, genetically encoded probes and sensors to achieve extraordinary Ca2+ imaging in plants and animals.

15.
Plant J ; 109(1): 241-260, 2022 01.
Article in English | MEDLINE | ID: mdl-34748255

ABSTRACT

Calcium (Ca2+ ) is widely recognized as a key second messenger in mediating various plant adaptive responses. Here we show that calcineurin B-like interacting protein kinase CIPK9 along with its interacting partner VDAC3 identified in the present study are involved in mediating plant responses to methyl viologen (MV). CIPK9 physically interacts with and phosphorylates VDAC3. Co-localization, co-immunoprecipitation, and fluorescence resonance energy transfer experiments proved their physical interaction in planta. Both cipk9 and vdac3 mutants exhibited a tolerant phenotype against MV-induced oxidative stress, which coincided with the lower-level accumulation of reactive oxygen species in their roots. In addition, the analysis of cipk9vdac3 double mutant and VDAC3 overexpressing plants revealed that CIPK9 and VDAC3 were involved in the same pathway for inducing MV-dependent oxidative stress. The response to MV was suppressed by the addition of lanthanum chloride, a non-specific Ca2+ channel blocker indicating the role of Ca2+ in this pathway. Our study suggest that CIPK9-VDAC3 module may act as a key component in mediating oxidative stress responses in Arabidopsis.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Protein Serine-Threonine Kinases/metabolism , Voltage-Dependent Anion Channels/metabolism , Arabidopsis/drug effects , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Gene Expression Regulation, Plant , Lanthanum/pharmacology , Oxidative Stress , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Voltage-Dependent Anion Channels/genetics
16.
Methods Mol Biol ; 2392: 17-33, 2022.
Article in English | MEDLINE | ID: mdl-34773612

ABSTRACT

Classical restriction fragment length polymorphism (RFLP) and sequencing are labor-intensive and expensive methods to study single base changes, whereas polymerase chain reaction amplification of specific alleles (PASA) or allele-specific polymerase chain reaction (ASPCR) is a PCR-based application that allows direct detection of any point mutation by analyzing the PCR products in an ethidium bromide-stained agarose or polyacrylamide gel. PASA is based on oligonucleotide primers containing one or more 3' mismatch with the target DNA making it refractory to primer extension by Thermus aquaticus DNA polymerase lacking the 3' to 5' exonuclease proofreading activity because of which it is also called amplification refractory mutation system-PCR (ARMS-PCR). This technique has found application in detection of allele, mutation, single-nucleotide polymorphisms (SNPs) causing genetic and infectious diseases. This chapter describes an approach of cohort PASA in context of genotyping single and double mutant worms generated to study the process of cell migration and axon outgrowth in C. elegans. Single worm-based cohort PASA allows genotyping for identification of single base mutations; particularly it is convenient method to detect mutations without a visible phenotype.


Subject(s)
Caenorhabditis elegans , Polymorphism, Single Nucleotide , Alleles , Animals , Caenorhabditis elegans/genetics , Genotype , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Taq Polymerase , Thermus
17.
Methods Mol Biol ; 2392: 161-171, 2022.
Article in English | MEDLINE | ID: mdl-34773622

ABSTRACT

Transgenic events are defined as exogenous DNA insertion in the genome through genetic transformation. It is a powerful means for the improvement of crop plants and to understand the gene function. Multiple DNA insertion events may occur at one or several chromosomal locations. One of the important tasks, after validation of the transformation of transgenic plants, is the identification of single copy in the transgenic. This means the insertion of exogenous DNA fragment only in a single locus in the genome. Southern blot hybridization is a convincing and reliable method, for estimation of copy number in transgenic lines but it is cumbersome and time-consuming process. One of the other well-known methods is quantitative polymerase chain reactions (qPCR), a simple and rapid method to identify copy number from a population of independent transgenic lines. In comparison to the Southern hybridization method, qPCR is simpler to perform, requires less DNA, lesser time and does not require any labeled probes. This method utilizes specific primers to amplify target transgenes and endogenous reference genes. Designing an appropriate and specific primer pair is a very crucial part of the estimation of the gene copy number. In this chapter, we have illustrated a detailed methodology for identification of the gene copy of the transgenic plants.


Subject(s)
Gene Dosage , Blotting, Southern , Plants, Genetically Modified/genetics , Real-Time Polymerase Chain Reaction , Transgenes
18.
Plant Cell Rep ; 40(11): 2111-2122, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34415375

ABSTRACT

Calcium (Ca2+) is a universal second messenger essential for the growth and development of plants in normal and stress situations. In plants, the proteins, CBL (calcineurin B-like) and CIPK (CBL-interacting protein kinase), form one of the important Ca2+ decoding complexes to decipher Ca2+ signals elicited by environmental challenges. Multiple interactors distinguish CBL and CIPK protein family members to form a signaling network for regulated perception and transduction of environmental signals, e.g., signals generated under nutrient stress conditions. Conservation of equilibrium in response to varying soil nutrient status is an important aspect for plant vigor and yield. Signaling processes have been reported to observe nutrient fluctuations as a signal responsible for regulated nutrient transport adaptation. Recent studies have identified downstream targets of CBL-CIPK modules as ion channels or transporters and their association in signaling nutrient disposal including potassium, nitrate, ammonium, magnesium, zinc, boron, and iron. Ca2+-CBL-CIPK pathway modulates ion transporters/channels and hence maintains a homeostasis of several important plant nutrients in the cytosol and sub-cellular compartments. In this article, we summarize recent literature to discuss the role of the Ca2+-CBL-CIPK pathway in cellular osmoregulation and homeostasis on exposure to nutrient excess or deprived soils. This further establishes a link between taking up the nutrient in the roots and its distribution and homeostasis during the generation of signal for the development and survival of plants.


Subject(s)
Plant Physiological Phenomena , Plant Proteins/metabolism , Protein Kinases/metabolism , Arabidopsis Proteins/metabolism , Calcium Signaling , Calcium-Binding Proteins/metabolism , Carbon/metabolism , Iron/metabolism , Magnesium/metabolism , Nitrogen/metabolism , Potassium/metabolism , Protein Serine-Threonine Kinases/metabolism , Soil/chemistry
19.
Front Physiol ; 12: 683920, 2021.
Article in English | MEDLINE | ID: mdl-34421635

ABSTRACT

The voltage-dependent anion channels (VDACs) are the most abundant proteins present on the outer mitochondrial membrane. They serve a myriad of functions ranging from energy and metabolite exchange to highly debatable roles in apoptosis. Their role in molecular transport puts them on the center stage as communicators between cytoplasmic and mitochondrial signaling events. Beyond their general role as interchangeable pores, members of this family may exhibit specific functions. Even after nearly five decades of their discovery, their role in plant systems is still a new and rapidly emerging field. The information on biochemical regulation of VDACs is limited. Various interacting proteins and post-translational modifications (PTMs) modulate VDAC functions, amongst these, phosphorylation is quite noticeable. In this review, we have tried to give a glimpse of the recent advancements in the biochemical/interactional regulation of plant VDACs. We also cover a critical analysis on the importance of PTMs in the functional regulation of VDACs. Besides, the review also encompasses numerous studies which can identify VDACs as a connecting link between Ca2+ and reactive oxygen species signaling in special reference to the plant systems.

20.
Plant Cell Rep ; 40(11): 2205-2223, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34250550

ABSTRACT

KEY MESSAGE: Overexpression of finger millet calmodulin imparts drought and salt tolerance in plants. Drought and salinity are major environmental stresses which affect crop productivity and therefore are major hindrance in feeding growing population world-wide. Calcium (Ca2+) signaling plays a crucial role during the plant's response to these stress stimuli. Calmodulin (CaM), a crucial Ca2+sensor, is involved in transducing the signal downstream in various physiological, developmental and stress responses by modulating a plethora of target proteins. The role of CaM has been well established in the model plant Arabidopsis thaliana for regulating various developmental processes, stress signaling and ion transport. In the current study, we investigate the CaM of Eleusine coracana (common name finger millet, known especially for its drought tolerance and superior Ca2+ content). In-silico analysis showed that Eleusine CaM (EcCaM) has greater similarity to rice CaM as compared to Arabidopsis CaM due to the presence of highly conserved four EF-hand domains. To decipher the in-planta function of EcCaM, we have adopted the gain-of-function approach by generating the 35S::EcCaM over-expression transgenic in Arabidopsis. Overexpression of EcCaM in Arabidopsis makes the plant tolerant to polyethylene glycol (PEG) induced drought and salt stress (NaCl) as demonstrated by post-germination based phenotypic assay, ion leakage, MDA and proline estimation, ROS detection under stressed and normal conditions. Moreover, EcCaM overexpression leads to hypersensitivity toward exogenously applied ABA at the seed germination stage. These findings reveal that EcCaM mediates tolerance to drought and salinity stress. Also, our results indicate that EcCaM is involved in modulating ABA signaling. Summarizing our results, we report for the first time that EcCaM is involved in modulating plants response to stress and this information can be used for the generation of future-ready crops that can tolerate a wide range of abiotic stresses.


Subject(s)
Arabidopsis/physiology , Calmodulin/genetics , Eleusine/genetics , Plant Proteins/genetics , Salt Tolerance/genetics , Abscisic Acid/metabolism , Abscisic Acid/pharmacology , Arabidopsis/anatomy & histology , Arabidopsis/drug effects , Arabidopsis/genetics , Calmodulin/chemistry , Calmodulin/metabolism , Chlorophyll/genetics , Chlorophyll/metabolism , Droughts , Eleusine/drug effects , Gene Expression Regulation, Plant , Oryza/chemistry , Plant Proteins/chemistry , Plant Proteins/metabolism , Plants, Genetically Modified , Polyethylene Glycols/pharmacology , Protein Domains , Reactive Oxygen Species/metabolism , Salinity
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