Kuninori Iwamoto

The receptor-like kinase SOL2 mediates CLE signaling in Arabidopsis

Arabidopsis sol2 mutants showed CLV3 peptide resistance. Twenty-six synthetic CLE peptides were examined in the clv1, clv2 and sol2 mutants. sol2 showed different levels of resistance to the various peptides, and the spectrum of peptide resistance was quite similar to that of clv2. SOL2 encoded a receptor-like kinase protein which is identical to CORYNE (CRN). GeneChip analysis revealed that the expression of several genes was altered in the sol2 root tip. Here, we suggest that SOL2, together with CLV2, plays an important role in the regulation of root meristem development through the CLE signaling pathway.

Involvement of Phytosulfokine in the Attenuation of Stress Response during the Transdifferentiation of Zinnia Mesophyll Cells into Tracheary Elements

Phytosulfokine (PSK) is a sulfated peptide hormone required for the proliferation and differentiation of plant cells. Here, we characterize the physiological roles of PSK in transdifferentiation of isolated mesophyll cells of zinnia (Zinnia elegans ‘Canary Bird’) into tracheary elements (TEs). Transcripts for a zinnia PSK precursor gene, ZePSK1, show two peaks of expression during TE differentiation; the first accumulation is transiently induced in response to wounding at 24 h of culture, and the second accumulation is induced in the final stage of TE differentiation and is dependent on endogenous brassinosteroids. Chlorate, a potent inhibitor of peptide sulfation, is successfully applied as an inhibitor of PSK action. Chlorate significantly suppresses TE differentiation. The chlorate-induced suppression of TE differentiation is overcome by exogenously applied PSK. In the presence of chlorate, expression of stress-related genes for proteinase inhibitors and a pathogenesis-related protein is enhanced and changed from a transient to a continuous pattern. On the contrary, administration of PSK significantly reduces the accumulation of transcripts for the stress-related genes. Even in the absence of auxin and cytokinin, addition of PSK suppresses stress-related gene expression. Microarray analysis reveals 66 genes down-regulated and 42 genes up-regulated in the presence of PSK. The large majority of down-regulated genes show significant similarity to various families of stress-related proteins, including chitinases, phenylpropanoid biosynthesis enzymes, 1-aminocyclopropane-1-carboxylic acid synthase, and receptor-like protein kinases. These results suggest the involvement of PSK in the attenuation of stress response and healing of wound-activated cells during the early stage of TE differentiation.

A Negative Feedback Loop Controlling bHLH Complexes Is Involved in Vascular Cell Division and Differentiation in the Root Apical Meristem.

Controlling cell division and differentiation in meristems is essential for proper plant growth. Two bHLH heterodimers consisting of LONESOME HIGHWAY (LHW) and TARGET OF MONOPTEROS 5 (TMO5)/TMO5-LIKE1 (T5L1) regulate periclinal cell division in vascular cells in the root apical meristem (RAM). In this study, we further investigated the functions of LHW-T5L1, finding that in addition to controlling cell division, this complex regulates xylem differentiation in the RAM via a novel negative regulatory system. LHW-T5L1 upregulated the thermospermine synthase gene ACAULIS5 (ACL5), as well as SUPPRESSOR OF ACAULIS5 LIKE3 (SACL3), which encodes a bHLH protein, in the RAM. The SACL3 promoter sequence contains a conserved upstream open reading frame (uORF), which blocked translation of the main SACL3 ORF in the absence of thermospermine. Thermospermine eliminated the negative effect of uORF and enhanced SACL3 production. Further genetic and molecular biological analyses indicated that ACL5 and SACL3 suppress the function of LHW-T5L1 through a protein-protein interaction between LHW and SACL3. Finally, we showed that a negative feedback loop consisting of LHW-T5L1, ACL5, SACL3, and LHW-SACL3 contributes to maintain RAM size and proper root growth. These findings suggest that a negative feedback loop regulates the LHW-T5L1 output level to coordinate cell division and differentiation in a cell-autonomous manner.

Comprehensive analysis of the regulatory roles of auxin in early transdifferentiation into xylem cells.

Auxin is essential for the formation of the vascular system. We previously reported that a polar auxin transport inhibitor, 1-N-naphthylphthalamic acid (NPA) decreased intracellular auxin levels and prevented tracheary element (TE) differentiation from isolated Zinnia mesophyll cells, but that additional auxin, 1-naphthaleneacetic acid (NAA) overcame this inhibition. To understand the role of auxin in gene regulation during TE differentiation, we performed microarray analysis of genes expressed in NPA-treated cells and NPA–NAA-treated cells. The systematic gene expression analysis revealed that NAA promoted the expression of genes related to auxin signaling and transcription factors that are known to be key regulators of differentiation of procambial and xylem precursor cells. NAA also promoted the expression of genes related to biosynthesis and metabolism of other plant hormones, such as cytokinin, gibberellin and brassinosteroid. Interestingly, detailed analysis showed that NAA rapidly induces the expression of auxin carrier gene homologues. It suggested a positive feedback loop for auxin-regulating vascular differentiation. Based on these results, we discuss the auxin function in early processes of transdifferentiation into TEs.

Expression and Genome-Wide Analysis of the Xylogen-Type Gene Family

In higher plants, many extracellular proteins are involved in developmental processes, including cell-cell signaling and cell wall construction. Xylogen is an extracellular arabinogalactan protein (AGP) isolated from Zinnia elegans xylogenic culture medium, which promotes xylem cell differentiation. Xylogen has a unique structure, containing a non-specific lipid transfer protein (nsLTP) domain and AGP domains. We searched for xylogen-type genes in the genomes of land plants, including Arabidopsis thaliana, to further our knowledge of xylogen-type genes as functional extracellular proteins in plants. We found that many xylogen-type genes, including 13 Arabidopsis genes, comprise a gene family in land plants, including Populus trichocarpa, Vitis vinifera, Lotus japonicus, Oryza sativa, Selaginella moellendorffii and Physcomitrella patens. The genes shared an N-terminal signal peptide sequence, a distinct nsLTP domain, one or more AGP domains and a glycosylphosphatidylinositol (GPI)-anchored sequence. We analyzed transgenic plants harboring promoter::GUS (β-glucuronidase) constructs to test expression of the 13 Arabidopsis xylogen-type genes, and detected a diversity of gene family members with related expression patterns. AtXYP2 was the best candidate as the Arabidopsis counterpart of the Zinnia xylogen gene. We observed two distinct expression patterns for several genes, with some anther specific and others preferentially expressed in the endodermis/pericycle. We conclude that xylogen-type genes, which may have diverse functions, form a novel chimeric AGP gene family with a distinct nsLTP domain.

Plant tissue cultures.

Plant tissue cultures are an efficient system to study cell wall biosynthesis in living cells in vivo. Tissue cultures also provide cells and culture medium where enzymes and cell wall polymers can easily be separated for further studies. Tissue cultures with tracheary element differentiation or extracellular lignin formation have provided useful information related to several aspects of xylem and lignin formation. In this chapter, methods for nutrient medium preparation, callus culture initiation, and its maintenance, as well as those for protoplast isolation and viability observation, are described. As a case study, we describe the establishment of a xylogenic culture of Zinnia elegans mesophyll cells.

LOB DOMAIN-CONTAINING PROTEIN 15 Positively Regulates Expression of VND7, a Master Regulator of Tracheary Elements

Xylem includes xylem parenchyma cells, fibers and tracheary elements. Differentiation of tracheary elements is an irreversible process that is controlled by the master regulator VASCULAR-RELATED NAC-DOMAIN 7 (VND7). Molecular events occurring downstream of VND7 are well understood, but little is known regarding upstream regulation of VND7. In this study, we identified LOB DOMAIN-CONTAINING PROTEIN 15 (LBD15)/ASYMMETRIC LEAVES2-LIKE (ASL11) as a regulator of VND7. LBD15 was expressed in immature vascular cells and positively regulated both VND7 expression and differentiation of tracheary elements. LBD15 directly associated with the upstream sequence of VND7 and positively regulated VND7 expression. A 25 bp upstream sequence was essential for VND7 expression in the elongation zone of Arabidopsis roots. Taken together with previous studies identifying LBD15 as a target of VND7, we propose that LBD15 acts in a positive feedback regulation system that promotes and accelerates VND7 expression during the initiation phase of tracheary element differentiation in roots.

Overexpression and cosuppression of xylem-related genes in an early xylem differentiation stage-specific manner by the AtTED4 promoter

Summary Tissue‐specific overexpression of useful genes, which we can design according to their cause‐and‐effect relationships, often gives valuable gain‐of‐function phenotypes. To develop genetic tools in woody biomass engineering, we produced a collection of Arabidopsis lines that possess chimeric genes of a promoter of an early xylem differentiation stage‐specific gene, Arabidopsis Tracheary Element Differentiation‐related 4 (AtTED4) and late xylem development‐associated genes, many of which are uncharacterized. The AtTED4 promoter directed the expected expression of transgenes in developing vascular tissues from young to mature stage. Of T2 lines examined, 42%, 49% and 9% were judged as lines with the nonrepeat type insertion, the simple repeat type insertion and the other repeat type insertion of transgenes. In 174 T3 lines, overexpression lines were confirmed for 37 genes, whereas only cosuppression lines were produced for eight genes. The AtTED4 promoter activity was high enough to overexpress a wide range of genes over wild‐type expression levels, even though the wild‐type expression is much higher than AtTED4 expression for several genes. As a typical example, we investigated phenotypes of pAtTED4::At5g60490 plants, in which both overexpression and cosuppression lines were included. Overexpression but not cosuppression lines showed accelerated xylem development, suggesting the positive role of At5g60490 in xylem development. Taken together, this study provides valuable results about behaviours of various genes expressed under an early xylem‐specific promoter and about usefulness of their lines as genetic tools in woody biomass engineering.