Characterization of Highbush Blueberry (Vaccinium corymbosum L.) Anthocyanin Biosynthesis Related MYBs and Functional Analysis of VcMYB Gene.

As one of the most important transcription factors regulating plant anthocyanin biosynthesis, MYB has attracted great attentions. In this study, we identified fifteen candidate anthocyanin biosynthesis related MYB (ABRM) proteins, including twelve R2R3-MYBs and three 1R-MYBs, from highbush blueberry. The subcellular localization prediction results showed that, with the exception of VcRVE8 (localized in chloroplast and nucleus), all of the blueberry ABRMs were nucleus-localized. The gene structure analysis revealed that the exon numbers of the blueberry ABRM genes varied greatly, ranging between one and eight. There are many light-responsive, phytohormone-responsive, abiotic stress-responsive and plant growth and development related cis-acting elements in the promoters of the blueberry ABRM genes. It is noteworthy that almost all of their promoters contain light-, ABA- and MeJA-responsive elements, which is consistent with the well-established results that anthocyanin accumulation and the expression of MYBs are influenced significantly by many factors, such as light, ABA and JA. The gene expression analysis revealed that VcMYB, VcMYB6, VcMYB23, VcMYBL2 and VcPH4 are expressed abundantly in blueberry fruits, and VcMYB is expressed the highest in the red, purple and blue fruits among all blueberry ABRMs. VcMYB shared high similarity with functionally proven ABRMs from many other plant species. The gene cloning results showed that VcMYB had three variable transcripts, but only the transient overexpression of VcMYB-1 promoted anthocyanin accumulation in the green fruits. Our study can provide a basis for future research on the anthocyanin biosynthesis related MYBs in blueberry.

The IPGA1-ANGUSTIFOLIA module regulates microtubule organisation and pavement cell shape in Arabidopsis.

Plant cells continuously experience mechanical stress resulting from the cell wall that bears internal turgor pressure. Cortical microtubules align with the predicted maximal tensile stress direction to guide cellulose biosynthesis and therefore results in cell wall reinforcement. We have previously identified Increased Petal Growth Anisotropy (IPGA1) as a putative microtubule-associated protein in Arabidopsis, but the function of IPGA1 remains unclear. Here, using the Arabidopsis cotyledon pavement cell as a model, we demonstrated that IPGA1 forms protein granules and interacts with ANGUSTIFOLIA (AN) to cooperatively regulate microtubule organisation in response to stress. Application of mechanical perturbations, such as cell ablation, led to microtubule reorganisation into aligned arrays in wild-type cells. This microtubule response to stress was enhanced in the IPGA1 loss-of-function mutant. Mechanical perturbations promoted the formation of IPGA1 granules on microtubules. We further showed that IPGA1 physically interacted with AN both in vitro and on microtubules. The ipga1 mutant alleles exhibited reduced interdigitated growth of pavement cells, with smooth shape. IPGA1 and AN had a genetic interaction in regulating pavement cell shape. Furthermore, IPGA1 genetically and physically interacted with the microtubule-severing enzyme KATANIN. We propose that the IPGA1-AN module regulates microtubule organisation and pavement cell shape.

GmPIN-dependent polar auxin transport is involved in soybean nodule development.

To overcome nitrogen deficiency, legume roots establish symbiotic interactions with nitrogen-fixing rhizobia that are fostered in specialized organs (nodules). Similar to other organs, nodule formation is determined by a local maximum of the phytohormone auxin at the primordium site. However, how auxin regulates nodule development remains poorly understood. Here, we found that in soybean, (Glycine max), dynamic auxin transport driven by PIN-FORMED (PIN) transporter GmPIN1 is involved in nodule primordium formation. GmPIN1 was specifically expressed in nodule primordium cells and GmPIN1 was polarly localized in these cells. Two nodulation regulators, (iso)flavonoids trigger expanded distribution of GmPIN1b to root cortical cells, and cytokinin rearranges GmPIN1b polarity. Gmpin1abc triple mutants generated with CRISPR-Cas9 showed the impaired establishment of auxin maxima in nodule meristems and aberrant divisions in the nodule primordium cells. Moreover, overexpression of GmPIN1 suppressed nodule primordium initiation. GmPIN9d, an ortholog of Arabidopsis thaliana PIN2, acts together with GmPIN1 later in nodule development to acropetally transport auxin in vascular bundles, fine-tuning the auxin supply for nodule enlargement. Our findings reveal how PIN-dependent auxin transport modulates different aspects of soybean nodule development and suggest that the establishment of auxin gradient is a prerequisite for the proper interaction between legumes and rhizobia.

Salicylic acid regulates PIN2 auxin transporter hyperclustering and root gravitropic growth via Remorin-dependent lipid nanodomain organisation in Arabidopsis thaliana.

To adapt to the diverse array of biotic and abiotic cues, plants have evolved sophisticated mechanisms to sense changes in environmental conditions and modulate their growth. Growth-promoting hormones and defence signalling fine tune plant development antagonistically. During host-pathogen interactions, this defence-growth trade-off is mediated by the counteractive effects of the defence hormone salicylic acid (SA) and the growth hormone auxin. Here we revealed an underlying mechanism of SA regulating auxin signalling by constraining the plasma membrane dynamics of PIN2 auxin efflux transporter in Arabidopsis thaliana roots. The lateral diffusion of PIN2 proteins is constrained by SA signalling, during which PIN2 proteins are condensed into hyperclusters depending on REM1.2-mediated nanodomain compartmentalisation. Furthermore, membrane nanodomain compartmentalisation by SA or Remorin (REM) assembly significantly suppressed clathrin-mediated endocytosis. Consequently, SA-induced heterogeneous surface condensation disrupted asymmetric auxin distribution and the resultant gravitropic response. Our results demonstrated a defence-growth trade-off mechanism by which SA signalling crosstalked with auxin transport by concentrating membrane-resident PIN2 into heterogeneous compartments.

Salicylic acid-mediated plasmodesmal closure via Remorin-dependent lipid organization.

Plasmodesmata (PD) are plant-specific membrane-lined channels that create cytoplasmic and membrane continuities between adjacent cells, thereby facilitating cell-cell communication and virus movement. Plant cells have evolved diverse mechanisms to regulate PD plasticity in response to numerous environmental stimuli. In particular, during defense against plant pathogens, the defense hormone, salicylic acid (SA), plays a crucial role in the regulation of PD permeability in a callose-dependent manner. Here, we uncover a mechanism by which plants restrict the spreading of virus and PD cargoes using SA signaling by increasing lipid order and closure of PD. We showed that exogenous SA application triggered the compartmentalization of lipid raft nanodomains through a modulation of the lipid raft-regulatory protein, Remorin (REM). Genetic studies, superresolution imaging, and transmission electron microscopy observation together demonstrated that Arabidopsis REM1.2 and REM1.3 are crucial for plasma membrane nanodomain assembly to control PD aperture and functionality. In addition, we also found that a 14-3-3 epsilon protein modulates REM clustering and membrane nanodomain compartmentalization through its direct interaction with REM proteins. This study unveils a molecular mechanism by which the key plant defense hormone, SA, triggers membrane lipid nanodomain reorganization, thereby regulating PD closure to impede virus spreading.

Dynamic regulation of plasmodesmatal permeability and its application to horticultural research.

Effective cell-to-cell communication allows plants to fine-tune their developmental processes in accordance with the prevailing environmental stimuli. Plasmodesmata (PD) are intercellular channels that span the plant cell wall and serve as cytoplasmic bridges to facilitate efficient exchange of signaling molecules between neighboring cells. The identification of PD-associated proteins and the subsequent elucidation of the regulation of PD structure have provided vital insights into the role of PD architecture in enforcing crucial cellular processes, including callose deposition, ER-Golgi-based secretion, cytoskeleton dynamics, membrane lipid raft organization, chloroplast metabolism, and cell wall formation. In this review, we summarize the emerging discoveries from recent studies that elucidated the regulatory mechanisms involved in PD biogenesis and the dynamics of PD opening-closure. Retrospectively, PD-mediated cell-to-cell communication has been implicated in diverse cellular and physiological processes that are fundamental for the development of horticultural plants. The potential application of PD biotechnological engineering represents a powerful approach for improving agronomic traits in horticultural crops in the future.

SPIKE1 Activates ROP GTPase to Modulate Petal Growth and Shape.

Plant organ growth and final shape rely on cell proliferation and, particularly, on cell expansion that largely determines the visible growth of plant organs. Arabidopsis (Arabidopsis thaliana) petals serve as an excellent model for dissecting the coordinated regulation of patterns of cell expansion and organ growth, but the molecular signaling mechanisms underlying this regulation remain largely unknown. Here, we demonstrate that during the late petal development stages, SPIKE1 (SPK1), encoding a guanine nucleotide exchange factor, activates Rho of Plants (ROP) GTPase proteins (ROP2, ROP4, and ROP6) to affect anisotropic expansion of epidermal cells in both petal blades and claws, thereby affecting anisotropic growth of the petal and the final characteristic organ shape. The petals of SPK1 knockdown mutants were significantly longer but narrower than those of the wild type, associated with increased anisotropic expansion of epidermal cells at late development stages. In addition, ROP2, ROP4, and ROP6 are activated by SPK1 to promote the isotropic organization of cortical microtubule arrays and thus inhibit anisotropic growth in the petal. Both knockdown of SPK1 and multiple rop mutants caused highly ordered cortical microtubule arrays that were transversely oriented relative to the axis of cell elongation after development stage 11. Taken together, our results suggest a SPK1-ROP-dependent signaling module that influences anisotropic growth in the petal and defines the final organ shape.

Integrated Systems Biology Analysis of Transcriptomes Reveals Candidate Genes for Acidity Control in Developing Fruits of Sweet Orange (Citrus sinensis L. Osbeck).

Organic acids, such as citrate and malate, are important contributors for the sensory traits of fleshy fruits. Although their biosynthesis has been illustrated, regulatory mechanisms of acid accumulation remain to be dissected. To provide transcriptional architecture and identify candidate genes for citrate accumulation in fruits, we have selected for transcriptome analysis four varieties of sweet orange (Citrus sinensis L. Osbeck) with varying fruit acidity, Succari (acidless), Bingtang (low acid), and Newhall and Xinhui (normal acid). Fruits of these varieties at 45 days post anthesis (DPA), which corresponds to Stage I (cell division), had similar acidity, but they displayed differential acid accumulation at 142 DPA (Stage II, cell expansion). Transcriptomes of fruits at 45 and 142 DPA were profiled using RNA sequencing and analyzed with three different algorithms (Pearson correlation, gene coexpression network and surrogate variable analysis). Our network analysis shows that the acid-correlated genes belong to three distinct network modules. Several of these candidate fruit acidity genes encode regulatory proteins involved in transport (such as AHA10), degradation (such as APD2) and transcription (such as AIL6) and act as hubs in the citrate accumulation gene networks. Taken together, our integrated systems biology analysis has provided new insights into the fruit citrate accumulation gene network and led to the identification of candidate genes likely associated with the fruit acidity control.