Xiangdong Fu

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.

Auxin promotes Arabidopsis root growth by modulating gibberellin response

The growth of plant organs is influenced by a stream of the phytohormone auxin that flows from the shoot apex to the tip of the root. However, until now it has not been known how auxin regulates the cell proliferation and enlargement that characterizes organ growth. Here we show that auxin controls the growth of roots by modulating cellular responses to the phytohormone gibberellin (GA). GA promotes the growth of plants by opposing the effects of nuclear DELLA protein growth repressors, one of which is Arabidopsis RGA (for repressor of gal-3). GA opposes the action of several DELLA proteins by destabilizing them, reducing both the concentration of detectable DELLA proteins and their growth-restraining effects. We also show that auxin is necessary for GA-mediated control of root growth, and that attenuation of auxin transport or signalling delays the GA-induced disappearance of RGA from root cell nuclei. Our observations indicate that the shoot apex exerts long-distance control on the growth of plant organs through the effect of auxin on GA-mediated DELLA protein destabilization.

Natural variation at the DEP1 locus enhances grain yield in rice

Grain yield is controlled by quantitative trait loci (QTLs) derived from natural variations in many crop plants. Here we report the molecular characterization of a major rice grain yield QTL that acts through the determination of panicle architecture. The dominant allele at the DEP1 locus is a gain-of-function mutation causing truncation of a phosphatidylethanolamine-binding protein-like domain protein. The effect of this allele is to enhance meristematic activity, resulting in a reduced length of the inflorescence internode, an increased number of grains per panicle and a consequent increase in grain yield. This allele is common to many Chinese high-yielding rice varieties and likely represents a relatively recent introduction into the cultivated rice gene pool. We also show that a functionally equivalent allele is present in the temperate cereals and seems to have arisen before the divergence of the wheat and barley lineages.

Control of grain size, shape and quality by OsSPL16 in rice

Grain size and shape are important components of grain yield and quality and have been under selection since cereals were first domesticated. Here, we show that a quantitative trait locus GW8 is synonymous with OsSPL16, which encodes a protein that is a positive regulator of cell proliferation. Higher expression of this gene promotes cell division and grain filling, with positive consequences for grain width and yield in rice. Conversely, a loss-of-function mutation in Basmati rice is associated with the formation of a more slender grain and better quality of appearance. The correlation between grain size and allelic variation at the GW8 locus suggests that mutations within the promoter region were likely selected in rice breeding programs. We also show that a marker-assisted strategy targeted at elite alleles of GS3 and OsSPL16 underlying grain size and shape can be effectively used to simultaneously improve grain quality and yield.

Gibberellin regulates Arabidopsis floral development via suppression of DELLA protein function.

The phytohormone gibberellin (GA) regulates the development and fertility of Arabidopsis flowers. The mature flowers of GA-deficient mutant plants typically exhibit reduced elongation growth of petals and stamens. In addition, GA-deficiency blocks anther development, resulting in male sterility. Previous analyses have shown that GA promotes the elongation of plant organs by opposing the function of the DELLA proteins, a family of nuclear growth repressors. However, it was not clear that the DELLA proteins are involved in the GA-regulation of stamen and anther development. We show that GA regulates cell elongation rather than cell division during Arabidopsis stamen filament elongation. In addition, GA regulates the cellular developmental pathway of anthers leading from microspore to mature pollen grain. Genetic analysis shows that the Arabidopsis DELLA proteins RGA and RGL2 jointly repress petal, stamen and anther development in GA-deficient plants, and that this function is enhanced by RGL1 activity. GA thus promotes Arabidopsis petal, stamen and anther development by opposing the function of the DELLA proteins RGA, RGL1 and RGL2.

The Arabidopsis Mutant sleepy1gar2-1 Protein Promotes Plant Growth by Increasing the Affinity of the SCFSLY1 E3 Ubiquitin Ligase for DELLA Protein Substrates

DELLA proteins restrain the cell proliferation and enlargement that characterizes the growth of plant organs. Gibberellin stimulates growth via 26S proteasome–dependent destruction of DELLAs, thus relieving DELLA-mediated growth restraint. Here, we show that the Arabidopsis thaliana sleepy1gar2-1 (sly1gar2-1) mutant allele encodes a mutant subunit (sly1gar2-1) of an SCFSLY1 E3 ubiquitin ligase complex. SLY1 (the wild-type form) and sly1gar2-1 both confer substrate specificity on this complex via specific binding to the DELLA proteins. However, sly1gar2-1 interacts more strongly with the DELLA target than does SLY1. In addition, the strength of the SCFSLY1–DELLA interaction is increased by target phosphorylation. Growth-promoting DELLA destruction is dependent on SLY1 availability, on the strength of the interaction between SLY1 and the DELLA target, and on promotion of the SCFSLY1–DELLA interaction by DELLA phosphorylation.

Phosphate Starvation Root Architecture and Anthocyanin Accumulation Responses Are Modulated by the Gibberellin-DELLA Signaling Pathway in Arabidopsis

Phosphate (Pi) is a macronutrient that is essential for plant growth and development. However, the low mobility of Pi impedes uptake, thus reducing availability. Accordingly, plants have developed physiological strategies to cope with low Pi availability. Here, we report that the characteristic Arabidopsis thaliana Pi starvation responses are in part dependent on the activity of the nuclear growth-repressing DELLA proteins (DELLAs), core components of the gibberellin (GA)-signaling pathway. We first show that multiple shoot and root Pi starvation responses can be repressed by exogenous GA or by mutations conferring a substantial reduction in DELLA function. In contrast, mutants having enhanced DELLA function exhibit enhanced Pi starvation responses. We also show that Pi deficiency promotes the accumulation of a green fluorescent protein-tagged DELLA (GFP-RGA [repressor of ga1-3]) in root cell nuclei. In further experiments, we show that Pi starvation causes a decrease in the level of bioactive GA and associated changes in the levels of gene transcripts encoding enzymes of GA metabolism. Finally, we show that the GA-DELLA system regulates the increased root hair length that is characteristic of Pi starvation. In conclusion, our results indicate that DELLA-mediated signaling contributes to the anthocyanin accumulation and root architecture changes characteristic of Pi starvation responses, but do not regulate Pi starvation-induced changes in Pi uptake efficiency or the accumulation of selected Pi starvation-responsive gene transcripts. Pi starvation causes a reduction in bioactive GA level, which, in turn, causes DELLA accumulation, thus modulating several adaptively significant plant Pi starvation responses.

Gibberellin-mediated proteasome-dependent degradation of the barley DELLA protein SLN1 repressor

DELLA proteins are nuclear repressors of plant gibberellin (GA) responses. Here, we investigate the properties of SLN1, a DELLA protein from barley that is destabilized by GA treatment. Using specific inhibitors of proteasome function, we show that proteasome-mediated protein degradation is necessary for GA-mediated destabilization of SLN1. We also show that GA responses, such as the aleurone α-amylase response and seedling leaf extension growth, require proteasome-dependent GA-mediated SLN1 destabilization. In further experiments with protein kinase and protein phosphatase inhibitors, we identify two additional signaling steps that are necessary for GA response and for GA-mediated destabilization of SLN1. Thus, GA signaling involves protein phosphorylation and dephosphorylation steps and promotes the derepression of GA responses via proteasome-dependent destabilization of DELLA repressors.

DELLAs Contribute to Plant Photomorphogenesis

Plant morphogenesis is profoundly influenced by light (a phenomenon known as photomorphogenesis). For example, light inhibits seedling hypocotyl growth via activation of phytochromes and additional photoreceptors. Subsequently, information is transmitted through photoreceptor-linked signal transduction pathways and used (via previously unknown mechanisms) to control hypocotyl growth. Here we show that light inhibition of Arabidopsis (Arabidopsis thaliana) hypocotyl growth is in part dependent on the DELLAs (a family of nuclear growth-restraining proteins that mediate the effect of the phytohormone gibberellin [GA] on growth). We show that light inhibition of growth is reduced in DELLA-deficient mutant hypocotyls. We also show that light activation of phytochromes promotes the accumulation of DELLAs. A green fluorescent protein (GFP)-tagged DELLA (GFP-RGA) accumulates in elongating cells of light-grown, but not dark-grown, transgenic wild-type hypocotyls. Furthermore, transfer of seedlings from light to dark (or vice versa) results in rapid changes in hypocotyl GFP-RGA accumulation, changes that are paralleled by rapid alterations in the abundance in hypocotyls of transcripts encoding enzymes of GA metabolism. These observations suggest that light-dependent changes in hypocotyl GFP-RGA accumulation are a consequence of light-dependent changes in bioactive GA level. Finally, we show that GFP accumulation and quantitative modulation of hypocotyl growth is proportionate with light energy dose (the product of exposure duration and fluence rate). Hence, DELLAs inhibit hypocotyl growth during the light phase of the day-night cycle via a mechanism that is quantitatively responsive to natural light variability. We conclude that DELLAs are a major component of the adaptively significant mechanism via which light regulates plant growth during photomorphogenesis.

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.