A ROP GTPase-Dependent Auxin Signaling Pathway Regulates the Subcellular Distribution of PIN2 in Arabidopsis Roots

SummaryPIN-FORMED (PIN) protein-mediated auxin polar transport is critically important for development, pattern formation, and morphogenesis in plants. Auxin has been implicated in the regulation of polar auxin transport by inhibiting PIN endocytosis [1, 2], but how auxin regulates this process is poorly understood. Our genetic screen identified the Arabidopsis SPIKE1 (SPK1) gene whose loss-of-function mutations increased lateral root density and retarded gravitropic responses, as do pin2 knockout mutations [3]. SPK1 belongs to the conserved DHR2-Dock family of Rho guanine nucleotide exchange factors [4–6]. The spk1 mutations induced PIN2 internalization that was not suppressed by auxin, as did the loss-of-function mutations for Rho-like GTPase from Plants 6 (ROP6)-GTPase or its effector RIC1. Furthermore, SPK1 was required for auxin induction of ROP6 activation. Our results have established a Rho GTPase-based auxin signaling pathway that maintains PIN2 polar distribution to the plasma membrane via inhibition of its internalization in Arabidopsis roots. Our findings provide new insights into signaling mechanisms that underlie the regulation of the dynamic trafficking of PINs required for long-distance auxin transport and that link auxin signaling to PIN-mediated pattern formation and morphogenesis

Cell Surface- and Rho GTPase-Based Auxin Signaling Controls Cellular Interdigitation in Arabidopsis

Auxin is a multi-functional hormone essential for plant development and pattern formation. A nuclear auxin signaling system controlling auxin-induced gene expression is well established, but cytoplasmic auxin signaling as in its coordination of cell polarization is unexplored. We found a cytoplasmic auxin signaling mechanism that modulates the interdigitated growth of Arabidopsis leaf epidermal pavement cells (PCs), which develop interdigitated lobes and indentations to form a puzzle-piece shape in a two-dimensional plane. PC interdigitation is compromised in leaves deficient in either auxin biosynthesis or its export mediated by PINFORMED 1 localized at the lobe tip. Auxin coordinately activates two Rho GTPases, ROP2 and ROP6, which promote the formation of complementary lobes and indentations, respectively. Activation of these ROPs by auxin occurs within 30 seconds and depends on AUXIN-BINDING PROTEIN 1. These findings reveal Rho GTPase-based novel auxin signaling mechanisms, which modulate the spatial coordination of cell expansion across a field of cells

Uniform auxin triggers the Rho GTPase-dependent formation of interdigitation patterns in pavement cells

The investigation of Rho-family GTPases has uncovered mechanisms for spatiotemporal control of cellular processes such as cell polarization, movement, morphogenesis and cell division. Now Rho GTPase plays another leading role in the discovery of a new signaling mechanism for auxin, a multifunctional hormone that regulates pattern formation in plants. Arabidopsis leaf epidermal pavement cells (PCs) develop the puzzle-piece cell shape with interlocking lobes and indentations via interdigitated cellular growth.1 Through the ABP1 (Auxin Binding Protein 1) cell surface receptor, auxin coordinately activates 2 mutually exclusive Rho GTPase signaling pathways that are activated in the complementary lobing and indenting sides of adjacent cells: the ROP2 pathway for lobe formation and the ROP6 pathway for promoting indentation. This new signaling mechanism also involves ROP2-dependent polar accumulation of PIN1 in the plasma membrane, a member of the PIN auxin efflux carrier family that is critical for the formation of various developmental patterns including the PC interdigitation pattern. This Rho-dependent auxin signaling mechanism explains how interdigitated cellular growth is coordinated. In this extra view, we propose that the same mechanism can also explain how a uniform auxin signal initiates the formation of the interdigitated pattern

FvNST1b NAC Protein Induces Secondary Cell Wall Formation in Strawberry

Secondary cell wall thickening plays a crucial role in plant growth and development. Diploid woodland strawberry (Fragaria vesca) is an excellent model for studying fruit development, but its molecular control of secondary wall thickening is largely unknown. Previous studies have shown that Arabidopsis NAC secondary wall thickening promoting factor1 (AtNST1) and related proteins are master regulators of xylem fiber cell differentiation in multiple plant species. In this study, a NST1-like gene, FvNST1b, was isolated and characterized from strawberry. Sequence alignment and phylogenetic analysis showed that the FvNST1b protein contains a highly conserved NAC domain, and it belongs to the same family as AtNST1. Overexpression of FvNST1b in wild-type Arabidopsis caused extreme dwarfism, induced ectopic thickening of secondary walls in various tissues, and upregulated the expression of genes related to secondary cell wall synthesis. In addition, transient overexpression of FvNST1b in wild-type Fragaria vesca fruit produced cells resembling tracheary elements. These results suggest that FvNST1b positively regulates secondary cell wall formation as orthologous genes from other species

Ectopic Expression of <i>FvVND4c</i> Promotes Secondary Cell Wall Thickening and Flavonoid Accumulation in <i>Fragaria vesca</i>

Secondary cell wall (SCW) thickening has a significant effect on the growth and development of plants, as well as in the resistance to various biotic and abiotic stresses. Lignin accounts for the strength of SCW. It is synthesized through the phenylpropanoid pathway that also leads to flavonoid synthesis. The coupling strategies for lignin and flavonoid syntheses are diverse in plants. How their syntheses are balanced by transcriptional regulation in fleshy fruits is still unclear. The diploid strawberry (Fragaria vesca) is a model for fleshy fruits research due to its small genome and wide scope of genetic transformation. SCW thickening is regulated by a multilevel transcriptional regulatory network wherein vascular-related NAC domains (VNDs) act as key regulators. In this study, we systematically characterized VNDs in Fragaria vesca and explored their functions. The overexpression of FvVND4c in diploid strawberry fruits resulted in SCW thickening and fruit color changes accompanied with the accumulation of lignin and flavonoids. Genes related to these phenotypes were also induced upon FvVND4c overexpression. Among the induced genes, we found FvMYB46 to be a direct downstream regulator of FvVND4c. The overexpression of FvMYB46 resulted in similar phenotypes as FvVND4c, except for the color change. Transcriptomic analyses suggest that both FvVND4c and FvMYB46 act on phenylpropanoid and flavonoid biosynthesis pathways, and induce lignin synthesis for SCW. These results suggest that FvVND4c and FvMYB46 cooperatively regulate SCW thickening and flavonoid accumulation in Fragaria vesca

Discovery of a cis-regulatory element SaeM involved in dynamic regulation of synergid-specific MYB98

MYB98 is a key regulator of the genetic network behind pollen tube attraction toward the female gametophyte. MYB98 is specifically expressed in the synergid cells (SCs), a female gametophyte component cells specialized for pollen tube attraction. However, it had not been clear how exactly MYB98 achieves this specific expression pattern. In the current study, we have determined that a normal SC-specific expression of MYB98 is dependent on a 16-bp-long cis-regulatory element, CATTTACACATTAAAA, freshly named as the “Synergid-specificActivationElement ofMYB98” (SaeM). An 84 bp fragment harboring SaeM in the middle was sufficient to drive exclusively SC-specific expression. The element was present in a significantly large proportion of SC-specific gene promoters and in the promoter of MYB98 homologous genes in the Brassicaceae (pMYB98s). Significance of such family-wide SaeM-like element conservation in exclusive SC-specific expression was confirmed by the Arabidopsis-like activation feature of Brassica oleracea–derived pMYB98 and absence of such feature of pMYB98 derived from a non-Brassicaceae member Prunus persica. Additionally, the yeast-one-hybrid assay showed that the SaeM can be recognized by ANTHOCYANINLESS2 (ANL2) and DAP-seq data further suggested for additional three ANL2 homologs targeting the similar cis-element. Overall, our study has concluded that SaeM plays a crucial role in driving exclusively SC-specific expression of MYB98 and strongly suggests for the involvement of ANL2 and its homologs in its dynamic regulation in planta. Future study on the transcription factors is expected to shed more light on the mechanism behind the process

ROP GTPase-Dependent Actin Microfilaments Promote PIN1 Polarization by Localized Inhibition of Clathrin-Dependent Endocytosis

Cell polarization via asymmetrical distribution of structures or molecules is essential for diverse cellular functions and development of organisms, but how polarity is developmentally controlled has been poorly understood. In plants, the asymmetrical distribution of the PIN-FORMED (PIN) proteins involved in the cellular efflux of the quintessential phytohormone auxin plays a central role in developmental patterning, morphogenesis, and differential growth. Recently we showed that auxin promotes cell interdigitation by activating the Rho family ROP GTPases in leaf epidermal pavement cells. Here we found that auxin activation of the ROP2 signaling pathway regulates the asymmetric distribution of PIN1 by inhibiting its endocytosis. ROP2 inhibits PIN1 endocytosis via the accumulation of cortical actin microfilaments induced by the ROP2 effector protein RIC4. Our findings suggest a link between the developmental auxin signal and polar PIN1 distribution via Rho-dependent cytoskeletal reorganization and reveal the conservation of a design principle for cell polarization that is based on Rho GTPase-mediated inhibition of endocytosis

Functional Analyses of LONELY GUY Cytokinin-Activating Enzymes Reveal the Importance of the Direct Activation Pathway in Arabidopsis[W][OA]

Cytokinins play crucial roles in diverse aspects of plant growth and development. Spatiotemporal distribution of bioactive cytokinins is finely regulated by metabolic enzymes. LONELY GUY (LOG) was previously identified as a cytokinin-activating enzyme that works in the direct activation pathway in rice (Oryza sativa) shoot meristems. In this work, nine Arabidopsis thaliana LOG genes (At LOG1 to LOG9) were predicted as homologs of rice LOG. Seven At LOGs, which are localized in the cytosol and nuclei, had enzymatic activities equivalent to that of rice LOG. Conditional overexpression of At LOGs in transgenic Arabidopsis reduced the content of N6-(Δ2-isopentenyl)adenine (iP) riboside 5′-phosphates and increased the levels of iP and the glucosides. Multiple mutants of At LOGs showed a lower sensitivity to iP riboside in terms of lateral root formation and altered root and shoot morphology. Analyses of At LOG promoter:β-glucuronidase fusion genes revealed differential expression of LOGs in various tissues during plant development. Ectopic overexpression showed pleiotropic phenotypes, such as promotion of cell division in embryos and leaf vascular tissues, reduced apical dominance, and a delay of leaf senescence. Our results strongly suggest that the direct activation pathway via LOGs plays a pivotal role in regulating cytokinin activity during normal growth and development in Arabidopsis