Both tracheary elements and fiber cells undergo programmed cell death (PCD) during xylem development. In this study we investigated the role of papain-like cysteine protease CEP1 in PCD in the xylem of Arabidopsis. CEP1 was located in the cell wall of xylem cells, and CEP1 expression levels in inflorescence stems increased during stem maturation. cep1 mutant plants exhibited delayed stem growth and reduced xylem cell number compared to wild-type plants. Transmission electron microscopy demonstrated that organelle degradation was delayed during PCD, and thicker secondary walls were present in fiber cells and tracheary elements of the cep1 mutant. Transcriptional analyses of the maturation stage of the inflorescence stem revealed that genes involved in the biosynthesis of secondary wall components, including cellulose, hemicellulose, and lignin, as well as wood-associated transcriptional factors, were up-regulated in the cep1 mutant. These results suggest that CEP1 is directly involved in the clearing of cellular content during PCD and regulates secondary wall thickening during xylem development.

Wood constitutes the largest reservoir of terrestrial biomass. Composed of xylem, it arises from one side of the vascular cambium, a bifacial stem cell niche that also produces phloem on the opposing side. It is currently unknown which molecular factors endow cambium stem cell identity. Here we show that TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF) ligand–activated PHLOEM INTERCALATED WITH XYLEM (PXY) receptors promote the expression of CAMBIUM-EXPRESSED AINTEGUMENTA-LIKE (CAIL) transcription factors to define cambium stem cell identity in the Arabidopsis root. By sequestrating the phloem-originated TDIF, xylem-expressed PXY confines the TDIF signaling front, resulting in the activation of CAIL expression and stem cell identity in only a narrow domain. Our findings show how signals emanating from cells on opposing sides ensure robust yet dynamically adjustable positioning of a bifacial stem cell layer.

Studying the developmental genetics of plant organs requires following gene expression in specific tissues. To facilitate this, we have developed dual expression anatomy lines, which incorporate a red plasma membrane marker alongside a fluorescent reporter for a gene of interest in the same vector. Here, we adapted the GreenGate cloning vectors to create two destination vectors showing strong marking of cell membranes in either the whole root or specifically in the lateral roots. This system can also be used in both embryos and whole seedlings. As proof of concept, we follow both gene expression and anatomy in Arabidopsis (Arabidopsis thaliana) during lateral root organogenesis for a period of over 24 h. Coupled with the development of a flow cell and perfusion system, we follow changes in activity of the DII auxin sensor following application of auxin.

Summary: In plant development, key receptor kinases are often active in disparate cell types, with each requiring vastly different signalling outputs. The ERECTA (ER) receptor kinase and its homologs ERL1 and ERL2 exemplify this pleiotropy. In Arabidopsis , they influence stomatal patterning, shoot meristem function, ovule morphogenesis, xylem fiber differentiation, and cell division in the vascular cambium ^1–6 . Such diverse expression and functionality raises the question of how ER signalling can specify such distinct cell behaviours. One mechanism is via cell-type specific interactions with co-receptors, ligands, or other proteins that modulate signalling. However, little is known about ER interactors in the vascular cambium, a bifacial stem cell niche that generates phloem and xylem ( Figure 1A ). Combinatorial mutations between ER, ERL1 and ERL2 and receptor kinases of a second family, PXY, PXL1 , and PXL2 , show severe cambial defects ^5,7 , but the mechanism underpinning these phenotypes is not known. Here we discovered that PXY proteins form protein complexes with ER and ERL2. PXY signalling can be manipulated by altering levels of its cognate ligand, TDIF. In genetic analysis, plant lines in which TDIF levels were altered had dramatic phenotypic changes that required the presence of ER or ERL2. Our results demonstrate that PXY signalling mediated cambium regulation depends on ER signalling and explains ER function in the cambium. Because the cambium produces xylem, which constitutes the wood in vascular plants, our findings position PXY-ER complexes at the centre of the accumulation of this versatile biomaterial and essential carbon sink.

Protein turnover is essential for cellular metabolism, organelle biogenesis, stress adaptation, and ultimately the viability of cells and tissues. Papain-like cysteine proteases (PLCPs) are one of the vital components in protein degradation. PLCPs have been reported to act in senescence-associated proteolysis, but their roles in vegetative growth remain unclear. We identified PtCP1, an AALP-like PLCP in Populus tomentosa, localized to the vacuole and acid-triggered activated. CRISPR/Cas9-generated loss-of-function mutant (d7) showed dwarfism and non-stomatal photosynthetic limitations. On the other hand, the gain-of-function line (EM, deleted ERFNIN domain) exhibited accelerated growth and enhanced photosynthetic parameters. We showed d7 had the accumulation of Rubisco, which was the most important protein in photosynthetic carbon fixation. Transcriptomics revealed dysregulated carbon metabolism in d7. This data supported PtCP1-mediated proteolysis regulated photosynthetic carbon assimilation via altered Rubisco turnover, and then it increased the biomass accumulation during vegetative growth in woody plants.

Wood is the greatest reservoir of terrestrial biomass and an essential carbon sink. Formed of xylem, it is derived from the cambium, a meristematic zone within plant stems from which phloem also forms. In Arabidopsis, cell division within the cambium is promoted by three major factors: auxin, cytokinin, and the TDIF-PXY ligand-receptor pair. Meristems and other stem cell populations are typically regulated by a balance between cell division-promoting factors and those that repress cell division to control meristem size, however few factors with cambium-repressing activity are known. Here we combined transcriptomics and transcriptional network analysis, which led to identification of related homeodomain zinc-finger transcription factors, ATHB23, ATHB30, and ATHB34, that repress cambium activity. These factors inhibit cambium activity by directly binding of promoters from a subset of auxin, cytokinin and TDIF-PXY transcriptional target genes, resulting in attenuation of their transcription. Our findings thus reveal a new mechanism underpinning balanced cambium activity.