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Targeting heterologous multi-transmembrane domain (TMD) proteins to plant chloroplasts requires sequences in addition to the chloroplast transit peptide (cTP). The N-terminal domain (N-region), located C-terminal to the cTP in chloroplast inner envelope membrane proteins, is an essential region for import. However, it was unclear if the N-region functions solely as a spacer sequence to facilitate cTP access or if it plays an active role in the import process. This study addresses the N-region’s role by using combinations of cTPs and N-regions from Arabidopsis chloroplast inner envelope membrane proteins to direct the cyanobacterial protein SbtA to the chloroplast. We find that the sequence context of the N-region affects the chloroplast import efficiency of SbtA, with particular sequences mis-targeting the protein to different cellular sub-compartments. Additionally, specific cTP and N-region pairs exhibit varying targeting efficiencies for different heterologous proteins. Substituting individual N-region motifs did not significantly alter the chloroplast targeting efficiency of a particular cTP and N-region pair. We conclude that the N-region exhibits contextual functioning and potentially functional redundancy in motifs.Key message: The N-region, a sequence stretch C-terminal to the transit peptide (cTP), is necessary for the chloroplast import of multi-transmembrane domain proteins. The N-region sequence context provided to the cTP influences the chloroplast import efficiency of a heterologous multi-TMD protein. A particular cTP and N-region pair exhibits varying import efficiencies for different cargo proteins but is not dependent on the cargo size. Substituting amino acids in a specific N-region did not significantly alter the protein import efficiency, which suggests functional redundancy in N-region motifs. [ABSTRACT FROM AUTHOR]

This work aimed to design a synthetic salt-inducible promoter using a cis-engineering approach. The designed promoter (PS) comprises a minimal promoter sequence for basal-level expression and upstream cis-regulatory elements (CREs) from promoters of salinity-stress-induced genes. The copy number, spacer lengths, and locations of CREs were manually determined based on their occurrence within native promoters. The initial activity profile of the synthesized PS promoter in transiently transformed N. tabacum leaves shows a seven-fold, five-fold, and four-fold increase in reporter GUS activity under salt, drought, and abscisic acid stress, respectively, at the 24-h interval, compared to the constitutive CaMV35S promoter. Analysis of gus expression in stable Arabidopsis transformants showed that the PS promoter induces over a two-fold increase in expression under drought or abscisic acid stress and a five-fold increase under salt stress at 24- and 48-h intervals, compared to the CaMV35S promoter. The promoter PS exhibits higher and more sustained activity under salt, drought, and abscisic acid stress compared to the constitutive CaMV35S. [ABSTRACT FROM AUTHOR]

Key Message: Plasma membrane-localized AtAVT6D importing aspartic acid can be targeted to develop plants with enhanced osmotic and nitrogen-starvation tolerance. AtAVT6D promoter can be exploited as a stress-inducible promoter for genetic improvements to raise stress-resilient crops. The AtAVT6 family of amino acid transporters in Arabidopsis thaliana has been predicted to export amino acids like aspartate and glutamate. However, the functional characterization of these amino acid transporters in plants remains unexplored. The present study investigates the expression patterns of AtAVT6 genes in different tissues and under various abiotic stress conditions using quantitative Real-time PCR. The expression analysis demonstrated that the member AtAVT6D was significantly induced in response to phytohormone ABA and stresses like osmotic and drought. The tissue-specific expression analysis showed that AtAVT6D was strongly expressed in the siliques. Taking together these results, we can speculate that AtAVT6D might play a vital role in silique development and abiotic stress tolerance. Further, subcellular localization study showed AtAVT6D was localized to the plasma membrane. The heterologous expression of AtAVT6D in yeast cells conferred significant tolerance to nitrogen-deficient and osmotic stress conditions. The Xenopus oocyte studies revealed that AtAVT6D is involved in the uptake of Aspartic acid. While overexpression of AtAVT6D resulted in smaller siliques in Arabidopsis thaliana. Additionally, transient expression studies were performed with the full-length AtAVT6D promoter and its deletion constructs to study the effect of ACGT-N24-ACGT motifs on the reporter gene expression in response to abiotic stresses and ABA treatment. The fluorometric GUS analyses revealed that the promoter deletion construct-2 (Pro.C2) possessing a single copy of ACGT-N24-ACGT motif directed the strongest GUS expression under all the abiotic conditions tested. These results suggest that Pro.C2 can be used as a stress-inducible promoter to drive a significant transgene expression. [ABSTRACT FROM AUTHOR]

Glutathione-S transferase (GST) is a most ancient protein superfamily of multipurpose roles and evolved principally from gene duplication of an ancestral GSH binding protein. They have implemented in diverse plant functions such as detoxification of xenobiotic, secondary metabolism, growth and development, and majorly against biotic and abiotic stresses. The vital structural features of GSTs like highly divergent functional topographies, conserved integrated architecture with separate binding pockets for substrates and ligand, the stringent structural fidelity with high Tm values (50º–60º), and stress-responsive cis-regulatory elements in the promoter region offer this protein as most flexible plant protein for plant breeding approaches, biotechnological applications, etc. This review article summarizes the recent information of GST evolution, and their distribution and structural features with emphasis on the assorted roles of Ser and Cys GSTs with the signature motifs in their active sites, alongside their recent biotechnological application in the area of agriculture, environment, and nanotechnology have been highlighted. [ABSTRACT FROM AUTHOR]

Key message: The review describes the importance of amino acid transporters in plant growth, development, stress tolerance, and productivity. The promoter analysis provides valuable insights into their functionality leading to agricultural benefits. Arabidopsis thaliana genome is speculated to possess more than 100 amino acid transporter genes. This large number suggests the functional significance of amino acid transporters in plant growth and development. The current article summarizes the substrate specificity, cellular localization, tissue-specific expression, and expression of the amino acid transporter genes in response to environmental cues. However, till date functionality of a majority of amino acid transporter genes in plant development and stress tolerance is unexplored. Considering, that gene expression is mainly regulated by the regulatory motifs localized in their promoter regions at the transcriptional levels. The promoter regions (~ 1-kbp) of these amino acid transporter genes were analysed for the presence of cis-regulatory motifs responsive to developmental and external cues. This analysis can help predict the functionality of known and unexplored amino acid transporters in different tissues, organs, and various growth and development stages and responses to external stimuli. Furthermore, based on the promoter analysis and utilizing the microarray expression data we have attempted to identify plausible candidates (listed below) that might be targeted for agricultural benefits. [ABSTRACT FROM AUTHOR]

RuBisCO (Ribulose 1,5 bisphosphate carboxylase/oxygenase) by virtue of its dual specificity towards oxygen and carbon dioxide is an important rate-limiting step in photosynthesis and is believed to be the key factor for limited productivity of higher plants and algae. The photoautotrophic growth rate of cyanobacteria is a culmination of several factors including, rates of photosynthetic reactions, stress combating mechanisms and basic biomass generation metabolism in combination with optimal nutrient availability, irradiance, gaseous environment, etc. In case of cyanobacteria, the effect of RuBisCO in affecting the multiplication rate has been observed to show varied response. The current paper presents the RuBisCO activity of an early diverging cyanobacterium, Gloeobacter violaceus PCC 7421 and also compares the growth rates and RuBisCO activity of various cyanobacteria. A spectrophotometric estimation in a coupled enzyme assay system of the heterologous expressed G. violaceus PCC 7421 RuBisCO in E. coli, upon purification, revealed a carboxylation activity of LSu to be 5 nMol of phosphoglycerate min−1 mg−1 of protein, which is in coherence with the organism's slow growth rate. Further, the in vitro complementation of RbcL with RbcS in presence of RbcX of G. violaceus facilitated partial reconstitution of the protein and was hence found to cause a four-fold enhancement in its specific activity. The unique characteristics of the primitive cyanobacteria, such as, absence of thylakoids, lack of several photosystem constituting genes, slow carboxylation rate, pose limitation for its rapid multiplication. The RuBisCO carboxylation rate is observed as not the sole but an important parameter for obtaining optimal cell multiplication rates in photo-autotrophically multiplying cyanobacteria. [ABSTRACT FROM AUTHOR]

Abiotic and biotic stresses adversely affect plant growth and development and eventually result in less yield and threaten food security worldwide. In plants, several studies have been carried out to understand molecular responses to abiotic and biotic stresses. However, the complete circuitry of stress-responsive genes that plants utilise in response to those environmental stresses are still unknown. The protein phosphatase 2A (PP2A) gene has been known to have a crucial role in abiotic and biotic stresses; but how it regulates the stress response in plants is still not known completely. In this study, we constructed gene co-expression networks of PP2A genes with stress-responsive gene datasets from cold, drought, heat, osmotic, genotoxic, salt, and wounding stresses to unveil their relationships with the PP2A under different conditions of stress. The graph analysis identified 13 hub genes and several influential genes based on closeness centrality score (CCS). Our findings also revealed the count of unique genes present in different settings of stresses and subunits. We also formed clusters of influential genes based on the stress, CCS, and co-expression value. Analysis of cis-regulatory elements (CREs), recurring in promoters of these genes was also performed. Our study has led to the identification of 16 conserved CREs. [ABSTRACT FROM AUTHOR]

Agricultural yields have kept pace with the rising demands in the recent past as a result of the breeding and improved farming practices, but these practices alone will not be able to meet the demands of the future. The focus is now on the enhancement of the photosynthetic machinery. In photosynthesis, the rate limiting step is the one catalyzed by RuBisCO- Ribulose-1,5-bisphosphate carboxylase/oxygenase (4.1.1.39), which, because of its loose specificity and low turnover rate, is the primary target of most research programs directed towards improved photosynthesis. The other avenues of photosynthetic machinery that are under investigation to enhance it include-improved stomatal regulation and membrane permeability, RuBisCO with high specificity for CO and higher catalytic turnover; bypass of photorespiration and introduction of carbon concentrating mechanism (CCM) into the C plants. Carbon concentrating mechanisms cause accumulation of carbon dioxide in vicinity of RuBisCO producing a high CO/O ratio and hence an environment more suitable for carboxylation reactions than oxygenation reactions. This article includes the basic details of the major naturally occurring CCMs in various photosynthetic organisms to identify the knowledge gaps in each which could help study the prospects of its possible introduction into a non-native system as C plants which are devoid of any CCM. [ABSTRACT FROM AUTHOR]

This review provides an insight into the regulation of the carbon concentrating mechanisms (CCMs) in lower organisms like cyanobacteria, proteobacteria, and algae. CCMs evolved as a mechanism to concentrate CO at the site of primary carboxylating enzyme Ribulose-1, 5-bisphosphate carboxylase oxygenase (Rubisco), so that the enzyme could overcome its affinity towards O which leads to wasteful processes like photorespiration. A diverse set of CCMs exist in nature, i.e., carboxysomes in cyanobacteria and proteobacteria; pyrenoids in algae and diatoms, the C system, and Crassulacean acid metabolism in higher plants. Prime regulators of CCM in most of the photosynthetic autotrophs belong to the LysR family of transcriptional regulators, which regulate the activity of the components of CCM depending upon the ambient CO concentrations. Major targets of these regulators are carbonic anhydrase and inorganic carbon uptake systems (CO and HCO transporters) whose activities are modulated either at transcriptional level or by changes in the levels of their co-regulatory metabolites. The article provides information on the localization of the CCM components as well as their function and participation in the development of an efficient CCM. Signal transduction cascades leading to activation/inactivation of inducible CCM components on perception of low/high CO stimuli have also been brought into picture. A detailed study of the regulatory components can aid in identifying the unraveled aspects of these mechanisms and hence provide information on key molecules that need to be explored to further provide a clear understanding of the mechanism under study. [ABSTRACT FROM AUTHOR]

The oxygenase reaction catalyzed by RuBisCO became an issue only after the evolution of the oxygenic photosynthesis in cyanobacteria. Several strategies were developed by autotrophic organisms as an evolutionary response to increase oxygen levels to help RuBisCO maximize its net carboxylation rate. One of the crucial advancements in this context was the development of more efficient inorganic carbon transporters which could help in increasing the influx of inorganic carbon (Ci) at the site of CO2 fixation.We conducted a survey to find out the genes coding for cyanobacterial Ci transporters in 40 cyanobacterial phyla with respect to transporters present in Gloeobacter violaceous PCC 7421, an early-diverging cyanobacterium. An attempt was also made to correlate the prevalence of the kind of transporter present in the species with its habitat. Basically, two types of cyanobacterial inorganic carbon transporters exist, i.e. bicarbonate transporters and CO2-uptake systems. The transporters also show variation in context to their structure as some exist as single subunit proteins (BicA and SbtA), while others exist as multisubunit proteins (namely BCT1, NdhI3 and NdhI4). The phylogeny and distribution of the former have been extensively studied and the present analysis provides an insight into the latter ones. The in silico analysis of the genes under study revealed that their distribution was greatly influenced by the habitat and major environmental changes such as the great oxidation event (GOE) in the course of their evolution. [ABSTRACT FROM AUTHOR]

dvances in systematic computational biology and rapid elucidation of synergistic interplay between cis and trans factors governing transcriptional control have facilitated functional annotation of gene networks. The generation of data through deconstructive, reconstructive and database assisted promoter studies, and its integration to principles of synthetic engineering has started an era of designer promoters. Exploration of natural promoter architecture and the concept of cis engineering have not only enabled fine tuning of single or multiple transgene expression in response to perturbations in the chemical, physiological and environmental stimuli but also provided researchers with a unique answer to various problems in crop improvement in the form of bidirectional promoters. [ABSTRACT FROM AUTHOR]

To design, synthetic promoters leading to stress-specific induction of a transgene, the study of cis-regulatory elements is of great importance. Cis-regulatory elements play a major role in regulating the gene expression spatially and temporally at the transcriptional level. The present work focuses on one of the important cis-regulatory element, W-box having TGAC as a core motif which serves as a binding site for the members of the WRKY transcription factor family. In the present study, we have analyzed the occurrence frequency of TGAC core motifs for varying spacer lengths (ranging from 0 to 30 base pairs) across the Arabidopsis thaliana genome in order to determine the biological and functional significance of these conserved sequences. Further, the available microarray data was used to determine the role of TGAC motif in abiotic stresses namely salinity, osmolarity and heat. It was observed that TGAC motifs with spacer sequences like TGACCCATTTTGAC and TGACCCATGAATTTTGAC had a significant deviation in frequency and were thought to be favored for transcriptional bindings. The microarray data analysis revealed the involvement of TGAC motif mainly with genes down-regulated under abiotic stress conditions. These results were further confirmed by the transient expression studies with promoter-reporter cassettes carrying TGAC and TGAC-ACGT variant motifs with spacer lengths of 5 and 10. [ABSTRACT FROM AUTHOR]