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.

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.

Receptor-like kinases (RLKs) are instrumental in regulating plant cell surface sensing and vascular tissue differentiation. Wall-associated kinases (WAKs) are a unique group of RLKs that have been identified as key cell wall integrity (CWI) sensors. WAK signaling is suggested to be activated during growth in response to cell expansion or when the cell wall is damaged, for example, during pathogen attack. WAKs are proposed to interact with pectins or pectin fragments that are enriched in primary walls. Secondary walls have low levels of pectins, yet recent studies have shown important functions of WAKs during secondary wall development. Several wak mutants show defects in secondary wall thickening of the xylem vessels and fibers or the development of vascular bundles. This review will discuss the recent advances in our understanding of WAK functions during plant development and responses to abiotic stresses and the regulation of vascular tissue secondary wall development.

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) play an important role in a variety of plant biological processes including growth, stress response, morphogenesis, signaling, and cell wall biosynthesis. The GPI anchor contains a lipid-linked glycan backbone that is synthesized in the endoplasmic reticulum (ER) where it is subsequently transferred to the C-terminus of proteins containing a GPI signal peptide by a GPI transamidase. Once the GPI anchor is attached to the protein, the glycan and lipid moieties are remodeled. In mammals and yeast, this remodeling is required for GPI-APs to be included in Coat Protein II-coated vesicles for their ER export and subsequent transport to the cell surface. The first reaction of lipid remodeling is the removal of the acyl chain from the inositol group by Bst1p (yeast) and Post-GPI Attachment to Proteins Inositol Deacylase 1 (PGAP1, mammals). In this work, we have used a loss-of-function approach to study the role of PGAP1/Bst1 like genes in plants. We have found that Arabidopsis (Arabidopsis thaliana) PGAP1 localizes to the ER and likely functions as the GPI inositol-deacylase that cleaves the acyl chain from the inositol ring of the GPI anchor. In addition, we show that PGAP1 function is required for efficient ER export and transport to the cell surface of GPI-APs.

Wall-associated kinases/kinase-likes (WAKs/WAKLs) are plant cell surface sensors. A variety of studies have revealed the important functions of WAKs/WAKLs in regulating cell expansion and defense in cells with primary cell walls. Less is known about their roles during the development of the secondary cell walls (SCWs) that are present in xylem vessel (XV) and interfascicular fiber (IF) cells. In this study, we used RNA-seq data to screen Arabidopsis thaliana WAKs/WAKLs members that may be involved in SCW development and identified WAKL8 as a candidate. We obtained T-DNA insertion mutants wakl8-1 (inserted at the promoter region) and wakl8-2 (inserted at the first exon) and compared the phenotypes to wild-type (WT) plants. Decreased WAKL8 transcript levels in stems were found in the wakl8-2 mutant plants, and the phenotypes observed included reduced stem length and thinner walls in XV and IFs compared with those in the WT plants. Cell wall analysis showed no significant changes in the crystalline cellulose or lignin content in mutant stems compared with those in the WT. We found that WAKL8 had alternative spliced versions predicted to have only extracellular regions, which may interfere with the function of the full-length version of WAKL8. Our results suggest WAKL8 can regulate SCW thickening in Arabidopsis stems.

The clustered, regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system is the most widely used gene-editing tool to date. However, its application in the genetic improvement of forestry trees has been largely limited. Here, we first established a highly efficient multi-target editing system in the magnoliid woody plant Liriodendron tulipifera. Using phytoene desaturase gene (PDS) as an example, we systematically compared CRISPR/Cas9 and CRSPR/Cpf1 expression systems for loss-of-function analysis and conducted genetic transformations using transient and stable transformation. Ultimately, our findings indicated that the CRISPR/Cas9 system, when applied to transformation based on single-cell-originated somatic embryogenesis, yielded the highest gene-editing efficiency, with mutation rates of nearly 100%. Furthermore, we obtained a total of 137 regeneration plantlets via somatic embryogenesis, of which 82.48% exhibited an albino phenotype. The Illumina sequencing results of albino seedlings and the callus tissue obtained from dedifferentiation of mutant plants revealed that the mutation at the T1 target site was homozygous. These results indicate that CRISPR/Cas9-based multiplex genome-editing technology can not only accelerate the identification of gene function but also be incorporated into the genetic improvement and breeding of tulip trees, supporting the scale propagation of genome-edited plantlets via somatic embryogenesis.