State Key Laboratory of Plant Physiology and Biochemistry

The agriGO platform, which has been serving the scientific community for >10 years, specifically focuses on gene ontology (GO) enrichment analyses of plant and agricultural species. We continuously maintain and update the databases and accommodate the various requests of our global users. Here, we present our updated agriGO that has a largely expanded number of supporting species (394) and datatypes (865). In addition, a larger number of species have been classified into groups covering crops, vegetables, fish, birds and insects closely related to the agricultural community. We further improved the computational efficiency, including the batch analysis and P-value distribution (PVD), and the user-friendliness of the web pages. More visualization features were added to the platform, including SEACOMPARE (cross comparison of singular enrichment analysis), direct acyclic graph (DAG) and Scatter Plots, which can be merged by choosing any significant GO term. The updated platform agriGO v2.0 is now publicly accessible at http://systemsbiology.cau.edu.cn/agriGOv2/.

Gene Ontology (GO), the de facto standard in gene functionality description, is used widely in functional annotation and enrichment analysis. Here, we introduce agriGO, an integrated web-based GO analysis toolkit for the agricultural community, using the advantages of our previous GO enrichment tool (EasyGO), to meet analysis demands from new technologies and research objectives. EasyGO is valuable for its proficiency, and has proved useful in uncovering biological knowledge in massive data sets from high-throughput experiments. For agriGO, the system architecture and website interface were redesigned to improve performance and accessibility. The supported organisms and gene identifiers were substantially expanded (including 38 agricultural species composed of 274 data types). The requirement on user input is more flexible, in that user-defined reference and annotation are accepted. Moreover, a new analysis approach using Gene Set Enrichment Analysis strategy and customizable features is provided. Four tools, SEA (Singular enrichment analysis), PAGE (Parametric Analysis of Gene set Enrichment), BLAST4ID (Transfer IDs by BLAST) and SEACOMPARE (Cross comparison of SEA), are integrated as a toolkit to meet different demands. We also provide a cross-comparison service so that different data sets can be compared and explored in a visualized way. Lastly, agriGO functions as a GO data repository with search and download functions; agriGO is publicly accessible at http://bioinfo.cau.edu.cn/agriGO/.

BACKGROUND: To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required. RESULTS: We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation. CONCLUSIONS: We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.

Contents Summary 523 I. Introduction 523 II. Sensing salt stress 524 III. Ion homeostasis regulation 524 IV. Metabolite and cell activity responses to salt stress 527 V. Conclusions and perspectives 532 Acknowledgements 533 References 533 SUMMARY: Excess soluble salts in soil (saline soils) are harmful to most plants. Salt imposes osmotic, ionic, and secondary stresses on plants. Over the past two decades, many determinants of salt tolerance and their regulatory mechanisms have been identified and characterized using molecular genetics and genomics approaches. This review describes recent progress in deciphering the mechanisms controlling ion homeostasis, cell activity responses, and epigenetic regulation in plants under salt stress. Finally, we highlight research areas that require further research to reveal new determinants of salt tolerance in plants.

Salt stress is a major environmental factor limiting plant growth and productivity. A better understanding of the mechanisms mediating salt resistance will help researchers design ways to improve crop performance under adverse environmental conditions. Salt stress can lead to ionic stress, osmotic stress and secondary stresses, particularly oxidative stress, in plants. Therefore, to adapt to salt stress, plants rely on signals and pathways that re-establish cellular ionic, osmotic, and reactive oxygen species (ROS) homeostasis. Over the past two decades, genetic and biochemical analyses have revealed several core stress signaling pathways that participate in salt resistance. The Salt Overly Sensitive signaling pathway plays a key role in maintaining ionic homeostasis, via extruding sodium ions into the apoplast. Mitogen-activated protein kinase cascades mediate ionic, osmotic, and ROS homeostasis. SnRK2 (sucrose nonfermenting 1-related protein kinase 2) proteins are involved in maintaining osmotic homeostasis. In this review, we discuss recent progress in identifying the components and pathways involved in the plant's response to salt stress and their regulatory mechanisms. We also review progress in identifying sensors involved in salt-induced stress signaling in plants.

Arabidopsis mutants produced by constitutive overexpression of the CRISPR/Cas9 genome editing system are usually mosaics in the T1 generation. In this study, we used egg cell-specific promoters to drive the expression of Cas9 and obtained non-mosaic T1 mutants for multiple target genes with high efficiency. Comparisons of 12 combinations of eight promoters and two terminators found that the efficiency of the egg cell-specific promoter-controlled CRISPR/Cas9 system depended on the presence of a suitable terminator, and the composite promoter generated by fusing two egg cell-specific promoters resulted in much higher efficiency of mutation in the T1 generation compared with the single promoters.

Contents Summary I. Introduction II. Cold stress and physiological responses in plants III. Sensing of cold signals in plants IV. Messenger molecules involved in cold signal transduction V. Cold signal transduction in plants VI. Conclusions and perspectives Acknowledgements References SUMMARY: Cold stress is a major environmental factor that seriously affects plant growth and development, and influences crop productivity. Plants have evolved a series of mechanisms that allow them to adapt to cold stress at both the physiological and molecular levels. Over the past two decades, much progress has been made in identifying crucial components involved in cold-stress tolerance and dissecting their regulatory mechanisms. In this review, we summarize recent major advances in our understanding of cold signalling and put forward open questions in the field of plant cold-stress responses. Answering these questions should help elucidate the molecular mechanisms underlying plant tolerance to cold stress.

The phytohormone ethylene regulates multiple aspects of plant growth and development and responses to environmental stress. However, the exact role of ethylene in freezing stress remains unclear. Here, we report that ethylene negatively regulates plant responses to freezing stress in Arabidopsis thaliana. Freezing tolerance was decreased in ethylene overproducer1 and by the application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid but increased by the addition of the ethylene biosynthesis inhibitor aminoethoxyvinyl glycine or the perception antagonist Ag+. Furthermore, ethylene-insensitive mutants, including etr1-1, ein4-1, ein2-5, ein3-1, and ein3 eil1, displayed enhanced freezing tolerance. By contrast, the constitutive ethylene response mutant ctr1-1 and EIN3-overexpressing plants exhibited reduced freezing tolerance. Genetic and biochemical analyses revealed that EIN3 negatively regulates the expression of CBFs and type-A Arabidopsis response regulator5 (ARR5), ARR7, and ARR15 by binding to specific elements in their promoters. Overexpression of these ARR genes enhanced the freezing tolerance of plants. Thus, our study demonstrates that ethylene negatively regulates cold signaling at least partially through the direct transcriptional control of cold-regulated CBFs and type-A ARR genes by EIN3. Our study also provides evidence that type-A ARRs function as key nodes to integrate ethylene and cytokinin signaling in regulation of plant responses to environmental stress.

As one of the most important mineral nutrient elements, potassium (K(+)) participates in many plant physiological processes and determines the yield and quality of crop production. In this review, we summarize K(+) signaling processes and K(+) transport regulation in higher plants, especially in plant responses to K(+)-deficiency stress. Plants perceive external K(+) fluctuations and generate the initial K(+) signal in root cells. This signal is transduced into the cytoplasm and encoded as Ca(2+) and reactive oxygen species signaling. K(+)-deficiency-induced signals are subsequently decoded by cytoplasmic sensors, which regulate the downstream transcriptional and posttranslational responses. Eventually, plants produce a series of adaptive events in both physiological and morphological alterations that help them survive K(+) deficiency.

Eukaryotic genomes are pervasively transcribed. Besides protein-coding RNAs, there are different types of non-coding RNAs that modulate complex molecular and cellular processes. RNA sequencing technologies and bioinformatics methods greatly promoted the study of ncRNAs, which revealed ncRNAs' essential roles in diverse aspects of biological functions. As important key players in gene regulatory networks, ncRNAs work with other biomolecules, including coding and non-coding RNAs, DNAs and proteins. In this review, we discuss the distinct types of ncRNAs, including housekeeping ncRNAs and regulatory ncRNAs, their versatile functions and interactions, transcription, translation, and modification. Moreover, we summarize the integrated networks of ncRNA interactions, providing a comprehensive landscape of ncRNAs regulatory roles.

Previous research has demonstrated that AtPHR1 plays a central role in phosphate (Pi)-starvation signaling in Arabidopsis thaliana. In this work, two OsPHR genes from rice (Oryza sativa) were isolated and designated as OsPHR1 and OsPHR2 based on amino acid sequence homology to AtPHR1. Their functions in Pi signaling in rice were investigated using transgenic plants. Our results showed that both OsPHR1 and OsPHR2 are involved in Pi-starvation signaling pathway by regulation of the expression of Pi-starvation-induced genes, whereas only OsPHR2 overexpression results in the excessive accumulation of Pi in shoots under Pi-sufficient conditions. Under Pi-sufficient conditions, overexpression of OsPHR2 mimics Pi-starvation stress in rice with enhanced root elongation and proliferated root hair growth, suggesting the involvement of OsPHR2 in Pi-dependent root architecture alteration by both systematic and local pathways. In OsPHR2-overexpression plants, some Pi transporters were up-regulated under Pi-sufficient conditions, which correlates with the strongly increased content of Pi. The mechanism behind the OsPHR2 regulated Pi accumulation will provide useful approaches to develop smart plants with high Pi efficiency.

Abstract Stomata, the pores formed by a pair of guard cells, are the main gateways for water transpiration and photosynthetic CO 2 exchange, as well as pathogen invasion in land plants. Guard cell movement is regulated by a combination of environmental factors, including water status, light, CO 2 levels and pathogen attack, as well as endogenous signals, such as abscisic acid and apoplastic reactive oxygen species (ROS). Under abiotic and biotic stress conditions, extracellular ROS are mainly produced by plasma membrane‐localized NADPH oxidases, whereas intracellular ROS are produced in multiple organelles. These ROS form a sophisticated cellular signaling network, with the accumulation of apoplastic ROS an early hallmark of stomatal movement. Here, we review recent progress in understanding the molecular mechanisms of the ROS signaling network, primarily during drought stress and pathogen attack. We summarize the roles of apoplastic ROS in regulating stomatal movement, ABA and CO 2 signaling, and immunity responses. Finally, we discuss ROS accumulation and communication between organelles and cells. This information provides a conceptual framework for understanding how ROS signaling is integrated with various signaling pathways during plant responses to abiotic and biotic stress stimuli.

Many biochemical approaches show functions of calcium-dependent protein kinases (CDPKs) in abscisic acid (ABA) signal transduction, but molecular genetic evidence linking defined CDPK genes with ABA-regulated biological functions at the whole-plant level has been lacking. Here, we report that ABA stimulated two homologous CDPKs in Arabidopsis thaliana, CPK4 and CPK11. Loss-of-function mutations of CPK4 and CPK11 resulted in pleiotropic ABA-insensitive phenotypes in seed germination, seedling growth, and stomatal movement and led to salt insensitivity in seed germination and decreased tolerance of seedlings to salt stress. Double mutants of the two CDPK genes had stronger ABA- and salt-responsive phenotypes than the single mutants. CPK4- or CPK11-overexpressing plants generally showed inverse ABA-related phenotypes relative to those of the loss-of-function mutants. Expression levels of many ABA-responsive genes were altered in the loss-of-function mutants and overexpression lines. The CPK4 and CPK11 kinases both phosphorylated two ABA-responsive transcription factors, ABF1 and ABF4, in vitro, suggesting that the two kinases may regulate ABA signaling through these transcription factors. These data provide in planta genetic evidence for the involvement of CDPK/calcium in ABA signaling at the whole-plant level and show that CPK4 and CPK11 are two important positive regulators in CDPK/calcium-mediated ABA signaling pathways.

Protein kinases are major players in various signal transduction pathways. Understanding the molecular mechanisms behind plant responses to biotic and abiotic stresses has become critical for developing and breeding climate-resilient crops. In this review, we summarize recent progress on understanding plant drought, salt, and cold stress responses, with a focus on signal perception and transduction by different protein kinases, especially sucrose nonfermenting1 (SNF1)-related protein kinases (SnRKs), mitogen-activated protein kinase (MAPK) cascades, calcium-dependent protein kinases (CDPKs/CPKs), and receptor-like kinases (RLKs). We also discuss future challenges in these research fields.

H3K27 methylation mediated by the histone methyltransferase complex PRC2 is critical for transcriptional regulation, Polycomb silencing, Drosophila segmentation, mammalian X chromosome inactivation, and cancer. PRC2-mediated H3K27 methylation can spread along the chromatin and propagate the repressive chromatin environment; thus, chromatin components that antagonize the activity of PRC2 are important for restraining Polycomb silencing. Here we report that in HeLa cells, H3 histones unmethylated at Lys-36 are mostly methylated at Lys-27, with the exception of newly synthesized H3. In addition, K27me3 rarely co-exists with K36me2 or K36me3 on the same histone H3 polypeptide. Moreover, PRC2 activity is greatly inhibited on nucleosomal substrates with preinstalled H3K36 methylation. These findings collectively identify H3K36 methylation as a chromatin component that restricts the PRC2-mediated spread of H3K27 methylation. Finally, we provide evidence that the controversial histone lysine methyltransferase Ash1, a known Trithorax group protein that antagonizes Polycomb silencing in vivo, is an H3K36-specific dimethylase, not an H3K4 methylase, further supporting the role of H3K36 methylation in antagonizing PRC2-mediated H3K27 methylation.

Reactive oxygen species (ROS) are widely produced in different cellular compartments under both biotic and abiotic stress conditions. ROS play a central role in plant signaling and regulate diverse cellular processes. Recent advances are shedding new light on sophisticated mechanisms controlling ROS biogenesis and signaling in plant immunity. In this review, we summarize our current understanding of the regulation of apoplastic ROS production in response to microbial molecular patterns and draw comparison with abscisic acid (ABA)-induced apoplastic ROS. We also discuss how ROS act as signal molecules to regulate cellular activities using stomatal movement as an example.

We isolated two T-DNA insertion mutants of Arabidopsis thaliana GLUTATHIONE PEROXIDASE3 (ATGPX3) that exhibited a higher rate of water loss under drought stress, higher sensitivity to H(2)O(2) treatment during seed germination and seedling development, and enhanced production of H(2)O(2) in guard cells. By contrast, lines engineered to overexpress ATGPX3 were less sensitive to drought stress than the wild type and displayed less transpirational water loss, which resulted in higher leaf surface temperature. The atgpx3 mutation also disrupted abscisic acid (ABA) activation of calcium channels and the expression of ABA- and stress-responsive genes. ATGPX3 physically interacted with the 2C-type protein phosphatase ABA INSENSITIVE2 (ABI2) and, to a lesser extent, with ABI1. In addition, the redox states of both ATGPX3 and ABI2 were found to be regulated by H(2)O(2). The phosphatase activity of ABI2, measured in vitro, was reduced approximately fivefold by the addition of oxidized ATGPX3. The reduced form of ABI2 was converted to the oxidized form by the addition of oxidized ATGPX3 in vitro, which might mediate ABA and oxidative signaling. These results suggest that ATGPX3 might play dual and distinctive roles in H(2)O(2) homeostasis, acting as a general scavenger and specifically relaying the H(2)O(2) signal as an oxidative signal transducer in ABA and drought stress signaling.

Plant phosphate (Pi) transporters mediate the uptake and translocation of this nutrient within plants. A total of 13 sequences in the rice (Oryza sativa) genome can be identified as belonging to the Pi transporter (Pht1) family. Here, we report on the expression patterns, biological properties and the physiological roles of two members of the family: OsPht1;2 (OsPT2) and OsPht1;6 (OsPT6). Expression of both genes increased significantly under Pi deprivation in roots and shoots. By using transgenic rice plants expressing the GUS reporter gene, driven by their promoters, we detected that OsPT2 was localized exclusively in the stele of primary and lateral roots, whereas OsPT6 was expressed in both epidermal and cortical cells of the younger primary and lateral roots. OsPT6, but not OsPT2, was able to complement a yeast Pi uptake mutant in the high-affinity concentration range. Xenopus oocytes injected with OsPT2 mRNA showed increased Pi accumulation and a Pi-elicited depolarization of the cell membrane electrical potential, when supplied with mM external concentrations. Both results show that OsPT2 mediated the uptake of Pi in oocytes. In transgenic rice, the knock-down of either OsPT2 or OsPT6 expression by RNA interference significantly decreased both the uptake and the long-distance transport of Pi from roots to shoots. Taken together, these data suggest OsPT6 plays a broad role in Pi uptake and translocation throughout the plant, whereas OsPT2 is a low-affinity Pi transporter, and functions in translocation of the stored Pi in the plant.

To cope with growth in low-phosphate (Pi) soils, plants have evolved adaptive responses that involve both developmental and metabolic changes. Phosphate Starvation Response 1 (PHR1) and related transcription factors play a central role in the control of Pi starvation responses (PSRs). How Pi levels control PHR1 activity, and thus PSRs, remains to be elucidated. Here, we identify a direct Pi-dependent inhibitor of PHR1 in Arabidopsis, SPX1, a nuclear protein that shares the SPX domain with yeast Pi sensors and with several Pi starvation signaling proteins from plants. Double mutation of SPX1 and of a related gene, SPX2, resulted in molecular and physiological changes indicative of increased PHR1 activity in plants grown in Pi-sufficient conditions or after Pi refeeding of Pi-starved plants but had only a limited effect on PHR1 activity in Pi-starved plants. These data indicate that SPX1 and SPX2 have a cellular Pi-dependent inhibitory effect on PHR1. Coimmunoprecipitation assays showed that the SPX1/PHR1 interaction in planta is highly Pi-dependent. DNA-binding and pull-down assays with bacterially expressed, affinity-purified tagged SPX1 and ΔPHR1 proteins showed that SPX1 is a competitive inhibitor of PHR1 binding to its recognition sequence, and that its efficiency is highly dependent on the presence of Pi or phosphite, a nonmetabolizable Pi analog that can repress PSRs. The relative strength of the SPX1/PHR1 interaction is thus directly influenced by Pi, providing a link between Pi perception and signaling.

Auxin transport affects a variety of important growth and developmental processes in plants, including the regulation of shoot and root branching. The asymmetrical localization of auxin influx and efflux carriers within the plasma membrane establishes the auxin gradient and facilitates its transport. REH1, a rice EIR1 (Arabidopsis ethylene insensitive root 1)-like gene, is a putative auxin efflux carrier. Phylogenetic analysis of 32 members of the PIN family, taken from across different species, showed that in terms of evolutionary relationship, OsPIN1 is closer to the PIN1 family than to the PIN2 family. It is, therefore, renamed as OsPIN1 in this study. OsPIN1 was expressed in the vascular tissues and root primordial in a manner similar to AtPIN1. Adventitious root emergence and development were significantly inhibited in the OsPIN1 RNA interference (RNAi) transgenic plants, which was similar to the phenotype of NPA (N-1-naphthylphalamic acid, an auxin-transport inhibitor)-treated wild-type plants. alpha-naphthylacetic acid (alpha-NAA) treatment was able to rescue the mutated phenotypes occurring in the RNAi plants. Overexpression or suppression of the OsPIN1 expression through a transgenic approach resulted in changes of tiller numbers and shoot/root ratio. Taken together, these data suggest that OsPIN1 plays an important role in auxin-dependent adventitious root emergence and tillering.