Misuk Cho

P-Glycoprotein4 Displays Auxin Efflux Transporter–Like Action in Arabidopsis Root Hair Cells and Tobacco Cells

ATP binding cassette (ABC) transporters transport diverse substrates across membranes in various organisms. However, plant ABC transporters have only been scantily characterized. By taking advantage of the auxin-sensitive Arabidopsis thaliana root hair cell and tobacco (Nicotiana tabacum) suspension cell systems, we show here that Arabidopsis P-glycoprotein4 (PGP4) displays auxin efflux activity in plant cells. Root hair cell–specific overexpression of PGP4 (PGP4ox) and known auxin efflux transporters, such as PGP1, PGP19, and PIN-FORMEDs, decreased root hair elongation, whereas overexpression of the influx transporter AUXIN-RESISTANT1 enhanced root hair length. PGP4ox-mediated root hair shortening was rescued by the application of auxin or an auxin efflux inhibitor. These results indicate that the increased auxin efflux activity conferred by PGP4 reduces auxin levels in the root hair cell and consequently inhibits root hair elongation. PGP4ox in tobacco suspension cells also increased auxin efflux. PGP4 proteins were targeted to the plasma membrane of Arabidopsis root hair cells and tobacco cells without any clear subcellular polarity. Brefeldin A partially interfered with the trafficking of PGP4 reversibly, and this was rescued by pretreatment with auxin. These results suggest that PGP4 is an auxin efflux transporter in plants and that its trafficking to the plasma membrane involves both BFA-sensitive and -insensitive pathways.

cis-Element- and Transcriptome-Based Screening of Root Hair-Specific Genes and Their Functional Characterization in Arabidopsis

Understanding the cellular differentiation of multicellular organisms requires the characterization of genes whose expression is modulated in a cell type-specific manner. The Arabidopsis (Arabidopsis thaliana) root hair cell is one model for studying cellular differentiation. In this study, root hair cell-specific genes were screened by a series of in silico and experimental filtration procedures. This process included genome-wide screening for genes with a root hair-specific cis-element in their promoters, filtering root-specific genes from the root hair-specific cis-element-containing genes, further filtering of genes that were suppressed in root hair-defective plant lines, and experimental confirmation by promoter assay. These procedures revealed 19 root hair-specific genes, including many protein kinases and cell wall-related genes, most of which have not been characterized thus far. Functional analyses of these root hair-specific genes with loss-of-function mutants and overexpressing transformants revealed that they play roles in hair growth and morphogenesis. This study demonstrates that a defined cis-element can serve as a filter to screen certain cell type-specific genes and implicates many new root hair-specific genes in root hair development.

Functional Conservation of a Root Hair Cell-Specific cis-Element in Angiosperms with Different Root Hair Distribution Patterns

Vascular plants develop distinctive root hair distribution patterns in the root epidermis, depending on the taxon. The three patterns, random (Type 1), asymmetrical cell division (Type 2), and positionally cued (Type 3), are controlled by different upstream fate-determining factors that mediate expression of root hair cell-specific genes for hair morphogenesis. Here, we address whether these root hair genes possess a common transcriptional regulatory module (cis-element) determining cell-type specificity despite differences in the final root hair pattern. We identified Arabidopsis thaliana expansinA7 (At EXPA7) orthologous (and paralogous) genes from diverse angiosperm species with different hair distribution patterns. The promoters of these genes contain conserved root hair–specific cis-elements (RHEs) that were functionally verified in the Type-3 Arabidopsis root. The promoter of At EXPA7 (Type-3 pattern) also showed hair cell–specific expression in the Type 2 rice (Oryza sativa) root. Root hair–specific genes other than EXPAs also carry functionally homologous RHEs in their promoters. The RHE core consensus was established by a multiple alignment of functionally characterized RHEs from different species and by high-resolution analysis of At EXPA7 RHE1. Our results suggest that this regulatory module of root hair–specific genes has been conserved across angiosperms despite the divergence of upstream fate-determining machinery.

Differential Auxin-Transporting Activities of PIN-FORMED Proteins in Arabidopsis Root Hair Cells

The Arabidopsis (Arabidopsis thaliana) genome includes eight PIN-FORMED (PIN) members that are molecularly diverged. To comparatively examine their differences in auxin-transporting activity and subcellular behaviors, we expressed seven PIN proteins specifically in Arabidopsis root hairs and analyzed their activities in terms of the degree of PIN-mediated root hair inhibition or enhancement and determined their subcellular localization. Expression of six PINs (PIN1–PIN4, PIN7, and PIN8) in root hair cells greatly inhibited root hair growth, most likely by lowering auxin levels in the root hair cell by their auxin efflux activities. The auxin efflux activity of PIN8, which had not been previously demonstrated, was further confirmed using a tobacco (Nicotiana tabacum) cell assay system. In accordance with these results, those PINs were localized in the plasma membrane, where they likely export auxin to the apoplast and formed internal compartments in response to brefeldin A. These six PINs conferred different degrees of root hair inhibition and sensitivities to auxin or auxin transport inhibitors. Conversely, PIN5 mostly localized to internal compartments, and its expression in root hair cells rather slightly stimulated hair growth, implying that PIN5 enhanced internal auxin availability. These results suggest that different PINs behave differentially in catalyzing auxin transport depending upon their molecular activity and subcellular localization in the root hair cell.

HY5 regulates anthocyanin biosynthesis by inducing the transcriptional activation of the MYB75/PAP1 transcription factor in Arabidopsis

Several positive transcription factors regulate Arabidopsis anthocyanin biosynthesis. HY5, a component of light‐signaling pathways, and PAP1, an R2R3‐MYB transcription factor, share common regulatory targets on anthocyanin biosynthesis genes. The epistatic interactions between the two transcription factors are currently unknown. To address this problem, we analyzed crosses between hy5 and pap1 mutants (hy5pap1) or pap1D overexpressors (hy5pap1D), performed chromatin immunoprecipitation‐qPCR, and determined the PAP1 promoter region through deletion analysis. The results show that HY5 regulates PAP1 expression via direct binding to G‐ and ACE‐boxes in the promoter region, which suggests bifurcate regulation of anthocyanin biosynthesis by HY5 via transcriptional activation of PAP1.

The function of ABCB transporters in auxin transport

Plant ATP-binding cassette (ABC) transporters consist of largest family members among many other membrane transporters and have been implicated in various functions such as detoxification, disease resistance and transport of diverse substrates. Of the ABC-B/multi-drug resistance/P-glycoprotein (ABCB/MDR/PGP) subfamily, at least five members have been reported to mediate cellular transport of auxin or auxin derivatives. Although single mutant phenotypes of these genes are milder than PIN-FORMED (PIN) mutants, those ABCBs significantly contribute for the directional auxin movement in the tissue-level auxin-transporting assay. Uniformly localized ABCB proteins in the plasma membrane (PM) are generaly found in different plant species and stably retained regardless of internal and external signals. This implies that these ABCB proteins may play as basal auxin transporters.

Root hair-specific EXPANSIN B genes have been selected for graminaceae root hairs

Cell differentiation ultimately relies on the regulation of cell type-specific genes. For a root hair cell to undergo morphogenesis, diverse cellular processes including cell-wall loosening must occur in a root hair cell-specific manner. Previously, we identified and characterized root hairspecific cis-elements (RHE) from the genes encoding the cell wall-loosening protein EXPANSIN A (EXPA) which functions preferentially on dicot cell walls. This study reports two root hair-specific grass EXPB genes that contain RHEs. These genes are thought to encode proteins that function more efficiently on grass cell walls. The proximal promoter regions of two orthologous EXPB genes from rice (Oryza sativa; OsEXPB5) and barley (Hordeum vulgare; HvEXPB1) included RHE motifs. These promoters could direct root hair-specific expression of green fluorescent protein (GFP) in the roots of rice and Arabidopsis (Arabidopsis thaliana). Promoter deletion analyses demonstrated that the RHE motifs are necessary for root hairspecific expression of these EXPB promoters. Phylogenetic analysis of EXP protein sequences indicated that grass EXPBs are the only orthologs to these root hair-specific EXPBs, separating dicot EXPBs to distal branches of the tree. These results suggest that RHE-containing root hair-specific EXPB genes have evolved for grass-specific cell wall modification during root hair morphogenesis.

ATP-Binding Cassette B4, an Auxin-Efflux Transporter, Stably Associates with the Plasma Membrane and Shows Distinctive Intracellular Trafficking from That of PIN-FORMED Proteins

Intracellular trafficking of auxin transporters has been implicated in diverse developmental processes in plants. Although the dynamic trafficking pathways of PIN-FORMED auxin efflux proteins have been studied intensively, the trafficking of ATP-binding cassette protein subfamily B proteins (ABCBs; another group of auxin efflux carriers) still remains largely uncharacterized. In this study, we address the intracellular trafficking of ABCB4 in Arabidopsis (Arabidopsis thaliana) root epidermal cells. Pharmacological analysis showed that ABCB4 barely recycled between the plasma membrane and endosomes, although it slowly endocytosed via the lytic vacuolar pathway. Fluorescence recovery after photobleaching analysis revealed that ABCB4 is strongly retained in the plasma membrane, further supporting ABCB4’s nonrecycling property. The endocytosis of ABCB4 was not dependent on the GNOM-LIKE1 function, and the sensitivity of ABCB4 to brefeldin A required guanine nucleotide exchange factors for adenosyl ribosylation factor other than GNOM. These characteristics of intracellular trafficking of ABCB4 are well contrasted with those of PIN-FORMED proteins, suggesting that ABCB4 may be a basic and constitutive auxin efflux transporter for cellular auxin homeostasis.

Calcium dependent sucrose uptake links sugar signaling to anthocyanin biosynthesis in Arabidopsis.

Sugars enhance light signaling-induced anthocyanin accumulation in Arabidopsis seedlings via differential regulation of several positive and negative transcription factors. Ca(2+) plays a role as a second messenger in sugar signaling in grape and wheat. However, whether anthocyanin pigmentation is modulated by changes in intracellular Ca(2+) level in Arabidopsis is not known. Here, we used a pharmaceutical approach that Ca(2+) antagonists strongly interfered with sucrose uptake and anthocyanin accumulation by downregulating the expression of sucrose transporter 1 (SUC1) and transcriptional regulatory factors, such as PAP1. Time course analysis of the effect of Ca(2+) antagonists showed the early inhibition of sucrose-induced sugar uptake leading to decreased anthocyanin accumulation, indicating that Ca(2+) signals play a role in sugar uptake rather than in anthocyanin biosynthesis. An early increase in cytosolic Ca(2+) level in Arabidopsis roots in response to sucrose feeding was significantly inhibited by Ca(2+) antagonists. Taken together, these results indicate that sucrose-induced sugar uptake in Arabidopsis is modulated by changes in endogenous Ca(2+) levels, which in turn regulate anthocyanin accumulation.

Auxin-signaling: short and long

Long-standing major questions in auxin biology are now being answered through the latest discoveries and characterizations of auxin receptors and transporters. An F-box protein TIR1 and its close homologs are emerging as potent auxin receptors, which directly modulate the degradation of transcriptional repressors for auxin-responsive genes. The membrane proteins for polar auxin transport, intuited by Darwin almost 130 years ago, have been characterized over the past decade and implicated in diverse aspects of auxin-mediated plant development. This growth regulator is now considered to be a plant equivalent of morphogen because of how crucial the formation of its transporter-associated concentration gradient is to the patterning processes of plants. Such long-distance auxin-signaling from the source to the target cell via transporters has helped advance our understanding of plant development as a holistic system. Here, we summarize recent achievements in the study of molecular and long-distance signaling mechanisms for auxin, and discuss their biological meaning.