A COMPASS histone H3K4 trimethyltransferase pentamer transactivates drought tolerance and growth/biomass production in Populus trichocarpa.

Histone H3 lysine-4 trimethylation (H3K4me3) activating drought-responsive genes in plants for drought adaptation has long been established, but the underlying regulatory mechanisms are unknown. Here, using yeast two-hybrid, bimolecular fluorescence complementation, biochemical analyses, transient and CRISPR-mediated transgenesis in Populus trichocarpa, we unveiled in this adaptation a regulatory interplay between chromatin regulation and gene transactivation mediated by an epigenetic determinant, a PtrSDG2-1-PtrCOMPASS (complex proteins associated with Set1)-like H3K4me3 complex, PtrSDG2-1-PtrWDR5a-1-PtrRbBP5-1-PtrAsh2-2 (PtrSWRA). Under drought conditions, a transcription factor PtrAREB1-2 interacts with PtrSWRA, forming a PtrSWRA-PtrAREB1-2 pentamer, to recruit PtrSWRA to specific promoter elements of drought-tolerant genes, such as PtrHox2, PtrHox46, and PtrHox52, for depositing H3K4me3 to promote and maintain activated state of such genes for tolerance. CRISPR-edited defects in the pentamer impaired drought tolerance and elevated expression of PtrHox2, PtrHox46, or PtrHox52 improved the tolerance as well as growth in P. trichocarpa. Our findings revealed the identity of the underlying H3K4 trimethyltransferase and its interactive arrangement with the COMPASS for catalysis specificity and efficiency. Furthermore, our study uncovered how the H3K4 trimethyltransferase-COMPASS complex is recruited to the effector genes for elevating H3K4me3 marks for improved drought tolerance and growth/biomass production in plants.

Intracardiac thrombosis and pulmonary thromboembolism during liver transplantation: A systematic review and meta-analysis.

Intracardiac thrombosis and/or pulmonary thromboembolism (ICT/PE) is a rare but devastating complication during liver transplantation. Its pathophysiology remains poorly understood, and successful treatment remains a challenge. This systematic review summarizes the available published clinical data regarding ICT/PE during liver transplantation. Databases were searched for all publications reporting on ICT/PE during liver transplantation. Data collected included its incidence, patient characteristics, the timing of diagnosis, treatment strategies, and patient outcomes. This review included 59 full-text citations. The point prevalence of ICT/PE was 1.42%. Thrombi were most often diagnosed during the neohepatic phase, particularly at allograft reperfusion. Intravenous heparin was effective in preventing early-stage thrombus from progressing further and restoring hemodynamics in 76.32% of patients it was utilized for; however, the addition of tissue plasminogen activator or sole use of tissue plasminogen activator offered diminishing returns. Despite all resuscitation efforts, the in-hospital mortality rate of an intraoperative ICT/PE was 40.42%, with nearly half of these patients dying intraoperatively. The results of our systematic review are an initial step for providing clinicians with data that can help identify higher-risk patients. The clinical implications of our results warrant the development of identification and management strategies for the timely and effective treatment of these tragic occurrences during liver transplantation.

A novel synthetic-genetic-array-based yeast one-hybrid system for high discovery rate and short processing time.

Eukaryotic gene expression is often tightly regulated by interactions between transcription factors (TFs) and their DNA cis targets. Yeast one-hybrid (Y1H) is one of the most extensively used methods to discover these interactions. We developed a high-throughput meiosis-directed yeast one-hybrid system using the Magic Markers of the synthetic genetic array analysis. The system has a transcription factor-DNA interaction discovery rate twice as high as the conventional diploid-mating approach and a processing time nearly one-tenth of the haploid-transformation method. The system also offers the highest accuracy in identifying TF-DNA interactions that can be authenticated in vivo by chromatin immunoprecipitation. With these unique features, this meiosis-directed Y1H system is particularly suited for constructing novel and comprehensive genome-scale gene regulatory networks for various organisms.

Dimerization of PtrMYB074 and PtrWRKY19 mediates transcriptional activation of PtrbHLH186 for secondary xylem development in Populus trichocarpa.

Wood formation is controlled by transcriptional regulatory networks (TRNs) involving regulatory homeostasis determined by combinations of transcription factor (TF)-DNA and TF-TF interactions. Functions of TF-TF interactions in wood formation are still in the early stages of identification. PtrMYB074 is a woody dicot-specific TF in a TRN for wood formation in Populus trichocarpa. Here, using yeast two-hybrid and bimolecular fluorescence complementation, we conducted a genome-wide screening for PtrMYB074 interactors and identified 54 PtrMYB074-TF pairs. Of these pairs, 53 are novel. We focused on the PtrMYB074-PtrWRKY19 pair, the most highly expressed and xylem-specific interactor, and its direct transregulatory target, PtrbHLH186, the xylem-specific one of the pair's only two direct TF target genes. Using transient and CRISPR-mediated transgenesis in P. trichocarpa coupled with chromatin immunoprecipitation and electrophoretic mobility shift assays, we demonstrated that PtrMYB074 is recruited by PtrWRKY19 and that the PtrMYB074-PtrWRKY19 dimers are required to transactive PtrbHLH186. Overexpressing PtrbHLH186 in P. trichocarpa resulted in retarded plant growth, increased guaiacyl lignin, a higher proportion of smaller stem vessels and strong drought-tolerant phenotypes. Knowledge of the PtrMYB074-PtrWRKY19-PtrbHLH186 regulation may help design genetic controls of optimal growth and wood formation to maximize beneficial wood properties while minimizing negative effects on growth.

Transcriptional reprogramming of xylem cell wall biosynthesis in tension wood.

Tension wood (TW) is a specialized xylem tissue developed under mechanical/tension stress in angiosperm trees. TW development involves transregulation of secondary cell wall genes, which leads to altered wood properties for stress adaptation. We induced TW in the stems of black cottonwood (Populus trichocarpa, Nisqually-1) and identified two significantly repressed transcription factor (TF) genes: class B3 heat-shock TF (HSFB3-1) and MYB092. Transcriptomic analysis and chromatin immunoprecipitation (ChIP) were used to identify direct TF-DNA interactions in P. trichocarpa xylem protoplasts overexpressing the TFs. This analysis established a transcriptional regulatory network in which PtrHSFB3-1 and PtrMYB092 directly activate 8 and 11 monolignol genes, respectively. The TF-DNA interactions were verified for their specificity and transactivator roles in 35 independent CRISPR-based biallelic mutants and overexpression transgenic lines of PtrHSFB3-1 and PtrMYB092 in P. trichocarpa. The gene-edited trees (mimicking the repressed PtrHSFB3-1 and PtrMYB092 under tension stress) have stem wood composition resembling that of TW during normal growth and under tension stress (i.e., low lignin and high cellulose), whereas the overexpressors showed an opposite effect (high lignin and low cellulose). Individual overexpression of the TFs impeded lignin reduction under tension stress and restored high levels of lignin biosynthesis in the TW. This study offers biological insights to further uncover how metabolism, growth, and stress adaptation are coordinately regulated in trees.

Hierarchical Transcription Factor and Chromatin Binding Network for Wood Formation in Black Cottonwood (Populus trichocarpa).

Wood remains the world's most abundant and renewable resource for timber and pulp and is an alternative to fossil fuels. Understanding the molecular regulation of wood formation can advance the engineering of wood for more efficient material and energy productions. We integrated a black cottonwood (Populus trichocarpa) wood-forming cell system with quantitative transcriptomics and chromatin binding assays to construct a transcriptional regulatory network (TRN) directed by a key transcription factor (TF), PtrSND1-B1 (secondary wall-associated NAC-domain protein). The network consists of four layers of TF-target gene interactions with quantitative regulatory effects, describing the specificity of how the regulation is transduced through these interactions to activate cell wall genes (effector genes) for wood formation. PtrSND1-B1 directs 57 TF-DNA interactions through 17 TFs transregulating 27 effector genes. Of the 57 interactions, 55 are novel. We tested 42 of these 57 interactions in 30 genotypes of transgenic P. trichocarpa and verified that ∼90% of the tested interactions function in vivo. The TRN reveals common transregulatory targets for distinct TFs, leading to the discovery of nine TF protein complexes (dimers and trimers) implicated in regulating the biosynthesis of specific types of lignin. Our work suggests that wood formation may involve regulatory homeostasis determined by combinations of TF-DNA and TF-TF (protein-protein) regulations.

The AREB1 Transcription Factor Influences Histone Acetylation to Regulate Drought Responses and Tolerance in Populus trichocarpa.

Plants develop tolerance to drought by activating genes with altered levels of epigenetic modifications. Specific transcription factors are involved in this activation, but the molecular connections within the regulatory system are unclear. Here, we analyzed genome-wide acetylated lysine residue 9 of histone H3 (H3K9ac) enrichment and examined its association with transcriptomes in Populus trichocarpa under drought stress. We revealed that abscisic acid-Responsive Element (ABRE) motifs in promoters of the drought-responsive genes PtrNAC006, PtrNAC007, and PtrNAC120 are involved in H3K9ac enhancement and activation of these genes. Overexpressing these PtrNAC genes in P trichocarpa resulted in strong drought-tolerance phenotypes. We showed that the ABRE binding protein PtrAREB1-2 binds to ABRE motifs associated with these PtrNAC genes and recruits the histone acetyltransferase unit ADA2b-GCN5, forming AREB1-ADA2b-GCN5 ternary protein complexes. Moreover, this recruitment enables GCN5-mediated histone acetylation to enhance H3K9ac and enrich RNA polymerase II specifically at these PtrNAC genes for the development of drought tolerance. CRISPR editing or RNA interference-mediated downregulation of any of the ternary members results in highly drought-sensitive P trichocarpa Thus, the combinatorial function of the ternary proteins establishes a coordinated histone acetylation and transcription factor-mediated gene activation for drought response and tolerance in Populus species.

Student Self-perception on Digital Literacy in STEM Blended Learning Environments.

As students transition into tertiary blended learning environments, their digital literacy in terms of technical capabilities have potential to impact on their access to digital resources. The first foundational year of STEM degrees includes compulsory courses across a broad range of scientific areas, each of which incorporates online technology in a discipline-specific manner. Given the diversity of online resources that STEM students need to access across their first-year coursework, this study applies learning analytical methods to determine whether students' perceived level of digital literacy has an effect on their navigation of learning management systems (LMS) and overall academic performance. The frequency and nature of LMS interactivity were examined across four first-year STEM courses offered in the same semester at a single institution, using a K-means cluster analysis to group student responses. It was observed that high achieving students accessed LMS resources more frequently than mid or low-achieving students across all four STEM courses. Students' perceived level of digital literacy was collected via survey (n = 282), and students were sorted high (n = 106) and low-level (n = 176) of perceived digital literacy-HDL and LDL, respectively. HDL students were not consistently found in the high-achieving academic group and did not perform better in their overall grade when compared to LDL students. LDL students were observed to perform better in specific online assessment tasks, which may be attributed to their increased frequency of LMS interactivity. These findings highlight the delicate balance between students' perceived level of digital literacy, motivation for engaging with online learning environments, and academic performance.

CRISPR-Cas9 editing of CAFFEOYL SHIKIMATE ESTERASE 1 and 2 shows their importance and partial redundancy in lignification in Populus tremula × P. alba.

Lignins are cell wall-located aromatic polymers that provide strength and hydrophobicity to woody tissues. Lignin monomers are synthesized via the phenylpropanoid pathway, wherein CAFFEOYL SHIKIMATE ESTERASE (CSE) converts caffeoyl shikimate into caffeic acid. Here, we explored the role of the two CSE homologs in poplar (Populus tremula × P. alba). Reporter lines showed that the expression conferred by both CSE1 and CSE2 promoters is similar. CRISPR-Cas9-generated cse1 and cse2 single mutants had a wild-type lignin level. Nevertheless, CSE1 and CSE2 are not completely redundant, as both single mutants accumulated caffeoyl shikimate. In contrast, the cse1 cse2 double mutants had a 35% reduction in lignin and associated growth penalty. The reduced-lignin content translated into a fourfold increase in cellulose-to-glucose conversion upon limited saccharification. Phenolic profiling of the double mutants revealed large metabolic shifts, including an accumulation of p-coumaroyl, 5-hydroxyferuloyl, feruloyl and sinapoyl shikimate, in addition to caffeoyl shikimate. This indicates that the CSEs have a broad substrate specificity, which was confirmed by in vitro enzyme kinetics. Taken together, our results suggest an alternative path within the phenylpropanoid pathway at the level of the hydroxycinnamoyl-shikimates, and show that CSE is a promising target to improve plants for the biorefinery.

MYB Transcription Factor161 Mediates Feedback Regulation of Secondary wall-associated NAC-Domain1 Family Genes for Wood Formation.

Wood formation is a complex process that involves cell differentiation, cell expansion, secondary wall deposition, and programmed cell death. We constructed a four-layer wood formation transcriptional regulatory network (TRN) in Populus trichocarpa (black cottonwood) that has four Secondary wall-associated NAC-Domain1 (PtrSND1) transcription factor (TF) family members as the top-layer regulators. We characterized the function of a MYB (PtrMYB161) TF in this PtrSND1-TRN, using transgenic P trichocarpa cells and whole plants. PtrMYB161 is a third-layer regulator that directly transactivates five wood formation genes. Overexpression of PtrMYB161 in P. trichocarpa (OE-PtrMYB161) led to reduced wood, altered cell type proportions, and inhibited growth. Integrative analysis of wood cell-based chromatin-binding assays with OE-PtrMYB161 transcriptomics revealed a feedback regulation system in the PtrSND1-TRN, where PtrMYB161 represses all four top-layer regulators and one second-layer regulator, PtrMYB021, possibly affecting many downstream TFs in, and likely beyond, the TRN, to generate the observed phenotypic changes. Our data also suggested that the PtrMYB161's repressor function operates through interaction of the base PtrMYB161 target-binding system with gene-silencing cofactors. PtrMYB161 protein does not contain any known negative regulatory domains. CRISPR-based mutants of PtrMYB161 in P. trichocarpa exhibited phenotypes similar to the wild type, suggesting that PtrMYB161's activator functions are redundant among many TFs. Our work demonstrated that PtrMYB161 binds to multiple sets of target genes, a feature that allows it to function as an activator as well as a repressor. The balance of the two functions may be important to the establishment of regulatory homeostasis for normal growth and development.

Modeling cross-regulatory influences on monolignol transcripts and proteins under single and combinatorial gene knockdowns in Populus trichocarpa.

Accurate manipulation of metabolites in monolignol biosynthesis is a key step for controlling lignin content, structure, and other wood properties important to the bioenergy and biomaterial industries. A crucial component of this strategy is predicting how single and combinatorial knockdowns of monolignol specific gene transcripts influence the abundance of monolignol proteins, which are the driving mechanisms of monolignol biosynthesis. Computational models have been developed to estimate protein abundances from transcript perturbations of monolignol specific genes. The accuracy of these models, however, is hindered by their inability to capture indirect regulatory influences on other pathway genes. Here, we examine the manifestation of these indirect influences on transgenic transcript and protein abundances, identifying putative indirect regulatory influences that occur when one or more specific monolignol pathway genes are perturbed. We created a computational model using sparse maximum likelihood to estimate the resulting monolignol transcript and protein abundances in transgenic Populus trichocarpa based on targeted knockdowns of specific monolignol genes. Using in-silico simulations of this model and root mean square error, we showed that our model more accurately estimated transcript and protein abundances, in comparison to previous models, when individual and families of monolignol genes were perturbed. We leveraged insight from the inferred network structure obtained from our model to identify potential genes, including PtrHCT, PtrCAD, and Ptr4CL, involved in post-transcriptional and/or post-translational regulation. Our model provides a useful computational tool for exploring the cascaded impact of single and combinatorial modifications of monolignol specific genes on lignin and other wood properties.

Simple Organic Carbon Sources and High Diversity Inocula Enhance Microbial Bioneutralization of Alkaline Bauxite Residues.

Bioneutralization of pH by microbial fermentation of added carbon substrates is a promising new method for remediation of the 1.7 GT/yr of alkaline mining tailings produced globally. Here, we present the first study to systematically compare and optimize the efficacy of microbial inocula of varying diversities, structures, and provenance and organic carbon substrates of varying complexities on the rate and extent of pH bioneutralization in alkaline bauxite residue tailings. Laboratory-scale bioreactors inoculated with soda lake sediments or with monosaccharide substrates added had a significantly lower minimum pH (<8) and a significantly higher maximum rate of pH neutralization (>0.02 µmol H+ day-1) and achieved these in significantly less time (<26 days) compared to bioreactors with other inocula or substrates. The soda lake sediment introduced a significantly higher-diversity microbial community with a distinct structure (dominated by Euryarchaeota and Bacteroidetes, rather than Acidobacteria and Actinobacteria), supporting higher acetate and formate-yielding fermentation pathways compared to other inocula. The strong performance of monosaccharides is attributed to widespread microbial capacity for efficient fermentation. Using either monosaccharide carbon substrates or soda lake sediments is recommended to maximize bioneutralization efficiency at the industrial scale.

Clinicopathological features and outcome of esophageal neuroendocrine tumor: A retrospective multicenter survey by the digestive endoscopy society of Taiwan.

BACKGROUND & AIMS: Esophageal neuroendocrine tumors (NET) are very rare and mostly carcinomic, carrying poor prognosis. There is still no guideline or consensus on the treatment for esophageal NET. METHODS: Patients with histologically-proven esophageal neuroendocrine tumor were recruited from 9 hospitals in Taiwan between 2002 and 2017. Clinical, laboratory, radiological, endoscopic, pathological data, treatment strategies, follow-up periods, and survivals were collected retrospectively. RESULTS: In total, 39 esophageal NET were analyzed and 38 were neuroendocrine carcinoma (NEC). Sixteen (41%) patients had mixed components with either adenocarcinoma (N = 9, 23%) or squamous-cell carcinoma (SCC) (N = 7, 18%). 64.1% of the patients experienced dysphagia and ulcerative mass was the most comment endoscopic finding. There was a higher proportion of drinkers (54.1%), betel chewers (21.6%) and smokers (64.9%) among the patients than in the general population in Taiwan. Five patients (12.8%) had been diagnosed with other cancers. Definite chemoradiotherapy (N = 14, 35.9%) and surgery (N = 7, 17.9%) were the major treatment. Patients with Ki-67% above the median level (50%) in the tumors tended to have worse survival (P = 0.06). However, presence of mixed component was not a significant survival predictor in our study (P = 0.56). CONCLUSION: Mixed component of an esophageal NET is commonly observed. Staged workup and the principle of treatment can follow that for the common cancer type of esophagus. The risk factors and behaviors of esophageal NEC in Taiwan seem to be similar to that of esophageal SCC.

Reciprocal cross-regulation of VND and SND multigene TF families for wood formation in Populus trichocarpa.

Secondary cell wall (SCW) biosynthesis is the biological process that generates wood, an important renewable feedstock for materials and energy. NAC domain transcription factors, particularly Vascular-Related NAC-Domain (VND) and Secondary Wall-Associated NAC Domain (SND) proteins, are known to regulate SCW differentiation. The regulation of VND and SND is important to maintain homeostasis for plants to avoid abnormal growth and development. We previously identified a splice variant, PtrSND1-A2IR , derived from PtrSND1-A2 as a dominant-negative regulator, which suppresses the transactivation of all PtrSND1 family members. PtrSND1-A2IR also suppresses the self-activation of the PtrSND1 family members except for its cognate transcription factor, PtrSND1-A2, suggesting the existence of an unknown factor needed to regulate PtrSND1-A2 Here, a splice variant, PtrVND6-C1IR , derived from PtrVND6-C1 was discovered that suppresses the protein functions of all PtrVND6 family members. PtrVND6-C1IR also suppresses the expression of all PtrSND1 members, including PtrSND1-A2, demonstrating that PtrVND6-C1IR is the previously unidentified regulator of PtrSND1-A2 We also found that PtrVND6-C1IR cannot suppress the expression of its cognate transcription factor, PtrVND6-C1PtrVND6-C1 is suppressed by PtrSND1-A2IR Both PtrVND6-C1IR and PtrSND1-A2IR cannot suppress their cognate transcription factors but can suppress all members of the other family. The results indicate that the splice variants from the PtrVND6 and PtrSND1 family may exert reciprocal cross-regulation for complete transcriptional regulation of these two families in wood formation. This reciprocal cross-regulation between families suggests a general mechanism among NAC domain proteins and likely other transcription factors, where intron-retained splice variants provide an additional level of regulation.

Calibration of free-space and fiber-coupled single-photon detectors.

We measure the detection efficiency of single-photon detectors at wavelengths near 851 nm and 1533.6 nm. We investigate the spatial uniformity of one free-space-coupled single-photon avalanche diode and present a comparison between fusion-spliced and connectorized fiber-coupled single-photon detectors. We find that our expanded relative uncertainty for a single measurement of the detection efficiency is as low as 0.70% for fiber-coupled measurements at 1533.6 nm and as high as 1.78% for our free-space characterization at 851.7 nm. The detection-efficiency determination includes corrections for afterpulsing, dark count, and count-rate effects of the single-photon detector with the detection efficiency interpolated to operation at a specified detected count rate.

CAD1 and CCR2 protein complex formation in monolignol biosynthesis in Populus trichocarpa.

Lignin is the major phenolic polymer in plant secondary cell walls and is polymerized from monomeric subunits, the monolignols. Eleven enzyme families are implicated in monolignol biosynthesis. Here, we studied the functions of members of the cinnamyl alcohol dehydrogenase (CAD) and cinnamoyl-CoA reductase (CCR) families in wood formation in Populus trichocarpa, including the regulatory effects of their transcripts and protein activities on monolignol biosynthesis. Enzyme activity assays from stem-differentiating xylem (SDX) proteins showed that RNAi suppression of PtrCAD1 in P. trichocarpa transgenics caused a reduction in SDX CCR activity. RNAi suppression of PtrCCR2, the only CCR member highly expressed in SDX, caused a reciprocal reduction in SDX protein CAD activities. The enzyme assays of mixed and coexpressed recombinant proteins supported physical interactions between PtrCAD1 and PtrCCR2. Biomolecular fluorescence complementation and pull-down/co-immunoprecipitation experiments supported a hypothesis of PtrCAD1/PtrCCR2 heterodimer formation. These results provide evidence for the formation of PtrCAD1/PtrCCR2 protein complexes in monolignol biosynthesis in planta.

Induction of microvascular network growth in the mouse mesentery.

OBJECTIVE: Motivated by observations of mesenteries harvested from mice treated with tamoxifen dissolved in oil for inducible gene mutation studies, the objective of this study was to demonstrate that microvascular growth can be induced in the avascular mouse mesentery tissue. METHODS: C57BL/6 mice were administered an IP injection for five consecutive days of: saline, sunflower oil, tamoxifen dissolved in sunflower oil, corn oil, or peanut oil. RESULTS: Twenty-one days post-injection, zero tissues from saline group contained branching microvascular networks. In contrast, all tissues from the three oils and tamoxifen groups contained vascular networks with arterioles, venules, and capillaries. Smooth muscle cells and pericytes were present in their expected locations and wrapping morphologies. Significant increases in vascularized tissue area and vascular density were observed when compared to saline group, but sunflower oil and tamoxifen group were not significantly different. Vascularized tissues also contained LYVE-1-positive and Prox1-positive lymphatic networks, indicating that lymphangiogenesis was stimulated. When comparing the different oils, vascularized tissue area and vascular density of sunflower oil were significantly higher than corn and peanut oils. CONCLUSIONS: These results provide novel evidence supporting that induction of microvascular network growth into the normally avascular mouse mesentery is possible.

Local axonal protection by WldS as revealed by conditional regulation of protein stability.

The expression of the mutant Wallerian degeneration slow (WldS) protein significantly delays axonal degeneration from various nerve injuries and in multiple species; however, the mechanism for its axonal protective property remains unclear. Although WldS is localized predominantly in the nucleus, it also is present in a smaller axonal pool, leading to conflicting models to account for the WldS fraction necessary for axonal protection. To identify where WldS activity is required to delay axonal degeneration, we adopted a method to alter the temporal expression of WldS protein in neurons by chemically regulating its protein stability. We demonstrate that continuous WldS activity in the axonal compartment is both necessary and sufficient to delay axonal degeneration. Furthermore, by specifically increasing axonal WldS expression postaxotomy, we reveal a critical period of 4-5 h postinjury during which the course of Wallerian axonal degeneration can be halted. Finally, we show that NAD(+), the metabolite of WldS/nicotinamide mononucleotide adenylyltransferase enzymatic activity, is sufficient and specific to confer WldS-like axon protection and is a likely molecular mediator of WldS axon protection. The results delineate a therapeutic window in which the course of Wallerian degeneration can be delayed even after injures have occurred and help narrow the molecular targets of WldS activity to events within the axonal compartment.

Phosphorylation is an on/off switch for 5-hydroxyconiferaldehyde O-methyltransferase activity in poplar monolignol biosynthesis.

Although phosphorylation has long been known to be an important regulatory modification of proteins, no unequivocal evidence has been presented to show functional control by phosphorylation for the plant monolignol biosynthetic pathway. Here, we present the discovery of phosphorylation-mediated on/off regulation of enzyme activity for 5-hydroxyconiferaldehyde O-methyltransferase 2 (PtrAldOMT2), an enzyme central to monolignol biosynthesis for lignification in stem-differentiating xylem (SDX) of Populus trichocarpa. Phosphorylation turned off the PtrAldOMT2 activity, as demonstrated in vitro by using purified phosphorylated and unphosphorylated recombinant PtrAldOMT2. Protein extracts of P. trichocarpa SDX, which contains endogenous kinases, also phosphorylated recombinant PtrAldOMT2 and turned off the recombinant protein activity. Similarly, ATP/Mn(2+)-activated phosphorylation of SDX protein extracts reduced the endogenous SDX PtrAldOMT2 activity by ∼ 60%, and dephosphorylation fully restored the activity. Global shotgun proteomic analysis of phosphopeptide-enriched P. trichocarpa SDX protein fractions identified PtrAldOMT2 monophosphorylation at Ser(123) or Ser(125) in vivo. Phosphorylation-site mutagenesis verified the PtrAldOMT2 phosphorylation at Ser(123) or Ser(125) and confirmed the functional importance of these phosphorylation sites for O-methyltransferase activity. The PtrAldOMT2 Ser(123) phosphorylation site is conserved across 93% of AldOMTs from 46 diverse plant species, and 98% of the AldOMTs have either Ser(123) or Ser(125). PtrAldOMT2 is a homodimeric cytosolic enzyme expressed more abundantly in syringyl lignin-rich fiber cells than in guaiacyl lignin-rich vessel cells. The reversible phosphorylation of PtrAldOMT2 is likely to have an important role in regulating syringyl monolignol biosynthesis of P. trichocarpa.

Tissue and cell-type co-expression networks of transcription factors and wood component genes in Populus trichocarpa.

MAIN CONCLUSION: Co-expression networks based on transcriptomes of Populus trichocarpa major tissues and specific cell types suggest redundant control of cell wall component biosynthetic genes by transcription factors in wood formation. We analyzed the transcriptomes of five tissues (xylem, phloem, shoot, leaf, and root) and two wood forming cell types (fiber and vessel) of Populus trichocarpa to assemble gene co-expression subnetworks associated with wood formation. We identified 165 transcription factors (TFs) that showed xylem-, fiber-, and vessel-specific expression. Of these 165 TFs, 101 co-expressed (correlation coefficient, r > 0.7) with the 45 secondary cell wall cellulose, hemicellulose, and lignin biosynthetic genes. Each cell wall component gene co-expressed on average with 34 TFs, suggesting redundant control of the cell wall component gene expression. Co-expression analysis showed that the 101 TFs and the 45 cell wall component genes each has two distinct groups (groups 1 and 2), based on their co-expression patterns. The group 1 TFs (44 members) are predominantly xylem and fiber specific, and are all highly positively co-expressed with the group 1 cell wall component genes (30 members), suggesting their roles as major wood formation regulators. Group 1 TFs include a lateral organ boundary domain gene (LBD) that has the highest number of positively correlated cell wall component genes (36) and TFs (47). The group 2 TFs have 57 members, including 14 vessel-specific TFs, and are generally less correlated with the cell wall component genes. An exception is a vessel-specific basic helix-loop-helix (bHLH) gene that negatively correlates with 20 cell wall component genes, and may function as a key transcriptional suppressor. The co-expression networks revealed here suggest a well-structured transcriptional homeostasis for cell wall component biosynthesis during wood formation.