SummaryCellular sugar starvation and/or energy deprivation (i.e., energy stress) serves as an important signaling cue for live cells to survive under nutrient-limited conditions. When exposed to cellular energy stress (ES) conditions, plants reconfigure metabolic pathways and rebalance energy status while restricting vegetative organ growth. Despite the vital importance of this growth restriction under cellular ES, the regulatory mechanisms underlying the response remain largely elusive in plants. Using plant cell- and systems-based functional analyses coupled with extended genetic validation, we show that cellular energy stress-activated SNF1-realted protein kinase 1 (SnRK1.1) directly interacts with and phosphorylates E2Fa transcription factor, a critical cell cycle regulator. Phosphorylation of E2Fa by SnRK1.1 leads to its proteasome-mediated protein degradation, resulting in S-phase repression and organ growth restriction. Our findings show that the evolutionarily conserved energy sensor SnRK1.1 dynamically adjusts cell proliferation and vegetative organ growth for the plants to cope with constantly fluctuating environments.

The evolutionarily conserved target of rapamycin (TOR) kinase acts as a master regulator that coordinates cell proliferation and growth by integrating nutrient, energy, hormone and stress signals in all eukaryotes 1 , 2 . Research has focused mainly on TOR-regulated translation, but how TOR orchestrates the global transcriptional network remains unclear. Here we identify ethylene-insensitive protein 2 (EIN2), a central integrator 3 – 5 that shuttles between the cytoplasm and the nucleus, as a direct substrate of TOR in Arabidopsis thaliana . Glucose-activated TOR kinase directly phosphorylates EIN2 to prevent its nuclear localization. Notably, the rapid global transcriptional reprogramming that is directed by glucose–TOR signalling is largely compromised in the ein2-5 mutant, and EIN2 negatively regulates the expression of a wide range of target genes of glucose-activated TOR that are involved in DNA replication, cell wall and lipid synthesis and various secondary metabolic pathways. Chemical, cellular and genetic analyses reveal that cell elongation and proliferation processes that are controlled by the glucose–TOR–EIN2 axis are decoupled from canonical ethylene–CTR1–EIN2 signalling, and mediated by different phosphorylation sites. Our findings reveal a molecular mechanism by which a central signalling hub is shared but differentially modulated by diverse signalling pathways using distinct phosphorylation codes that can be specified by upstream protein kinases.

To overcome high temperature stress, plants have developed transcriptional cascades which express a large amount of chaperone proteins called heat shock proteins (HSPs). In our recent publication, we reported that STABILIZED1, as an U5-snRNP-interacting protein, is involved in the splicing of heat shock factor (HSF) and HSP transcripts during high temperature stress. This indicates that not only transcriptional regulation, but also post-transcriptional regulation by STA1, is essential for the full activation of HSF-HSP cascades and for thermotolerance. Here, we observed that the splicing of HSP transcripts was induced independent of STA1 at room temperature after heat acclimation, indicating that STA1 acts as a high temperature-specific splicing factor for the splicing of HSP transcripts. Our findings suggest the molecular mechanism for how HSF and HSP transcripts are spliced well under high temperature stress that blocks the splicing of overall transcripts.

AN1/A20-like Zinc finger family proteins are evolutionarily conserved regulatory components in eukaryotic signaling circuits. In Arabidopsis thaliana, the AN1/A20 Zinc finger family is encoded as 14 members in the genome and collectively referred to as stress-associated proteins (SAPs). Here we described AtSAP5 localized to the nucleus, and played a role in heat-responsive gene regulation together with MBF1c. Seedling survival assay of sap5 and mbf1c demonstrated consistent effects of AtSAP5 and MBF1C in response to two-step heat treatment, supporting their function in heat stress tolerance. Our findings yield an insight in A20/AN1-like Zinc finger protein AtSAP5 functions in plant adaptability under high temperature.

Photomorphogenesis is an essential program in plant development. This process is effected by the balanced cooperation of many factors under light and dark conditions. In a previous study, we showed that MYB hypocotyl elongation-related (MYBH) is involved in cell elongation. To expand our understanding of MYBH function, we performed a yeast two-hybrid assay and identified an MYB-like Domain transcription factor (MYBD). In this study, we investigated the function of MYBD, which is an MYBH homolog involved in anthocyanin accumulation. MYBD expression increased in response to light or cytokinin, and MYBD enhanced anthocyanin biosynthesis via repression of MYBL2, which encodes a transcription factor that has a negative effect on this process. In addition, MYBD binding in vivo to the MYBL2 promoter and the lower level of histone H3K9 acetylation at the upstream region of MYBL2 in MYBD over-expressing plants in comparison with wild-type plants imply that MYBD represses MYBL2 expression via an epigenetic mechanism. HY5 directly binds to the MYBD promoter, which indicates that MYBD acts on HY5-downstream in light-or cytokinintriggered signaling pathways, leading to anthocyanin accumulation. Our results suggest that, although MYBD and MYBH are homologs, they act in opposite ways during plant photomorphogenesis, and these functions should be examined in further studies.

Terrestrial plants are exposed to complex stresses of high salt-induced abscisic acid (ABA) and submergence-induced hypoxia when seawater floods fields. Many studies have investigated plant responses to individual stress conditions, but not so much for coupled or sequentially imposed stresses. We examined molecular regulatory mechanisms of gene expression underlying the cellular responses involved in crosstalk between salt and hypoxia stresses. Salt/ABA- and AtMYC2-dependent induction of a synthetic ABA-responsive element and the native RD22 promoters were utilized in our cell-based functional assays. Such promoter-based reporter induction was largely inhibited by hypoxia and hypoxia-inducible AKIN10 activity. Biochemical analyses showed that AKIN10 negatively modulates AtMYC2 protein accumulation via proteasome activity upon AKIN10 kinase activity-dependent protein modification. Further genetic analysis using transgenic plants expressing AKIN10 provided evidence that AKIN10 activity undermined AtMYC2-dependent salt tolerance. Our findings unravel a novel molecular interaction between the key signalling constituents leading crosstalk between salt and hypoxia stresses in Arabidopsis thaliana under the detrimental condition of submergence in saltwater.

Ethylene was the first plant hormone for which a receptor-dependent signalling pathway was established. The signal transduction pathway is framed around genetically identified factors encompassing membrane receptors through to intra-cellular regulators and then nuclear transcription factors. Recently, the cellular and biochemical connections among these genetic factors have been characterized to reveal a complex and intertwined signalling scheme. For ethylene signalling, the short-lived ETHYLENE INSENSITIVE2 (EIN2) and ETHYLENE INSENSITIVE3 (EIN3) proteins play central roles, and so regulation of the turnover of these proteins by specific E3 ligases serves as a key regulatory step in the pathway. Two antagonistic mitogen-activated protein kinase (MAPK) cascades that modify EIN3 appear to mediate intra-cellular signalling from membrane receptors to nuclear transcription factors. The identification of further genomic and genetic components and elucidation of their cellular and biochemical roles in EIN2 and EIN3 regulation are thus required to complete the whole ethylene signal transduction pathway. Such a comprehensive understanding of ethylene signal transduction will provide useful information for the selection of genes that can enhance plant adaptations to unfavourable environmental conditions and so secure food production and plant-based renewable bio-energy for human society.

In higher plants, the regulation of anthocyanin synthesis by various factors including light, sugars and hormones is mediated by numerous regulatory factors acting at the transcriptional level. Here, the association between sucrose and the plant hormone, cytokinin, in the presence of light was investigated to elucidate cytokinin signaling cascades leading to the transcriptional activation of anthocyanin biosynthesis genes in Arabidopsis seedlings. We showed that cytokinin enhances anthocyanin content and transcript levels of sugar inducible structural gene UDPglucose: flavonoid 3-O-glucosyl transferase (UF3GT) and regulatory gene PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1). Genetic analysis showed that cytokinin signaling modulates sugar-induced anthocyanin biosynthesis through a two-component signaling cascade involving the type-B response regulators ARR1, ARR10 and ARR12 in a redundant manner. Genetic, physiological and molecular biological approaches demonstrated that cytokinin enhancement is partially dependent on phytochrome and cryptochrome downstream component HY5, but mainly on photosynthetic electron transport. Taken together, we suggest that cytokinin acts down-stream of the photosynthetic electron transport chain in which the plastoquinone redox poise is modulated by sugars in a photoreceptor independent manner.

Abscisic acid (ABA) is a phytohormone that positively regulates seed dormancy and stress tolerance. PYL/RCARs were identified an intracellular ABA receptors regulating ABA-dependent gene expression in Arabidopsis thaliana. However, their function in monocot species has not been characterized yet. Herein, it is demonstrated that PYL/RCAR orthologues in Oryza sativa function as a positive regulator of the ABA signal transduction pathway. Transgenic rice plants expressing OsPYL/RCAR5, a PYL/RCAR orthologue of rice, were found to be hypersensitive to ABA during seed germination and early seedling growth. A rice ABA signalling unit composed of OsPYL/RCAR5, OsPP2C30, SAPK2, and OREB1 for ABA-dependent gene regulation was further identified, via interaction assays and a transient gene expression assay. Thus, a core signalling unit for ABA-responsive gene expression modulating seed germination and early seedling growth in rice has been unravelled. This study provides substantial contributions toward understanding the ABA signal transduction pathway in rice.

Sucrose-nonfermentation1-related protein kinase1 (SnRK1) is an evolutionarily conserved energy sensor protein that regulates gene expression in response to energy depletion in plants. Efforts to elucidate the functions and mechanisms of this protein kinase are hampered, however, by inherent growth defects of snrk1-null mutant plants. To overcome these limitations and study SnRK1 functions in vivo, we applied a method combining transient expression in leaf mesophyll protoplasts and stable expression in transgenic plants. We found that both rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana) SnRK1 activities critically influence stress-inducible gene expression and the induction of stress tolerance. Genetic, molecular, and chromatin immunoprecipitation analyses further revealed that the nuclear SnRK1 modulated target gene transcription in a submergence-dependent manner. From early seedling development through late senescence, SnRK1 activities appeared to modulate developmental processes in the plants. Our findings offer insight into the regulatory functions of plant SnRK1 in stress-responsive gene regulation and in plant growth and development throughout the life cycle.

Despite its relevance for agricultural production, environmental stress-induced growth inhibition, which is responsible for significant yield reductions, is only poorly understood. Here, we investigated the molecular mechanisms underlying cell cycle inhibition in young proliferating leaves of the model plant Arabidopsis thaliana when subjected to mild osmotic stress. A detailed cellular analysis demonstrated that as soon as osmotic stress is sensed, cell cycle progression rapidly arrests, but cells are kept in a latent ambivalent state allowing a quick recovery (pause). Remarkably, cell cycle arrest coincides with an increase in 1-aminocyclopropane-1-carboxylate levels and the activation of ethylene signaling. Our work showed that ethylene acts on cell cycle progression via inhibition of cyclin-dependent kinase A activity independently of EIN3 transcriptional control. When the stress persists, cells exit the mitotic cell cycle and initiate the differentiation process (stop). This stop is reflected by early endoreduplication onset, in a process independent of ethylene. Nonetheless, the potential to partially recover the decreased cell numbers remains due to the activity of meristemoids. Together, these data present a conceptual framework to understand how environmental stress reduces plant growth.

Light is the ultimate energy source for photo-autotrophs on earth. For green plants, however, it can also be toxic under certain stressful environmental conditions and at critical developmental stages. Anthocyanins, a class of flavonoids, act as an effective screening mechanism that allows plant survival and proliferation under occasional periods of harmful irradiation through modulation of light absorption. Apart from light-sensing through photoreceptors such as phytochrome and cryptochrome, plants use the photosynthetic electron transfer (PET) chain to integrate light information. The redox status of the plastoquinone (PQ) pool of the PET chain regulates anthocyanin biosynthesis genes, together with the plant hormone ethylene and plant hormone-like sugars. A complex signaling apparatus in acyanic cells appears to transduce information to cyanic cells to regulate anthocyanin production through an intercellular signaling pathway that remains largely uncharacterized. This review will highlight recent advances in this field and their implications for the regulation of anthocyanin pigmentation.

Dof proteins are transcription factors that have a conserved single zinc finger DNA-binding domain. In this study, we isolated an activation tagging mutant Dof5.1-D exhibiting an upward-curling leaf phenotype due to enhanced expression of the REV gene that is required for establishing adaxial-abaxial polarity. Dof5.1-D plants also had reduced transcript levels for IAA6 and IAA19 genes, indicating an altered auxin biosynthesis in Dof5.1-D. An electrophoretic mobility shift assay using the Dof5.1 DNA-binding motif and the REV promoter region indicated that the DNA-binding domain of Dof5.1 binds to a TAAAGT motif located in the 5'-distal promoter region of the REV promoter. Further, transient and chromatin immunoprecipitation assays verified binding activity of the Dof5.1 DNA-binding motif with the REV promoter. Consistent with binding assays, constitutive over-expression of the Dof5.1 DNA-binding domain in wild-type plants caused a downward-curling phenotype, whereas crossing Dof5.1-D to a rev mutant reverted the upward-curling phenotype of the Dof5.1-D mutant leaf to the wild-type. These results suggest that the Dof5.1 protein directly binds to the REV promoter and thereby regulates adaxial-abaxial polarity.

All living organisms operate a variety of metabolic pathways that enable them to be self-sustainable with basic nutrients. To maintain the balance of metabolite and energy levels, organisms have developed sophisticated sensing and signaling mechanisms that underlie the physiological responses to

The gaseous plant hormone ethylene acts as a pivotal mediator to respond to and coordinate internal and external cues in modulating plant growth dynamics and developmental programs. Genetic analysis of Arabidopsis thaliana has been used to identify key components and to build a linear ethylene-signaling pathway from the receptors through to the nuclear transcription factors. Studies applying integrative approaches have revealed new regulators, molecular connections and mechanisms in ethylene signaling and unexpected links to other plant hormones. Here, we review and discuss recent discoveries about the functional mode of ethylene receptor complexes, dual mitogen-activated protein kinase cascade signaling, stability control of the master nuclear transcription activator ETHYLENE INSENSITIVE 3 (EIN3), and the contextual relationships between ethylene and other plant hormones, such as auxin and gibberellins, in organ-specific growth regulation.

The desire to understand and control plant diseases has been a major drive to study host-microbe interactions. In addition to the molecular basis of diseases and disease resistance, another fundamental question is how plants manage to stay healthy and survive constant exposure to a wide range of microbes. The innate immune response is the first line of defense, and is likely critical for the survival and fitness of plants in the ubiquitous presence of microbes. Research in the past decade has revealed remarkable convergent evolution in the recognition of pathogenor microbe-associated molecular patterns (PAMPs or MAMPs) and the activation of innate immune responses in plants, insects and mammals (Nürnberger et al., 2004; Ausubel, 2005; Zipfel and Felix, 2005; Akira et al., 2006; Ferrandon et al., 2007). We are interested in dissecting the plant signal transduction networks that are responding to or manipulated by microbial signals and effectors. For a robust innate immune system, both signaling specificity and complexity are required. Currently, the sequenced plant model Arabidopsis thaliana provides the most advanced and sophisticated molecular, cellular, genetic, genomic, proteomic, and bioinformatic tools for studying signaling mechanisms and transcription controls in plant innate immunity. The primary cell assays using protoplasts freshly isolated from leaves support highthroughput and versatile analyses of diverse MAMP and microbial effector signal responses (Asai et al., 2002; He et al., 2006a, 2006b; Yoo et al., 2007). The virus-induced gene silencing (VIGS) method (Burch-Smith et al., 2006), inducible RNAi capability, saturating insertional mutant resources (Sessions et al., 2002; Alonso et al., 2003), and whole-genome microarray databases