Oxidation-reduction process of Arabidopsis thaliana roots induced by bisphenol compounds based on RNA-seq analysis.

Bisphenol compounds (BPs) have various industrial uses and can enter the environment through various sources. To evaluate the ecotoxicity of BPs and identify potential gene candidates involved in the plant toxicity, Arabidopsis thaliana was exposed to bisphenol A (BPA), BPB, BPE, BPF, and BPS at 1, 3, 10 mg/L for a duration of 14 days, and their growth status were monitored. At day 14, roots and leaves were collected for internal BPs exposure concentration detection, RNA-seq (only roots), and morphological observations. As shown in the results, exposure to BPs significantly disturbed root elongation, exhibiting a trend of stimulation at low concentration and inhibition at high concentration. Additionally, BPs exhibited pronounced generation of reactive oxygen species, while none of the pollutants caused significant changes in root morphology. Internal exposure concentration analysis indicated that BPs tended to accumulate in the roots, with BPS exhibiting the highest level of accumulation. The results of RNA-seq indicated that the shared 211 differently expressed genes (DEGs) of these 5 exposure groups were enriched in defense response, generation of precursor metabolites, response to organic substance, response to oxygen-containing, response to hormone, oxidation-reduction process and so on. Regarding unique DEGs in each group, BPS was mainly associated with the redox pathway, BPB primarily influenced seed germination, and BPA, BPE and BPF were primarily involved in metabolic signaling pathways. Our results provide new insights for BPs induced adverse effects on Arabidopsis thaliana and suggest that the ecological risks associated with BPA alternatives cannot be ignored.

PCIS1, encoded by a pentatricopeptide protein co-expressed gene, is required for splicing of three mitochondrial nad transcripts in angiosperms.

Group II introns are large catalytic RNAs, which reside mainly within genes encoding respiratory complex I (CI) subunits in angiosperms' mitochondria. Genetic and biochemical analyses led to the identification of many nuclear-encoded factors that facilitate the splicing of the degenerated organellar introns in plants. Here, we describe the analysis of the PPR Co-expressed Intron Splicing1 (PCIS1) factor, which was identified in-silico by its co-expression pattern with many PPR proteins. PCIS1 is well conserved in land plants but has no sequence similarity with any known protein motifs. PCIS1 mutant lines are arrested in embryogenesis and can be maintained by the temporal expression of the gene under the embryo-specific ABI3 promoter. The pABI3::PCIS1 mutant plants display low germination and stunted growth phenotypes. RNA-seq and RT-qPCR analyses of wild type and mutant plants indicated that PCIS1 is a novel splicing cofactor that is pivotal for the maturation of several nad transcripts in Arabidopsis mitochondria. These phenotypes are tightly associated with respiratory complex I defects and altered plant growth. Our data further emphasizes the key roles of nuclear-encoded cofactors that regulate the maturation and expression of mitochondrial transcripts for the biogenesis of the oxidative phosphorylation (OXPHOS) system, and hence for plant physiology. The discovery of novel splicing factors other than typical RNA-binding proteins suggests further complexity of splicing mechanisms in plant mitochondria.

Glucosinolate derivatives as antifungals: A review.

Fungal infections are becoming a severe threat to the security of global public health due to the extensive use of antibiotic medications and the rise in immune-deficient patients globally. Additionally, there is an increase in the development of fungus resistance to available antifungal medications. It is necessary to focus on the development of new antifungal medications in order to address these problems. The wide range of chemical structures, low cost, high availability, high antimicrobial action, and lack of adverse effects are the characteristics of plant secondary metabolites. In order to find and develop new antifungal medications, plant secondary metabolites like glucosinolate (GSL) derivatives are crucial sources of information. These natural compounds are enzymatically transformed into isothiocyanates (ITCs), nitriles, epithionitriles, oxazolidin-2-thion, and thiocyanate when they get mechanically damaged. The current review offers a thorough understanding of how isothiocyanates affect fungi with detailed mechanism. Along with this antifungal activity of nitriles, epithionitriles, oxazolidin-2-thion, and thiocyanate are mentioned. The review summarizes our present understanding of the following subjects: role of isothiocyanate by inhibiting aflatoxin biosynthesis, effect of isothiocyanate on transcriptomes, isothiocyanate targets cell membrane, role of isothiocyanate in efflux, and the role of isothiocyanate in synergistic activity. Antifungal activity of nitrile, epithionitrile, oxazolidine-2-thion, and thiocyanate is mentioned. Cytotoxicity study and clinical trials data were also added. More extensive studies will be needed in this field to assess safety concerns and clinical efficacies of GSL derivatives.

Analysis of Guard Cell Readouts Using Arabidopsis thaliana Isolated Epidermal Peels.

Stomata are pores surrounded by a pair of specialized cells, called guard cells, that play a central role in plant physiology through the regulation of gas exchange between plants and the environment. Guard cells have features like cell-autonomous responses and easily measurable readouts that have turned them into a model system to study signal transduction mechanisms in plants. Here, we provide a detailed protocol to analyze different physiological responses specifically in guard cells. We describe, in detail, the steps and conditions to isolate epidermal peels with tweezers and to analyze i) stomatal aperture in response to different stimuli, ii) cytosolic parameters such as hydrogen peroxide (H2O2), glutathione redox potential (E GSH), and MgATP-2 in vivo dynamics using fluorescent biosensors, and iii) gene expression in guard cell-enriched samples. The importance of this protocol lies in the fact that most living cells on epidermal peels are guard cells, enabling the preparation of guard cell-enriched samples. Key features • Isolation of epidermal peels as a monolayer enriched in guard cells • Measurement of cytosolic guard cell signaling component dynamics in isolated epidermal peels through fluorescent biosensor analysis • Gene expression analysis of guard cell-enriched isolated tissue.

Assays to Study Mitophagy in Plants.

Like most eukaryotic cells, mitophagy is essential in plant development and stress response. Several recent studies have revealed proteins that regulate this process, such as Friendly (FMT) and TraB family proteins (TRB), which are plant-unique mitophagy regulators so far. Here, we describe methods for studying mitophagy activity in plants through conventional microscopy and the use of loss-of-function mutants, such as using transgenic mitochondrial marker lines followed by image analysis, chemical inhibitor treatment, and plant phenotype studies. These methods can be used in combination to identify the putative mitophagy regulators and understand their functions in mitochondrial-related activities in plants.

Genetic Screening of Factors in the Plant Protein Secretion.

The endomembrane system in plants is composed of interconnected membrane organelles that contribute to intracellular structure and function. These organelles include the endoplasmic reticulum (ER), Golgi apparatus, vacuole, trans-Golgi network, and prevacuolar compartment or multivesicular body. Through vesicle-mediated transport, secreted proteins are synthesized in the ER and subsequently transported along the secretory pathway to the vacuole or outside of cells to fulfill specialized functions. Genetic screening is a crucial method for studying plant protein secretion. It entails identifying phenotypic differences resulting from genetic mutations, such as ethyl methanesulfonate, T-DNA insertion, and RNAi, to investigate gene function and discover mutants with specific traits or gene functions. Significant progress has been achieved in the study of plant protein secretion through genetic screening. In this protocol, we provide a step-by-step guide to studying the protein secretion pathway using a genetic screen approach. We use the example of the free 1 suppressor of Arabidopsis thaliana and oil body mutants of Marchantia polymorpha. Additionally, we offer an overview of genetic screening and briefly summarize the emerging technologies in the field of protein secretion research.

PASTICCINO2 interacting with Golgi Anti-Apoptotic Proteins resists endoplasmic reticulum stress dependent on very long-chain fatty acids.

The endoplasmic reticulum (ER) is crucial for maintaining cell homeostasis because it is the primary site for synthesizing secreted and transmembrane proteins and lipids. The unfolded protein response (UPR) is activated to restore ER homeostasis under ER stress. However, the relationship between lipids and the ER stress response in plants is not well understood. Arabidopsis Golgi anti-apoptotic proteins (GAAPs) are involved in resisting ER stress. To elucidate the function of GAAPs, PASTICCINO2 (PAS2), involved in very long-chain fatty acid (VLCFA) synthesis, was found to interact with GAAPs and IRE1. Single pas2 and gaap1/gaap2pas2 double mutants exhibited increased seedling damage and impaired UPR response under chronic ER stress. Site mutation combined with genetic analysis revealed that the role of PAS2 in resisting ER stress depended on its VLCFA synthesis domain. VLCFA contents were upregulated under ER stress, which required GAAPs. Exogenous VLCFAs partially restored the defect in UPR upregulation caused by PAS2 or GAAP mutations under chronic ER stress. These findings demonstrate that the association of PAS2 with GAAPs confers plant resistance to ER stress by regulating VLCFA synthesis and the UPR. This provides a basis for further studies on the connection between lipids and cell fate decisions under stress.

BRASSINOSTEROID-SIGNALING KINASE 1 modulates OPEN STOMATA 1 phosphorylation and contributes to stomatal closure and plant immunity.

Stomatal movement plays a critical role in plant immunity by limiting the entry of pathogens. OPEN STOMATA 1 (OST1) is a key component that mediates stomatal closure in plants, however, how OST1 functions in response to pathogens is not well understood. RECEPTOR-LIKE KINASE 902 (RLK902) phosphorylates BRASSINOSTEROID-SIGNALING KINASE 1 (BSK1) and positively modulates plant resistance. In this study, by a genome-wide phosphorylation analysis, we found that the phosphorylation of BSK1 and OST1 was missing in the rlk902 mutant compared with the wild-type plants, indicating a potential connection between the RLK902-BSK1 module and OST1-mediated stomatal closure. We showed that RLK902 and BSK1 contribute to stomatal immunity, as the stomatal closure induced by the bacterial pathogen Pto DC3000 was impaired in rlk902 and bsk1-1 mutants. Stomatal immunity mediated by RLK902 was dependent on BSK1 phosphorylation at Ser230, a key phosphorylation site for BSK1 functions. Several phosphorylation sites of OST1 were important for RLK902- and BSK1-mediated stomatal immunity. Interestingly, the phosphorylation of Ser171 and Ser175 in OST1 contributed to the stomatal immunity mediated by RLK902 but not by BSK1, while phosphorylation of OST1 at Ser29 and Thr176 residues was critical for BSK1-mediated stomatal immunity. Taken together, these results indicate that RLK902 and BSK1 contribute to disease resistance via OST1-mediated stomatal closure. This work revealed a new function of BSK1 in activating stomatal immunity, and the role of RLK902-BSK1 and OST1 module in regulating pathogen-induced stomatal movement.

Evolution of the basic Helix-Loop-Helix transcription factor SPATULA and its role in gynoecium development.

BACKGROUND AND AIMS: SPATULA (SPT) encodes a basic Helix-Loop-Helix transcription factor in Arabidopsis thaliana that functions in the development of the style, stigma and replum tissues, all of which arise from the carpel margin meristem (CMM) of the gynoecium. Here, we use a comparative approach to investigate the evolutionary history of SPT and identify changes that potentially contributed to its role in gynoecium development. METHODS: We investigate SPT's molecular and functional evolution using phylogenetic reconstruction, yeast-2-hybrid analyses of protein-protein interactions, microarray-based analyses of protein-DNA interactions, plant transformation assays, RNA in-situ hybridization, and in-silico analyses of promoter sequences. KEY RESULTS: We demonstrate the SPT lineage to have arisen at the base of euphyllophytes from a clade of potentially light-regulated transcription factors through gene duplication followed by the loss of an Active Phytochrome Binding (APB) domain. We also clarify the more recent evolutionary history of SPT and its paralog ALCATRAZ (ALC), which appear to have arisen through a large-scale duplication within Brassicales. We find that SPT orthologs from diverse groups of seed plants share strikingly similar capacities for protein-protein and protein-DNA interactions, and that SPT coding regions from a wide taxonomic range of plants are able to complement loss-of-function spt mutations in transgenic Arabidopsis. However, the expression pattern of SPT appears to have evolved significantly within angiosperms, and we identify structural changes in SPT's promoter region that correlate with the acquisition of high expression levels in tissues arising from the CMM in Brassicaeae. CONCLUSIONS: We conclude that changes to SPT's expression pattern made a major contribution to the evolution of its developmental role in the gynoecium of Brassicaeae. By contrast, the main biochemical capacities of SPT, as well as many of its immediate transcriptional targets, appear to have been conserved at least since the base of living angiosperms.

BAPID suppresses the inhibition of BRM on Di19-PR module in response to drought.

Drought is one of the most important abiotic stresses, and seriously threatens plant development and productivity. Increasing evidence indicates that chromatin remodelers are pivotal for plant drought response. However, molecular mechanisms of chromatin remodelers-mediated plant drought responses remain obscure. In this study, we found a novel interactor of BRM called BRM-associated protein involved in drought response (BAPID), which interacted with SWI/SNF chromatin remodeler BRM and drought-induced transcription factor Di19. Our findings demonstrated that BAPID acted as a positive drought regulator since drought tolerance was increased in BAPID-overexpressing plants, but decreased in BAPID-deficient plants, and physically bound to PR1, PR2, and PR5 promoters to mediate expression of PR genes to defend against dehydration stress. Genetic approaches demonstrated that BRM acted epistatically to BAPID and Di19 in drought response in Arabidopsis. Furthermore, the BAPID protein-inhibited interaction between BRM and Di19, and suppressed the inhibition of BRM on the Di19-PR module by mediating the H3K27me3 deposition at PR loci, thus changing nucleosome accessibility of Di19 and activating transcription of PR genes in response to drought. Our results shed light on the molecular mechanism whereby the BAPID-BRM-Di19-PRs pathway mediates plant drought responses. We provide data improving our understanding of chromatin remodeler-mediated plant drought regulation network.

The role of lysophosphatidic acid acyltransferase 1 in reproductive growth of Arabidopsis thaliana.

Lysophosphatidic acid acyltransferase1 (LPAT1) catalyzes the second step of de novo glycerolipid biosynthesis in chloroplasts. However, the embryonic-lethal phenotype of the knockout mutant suggested an unknown role for LPAT1 in non-photosynthetic reproductive organs. Reciprocal genetic crossing of the lpat1-1 heterozygous line suggested a female gametophytic defect of the lpat1-1 knockout mutant. By suppressing LPAT1 specifically during seed development, we showed that LPAT1 suppression affected silique growth and seed production. Glycerolipid analysis of the LPAT1 knockdown lines revealed a pronounced decrease of phosphatidylcholine (PC) content in mature siliques along with an altered polyunsaturation level of the polar glycerolipids. In seeds, the acyl composition of triacylglycerol (TAG) was altered albeit not the content. These results indicate that plastidic LPAT1 plays an important role in reproductive growth and extraplastidic glycerolipid metabolism involving PC and TAG.

Non-specific phospholipase C3 is involved in endoplasmic reticulum stress tolerance in Arabidopsis.

Non-specific phospholipase C (NPC) is an emerging family of lipolytic enzymes unique to plants and bacteria that play crucial roles in growth and stress responses. Among six copies of NPC isoforms found in Arabidopsis, the role of NPC3 remains elusive to date. Here, we show that NPC3 is a functional non-specific phospholipase C involved in tolerance to tunicamycin (TM)-induced endoplasmic reticulum (ER) stress through the synthesis of phosphocholine (PCho), a reaction product of NPC3. The npc3 mutant exhibited reduced sensitivity to TM treatment. Recombinant NPC3 possessed pronounced phospholipase C activity that hydrolyses phosphatidylcholine (PC). The hyposensitivity of npc3 to TM treatment was complemented by exogenous PCho, suggesting that NPC3-catalysed PCho production is involved in TM-induced ER stress tolerance. NPC3 was localized at the ER and was predominantly expressed in the roots, and it was further induced by TM-induced ER stress. Intriguingly, npc3 mutants showed a markedly reduced PCho content in shoots under ER stress. Our results indicate that ER stress induces NPC3 to produce PCho, which is involved in TM-induced ER stress tolerance.

Caught between two states: The compromise in acclimation of photosynthesis, transpiration and mesophyll conductance to different amplitudes of fluctuating irradiance.

While dynamic regulation of photosynthesis in fluctuating light is increasingly recognized as an important driver of carbon uptake, acclimation to realistic irradiance fluctuations is still largely unexplored. We subjected Arabidopsis thaliana (L.) wild-type and jac1 mutants to irradiance fluctuations with distinct amplitudes and average irradiance. We examined how irradiance fluctuations affected leaf structure, pigments and physiology. A wider amplitude of fluctuations produced a stronger acclimation response. Large reductions of leaf mass per area under fluctuating irradiance framed our interpretation of changes in photosynthetic capacity and mesophyll conductance as measured by three separate methods, in that photosynthetic investment increased markedly on a mass basis, but only a little on an area basis. Moreover, thermal imagery showed that leaf transpiration was four times higher under fluctuating irradiance. Leaves growing under fluctuating irradiance, although thinner, maintained their photosynthetic capacity, as measured through light- and CO2-response curves; suggesting their photosynthesis may be more cost-efficient than those under steady light, but overall may incur increased maintenance costs. This is especially relevant for plant performance globally because naturally fluctuating irradiance creates conflicting acclimation cues for photosynthesis and transpiration that may hinder progress towards ensuring food security under climate-related extremes of water stress.

Primary multistep phosphorelay activation comprises both cytokinin and abiotic stress responses: Insights from comparative analysis of Brassica type-A response regulators.

Multistep phosphorelay (MSP) signaling integrates hormonal and environmental signals to control both plant development and adaptive responses. The type-A RESPONSE REGULATORs (RRAs), the downstream members of the MSP cascade and cytokinin primary response genes are supposed to mediate primarily the negative feedback regulation of (cytokinin-induced) MSP signaling. However, the transcriptional data suggest the involvement of RRAs in stress-related responses as well. By employing evolutionary conservation with the well-characterized Arabidopsis thaliana RRAs, we identified 5 and 38 novel putative RRAs in Brassica oleracea and Brassica napus, respectively. Our phylogenetic analysis suggests the existence of gene-specific selective pressure, maintaining the homologs of ARR3, ARR6, and ARR16 as singletons during the evolution of Brassicaceae. We categorized RRAs based on the kinetics of their cytokinin-mediated upregulation and observed both similarities and specificities in this type of response across Brassicaceae species. Using bioinformatic analysis and experimental data demonstrating the cytokinin and abiotic stress responsiveness of A. thaliana-derived TCSv2 reporter, we unveil the mechanistic conservation of cytokinin- and stress-mediated upregulation of RRAs in Brassica rapa and Brassica napus. Notably, we identify partial cytokinin dependency of cold stress-induced RRA transcription, thus corroborating the role of cytokinin signaling in the crop adaptive responses.

Heavy metal toxicity leads to accumulation of insoluble proteins and induces endoplasmic reticulum stress-specific unfolded protein response in Arabidopsis thaliana.

Unfolded protein accumulation in the endoplasmic reticulum (ER) triggers ER stress, leading to a unique transcriptomic response called unfolded protein response (UPR). While ER stress is linked to various environmental stresses, its role in plant responses to heavy metal toxicity remains unclear. This study aimed to elucidate if heavy metals Fe, Zn, Cu, and As induce ER stress in plants. For this purpose, Arabidopsis thaliana seedlings were treated with Fe (200, 400 µM), Zn (500, 700 µM), Cu (25, 50 µM), and As (250, 500 µM) for 7 days, which resulted in 50-70% decrease in plant growth. All treatments increased insoluble protein levels, indicating unfolded protein accumulation, with the highest induction observed for 50 µM Cu treatment (fivefold). Expressions of genes involved in the perception and signaling of ER stress (IRE1, bZIP28, bZIP60, bZIP17) indicate that Zn toxicity specifically induces bZIP28 but not the IRE1 branch of UPR. All metals except Fe also induced genes associated with protein folding in the ER (BIP1, BIP3, and CNX) and ER-associated protein degradation (ERAD) (HRD1). This finding indicates Zn, Cu, and As but not Fe cause ER stress in plants. Furthermore, increased expression of ER oxidoreductase 1 (ERO1) suggests that metal toxicity also disrupts oxidative protein folding in the ER lumen. This study enhances our understanding of the intricate interplay between essential nutrients, metal toxicity, protein folding machinery, and ER stress, demonstrating that heavy metal toxicity has an ER stress component in plants alongside its established effects on energy metabolism, membrane integrity, and oxidative stress.

VIP1 and its close homologs confer mechanical stress tolerance in Arabidopsis leaves.

VIP1, an Arabidopsis thaliana basic leucine zipper transcription factor, and its close homologs are imported from the cytoplasm to the nucleus when cells are exposed to mechanical stress. They bind to AGCTG (G/T) and regulate mechanical stress responses in roots. However, their role in leaves is unclear. To clarify this, mutant lines (QM1 and QM2) that lack the functions of VIP1 and its close homologs (bZIP29, bZIP30 and PosF21) were generated. Brushing more severely damaged QM1 and QM2 leaves than wild-type leaves. Genes regulating stress responses and cell wall properties were downregulated in brushed QM2 leaves and upregulated in brushed VIP1-GFP-overexpressing (VIP1-GFPox) leaves compared to wild-type leaves in a transcriptome analysis. The VIP1-binding sequence AGCTG (G/T) was enriched in the promoters of genes downregulated in brushed QM2 leaves compared to wild-type leaves and in those upregulated in brushed VIP1-GFPox leaves. Calmodulin-binding transcription activators (CAMTAs) are known regulators of mechanical stress responses, and the CAMTA-binding sequence CGCGT was enriched in the promoters of genes upregulated in the brushed QM2 leaves and in those downregulated in the brushed VIP1-GFPox leaves. These findings suggest that VIP1 and its homologs upregulate genes via AGCTG (G/T) and influence CAMTA-dependent gene expression to enhance mechanical stress tolerance in leaves.

CNGC15-DMI1 Gating in Nuclear Calcium Signaling: Opening New Questions and Closing Controversies.

Nuclear Ca²âº signaling is crucial for symbiotic interactions between legumes and beneficial microbes, such as rhizobia and arbuscular mycorrhizal fungi. Key to generating repetitive nuclear Ca²âº oscillations are the ion channels DMI1 and CNGC15. Despite over 20 years of research on symbiotic nuclear Ca²âº spiking, important questions remain, including the exact function of the DMI1 channel. This review highlights recent developments that have filled knowledge gaps regarding the regulation of CNGC15 and its interplay with DMI1. We also explore new insights into the evolutionary conservation of DMI1-induced symbiotic nuclear Ca²âº oscillations and the roles of CNGC15 and DMI1 beyond symbiosis, such as in nitrate signaling, and discuss new questions this raises. As we delve deeper into the regulatory mechanisms and evolutionary history of these ion channels, we move closer to fully understanding the roles of nuclear Ca²âº signaling in plant life.

Temporal and spatial frameworks supporting plant responses to vegetation proximity.

After perception of vegetation proximity by phytochrome photoreceptors, shade-avoider plants initiate a set of responses known as the Shade Avoidance Syndrome (SAS). Shade perception by the phytochrome B (phyB) photoreceptor unleashes the PHYTOCHROME INTERACTING FACTORs (PIFs) and initiates SAS responses. In Arabidopsis (Arabidopsis thaliana) seedlings, shade perception involves rapid and massive changes in gene expression, increases auxin production, and promotes hypocotyl elongation. Other components, such as phyA and ELONGATED HYPOCOTYL 5 (HY5), also participate in the shade regulation of the hypocotyl elongation response by repressing it. However, why and how so many regulators with either positive or negative activities modulate the same response remain unclear. Our physiological, genetic, cellular, and transcriptomic analyses showed that (1) these components are organized into two main branches or modules and (2) the connection between them is dynamic and changes with the time of shade exposure. We propose a model for the regulation of shade-induced hypocotyl elongation in which the temporal and spatial functional importance of the various SAS regulators analyzed here helps to explain the co-existence of differentiated regulatory branches with overlapping activities.

Transcriptomic landscape of seedstick in Arabidopsis thaliana funiculus after fertilisation.

BACKGROUND: In Angiosperms, the continuation of plant species is intricately dependent on the funiculus multifaceted role in nutrient transport, mechanical support, and dehiscence of seeds. SEEDSTICK (STK) is a MADS-box transcription factor involved in seed size and abscission, and one of the few genes identified as affecting funiculus growth. Given the importance of the funiculus to a correct seed development, allied with previous phenotypic observations of stk mutants, we performed a transcriptomic analysis of stk funiculi from floral stage 17, using RNA-sequencing, to infer on the deregulated networks of genes. RESULTS: The generated dataset of differentially expressed genes was enriched with cell wall biogenesis, cell cycle, sugar metabolism and transport terms, all in accordance with stk phenotype observed in funiculi from floral stage 17. We selected eight differentially expressed genes for transcriptome validation using qPCR and/or promoter reporter lines. Those genes were involved with abscission, seed development or novel functions in stk funiculus, such as hormones/secondary metabolites transport. CONCLUSION: Overall, the analysis performed in this study allowed delving into the STK-network established in Arabidopsis funiculus, fulfilling a literature gap. Simultaneously, our findings reinforced the reliability of the transcriptome, making it a valuable resource for candidate genes selection for functional genetic studies in the funiculus. This will enhance our understanding on the regulatory network controlled by STK, on the role of the funiculus and how seed development may be affected by them.

The non-canonically organized members of MIR395 gene family in Brassica juncea are associated with developmentally regulated, sulfate-stress responsive bidirectional promoters that exhibit orientation-dependent differential transcriptional activity.

Several MICRORNA genes belonging to same family or different families are often found in homologous or non-homologous clusters. Among the various classes, head-to-head arranged genes form one of the largest categories of non-canonically organized genes. Such head-to-head arranged, non-canonically organized genes possibly share cis-regulatory region with the intergenic sequence having the potential to function as bi-directional promoter (BDP). The transcriptional regulation of head-to-head arranged genes, especially with bidirectional promoters, remains an enigma. In the past, bidirectional promoters have been characterized for a small set of protein-coding gene pairs in plants; however, to the best of our knowledge, no such study has been carried so far for MICRORNA genes. The present study thus functionally characterizes bidirectional promoters associated with members of MIR395 family, which is evolutionary conserved and is most frequently occurring cluster across plant kingdom. In Arabidopsis thaliana, the MIR395 gene family contains six members with two head-to-head arranged gene pairs- MIR395A-B and MIR395E-F. This organization was found to be conserved at seven loci for MIR395A-B, and eleven loci for MIR395E-F in five Brassica sps. Sequence analysis of the putative bidirectional promoters revealed variation in length, GC content and distribution of strict TATA-box. Comparatively higher level of conservation at both the ends of the bidirectional promoters, corresponding to ca. 250 bp upstream of 5'end of the respective MIRNA precursor, was observed. These conserved regions harbour several abiotic stress (nutrient, salt, drought) and hormone (ABA, ethylene) responsive cis-motifs. Functional characterization of putative bidirectional promoters associated with MIR395A-B and MIR395E-F from Arabidopsis and their respective orthologs from Brassica juncea (Bj_A08 MIR395A-B, Bj_B03 MIR395A-B, Bj_A07.1 MIR395E-F and Bj_A07.2 MIR395E-F) was carried out using a dual-reporter vector with ß-glucuronidase (GUS) and Green Fluorescent Protein (GFP). Analysis of transcriptional regulation of the two reporter genes - GUS and GFP during developmental stages confirmed their bidirectional nature. Orientation-dependent differential reporter activity indicated asymmetric nature of the promoters. Comparison of the reporter activity amongst orthologs, paralogs and homeologs revealed regulatory diversification, an outcome expected in polyploid genomes. Interestingly, reporter gene activities driven by selected bidirectional promoters were also observed in anther and siliques apart vegetative tissues indicating role of miR395 in anther and fruit development. Finally, we evaluated the activity of reporter genes driven under transcriptional regulation of bidirectional promoters under normal and sulfate-deprived conditions which revealed asymmetric inducibility under sulfate-starvation, in agreement with the known role of miR395 in sulfate homeostasis.