Warm springs alter timing but not total growth of temperate deciduous trees.

As the climate changes, warmer spring temperatures are causing earlier leaf-out1-3 and commencement of CO2 uptake1,3 in temperate deciduous forests, resulting in a tendency towards increased growing season length3 and annual CO2 uptake1,3-7. However, less is known about how spring temperatures affect tree stem growth8,9, which sequesters carbon in wood that has a long residence time in the ecosystem10,11. Here we show that warmer spring temperatures shifted stem diameter growth of deciduous trees earlier but had no consistent effect on peak growing season length, maximum growth rates, or annual growth, using dendrometer band measurements from 440 trees across two forests. The latter finding was confirmed on the centennial scale by 207 tree-ring chronologies from 108 forests across eastern North America, where annual ring width was far more sensitive to temperatures during the peak growing season than in the spring. These findings imply that any extra CO2 uptake in years with warmer spring temperatures4,5 does not significantly contribute to increased sequestration in long-lived woody stem biomass. Rather, contradicting projections from global carbon cycle models1,12, our empirical results imply that warming spring temperatures are unlikely to increase woody productivity enough to strengthen the long-term CO2 sink of temperate deciduous forests.

A transcriptome-based association study of growth, wood quality, and oleoresin traits in a slash pine breeding population.

Slash pine (Pinus elliottii Engelm.) is an important timber and resin species in the United States, China, Brazil and other countries. Understanding the genetic basis of these traits will accelerate its breeding progress. We carried out a genome-wide association study (GWAS), transcriptome-wide association study (TWAS) and weighted gene co-expression network analysis (WGCNA) for growth, wood quality, and oleoresin traits using 240 unrelated individuals from a Chinese slash pine breeding population. We developed high quality 53,229 single nucleotide polymorphisms (SNPs). Our analysis reveals three main results: (1) the Chinese breeding population can be divided into three genetic groups with a mean inbreeding coefficient of 0.137; (2) 32 SNPs significantly were associated with growth and oleoresin traits, accounting for the phenotypic variance ranging from 12.3% to 21.8% and from 10.6% to 16.7%, respectively; and (3) six genes encoding PeTLP, PeAP2/ERF, PePUP9, PeSLP, PeHSP, and PeOCT1 proteins were identified and validated by quantitative real time polymerase chain reaction for their association with growth and oleoresin traits. These results could be useful for tree breeding and functional studies in advanced slash pine breeding program.

Increased wood biomass growth is associated with lower wood density in Quercus petraea (Matt.) Liebl. saplings growing under elevated CO2.

Atmospheric carbon dioxide (CO2) has increased substantially since the industrial revolution began, and physiological responses to elevated atmospheric CO2 concentrations reportedly alter the biometry and wood structure of trees. Additionally, soil nutrient availability may play an important role in regulating these responses. Therefore, in this study, we grew 288 two-year-old saplings of sessile oak (Quercus petraea (Matt.) Liebl.) in lamellar glass domes for three years to evaluate the effects of CO2 concentrations and nutrient supply on above- and belowground biomass, wood density, and wood structure. Elevated CO2 increased above- and belowground biomass by 44.3% and 46.9%, respectively. However, under elevated CO2 treatment, sapling wood density was markedly lower (approximately 1.7%), and notably wider growth rings-and larger, more efficient conduits leading to increased hydraulic conductance-were observed. Moreover, despite the vessels being larger in saplings under elevated CO2, the vessels were significantly fewer (p = 0.023). No direct effects of nutrient supply were observed on biomass growth, wood density, or wood structure, except for a notable decrease in specific leaf area. These results suggest that, although fewer and larger conduits may render the xylem more vulnerable to embolism formation under drought conditions, the high growth rate in sessile oak saplings under elevated CO2 is supported by an efficient vascular system and may increase biomass production in this tree species. Nevertheless, the decreased mechanical strength, indicated by low density and xylem vulnerability to drought, may lead to earlier mortality, offsetting the positive effects of elevated CO2 levels in the future.

Wood Formation under Severe Drought Invokes Adjustment of the Hormonal and Transcriptional Landscape in Poplar.

Drought is a severe environmental stress that exerts negative effects on plant growth. In trees, drought leads to reduced secondary growth and altered wood anatomy. The mechanisms underlying wood stress adaptation are not well understood. Here, we investigated the physiological, anatomical, hormonal, and transcriptional responses of poplar to strong drought. Drought-stressed xylem was characterized by higher vessel frequencies, smaller vessel lumina, and thicker secondary fiber cell walls. These changes were accompanied by strong increases in abscisic acid (ABA) and antagonistic changes in salicylic acid in wood. Transcriptional evidence supported ABA biosynthesis and signaling in wood. Since ABA signaling activates the fiber-thickening factor NST1, we expected upregulation of the secondary cell wall (SCW) cascade under stress. By contrast, transcription factors and biosynthesis genes for SCW formation were down-regulated, whereas a small set of cellulose synthase-like genes and a huge array of genes involved in cell wall modification were up-regulated in drought-stressed wood. Therefore, we suggest that ABA signaling monitors normal SCW biosynthesis and that drought causes a switch from normal to "stress wood" formation recruiting a dedicated set of genes for cell wall biosynthesis and remodeling. This proposition implies that drought-induced changes in cell wall properties underlie regulatory mechanisms distinct from those of normal wood.

PopulusPtERF85 Balances Xylem Cell Expansion and Secondary Cell Wall Formation in Hybrid Aspen.

Secondary growth relies on precise and specialized transcriptional networks that determine cell division, differentiation, and maturation of xylem cells. We identified a novel role for the ethylene-induced Populus Ethylene Response Factor PtERF85 (Potri.015G023200) in balancing xylem cell expansion and secondary cell wall (SCW) formation in hybrid aspen (Populus tremula x tremuloides). Expression of PtERF85 is high in phloem and cambium cells and during the expansion of xylem cells, while it is low in maturing xylem tissue. Extending PtERF85 expression into SCW forming zones of woody tissues through ectopic expression reduced wood density and SCW thickness of xylem fibers but increased fiber diameter. Xylem transcriptomes from the transgenic trees revealed transcriptional induction of genes involved in cell expansion, translation, and growth. The expression of genes associated with plant vascular development and the biosynthesis of SCW chemical components such as xylan and lignin, was down-regulated in the transgenic trees. Our results suggest that PtERF85 activates genes related to xylem cell expansion, while preventing transcriptional activation of genes related to SCW formation. The importance of precise spatial expression of PtERF85 during wood development together with the observed phenotypes in response to ectopic PtERF85 expression suggests that PtERF85 contributes to the transition of fiber cells from elongation to secondary cell wall deposition.

Manipulating phloem transport affects wood formation but not local nonstructural carbon reserves in an evergreen conifer.

How variations in carbon supply affect wood formation remains poorly understood in particular in mature forest trees. To elucidate how carbon supply affects carbon allocation and wood formation, we attempted to manipulate carbon supply to the cambial region by phloem girdling and compression during the mid- and late-growing season and measured effects on structural development, CO2 efflux and nonstructural carbon reserves in stems of mature white pines. Wood formation and stem CO2 efflux varied with a location relative to treatment (i.e., above or below the restriction). We observed up to twice as many tracheids formed above versus below the treatment after the phloem transport manipulation, whereas the cell-wall area decreased only slightly below the treatments, and cell size did not change relative to the control. Nonstructural carbon reserves in the xylem, needles and roots were largely unaffected by the treatments. Our results suggest that low and high carbon supply affects wood formation, primarily through a strong effect on cell proliferation, and respiration, but local nonstructural carbon concentrations appear to be maintained homeostatically. This contrasts with reports of decoupling of source activity and wood formation at the whole-tree or ecosystem level, highlighting the need to better understand organ-specific responses, within-tree feedbacks, as well as phenological and ontogenetic effects on sink-source dynamics.

MYB Transcription Factors and Its Regulation in Secondary Cell Wall Formation and Lignin Biosynthesis during Xylem Development.

The secondary wall is the main part of wood and is composed of cellulose, xylan, lignin, and small amounts of structural proteins and enzymes. Lignin molecules can interact directly or indirectly with cellulose, xylan and other polysaccharide molecules in the cell wall, increasing the mechanical strength and hydrophobicity of plant cells and tissues and facilitating the long-distance transportation of water in plants. MYBs (v-myb avian myeloblastosis viral oncogene homolog) belong to one of the largest superfamilies of transcription factors, the members of which regulate secondary cell-wall formation by promoting/inhibiting the biosynthesis of lignin, cellulose, and xylan. Among them, MYB46 and MYB83, which comprise the second layer of the main switch of secondary cell-wall biosynthesis, coordinate upstream and downstream secondary wall synthesis-related transcription factors. In addition, MYB transcription factors other than MYB46/83, as well as noncoding RNAs, hormones, and other factors, interact with one another to regulate the biosynthesis of the secondary wall. Here, we discuss the biosynthesis of secondary wall, classification and functions of MYB transcription factors and their regulation of lignin polymerization and secondary cell-wall formation during wood formation.

A computer-aided method for identifying the presence of softwood growth ring boundaries.

The objective of this study was to develop a computer-aided method to quantify the obvious degree of growth ring boundaries of softwood species, based on data analysis with some image processing technologies. For this purpose, a 5× magnified cross-section color micro-image of softwood was cropped into 20 sub-images, and then every image was binarized as a gray image according to an automatic threshold value. After that, the number of black pixels in the gray image was counted row by row and the number of black pixels was binarized to 0 or 100. Finally, a transition band from earlywood to latewood on the sub-image was identified. If everything goes as planned, the growth ring boundaries of the sub-image would be distinct. Otherwise would be indistinct or absent. If more than 50% sub-images are distinct, with the majority voting method, the growth ring boundaries of softwood would be distinct, otherwise would be indistinct or absent. The proposed method has been visualized as a growth-ring-boundary detecting system based on the .NET Framework. A sample of 100 micro-images (see S1 Fig via https://github.com/senly2019/Lin-Qizhao/) of softwood cross-sections were selected for evaluation purposes. In short, this detecting system computes the obvious degree of growth ring boundaries of softwood species by image processing involving image importing, image cropping, image reading, image grayscale, image binarization, data analysis. The results showed that the method used avoided mistakes made by the manual comparison method of identifying the presence of growth ring boundaries, and it has a high accuracy of 98%.

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.

Multi-volume modeling of Eucalyptus trees using regression and artificial neural networks.

The stem volume of commercial trees is an important variable that assists in decision making and economic analysis in forest management. Wood from forest plantations can be used for several purposes, which makes estimating multi-volumes for the same tree a necessary task. Defining its exploitation and use potential, such as the total and merchantable volumes (up to a minimum diameter of interest), with or without bark, is a possible work. The goal of this study was to use different strategies to model multi-volumes of the stem of eucalyptus trees. The data came from rigorous scaling of 460 felled trees stems from four eucalyptus clones in high forest and coppice regimes. The diameters were measured at different heights, with the volume of the sections obtained by the Smalian method. Data were randomly separated into fit and validation data. The single multi-volume model, volume-specific models, and the training of artificial neural networks (ANNs) were fitted. The evaluation criteria of the models were: coefficient of determination, root mean square error, mean absolute error, mean bias error, as well as graphical analysis of observed and estimated values and distribution of residuals. Additionally, the t-test (α = 0.05) was performed between the volume obtained in the rigorous scaling and estimated by each strategy with the validation data. Results showed that the strategies used to model different tree stem volumes are efficient. The actual and estimated volumes showed no differences. The multi-volume model had the most considerable advantage in volume estimation practicality, while the volume-specific models were more efficient in the accuracy of estimates. Given the conditions of this study, the ANNs are more suitable than the regression models in the estimation of multi-volumes of eucalyptus trees, revealing greater accuracy and practicality.

Damage severity of wood-destroying insects according to the Bevan damage classification system in log depots of Northwest Turkey.

The aim of the study was to determine damage severity of wood-destroying insects on logs stored in forest depots. The Bevan damage classification (BDC) system, developed in 1987, was utilized to determine damage severity in log depots in 21 locations throughout seven provinces in Turkey. Pheromone traps were placed in those locations at the beginning of April in 2015 and 2016. Furthermore some stored wood within the log depots were checked and split into small pieces to collect insects that damage wood. The BDC system was used for the first time to measure the severity of insect damage in log depots. Twenty-eight families, 104 genera and 123 species were identified in this study. Based on the BDC system, the highest damage was found from the Cerambycidae and Buprestidae families. Arhopalus rusticus was determined as the insect responsible for the highest amount of damage with 8.8% severity rating in the pheromone-trapped insects group. When the stored wood material was considered, Hylotrupes bajulus was found to be the cause of the highest damage. The lowest damage values were among the predator insects (Cleridae, Trogossitidae, Cantharidae) and those feeding on fungi colonized on the wood (Mordellidae, Cerylonidae, Nitidulidae). Some other predator insects of the Tenebrionidae family (Uloma cypraea, Uloma culinaris, Menephilus cylindricus) and Elateridae family (Lacon punctatus, Ampedus sp.) exhibited relatively higher damage severity values since they had built tunnels and made holes in the stored wood material. When the environmental factors were considered, the Buprestidae family exhibited a very strong positive relationship (p < 0.005) with insect frequency distribution (r = 0.922), number of species (r = 0.879) and insect density (r = 0.942). Both families showed the highest number and frequency during July and August, highlighting the importance of insect control and management during these months.

Photoperiod and temperature as dominant environmental drivers triggering secondary growth resumption in Northern Hemisphere conifers.

Wood formation consumes around 15% of the anthropogenic CO2 emissions per year and plays a critical role in long-term sequestration of carbon on Earth. However, the exogenous factors driving wood formation onset and the underlying cellular mechanisms are still poorly understood and quantified, and this hampers an effective assessment of terrestrial forest productivity and carbon budget under global warming. Here, we used an extensive collection of unique datasets of weekly xylem tissue formation (wood formation) from 21 coniferous species across the Northern Hemisphere (latitudes 23 to 67°N) to present a quantitative demonstration that the onset of wood formation in Northern Hemisphere conifers is primarily driven by photoperiod and mean annual temperature (MAT), and only secondarily by spring forcing, winter chilling, and moisture availability. Photoperiod interacts with MAT and plays the dominant role in regulating the onset of secondary meristem growth, contrary to its as-yet-unquantified role in affecting the springtime phenology of primary meristems. The unique relationships between exogenous factors and wood formation could help to predict how forest ecosystems respond and adapt to climate warming and could provide a better understanding of the feedback occurring between vegetation and climate that is mediated by phenology. Our study quantifies the role of major environmental drivers for incorporation into state-of-the-art Earth system models (ESMs), thereby providing an improved assessment of long-term and high-resolution observations of biogeochemical cycles across terrestrial biomes.

MicroRNA comparison between poplar and larch provides insight into the different mechanism of wood formation.

KEY MESSAGE: MiRNA transcriptome analysis of different tissues in poplar and larch suggests variant roles of miRNAs in regulating wood formation between two kinds of phyla. Poplar and larch belong to two different phyla. Both are ecological woody species and major resources for wood-related industrial applications. However, wood properties are different between these two species and the molecular basis is largely unknown. In this study, we performed high-throughput sequencing of microRNAs (miRNAs) in the three tissues, xylem, phloem and leaf of Populus alba × Populus glandulosa and Larix kaempferi. Differentially expressed miRNA (DEmiRNA) analysis identified 85 xylem-specific miRNAs in P. alba × P. glandulosa and 158 xylem-specific miRNAs in L. kaempferi. Among 36 common miRNAs, 12 were conserved between the two species. GO and KEGG analyses of the miRNA target genes showed similar metabolism in two species. Through KEGG and BLASTN, we predicted target genes of xylem differentially expressed (DEmiRNA) in the wood formation-related pathways and located DEmiRNAs in these pathways. A network was built for wood formation-related DEmiRNAs, their target genes and orthologous genes in Arabidopsis thaliana. Comparison of DEmiRNA and target gene annotation between P. alba × P. glandulosa and L. kaempferi suggested the different functions of DEmiRNAs and divergent mechanism in wood formation between two species, providing knowledge to understand wood formation mechanism in gymnosperm and angiosperm woody plants.

Growth Features of Castanea sativa Mill. in Forest Stands Infected by Cryphoneciria parasitica (Murrill) М. Е. Barr. in Abkhazia.

Currently, the forest plantations of Castanea sativa Mill. are largely damaged by phytopathogenic organisms, of which Cryphoneciria parasitica (Murrill) М. Е. Barr. has the most negative impact. The spread of this type of disease reduces the vital state and significantly reduces the life expectancy of C. sativa trees. For the first time, using dendrochronological analysis, the specificity of distribution and degree of phytopathogenic damage to C. sativa trunk tissues were assessed. It was revealed that in its lower part the dieback of the peripheral layers of wood occurs fairly evenly with a total degradation of cellular structures in the amount of 18-20%. In the middle segments of the trunk this figure increases almost twice. And the greatest degree of wood dieback is observed in the upper part of damaged trees. It was found that climatic changes of the last decades determine the strengthening of destructive processes in the woodland of C. sativa of the Caucasus.

Effect of number of annual rings and tree ages on genomic predictive ability for solid wood properties of Norway spruce.

BACKGROUND: Genomic selection (GS) or genomic prediction is considered as a promising approach to accelerate tree breeding and increase genetic gain by shortening breeding cycle, but the efforts to develop routines for operational breeding are so far limited. We investigated the predictive ability (PA) of GS based on 484 progeny trees from 62 half-sib families in Norway spruce (Picea abies (L.) Karst.) for wood density, modulus of elasticity (MOE) and microfibril angle (MFA) measured with SilviScan, as well as for measurements on standing trees by Pilodyn and Hitman instruments. RESULTS: GS predictive abilities were comparable with those based on pedigree-based prediction. Marker-based PAs were generally 25-30% higher for traits density, MFA and MOE measured with SilviScan than for their respective standing tree-based method which measured with Pilodyn and Hitman. Prediction accuracy (PC) of the standing tree-based methods were similar or even higher than increment core-based method. 78-95% of the maximal PAs of density, MFA and MOE obtained from coring to the pith at high age were reached by using data possible to obtain by drilling 3-5 rings towards the pith at tree age 10-12. CONCLUSIONS: This study indicates standing tree-based measurements is a cost-effective alternative method for GS. PA of GS methods were comparable with those pedigree-based prediction. The highest PAs were reached with at least 80-90% of the dataset used as training set. Selection for trait density could be conducted at an earlier age than for MFA and MOE. Operational breeding can also be optimized by training the model at an earlier age or using 3 to 5 outermost rings at tree age 10 to 12 years, thereby shortening the cycle and reducing the impact on the tree.

Water lily (Nymphaea thermarum) genome reveals variable genomic signatures of ancient vascular cambium losses.

For more than 225 million y, all seed plants were woody trees, shrubs, or vines. Shortly after the origin of angiosperms ∼140 million y ago (MYA), the Nymphaeales (water lilies) became one of the first lineages to deviate from their ancestral, woody habit by losing the vascular cambium, the meristematic population of cells that produces secondary xylem (wood) and phloem. Many of the genes and gene families that regulate differentiation of secondary tissues also regulate the differentiation of primary xylem and phloem, which are produced by apical meristems and retained in nearly all seed plants. Here, we sequenced and assembled a draft genome of the water lily Nymphaea thermarum, an emerging system for the study of early flowering plant evolution, and compared it to genomes from other cambium-bearing and cambium-less lineages (e.g., monocots and Nelumbo). This revealed lineage-specific patterns of gene loss and divergence. Nymphaea is characterized by a significant contraction of the HD-ZIP III transcription factors, specifically loss of REVOLUTA, which influences cambial activity in other angiosperms. We also found the Nymphaea and monocot copies of cambium-associated CLE signaling peptides display unique substitutions at otherwise highly conserved amino acids. Nelumbo displays no obvious divergence in cambium-associated genes. The divergent genomic signatures of convergent loss of vascular cambium reveals that even pleiotropic genes can exhibit unique divergence patterns in association with independent events of trait loss. Our results shed light on the evolution of herbaceousness-one of the key biological innovations associated with the earliest phases of angiosperm evolution.

The effect of soil nutrients and moisture during ontogeny on apparent wood density of Eucalyptus grandis.

Knowledge of the effect of soil nutrients, such as K and Na and their interaction with water availability, on the growth and wood properties of the eucalypts, is needed to increase the productivity of commercial plantation forests in Brazil that generate employment and taxes. The present study evaluates the apparent wood density (at 12% wood moisture) of Eucalyptus grandis trees at 12, 24, 36 and 48 months old under ambient and lower than ambient (66%) rainfall conditions and K and Na nutrient enrichment. The treatments were two water availability (100 and 66% of the rainfall) and the three nutrient treatments were: K (4.5 kmol/ha), Na (4.5 kmol/ha) and a control (natural conditions). The apparent wood density of samples at breast height (1.3 m) was determined by X-ray densitometry and digital images. Increased nutrients at all four ages and water availability at 36 and 48 months reduced apparent wood density in E. grandis trees, however, effects of nutrients are lower under water availability reduction. The radial profile of wood density was higher in four-year-old trees, but there was radial variation in apparent wood density at all ages. These findings predict that, under water stress, apparent wood density will not decline in commercial E. grandis plantations fertilized with potassium. The use of sodium, as a substitute of potassium, should consider their negative impacts on wood density of Eucalyptus grandis trees.

Identification and characterization of circular RNAs during wood formation of poplars in acclimation to low nitrogen availability.

MAIN CONCLUSION: Circular RNA (circRNA) identification and expression profiles, and construction of circRNAs-miRNAs-mRNAs networks indicates that circRNAs are involved in wood formation of poplars in acclimation to low nitrogen availability. Circular RNAs (circRNAs) are covalently closed non-coding RNAs that play pivotal roles in various biological processes. However, circRNAs' roles in wood formation of poplars in acclimation to low nitrogen (N) availability are currently unknown. Here, we undertook a systematic identification and characterization of circRNAs in the wood of Populus × canescens exposed to either 50 (low N) or 500 (normal N) µM NH4NO3 using rRNA-depleted RNA-sequencing. A total of 2,509 unique circRNAs were identified, and 163 (ca. 6.5%) circRNAs were significantly differentially expressed (DE) under low N condition. We observed a positive correlation between the expression patterns of DE circRNAs and their hosting protein-coding genes. Moreover, circRNAs-miRNAs-mRNAs' networks were identified in the wood of poplars under low N availability. For instance, upregulated several circRNAs, such as circRNA1226, circRNA 1732, and circRNA392 induced increases in nuclear factor Y, subunit A1-A (NFYA1-A), NFYA1-B, and NFYA10 transcript levels via the mediation of miR169b members, which is in line with reduced xylem width and cell layers of the xylem in the wood of low N-supplied poplars. Upregulation of circRNA1006, circRNA1344, circRNA1941, circRNA901, and circRNA146 caused increased transcript level of MYB61 via the mediation of a miR5021 member, corresponding well to the higher lignin concentration in the wood of low N-treated poplars. Overall, these results indicated that DE circRNAs play an essential role in regulating gene expression via circRNAs-miRNAs-mRNAs' networks to modulate wood anatomical and chemical properties of poplars in acclimation to low N availability.

KNAT2/6b, a class I KNOX gene, impedes xylem differentiation by regulating NAC domain transcription factors in poplar.

Wood formation is the terminal differentiation of xylem mother cells derived from cambial initials, and negative regulators play important roles in xylem differentiation. The molecular mechanism of the negative regulator of xylem differentiation PagKNAT2/6b was investigated. PagKNAT2/6b is an ortholog of Arabidopsis KNAT2 and KNAT6 that is highly expressed in phloem and xylem. Compared to nontransgenic control plants, transgenic poplar plants overexpressing PagKNAT2/6b present with altered vascular patterns, characterized by decreased secondary xylem with thin cell walls containing less cellulose, xylose and lignin. RNA sequencing analyses revealed that differentially expressed genes are enriched in xylem differentiation and secondary wall synthesis functions. Expression of NAM/ATAF/CUC (NAC) domain genes including PagSND1-A1, PagSND1-A2, PagSND1-B2 and PagVND6-C1 is downregulated by PagKNAT2/6b, while PagXND1a is directly upregulated. Accordingly, the dominant repression form of PagKNAT2/6b leads to increased xylem width per stem diameter through downregulation of PagXND1a. PagKNAT2/6b can inhibit cell differentiation and secondary wall deposition during wood formation in poplar by modulating the expression of NAC domain transcription factors. Direct activation of PagXND1a by PagKNAT2/6b is a key node in the negative regulatory network of xylem differentiation by KNOXs.

Dual regulation of xylem formation by an auxin-mediated PaC3H17-PaMYB199 module in Populus.

Wood (secondary xylem) formation in tree species is dependent on auxin-mediated vascular cambium activity in stems. However, the complex regulatory networks underlying xylem formation remain elusive. Xylem development in Populus was characterized based on microscopic observations of stem sections in transgenic plants. Transcriptomic, quantitative real-time PCR, chromatin immunoprecipitation PCR, and electrophoretic mobility shift assay analyses were conducted to identify target genes involved in xylem development. Yeast two-hybrid, pull-down, bimolecular fluorescence complementation, and co-immunoprecipitation assays were used to validate protein-protein interactions. PaC3H17 and its target PaMYB199 were found to be predominantly expressed in the vascular cambium and developing secondary xylem in Populus stems and play opposite roles in controlling cambial cell proliferation and secondary cell wall thickening through an overlapping pathway. Further, PaC3H17 interacts with PaMYB199 to form a complex, attenuating PaMYB199-driven suppression of its xylem targets. Exogenous auxin application enhances the dual control of the PaC3H17-PaMYB199 module during cambium division, thereby promoting secondary cell wall deposition. Dual regulation of xylem formation by an auxin-mediated PaC3H17-PaMYB199 module represents a novel regulatory mechanism in Populus, increasing our understanding of the regulatory networks involved in wood formation.