Chengsong Zhao

The Xylem and Phloem Transcriptomes from Secondary Tissues of the Arabidopsis Root-Hypocotyl

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.

Plant proteolytic enzymes: possible roles during programmed cell death

Proteolytic enzymes are known to be associated with developmentally programmed cell death during organ senescence and tracheary element differentiation. Recent evidence also links proteinases with some types of pathogen- and stress-induced cell suicide. The precise roles of proteinases in these and other plant programmed cell death processes are not understood, however. To provide a framework for consideration of the importance of proteinases during plant cell suicide, characteristics of the best-known proteinases from plants including subtilisin-type and papain-type enzymes, phytepsins, metalloproteinases and the 26S proteasome are summarized. Examples of serine, cysteine, aspartic, metallo- and threonine proteinases linked to animal programmed cell death are cited and the potential for plant proteinases to act as mediators of signal transduction and as effectors of programmed cell death is discussed.

Arabidopsis as a Model for Investigating Gene Activity and Function in Vascular Tissues

ABSTRACT Using the zinnia mesophyll cell system for tracheary element differentiation, members of at least three families of mechanistically distinct (serine, cysteine and threonine) proteases have been implicated in the regulation of tracheary element differentiation. We are currently using Arabidopsis to facilitate genetic analysis of the roles of proteolytic enzymes during vascular tissue differentiation. Although not typically considered as a model for secondary growth, Arabidopsis forms a true cambium and produces a relatively large amount of secondary xylem and phloem within the root and hypocotyl. This potential for secondary growth is best realized under conditions that delay senescence, i.e., inflorescence removal and growth at low population density. After growing Arabidopsis under these conditions for at least eight weeks it is possible to isolate biochemical quantities of xylem and phloem for analysis of tissue-specific protease activities. After dissecting approximately 300 root-hypocotyl segments into xylem and bark fractions we isolated RNA for the construction of the first xylem and bark cDNA libraries from Arabidopsis. Using gene-specific primers and degenerate primers we screened the libraries by PCR and amplified fragments from seven protease genes including members of serine, cysteine, and aspartic acid protease families. Expression for three of these genes (XCP1, XCP2, and XSP1) is xylem-specific. XCP1 and XCP2 are predicted to encode papain-like cysteine proteases and XSP1 is predicted to encode a subtilisin-like serine protease. To identify additional genes with potential importance to vascular tissue differentiation and physiology, we analyzed 1,000 (500 from xylem and 500 from bark) ESTs.

The Arabidopsis Myb genes MYR1 and MYR2 are redundant negative regulators of flowering time under decreased light intensity.

Changes in the duration, quality and intensity of light affect flowering time. Compared with the effects of light duration and quality, less is known about the effects of light intensity on flowering. Here we describe two paralogous single Myb domain genes, MYB-RELATED PROTEIN 1 (MYR1) and MYB-RELATED PROTEIN 2 (MYR2), and their roles as repressors of responses to decreased light intensity in Arabidopsis. Homozygous myr1 myr2 double mutants flowered early under low light intensities. Additionally, myr1 myr2 mutants exhibited increases in petiole length, leaf angle and apical dominance. Genetic analyses involving mutants in the long-day, gibberellin (GA) and phyB flowering pathways indicated that all aspects of the myr1 myr2 phenotype required GA biosynthesis. The early-flowering phenotype of myr1 myr2 also required FLOWERING LOCUS T, and myr1 myr2 mutants showed an epistatic interaction with the phyB-9 mutant. Over-expression of MYR1 or MYR2 produced GA-deficiency symptoms that were rescued by application of gibberellic acid (GA₃). Loss of MYR1 and MYR2 function was associated with a twofold increase in GA20ox2 expression and a 30% increase in GA₄ levels, while over-expression of MYR2 led to a threefold decrease in GA20ox2 expression and a 50% decrease in GA₄ levels. Considered together, these results suggest that the ability of MYR1 and MYR2 to repress flowering and organ elongation is at least partly due to their negative effect on levels of bioactive GA.

Alternative splicing of Myb-related genes MYR1 and MYR2 may modulate activities through changes in dimerization, localization, or protein folding

Arabidopsis genes MYR1 and MYR2 are regulators of flowering time under low light intensity. These Myb-related genes are expressed as alternative splice variants affected in their coiled-coil and DNA-binding domains. We tested whether alternative splicing could affect dimerization and localization of MYR1 and MYR2, thereby potentially affecting their activity. Using MYR1 as a model for variants within the coiled-coil region, we detected 2 types of homodimers. For MYR2, alternative splicing in the DNA-binding Myb-like domain abolished the ability of MYR2 to dimerize. Alternative splicing in the coiled-coil domain did not affect nuclear localization, as determined by transient expression in tobacco, while alternative splicing in the DNA-binding domain of MYR2 yielded a distinct intranuclear localization pattern that may reflect changes in phosphorylation-dependent protein folding. Thus alternative splicing of these genes may result in changes in dimerization or protein folding resulting in changes in activity and abundance of MYR1 or MYR2 protein.

XYLEM NAC DOMAIN1, an angiosperm NAC transcription factor, inhibits xylem differentiation through conserved motifs that interact with RETINOBLASTOMA‐RELATED

The Arabidopsis thaliana gene XYLEM NAC DOMAIN1 (XND1) is upregulated in xylem tracheary elements. Yet overexpression of XND1 blocks differentiation of tracheary elements. The molecular mechanism of XND1 action was investigated. Phylogenetic and motif analyses indicated that XND1 and its homologs are present only in angiosperms and possess a highly conserved C-terminal region containing linear motifs (CKII-acidic, LXCXE, E2FTD -like and LXCXE-mimic) predicted to interact with the cell cycle and differentiation regulator RETINOBLASTOMA-RELATED (RBR). Protein-protein interaction and functional analyses of XND1 deletion mutants were used to test the importance of RBR-interaction motifs. Deletion of either the LXCXE or the LXCXE-mimic motif reduced both the XND1-RBR interaction and XND1 efficacy as a repressor of differentiation, with loss of the LXCXE motif having the strongest negative impacts. The function of the XND1 C-terminal domain could be partially replaced by RBR fused to the N-terminal domain of XND1. XND1 also transactivated gene expression in yeast and plants. The properties of XND1, a transactivator that depends on multiple linear RBR-interaction motifs to inhibit differentiation, have not previously been described for a plant protein. XND1 harbors an apparently angiosperm-specific combination of interaction motifs potentially linking the general differentiation regulator RBR with a xylem-specific pathway for inhibition of differentiation.

Populus biomass protein-protein interactions and their functions

Proteins are molecular machines that play roles in almost all biological activities through interactions with other molecules such as carbohydrates, lipids, nucleic acids and other proteins. We are mapping protein-protein interactions relevant to woody biomass production by focusing on proteins co-expressed in poplar secondary xylem. In addition to revealing novel regulatory mechanisms important to woody biomass production, mapping the poplar protein-protein interactome will provide fundamental information relevant to xylem differentiation and secondary growth. We have cloned a portion of the poplar biomass ORFeome, specifically 374 ORFs that are upregulated in xylem versus phloem, for use in protein-protein interaction research. Here we summarize the techniques we are using to discover and study protein-protein interactions and results to date. 1. Yeast two-hybrid (Y2H) binary assays: 108,205 Y2H binary assays involving over 300 biomass ORFeome members have been performed and 11 interaction pairs identified. The proportional yield from our binary screen is similar to that represented by the current preliminary binary screen data from the Arabidopsis interactome project. 2. Y2H cDNA library screening: We have used 40 bait proteins to screen our poplar xylem cDNA prey library, and a total of 60 biomass ORFeome members comprising putative regulators of lignocellulose synthesis will be screened in 2011. For the 26 ORFs that are completely through the library screen, we have identified 44 unique interacting sequences. Thus far, the proportional yield of interactors for proteins catalyzing metabolic reactions (such as cellulose synthase, PB138) is much lower than that for regulatory proteins (such as NIMA kinase, PB223). Results from these Y2H screens are available at our project website (http://xylome.vbi.vt.edu/index.html). Additional website information includes a project overview, project objectives, progress to date, sequences for all ORFs and primers, clone availability, and Y2H protocols. 3. Confirmation of selected protein-protein interactions identified by Y2H screens: Selected Y2H interactions are being confirmed by independent methods including bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation/affinity purification using plant transient or stable expression systems. We have developed improved vectors and protocols for reducing background fluorescence during BiFC experiments. 4. Functional analysis: We are characterizing the functions of selected interacting pairs in both poplar and Arabidopsis by ectopically expressing or suppressing genes singly and in combination. For ectopic and/or over-expression experiments we are using 35S, SUC2 (phloem specific) and XCP2 (xylem specific) promoters. Co-expression of 35S::PB15 and 35S::PB129 in Arabidopsis resulted in expanded interfascicular regions containing enlarged fibers compared to fibers in wild-type plant interfascicular regions of the inflorescence stem. Importantly, this phenotype was not observed in transgenics overexpressing just one of these genes, showing the potential of novel interactome data to be translated into alteration of wood phenotypes.Arabidopsis mutants carrying T-DNA insertions within Arabidopsis orthologs of PB15 and PB129 are also being evaluated, and Populus RNA interference (RNAi) lines have been generated to study the effects of silencing PB15 and/or PB129 and their paralogs. 5. Transgenic poplar field trial: We prepared transgenic poplar overexpressing 11 biomass genes as tandem affinity purification-(TAPa)-tagged fusions and established these in a replicated field trial, which will be evaluated for overexpression phenotypes and serve as a source of xylem for identification of co-purified proteins.

Identification of new protein–protein and protein–DNA interactions linked with wood formation in Populus trichocarpa

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.

DIVARICATA AND RADIALIS INTERACTING FACTOR (DRIF) also interacts with WOX and KNOX proteins associated with wood formation in Populus trichocarpa

DIVARICATA AND RADIALIS INTERACTING FACTOR (DRIF) from snapdragon (Antirrhinum majus) is a MYB/SANT protein that interacts with related MYB/SANT proteins, RADIALIS and DIVARICATA, through its N-terminal MYB/SANT domain. In addition to the MYB/SANT domain, DRIF contains a C-terminal domain of unknown function (DUF3755). Here we describe novel protein-protein interactions involving a poplar (Populus trichocarpa) homolog of DRIF, PtrDRIF1. In addition to interacting with poplar homologs of RADIALIS (PtrRAD1) and DIVARICATA (PtrDIV4), PtrDRIF1 interacted with members of other families within the homeodomain-like superfamily, including PtrWOX13c, a WUSCHEL-RELATED HOMEOBOX protein, and PtrKNAT7, a KNOTTED1-LIKE HOMEOBOX protein. PtrRAD1 and PtrDIV4 interacted with the MYB/SANT-containing N-terminal portion of PtrDRIF1, whereas DUF3755 was both necessary and sufficient for interactions with PtrWOX13c and PtrKNAT7. Of the two MYB/SANT domains present in PtrDIV4, only the N-terminal MYB/SANT domain interacted with PtrDRIF1. GFP-PtrDRIF1 expressed alone or with PtrRAD1 localized to the cytoplasm, whereas co-expression of GFP-PtrDRIF1 with PtrDIV4, PtrWOX13c or PtrKNAT7 resulted in nuclear localization of GFP-PtrDRIF1. Modified yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiments using PtrDRIF1 as a bridge protein revealed that PtrDRIF1 simultaneously interacted with PtrRAD1 and PtrWOX13c, but could not form a heterotrimeric complex when PtrDIV4 was substituted for PtrRAD1. Moreover, a Y2H competition assay indicated that PtrKNAT7 inhibits the interaction between PtrRAD1 and PtrDRIF1. The discovery of an additional protein-protein interaction domain in DRIF proteins, DUF3755, and its ability to form heterodimers and heterotrimers involving MYB/SANT and wood-associated homeodomain proteins, implicates DRIF proteins as mediators of a broader array of processes than previously reported.