The growth of secondary xylem and phloem depends on the division of cells in the vascular cambium and results in an increase in the diameter of the root and stem. Very little is known about the genetic mechanisms that control cambial activity and the differentiation of secondary xylem and phloem cell types. To begin to identify new genes required for vascular cell differentiation and function, we performed genome-wide expression profiling of xylem and phloem-cambium isolated from the root-hypocotyl of Arabidopsis (Arabidopsis thaliana). Gene expression in the remaining nonvascular tissue was also profiled. From these transcript profiles, we assembled three sets of genes with expression significantly biased toward xylem, phloem-cambium, or nonvascular tissue. We also assembled three two-tissue sets of genes with expression significantly biased toward xylem/phloem-cambium, xylem/nonvascular, or phloem-cambium/nonvascular tissues. Localizations predicted by transcript profiles were supported by results from promoter-reporter and reverse transcription-polymerase chain reaction experiments with nine xylem- or phloem-cambium-biased genes. An analysis of the members of the phloem-cambium gene set suggested that some genes involved in regulating primary meristems are also regulators of the cambium. Secondary phloem was implicated in the synthesis of auxin, glucosinolates, cytokinin, and gibberellic acid. Transcript profiles also supported the importance of class III HD ZIP and KANADI transcription factors as regulators of radial patterning during secondary growth, and identified several members of the G2-like, NAC, AP2, MADS, and MYB transcription factor families that may play roles as regulators of xylem or phloem cell differentiation and activity.

SummaryEstablishing the mechanisms regulating the autolysis of xylem tracheary elements (TEs) is important for understanding this programmed cell death process. These data demonstrate that two paralogous Arabidopsis thaliana proteases, XYLEM CYSTEINE PROTEASE1 (XCP1) and XCP2, participated in micro-autolysis within the intact central vacuole before mega-autolysis was initiated by tonoplast implosion. The data acquisition was aided by the predictable pattern of seedling root xylogenesis, the availability of single and double total knockout T-DNA lines, anti-sera that recognized XCP1 and XCP2, and the microwave-assisted processing of whole seedlings prior to immunolabeling and observation in the transmission electron microscope. During secondary wall thickening, XCP1 and XCP2 (in wild type), XCP1 (in xcp2 seedlings) or XCP2 (in xcp1 seedlings) were imported into the TE central vacuole. Both XCP1 and XCP2 heavily labeled dense aggregates of material within the vacuole. However, because of XCP1 deficiency in xcp1 and xcp1 xcp2 TEs, non-degraded cellular remnants first accumulated in the vacuole and then persisted in the TE lumen (longer than in the wild type) after the final mega-autolysis was otherwise complete. This delayed TE clearing phenotype in xcp1 was rescued by complementation with wild-type XCP1. Although TEs in the xcp2 single knock-out cleared comparably with wild type, the non-degraded remnants in xcp1 xcp2 TEs were more densely packed than in xcp1 TEs. Therefore, XCP2 has a minor but distinct role in micro-autolysis. After tonoplast implosion, XCP1 and XCP2 remained associated with disintegrating cellular material as mega-autolysis, aided by additional lytic enzymes, destroyed the bulk of the cellular contents.

SummaryThe class I KNOX homeodomain transcription factor ARBORKNOX1 (ARK1) is a key regulator of vascular cambium maintenance and cell differentiation in Populus. Currently, basic information is lacking concerning the distribution, functional characteristics, and evolution of ARK1 binding in the Populus genome.Here, we used chromatin immunoprecipitation sequencing (ChIP-seq) technology to identify ARK1 binding loci genome-wide in Populus. Computational analyses evaluated the distribution of ARK1 binding loci, the function of genes associated with bound loci, the effect of ARK1 binding on transcript levels, and evolutionary conservation of ARK1 binding loci.ARK1 binds to thousands of loci which are highly enriched proximal to the transcriptional start sites of genes of diverse functions. ARK1 target genes are significantly enriched in paralogs derived from the whole-genome salicoid duplication event. Both ARK1 and a maize (Zea mays) homolog, KNOTTED1, preferentially target evolutionarily conserved genes. However, only a small portion of ARK1 target genes are significantly differentially expressed in an ARK1 over-expression mutant.This study describes the functional characteristics and evolution of DNA binding by a transcription factor in an undomesticated tree, revealing complexities similar to those shown for transcription factors in model animal species.

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Cellular processes, such as signal transduction and cell wall deposition, are organized by macromolecule interactions. Experimentally determined protein-protein interactions (PPIs) and protein-DNA interactions (PDIs) relevant to woody plant development are sparse. To begin to develop a Populus trichocarpa Torr. & A. Gray wood interactome, we applied the yeast-two-hybrid (Y2H) assay in different ways to enable the discovery of novel PPIs and connected networks. We first cloned open reading frames (ORFs) for 361 genes markedly upregulated in secondary xylem compared with secondary phloem and performed a binary Y2H screen with these proteins. By screening a xylem cDNA library for interactors of a subset of these proteins and then recapitulating the process by using a subset of the interactors as baits, we ultimately identified 165 PPIs involving 162 different ORFs. Thirty-eight transcription factors (TFs) included in our collection of P. trichocarpa wood ORFs were used in a Y1H screen for binding to promoter regions of three genes involved in lignin biosynthesis resulting in 40 PDIs involving 20 different TFs. The network incorporating both the PPIs and PDIs included 14 connected subnetworks, with the largest having 132 members. Protein-protein interactions and PDIs validated previous reports and also identified new candidate wood formation proteins and modules through their interactions with proteins and promoters known to be involved in secondary cell wall synthesis. Selected examples are discussed including a PPI between Mps one binder (MOB1) and a mitogen-activated protein kinase kinase kinase kinase (M4K) that was further characterized by assays confirming the PPI as well as its effect on subcellular localization. Mapping of published transcriptomic data showing developmentally detailed expression patterns across a secondary stem onto the network supported that the PPIs and PDIs are relevant to wood formation, and also illustrated that wood-associated interactions involve gene products that are not upregulated in secondary xylem.