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

The Arabidopsis Zinc Finger Protein 3 interferes with ABA 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

E2FA and E2FB transcription factors coordinate cell proliferation with seed maturation

The E2F transcription factors and the RETINOBLASTOMA RELATED (RBR) repressor protein are principal regulators coordinating cell proliferation with differentiation, but their role during seed development is little understood. We show that in the fully developed 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 (ABI3), FUSCA 3 (FUS3) and WRINKLED 1 (WRI1) are upregulated in the e2fab double mutant embryo. In accordance, E2FA directly regulates LEC2, and mutation at the consensus E2F-binding site in LEC2 promoter de-represses its activity during the proliferative stage of seed development. Additionally, 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 program until the cell proliferation phase is completed

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

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. 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 two-hybrid and bimolecular fluorescence complementation (BiFC) 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, Ser309 being the major phosphorylation site. Activation of the MPK3 and MPK6 MAPK pathway led to the transcriptional activation of the heat-shock protein gene HSP17.6A. In agreement that mutation of Ser309 to alanine strongly diminished phosphorylation of HSFA4A, it also strongly reduced the transcriptional activation of HSP17.6A. These data suggest that HSFA4A is a substrate of the MPK3/6 signalling and it regulates stress responses in Arabidopsis

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 H2O2 level 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 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 temperatures, salinity, water deprivation or heavy metals. Their function in stress combinations is however not known. The 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 combination of these conditions. Fast translocation of HSFA4A-YFP protein from cytosol to nuclei takes place in salt-treated cells. HSFA4A can be phosphorylated not only by MAP kinases MPK3/6 but also by MPK4 and Ser309 is the dominant MAPK phosphorylation site. In vivo data suggest that HSFA4A can be substrate of other kinases as well. Changing Ser309 to Asp or Ala has altered 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

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 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 DIMERISATION PARTNER A, further elevates 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 the 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

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