Liu-Min Fan

Coordinated regulation of Arabidopsis thaliana development by light and gibberellins

Light and gibberellins (GAs) mediate many essential and partially overlapping plant developmental processes. DELLA proteins are GA-signalling repressors that block GA-induced development. GA induces degradation of DELLA proteins via the ubiquitin/proteasome pathway, but light promotes accumulation of DELLA proteins by reducing GA levels. It was proposed that DELLA proteins restrain plant growth largely through their effect on gene expression. However, the precise mechanism of their function in coordinating GA signalling and gene expression remains unknown. Here we characterize a nuclear protein interaction cascade mediating transduction of GA signals to the activity regulation of a light-responsive transcription factor. In the absence of GA, nuclear-localized DELLA proteins accumulate to higher levels, interact with phytochrome-interacting factor 3 (PIF3, a bHLH-type transcription factor) and prevent PIF3 from binding to its target gene promoters and regulating gene expression, and therefore abrogate PIF3-mediated light control of hypocotyl elongation. In the presence of GA, GID1 proteins (GA receptors) elevate their direct interaction with DELLA proteins in the nucleus, trigger DELLA protein’s ubiquitination and proteasome-mediated degradation, and thus release PIF3 from the negative effect of DELLA proteins.

Nitric Oxide Signaling in Plant Responses to Abiotic Stresses

Nitric oxide (NO) plays important roles in diverse physiological processes in plants. NO can provoke both beneficial and harmful effects, which depend on the concentration and location of NO in plant cells. This review is focused on NO synthesis and the functions of NO in plant responses to abiotic environmental stresses. Abiotic stresses mostly induce NO production in plants. NO alleviates the harmfulness of reactive oxygen species, and reacts with other target molecules, and regulates the expression of stress responsive genes under various stress conditions.

Biochemical insights on degradation of Arabidopsis DELLA proteins gained from a cell-free assay system.

The phytohormone gibberellic acid (GA) regulates diverse aspects of plant growth and development. GA responses are triggered by the degradation of DELLA proteins, which function as repressors in GA signaling pathways. Recent studies in Arabidopsis thaliana and rice (Oryza sativa) have implied that the degradation of DELLA proteins occurred via the ubiquitin-proteasome system. Here, we developed an Arabidopsis cell-free system to recapitulate DELLA protein degradation in vitro. Using this cell-free system, we documented that Lys-29 of ubiquitin is the major site for ubiquitin chain formation to mediate DELLA protein degradation. We also confirmed the specific roles of GA receptors and multisubunit E3 ligase components in regulating DELLA protein degradation. In addition, blocking DELLA degradation with a PP1/PP2A phosphatase inhibitor in our cell-free assay suggested that degradation of DELLA proteins required protein Ser/Thr dephosphorylation activity. Furthermore, our data revealed that the LZ domain of Arabidopsis DELLA proteins is essential for both their stability and activity. Thus, our in vitro degradation system provides biochemical insights into the regulation of DELLA protein degradation. This in vitro assay system could be widely adapted for dissecting cellular signaling pathways in which regulated proteolysis is a key recurrent theme.

DELLA-mediated PIF degradation contributes to coordination of light and gibberellin signalling in Arabidopsis

Light and gibberellins (GAs) antagonistically regulate hypocotyl elongation in plants. It has been demonstrated that DELLAs, which are negative regulators of GA signalling, inhibit phytochrome-interacting factors 3 and 4 (PIF3 and PIF4) by sequestering their DNA-recognition domains. However, it is unclear whether there are other mechanisms of regulatory crosstalk between DELLAs and PIFs. Here, we demonstrate that DELLAs negatively regulate the abundance of four PIF proteins through the ubiquitin–proteasome system. Reduction of PIF3 protein abundance by DELLAs correlates closely with reduced hypocotyl elongation. Both sequestration and degradation of PIF3 by DELLAs contribute to a reduction in PIF3 binding to its target genes. Thus, we show that promotion of PIF degradation by DELLAs is required to coordinate light and GA signals, and the dual regulation of transcription factors by DELLAs by both sequestration and degradation may be a general mechanism.

Arabidopsis DET1 Represses Photomorphogenesis in Part by Negatively Regulating DELLA Protein Abundance in Darkness

Arabidopsis De-etiolated 1 (DET1) is one of the key repressors that maintain the etiolated state of seedlings in darkness. The plant hormone gibberellic acid (GA) also participates in this process, and plants deficient in GA synthesis or signaling show a partially de-etiolated phenotype in darkness. However, how DET1 and the GA pathway work in concert in repressing photomorphogenesis remains largely unknown. In this study, we found that the abundance of DELLA proteins in det1-1 was increased in comparison with that in the wild-type plants. Mutation in DET1 changed the sensitivity of hypocotyl elongation of mutant seedlings to GA and paclobutrazol (PAC), an inhibitor of GA synthesis. However, we did not find obvious differences between det1-1 and wild-type plants with regard to the bioactive GA content or the GA signaling upstream of DELLAs. Genetic data showed that removal of several DELLA proteins suppressed the det1-1 mutant phenotype more obviously than GA treatment, indicating that DET1 can regulate DELLA proteins via some other mechanisms. In addition, a large-scale transcriptomic analysis revealed that DET1 and DELLAs play antagonistic roles in regulating expression of photosynthetic and cell elongation-related genes in etiolated seedlings. Taken together, our results show that DET1 represses photomorphogenesis in darkness in part by reducing the abundance of DELLA proteins.

Actin Dynamics Regulates Voltage-Dependent Calcium-Permeable Channels of the Vicia faba Guard Cell Plasma Membrane

Free cytosolic Ca(2+) ([Ca(2+)](cyt)) is an ubiquitous second messenger in plant cell signaling, and [Ca(2+)](cyt) elevation is associated with Ca(2+)-permeable channels in the plasma membrane and endomembranes regulated by a wide range of stimuli. However, knowledge regarding Ca(2+) channels and their regulation remains limited in planta. A type of voltage-dependent Ca(2+)-permeable channel was identified and characterized for the Vicia faba L. guard cell plasma membrane by using patch-clamp techniques. These channels are permeable to both Ba(2+) and Ca(2+), and their activities can be inhibited by micromolar Gd(3+). The unitary conductance and the reversal potential of the channels depend on the Ca(2+) or Ba(2+) gradients across the plasma membrane. The inward whole-cell Ca(2+) (Ba(2+)) current, as well as the unitary current amplitude and NP(o) of the single Ca(2+) channel, increase along with the membrane hyperpolarization. Pharmacological experiments suggest that actin dynamics may serve as an upstream regulator of this type of calcium channel of the guard cell plasma membrane. Cytochalasin D, an actin polymerization blocker, activated the NPo of these channels at the single channel level and increased the current amplitude at the whole-cell level. But these channel activations and current increments could be restrained by pretreatment with an F-actin stabilizer, phalloidin. The potential physiological significance of this regulatory mechanism is also discussed.

CHD3 chromatin-remodeling factor PICKLE regulates floral transition partially via modulating LEAFY expression at the chromatin level in Arabidopsis

PICKLE (PKL), a putative CHD3 chromatin remodeling factor, has been suggested to be involved in multiple processes in Arabidopsis. Here, we confirmed the late-flowering phenotype caused by pkl mutation with pkl mutants in two different ecotypes, and investigated the possible mechanisms that account for PKL regulation of flowering time. Quantitative RT-PCR and RNA-seq assays showed that expression of the LEAFY gene (LFY) and a number of LFY-regulated floral homeotic genes were down-regulated in seedlings of the pkl mutants. As predicted, overexpression of LFY restored normal flowering time of pkl mutants. Our results suggest that PKL may be involved in regulating flowering time via LFY expression. To uncover the underlying mechanism, ChIP-PCR using anti-PKL was performed on materials from three developmental stages of seedlings. Our results showed that PKL associated with the genomic sequences of LFY, particularly at 10-day and 25-day after germination. We also showed that loss of PKL affected H3K27me3 level at the promoter of LFY. Taken together, our data suggest that transcriptional regulation of LFY at the chromatin level by PKL may at least partially account for the late-flowering phenotype of pkl mutants.

Natural variation of H3K27me3 modification in two Arabidopsis accessions and their hybrid

Histone modifications affect gene expression, but the mechanism and biological consequence of natural variation in histone modifications remain unclear. Here, we generated genome-wide integrated maps of H3K27me3 modification and transcriptome for Col, C24 and their F1 hybrid. A total of 1,828 genomic regions showing variation in H3K27me3 modification between Col and C24 were identified, most of which were associated with genic regions. Natural variation of H3K27me3 modification between parents could result in allelic bias of H3K27me3 in hybrids. Furthermore, we found that H3K27me3 variation between Col and C24 was negatively correlated with gene expression differences between two accessions, especially with those arising from the cis-effect. Importantly, mutation of CLF, an Arabidopsis methyltransferase for H3K27, altered gene expression patterns between the parents. Together, these data provide insights into natural variation of histone modifications and their association with gene expression differences between Arabidopsis ecotypes.

Phosphorylation and negative regulation of CONSTITUTIVELY PHOTOMORPHOGENIC 1 by PINOID in Arabidopsis

Significance As an E3 ubiquitin ligase, CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) directly ubiquitinates and triggers the degradation of various downstream targets and acts as a central repressor of light signaling. In this study, our data reveal that a Ser/Thr kinase, PINOID (PID), promotes photomorphogenic development. PID directly interacts with COP1 and phosphorylates it at the site of Ser20, thereby repressing the activity of COP1 in plants. Thus, our findings provide insight into the precise regulatory mechanism in maintaining appropriate COP1 activity in response to dynamically changing light conditions in plants. CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) plays crucial roles in various cellular processes via its E3 ubiquitin ligase activity in organisms, ranging from fungi to humans. As a key component in regulating various biological events, COP1 itself is precisely controlled at multiple layers. Here, we report a negative regulator of COP1, PINOID (PID), which positively mediates photomorphogenic development. Specifically, PID genetically and physically interacts with COP1 and directly phosphorylates COP1 at Ser20. As a result, this posttranslational modification serves to repress COP1 activity and promote photomorphogenesis. Our findings signify a key regulatory mechanism for precisely maintaining COP1 activity, thereby ensuring appropriate development in plants.

B-BOX DOMAIN PROTEIN28 Negatively Regulates Photomorphogenesis by Repressing the Activity of Transcription Factor HY5 and Undergoes COP1-Mediated Degradation

The B-box protein BBX28 negatively regulates photomorphogenic development by functioning as a key factor in the CONSTITUTIVELY PHOTOMORPHOGENIC1-HY5 regulatory hub. Plants have evolved a delicate molecular system to fine-tune their growth and development in response to dynamically changing light environments. In this study, we found that BBX28, a B-box domain protein, negatively regulates photomorphogenic development in a dose-dependent manner in Arabidopsis thaliana. BBX28 interferes with the binding of transcription factor HY5 to the promoters of its target genes through physical interactions, thereby repressing its activity and negatively affecting HY5-regulated gene expression. In darkness, BBX28 associates with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) and undergoes COP1-mediated degradation via the 26S proteasome system. Collectively, these results demonstrate that BBX28 acts as a key factor in the COP1-HY5 regulatory hub by maintaining proper HY5 activity to ensure normal photomorphogenic development in plants.