Secondary Wall Deposition Research Articles

Molecular understanding of wood formation in trees

Trees convert and store the majority of their photosynthetic products in wood which is an essential renewable resource much in demand by human society. Formation of wood follows a process of consecutive cell developmental stages, from vascular cambium proliferation, cell expansion and differentiation, secondary cell wall deposition to programmed cell death, which is controlled by the functionality of complex molecular networks. What are the molecular networks involved in wood formation? How do the molecular networks act in a way to generate wood tissue during tree growth? What are the regulatory modules that lead to the formation of various wood characteristics? The answers to these questions are fundamental to understanding how trees grow, as well as how we can genetically engineer trees with desired properties of wood for human needs. In recent years, a great deal of interest has been invested in the elucidation of wood formation at the molecular level. This review summarizes the current state of understanding of the molecular process that guides wood formation in trees.

Distinct and Overlapping Functions of Miscanthus sinensis MYB Transcription Factors SCM1 and MYB103 in Lignin Biosynthesis.

Cell wall recalcitrance is a major constraint for the exploitation of lignocellulosic biomass as a renewable resource for energy and bio-based products. Transcriptional regulators of the lignin biosynthetic pathway represent promising targets for tailoring lignin content and composition in plant secondary cell walls. However, knowledge about the transcriptional regulation of lignin biosynthesis in lignocellulosic feedstocks, such as Miscanthus, is limited. In Miscanthus leaves, MsSCM1 and MsMYB103 are expressed at growth stages associated with lignification. The ectopic expression of MsSCM1 and MsMYB103 in N. benthamiana leaves was sufficient to trigger secondary cell wall deposition with distinct sugar and lignin compositions. Moreover, RNA-seq analysis revealed that the transcriptional responses to MsSCM1 and MsMYB103 overexpression showed an extensive overlap with the response to the NAC master transcription factor MsSND1, but were distinct from each other, underscoring the inherent complexity of secondary cell wall formation. Furthermore, conserved and previously described promoter elements as well as novel and specific motifs could be identified from the target genes of the three transcription factors. Together, MsSCM1 and MsMYB103 represent interesting targets for manipulations of lignin content and composition in Miscanthus towards a tailored biomass.

Comparative Transcriptomic Analysis Identifies Key Cellulose Synthase Genes (CESA) and Cellulose Synthase-like Genes (CSL) in Fast Growth Period of Flax Stem (Linum Usitatissimum L.)

ABSTRACT The cellulose synthase gene superfamily is vital for cell wall biogenesis during plant growth, particularly for flax fiber development. This study performed asequencing search of key CESA and CSL genes from several flax stem parts at different fiber development stages by comparing RNA-Seq. Quantitative RT-PCR was used to validate the expression of these genes. This study revealed that CESA4 genes (Lus10008225.g and Lus10008226.g), CESA6 genes (Lus10006161.g and Lus10041063.g), CESA8 genes (Lus10007296.g and Lus10029245.g), CSLD4 gene (Lus10026568.g), CSLE1 (Lus10016625.g) and CSLG genes (Lus10023056.g and Lus10023057.g) were specifically expressed in stem below the snap point where fibers is increased amounts of secondary cell wall deposition. LusCESA4 genes, LusCESA8, genes and LusCSLD4 gene were specifically expressed in fiber development stage during the fast growth period of flax plants. Based on GO and KEGG analyses, it was found that genes involved in pathways of cellulose microfibril organization, galactosyl transferase activity and galactose metabolism were specifically enriched in the stem tissue of the fiber development stage. Other genes involved in cellulose biosynthesis and cell wall development were also analyzed and discussed. The results will provide an important foundation for understanding fiber cell wall biogenesis, particularly the roles of LusCESAs and LusCSLs in the process of fiber development.

The Effects of Turnip Mosaic Virus Infections on the Deposition of Secondary Cell Walls and Developmental Defects in Arabidopsis Plants Are Virus-Strain Specific.

Two isolates of Turnip mosaic virus (UK 1 and JPN 1), representative of two different viral strains, induced differential alterations on secondary cell wall (SCW) development in Arabidopsis thaliana, suggesting cell-type specific effects of these viral infections. These potential effects were analyzed in inflorescence stems and flowers of infected plants, together with other possible cellular effects of the infections. Results obtained from macroscopic and histochemical analyses showed that infection with either virus significantly narrowed stem area, but defects in SCW were only found in JPN 1 infections. In flowers, reduced endothecium lignification was also found for JPN 1, while UK 1 infections induced severe floral cell and organ development alterations. A transcriptomic analysis focused on genes controlling and regulating SCW formation also showed notable differences between both viral isolates. UK 1 infections induced a general transcriptional decrease of most regulatory genes, whereas a more complex pattern of alterations was found in JPN 1 infections. The role of the previously identified viral determinant of most developmental alterations, the P3 protein, was also studied through the use of viral chimeras. No SCW alterations or creeping habit growth were found in infections by the chimeras, indicating that if the P3 viral protein is involved in the determination of these symptoms, it is not the only determinant. Finally, considerations as to the possibility of a taxonomical reappraisal of these TuMV viral strains are provided.

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.

Functional Characterisation of the Poplar Atypical Aspartic Protease Gene PtAP66 in Wood Secondary Cell Wall Deposition

Secondary cell wall (SCW) deposition is an important process during wood formation. Although aspartic proteases (APs) have been reported to have regulatory roles in herbaceous plants, the involvement of atypical APs in SCW deposition in trees has not been reported. In this study, we characterised the Populus trichocarpa atypical AP gene PtAP66, which is involved in wood SCW deposition. Transcriptome data from the AspWood resource showed that in the secondary xylem of P. trichocarpa, PtAP66 transcripts increased from the vascular cambium to the xylem cell expansion region and maintained high levels in the SCW formation region. Fluorescent signals from transgenic Arabidopsis plant roots and transiently transformed P. trichocarpa leaf protoplasts strongly suggested that the PtAP66-fused fluorescent protein (PtAP66-GFP or PtAP66-YFP) localised in the plasma membrane. Compared with the wild-type plants, the Cas9/gRNA-induced PtAP66 mutants exhibited reduced SCW thickness of secondary xylem fibres, as suggested by the scanning electron microscopy (SEM) data. In addition, wood composition assays revealed that the cellulose content in the mutants decreased by 4.90–5.57%. Transcription analysis further showed that a loss of PtAP66 downregulated the expression of several SCW synthesis-related genes, including cellulose and hemicellulose synthesis enzyme-encoding genes. Altogether, these findings indicate that atypical PtAP66 plays an important role in SCW deposition during wood formation.

Functional Analysis of Poplar Sombrero-Type NAC Transcription Factors Yields a Strategy to Modify Woody Cell Wall Properties.

Woody cells generate lignocellulosic biomass, which is a promising sustainable bioresource for wide industrial applications. Woody cell differentiation in vascular plants, including the model plant poplar (Populus trichocarpa), is regulated by a set of NAC family transcription factors, the VASCULAR-RELATED NAC-DOMAIN (VND), NAC SECONDARY CELL WALL THICKENING PROMOTING FACTOR (NST)/SND, and SOMBRERO (SMB) (VNS)-related proteins, but the precise contributions of each VNS protein to wood quality are unknown. Here, we performed a detailed functional analysis of the poplar SMB-type VNS proteins PtVNS13-PtVNS16. PtVNS13-PtVNS16 were preferentially expressed in the roots of young poplar plantlets, similar to the Arabidopsis thalianaSMB gene. PtVNS13 and PtVNS14, as well as the NST-type PtVNS11, suppressed the abnormal root cap phenotype of the Arabidopsis sombrero-3 mutant, whereas the VND-type PtVNS07 gene did not, suggesting a functional gap between SMB- or NST-type VNS proteins and VND-type VNS proteins. Overexpressing PtVNS13-PtVNS16 in Arabidopsis seedlings and poplar leaves induced ectopic xylem-vessel-like cells with secondary wall deposition, and a transient expression assay showed that PtVNS13-16 transactivated woody-cell-related genes. Interestingly, although any VNS protein rescued the pendant stem phenotype of the Arabidopsis nst1-1 nst3-1 mutant, the resulting inflorescence stems exhibited distinct cell wall properties: poplar VNS genes generated woody cell walls with higher enzymatic saccharification efficiencies compared with Arabidopsis VNS genes. Together, our data reveal clear functional diversity among VNS proteins in woody cell differentiation and demonstrate a novel VNS-based strategy for modifying woody cell wall properties toward enhanced utilization of woody biomass.

Xylem vessel-specific SND5 and its homologs regulate secondary wall biosynthesis through activating secondary wall NAC binding elements.

Secondary cell wall biosynthesis has been shown to be regulated by a suite of transcription factors. Here, we identified a new xylem vessel-specific NAC domain transcription factor, secondary wall-associated NAC domain protein5 (SND5), in Arabidopsis thaliana and studied its role in regulating secondary wall biosynthesis. We showed that the expression of SND5 and its close homolog, SND4/ANAC075, was specifically associated with secondary wall-containing cells and dominant repression of their functions severely reduced secondary wall thickening in these cells. Overexpression of SND4/5 as well as their homologs SND2/3 fused with the activation domain of the viral protein VP16 led to ectopic secondary wall deposition in cells that are normally parenchymatous. SND2/3/4/5 regulated the expression of the same downstream target genes as do the secondary wall NAC master switches (SWNs) by binding to and activating the secondary wall NAC binding elements (SNBEs). Furthermore, we demonstrated that the poplar (Populus trichocarpa) orthologs of SND2/3/4/5 also activated SNBEs and regulated secondary wall biosynthesis during wood formation. Together, these findings indicate that SND2/3/4/5 and their poplar orthologs regulate the expression of secondary wall-associated genes through activating SNBEs and they are positioned at an upper level in the SWN-mediated transcriptional network.

Sucrose enhanced reactive oxygen species generation promotes cotton fibre initiation and secondary cell wall deposition.

Cotton fiber is a single cell that derived from the ovule epidermis. Fiber cell initiation and elongation determine cotton fiber yield and quality. Sucrose is the most important substrate for cellulose synthesis and energy production, as well as provides turgor pressure to promote fiber elongation. However, varieties with high-sugar content in fibers usually have short fibers, and the mechanism behind this is unclear.

Construction of a Full-Length cDNA Over-Expressing Library to Identify Valuable Genes from Populus tomentosa.

Poplar wood is the main source of renewable biomass energy worldwide, and is also considered to be a model system for studying woody plants. The Full-length cDNA Over-eXpressing (FOX) gene hunting system is an effective method for generating gain-of-function mutants. Large numbers of novel genes have successfully been identified from many herbaceous plants according to the phenotype of gain-of-function mutants under normal or abiotic stress conditions using this system. However, the system has not been used for functional gene identification with high-throughput mutant screening in woody plants. In this study, we constructed a FOX library from the Chinese white poplar, Populus tomentosa. The poplar cDNA library was constructed into the plant expression vector pEarleyGate101 and further transformed into Arabidopsis thaliana (thale cress). We collected 1749 T1 transgenic plants identified by PCR. Of these, 593 single PCR bands from different transgenic lines were randomly selected for sequencing, and 402 diverse sequences of poplar genes were isolated. Most of these genes were involved in photosynthesis, environmental adaptation, and ribosome biogenesis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation. We characterized in detail two mutant lines carrying PtoCPCa or PtoWRKY13 cDNA insertions. Phenotypic characterization showed that overexpression of these genes in A. thaliana affected trichome development or secondary cell wall (SCW) deposition, respectively. Together, the Populus-FOX-Arabidopsis library generated in our experiments will be helpful for efficient discovery of novel genes in poplar.

Environmental and developmental factors driving xylem anatomy and micro-density in black spruce.

Wood density is the product of carbon allocation for structural growth and reflects the trade-off between mechanical support and water conductivity. We tested a conceptual framework based on the assumption that micro-density depends on direct and indirect relationships with endogenous and exogenous factors. The dynamics of wood formation, including timings and rates of cell division, cell enlargement, and secondary wall deposition, were assessed from microcores collected weekly between 2002 and 2016 from five black spruce stands located along a latitudinal gradient in Quebec, Canada. Cell anatomy and micro-density were recorded by anatomical analyses and X-ray measurements. Our structural equation model explained 80% of micro-density variation within the tree-ring with direct effects of wall thickness (σ=0.61), cell diameter (σ=-0.51), and photoperiod (σ=-0.26). Wood formation dynamics had an indirect effect on micro-density. Micro-density increased under longer periods of cell-wall deposition and shorter durations of enlargement. Our results fill a critical gap in understanding the relationships underlying micro-density variation in conifers. We demonstrated that short-term responses to environmental variations could be overridden by plastic responses that modulate cell differentiation. Our results point to wood formation dynamics as a reliable predictor of carbon allocation in trees.

Effect of Exogenously Applied 24-Epibrassinolide and Brassinazole on Xylogenesis and Microdistribution of Cell Wall Polymers in Leucaena leucocephala (Lam) De Wit

Plant growth regulators play a key role in cell wall structure and chemistry of woody plants. Understanding of these regulatory signals is important in advanced research on wood quality improvement in trees. The present study is aimed to investigate the influence of exogenous application of 24-epibrassinolide (EBR) and brassinosteroid inhibitor, brassinazole (BRZ) on wood formation and spatial distribution of cell wall polymers in the xylem tissue of Leucaena leucocephala using light and immuno electron microscopy methods. Brassinazole caused a decrease in cambial activity, xylem differentiation, length and width of fibres, vessel element width and radial extent of xylem suggesting brassinosteroid inhibition has a concomitant impact on cell elongation, expansion and secondary wall deposition. Histochemical studies of 24-epibrassinolide treated plants showed an increase in syringyl lignin content in the xylem cell walls. Fluorescence microscopy and transmission electron microscopy studies revealed the inhomogenous pattern of lignin distribution in the cell corners and middle lamellae region of BRZ treated plants. Immunolocalization studies using LM10 and LM 11 antibodies have shown a drastic change in the micro-distribution pattern of less substituted and highly substituted xylans in the xylem fibres of plants treated with EBR and BRZ. In conclusion, present study demonstrates an important role of brassinosteroid in plant development through regulating xylogenesis and cell wall chemistry in higher plants.

Differentiation of Tracheary Elements in Sugarcane Suspension Cells Involves Changes in Secondary Wall Deposition and Extensive Transcriptional Reprogramming.

Plant lignocellulosic biomass, mostly composed of polysaccharide-rich secondary cell walls (SCWs), provides fermentable sugars that may be used to produce biofuels and biomaterials. However, the complex chemical composition and physical structure of SCWs hinder efficient processing of plant biomass. Understanding the molecular mechanisms underlying SCW deposition is, thus, essential to optimize bioenergy feedstocks. Here, we establish a xylogenic culture as a model system to study SCW deposition in sugarcane; the first of its kind in a C4 grass species. We used auxin and brassinolide to differentiate sugarcane suspension cells into tracheary elements, which showed metaxylem-like reticulate or pitted SCW patterning. The differentiation led to increased lignin levels, mainly caused by S-lignin units, and a rise in p-coumarate, leading to increased p-coumarate:ferulate ratios. RNAseq analysis revealed massive transcriptional reprogramming during differentiation, with upregulation of genes associated with cell wall biogenesis and phenylpropanoid metabolism and downregulation of genes related to cell division and primary metabolism. To better understand the differentiation process, we constructed regulatory networks of transcription factors and SCW-related genes based on co-expression analyses. Accordingly, we found multiple regulatory modules that may underpin SCW deposition in sugarcane. Our results provide important insights and resources to identify biotechnological strategies for sugarcane biomass optimization.

Histone methyltransferase ATX1 dynamically regulates fiber secondary cell wall biosynthesis in Arabidopsis inflorescence stem.

Secondary wall thickening in the sclerenchyma cells is strictly controlled by a complex network of transcription factors in vascular plants. However, little is known about the epigenetic mechanism regulating secondary wall biosynthesis. In this study, we identified that ARABIDOPSIS HOMOLOG of TRITHORAX1 (ATX1), a H3K4-histone methyltransferase, mediates the regulation of fiber cell wall development in inflorescence stems of Arabidopsis thaliana. Genome-wide analysis revealed that the up-regulation of genes involved in secondary wall formation during stem development is largely coordinated by increasing level of H3K4 tri-methylation. Among all histone methyltransferases for H3K4me3 in Arabidopsis, ATX1 is markedly increased during the inflorescence stem development and loss-of-function mutant atx1 was impaired in secondary wall thickening in interfascicular fibers. Genetic analysis showed that ATX1 positively regulates secondary wall deposition through activating the expression of secondary wall NAC master switch genes, SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1) and NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1). We further identified that ATX1 directly binds the loci of SND1 and NST1, and activates their expression by increasing H3K4me3 levels at these loci. Taken together, our results reveal that ATX1 plays a key role in the regulation of secondary wall biosynthesis in interfascicular fibers during inflorescence stem development of Arabidopsis.

Auxin Directly Upregulates GhRAC13 Expression to Promote the Onset of Secondary Cell Wall Deposition in Cotton Fibers.

Cotton fibers are single cells that show a relatively independent developmental process of cell differentiation, elongation, and secondary wall deposition. Auxin promotes fiber cell protrusion from the surface of the ovule. However, the role of auxin at other stages of cotton fiber development remains largely unknown. To gain a deeper insight into this aspect, we measured indoleacetic acid (IAA) content in developing fibers. Results showed an increase in IAA content at the transition stage from elongation to secondary cell wall deposition. Subsequently, we investigated the differences between two transgenic cottons that show upregulated and downregulated fiber auxin levels, respectively. In planta analysis revealed that, in addition to promoting cell elongation, auxin regulated the time of initiation of reactive oxygen species (ROS) production and secondary wall deposition in cotton fibers. This was closely correlated with the upregulated expression of GhRAC13, which regulates ROS-triggered cellulose synthesis. We found multiple putative auxin-responsive elements existed within the promoter region of GhRAC13, and IAA could induce proGhRAC13 activity. The dual-luciferase reporter assay further proved the activation of proGhRAC13 by GhARF5, an auxin-signaling activator. Altogether, our results suggest a role of auxin in promoting the onset of secondary growth by directly upregulating GhRAC13 expression in cotton fibers.

XND1 Regulates Secondary Wall Deposition in Xylem Vessels through the Inhibition of VND Functions.

Secondary wall deposition in xylem vessels is activated by Vascular-Related NAC Domain proteins (VNDs) that belong to a group of secondary wall NAC (SWN) transcription factors. By contrast, Xylem NAC Domain1 (XND1) negatively regulates secondary wall deposition in xylem vessels when overexpressed. The mechanism by which XND1 exerts its functions remains elusive. We employed the promoter of the fiber-specific Secondary Wall-Associated NAC Domain1 (SND1) gene to ectopically express XND1 in fiber cells to investigate its mechanism of action on secondary wall deposition. Ectopic expression of XND1 in fiber cells severely diminished their secondary wall deposition and drastically reduced the expression of SWN-regulated downstream transcription factors and secondary wall biosynthetic genes but not that of the SWN genes themselves. Transactivation analyses revealed that XND1 specifically inhibited SWN-activated expression of these downstream genes but not their MYB46-activated expression. Both the NAC domain and the C-terminus of XND1 were required for its inhibitory function and its NAC domain interacted with the DNA-binding domains of SWNs. XND1 was shown to be localized in the cytoplasm and the nucleus and its co-expression with VND6 resulted in the cytoplasmic sequestration of VND6. Furthermore, the C-terminus of XND1 was indispensable for the XND1-mediated cytoplasmic retention of VND6 and its fusion to VND6 was able to direct VND6 to the cytoplasm and render it unable to activate the gene expression. Since the XND1 gene is specifically expressed in xylem cells, these results indicate that XND1 acts through inhibiting VND functions to negatively regulate secondary wall deposition in xylem vessels.

Earlywood structure of evergreen conifers near forest line is habitat driven but latewood depends on species and seasons

Near upper forest line, values of conifer wood anatomical traits are species-specific, but relationships among traits are common. Growing season temperature significantly impacts wood anatomy only in its extremes. Quantitative wood anatomy can provide detailed insight into adaptation of trees to changing environment, especially on the borders of species distribution ranges. This study investigated wood anatomy of Pinus sylvestris L., Pinus sibirica Du Tour, and Picea obovata Ledeb. near the forest line in the Western Sayan Mountains, where local climate changes rapidly. Anatomical traits reflecting three developmental stages of conifer tracheids (division = cell number, cell enlargement = radial diameter, and secondary wall deposition = cell wall thickness) were calculated for earlywood, latewood and total tree ring over 50 years. Similar earlywood anatomical structure and low between-trait correlations (r = 0.21…67) were observed in all species, which supports prevalence of external impact on its formation, i.e. that shared habitat, climate, and similar habitus provide common trade-off between hydraulic efficiency and safety. Also, stronger nonlinearity of relationship between cell number and radial diameter in earlywood decreased correlations between them. In latewood, anatomical traits are strongly interconnected (r = 0.63…93) for all species. However, Siberian pine has significantly less pronounced latewood; later switch from earlywood and different strategy of carbon allocation are proposed as possible reasons. Length of vegetative season and sum of temperatures above thresholds 5 °C and 8 °C have no significant correlations with anatomical traits, but extremes of these temperature variables led to forming more pronounced latewood (higher proportion of latewood cells with thicker walls) during warm/long vegetative seasons than during short/cool ones.

Comparative transcriptome analyses define genes and gene modules differing between two Populus genotypes with contrasting stem growth rates

BackgroundWood provides an important biomass resource for biofuel production around the world. The radial growth of tree stems is central to biomass production for forestry and biofuels, but it is challenging to dissect genetically because it is a complex trait influenced by many genes. In this study, we adopted methods of physiology, transcriptomics and genetics to investigate the regulatory mechanisms of tree radial growth and wood development.ResultsPhysiological comparison showed that two Populus genotypes presented different rates of radial growth of stems and accumulation of woody biomass. A comparative transcriptional network approach was used to define and characterize functional differences between two Populus genotypes. Analyses of transcript profiles from wood-forming tissue of the two genotypes showed that 1542, 2295 and 2110 genes were differentially expressed in the pre-growth, fast-growth and post-growth stages, respectively. The co-expression analyses identified modules of co-expressed genes that displayed distinct expression profiles. Modules were further characterized by correlating transcript levels with genotypes and physiological traits. The results showed enrichment of genes that participated in cell cycle and division, whose expression change was consistent with the variation of radial growth rates. Genes related to secondary vascular development were up-regulated in the faster-growing genotype in the pre-growth stage. We characterized a BEL1-like (BELL) transcription factor, PeuBELL15, which was up-regulated in the faster-growing genotype. Analyses of transgenic Populus overexpressing as well as CRISPR/Cas9-induced mutants for BELL15 showed that PeuBELL15 improved accumulation of glucan and lignin, and it promoted secondary vascular growth by regulating the expression of genes relevant for cellulose synthases and lignin biosynthesis.ConclusionsThis study illustrated that active division and expansion of vascular cambium cells and secondary cell wall deposition of xylem cells contribute to stem radial increment and biomass accumulation, and it identified relevant genes for these complex growth traits, including a BELL transcription factor gene PeuBELL15. This provides genetic resources for improving and breeding elite genotypes with fast growth and high wood biomass.

The Osmotin-Like Protein Gene PdOLP1 Is Involved in Secondary Cell Wall Biosynthesis during Wood Formation in Poplar.

Osmotin-like proteins (OLPs) mediate defenses against abiotic and biotic stresses and fungal pathogens in plants. However, no OLPs have been functionally elucidated in poplar. Here, we report an osmotin-like protein designated PdOLP1 from Populus deltoides (Marsh.). Expression analysis showed that PdOLP1 transcripts were mainly present in immature xylem and immature phloem during vascular tissue development in P. deltoides. We conducted phenotypic, anatomical, and molecular analyses of PdOLP1-overexpressing lines and the PdOLP1-downregulated hybrid poplar 84K (Populus alba × Populus glandulosa) (Hybrid poplar 84K PagOLP1, PagOLP2, PagOLP3 and PagOLP4 are highly homologous to PdOLP1, and are downregulated in PdOLP1-downregulated hybrid poplar 84K). The overexpression of PdOLP1 led to a reduction in the radial width and cell layer number in the xylem and phloem zones, in expression of genes involved in lignin biosynthesis, and in the fibers and vessels of xylem cell walls in the overexpressing lines. Additionally, the xylem vessels and fibers of PdOLP1-downregulated poplar exhibited increased secondary cell wall thickness. Elevated expression of secondary wall biosynthetic genes was accompanied by increases in lignin content, dry weight biomass, and carbon storage in PdOLP1-downregulated lines. A PdOLP1 coexpression network was constructed and showed that PdOLP1 was coexpressed with a large number of genes involved in secondary cell wall biosynthesis and wood development in poplar. Moreover, based on transcriptional activation assays, PtobZIP5 and PtobHLH7 activated the PdOLP1 promoter, whereas PtoBLH8 and PtoWRKY40 repressed it. A yeast one-hybrid (Y1H) assay confirmed interaction of PtoBLH8, PtoMYB3, and PtoWRKY40 with the PdOLP1 promoter in vivo. Together, our results suggest that PdOLP1 is a negative regulator of secondary wall biosynthesis and may be valuable for manipulating secondary cell wall deposition to improve carbon fixation efficiency in tree species.

The class II KNOX transcription factors KNAT3 and KNAT7 synergistically regulate monolignol biosynthesis in Arabidopsis.

The function of the transcription factor KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) is still unclear since it appears to be either a negative or a positive regulator for secondary cell wall deposition with its loss-of-function mutant displaying thicker interfascicular and xylary fiber cell walls but thinner vessel cell walls in inflorescence stems. To explore the exact function of KNAT7, class II KNOTTED1-LIKE HOMEOBOX (KNOX II) genes in Arabidopsis including KNAT3, KNAT4, and KNAT5 were studied together. By chimeric repressor technology, we found that both KNAT3 and KNAT7 repressors exhibited a similar dwarf phenotype. Both KNAT3 and KNAT7 genes were expressed in the inflorescence stems and the knat3 knat7 double mutant exhibited a dwarf phenotype similar to the repressor lines. A stem cross-section of knat3 knat7 displayed an enhanced irregular xylem phenotype as compared with the single mutants, and its cell wall thickness in xylem vessels and interfascicular fibers was significantly reduced. Analysis of cell wall chemical composition revealed that syringyl lignin was significantly decreased while guaiacyl lignin was increased in the knat3 knat7 double mutant. Coincidently, the knat3 knat7 transcriptome showed that most lignin pathway genes were activated, whereas the syringyl lignin-related gene Ferulate 5-Hydroxylase (F5H) was down-regulated. Protein interaction analysis revealed that KNAT3 and KNAT7 can form a heterodimer, and KNAT3, but not KNAT7, can interact with the key secondary cell wall formation transcription factors NST1/2, which suggests that the KNAT3-NST1/2 heterodimer complex regulates F5H to promote syringyl lignin synthesis. These results indicate that KNAT3 and KNAT7 synergistically work together to promote secondary cell wall biosynthesis.