Xiushan Li

FERONIA interacts with ABI2-type phosphatases to facilitate signaling cross-talk between abscisic acid and RALF peptide in Arabidopsis

Significance Receptor-like kinase FERONIA (FER) not only serves as a receptor for growth-regulating rapid alkalinization factor (RALF) peptide but also acts as an important node in a variety of other signaling pathways, including plant responses to hormones, pathogens, and abiotic stresses. However, the mechanism underlying FER actions in these signaling cross-talks remain largely unknown. Our previous work identified a molecular relay that allows FER to inhibit abscisic acid (ABA) response through activation of a small G protein to enhance the activity of the clade A protein phosphatase type 2C (PP2C) ABA Insensitive 2 (ABI2), a repressor of ABA response. In this study, we found that ABI2 can directly interact and dephosphorylate FER, providing a feedback mechanism for RALF activation of FER. Receptor-like kinase FERONIA (FER) plays a crucial role in plant response to small molecule hormones [e.g., auxin and abscisic acid (ABA)] and peptide signals [e.g., rapid alkalinization factor (RALF)]. It remains unknown how FER integrates these different signaling events in the control of cell growth and stress responses. Under stress conditions, increased levels of ABA will inhibit cell elongation in the roots. In our previous work, we have shown that FER, through activation of the guanine nucleotide exchange factor 1 (GEF1)/4/10-Rho of Plant 11 (ROP11) pathway, enhances the activity of the phosphatase ABA Insensitive 2 (ABI2), a negative regulator of ABA signaling, thereby inhibiting ABA response. In this study, we found that both RALF and ABA activated FER by increasing the phosphorylation level of FER. The FER loss-of-function mutant displayed strong hypersensitivity to both ABA and abiotic stresses such as salt and cold conditions, indicating that FER plays a key role in ABA and stress responses. We further showed that ABI2 directly interacted with and dephosphorylated FER, leading to inhibition of FER activity. Several other ABI2-like phosphatases also function in this pathway, and ABA-dependent FER activation required PYRABACTIN RESISTANCE (PYR)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR)–A-type protein phosphatase type 2C (PP2CA) modules. Furthermore, suppression of RALF1 gene expression, similar to disruption of the FER gene, rendered plants hypersensitive to ABA. These results formulated a mechanism for ABA activation of FER and for cross-talk between ABA and peptide hormone RALF in the control of plant growth and responses to stress signals.

Receptor kinase complex transmits RALF peptide signal to inhibit root growth in Arabidopsis

Significance Receptor-like kinase FERONIA (FER) is a versatile regulator of cell growth under both normal and stress environments. FER binds its peptide ligand, rapid alkalinization factor 1 (RALF1), and triggers downstream events to inhibit cell growth in primary roots. However, the mechanism of RALF1 reception by FER is still largely unknown. In this study, we identified a receptor-like cytoplasmic kinase (RPM1-induced protein kinase, RIPK) that directly interacts with and is phosphorylated by FER in a RALF1 peptide-dependent manner. The defects of fer-4 mutant in RALF1 response and root hair development are mimicked by ripk loss-of-function but partially compensated by RIPK overexpression. These and other data suggest that formation of the FER–RIPK complex serves as a crucial step in the RALF1 signaling pathway. A number of hormones work together to control plant cell growth. Rapid Alkalinization Factor 1 (RALF1), a plant-derived small regulatory peptide, inhibits cell elongation through suppression of rhizosphere acidification in plants. Although a receptor-like kinase, FERONIA (FER), has been shown to act as a receptor for RALF1, the signaling mechanism remains unknown. In this study, we identified a receptor-like cytoplasmic kinase (RPM1-induced protein kinase, RIPK), a plasma membrane-associated member of the RLCK-VII subfamily, that is recruited to the receptor complex through interacting with FER in response to RALF1. RALF1 triggers the phosphorylation of both FER and RIPK in a mutually dependent manner. Genetic analysis of the fer-4 and ripk mutants reveals RIPK, as well as FER, to be required for RALF1 response in roots. The RALF1–FER–RIPK interactions may thus represent a mechanism for peptide signaling in plants.

Molecular character of a phosphatase 2C (PP2C) gene relation to stress tolerance in Arabidopsis thaliana

Protein phosphatases type 2C (PP2Cs) from group A, which includes the ABI1/HAB1 and PP2CA branches, are key negative regulators of ABA signaling. HAI-1 gene had been shown to affect both seed and vegetative responses to ABA, which is one of PP2Cs clade A in Arabidopsis thaliana. Transgenic plants containing pHAI-1::GUS (β-glucuronidase) displayed GUS activity existing in the vascular system of leave veins, stems and petioles. Green fluorescent protein fused HAI-1 (HAI-1-GFP) was found in the nucleus through transient transformation assays with onion epidermal cells. The water-loss assays indicated the loss-of-function mutants did not show symptoms of wilting and they had still turgid green rosette leaves. The assays of seed germination by exogenous ABA and NaCl manifested that the loss-of-function mutants displayed higher insensitivity than wild-type plants. Taken together, the final results suggest that the HAI-1 (AT5G59220) encoded a nuclear protein and it can be highly induced by ABA and wound in Arabidposis, the stress-tolerance phenotype showed a slightly improvement when HAI-1 gene was disrupted.

Dwarfism in Brassica napus L. induced by the over-expression of a gibberellin 2-oxidase gene from Arabidopsis thaliana

Gibberellins (GAs) are endogenous hormones that play an important role in regulating plant stature by increasing cell division and elongation in stem internodes. The GA2-oxidase gene from Arabidopsis thaliana (AtGA2ox8) was introduced into Brassica napus L. by Agrobacterium-mediated floral-dip transformation with the aim of decreasing the amount of bioactive GA and hence reducing plant stature. As anticipated, the transgenic plants exhibited dwarf phenotype. Compared with the wild type, the transgenic plants had increased primary branches (by 14.1–15.3%) and siliques (by 10.8–15.2%), which resulted in a significant increase in the seed yield (by 9.6–12.4%). Moreover, the contents of anthocyanin in leaves of 60-day-old transgenic plants was about 9.4-fold higher in winter and about 6.8-fold higher in summer than the wild type. These excellent agronomic traits of the transgenic plants could not only improve the lodging resistance and seed yields, but also protect them against stress. Therefore, the over-expression of AtGA2ox8 might be used to produce dwarf varieties and increase seed yield in Brassica napus L.

AtWNK9 is regulated by ABA and dehydration and is involved in drought tolerance in Arabidopsis.

WNK (with no lysine [K]) kinases play important regulatory roles in flowering, as well as salt and osmotic stress tolerance in plants. Here, we report that AtWNK9, a member of the Arabidopsis WNK gene family, was induced by exogenous abscisic acid (ABA) treatment and dehydration stress. Overexpression of AtWNK9 from the cauliflower mosaic virus 35S promoter in Arabidopsis resulted in increased sensitivity to ABA, strong inhibition of primary root elongation, increased proline accumulation, reduced stomatal aperture, and a reduced rate of water loss. In addition, plant survival under drought stress was improved compared to wild type. In contrast, a mutant with a T-DNA insertion in AtWNK9 showed reduced ABA sensitivity and an increased rate of water loss; further, it showed increased susceptibility to drought stress. The transcription of a number of ABA signaling components, including ABI1, ERA1, ABI3, and ABF3, was up-regulated in AtWNK9 transgenic plants and down-regulated in the wnk9 mutant in response to ABA. Some ABA-responsive and biosynthetic genes, as well as other drought-related genes, were altered at various levels in AtWNK9 transgenic plants and wnk9 mutants under dehydration stress. Overall, these findings suggest that AtWNK9 plays a positive role in ABA signaling and improves drought tolerance in transgenic Arabidopsis.

Arabidopsis casein kinase 1-like 2 involved in abscisic acid signal transduction pathways

In this study, we isolated a homozygous T-DNA insertion mutant line, ckl2, of the casein kinase 1-Like 2 (CKL2) gene in Arabidopsis thaliana. Through analysis of the germination ratio, root length, water loss rate, and stomatal aperture, we found that the ckl2 mutants showed an abscisic acid (ABA)-hyposensitive phenotype in all tests. CKL2 was also found to mediate the expression of ABA-upregulated genes in seeds and seedlings. CKL2 was expressed in all tissues, especially in the leaf and leaf stalk, and could be induced by ABA. A proline accumulation experiment showed that the Pro content in ckl2 was lower than that of the wild type. The above results demonstrate that CKL2 was required for ABA-regulated seed germination, root growth, and gene expression, suggesting that CKL2 positively mediates ABA signaling in Arabidopsis.

RPN1a negatively regulates ABA signaling in Arabidopsis

The 26S proteasome selectively regulates key abscisic acid (ABA) signaling proteins, but the physiological functions and mechanisms of RPN1a (a subunit of the 26S proteasome) in ABA signaling remain largely unknown. In this study, we found that the mRNA expression of RPN1a was suppressed by ABA treatment, and that RPN1a protein was expressed abundantly in guard cells. In the presence of ABA, rpn1a mutants showed rapid stomatal closure, low water loss, delayed germination, and inhibited root elongation. In addition, the transcripts of key ABA signaling genes, including ABI5, RD22, RD29A, and RD29B, were upregulated in rpn1a mutant plants in response to ABA. Furthermore, the ABI5 protein level was higher in rpn1a mutants subjected to ABA treatment. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that RPN1a interacts with ABI1. Overall, these findings suggest that RPN1a negatively regulates ABA signaling in Arabidopsis.

RPN1a, a subunit of the 26S proteasome, controls trichome development in Arabidopsis.

The ubiquitin-mediated 26S proteasome pathway (UPS) is of great importance to plant growth and development. Previously research showed that a subunit of the 26S proteasome, named RPN1a, was involved in trichomes branching in Arabidopsis. Mutation in RPN1a give rise to more trichome branches on leaves. Here, we found that T-DNA insertion mutation in RPN1a resulted in increased trichome branches on main stem, and trichome number on rosette leaves and the main stem compared with the wild type plant. Expression analysis results showed that the transcription levels of ZFP6, ZFP5, GIS, GL1, GL2, GL3, TTG1 and MYB23, which promote trichome initiation, were up-regulated in the rpn1a mutant, and expression of FRC4, which is responsible for increased trichome branching, was also increased in the rpn1a mutant. Moreover, the mRNA expression level of RPN1a was significantly repressed by GA (gibberellin) and CK (cytokinin) treatment, which are two important phytohormones that play essential roles in trichome development. These results demonstrate that RPN1a may be involved in trichome development through the GA and CK signaling pathways.

The genetic and physiological analysis of late-flowering phenotype of T-DNA insertion mutants of AtCAL1 and AtCAL2 in Arabidopsis

The homozygous T-DNA mutants of AtCAL1 (Rat1) and AtCAL2 (Rat2) were obtained. The double mutant of Rat2/Rat1RNAi was constructed which showed obvious late-flowering phenotype from others. The expression of various flowering-related genes was studied among mutants and wild-type plants by quantitative RT–PCR. The double mutant plants showed the shortest root length compared with T-DNA insertion mutants and wild type plants under red light, blue light, and white light. The double mutants showed hypersensitivity to NaCl and ABA. However, these mutants had no effect on stomatal closure by ABA.

EBP1 nuclear accumulation negatively feeds back on FERONIA-mediated RALF1 signaling

FERONIA (FER), a plasma membrane receptor-like kinase, is a central regulator of cell growth that integrates environmental and endogenous signals. A peptide ligand rapid alkalinization factor 1 (RALF1) binds to FER and triggers a series of downstream events, including inhibition of Arabidopsis H+-ATPase 2 activity at the cell surface and regulation of gene expression in the nucleus. We report here that, upon RALF1 binding, FER first promotes ErbB3-binding protein 1 (EBP1) mRNA translation and then interacts with and phosphorylates the EBP1 protein, leading to EBP1 accumulation in the nucleus. There, EBP1 associates with the promoters of previously identified RALF1-regulated genes, such as CML38, and regulates gene transcription in response to RALF1 signaling. EBP1 appears to inhibit the RALF1 peptide response, thus forming a transcription–translation feedback loop (TTFL) similar to that found in circadian rhythm control. The plant RALF1-FER-EBP1 axis is reminiscent of animal epidermal growth factor receptor (EGFR) signaling, in which EGF peptide induces EGFR to interact with and phosphorylate EBP1, promoting EBP1 nuclear accumulation to control cell growth. Thus, we suggest that in response to peptide signals, plant FER and animal EGFR use the conserved key regulator EBP1 to control cell growth in the nucleus.