The canonical E2Fs together with RETINOBLASTOMA-RELATED are required to establish quiescence during plant development.

Maintaining stable and transient quiescence in differentiated and stem cells, respectively, requires repression of the cell cycle. The plant RETINOBLASTOMA-RELATED (RBR) has been implicated in stem cell maintenance, presumably by forming repressor complexes with E2F transcription factors. Surprisingly we find that mutations in all three canonical E2Fs do not hinder the cell cycle, but similarly to RBR silencing, result in hyperplasia. Contrary to the growth arrest that occurs when exit from proliferation to differentiation is inhibited upon RBR silencing, the e2fabc mutant develops enlarged organs with supernumerary stem and differentiated cells as quiescence is compromised. While E2F, RBR and the M-phase regulatory MYB3Rs are part of the DREAM repressor complexes, and recruited to overlapping groups of targets, they regulate distinct sets of genes. Only the loss of E2Fs but not the MYB3Rs interferes with quiescence, which might be due to the ability of E2Fs to control both G1-S and some key G2-M targets. We conclude that collectively the three canonical E2Fs in complex with RBR have central roles in establishing cellular quiescence during organ development, leading to enhanced plant growth.

TOR represses stress responses through global regulation of H3K27 trimethylation in plants.

Target of rapamycin (TOR) functions as a central sensory hub linking a wide range of external stimuli to gene expression. The mechanisms underlying stimulus-specific transcriptional reprogramming by TOR remain elusive. Here, we describe an in silico analysis in Arabidopsis demonstrating that TOR-repressed genes are associated with either bistable or silent chromatin states. Both states regulated by the TOR signaling pathway are associated with a high level of histone H3K27 trimethylation (H3K27me3) deposited by CURLY LEAF in a specific context with LIKE HETEROCHROMATIN PROTEIN1. The combination of the two epigenetic histone modifications H3K4me3 and H3K27me3 implicates a bistable feature that alternates between an 'on' and an 'off' state, allowing rapid transcriptional changes upon external stimuli. The chromatin remodeler SWI2/SNF2 ATPase BRAHMA activates TOR-repressed genes only at bistable chromatin domains to rapidly induce biotic stress responses. Here, we demonstrate both in silico and in vivo that TOR represses transcriptional stress responses through global maintenance of H3K27me3.

TOR senses and regulates spermidine metabolism during seedling establishment and growth in maize and Arabidopsis.

Spermidine (Spd) is a nitrogen sink and signaling molecule that plays pivotal roles in eukaryotic cell growth and must be finetuned to meet various energy demands. In eukaryotes, target of rapamycin (TOR) is a central nutrient sensor, especially N, and a master-regulator of growth and development. Here, we discovered that Spd stimulates the growth of maize and Arabidopsis seedlings through TOR signaling. Inhibition of Spd biosynthesis led to TOR inactivation and growth defects. Furthermore, disruption of a TOR complex partner RAPTOR1B abolished seedling growth stimulation by Spd. Strikingly, TOR activated by Spd promotes translation of key metabolic enzyme upstream open reading frame (uORF)-containing mRNAs, PAO and CuAO, by facilitating translation reinitiation and providing feedback to polyamine metabolism and TOR activation. The Spd-TOR relay protected young-age seedlings of maize from expeditious stress heat shock. Our results demonstrate Spd is an upstream effector of TOR kinase in planta and provide its potential application for crop protection.

ErbB-3 BINDING PROTEIN 1 Regulates Translation and Counteracts RETINOBLASTOMA RELATED to Maintain the Root Meristem.

The ErbB-3 BINDING PROTEIN 1 (EBP1) drives growth, but the mechanism of how it acts in plants is little understood. Here, we show that EBP1 expression and protein abundance in Arabidopsis (Arabidopsis thaliana) are predominantly confined to meristematic cells and are induced by sucrose and partially dependent on TARGET OF RAPAMYCIN (TOR) kinase activity. Consistent with being downstream of TOR, silencing of EBP1 restrains, while overexpression promotes, root growth, mostly under sucrose-limiting conditions. Inducible overexpression of RETINOBLASTOMA RELATED (RBR), a sugar-dependent transcriptional repressor of cell proliferation, depletes meristematic activity and causes precocious differentiation, which is attenuated by EBP1. To understand the molecular mechanism, we searched for EBP1- and RBR-interacting proteins by affinity purification and mass spectrometry. In line with the double-stranded RNA-binding activity of EBP1 in human (Homo sapiens) cells, the overwhelming majority of EBP1 interactors are part of ribonucleoprotein complexes regulating many aspects of protein synthesis, including ribosome biogenesis and mRNA translation. We confirmed that EBP1 associates with ribosomes and that EBP1 silencing hinders ribosomal RNA processing. We revealed that RBR also interacts with a set of EBP1-associated nucleolar proteins as well as factors that function in protein translation. This suggests EBP1 and RBR act antagonistically on common processes that determine the capacity for translation to tune meristematic activity in relation to available resources.

γ-Tubulin interacts with E2F transcription factors to regulate proliferation and endocycling in Arabidopsis.

γ-Tubulin is associated with microtubule nucleation, but evidence is accumulating in eukaryotes that it also functions in nuclear processes and in cell division control independently of its canonical role. We found that in Arabidopsis thaliana, γ-tubulin interacts specifically with E2FA, E2FB, and E2FC transcription factors both in vitro and in vivo. The interaction of γ-tubulin with the E2Fs is not reduced in the presence of their dimerization partners (DPs) and, in agreement, we found that γ-tubulin interaction with E2Fs does not require the dimerization domain. γ-Tubulin associates with the promoters of E2F-regulated cell cycle genes in an E2F-dependent manner, probably in complex with the E2F-DP heterodimer. The up-regulation of E2F target genes PCNA, ORC2, CDKB1;1, and CCS52A under γ-tubulin silencing suggests a repressive function for γ-tubulin at G1/S and G2/M transitions, and the endocycle, which is consistent with an excess of cell division in some cells and enhanced endoreduplication in others in the shoot and young leaves of γ-tubulin RNAi plants. Altogether, our data show ternary interaction of γ-tubulin with the E2F-DP heterodimer and suggest a repressive role for γ-tubulin with E2Fs in controlling mitotic activity and endoreduplication during plant development.

E2FB Interacts with RETINOBLASTOMA RELATED and Regulates Cell Proliferation during Leaf Development.

Cell cycle entry and quiescence are regulated by the E2F transcription factors in association with RETINOBLASTOMA-RELATED (RBR). E2FB is considered to be a transcriptional activator of cell cycle genes, but its function during development remains poorly understood. Here, by studying E2FB-RBR interaction, E2F target gene expression, and epidermal cell number and shape in e2fb mutant and overexpression lines during leaf development in Arabidopsis (Arabidopsis thaliana), we show that E2FB in association with RBR plays a role in the inhibition of cell proliferation to establish quiescence. In young leaves, both RBR and E2FB are abundant and form a repressor complex that is reinforced by an autoregulatory loop. Increased E2FB levels, either by expression driven by its own promoter or ectopically together with DIMERIZATION PARTNER A, further elevate the amount of this repressor complex, leading to reduced leaf cell number. Cell overproliferation in e2fb mutants and in plants overexpressing a truncated form of E2FB lacking the RBR binding domain strongly suggested that RBR repression specifically acts through E2FB. The increased number of small cells below the guard cells and of fully developed stomata indicated that meristemoids preferentially hyperproliferate. As leaf development progresses and cells differentiate, the amount of RBR and E2FB gradually declined. At this stage, elevation of E2FB level can overcome RBR repression, leading to reactivation of cell division in pavement cells. In summary, E2FB in association with RBR is central to regulating cell proliferation during organ development to determine final leaf cell number.

E2FA and E2FB transcription factors coordinate cell proliferation with seed maturation.

The E2F transcription factors and the RETINOBLASTOMA-RELATED repressor protein are principal regulators coordinating cell proliferation with differentiation, but their role during seed development is little understood. We show that in fully developed Arabidopsis thaliana embryos, cell number was not affected either in single or double mutants for the activator-type E2FA and E2FB Accordingly, these E2Fs are only partially required for the expression of cell cycle genes. In contrast, the expression of key seed maturation genes LEAFY COTYLEDON 1/2 (LEC1/2), ABSCISIC ACID INSENSITIVE 3, FUSCA 3 and WRINKLED 1 is upregulated in the e2fab double mutant embryo. In accordance, E2FA directly regulates LEC2, and mutation at the consensus E2F-binding site in the LEC2 promoter de-represses its activity during the proliferative stage of seed development. In addition, the major seed storage reserve proteins, 12S globulin and 2S albumin, became prematurely accumulated at the proliferating phase of seed development in the e2fab double mutant. Our findings reveal a repressor function of the activator E2Fs to restrict the seed maturation programme until the cell proliferation phase is completed.

The mitogen-activated protein kinase 4-phosphorylated heat shock factor A4A regulates responses to combined salt and heat stresses.

Heat shock factors regulate responses to high temperature, salinity, water deprivation, or heavy metals. Their function in combinations of stresses is, however, not known. Arabidopsis HEAT SHOCK FACTOR A4A (HSFA4A) was previously reported to regulate responses to salt and oxidative stresses. Here we show, that the HSFA4A gene is induced by salt, elevated temperature, and a combination of these conditions. Fast translocation of HSFA4A tagged with yellow fluorescent protein from cytosol to nuclei takes place in salt-treated cells. HSFA4A can be phosphorylated not only by mitogen-activated protein (MAP) kinases MPK3 and MPK6 but also by MPK4, and Ser309 is the dominant MAP kinase phosphorylation site. In vivo data suggest that HSFA4A can be the substrate of other kinases as well. Changing Ser309 to Asp or Ala alters intramolecular multimerization. Chromatin immunoprecipitation assays confirmed binding of HSFA4A to promoters of target genes encoding the small heat shock protein HSP17.6A and transcription factors WRKY30 and ZAT12. HSFA4A overexpression enhanced tolerance to individually and simultaneously applied heat and salt stresses through reduction of oxidative damage. Our results suggest that this heat shock factor is a component of a complex stress regulatory pathway, connecting upstream signals mediated by MAP kinases MPK3/6 and MPK4 with transcription regulation of a set of stress-induced target genes.

Cell cycle control by the target of rapamycin signalling pathway in plants.

Cells need to ensure a sufficient nutrient and energy supply before committing to proliferate. In response to positive mitogenic signals, such as light, sugar availability, and hormones, the target of rapamycin (TOR) signalling pathway promotes cell growth that connects to the entry and passage through the cell division cycle via multiple signalling mechanisms. Here, we summarize current understanding of cell cycle regulation by the RBR-E2F regulatory hub and the DREAM-like complexes, and highlight possible functional relationships between these regulators and TOR signalling. A genetic screen recently uncovered a downstream signalling component to TOR that regulates cell proliferation, YAK1, a member of the dual specificity tyrosine phosphorylation-regulated kinase (DYRK) family. YAK1 activates the plant-specific SIAMESE-RELATED (SMR) cyclin-dependent kinase inhibitors and therefore could be important to regulate both the CDKA-RBR-E2F pathway to control the G1/S transition and the mitotic CDKB1;1 to control the G2/M transition. TOR, as a master regulator of both protein synthesis-driven cell growth and cell proliferation is also central for cell size homeostasis. We conclude the review by briefly highlighting the potential applications of combining TOR and cell cycle knowledge in the context of ensuring future food security.

Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control.

The rapidly proliferating cells in plant meristems must be protected from genome damage. Here, we show that the regulatory role of the Arabidopsis RETINOBLASTOMA RELATED (RBR) in cell proliferation can be separated from a novel function in safeguarding genome integrity. Upon DNA damage, RBR and its binding partner E2FA are recruited to heterochromatic γH2AX-labelled DNA damage foci in an ATM- and ATR-dependent manner. These γH2AX-labelled DNA lesions are more dispersedly occupied by the conserved repair protein, AtBRCA1, which can also co-localise with RBR foci. RBR and AtBRCA1 physically interact in vitro and in planta Genetic interaction between the RBR-silenced amiRBR and Atbrca1 mutants suggests that RBR and AtBRCA1 may function together in maintaining genome integrity. Together with E2FA, RBR is directly involved in the transcriptional DNA damage response as well as in the cell death pathway that is independent of SOG1, the plant functional analogue of p53. Thus, plant homologs and analogues of major mammalian tumour suppressor proteins form a regulatory network that coordinates cell proliferation with cell and genome integrity.

The role of Arabidopsis glutathione transferase F9 gene under oxidative stress in seedlings.

Arabidopsis thaliana contains 54 soluble glutathione transferases (GSTs, EC 2.5.1.18), which are thought to play major roles in oxidative stress responses, but little is known about the function of individual isoenzymes. The role of AtGST phi 9 (GSTF9) in the salt- and salicylic acid response was investigated using 2-week-old Atgstf9 and wild type (Wt) plants. Atgstf9 mutants accumulated more ascorbic acid (AsA) and glutathione (GSH) and had decreased glutathione peroxidase (GPOX) activity under control conditions. Treatment of 2-week-old seedlings with 10⁻7 M salicylic acid (SA) for 48 h resulted in elevated H2O2level and enhanced GST activity in Atgstf9 plants, 10⁻5 M SA treatment enhanced the malondialdehyde and dehydroascorbate contents compared to Wt. 50 and 150 mM NaCl increased the GST activity, AsA and GSH accumulation in Atgstf9 seedlings more pronounced than in Wt plants. We found that the Atgstf9 mutants had altered redox homeostasis under control and stress conditions, in which elevated AsA and GSH levels and modified GST and GPOX activities may play significant role. The half-cell potential values calculated from the concentration of GSH and GSSG indicate that this GST isoenzyme has an important role in the salt stress response.

The low oxygen, oxidative and osmotic stress responses synergistically act through the ethylene response factor VII genes RAP2.12, RAP2.2 and RAP2.3.

The ethylene response factor VII (ERF-VII) transcription factor RELATED TO APETALA2.12 (RAP2.12) was previously identified as an activator of the ALCOHOL DEHYDROGENASE1 promoter::luciferase (ADH1-LUC) reporter gene. Here we show that overexpression of RAP2.12 and its homologues RAP2.2 and RAP2.3 sustains ABA-mediated activation of ADH1 and activates hypoxia marker genes under both anoxic and normoxic conditions. Inducible expression of all three RAP2s conferred tolerance to anoxia, oxidative and osmotic stresses, and enhanced the sensitivity to abscisic acid (ABA). Consistently, the rap2.12-2 rap2.3-1 double mutant showed hypersensitivity to both submergence and osmotic stress. These findings suggest that the three ERF-VII-type transcription factors play roles in tolerance to multiple stresses that sequentially occur during and after submergence in Arabidopsis. Oxygen-dependent degradation of RAP2.12 was previously shown to be mediated by the N-end rule pathway. During submergence the RAP2.12, RAP2.2 and RAP2.3 are stabilized and accumulates in the nucleus affecting the transcription of stress response genes. We conclude that the stabilized RAP2 transcription factors can prolong the ABA-mediated activation of a subset of osmotic responsive genes (e.g. ADH1). We also show that RAP2.12 protein level is affected by the REALLY INTERESTING GENE (RING) domain containing SEVEN IN ABSENTIA of Arabidopsis thaliana 2 (SINAT2). Silencing of SINAT1/2 genes leads to enhanced RAP2.12 abundance independently of the presence or absence of its N-terminal degron. Taken together, our results suggest that RAP2.12 and its homologues RAP2.2 and RAP2.3 act redundantly in multiple stress responses. Alternative protein degradation pathways may provide inputs to the RAP2 transcription factors for the distinct stresses.

Exogenous salicylic acid-triggered changes in the glutathione transferases and peroxidases are key factors in the successful salt stress acclimation of Arabidopsis thaliana.

Salicylic acid (SA) applied exogenously is a potential priming agent during abiotic stress. In our experiments, the priming effect of SA was tested by exposing Arabidopsis thaliana (L.) Heynh. plants to 2-week-long 10-9-10-5 M SA pretreatments in a hydroponic medium, followed by 1 week of 100mM NaCl stress. The levels of reactive oxygen species and H2O2, changes in antioxidant enzyme activity and the expression of selected glutathione transferase (GST) genes were investigated. Although 10-9-10-7 M SA pretreatment insufficiently induced defence mechanisms during the subsequent salt stress, 2-week pretreatments with 10-6 and 10-5 M SA alleviated the salinity-induced H2O2 and malondialdehyde accumulation, and increased superoxide dismutase, guaiacol peroxidase, GST and glutathione peroxidase (GPOX) activity. Our results indicate that long-term 10-6 and 10-5 M SA treatment mitigated the salt stress injury in this model plant. Enhanced expression of AtGSTU19 and AtGSTU24 may be responsible for the induced GST and GPOX activity, which may play an important role in acclimation. Modified GST expression suggested altered signalling in SA-hardened plants during salt stress. The hydroponic system applied in our experiments proved to be a useful tool for studying the effects of sequential treatments in A. thaliana.

The Arabidopsis ZINC FINGER PROTEIN3 Interferes with Abscisic Acid and Light Signaling in Seed Germination and Plant Development.

Seed germination is controlled by environmental signals, including light and endogenous phytohormones. Abscisic acid (ABA) inhibits, whereas gibberellin promotes, germination and early seedling development, respectively. Here, we report that ZFP3, a nuclear C2H2 zinc finger protein, acts as a negative regulator of ABA suppression of seed germination in Arabidopsis (Arabidopsis thaliana). Accordingly, regulated overexpression of ZFP3 and the closely related ZFP1, ZFP4, ZFP6, and ZFP7 zinc finger factors confers ABA insensitivity to seed germination, while the zfp3 zfp4 double mutant displays enhanced ABA susceptibility. Reduced expression of several ABA-induced genes, such as RESPONSIVE TO ABSCISIC ACID18 and transcription factor ABSCISIC ACID-INSENSITIVE4 (ABI4), in ZFP3 overexpression seedlings suggests that ZFP3 negatively regulates ABA signaling. Analysis of ZFP3 overexpression plants revealed multiple phenotypic alterations, such as semidwarf growth habit, defects in fertility, and enhanced sensitivity of hypocotyl elongation to red but not to far-red or blue light. Analysis of genetic interactions with phytochrome and abi mutants indicates that ZFP3 enhances red light signaling by photoreceptors other than phytochrome A and additively increases ABA insensitivity conferred by the abi2, abi4, and abi5 mutations. These data support the conclusion that ZFP3 and the related ZFP subfamily of zinc finger factors regulate light and ABA responses during germination and early seedling development.

The heat shock factor A4A confers salt tolerance and is regulated by oxidative stress and the mitogen-activated protein kinases MPK3 and MPK6.

Heat shock factors (HSFs) are principal regulators of plant responses to several abiotic stresses. Here, we show that estradiol-dependent induction of HSFA4A confers enhanced tolerance to salt and oxidative agents, whereas inactivation of HSFA4A results in hypersensitivity to salt stress in Arabidopsis (Arabidopsis thaliana). Estradiol induction of HSFA4A in transgenic plants decreases, while the knockout hsfa4a mutation elevates hydrogen peroxide accumulation and lipid peroxidation. Overexpression of HSFA4A alters the transcription of a large set of genes regulated by oxidative stress. In yeast (Saccharomyces cerevisiae) two-hybrid and bimolecular fluorescence complementation assays, HSFA4A shows homomeric interaction, which is reduced by alanine replacement of three conserved cysteine residues. HSFA4A interacts with mitogen-activated protein kinases MPK3 and MPK6 in yeast and plant cells. MPK3 and MPK6 phosphorylate HSFA4A in vitro on three distinct sites, serine-309 being the major phosphorylation site. Activation of the MPK3 and MPK6 mitogen-activated protein kinase pathway led to the transcriptional activation of the HEAT SHOCK PROTEIN17.6A gene. In agreement that mutation of serine-309 to alanine strongly diminished phosphorylation of HSFA4A, it also strongly reduced the transcriptional activation of HEAT SHOCK PROTEIN17.6A. These data suggest that HSFA4A is a substrate of the MPK3/MPK6 signaling and that it regulates stress responses in Arabidopsis.

Transformation using controlled cDNA overexpression system.

The controlled cDNA overexpression system (COS) was developed to identify novel regulatory genes in model plants as well as in other species that might have a particular valuable trait. The COS system (Papdi et al. Plant Physiol 147:528-542, 2008) is composed of a random cDNA library prepared in a T-DNA plant expression vector, under the control of the estradiol-inducible XVE promoter. Large-scale genetic transformation of Arabidopsis thaliana generates a transgenic plant population with randomly inserted cDNA clones. Overexpression of the inserted cDNA can create selectable phenotypes, allowing the facile identification and cloning of the responsible genes. Here we describe protocols to create and use the COS system for diverse purposes in plant biology.

Genetic screens to identify plant stress genes.

A powerful means to learn about gene functions in a developmental or physiological context in an organism is to isolate the corresponding mutants with altered phenotypes. Diverse mutagenic agents, including chemical and biological, have been widely employed, and each comes with its own advantages and inconveniences. For Arabidopsis thaliana, whose genome sequence is publicly available, the reliance of reverse genetics to understand the relevant roles of genes particularly those coding for proteins in growth and development is now a common practice. Identifying multiple alleles at each locus is important because they can potentially reveal epistatic relationship in a signaling pathway or components belonging to a common signaling complex by their synergistic or even allele-specific enhancement of the phenotypic severity. In this article, we describe mutagenesis by using ethyl methanesulfonate (EMS) and transfer (T)-DNA-mediated insertion or activation tagging as applied to the most widely used genetic plant model A. thaliana. Also, we demonstrate the utility of several genetic screening approaches to dissect adaptive responses to various abiotic stresses.

Genetic technologies for the identification of plant genes controlling environmental stress responses.

Abiotic conditions such as light, temperature, water availability and soil parameters determine plant growth and development. The adaptation of plants to extreme environments or to sudden changes in their growth conditions is controlled by a well balanced, genetically determined signalling system, which is still far from being understood. The identification and characterisation of plant genes which control responses to environmental stresses is an essential step to elucidate the complex regulatory network, which determines stress tolerance. Here, we review the genetic approaches, which have been used with success to identify plant genes which control responses to different abiotic stress factors. We describe strategies and concepts for forward and reverse genetic screens, conventional and insertion mutagenesis, TILLING, gene tagging, promoter trapping, activation mutagenesis and cDNA library transfer. The utility of the various genetic approaches in plant stress research we review is illustrated by several published examples.

Functional identification of Arabidopsis stress regulatory genes using the controlled cDNA overexpression system.

Responses to environmental stresses in higher plants are controlled by a complex web of abscisic acid (ABA)-dependent and independent signaling pathways. To perform genetic screens for identification of novel Arabidopsis (Arabidopsis thaliana) loci involved in the control of abiotic stress responses, a complementary DNA (cDNA) expression library was created in a Gateway version of estradiol-inducible XVE binary vector (controlled cDNA overexpression system [COS]). The COS system was tested in three genetic screens by selecting for ABA insensitivity, salt tolerance, and activation of a stress-responsive ADH1-LUC (alcohol dehydrogenase-luciferase) reporter gene. Twenty-seven cDNAs conferring dominant, estradiol-dependent stress tolerance phenotype, were identified by polymerase chain reaction amplification and sequence analysis. Several cDNAs were recloned into the XVE vector and transformed recurrently into Arabidopsis, to confirm that the observed conditional phenotypes were due to their estradiol-dependent expression. Characterization of a cDNA conferring insensitivity to ABA in germination assays has identified the coding region of heat shock protein HSP17.6A suggesting its implication in ABA signal transduction. Screening for enhanced salt tolerance in germination and seedling growth assays revealed that estradiol-controlled overexpression of a 2-alkenal reductase cDNA confers considerable level of salt insensitivity. Screening for transcriptional activation of stress- and ABA-inducible ADH1-LUC reporter gene has identified the ERF/AP2-type transcription factor RAP2.12, which sustained high-level ADH1-LUC bioluminescence, enhanced ADH1 transcription rate, and increased ADH enzyme activity in the presence of estradiol. These data illustrate that application of the COS cDNA expression library provides an efficient strategy for genetic identification and characterization of novel regulators of abiotic stress responses.