Deep Imaging Analysis in VISUAL Reveals the Role of YABBY Genes in Vascular Stem Cell Fate Determination.

Stem cells undergo cell division and differentiation to ensure organized tissue development. Because plant cells are immobile, plant stem cells ought to decide their cell fate prior to differentiation, to locate specialized cells in the correct position. In this study, based on a chemical screen, we isolated a novel secondary cell wall indicator BF-170, which binds to lignin and can be used to image in vitro and in situ xylem development. Use of BF-170 to observe the vascular differentiation pattern in the in vitro vascular cell induction system, VISUAL, revealed that adaxial mesophyll cells of cotyledons predominantly generate ectopic xylem cells. Moreover, phloem cells are abundantly produced on the abaxial layer, suggesting the involvement of leaf adaxial-abaxial polarity in determining vascular cell fate. Analysis of abaxial polarity mutants highlighted the role of YAB3, an abaxial cell fate regulator, in suppressing xylem and promoting phloem differentiation on the abaxial domains in VISUAL. Furthermore, YABBY family genes affected in vivo vascular development during the secondary growth. Our results denoted the possibility that such mediators of spatial information contribute to correctly determine the cell fate of vascular stem cells, to conserve the vascular pattern of land plants.

Cargo-dependent and cell wall-associated xylem transport in Arabidopsis.

Sap molecules are transported by xylem flow throughout the whole plant body. Factors regulating the xylem transport of different molecules remain to be identified. We used fluorophores to visualize xylem transport from roots to leaves in Arabidopsis thaliana. Several previously established Arabidopsis lines with modified xylem cell walls were used to determine the contribution of xylem cell walls to xylem transport. Fluorophores underwent xylem flow-dependent transport from roots to leaves within 20 min. A comparison of rhodamine, fluorescein and three fluorescently labeled CLV3/ESR-related (CLE) peptides revealed cargo-dependent xylem transport patterns in terms of leaf position and vein order. Only minor changes in amino acid sequence were sufficient to alter the xylem transport patterns of the labeled CLE peptides. We found that the xylem transport pattern of fluorescein was affected in Arabidopsis lines with modified AtXYN1, LAC4 or CCoAOMT1 expression. In these lines, application of a defense inducer, pipecolic acid, to roots resulted in altered defense response patterns in leaves, whereas all the lines showed wild-type-like responses when pipecolic acid was sprayed onto leaves. The combined results reveal a finely controlled cargo-dependent xylem transport and suggest that the xylem cell wall structure is crucial for this transport system.

Secondary cell wall patterning during xylem differentiation.

Xylem cell differentiation involves temporal and spatial regulation of secondary cell wall deposition. The cortical microtubules are known to regulate the spatial pattern of the secondary cell wall by orientating cellulose deposition. However, it is largely unknown how the microtubule arrangement is regulated during secondary wall formation. Recent findings of novel plant microtubule-associated proteins in developing xylem vessels shed new light on the regulation mechanism of the microtubule arrangement leading to secondary wall patterning. In addition, in vitro culture systems allow the dynamics of microtubules and microtubule-associated proteins during secondary cell wall formation to be followed. Therefore, this review focuses on novel aspects of microtubule dynamics leading to secondary cell wall patterning with a focus on microtubule-associated proteins.

Isolation and characterization of a novel peroxidase gene ZPO-C whose expression and function are closely associated with lignification during tracheary element differentiation.

In an attempt to elucidate the regulatory mechanism of vessel lignification, we isolated ZPO-C, a novel peroxidase gene of Zinnia elegans that is expressed specifically in differentiating tracheary elements (TEs). The ZPO-C transcript was shown to accumulate transiently at the time of secondary wall thickening of TEs in xylogenic culture of Zinnia cells. In situ hybridization indicated specific accumulation of the ZPO-C transcript in immature vessels in Zinnia seedlings. Immunohistochemical analysis using anti-ZPO-C antibody showed that the ZPO-C protein is abundant in TEs, especially at their secondary walls. For enzymatic characterization of ZPO-C, 6 x His-tagged ZPO-C was produced in tobacco cultured cells and purified. The ZPO-C:6 x His protein had a peroxidase activity preferring sinapyl alcohol as well as coniferyl alcohol as a substrate, with a narrow pH optimum around 5.25. The peroxidase activity required calcium ion and was elevated by increasing Ca2+ concentration in the range of 0-10 mM. An Arabidopsis homolog of ZPO-C, At5g51890, was examined for expression patterns with transgenic plants carrying a yellow fluorescent protein (YFP) gene under the control of the At5g51890 promoter. The YFP fluorescence localization demonstrated vessel-specific expression of At5g51890 in the Arabidopsis roots. Taken collectively, our results strongly suggest that ZPO-C and its homologs play an important role in lignification of secondary cell walls in differentiating TEs.

Involvement of extracellular dilignols in lignification during tracheary element differentiation of isolated Zinnia mesophyll cells.

During differentiation of isolated Zinnia mesophyll cells into tracheary elements (TEs), lignification on TEs progresses by supply of monolignols not only from TEs themselves but also from surrounding xylem parenchyma-like cells through the culture medium. However, how lignin polymerizes from the secreted monolignols has not been resolved. In this study, we analyzed phenol compounds in culture medium with reversed-phase HPLC, gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry, and found 12 phenolic compounds including coniferyl alcohol and four dilignols, i.e. erythro-guaiacylglycerol-beta-coniferyl ether, threo-guaiacylglycerol-beta-coniferyl ether, dehydrodiconiferyl alcohol and pinoresinol, in the medium in which TEs were developing. Coniferyl alcohol applied to TE-inductive cultures during TE formation rapidly disappeared from the medium, and caused a sudden increase in dilignols. Addition of the dilignols promoted lignification of TEs in which monolignol biosynthesis was blocked by an inhibitor of phenylalanine anmmonia-lyase (PAL), L-alpha-aminooxy-beta-phenylpropionic acid (AOPP). These results suggested that dilignols can act as intermediates of lignin polymerization.

The ATE genes are responsible for repression of transdifferentiation into xylem cells in Arabidopsis.

We isolated three recessive mutants of Arabidopsis (Arabidopsis thaliana) showing ectopic expression of the xylem-specific marker, pAtxyn3::YFP. Genetic analysis indicated that the phenotypes were caused by mutations in three different genes, designated Abnormal Tracheary Element formation-related gene expression (ate1-3). The ate1 mutants showed a normal DR5::GUS gene expression pattern, and the ate1 mutation did not affect the abnormal vascular pattern formation in the van3 and pin1 mutants, indicating that the ate1 mutation does not affect the vascular pattern organization governed by auxin. The ate mutants showed ectopic lignin deposition, patterned secondary wall thickenings, and cell death, which are characteristic of mature tracheary elements (TEs) in cells ectopically expressing the pAtxyn3::YFP gene. Ectopic TE formation was rapidly induced in parenchymal tissue of the ate mutants in a TE-inducible system with excised hypocotyl. Furthermore, reverse transcription-polymerase chain reaction experiments showed that the expression of TE formation-related genes is up-regulated in the ate mutants. The ate1 mutation also caused ectopic expression of another xylem-specific marker gene, pAt3g62160::YFP. Overall, our results suggest that the ATE genes are responsible for the in situ repression of transdifferentiation into TEs in Arabidopsis and could be participants in the transdifferentiation-masking system.

Immunocytochemical localization of polygalacturonase during tracheary element differentiation in Zinnia elegans.

Polygalacturonase (PG) is a cell wall-associated protein that degrades pectin. A ZePG1 cDNA encoding a putative PG was isolated from Zinnia elegens L. and a rabbit antibody specific to the ZePG1 protein was generated. The level of the ZePG1 protein was up-regulated when tracheary element differentiation was initiated. Using gold-labeled secondary antibodies for light and electron microscopy, ZePG1 protein was localized in cultured Zinnia cells. This protein was preferentially distributed on tracheary elements (TEs). At the subcellular level, the protein was localized on secondary wall thickenings, primary walls, Golgi bodies and vesicles. Thus, the putative role of the ZePG1 protein might be the degradation of pectic substances before lignification. Some non-TE cells also accumulated ZePG1 protein on primary walls, Golgi bodies and vesicles. The accumulation of ZePG1 protein on primary walls seems to be at the elongating tips of non-TE cells. In plants, ZePG1 protein was localized on the secondary wall thickenings of differentiating TEs and phloem regions. These results suggest that the expression of the ZePG1 protein is highly regulated both spatially and temporally during in vitro and in situ TE differentiation.

Visualization by comprehensive microarray analysis of gene expression programs during transdifferentiation of mesophyll cells into xylem cells.

Plants have a unique transdifferentiation mechanism by which differentiated cells can initiate a new program of differentiation. We used a comprehensive analysis of gene expression in an in vitro zinnia (Zinnia elegans L.) culture model system to gather fundamental information about the gene regulation underlying the transdifferentiation of plant cells. In this model, photosynthetic mesophyll cells isolated from zinnia leaves transdifferentiate into xylem cells in a morphogenic process characterized by features such as secondary-wall formation and programmed cell death. More than 8,000 zinnia cDNA clones were isolated from an equalized cDNA library prepared from cultured cells transdifferentiating into xylem cells. Microarray analysis using these cDNAs revealed several types of unique gene regulation patterns, including: the transient expression of a set of genes during cell isolation, presumably induced by wounding; a rapid reduction in the expression of photosynthetic genes and the rapid induction of protein synthesis-associated genes during the first stage; the preferential induction of auxin-related genes during the subsequent stage; and the transient induction of genes closely associated with particular morphogenetic events, including cell-wall formation and degradation and programmed cell death during the final stage. This analysis also revealed a number of previously uncharacterized genes encoding proteins that function in signal transduction, such as protein kinases and transcription factors that are expressed in a stage-specific manner. These findings provide new clues to the molecular mechanisms of both plant transdifferentiation and wood formation.