Xylem consists of three types of cells: tracheary elements (TEs), parenchyma cells, and fiber cells. TE differentiation includes two essential processes, programmed cell death (PCD) and secondary cell wall formation. These two processes are tightly coupled. However, little is known about the molecular mechanisms underlying these processes. Here, we show that VASCULAR-RELATED NAC-DOMAIN6 (VND6), a master regulator of TEs, regulates some of the downstream genes involved in these processes in a coordinated manner. We first identified genes that are expressed downstream of VND6 but not downstream of SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1), a master regulator of xylem fiber cells, using transformed suspension culture cells in microarray experiments. We found that VND6 and SND1 governed distinct aspects of xylem formation, whereas they regulated a number of genes in common, specifically those related to secondary cell wall formation. Genes involved in TE-specific PCD were upregulated only by VND6. Moreover, we revealed that VND6 directly regulated genes that harbor a TE-specific cis-element, TERE, in their promoters. Thus, we found that VND6 is a direct regulator of genes related to PCD as well as to secondary wall formation.

A specifically patterned cell wall is a determinant of plant cell shape. Yet, the precise mechanisms that underlie initiation of cell wall patterning remain elusive. By using a reconstitution assay, we revealed that ROPGEF4 (Rho of plant guanine nucleotide exchange factor 4) and ROPGAP3 [ROP guanosine triphosphatase (GTPase)-activating protein 3] mediate local activation of the plant Rho GTPase ROP11 to initiate distinct pattern of secondary cell walls in xylem cells. The activated ROP11 recruits MIDD1 to induce local disassembly of cortical microtubules. Conversely, cortical microtubules eliminate active ROP11 from the plasma membrane through MIDD1. Such a mutual inhibitory interaction between active ROP domains and cortical microtubules establishes the distinct pattern of secondary cell walls. This Rho-based regulatory mechanism shows how plant cells initiate and control cell wall patterns to form various cell shapes.

Higher organisms possess mechanisms to maintain stem cells' proliferative and pluripotent states in stem cell niches [1]. Plants possess two types of stem cell niches in the root and shoot apical meristems, where regulatory interactions exist between stem cells and organizing cells. Recent studies provided new insights into the molecular mechanism of stem cell maintenance [2-4]. However, earlier and more essential developmental events such as the acquisition of stem cell proliferative activity are still unknown. In vascular tissues, procambial cells function as stem cells and differentiate into xylem, phloem, and procambium. Procambial cell proliferation starts at root apical meristem (RAM) postembryonically; therefore, procambial cell development in RAM is a good model for investigating the regulation of stem cell proliferation. LONESOME HIGHWAY (LHW) and TARGET OF MONOPTEROS5 (TMO5), as well as its homolog, TMO5-LIKE1 (T5L1), encode bHLH proteins that function as heterodimers (LHW-TMO5 and LHW-T5L1) in vascular tissue organization [5-7]. LHW-T5L1 promotes vascular-cell-specific proliferation in RAM [7]. Here, we demonstrate that LHW-T5L1 promotes expression of key cytokinin production genes, including LONELY GUY3 (LOG3) and LOG4, in xylem precursor cells, resulting in elevated cytokinin levels in the surrounding cells. LHW-T5L1 can also promote expression of AHP6, which suppresses cytokinin signaling and then maintains xylem precursor cells at a nondividing state. Our results indicate that LHW-T5L1 establishes xylem precursor cells as a signal center that promotes procambial-cell-specific proliferation through cytokinin response.

Plant cells have evolved cortical microtubules, in a two-dimensional space beneath the plasma membrane, that regulate patterning of cellulose deposition. Although recent studies have revealed that several microtubule-associated proteins facilitate self-organization of transverse cortical microtubules, it is still unknown how diverse patterns of cortical microtubules are organized in different xylem cells, which are the major components of wood. Using our newly established in vitro xylem cell differentiation system, we found that a novel microtubule end-tracking protein, microtubule depletion domain 1 (MIDD1), was anchored to distinct plasma membrane domains and promoted local microtubule disassembly, resulting in pits on xylem cell walls. The introduction of RNA interference for MIDD1 resulted in the failure of local microtubule depletion and the formation of secondary walls without pits. Conversely, the overexpression of MIDD1 reduced microtubule density. MIDD1 has two coiled-coil domains for the binding to microtubules and for the anchorage to plasma membrane domains, respectively. Combination of the two coils caused end tracking of microtubules during shrinkage and suppressed their rescue events. Our results indicate that MIDD1 integrates spatial information in the plasma membrane with cortical microtubule dynamics for determining xylem cell wall pattern.

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Plant cortical microtubule arrays determine the cell wall deposition pattern and proper cell shape and function. Although various microtubule-associated proteins regulate the cortical microtubule array, the mechanisms underlying marked rearrangement of cortical microtubules during xylem differentiation are not fully understood. Here, we show that local Rho of Plant (ROP) GTPase signaling targets an Arabidopsis thaliana kinesin-13 protein, Kinesin-13A, to cortical microtubules to establish distinct patterns of secondary cell wall formation in xylem cells. Kinesin-13A was preferentially localized with cortical microtubules in secondary cell wall pits, areas where cortical microtubules are depolymerized to prevent cell wall deposition. This localization of Kinesin-13A required the presence of the activated ROP GTPase, MICROTUBULE DEPLETION DOMAIN1 (MIDD1) protein, and cortical microtubules. Knockdown of Kinesin-13A resulted in the formation of smaller secondary wall pits, while overexpression of Kinesin-13A enlarged their surface area. Kinesin-13A alone could depolymerize microtubules in vitro; however, both MIDD1 and Kinesin-13A were required for the depolymerization of cortical microtubules in vivo. These results indicate that Kinesin-13A regulates the formation of secondary wall pits by promoting cortical microtubule depolymerization via the ROP-MIDD1 pathway.

SummaryThe actin cytoskeleton of higher plants plays an essential role in plant morphogenesis and in maintaining various cellular activities. In this study we established a tobacco BY-2 cell line, stably transformed with a GFPfimbrin actin-binding domain (ABD) 2 construct, that allows visualization of actin microfilaments (MFs) in living cells. Using this cell line, designated BY-GF11, we performed time-sequential observations of MF dynamics during cell-cycle progression. Detailed analyses revealed the appearance of a broad MF band in the late G 2 phase that separated to form a structure corresponding to the so-called actin-depleted zone (ADZ) in mitosis. In BY-GF11, the MF structure at the cell cortex in mitosis appeared to form two bands rather than the ADZ. Measurements of fluorescent intensities of the cell cortex indicated an MF distribution that resembled two peaks, and we therefore named the structure MF 'twin peaks' (MFTP). The cell plate formed exactly within the valley between the MFTP at cytokinesis, and this cell-plate guidance was distorted by disruption of the MFTP by an inhibitor of actin polymerization. These results suggest that the MFTP originates from the broad MF band in the G 2 phase and functions as a marker of cytokinesis.

SUMMARYThe nuclear envelope (NE) is a highly active structure with a specific set of nuclear envelope proteins acting in diverse cellular events. SUN proteins are conserved NE proteins among eukaryotes. Although they form nucleocytoplasmic linkage complexes in metazoan cells, their functions in the plant kingdom are unknown. To understand the function of plant SUN proteins, in this study we first investigated the dynamics of Arabidopsis SUN proteins during mitosis in Arabidopsis roots and cultured cells. For this purpose, we performed dual and triple visualization of these proteins, microtubules, chromosomes, and endoplasmic reticulum (ER) in cultured cells, and observed their dynamics during mitosis using a high-speed spinning disk confocal microscope. The localizations of SUN proteins changed dynamically during mitosis, tightly coupled with NE dynamics. Moreover, NE re-formation marked with SUN proteins is temporally and spatially coordinated with plantspecific microtubule structures such as phragmoplasts. Finally, the analysis with gene knockdowns of AtSUN1 and AtSUN2 indicated that they are necessary for the maintenance and/or formation of polarized nuclear shape in root hairs. These results suggest that Arabidopsis SUN proteins function in the maintenance or formation of nuclear shape as components of the nucleocytoskeletal complex.