Journal of Experimental Botany Advance Access published March 5, 2015 Journal of Experimental Botany doi:10.1093/jxb/erv081

Review Paper

Molecular control of wood formation in trees Zheng-Hua Ye* and Ruiqin Zhong Department of Plant Biology, University of Georgia, Athens, GA 30602, USA *  To whom correspondence should be addressed. E-mail: [email protected]

Abstract Wood (also termed secondary xylem) is the most abundant biomass produced by plants, and is one of the most important sinks for atmospheric carbon dioxide. The development of wood begins with the differentiation of the lateral meristem, vascular cambium, into secondary xylem mother cells followed by cell expansion, secondary wall deposition, programmed cell death, and finally heartwood formation. Significant progress has been made in the past decade in uncovering the molecular players involved in various developmental stages of wood formation in tree species. Hormonal signalling has been shown to play critical roles in vascular cambium cell proliferation and a peptide–receptor–transcription factor regulatory mechanism similar to that controlling the activity of apical meristems is proposed to be involved in the maintenance of vascular cambium activity. It has been demonstrated that the differentiation of vascular cambium into xylem mother cells is regulated by plant hormones and HD-ZIP III transcription factors, and the coordinated activation of secondary wall biosynthesis genes during wood formation is mediated by a transcription network encompassing secondary wall NAC and MYB master switches and their downstream transcription factors. Most genes encoding the biosynthesis enzymes for wood components (cellulose, xylan, glucomannan, and lignin) have been identified in poplar and a number of them have been functionally characterized. With the availability of genome sequences of tree species from both gymnosperms and angiosperms, and the identification of a suite of wood-associated genes, it is expected that our understanding of the molecular control of wood formation in trees will be greatly accelerated. Key words:  Plant hormones, secondary wall, secondary xylem, transcriptional regulation, vascular cambium, wood formation.

Introduction Land plants fix about 56 billion metric tons of carbon every year and about half of that is stored in tree species (Field et al., 1998). Because the bulk of tree biomass is wood, it is an important reservoir of fixed carbon and, therefore, carbon storage in wood is crucial for balancing the atmospheric carbon dioxide level. Wood is also an abundant source of raw materials for a myriad of uses by humans, such as burning for energy, pulping, paper-making, construction, and potentially lignocellulosic biofuel production. To tailor wood for

our use, it is critical to dissect the molecular and biochemical mechanisms controlling wood formation. Knowledge gained from such studies can be applied to genetically modify wood quantity and quality. Wood, which is also termed secondary xylem, is produced from the activity of vascular cambium that is composed of two meristematic initials: fusiform initials and ray initials. Fusiform initials generate axially oriented woody cells, termed tracheids, in gymnosperms, and vessels, fibres,

Abbreviations: ANT, AINTEGUMENTA; ARF, Auxin-Responsive Factor; ARK, ARBORKNOX; CesA, Cellulose synthase catalytic subunit; CLE, CLV3/Embryo surrounding region-related; CLV, CLAVATA; CRE1, Cytokinin Receptor1; CslA, Cellulose synthase-like A; ETR1, Ethylene Responsive1; FRA8, Fragile Fiber8; GID1, GIBBERELLIN INSENSITIVE DWARF1; GUX, Glucuronic Acid Substitution of Xylan; GXM, Glucuronoxylan methyltransferase; HB, homeobox; HK, Histidine Kinase; IFL1, INTERFASCICULAR FIBERLESS1; IRX, Irregular Xylem; PHB, PHABULOSA; PHV, PHAVOLUTA; PRE, PopREVOLUTA; PXY, Phloem Intercalated with Xylem; REV, REVOLUTA; RLK3, Receptor-Like Kinase3; RWA, Reduced Wall Acetylation; STM, SHOOT MERISTEMLESS; TDIF, Tracheary Element Differentiation Inhibition Factor; WND, Wood-Associated NAC-Domain Protein; WOX, WUSCHEL-related homeobox; WUS, WUSCHEL. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: [email protected]

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Received 7 November 2014; Revised 28 January 2015; Accepted 30 January 2015

Page 2 of 13  |  Ye and Zhong and parenchyma in angiosperms (Fig.  1), which provide mechanical strength to the plant body. Tracheids and vessels are also responsible for longitudinal conduction. Ray initials produce transversely oriented ray parenchyma (Fig. 1), which is responsible for transverse conduction and nutrient storage (Mauseth, 1988). Wood formation is a sequential developmental process, including differentiation of vascular cambium cells into secondary xylem mother cells, cell expansion, massive deposition of secondary walls, programmed cell death, and finally formation of heartwood (Fig. 2). A number of genes involved in vascular tissue differentiation and secondary wall biosynthesis have been uncovered using the herbaceous Arabidopsis model (Caño-Delgado et  al., 2010;

Schuetz et al., 2013; Zhong and Ye, 2015), but our focus here is a discussion of current understanding of genes known to regulate wood formation in tree species. For other aspects of wood formation, such as anatomical structure and chemistry of wood, as well as physiological, cytological, and biochemical changes associated with wood formation, see Fromm (2013).

Genomic studies of wood formation Transcriptome analyses in tree species have revealed that a suite of genes, including receptor kinases, transcription factors, and secondary wall biosynthesis genes, are highly Downloaded from http://jxb.oxfordjournals.org/ at University of Alabama at Birmingham on March 12, 2015

Fig. 1.  Comparison of gymnosperm and angiosperm wood anatomy. (A) Cross section of a stem of Pinus strobus showing annual rings of wood. (B) Tangential longitudinal section of the cambium zone of a stem of Liriodendron tulipifera showing the fusiform initials and the ray initials. (C, D) Cross (C) and tangential longitudinal (D) sections of wood of Pinus strobus (a gymnosperm species) showing the presence of tracheids and ray parenchyma. (E, F) Cross (E) and tangential longitudinal (F) sections of wood of L. tulipifera (an angiosperm species) showing the presence of vessels, xylary fibres, and ray parenchyma. fi, fusiform initial; rd, resin duct; ri, ray initial; rp, ray parenchyma; sp, secondary phloem; sx, secondary xylem; tr, tracheids; ve, vessel; xf, xylary fibre. Bar in (B) = 155 μm for (B) to (D). This figure is available in colour at JXB online.

Molecular control of wood formation  |  Page 3 of 13 2013) and Picea glauca (white spruce; Birol et al., 2013). The availability of these tree genome sequences together with an improvement of the methodologies used for generation of transgenic trees will enable researchers to directly employ tree species as models for studying wood formation.

Control of vascular cambium activity

expressed in wood-forming cells (Aspeborg et  al., 2005; Pavy et  al., 2008; Wang et  al., 2009; Wilkins et  al., 2009; Dharmawardhana et  al., 2010). These studies provide valuable resources for identifying putative genes involved in wood formation. Of particular note is the Populus Gene Expression (PopGenExpress) data set, which is generated by wholegenome transcriptome profiling of different tissues/organs of Populus trichocarpa, including secondary xylem, seedlings, young and mature leaves, roots, and male and female catkins (Wilkins et  al., 2009). The gene expression data are available in the Populus Electronic Fluorescent Pictograph (eFP) browser (http://bar.utoronto.ca/efppop/cgi-bin/efpWeb.cgi) and the transcript abundance data can be viewed in a simple, graphical format. With the sequencing of the genomes of increasing numbers of tree species from both gymnosperms and angiosperms, it is now possible to uncover the molecular mechanisms controlling the formation of both softwood and hardwood. So far, the genome sequences of four tree species have been released; these are the angiosperms Populus trichocarpa (Tuskan et al., 2006) and Eucalyptus grandis (Myburg et al., 2014), and the gymnosperms Picea abies (Norway spruce; Nystedt et  al.,

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Fig. 2.  The different developmental stages of wood formation and known factors involved in each stage. Genes known to be associated with these different stages of wood formation are listed in Table 1. This figure is available in colour at JXB online.

Since wood is differentiated from the vascular cambium, the activity of vascular cambium largely determines the rate of wood formation. It has been shown that vascular cambium activity is regulated by several plant hormones, including auxin, cytokinin, and ethylene (Fig. 2). Perturbation of auxin signalling by overexpression of a mutant form of PttIAA3 in transgenic poplar results in a reduction in cell division activity in vascular cambium (Nilsson et  al., 2008). The mutant form of PttIAA3 is presumably resistant to auxin-mediated degradation and thus constitutively represses the activation of auxin-responsive genes mediated by Auxin Responsive Factor (ARF). This finding indicates that auxin-mediated signalling is essential for vascular cambium activity (Table 1). Several cytokinin receptor genes from poplar (PtHK3a/ Histidine Kinase 3 and PtHK3b) and birch (BpCRE1/ Cytokinin Receptor1) exhibit a high level of expression in vascular cambium zones, and a reduction in cytokinin level by overexpression of a cytokinin catabolic gene, Arabidopsis cytokinin oxidase 2, in transgenic poplar leads to a decrease in the number of cambium cells and concomitantly a reduced stem diameter (Nieminen et al., 2008). Similarly, simultaneous mutations of four cytokinin biosynthesis genes encoding ATP/ADP isopentenyltransferases cause a loss of vascular cambium activity and a lack of secondary xylem in the hypocotyls of Arabidopsis (Matsumoto-Kitano et al., 2008). These results demonstrate that cytokinins are also critical regulators of vascular cambium activity (Table 1). It has been shown that exogenous application of ethylene stimulates vascular cambium activity, and that this stimulation is inhibited in transgenic poplar overexpressing a dominant negative mutant allele of the Arabidopsis ethylene receptor ETR1 (Ethylene Responsive1), which is insensitive to ethylene (Love et al., 2009). Consistent with a role of ethylene in stimulating vascular cambium activity, an ethylene biosynthesis gene, ACC oxidase (PttACO1), is highly expressed in developing secondary xylem of poplar, and its overexpression in transgenic poplar results in increased wood formation (Love et al., 2009; Table  1). Because auxin, cytokinin, and ethylene all stimulate vascular cambium activity, it is likely that these hormones crosstalk to coordinate their activities or that their signalling pathways converge to regulate common targets that control cell division in the vascular cambium. In Arabidopsis, it has been shown that auxin, cytokinin, and ethylene crosstalk to coordinate their regulation of various developmental processes. For example, both cytokinin and ethylene are involved in modulating auxin transport during lateral root development (Negi et al., 2008; Marhavy et al., 2014). Studies of vascular cambium-associated genes have revealed a conservation of the genetic mechanisms controlling

Page 4 of 13  |  Ye and Zhong Table 1.  Poplar genes involved in wood formation Genea Vascular cambium activity PttIAA3 PttACO1 PtHK3a, PtHK3b PttCLV1 PttRLK3

Description

Functions

References

Indole-3-Acetic Acid Inducible 3 involved in auxin signalling ACC oxidase 1, an ethylene biosynthesis gene Orthologues of Arabidopsis cytokinin receptor AHK3 Orthologue of Arabidopsis CLV1 receptor kinase Orthologue of Arabidopsis TDR receptor kinase

Overexpression of a mutant form of PttIAA3 reduces vascular cambium activity Overexpression results in increased vascular cambium activity and wood formation Preferential expression in vascular cambium

Nilsson et al., 2008

Preferential expression in vascular cambium

Schrader et al., 2004

Preferential expression in vascular cambium; its Arabidopsis orthologue controls cambium cell proliferation Preferential expression in vascular cambium; its Arabidopsis orthologue controls cambium cell proliferation Preferential expression in vascular cambium; its overexpression inhibits secondary xylem differentiation Preferential expression in vascular cambium; regulates vascular cambium activity and differentiation Preferential expression in vascular cambium

Schrader et al., 2004

Overexpression results in increased wood formation

Mauriat and Moritz, 2009

Regulates vascular patterning and secondary xylem differentiation Overexpression of a microRNA-resistant form causes delayed differentiation of secondary xylem and phloem fibres Regulates differentiation of secondary xylem and phloem

Robischon et al., 2011

Top-level master transcriptional switches controlling secondary wall biosynthesis

Zhong et al., 2010b, 2011; Ohtani et al., 2011

Second-level master transcriptional switches controlling secondary wall biosynthesis PtrWND-regulated downstream transcription factors; dominant repression of PtSND2 causes a reduction in secondary wall thickening in transgenic poplar wood PtrWND-regulated downstream transcription factors PtrWND-regulated downstream transcription factor PtrMYB152 overexpression in Arabidopsis causes increased lignification in secondary walls of fibres PtrWND-regulated downstream transcription factors PtrWND-regulated downstream transcription factors PtrWND-regulated downstream transcription factors PtrWND-regulated downstream transcription factors PtrWND-regulated downstream transcription factor

McCarthy et al., 2010; Zhong et al., 2013 Zhong et al., 2011; Wang et al., 2013

Orthologue of Arabidopsis WOX4

PttSTM/ARK1

Orthologue of Arabidopsis STM

PttKNOX1/ARK2, PttKNOX2, PttKNOX6

Orthologue of Arabidopsis KNATs

PttANT Secondary xylem differentiation PttGID1

Orthologuebof Arabidopsis ANT

PopREVOLUTA PopCORONA

PtrHB7

Orthologue of Arabidopsis gibberellin receptor GID1 Orthologueof Arabidopsis REV/IFL1 Orthologue of Arabidopsis CORONA/ATHB15 Orthologue of Arabidopsis ATHB8

Secondary wall deposition: transcriptional regulation PtrWNDs Wood-associated NAC domain transcription factors; orthologues of Arabidopsis SWNs PtrMYB2, PtrMYB3, PtrMYB20, Orthologues of Arabidopsis PtrMYB21 MYB46/MYB83 PtrNAC154 (PtSND2), PtrNAC156 Orthologues of Arabidopsis SND2

PtrNAC105, PtrNAC157

Orthologues of Arabidopsis SND3

PtrKNAT7 PtrMYB18, PtrMYB152

Orthologue of Arabidopsis KNAT7 Orthologues of Arabidopsis MYB20/MYB43 Orthologues of Arabidopsis MYB42/MYB85 Orthologues of Arabidopsis MYB58/MYB63 Orthologues of Arabidopsis MYB69 Orthologues of Arabidopsis MYB103 Orthologue of Arabidopsis LBD15

PtrMYB75, PtrMYB92, PtrMYB125, PtrMYB199 PtrMYB90, PtrMYB161, PtrMYB167, PtrMYB175 PtrMYB26, PtrMYB31, PtrMYB158, PtrMYB189 PtrMYB10, PtrMYB128 PtrLBD15

Nieminen et al., 2008

Schrader et al., 2004

Schrader et al., 2004; Groover et al., 2006 Schrader et al., 2004; Du et al., 2009 Schrader et al., 2004

Du et al., 2011

Zhu et al., 2013

Zhong et al., 2011 Zhong et al., 2011 Wang et al., 2014b Zhong et al., 2011 Zhong et al., 2011 Zhong et al., 2011 Zhong et al., 2011 Zhong et al., 2011

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PttHB3

Love et al., 2009

Molecular control of wood formation  |  Page 5 of 13 Table 1.  Continued Genea

Description

Functions

References

PopNAC122

Orthologue of Arabidopsis XND1

Overexpression in Arabidopsis inhibits xylem formation

Grant et al., 2010

Preferential expression in xylem; cellulose synthases for wood formation Overexpression causes a defect in cellulose synthesis in transgenic poplar wood Preferential expression in xylem; responsible for glucomannan biosynthesis Glycosyltransferases required for xylan backbone biosynthesis Glycosyltransferase required for biosynthesis of xylan-reducing end sequence Glycosyltransferases required for biosynthesis of xylan-reducing end sequence Glycosyltransferase required for biosynthesis of xylan-reducing end sequence Addition of methyl groups onto glucuronic acid side chains of xylan Preferential expression in xylem; Arabidopsis orthologues are required for xylan backbone biosynthesis Preferential expression in xylem; Arabidopsis orthologues are required for normal xylan biosynthesis Preferential expression in xylem; Arabidopsis orthologues are required for addition of glucuronic acid side chains in xylan Preferential expression in xylem; Arabidopsis orthologues are required for xylan acetylation Preferential expression in xylem; Arabidopsis orthologues are required for xylan acetylation Specific expression in developing xylem; biosynthesis enzymes for monolignols Specific expression in developing xylem; biosynthesis enzymes for monolignols Specific expression in developing xylem; biosynthesis enzymes for monolignols Specific expression in developing xylem; biosynthesis enzyme for monolignols

Suzuki et al., 2006

Specific expression in developing xylem; biosynthesis enzyme for monolignols Specific expression in developing xylem; biosynthesis enzymes for monolignols Specific expression in developing xylem; biosynthesis enzyme for monolignols Specific expression in developing xylem; biosynthesis enzymes for monolignols Specific expression in developing xylem; biosynthesis enzyme for monolignols Specific expression in developing xylem; biosynthesis enzyme for monolignols Preferential expression in developing xylem; enzymes for monolignol polymerization

Shi et al., 2010

Secondary wall deposition: biosynthesis of wood components PtCesA4, PtCesA7, PtCesA8, Orthologues of Arabidopsis PtCesA13, PtCesA17, PtCesA18 secondary wall CesAs PttCel9A1 Cellulase; orthologue of Arabidopsis Korrigan1 PtCslA1 Glucomannan synthase; orthologue of Arabidopsis CslAs PtrGT43A, PtrGT43B, PtrGT43C, Orthologues of Arabidopsis IRX9 PtrGT43D, PtrGT43E and IRX14 PoGT8 Orthologue of Arabidopsis IRX8

PoGT47C

Orthologues of Arabidopsis PARVUS Orthologue of Arabidopsis FRA8

PtrGXM1, PtrGXM2, PtrGXM3, PtrGXM4 Potri.001G068100, Potri.003G162000, Potri.015G116700

Glucuronoxylan methyltransferases

Potri.005G141300, Potri.007G047000

Orthologues of Arabidopsis IRX15/IRX15L

Potri.007G107200, Potri.005G061600, Potri.014G029900

Orthologues of Arabidopsis GUXs

Potri.001G352300, Potri.011G079400

Orthologues of Arabidopsis RWAs

Potri.008G069900, Potri.010G187600, Potri.010G187500 PtrPAL2, PtrPAL3, PtrPAL4, PtrPAL5

Orthologues of Arabidopsis ESK1

PtrC4H1, PtrC4H2

Cinnamate-4-hydroxylase

PtrCL3, PtrCL5

4-Coumarate:CoA ligase

PtrHCT1

p-HydroxycinnamoylCoA-quinate shikimate p-hydroxycinnamoyltransferase 4-Coumarate 3-hydroxylase

PtrC3H3

Orthologues of Arabidopsis IRX10/IRX10L

Phenylalanine ammonia-lyase

PtrCCoAOMT1, PtrCCoAOMT2, PtrCCoAOMT3 PtrCCR2

Caffeoyl-CoA O-methyltransferase

PtrCAld5H1, PtrCAld5H2

Coniferyl aldehyde 5-hydroxylase

PtrCOMT2

Caffeic acid O-methyltransferase

PtrCAD1

Cinnamyl alcohol dehydrogenase

PtrLAC2, PtrLAC6, PtrLAC7, PtrLAC11, PtrLAC14, PtrLAC17, PtrLAC23, PtrLAC24, PtrLAC25, PtrLAC27, PtrLAC30, PtrLAC49

Laccases

a

Cinnamoyl-CoA reductase

Po, Populus alba × tremula; Ptr/Pt/Pop, Populus trichocarpa; Ptt, Populus tremula × tremuloides.

Lee et al., 2011 Lee et al., 2011 Lee et al., 2009b Lee et al., 2009a Yuan et al., 2014 Wilkins et al., 2009

Wilkins et al., 2009

Wilkins et al., 2009

Wilkins et al., 2009 Wilkins et al., 2009 Shi et al., 2010 Shi et al., 2010 Shi et al., 2010 Shi et al., 2010

Shi et al., 2010 Shi et al., 2010 Shi et al., 2010 Shi et al., 2010 Shi et al., 2010 Lu et al., 2013

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PoGT8E, PoGT8F

Takahashi et al., 2009; Yu et al., 2014 Suzuki et al., 2006

Page 6 of 13  |  Ye and Zhong proliferation of vascular cambium cells and an inhibition of secondary xylem differentiation (Etchells and Turner, 2010), and T-DNA knockout mutations of TDR and WOX4 result in a disruption of the continuous cambium ring in the hypocotyls of Arabidopsis (Hirakawa et al., 2010). It has been proposed that TDIF from the phloem diffuses into the cambium zone, where it interacts with TDR and upregulates WOX4 expression, thereby leading to proliferation of vascular cambium cells and inhibition of differentiation of cambium cells into xylem cells (Etchells and Turner, 2010; Hirakawa et al., 2010).

Regulation of secondary xylem differentiation Vascular cambium cells undergo anticlinal divisions; the daughter cells produced on the inner side of the cambium differentiate into secondary xylem mother cells, and those produced towards the outside differentiate into secondary phloem mother cells (Fig. 2). The molecular mechanism underlying the precise spatial control of secondary xylem differentiation is not well understood. It has long been known that auxin, cytokinin, and brassinosteroid could induce xylem differentiation in cultured cells (Ohashi-Ito and Fukuda, 2010), indicating that the signalling pathways mediated by these hormones are involved in the initiation of secondary xylem differentiation. In addition, gibberellin signalling has been suggested to be involved in stimulating secondary xylem differentiation, because overexpression of the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1 (PttGID1) results in increased wood formation in transgenic poplar (Mauriat and Moritz, 2009; Table  1). It remains to be investigated how these hormonal signals are integrated to promote the differentiation of vascular cambium cells into secondary xylem mother cells. Transcriptome profiling of the vascular cambium zone of poplar stems has shown that in addition to the genes homologous to apical meristem regulators, several HD-ZIP III genes, PttHB8, PttHB9, and PttHB15, which are close homologues of Arabidopsis ATHB-8, PHAVOLUTA (PHV)/ ATHB-9, and CORONA/ATHB-15, respectively, are highly expressed in the cambium/developing xylem cells (Schrader et al., 2004; Table 1). Arabidopsis HD-ZIP III genes, ATHB8, PHV/ATHB-9, CORONA/ATHB-15, PHABULOSA (PHB)/ATHB-14, and INTERFASCICULAR FIBERLESS1 (IFL1)/REVOLUTA (REV), play important roles in regulating vascular patterning, organ polarity, polar auxin transport, and differentiation of primary xylem, secondary xylem, and interfascicular fibres (Prigge et al., 2005; Zhong and Ye, 1999, 2001, 2004). Overexpression and downregulation studies of poplar wood-associated HD-ZIP III genes have also revealed their roles in controlling vascular patterning and secondary xylem differentiation. Overexpression of a microRNAresistant form of PopREVOLUTA (PRE), an orthologue of Arabidopsis IFL1/REV, results in ectopic formation of vascular cambium and its associated secondary xylem in transgenic poplar (Robischon et al., 2011), a phenotype similar to that seen in the dominant Arabidopsis avb1 mutant, which

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the proliferation and maintenance of shoot apical meristem and vascular cambium (a lateral meristem). In Arabidopsis shoot apical meristem, WUSCHEL (WUS) together with the signalling peptide CLAVATA3 (CLV3) and the receptor kinase CLV1 form a peptide–receptor–transcription factor feedback regulatory loop that maintains the dynamic balance between meristematic cell division and differentiation. In addition, several other transcription factors, such as SHOOT MERISTEMLESS (STM), KNOX proteins (KNAT1 and KNAT6), and AINTEGUMENTA (ANT) also play important roles in the maintenance of shoot apical meristem (Ha et  al., 2010). Using tangential sectioning of vascular cambium cells of poplar stems coupled with microarray-based transcriptome profiling, it has been revealed that a number of regulatory genes, such as PttCLV1, PttRLK3 (ReceptorLike Kinase3), PttHB3 (WUSCHEL-related HomeoBox3), PttSTM, PttKNOX1, PttKNOX2, PttKNOX6, and PttANT, which are homologous to those involved in regulating shoot apical meristem in Arabidopsis, are highly expressed in vascular cambium cells (Schrader et  al., 2004; Table  1). Overexpression of PttSTM/ARBORKNOX1 (ARK1), an orthologue of Arabidopsis STM, and PttKNOX1/ ARBORKNOX2 (ARK2), an orthologue of Arabidopsis KNAT1, in transgenic poplar leads to an inhibition of secondary xylem differentiation (Groover et  al., 2006; Du et  al., 2009), indicating their potential role in regulating vascular cambium activity and secondary xylem differentiation. ARK1 has recently been shown to bind to promoter regions of many poplar genes with diverse functions (Liu et al., 2015), which provides an entry point for dissecting the mechanism of ARK1 regulation of wood formation in tree species. Functional study of Arabidopsis STM and KNAT1 has revealed their specific role in promoting differentiation of cambium cells into xylem fibres through transcriptional repression of two meristem boundary genes, BLADE-ONPETIOLE 1 (BOP1) and BOP2, during secondary growth in the Arabidopsis hypocotyl (Liebsch et al., 2014). The finding that PttCLV1 (a CLV1 orthologue), PttRLK3 (a CLV1 homologue), and PttHB3 (a WUS homologue) are expressed in the vascular cambium led to the proposal that a similar regulatory mechanism for controlling the activity of apical meristems was recruited to regulate the activity of the vascular cambium (Fig. 2). Although the exact functions of these poplar genes are currently unknown, their Arabidopsis orthologues have been shown to regulate vascular cambium activity. In Arabidopsis, the CLV3 signalling peptide homologue TDIF [Tracheary Element Differentiation Inhibition Factor, an active peptide derived from CLE41 (CLV3/ Embryo-surrounding region-related), CLE42, and CLE44], together with its receptor kinase TDR (TDIF Receptor, also called PXY/Phloem Intercalated with Xylem) and the WUS homologues WOX4/WOX14 (WUSCHEL-related homeobox), constitute a peptide–receptor–transcription factor signalling pathway controlling vascular cambium cell proliferation (Ito et al., 2006; Hirakawa et al., 2008, 2010; Etchells et al., 2013), which is analogous to the WUS/CLV feedback loop mechanism that maintains the stem cell population in apical meristems. Ectopic expression of CLE41 causes

Molecular control of wood formation  |  Page 7 of 13 produces a microRNA165-resistant form of IFL1/REV (Zhong and Ye, 2004). A delayed differentiation of secondary xylem and phloem fibres was observed in transgenic poplar overexpressing a microRNA-resistant form of PopCORONA, which is an orthologue of Arabidopsis Corona/ATHB-15 (Du et al., 2011). Furthermore, overexpression or downregulation of PtrHB7, an orthologue of ATHB-8, results in an alteration in the differentiation of cambium cells into secondary xylem and phloem (Zhu et  al., 2013). It will be interesting to further investigate how these wood-associated HD-ZIP III genes regulate the differentiation and patterning of secondary xylem during wood formation.

After differentiation of vascular cambium cells into secondary xylem mother cells, they undergo cell expansion followed by a massive deposition of secondary walls that are mainly composed of cellulose, hemicelluloses, and lignin (Fig.  2). The plant hormone gibberellin plays an important role during the expansion of secondary xylem cells in poplar. It has

Fig. 3.  The transcriptional network regulating secondary wall biosynthesis during wood formation in poplar. In this network, the top-level woodassociated NAC master switches directly activate the second-level MYB master switches and many other downstream transcription factors, all of which function concertedly to regulate the expression of genes involved in the biosynthesis of wood components. This figure is available in colour at JXB online.

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Regulation of cell expansion and secondary wall biosynthesis

been shown that the bioactive gibberellins GA1 and GA4 are predominantly concentrated in the zone of expanding xylem cells in poplar stems, as is the expression of the gibberellin signalling and response genes DELLA-like1 and GID-like1 (Israelsson et  al., 2005). GID1 is a gibberellin receptor and, upon activation by gibberellin, it targets the gibberellin signalling suppressor DELLA for degradation, which leads to transduction of gibberellin signals and gibberellin-stimulated responses (Hirano et al., 2008). A role of gibberellin in xylem cell expansion in poplar wood was further demonstrated by overexpression of GA 20-oxidase, a key enzyme in controlling gibberellin biosynthesis, which results in a significant increase in plant height and fibre length in wood (Eriksson et al., 2000). Deposition of secondary walls during wood formation requires coordinated expression of secondary wall biosynthesis genes, which is controlled by a secondary wall transcriptional network that is conserved in vascular plants (Zhong and Ye, 2014). In this transcriptional network, secondary wall NAC and secondary wall MYB transcription factors act as the top-level and second-level master switches, respectively, and together they activate a battery of downstream transcription factors and secondary wall biosynthesis genes (Zhong and Ye, 2014; Fig. 3). Wood-associated NAC master

Page 8 of 13  |  Ye and Zhong

Fig. 4.  Phylogenetic relationship of secondary wall NAC master switches from tree species in comparison to those from Arabidopsis (SND1/ NSTs/VNDs). The genes analysed were from Populus trichocarpa (Ptr), Eucalyptus grandis (Eucgr), Pinus taeda (PITA), and Picea abies (MA), and the sequences were retrieved from NCBI Genbank, Phytozome v9.1, the Dendrome database, and ConGenIE, respectively. The phylogenetic tree was constructed with the neighbour-joining algorithm and the bootstrap values resulted from 1000 replicates and are shown as percentages at the nodes. The 0.1 scale denotes 10% change.

PtrWNDs regulate the expression of a number of other wood-associated transcription factors (Ohtani et  al., 2011; Zhong et  al., 2011; Lin et  al., 2013), and four of them, PtrMYB2, PtrMYB3, PtrMYB20, and PtrMYB21, are direct targets of PtrWNDs (Table 1). PtrMYB2/3/20/21 are capable of activating the biosynthesis pathways of cellulose, xylan, and lignin, concomitantly leading to ectopic secondary wall deposition when overexpressed in Arabidopsis and poplar, and their dominant repression results in a reduction of secondary wall thickening in transgenic poplar wood (McCarthy et al., 2010; Zhong et al., 2013). PtMYB4 and PtMYB8 from pine and EgMYB2 from Eucalyptus, which are orthologues of PtrMYB2/3/20/21, are also able to activate the entire secondary wall biosynthesis programme when overexpressed (Patzlaff et al., 2003; Goicoechea et al., 2005; Bomal et al., 2008; Zhong et  al., 2010a). The available evidence demonstrates that in addition to the top-level NAC master switches, PtrMYB2/3/20/21, EgMYB2, and PtrMYB4/8 function as second-level master switches coordinating the activation of secondary wall biosynthesis genes during wood formation in trees (Fig. 3). In addition to PtrMYB2/3/20/21, a suite of other transcription factors have been found to be activated by PtrWNDs, indicating the complexity of the transcriptional network controlling wood formation (Zhong et  al., 2011) (Fig.  3). Among them, 24 are homologues of Arabidopsis secondary wall-associated genes activated by secondary wall NAC master switches (Table  1). Several of these PtrWND-regulated transcription factors and their orthologues in other tree species have been shown to be involved in regulating secondary wall biosynthesis: PtrNAC154 (an orthologue of Arabidopsis SND2), whose dominant repression causes reduced secondary wall thickening in fibres of transgenic poplar wood (Wang et  al., 2013), and its Eucalyptus orthologue, whose overexpression increases wood fibre cell area in transgenic Eucalyptus (Hussey et  al., 2011); PtrMYB152 (an orthologue of Arabidopsis MYB43), whose overexpression elevates lignin biosynthesis when overexpressed in Arabidopsis (Wang et  al., 2014b); PtrMYB28 (an orthologue of Arabidopsis MYB58/MYB63), which is able to activate lignin biosynthesis genes (Zhong and Ye, 2009); and pine PtMYB1 (an orthologue of Arabidopsis MYB85), whose overexpression in transgenic spruce leads to elevated lignin biosynthesis (Bomal et  al., 2008). Furthermore, a number of additional transcriptional factors, including PtrMYB18, PtrMYB75, PtrMYB74, PtrMYB121, PtrMYB128, PtrNAC150, PtrZF1, and PtrGATA8, have been shown to be able to activate the promoter activities of several biosynthesis genes for cellulose, xylan, and lignin, suggesting that they may also function as master switches controlling the entire secondary wall biosynthesis programme. It is likely that tree species have evolved to recruit additional master transcriptional switches to sustain a robust expression of secondary wall biosynthesis genes to ensure the deposition of a massive amount of secondary wall components in woody cells. Transactivation and electrophoretic mobility shift assays have revealed that PtrWNDs and EgWND1 activate their target gene expression through direct binding to the 19-bp

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switches from poplar (PtrWNDs), Eucalyptus (EgWND1), and spruce (PgNAC-7) have been functionally characterized (Zhong et  al., 2010a, b, 2011; Zhong and Ye, 2010; Ohtani et al., 2011; Bomal et al., 2014; Table 1). It has been demonstrated that when overexpressed, PtrWNDs are able to induce the expression of secondary wall biosynthesis genes and concomitantly lead to ectopic deposition of wood components, including cellulose, xylan, and lignin in transgenic poplar (Ohtani et al., 2011; Zhong et al., 2011). Furthermore, dominant repression of PtrWND functions causes a drastic reduction in secondary wall thickening in transgenic poplar wood (Zhong et al., 2011). EgWND1 overexpression in Arabidopsis also results in ectopic deposition of secondary walls (Zhong et  al., 2010a). These findings demonstrate that these woodassociated NACs are master transcriptional switches activating secondary wall biosynthesis during wood formation in trees (Fig. 3). Phylogenetic analysis of secondary wall NAC master switches in tree species indicates an expansion in the number of these genes in angiosperms (six in Eucalyptus and six pairs of duplicated ones in poplar) compared to gymnosperms (two in pine and spruce, respectively; Fig. 4), which correlates with the increased complexity of wood structure in angiosperms (composed of vessels and fibres) compared to that in gymnosperms (composed of tracheids; Fig. 1).

Molecular control of wood formation  |  Page 9 of 13

Biosynthesis of wood components Wood is mainly composed of cellulose, hemicelluloses (xylan and glucomannan), and lignin, the proportion of which varies among different species. For example, wood from Populus tremuloides consists of 48% cellulose, 24% glucuronoxylan, 3% glucomannan, and 21% lignin, and wood from Pinus strobus is made of 41% cellulose, 9% arabinoglucuronoxylan, 18% galactoglucomannan, and 29% lignin (Timell, 1967). It is envisaged that the deposition of secondary walls in the developing wood requires the catalytic activities of all the biosynthesis enzymes involved in the biosynthesis of wood components and, therefore, their genes must be coordinately expressed (Fig. 2). Transcriptome profiling of developing secondary xylem of poplar led to the identification of a number of xylem-specific glycosyltransferases (Aspeborg et  al., 2005), many of which have been demonstrated to participate

in the biosynthesis of cellulose, xylan, and glucomannan (Table  1). Among them, cellulose synthase (CesA) genes, which are orthologues of the three Arabidopsis secondary wall CesAs, are highly expressed in developing secondary xylem (Aspeborg et  al., 2005; Suzuki et  al., 2006), and overexpression of one of them causes co-suppression of the expression of wood-associated CesAs and a drastic reduction in cellulose content in transgenic poplar wood (Joshi et al., 2011). Several other proteins, such as sucrose synthases and cellulases, have been implicated in cellulose biosynthesis. Overexpression of a cotton sucrose synthase results in a slight elevation in cellulose content and an increased cellulose crystallinity in transgenic poplar wood, suggesting a possible association of sucrose synthase with cellulose biosynthesis (Coleman et al., 2009). Downregulation of sucrose synthase activities in transgenic poplar appears not to support a direct role of sucrose synthase in cellulose biosynthesis; instead, it is proposed that sucrose synthase is involved in supplying carbon for overall wood polymer biosynthesis (Gerber et al., 2014). Downregulation of a poplar cellulase, PttCel9A1, which is an orthologue of Arabidopsis Korrigan1 (Takahashi et  al., 2009), has been shown to cause a defect in cellulose biosynthesis in poplar wood (Yu et al., 2014). The biosynthesis of glucomannan is catalysed by members of cellulose synthase-like A  (CslA) family of glycosyltransferases, the functions of which are conserved in vascular plants (Liepman et al., 2007). One of the poplar CslA genes, PtrCslA1, is highly expressed in developing xylem and its recombinant protein exhibits glucomannan synthase activity, indicating its involvement in glucomannan biosynthesis during wood formation (Suzuki et al., 2006). Similarly, several CslAs from pine exhibit glucomannan synthase activities (Liepman et al., 2007). Xylan is the predominant hemicellulose in wood of angiosperms and it is made of a linear chain of β-1,4-linked xylosyl residues substituted with α-1,2-linked 4-O-methylglucuronic acid (MeGlcA) residues and acetylated at O-2 and/or O-3 (Timell, 1967). The reducing end of xylans from wood of gymnosperms and angiosperms contains a unique tetrasaccharide sequence composed of β-d-Xylp-(1→3)-α-l-Rhap(1→2)-α-d-GalpA-(1→4)-d-Xylp (Zhong and Ye, 2015). Among the xylem-specific glycosyltransferase genes identified from transcriptome profiling of developing wood of poplar (Aspeborg et  al., 2005), PoGT47C, PoGT8D, and PoGT8E/PoGT8F are orthologues of Arabidopsis FRA8 (Fragile Fiber8), IRX8 (Irregular Xylem8) and PARVUS, respectively, which are involved in the biosynthesis of xylanreducing end sequence (Lee et al., 2009a, b, 2011). It has been shown that PtrGT43A, PtrGT43B, and PtrGT43E are functional orthologues of Arabidopsis IRX9, and PtrGT43C and PtrGT43D are functional orthologues of Arabidopsis IRX14, all of which are required for xylan backbone biosynthesis during wood formation (Lee et al., 2011, 2012). The addition of methyl groups onto the GlcA side chains of xylan in poplar wood is catalysed by four DUF579 domain-containing glucuronoxylan methyltransferases, PtrGXM1, PtrGXM2, PtrGXM3, and PtrGXM4 (Yuan et al., 2014). Furthermore, a number of poplar orthologues of other Arabidopsis xylan

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secondary wall NAC-binding element (SNBE), (T/A) NN(C/T)(T/C/G)TNNNNNNNA(A/C)GN(A/C/T)(A/T) (Zhong et al., 2011), and PtrMYB2/3/20/21, EgMYB2, and PtMYB4 directly bind to and activate the 7-bp secondary wall MYB-responsive element (SMRE), ACC(A/T)A(A/C)(T/C) (Zhong et al., 2013). Because promoters of many PtrWNDregulated downstream transcription factors and secondary wall biosynthesis genes contain SNBE and/or SMRE sites (Zhong et al., 2013), it is proposed that the NAC and MYB master switches together with their downstream transcription factors act concertedly to activate the secondary wall biosynthesis programme during wood formation (Fig. 3). Regulation of secondary wall biosynthesis during wood formation not only involves transcriptional activators but also entails transcriptional repressors. Eucalyptus EgMYB1, an Arabidopsis MYB4 orthologue, represses the expression of secondary wall biosynthesis genes and inhibits secondary wall thickening in fibres when overexpressed in Arabidopsis and poplar, suggesting that it is a transcriptional repressor of secondary wall formation (Legay et  al., 2010). Poplar PopNAC122, an orthologue of Arabidopsis XND1, inhibits xylem formation and plant growth when overexpressed in Arabidopsis, indicating that like XND1, PopNAC122 may function to repress secondary wall biosynthesis during wood formation (Grant et al., 2010). RNA sequencing analysis of the xylem transcriptome in Populus trichocarpa revealed that about 36% of wood-expressed genes are alternatively spliced (Bao et al., 2013). Transcripts of one of the PtrWND genes, PtrWND1B, have an alternative splice form whose encoded protein contains the N-terminal DNA dimerization domain but lacks the DNA-binding and transcriptional activation domains (Li et  al., 2012). This alternative splice form of PtrWND1B has been shown to inhibit the transcriptional activity of PtrWNDs (Li et  al., 2012) and when overexpressed, cause a reduction in secondary wall thickening in the wood of transgenic poplar (Zhao et al., 2014). These findings indicate that the alternative splice form of PtrWND1B might act in a dominant-negative manner to inhibit secondary wall biosynthesis during wood formation.

Page 10 of 13  |  Ye and Zhong a gradual degradative process in both the nucleus and the cytoplasm before the loss of vacuolar integrity, a process different from that of vessels in which cell death is initiated by the loss of vacuolar integrity (Courtois-Moreau et al., 2009). Eventually, cell death of wood ray parenchyma together with tylosis formation, wood dehydration, and accumulation of heartwood substances converts sapwood into heartwood. The mechanism controlling heartwood formation is not well understood; several reports have shown upregulation of a number of genes during the transition from sapwood to heartwood, indicating that heartwood formation is a genetically controlled developmental process (Beritognolo et  al., 2002; Yang et al., 2004; Huang et al., 2009).

Reaction wood formation In a vertically grown tree stem, the vascular cambium undergoes uniform cell division and differentiation, which gives rise to the concentricity of wood. When a tree stem leans, the vascular cambium exhibits asymmetric activity, which results in the eccentricity of stems due to formation of reaction wood. Reaction wood in gymnosperms, termed compression wood, forms on the lower side of a leaning stem, and that in angiosperms, termed tension wood, forms on the upper side (Timell, 1967). Ethylene has been shown to be involved in tension wood formation. Several Ethylene Response Factors (ERFs), which mediate ethylene signalling, are induced in responsive to tension wood formation in poplar and, when overexpressed, they alter wood formation in transgenic trees (Vahala et  al., 2013). Disruption of ethylene signalling in transgenic poplar by overexpression of a dominant negative mutant allele of the Arabidopsis ethylene receptor ETR1 leads to inhibition of tension wood formation, demonstrating an essential role of ethylene in controlling the asymmetric activity of vascular cambium and formation of tension wood (Love et al., 2009). Transcriptome profiling of tension wood in poplar has uncovered a number of genes, including transcription factors and cell wall-related genes, that are upregulated in tension wood-forming tissues compared with normal woody tissues (Andersson-Gunneras et  al., 2006). Because tension wood is enriched in cellulose due to the deposition of a cellulose-rich gelatinous layer in fibres, uncovering the molecular mechanism controlling its formation may provide novel tools for generating cellulose-enriched wood tailored for biofuel production.

Programmed cell death and heartwood formation

Concluding remarks

After deposition of secondary walls, tracheids (gymnosperms), and vessels and fibres (angiosperms), in wood undergo programmed cell death (Fig. 2). A number of genes encoding proteases, nucleases, and autophage-related proteins have been shown to be upregulated during secondary xylem maturation in poplar, indicating their potential roles in regulating programmed cell death during wood formation (Courtois-Moreau et al., 2009). Cytological analysis of poplar wood has revealed that cell death in fibres involves

With the completion of genome sequencing of several tree species, it is an exciting time to study many aspects of wood formation in tree species, including the regulatory networks controlling vascular cambium cell proliferation and maintenance, secondary xylem differentiation, and reaction wood formation. The availability of genome sequences from both gymnosperms and angiosperms will make it feasible to perform comparative analysis of key genes controlling wood formation and discover what factors determine the

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biosynthesis genes are all preferentially expressed in developing wood (Wilkins et al., 2009), indicating that they are probably involved in xylan biosynthesis during wood formation (Table  1): IRX10/IRX10L, which are xylosyltransferases required for xylan backbone elongation; IRX15/IRX15L, which are essential for normal xylan biosynthesis; GUXs (Glucuronic Acid Substitution of Xylan), which are glucuronyltransferases catalysing the transfer of glucuronic acid side chains onto xylan; RWAs (Reduced Wall Acetylation), which are required for normal xylan acetylation; and ESKIMO1, which is an acetyltransferase mediating 2-O- and 3-O-monoacetylation of xylan (Zhong and Ye, 2015). Lignin is a polyphenolic polymer produced via oxidative polymerization of three monolignols, p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. Monolignols are synthesized through the phenylpropanoid biosynthesis pathway involving at least 10 enzymes. Comprehensive expression analysis in poplar of all candidate genes potentially participating in phenylpropanoid biosynthesis has shown that 18 of them are highly expressed in developing wood, and they are considered to be the core genes responsible for monolignol biosynthesis during wood formation (Shi et  al., 2010; Table  1). The kinetic properties of these core monolignol pathway enzymes have been comprehensively characterized and their endogenous amounts in developing wood have been quantitated, the data of which were used to construct a kinetic metabolic-flux model that could predict how perturbation of monolignol pathway enzymes may affect lignin content and composition (Wang et al., 2014a). Downregulation of lignin pathway genes has been shown to reduce lignin content in transgenic poplar wood, improving the efficiency of pulping (Pilate et al., 2002) and bioconversion to ethanol (Mansfield et al., 2012; Van Acker et al., 2014). Studies of monolignol transport in developing pine wood indicate that membrane transporters are involved in exporting monolignols from the cytosol into the cell wall for polymerization (Kaneda et al., 2008), which is consistent with the finding in Arabidopsis that ABC transporter activities mediate the transport of monolignols (Liu et al., 2011). Oxidative polymerization of monolignols is catalysed by peroxidases and laccases; overexpression of Ptr-miR397a, which targets a subset of laccase genes, leads to a reduction in laccase activities and a concomitant decrease in lignin content in transgenic poplar (Lu et al., 2013).

Molecular control of wood formation  |  Page 11 of 13 differentiation of vascular cambium cells into tracheids in gymnosperms and vessels and fibres in angiosperms (Fig. 1). Furthermore, many wood-associated genes have been identified through transcriptome profiling and proteomic analyses (Aspeborg et al., 2005; Pavy et al., 2008; Wang et al., 2009; Wilkins et  al., 2009; Dharmawardhana et  al., 2010; Song et  al., 2011) and their functional characterization promises to shed new light on the molecular mechanisms controlling wood formation. It can be foreseen that, with the available genomic and genetic tools, great leaps will be achieved in our understanding of the molecular control of wood formation in the coming years.

Funding

Acknowledgements We regret that many original articles on wood formation could not be cited due to space limitations.

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