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.

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.

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.