The cotton genome (Gossypium hirsutum) contains ~80K protein-coding genes, making precision breeding for complex traits a challenge. This study tested biology-informed approaches to improve genomic prediction (GP) accuracy for cotton fibre traits to help accelerate precision breeding of valuable traits. The foundational approach was the use of RNA-seq data from key time points during fibre development, namely fibre cells undergoing primary, transition, and secondary wall development. The test approaches included using a range of summary statistics from RNA-seq analysis such as gene Differential Expression (DE). The three test approaches included DE genes overall, target pairwise DE lists informed by gene functional annotation, and finally, gene-network-clusters created based on Partial Correlation and Information Theory (PCIT) as the prior information in Bayesian GP models. The most promising improvements in GP accuracy were at the level of ~ 5% increase by using PCIT-based gene-network clusters as the prior knowledge network neighbours of key genes, and for the traits of cotton fibre Elongation and Strength. These results indicate that there is scope to help improve precision breeding of target traits by incorporating biology-based inference into GP models, and points to specific approaches to achieve this.

Cultivated cotton plants are the world’s largest source of natural fibre, where yield and quality are key traits for this renewable and biodegradable commodity. The Gossypium hirsutum cotton genome contains ~80K protein-coding genes, making precision breeding of complex traits a challenge. This study tested approaches to improving the genomic prediction (GP) accuracy of valuable cotton fibre traits to help accelerate precision breeding. With a biology-informed basis, a novel approach was tested for improving GP for key cotton fibre traits with transcriptomics of key time points during fibre development, namely, fibre cells undergoing primary, transition, and secondary wall development. Three test approaches included weighting of SNPs in DE genes overall, in target DE gene lists informed by gene annotation, and in a novel approach of gene co-expression network (GCN) clusters created with partial correlation and information theory (PCIT) as the prior information in GP models. The GCN clusters were nucleated with known genes for fibre biomechanics, i.e., fasciclin-like arabinogalactan proteins, and cluster size effects were evaluated. The most promising improvements in GP accuracy were achieved by using GCN clusters for cotton fibre elongation by 4.6%, and strength by 4.7%, where cluster sizes of two and three neighbours proved most effective. Furthermore, the improvements in GP were due to only a small number of SNPs, in the order of 30 per trait using the GCN cluster approach. Non-trait-specific biological time points, and genes, were found to have neutral effects, or even reduced GP accuracy for certain traits. As the GCN clusters were generated based on known genes for fibre biomechanics, additional candidate genes were identified for fibre elongation and strength. These results demonstrate that GCN clusters make a specific and unique contribution in improving the GP of cotton fibre traits. The findings also indicate that there is room for incorporating biology-based GCNs into GP models of genomic selection pipelines for cotton breeding to help improve precision breeding of target traits. The PCIT-GCN cluster approach may also hold potential application in other crops and trees for enhancing breeding of complex traits.

White cotton is the dominant natural fibre, accounting for a $USD 36 billion share of the $USD 1.5 trillion global textile industry and, for many decades, has been dyed post-production. Modern cotton ginning and spinning processes require longer and stronger fibres, favouring superior white cotton varieties, which are more amenable to post-harvest dyeing (Gong et al., 2018; Vreeland, 1999). However, large quantities of synthetic dyes from textile dyeing released into the environment/waterways are harming the health of humans and other organisms (Manzoor and Sharma, 2020). Eco-friendly alternatives are urgently needed to reduce pollution and save water; coloured dye-free cotton could be a solution. While naturally coloured cotton has been known for more than 5000 years and occurs in all four species of cultivated cotton, that is Gossypium (G.) hirsutum, G. barbadense, G. herbaceum and G. arboretum (Murthy, 2001; Vreeland, 1999), these coloured varieties generally have low yield, poor fibre quality and variable and unstable colours (Rathinamoorthy and Parthiban, 2019; Vreeland, 1999). Although conventional breeding has improved the properties of some coloured cotton, quality and yield remain low compared with white cotton and colour range is limited (Murthy, 2001; Rathinamoorthy and Parthiban, 2019). Betalains are tyrosine-derived pigments found naturally in the order Caryophyllales of flowering plants, and fungi and bacteria (Timoneda et al., 2019). Betalains comprise two classes of compounds, that is yellow-orange betaxanthins and red-violet betacyanins. These compounds are synthesized through a series of enzymatic steps including hydroxylases, dioxygenases and glucosyl transferases that produce visible colours. As the Malvaceae typically do not produce betalains, we embarked on genetically engineering the betalain pathway in G. hirsutum, the world's largest plant-based fibre commodity. We designed constructs that included the coding sequences (CDS) of BvDODA1 (Beta vulgaris, GeneBank ID HQ656027.1), BvCYP76AD1 (HQ656023.1) and MjcDOPA5GT (Mirabilis jalapa, AB182643.1; Polturak et al., 2017; Timoneda et al., 2019; Figure 1a,b). The CDSs were optimized for Arabidopsis and synthesized by GeneArt (Table S1). Constructs (pAGM4723 vector) were assembled via Golden Gate cloning with a 2 × 35 S-driven kanamycin resistance gene. The betalain genes were driven by either a 2 × 35 S constitutive promoter or a ltp3/8K12 (LTP) mid-late-stage cotton fibre-specific promoter (Figure 1b, Table S1). Transgenic plants were generated through tissue culture transformation (Murray et al., 1999; Figure 1c). Coker 315-11 was used as the recipient of transformation by infection with Agrobacterium tumefaciens strain AGL1 containing different constructs. Transgenic and wild-type (controls) cotton plants were grown in a greenhouse at 28°C/20°C (day/night) with natural light. The pattern of betalain production differed strongly between these two promoters. Under the fibre-specific promoter, betalain pigments were successfully produced in cotton fibres and resulted in visibly pink fibres (Figure 1d–f). Plants were either green or partially purple (leaky expression in old tissues) and flowers resembled those of control plants at 0 days postanthesis (DPA; Figure S1). By contrast, the constitutive promoter-driven construct generated purple/violet plants with purple leaves, stems, boll coats and bracts (Figure S1). The flowers were pink at opening (0 DPA) while wild type had green leaves with creamy yellow flowers (Figure S1). However, constitutive promoter-driven constructs generated white fibres throughout development, similar to wild type, although ovules and mature seed interior were pink/purple (Figure S1). Despite strong betalain accumulation during fibre development in the fibre-specific lines, the colour faded to light brown/pink in the final days of boll maturation, when the bolls dried and opened (transgenic lines: 55–60 DPA vs wild type: 55 DPA; Figures 1e, S1). This suggests that vacuole-located betalain (Figure S2) was degraded during the final maturation stage. Cotton bolls were collected at around 10, 15, 20, 25, 30, 40, 50 and 60 DPA, and boll coats were either cut open or removed entirely followed by 48 h of freeze-drying, and the pink colour was retained (Figures 1f, S1). Mature fibre or freeze-dried immature fibre (>46 DPA) from controls and five independent T0 plants with the fibre-specific betalain expression were measured by Cottonscope for fibre quality. The results suggested the transgenic lines have the potential to present similar maturity ratio and fineness as the wild type (Line 44, Figure 1g). The seed numbers and fibre yield were less in the T0 compared with wild type, which is common to see in T0 generation (Figure S3). Subsequent generations of transgenic plants could potentially retain wild-type-like yield and fibre quality alongside strong pigment accumulation, noting that Coker 315 can be introgressed into modern elite varieties for yield and quality. New colours may be generated by crossing the betalain lines with existing naturally coloured genotypes (Ke et al., 2022). In conclusion, we present a novel example of introducing the exogenous multi-gene betalain pathway to generate a plant-made pink cotton fibre which remains pink until the very late stages of fibre development. Using the betalain pathway is advantageous because the pigment is stable over a wide pH range (Jackman and Smith, 1996), potentially improving colour stability and consistency. Future research could investigate reducing pigment degradation (potentially via cross-linking) and introducing new colours. We thank Lissette Perez and Dina Yulia for technical support, Drs Danny Llewellyn and Phil Hands for helpful advice and support, and the CSIRO Synthetic Traits Group for Golden Gate vectors and support. The authors declare no conflict of interest. This research was funded by the CSIRO SynBio Future Science Platform. MM, VR, CM and FP contributed to the conceptualization. MM, VR, CM, FP and XL contributed to the methodology. XL contributed to the formal analysis. SA, MM, XL, VR, CM and FP contributed to the investigation. XL contributed to the writing—original draft. XL, MM, CM, FP and VR contributed to the writing—review and editing. CM, FP and VR contributed to the supervision. FP, MM, CM and XL contributed to the project administration. FP and CM contributed to funding acquisition. The data are available in the article and accompanying supplementary material. Table S1 Betalain-pathway gene coding sequences (CDS) and LTP promoter. Figure S1 Images: cotton plants, seeds, mature bolls, freeze-dried immature bolls, stem sections and T1 plants. Figure S2 Betalain fluorescence. Figure S3 Yield of T0. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The role of glycoproteins as key cell surface molecules during development and stress is well established; yet, the relationship between their structural features and functional mechanisms is poorly defined. FASCICLIN-LIKE ARABINOGALACTAN PROTEINs (FLAs), which impact plant growth and development, are an excellent example of a glycoprotein family with a complex multidomain structure. FLAs combine globular fasciclin-like (FAS1) domains with regions that are intrinsically disordered and contain glycomotifs for directing the addition of O-linked arabinogalactan (AG) glycans. Additional posttranslational modifications on FLAs include N-linked glycans in the FAS1 domains, a cleaved signal peptide at the N terminus, and often a glycosylphosphatidylinositol (GPI) anchor signal sequence at the C terminus. The roles of glycosylation, the GPI anchor, and FAS1 domain functions in the polysaccharide-rich extracellular matrix of plants remain unclear, as do the relationships between them. In this study, we examined sequence-structure-function relationships of Arabidopsis (Arabidopsis thaliana) FLA11, demonstrated to have roles in secondary cell wall (SCW) development, by introducing domain mutations and functional specialization through domain swaps with FLA3 and FLA12. We identified FAS1 domains as essential for FLA function, differentiating FLA11/FLA12, with roles in SCW development, from FLA3, specific to flowers and involved in pollen development. The GPI anchor and AG glycosylation co-regulate the cell surface location and release of FLAs into cell walls. The AG glycomotif sequence closest to the GPI anchor (AG2) is a major feature differentiating FLA11 from FLA12. The results of our study show that the multidomain structure of different FLAs influences their subcellular location and biological functions during plant development.

Semidwarfing genes have greatly increased wheat yields globally, yet the widely used gibberellin (GA)-insensitive genes Rht-B1b and Rht-D1b have disadvantages for seedling emergence. Use of the GA-sensitive semidwarfing gene Rht13 avoids this pleiotropic effect. Here, we show that Rht13 encodes a nucleotide-binding site/leucine-rich repeat (NB-LRR) gene. A point mutation in the semidwarf Rht-B13b allele autoactivates the NB-LRR gene and causes a height reduction comparable with Rht-B1b and Rht-D1b in diverse genetic backgrounds. The autoactive Rht-B13b allele leads to transcriptional up-regulation of pathogenesis-related genes including class III peroxidases associated with cell wall remodeling. Rht13 represents a new class of reduced height (Rht) gene, unlike other Rht genes, which encode components of the GA signaling or metabolic pathways. This discovery opens avenues to use autoactive NB-LRR genes as semidwarfing genes in a range of crop species, and to apply Rht13 in wheat breeding programs using a perfect genetic marker.

Summary Secondary cell walls (SCWs) in stem xylem vessel and fibre cells enable plants to withstand the enormous compressive forces associated with upright growth. It remains unclear if xylem vessel and fibre cells can directly sense mechanical stimuli and modify their SCW during development. We provide evidence that Arabidopsis SCW‐specific Fasciclin‐Like Arabinogalactan‐proteins 11 (FLA11) and 12 (FLA12) are possible cell surface sensors regulating SCW development in response to mechanical stimuli. Plants overexpressing FLA11 (OE‐FLA11) showed earlier SCW development compared to the wild‐type (WT) and altered SCW properties that phenocopy WT plants under compression stress. By contrast, OE‐FLA12 stems showed higher cellulose content compared to WT plants, similar to plants experiencing tensile stress. fla11, OE‐FLA11, fla12, and OE‐FLA12 plants showed altered SCW responses to mechanical stress compared to the WT. Quantitative polymerase chain reaction (qPCR) and RNA‐seq analysis revealed the up‐regulation of genes and pathways involved in stress responses and SCW synthesis and regulation. Analysis of OE‐FLA11 nst1 nst3 plants suggests that FLA11 regulation of SCWs is reliant on classical transcriptional networks. Our data support the involvement of FLA11 and FLA12 in SCW sensing complexes to fine‐tune both the initiation of SCW development and the balance of lignin and cellulose synthesis/deposition in SCWs during development and in response to mechanical stimuli.

Background: Intersectionality contests that individuals have multiple characteristics in their identity that cannot be siloed or deemed exclusive to each other. Understanding and utilising an intersectional lens in organisations can increase inclusion of individuals and organisational performance. An educational package known as the Intersectionality Walk (IW) was developed by the authors, piloted, and evaluated in order to break down the commonly held descriptors of diversity silos that fragments inclusion, and to understand how various identity characteristics compound disadvantage. The paper outlines the need to transition from siloed views of diversity to a more intrinsic view of identity to achieve inclusivity. Methods: The IW was developed and trialled with a series of work-based scenarios and realistic multifaceted personas. Data collection occurred pre- and post-IW utilising a mixed methods approach. Responses to Likert scale surveys and open-ended questions were captured and analysed via inductive and grounded theory perspectives. Results: An improved awareness and understanding of individual knowledge, reflectivity and positionality relating to intersectionality and intersectional approaches was reported on completion of the IW. Furthermore, responses reported how and why organisations can approach and improve inclusivity via using intersectional approaches. Conclusions: The IW as an educational package has a positive impact and is a key linkage for all employers to build an inclusive culture and to harness the talent of all employees. Further research will occur to measure the implemented change in organisations following the IW.

The predominant Fascilin 1 (FAS1)-containing proteins in plants belong to the Fasciclin-Like Arabinogalactan-protein (FLA) family of extracellular glycoproteins. In addition to FAS1 domains, these multi-domain FLA proteins contain glycomotif regions predicted to direct addition of large arabinogalactan (AG) glycans and many contain signal sequences for addition of a glycosylphosphatidylinositol (GPI)-anchor to tether them to the plasma membrane. FLAs are proposed to play both structural and signaling functions by forming a range of interactions in the plant extracellular matrix, similar to FAS1-containing proteins in animals. FLA group B members contain two FAS1 domains and are not predicted to be GPI-anchored. None of the group B members have been functionally characterized or their sub-cellular location resolved, limiting understanding of their function. We investigated the group B FLA16 in Arabidopsis that is predominantly expressed in inflorescence tissues. FLA16 is the most highly expressed FLA in the stem after Group A members FLA11 and FLA12 that are stem specific. A FLA16-YFP fusion protein driven by the endogenous putative FLA16 promoter in wild type background showed expression in cells with secondary cell walls, and FLA16 displayed characteristics of cell wall glycoproteins with moderate glycosylation. Investigation of a fla16 mutant showed loss of FLA16 leads to reduced stem length and altered biomechanical properties, likely as a result of reduced levels of cellulose. Immuno-labeling indicated support for FLA16 location to the plasma-membrane and (apoplastic) cell wall of interfascicular stem fiber cells. Together these results indicate FLA16, a two-FAS1 domain FLAs, plays a role in plant secondary cell wall synthesis and function.

Semidwarfing genes have improved crop yield by reducing height, improving lodging resistance, and allowing plants to allocate more assimilates to grain growth. In wheat (Triticum aestivum), the Rht18 semidwarfing gene was identified and deployed in durum wheat before it was transferred into bread wheat, where it was shown to have agronomic potential. Rht18, a dominant and gibberellin (GA) responsive mutant, is genetically and functionally distinct from the widely used GA-insensitive semidwarfing genes Rht-B1b and Rht-D1b In this study, the Rht18 gene was identified by mutagenizing the semidwarf durum cultivar Icaro (Rht18) and generating mutants with a range of tall phenotypes. Isolating and sequencing chromosome 6A of these "overgrowth" mutants showed that they contained independent mutations in the coding region of GA2oxA9GA2oxA9 is predicted to encode a GA 2-oxidase that metabolizes GA biosynthetic intermediates into inactive products, effectively reducing the amount of bioactive GA (GA1). Functional analysis of the GA2oxA9 protein demonstrated that GA2oxA9 converts the intermediate GA12 to the inactive metabolite GA110 Furthermore, Rht18 showed higher expression of GA2oxA9 and lower GA content compared with its tall parent. These data indicate that the increased expression of GA2oxA9 in Rht18 results in a reduction of both bioactive GA content and plant height. This study describes a height-reducing mechanism that can generate new genetic diversity for semidwarfism in wheat by combining increased expression with mutations of specific amino acid residues in GA2oxA9.

Axial growth in plant stems requires a fine balance between elongation and stem mechanical reinforcement to ensure mechanical stability. Strength is provided by the plant cell wall, the deposition of which must be coordinated with cell expansion and elongation to ensure that integrity is maintained during growth. Coordination of these processes is critical and yet poorly understood. The plant-specific calpain, DEFECTIVE KERNEL1 (DEK1), plays a key role in growth coordination in leaves, yet its role in regulating stem growth has not been addressed. Using plants overexpressing the active CALPAIN domain of DEK1 (CALPAIN OE) and a DEK1 knockdown line (amiRNA-DEK1), we undertook morphological, biochemical, biophysical, and microscopic analyses of mature inflorescence stems. We identify a novel role for DEK1 in the maintenance of cell wall integrity and coordination of growth during inflorescence stem development. CALPAIN OE plants are significantly reduced in stature and have short, thickened stems, while amiRNA-DEK1 lines have weakened stems that are unable to stand upright. Microscopic analyses of the stems identify changes in cell size, shape and number, and differences in both primary and secondary cell wall thickness and composition. Taken together, our results suggest that DEK1 influences primary wall growth by indirectly regulating cellulose and pectin deposition. In addition, we observe changes in secondary cell walls that may compensate for altered primary cell wall composition. We propose that DEK1 activity is required for the coordination of stem strengthening with elongation during axial growth.

SummaryLateral Organ Boundaries Domain (LBD) proteins are plant‐specific transcription factors playing crucial roles in growth and development. However, the function of LBD proteins in Eucalyptus grandis remains largely unexplored. In this study, LBD genes in E. grandis were identified and characterized using bioinformatics approaches. Gene expression patterns in various tissues and the transcriptional responses of EgLBDs to exogenous hormones were determined by qRT‐PCR. Functions of the selected EgLBDs were studied by ectopically overexpressing in a hybrid poplar (Populus alba × Populus glandulosa). Expression levels of genes in the transgenic plants were investigated by RNA‐seq. Our results showed that there were forty‐six EgLBD members in the E. grandis genome and three EgLBDs displayed xylem‐ (EgLBD29) or phloem‐preferential expression (EgLBD22 and EgLBD37). Confocal microscopy indicated that EgLBD22, EgLBD29 and EgLBD37 were localized to the nucleus. Furthermore, we found that EgLBD22, EgLBD29 and EgLBD37 were responsive to the treatments of indol‐3‐acetic acid and gibberellic acid. More importantly, we demonstrated EgLBDs exerted different influences on secondary growth. Namely, 35S::EgLBD37 led to significantly increased secondary xylem, 35S::EgLBD29 led to greatly increased phloem fibre production, and 35S::EgLBD22 showed no obvious effects. We revealed that key genes related to gibberellin, ethylene and auxin signalling pathway as well as cell expansion were significantly up‐ or down‐regulated in transgenic plants. Our new findings suggest that LBD genes in E. grandis play important roles in secondary growth. This provides new mechanisms to increase wood or fibre production.

Upon publication of the original article [1], the authors had flagged that Fig. 1 had been published twice, as both Fig. 1 and Additional file 3.

The tiller inhibition gene (tin) that reduces tillering in wheat (Triticum aestivum) is also associated with large spikes, increased grain weight, and thick leaves and stems. In this study, comparison of near-isogenic lines (NILs) revealed changes in stem morphology, cell wall composition, and stem strength. Microscopic analysis of stem cross-sections and chemical analysis of stem tissue indicated that cell walls in tin lines were thicker and more lignified than in free-tillering NILs. Increased lignification was associated with stronger stems in tin plants. A candidate gene for tin was identified through map-based cloning and was predicted to encode a cellulose synthase-like (Csl) protein with homology to members of the CslA clade. Dinucleotide repeat-length polymorphism in the 5 'UTR region of the Csl gene was associated with tiller number in diverse wheat germplasm and linked to expression differences of Csl transcripts between NILs. We propose that regulation of Csl transcript and/or protein levels affects carbon partitioning throughout the plant, which plays a key role in the tin phenotype.

and poplar have been investigated, detailed examination of lignin deposition in agriculturally important crops such as cotton has been very limited. Lignin is a complex polymeric structure of phenolic compounds containing monolignols (coniferyl alcohol (guaiacyl, G-lignin monomer), sinapyl alcohol (sinapyl, S-lignin monomer), p-coumaryl alcohol (hydroxyphenyl, H-lignin monomer) and 5-OH coniferyl alcohol (5-hydroxyguaiacyl, 5-OH G-lignin monomer)) [1]. The key genes involved in lignin biosynthesis have been identified and functionally characterized in model plants allowing the realisation of lignin manipulation for potential useful application in plants (reviewed by Bedon and Legay) [2]. The lignin monomers are generally associated with the plant secondary cell wall (SCW) typified by specialised cells such as xylem vessels, xylem fibres in angiosperms, and tracheids in gymnosperms. The SCWs in these cells function to allow water transport via xylem vessels and tracheids, to provide mechanical support via tracheids, xylem fibres and phloem fibres, and to protect against biotic attack and abiotic stresses. The physical properties of lignin that contribute to its roles in different cell types is highly dependent on its quantity and quality, the latter determined by the ratio of the monolignols and how they are covalently linked. Cotton

Fasciclin-like arabinogalactan protein (FLA) families have been identified and characterised in key plant species, with some members exhibiting functional specialization. Here we identify the FLA family of Eucalyptus grandis, and investigate the roles of three single-FAS domain FLAs, with particular focus on secondary cell-wall formation and wood properties. We use various in-silico approaches to identify and characterise E. grandis genome FLAs, and perform phylogenetic comparisons with other species. For three key FLAs, we perform functional testing including promoter-reporter and overexpression transgenic approaches using eucalypts, poplar and tobacco. Of the 18 eucalypt FLAs identified, several were specifically and highly expressed in stems. The specificity to stem xylem vessel and fibre development was demonstrated with EniFLA1promoter:GUS studies in several species. Testing of select eucalypt FLAs resulted in altered wood development and properties, for example 35S:EgrFLA2 led to a 3 degree reduction in cellulose microfibril angle in eucalypt xylem fibres, and 35S:EgrFLA3 to a reduction in tobacco stem flexural strength. These results indicate that the eucalypt FLA family contains diverse members, and particular members with single FAS domains that are functionally specialized for secondary cell wall growth and properties.

•Arabidopsis inflorescence stems have wood-like biochemical and biophysical traits.•Many molecular/biophysical parallels exist between Arabidopsis and woody plants.•Cell wall proteins may be more important for wood structure than currently thought.•Long-day-grown inflorescences are useful for accession variation wood gene discovery.•CLE41/44-TDIF is conserved and may be a marker for wood formation capacity.

The natural trait variation in Arabidopsis thaliana (L.) Heynh. accessions is an important resource for understanding many biological processes but it is underexploited for wood-related properties. Twelve A. thaliana accessions from diverse geographical locations were examined for variation in secondary growth, biomechanical properties, cell wall glycan content, cellulose microfibril angle (MFA) and flowering time. The effect of daylength was also examined. Secondary growth in rosette and inflorescence stems was observed in all accessions. Organised cellulose microfibrils in inflorescence stems were found in plants grown under long and short days. A substantial range of phenotypic variation was found in biochemical and wood-related biophysical characteristics, particularly for tensile strength, tensile stiffness, MFA and some cell wall components. The four monosaccharides galactose, arabinose, rhamnose and fucose strongly correlated with each other as well as with tensile strength and MFA, consistent with mutations in arabinogalactan protein and fucosyl-and xyloglucan galactosyl-transferase genes that result in decreases in strength. Conversely, these variables showed negative correlations with lignin content. Our data support the notion that large-scale natural variation studies of wood-related biomechanical and biochemical properties of inflorescence stems will be useful for the identification of novel genes important for wood formation and quality, and therefore biomaterial and renewable biofuel production.

The ancient cell adhesion fasciclin (FAS) domain is found in bacteria, fungi, algae, insects and animals, and occurs in a large family of fasciclin-like arabinogalactan proteins (FLAs) in higher plants. Functional roles for FAS-containing proteins have been determined for insects, algae and vertebrates; however, the biological functions of the various higher-plant FLAs are not clear. Expression of some FLAs has been correlated with the onset of secondary-wall cellulose synthesis in Arabidopsis stems, and also with wood formation in the stems and branches of trees, suggesting a biological role in plant stems. We examined whether FLAs contribute to plant stem biomechanics. Using phylogenetic, transcript abundance and promoter-GUS fusion analyses, we identified a conserved subset of single FAS domain FLAs (group A FLAs) in Eucalyptus and Arabidopsis that have specific and high transcript abundance in stems, particularly in stem cells undergoing secondary-wall deposition, and that the phylogenetic conservation appears to extend to other dicots and monocots. Gene-function analyses revealed that Arabidopsis T-DNA knockout double mutant stems had altered stem biomechanics with reduced tensile strength and a reduced tensile modulus of elasticity, as well as altered cell-wall architecture and composition, with increased cellulose microfibril angle and reduced arabinose, galactose and cellulose content. Using materials engineering concepts, we relate the effects of these FLAs on cell-wall composition with stem biomechanics. Our results suggest that a subset of single FAS domain FLAs contributes to plant stem strength by affecting cellulose deposition, and to the stem modulus of elasticity by affecting the integrity of the cell-wall matrix.

Summary We used association studies to identify allelic variation in genes that influence wood fibre development in Eucalyptus nitens (Deane & Maiden). Genes selected for analysis were differentially expressed in wood with contrasting properties such as cellulose and lignin content, pulp yield and microfibril angle (MFA). Single nucleotide polymorphisms (SNPs) were identified by sequencing the candidate genes in a number of unrelated individuals. Selected SNPs were genotyped across 420 unrelated E. nitens trees from central Victorian populations and growing in a provenance trial at Meunna in north-western Tasmania. Significantly associated SNPs were genotyped across two other populations in northern Tasmania in order to validate associated SNPs. We have compiled a database of phenotypic information relating particularly to wood fibre properties for each individual in the association and validation populations. Associations between SNPs and wood properties were identified by comparing trait means in different SNP genotype classes. Several significantly associated SNPs identified in the Meunna population were validated in the other populations. The direction of the allele effect was reversed for two SNPs that were associated with kraft pulp yield. DNA markers identified in this research may be used to complement existing selection methods in breeding programs.