Sudden-Onset Blindness in a Child With Hypertensive Emergency: Unmasking of Chronic Kidney Disease.

A 10-year-old male presented with sudden-onset diminution of vision in both eyes. On systemic examination, he had severe hypertension, no pulse deficit, short stature, and no other focal neurological deficit. Dilated fundoscopy showed bilateral grade 4 hypertensive retinopathy with macular star formation. Detailed laboratory investigations revealed a stage 5 chronic kidney disease (CKD). We present this case to highlight this rare ocular manifestation of CKD in pediatric age group.

CIPK9 targets VDAC3 and modulates oxidative stress responses in Arabidopsis.

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.

Identification of Gene Copy Number in the Transgenic Plants by Quantitative Polymerase Chain Reaction (qPCR).

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.

VDACs: An Outlook on Biochemical Regulation and Function in Animal and Plant Systems.

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.

VDAC and its interacting partners in plant and animal systems: an overview.

Molecular trafficking between different subcellular compartments is the key for normal cellular functioning. Voltage-dependent anion channels (VDACs) are small-sized proteins present in the outer mitochondrial membrane, which mediate molecular trafficking between mitochondria and cytoplasm. The conductivity of VDAC is dependent on the transmembrane voltage, its oligomeric state and membrane lipids. VDAC acts as a convergence point to a diverse variety of mitochondrial functions as well as cell survival. This functional diversity is attained due to their interaction with a plethora of proteins inside the cell. Although, there are hints toward functional conservation/divergence between animals and plants; knowledge about the functional role of the VDACs in plants is still limited. We present here a comparative overview to provide an integrative picture of the interactions of VDAC with different proteins in both animals and plants. Also discussed are their physiological functions from the perspective of cellular movements, signal transduction, cellular fate, disease and development. This in-depth knowledge of the biological importance of VDAC and its interacting partner(s) will assist us to explore their function in the applied context in both plant and animal.

Arabidopsis Mitochondrial Voltage-Dependent Anion Channels Are Involved in Maintaining Reactive Oxygen Species Homeostasis, Oxidative and Salt Stress Tolerance in Yeast.

Voltage-dependent anion channels (VDACs) are conserved proteins of the mitochondria. We have functionally compared Arabidopsis VDACs using Saccharomyces cerevisiae Δpor1 and M3 yeast system. VDAC (1, 2, and 4) were able to restore Δpor1 growth in elevated temperature, in oxidative and salt stresses, whereas VDAC3 only partially rescued Δpor1 in these conditions. The ectopic expression of VDAC (1, 2, 3, and 4) in mutant yeast recapitulated the mitochondrial membrane potential thus, enabled it to maintain reactive oxygen species homeostasis. Overexpression of these VDACs (AtVDACs) in M3 strain did not display any synergistic or antagonistic activity with the native yeast VDAC1 (ScVDAC1). Collectively, our data suggest that Arabidopsis VDACs are involved in regulating respiration, reactive oxygen species homeostasis, and stress tolerance in yeast.

Genome analysis provides insight about pathogenesis of Indian strains of Rhizoctonia solani in rice.

The Rhizoctonia solani species complex is comprised of strains belonging to different anastomosis groups and causes diseases in several economically important crops, including rice. However, individuals within same anastomosis group exhibit distinct morphological and pathological differences on the same host. In this study, we have sequenced the genome of two aggressive Indian strains (BRS11 and BRS13) belonging to AG1-IA anastomosis group and compared them with the available genome of R. solani AG1-IA. We identified several SNPs and Indels in both of these genomes, in comparison to the AG1-IA genome. Furthermore, we observed expansion and emergence of orthogroups in these Indian strains and identified those potentially associated with pathogenesis. Amongst them, transposable elements, cell wall degrading enzymes, transcription factors, and oxalate decarboxylase were noteworthy. The current study unravels genetic variations and identifies genes that might account for pathogenicity variations amongst R. solani strains.

Alterations in plant sugar metabolism: signatory of pathogen attack.

MAIN CONCLUSION: This review summarizes the current understanding, future challenges and ongoing quest on sugar metabolic alterations that influence the outcome of plant-pathogen interactions. Intricate cellular and molecular events occur during plant-pathogen interactions. They cause major metabolic perturbations in the host and alterations in sugar metabolism play a pivotal role in governing the outcome of various kinds of plant-pathogen interactions. Sugar metabolizing enzymes and transporters of both host and pathogen origin get differentially regulated during the interactions. Both plant and pathogen compete for utilizing the host sugar metabolic machinery and in turn promote resistant or susceptible responses. However, the kind of sugar metabolism alteration that is beneficial for the host or pathogen is yet to be properly understood. Recently developed tools and methodologies are facilitating research to understand the intricate dynamics of sugar metabolism during the interactions. The present review elaborates current understanding, future challenges and ongoing quest on sugar metabolism, mobilization and regulation during various plant-pathogen interactions.

Identification of candidate pathogenicity determinants of Rhizoctonia solani AG1-IA, which causes sheath blight disease in rice.

Sheath blight disease is one of the predominant diseases of rice and it is caused by the necrotrophic fungal pathogen Rhizoctonia solani. The mechanistic insight about its widespread success as a broad host range pathogen is limited. In this study, we endeavor to identify pathogenicity determinants of R. solani during infection process in rice. Through RNAseq analysis, we identified a total of 65 and 232 R. solani (strain BRS1) genes to be commonly upregulated in three different rice genotypes (PB1, Tetep, and TP309) at establishment and necrotrophic phase, respectively. The induction of genes encoding extracellular protease, ABC transporter, and transcription factors were notable during establishment phase. While during necrotrophic phase, several CAZymes, sugar transporters, cellular metabolism, and protein degradation-related genes were prominently induced. We have also identified few putative secreted effector encoding genes that were upregulated during pathogenesis. The qPCR analysis further validated the phase-specific expression dynamics of some selected putative effectors and pathogenicity-associated genes. Overall, the present study reports identification of key genes and processes that might be crucial for R. solani pathogenesis. The ability to effectively damage host cell wall and survive in hostile plant environment by managing oxidative stress, cytotoxic compounds, etc. is being proposed to be important for pathogenesis of R. solani in rice. The functional characterization of these genes would provide key insights about this important pathosystem and facilitate development of strategies to control this devastating disease.

Alternative Splicing of CIPK3 Results in Distinct Target Selection to Propagate ABA Signaling in Arabidopsis.

Calcium (Ca2+) signaling is pivotal in transmission of information in the cell. Various Ca2+ sensing molecules work to sense and relay the encrypted messages to the intended targets in the cell to maintain this signal transduction. CBL-interacting protein kinases (CIPKs) are crucial components of Ca2+ signal transduction during various abiotic stresses. Although there are intron rich CIPKs in the plant genome but very little has been reported about their alternative splicing. Moreover the physiological significance of this event in the Ca2+ signaling is still elusive. Therefore in this study, we have selected CIPK3, which has highest number of splice variants amongst Arabidopsis CIPKs. Expression profiling of five splice variants of CIPK3 by qRT-PCR in four Arabidopsis thaliana ecotypes revealed preferential transcript accumulation but similar subcellular localization of the variants and interaction with similar CBLs. ABA and drought treatment resulted in the higher accumulation of the alternately spliced transcripts of CIPK3 in Arabidopsis ecotype Wassilewkija. The transcripts of CIPK3.1 and CIPK3.4 are relatively more induced compared to other alternative splice variants. Out of four splice variants studied, we found CIPK3.1 and CIPK3.2 showing preference for ABR1, a previously reported interactor of CIPK3. We conclude that the differential expression and choice of downstream partner by CIPK3-splice variants might be one of the mechanisms of Ca2+ mediated preferential regulation of ABA and other stress signals.

Alterations in rice chloroplast integrity, photosynthesis and metabolome associated with pathogenesis of Rhizoctonia solani.

Sheath blight disease is caused by a necrotrophic fungal pathogen Rhizoctonia solani and it continues to be a challenge for sustainable rice cultivation. In this study, we adopted a multi-pronged approach to understand the intricacies of rice undergoing susceptible interactions with R. solani. Extensive anatomical alteration, chloroplast localized ROS, deformed chloroplast ultrastructure along with decreased photosynthetic efficiency were observed in infected tissue. GC-MS based metabolite profiling revealed accumulation of glycolysis and TCA cycle intermediates, suggesting enhanced respiration. Several aromatic and aliphatic amino acids along with phenylpropanoid intermediates were also accumulated, suggesting induction of secondary metabolism during pathogenesis. Furthermore, alterations in carbon metabolism along with perturbation of hormonal signalling were highlighted in this study. The gene expression analysis including RNAseq profiling reinforced observed metabolic alterations in the infected tissues. In conclusion, the present study unravels key events associated during susceptible rice-R. solani interactions and identifies metabolites and transcripts that are accumulated in infected tissues.

Arabidopsis CBL interacting protein kinase 3 interacts with ABR1, an APETALA2 domain transcription factor, to regulate ABA responses.

Calcium (Ca2+) plays a vital role as a second messenger in several signaling pathways in plants. The calcineurin B-like proteins (CBLs) represent a family of plant calcium-binding proteins that function in propagating Ca2+ signals by interacting with CBL interacting protein kinases (CIPKs). Phosphorylation of CBL by CIPK is essential for the module to display full activity towards its target protein. Previous genetic analysis showed that the function of CBL9-CIPK3 module was implicated in negatively regulating seed germination and early development. In the present study, we have biochemically investigated the interaction of CBL9-CIPK3 module and our findings show that CBL9 is phosphorylated by CIPK3. Moreover, Abscisic acid repressor 1 (ABR1) is identified as the downstream target of CIPK3 and CIPK3-ABR1 function to regulate ABA responses during seed germination. Our study also indicates that the role of ABR1 is not limited to seed germination but it also regulates the ABA dependent processes in the adult stage of plant development. Combining our results, we conclude that the CBL9-CIPK3-ABR1 pathway functions to regulate seed germination and ABA dependent physiological processes in Arabidopsis.

A rice tonoplastic calcium exchanger, OsCCX2 mediates Ca2+/cation transport in yeast.

In plant cell, cations gradient in cellular compartments is maintained by synergistic action of various exchangers, pumps and channels. The Arabidopsis exchanger family members (AtCCX3 and AtCCX5) were previously studied and belong to CaCA (calcium cation exchangers) superfamily while none of the rice CCXs has been functionally characterized for their cation transport activities till date. Rice genome encode four CCXs and only OsCCX2 transcript showed differential expression under abiotic stresses and Ca(2+) starvation conditions. The OsCCX2 localized to tonoplast and suppresses the Ca(2+) sensitivity of K667 (low affinity Ca(2+) uptake deficient) yeast mutant under excess CaCl2 conditions. In contrast to AtCCXs, OsCCX2 expressing K667 yeast cells show tolerance towards excess Na(+), Li(+), Fe(2+), Zn(2+) and Co(2+) and suggest its ability to transport both mono as well as divalent cations in yeast. Additionally, in contrast to previously characterized AtCCXs, OsCCX2 is unable to complement yeast trk1trk2 double mutant suggesting inability to transport K(+) in yeast system. These finding suggest that OsCCX2 having distinct metal transport properties than previously characterized plant CCXs. OsCCX2 can be used as potential candidate for enhancing the abiotic stress tolerance in plants as well as for phytoremediation of heavy metal polluted soil.

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis.

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.

Comprehensive structural, interaction and expression analysis of CBL and CIPK complement during abiotic stresses and development in rice.

Calcium ion is involved in diverse physiological and developmental pathways. One of the important roles of calcium is a signaling messenger, which regulates signal transduction in plants. CBL (calcineurin B-like protein) is one of the calcium sensors that specifically interact with a family of serine-threonine protein kinases designated as CBL-interacting protein kinases (CIPKs). The coordination of these two gene families defines complexity of the signaling networks in several stimulus-response-coupling during various environmental stresses. In Arabidopsis, both of these gene families have been extensively studied. To understand in-depth mechanistic interplay of CBL-CIPK mediated signaling pathways, expression analysis of entire set of CBL and CIPK genes in rice genome under three abiotic stresses (salt, cold and drought) and different developmental stages (3-vegetative stages and 11-reproductive stages) were done using microarray expression data. Interestingly, expression analysis showed that rice CBLs and CIPKs are not only involved in the abiotic stress but their significant role is also speculated in the developmental processes. Chromosomal localization of rice CBL and CIPK genes reveals that only OsCBL7 and OsCBL8 shows tandem duplication among CBLs whereas CIPKs were evolved by many tandem as well as segmental duplications. Duplicated OsCIPK genes showed variable expression pattern indicating the role of gene duplication in the extension and functional diversification of CIPK gene family in rice. Arabidopsis SOS3/CBL4 related genes in rice (OsCBL4, OsCBL5, OsCBL7 and OsCBL8) were employed for interaction studies with rice and Arabidopsis CIPKs. OsCBLs and OsCIPKs are not only found structurally similar but likely to be functionally equivalent to Arabidopsis CBLs and CIPKs genes since SOS3/CBL4 related OsCBLs interact with more or less similarly to rice and Arabidopsis CIPKs and exhibited an interaction pattern comparable with Arabidopsis SOS3/CBL4.

Genome-wide expressional and functional analysis of calcium transport elements during abiotic stress and development in rice.

Ca²âº homeostasis is required to maintain a delicate balance of cytosolic Ca²âº during normal and adverse growth conditions. Various Ca²âº transporters actively participate to maintain this delicate balance especially during abiotic stresses and developmental events in plants. In this study, we present a genome-wide account, detailing expression profiles, subcellular localization and functional analysis of rice Ca²âº transport elements. Exhaustive in silico data mining and analysis resulted in the identification of 81 Ca²âº transport element genes, which belong to various groups such as Ca²âº-ATPases (pumps), exchangers, channels, glutamate receptor homologs and annexins. Phylogenetic analysis revealed that different Ca²âº transporters are evolutionarily conserved across different plant species. Comprehensive expression analysis by gene chip microarray and quantitative RT-PCR revealed that a substantial proportion of Ca²âº transporter genes were expressed differentially under abiotic stresses (salt, cold and drought) and reproductive developmental stages (panicle and seed) in rice. These findings suggest a possible role of rice Ca²âº transporters in abiotic stress and development triggered signaling pathways. Subcellular localization of Ca²âº transporters from different groups in Nicotiana benthamiana revealed their variable localization to different compartments, which could be their possible sites of action. Complementation of Ca²âº transport activity of K616 yeast mutant by Ca²âº-ATPase OsACA7 and involvement in salt tolerance verified its functional behavior. This study will encourage detailed characterization of potential candidate Ca²âº transporters for their functional role in planta.

Gene expression analysis of rice seedling under potassium deprivation reveals major changes in metabolism and signaling components.

Plant nutrition is one of the important areas for improving the yield and quality in crops as well as non-crop plants. Potassium is an essential plant nutrient and is required in abundance for their proper growth and development. Potassium deficiency directly affects the plant growth and hence crop yield and production. Recently, potassium-dependent transcriptomic analysis has been performed in the model plant Arabidopsis, however in cereals and crop plants; such a transcriptome analysis has not been undertaken till date. In rice, the molecular mechanism for the regulation of potassium starvation responses has not been investigated in detail. Here, we present a combined physiological and whole genome transcriptomic study of rice seedlings exposed to a brief period of potassium deficiency then replenished with potassium. Our results reveal that the expressions of a diverse set of genes annotated with many distinct functions were altered under potassium deprivation. Our findings highlight altered expression patterns of potassium-responsive genes majorly involved in metabolic processes, stress responses, signaling pathways, transcriptional regulation, and transport of multiple molecules including K(+). Interestingly, several genes responsive to low-potassium conditions show a reversal in expression upon resupply of potassium. The results of this study indicate that potassium deprivation leads to activation of multiple genes and gene networks, which may be acting in concert to sense the external potassium and mediate uptake, distribution and ultimately adaptation to low potassium conditions. The interplay of both upregulated and downregulated genes globally in response to potassium deprivation determines how plants cope with the stress of nutrient deficiency at different physiological as well as developmental stages of plants.

Plant omics: genome-wide analysis of ABA repressor1 (ABR1) related genes in rice during abiotic stress and development.

Rice is one of the world's most important food crops. Approximately 50% of rice production is affected by drought, an abiotic stress greatly impacting crop quality and yield. Agrigenomics research now offers the promise of understanding the drought stress impacts at a systems level. ABA repressor 1 (ABR1) is a member of the ethylene-responsive element-binding factor (ERF/AP2) superfamily of ERF transcription factors. We report here a global expression analysis of the rice ABR1-related genes where we identified the expression pattern of each Oryza sativa ERF (OsERF) during various developmental stages and abiotic stress treatments. The group X OsERFs, closely related genes orthologous to ABR1, exhibited significant differential expression profiles during certain stages of development and in response to abiotic stresses. We selected a subset of these genes and validated their observed expression profiles in response to abiotic stresses using quantitative RT-PCR. Moreover, we discovered that pairs of recently duplicated group X OsERFs display highly distinct expression profiles from one another. We determined the subcellular localization of two group X OsERF genes and observed localization to the nucleolus. To the best of our knowledge, this is the first report of localization of rice ERF protein to the nucleolus. This study also points out an overlap of expression under abiotic stress and reproductive developmental stages, indicating cross talk among different signaling pathways. This genome-wide expression analysis of rice ABR1 homologs paves the way for future functional analyses, with the goal to develop strategies to improve rice abiotic stress tolerance.

Comprehensive genomic analysis and expression profiling of phospholipase C gene family during abiotic stresses and development in rice.

BACKGROUND: Phospholipase C (PLC) is one of the major lipid hydrolysing enzymes, implicated in lipid mediated signaling. PLCs have been found to play a significant role in abiotic stress triggered signaling and developmental processes in various plant species. Genome wide identification and expression analysis have been carried out for this gene family in Arabidopsis, yet not much has been accomplished in crop plant rice. METHODOLOGY/PRINCIPAL FINDINGS: An exhaustive in-silico exploration of rice genome using various online databases and tools resulted in the identification of nine PLC encoding genes. Based on sequence, motif and phylogenetic analysis rice PLC gene family could be divided into phosphatidylinositol-specific PLCs (PI-PLCs) and phosphatidylcholine- PLCs (PC-PLC or NPC) classes with four and five members, respectively. A comparative analysis revealed that PLCs are conserved in Arabidopsis (dicots) and rice (monocot) at gene structure and protein level but they might have evolved through a separate evolutionary path. Transcript profiling using gene chip microarray and quantitative RT-PCR showed that most of the PLC members expressed significantly and differentially under abiotic stresses (salt, cold and drought) and during various developmental stages with condition/stage specific and overlapping expression. This finding suggested an important role of different rice PLC members in abiotic stress triggered signaling and plant development, which was also supported by the presence of relevant cis-regulatory elements in their promoters. Sub-cellular localization of few selected PLC members in Nicotiana benthamiana and onion epidermal cells has provided a clue about their site of action and functional behaviour. CONCLUSION/SIGNIFICANCE: The genome wide identification, structural and expression analysis and knowledge of sub-cellular localization of PLC gene family envisage the functional characterization of these genes in crop plants in near future.

Rice phospholipase A superfamily: organization, phylogenetic and expression analysis during abiotic stresses and development.

BACKGROUND: Phospholipase A (PLA) is an important group of enzymes responsible for phospholipid hydrolysis in lipid signaling. PLAs have been implicated in abiotic stress signaling and developmental events in various plants species. Genome-wide analysis of PLA superfamily has been carried out in dicot plant Arabidopsis. A comprehensive genome-wide analysis of PLAs has not been presented yet in crop plant rice. METHODOLOGY/PRINCIPAL FINDINGS: A comprehensive bioinformatics analysis identified a total of 31 PLA encoding genes in the rice genome, which are divided into three classes; phospholipase A(1) (PLA(1)), patatin like phospholipases (pPLA) and low molecular weight secretory phospholipase A(2) (sPLA(2)) based on their sequences and phylogeny. A subset of 10 rice PLAs exhibited chromosomal duplication, emphasizing the role of duplication in the expansion of this gene family in rice. Microarray expression profiling revealed a number of PLA members expressing differentially and significantly under abiotic stresses and reproductive development. Comparative expression analysis with Arabidopsis PLAs revealed a high degree of functional conservation between the orthologs in two plant species, which also indicated the vital role of PLAs in stress signaling and plant development across different plant species. Moreover, sub-cellular localization of a few candidates suggests their differential localization and functional role in the lipid signaling. CONCLUSION/SIGNIFICANCE: The comprehensive analysis and expression profiling would provide a critical platform for the functional characterization of the candidate PLA genes in crop plants.