Secondary Wall Formation Research Articles

Growing season and radial growth predicted for Fagus sylvatica under climate change

Climate scenarios for Slovenia suggest an increase in the mean annual temperature by 2 °C over the next six decades, associated with changes in the seasonal distribution of precipitation. European beech is an ecologically and economically important forest species in Europe, so it is important to understand the influence of changing conditions on its phenology and productivity for the upcoming years. We hypothesise that the ongoing warming and reduction in precipitation during the growing season will shorten the period of xylem development, thus limiting beech growth in the next decades. Xylem formation was monitored weekly from 2008 to 2016 at two sites in Slovenia. Onset and cessation of cell enlargement and secondary wall formation, as well as xylem growth, are used to evaluate climate-growth relationships by means of partial least squares regression and to predict xylem formation phenology and annual xylem increments under climate change scenarios. A positive correlation of spring phenological phases with March–May temperatures is found. In contrast, autumn phenological phases show a negative correlation with August and September temperatures, while high temperatures at the beginning of the year delay growth cessation. According to the selected climate change scenarios, phenological phases may advance by 2 days decade-1 in spring and delay by 1.5 days decade-1 in autumn. The duration of the growing season may increase by 20 days over the next six decades, resulting in 38 to 83% wider xylem increments. The growth of beech is expected to increase under a warming climate in the sites characterised by abundant water availability.

Proline-rich protein gene PdPRP regulates secondary wall formation in poplar

Proline-rich protein (PRP) is a plant cell wall associated protein. Its distinct patterns of regulation and localization studied in a number of plants indicate that it may play important roles in growth and development. However, the mechanism of how these genes control secondary cell wall development in tree species is largely unknown. Here, we report that a Populus deltoides (Marsh.) proline-rich protein gene PdPRP was preferentially expressed in immature/mature phloem and immature xylem in P. deltoides. PdPRP overexpression increased poplar plant height and diameter as well as the radial width of the phloem and xylem regions, facilitated secondary wall deposition, and induced expression of genes related to microfibril angle (MFA) and secondary wall biosynthesis. Downregulation of PdPRP retarded poplar growth, decreased the radial width of the secondary phloem and secondary xylem regions, reduced secondary wall thickening in fibers and vessels, and decreased the expression of genes related to MFA and secondary wall biosynthesis. These results suggest that PdPRP might positively regulate secondary cell wall formation by promoting secondary wall thickening and expansion in poplar. PdPRP-overexpressing poplar had a lower MFA, indicating that PdPRP may be useful for improving wood stiffness and properties in plants. Together, our results demonstrate that PdPRP is a proline-rich protein associated with cell wall development, playing a critical role in regulating secondary cell wall formation in poplar.

Gene regulatory networks for lignin biosynthesis in switchgrass (Panicum virgatum).

SummaryCell wall recalcitrance is the major challenge to improving saccharification efficiency in converting lignocellulose into biofuels. However, information regarding the transcriptional regulation of secondary cell wall biogenesis remains poor in switchgrass (Panicum virgatum), which has been selected as a biofuel crop in the United States. In this study, we present a combination of computational and experimental approaches to develop gene regulatory networks for lignin formation in switchgrass. To screen transcription factors (TFs) involved in lignin biosynthesis, we developed a modified method to perform co‐expression network analysis using 14 lignin biosynthesis genes as bait (target) genes. The switchgrass lignin co‐expression network was further extended by adding 14 TFs identified in this study, and seven TFs identified in previous studies, as bait genes. Six TFs (PvMYB58/63, PvMYB42/85, PvMYB4, PvWRKY12, PvSND2 and PvSWN2) were targeted to generate overexpressing and/or down‐regulated transgenic switchgrass lines. The alteration of lignin content, cell wall composition and/or plant growth in the transgenic plants supported the role of the TFs in controlling secondary wall formation. RNA‐seq analysis of four of the transgenic switchgrass lines revealed downstream target genes of the secondary wall‐related TFs and crosstalk with other biological pathways. In vitro transactivation assays further confirmed the regulation of specific lignin pathway genes by four of the TFs. Our meta‐analysis provides a hierarchical network of TFs and their potential target genes for future manipulation of secondary cell wall formation for lignin modification in switchgrass.

Pathway-specific genetic pretreatment strategy to improve bioenergy feedstock

The plant secondary wall is composed of a complex mixture of cellulose, hemicellulose (e.g., xylan), and lignin. The transcription factor MYB46 (At5g12870) has been reported as a central regulator of the secondary wall formation. However, constitutive overexpression of MYB46 results in a severe growth penalty, possibly due to ectopic lignification in the parenchymatous and photosynthetic cells. To test this hypothesis, we constitutively overexpressed MYB46 in the genetic background of a T-DNA insertion mutant 4cl1-2 of 4-coumarate-CoA ligase 1 (4CL1), a key lignin biosynthesis gene. As expected, the transgenic Arabidopsis plants 35S::MYB46/4cl1-2 grow normally, similarly to the 4cl1-2 mutant, with elevated expressions of three secondary wall cellulose synthase genes and xylan biosynthesis genes. Consistent with the gene expression changes, the immunohistological staining of stem tissues showed the increased accumulation of both cellulose and xylan contents in the 35S::MYB46/4cl1-2 plants. Further biochemical analyses confirmed that 35S::MYB46/4cl1-2 plants have higher level accumulations of crystalline cellulose and xylan in both leaf and stem tissues than wild-type control plants as well as the 4cl1-2 mutant, up to 1.18- and 1.13-fold respectively, with considerably reduced lignin content (up to 0.40-fold). Subsequent analysis of enzymatic glucose release from cell wall materials revealed that 35S::MYB46/4cl1-2 plants have higher yields up to 1.27-fold increase in both leaf and stem tissues than that of control plants and 4cl1-2 mutant. Our results showed that overexpression of MYB46 with simultaneous reduction of lignin biosynthesis significantly increases both fermentable sugar contents and cell wall digestibility without growth penalty associated with MYB46 overexpression.

Current Models for Transcriptional Regulation of Secondary Cell Wall Biosynthesis in Grasses

Secondary cell walls mediate many crucial biological processes in plants including mechanical support, water and nutrient transport and stress management. They also provide an abundant resource of renewable feed, fiber, and fuel. The grass family contains the most important food, forage, and biofuel crops. Understanding the regulatory mechanism of secondary wall formation in grasses is necessary for exploiting these plants for agriculture and industry. Previous research has established a detailed model of the secondary wall regulatory network in the dicot model species Arabidopsis thaliana. Grasses, branching off from the dicot ancestor 140–150 million years ago, display distinct cell wall morphology and composition, suggesting potential for a different secondary wall regulation program from that established for dicots. Recently, combined application of molecular, genetic and bioinformatics approaches have revealed more transcription factors involved in secondary cell wall biosynthesis in grasses. Compared with the dicots, grasses exhibit a relatively conserved but nevertheless divergent transcriptional regulatory program to activate their secondary cell wall development and to coordinate secondary wall biosynthesis with other physiological processes.

Wood formation in Norway spruce on a lowland site in Slovenia in 2015 and comparison with other conifers all over Europe

We present xylem formation in Norway spruce (Picea abies (L.) Karst.) in 2015 at Panška reka, near Ljubljana, Slovenia (ca. 46°00’N, 14°40’E, 400 m a.s.l.) with a temperate climate. The research was a part of a long-term project, which involves different sites and tree species. We measured the widths of cambium and the formation of xylem growth rings with differentiating cells in postcambial (PC) and secondary wall formation (SW) phases, and mature cells (MT). The results for 2015 were compared with those for previous years (2009–2011) in the same species and site, and with the published data from wood formation studies in conifers from all over Europe. The latter were used in two-factor regression models which confirmed the effects of latitude and altitude on the critical dates of onset, end, and duration of xylem production. The models thus helped us to predict/reconstruct phenology of wood formation in conifers in Europe.

An Uncanonical CCCH-Tandem Zinc-Finger Protein Represses Secondary Wall Synthesis and Controls Mechanical Strength in Rice

Secondary walls, which represent the bulk of biomass, have a large impact on plant growth and adaptation to environments. Secondary wall synthesis is switched and regulated by a sophisticated signaling transduction network. However, there is limited understanding of these regulatory pathways. Here, we report that ILA1-interacting protein 4 (IIP4) can repress secondary wall synthesis. IIP4 is a phosphorylation substrate of an Raf-like MAPKKK, but its function is unknown. By generating iip4 mutants and relevant transgenic plants, we found that lesions in IIP4 enhance secondary wall formation. Gene expression and transactivation activity assays revealed that IIP4 negatively regulates the expression of MYB61 and CESAs but does not bind their promoters. IIP4 interacts with NAC29/NAC31, the upstream regulators of secondary wall synthesis, and suppresses the downstream regulatory pathways in plants. Mutagenesis analyses showed that phosphomimic IIP4 proteins translocate from the nucleus to the cytoplasm, which releases interacting NACs and attenuates its repression function. Moreover, we revealed that IIPs are evolutionarily conserved and share unreported CCCH motifs, referred to as uncanonical CCCH-tandem zinc-finger proteins. Collectively, our study provides mechanistic insights into the control of secondary wall synthesis and presents an opportunity for improving relevant agronomic traits in crops.

Characterization of xylan in the early stages of secondary cell wall formation in tobacco bright yellow-2 cells

The major polysaccharides present in the primary and secondary walls surrounding plant cells have been well characterized. However, our knowledge of the early stages of secondary wall formation is limited. To address this, cell walls were isolated from differentiating xylem vessel elements of tobacco bright yellow-2 (BY-2) cells induced by VASCULAR-RELATED NAC-DOMAIN7 (VND7). The walls of induced VND7-VP16-GR BY-2 cells consisted of cellulose, pectic polysaccharides, hemicelluloses, and lignin, and contained more xylan and cellulose compared with non-transformed BY-2 and uninduced VND7-VP16-GR BY-2 cells. A reducing end sequence of xylan containing rhamnose and galaturonic acid- residues is present in the walls of induced, uninduced, and non-transformed BY-2 cells. Glucuronic acid residues in xylan from walls of induced cells are O-methylated, while those of xylan in non-transformed BY-2 and uninduced cells are not. Our results show that xylan changes in chemical structure and amounts during the early stages of xylem differentiation.

ZmNST3 and ZmNST4 are master switches for secondary wall deposition in maize (Zea mays L.)

Secondary walls are the most abundant biomass produced by plants, and they consist mainly of lignin, cellulose and hemicellulose. Understanding how secondary wall biosynthesis is regulated could potentially provide genetic tools for engineering biomass components, especially in maize and Sorghum bicolor. Although many works have focused on secondary wall biosynthesis in dicotyledons, little has been reported for these monocotyledons. In this study, we cloned two NAC transcriptional factor genes, ZmNST3 and ZmNST4, and analyzed their functions in maize secondary wall formation process. ZmNST3 and ZmNST4 were expressed specifically in secondary wall-forming cells, expression of ZmNST3/4 can restore the pendent phenotype of Arabidopsis nst1nst3 double mutant. ZmNST3/4-overexpressing Arabidopsis and maize displayed a thickened secondary wall in the stem, and knockdown maize showed defective secondary wall deposition. ZmNST3/4 could regulate the expression of ZmMYB109/128/149. Our results revealed that ZmNST3/4 are master switches of the maize secondary wall biosynthesis process and provides new evidence that the secondary wall regulatory pathway is conserved in different plant species.

MicroRNA expression profiles during cotton (Gossypium hirsutum L) fiber early development

The role of microRNAs (miRNAs) during cotton fiber development remains unclear. Here, a total of 54 miRNAs belonging to 39 families were selected to characterize miRNA regulatory mechanism in eight different fiber development stages in upland cotton cv BM-1. Among 54 miRNAs, 18 miRNAs were involved in cotton fiber initiation and eight miRNAs were related to fiber elongation and secondary wall biosynthesis. Additionally, 3,576 protein-coding genes were candidate target genes of these miRNAs, which are potentially involved in cotton fiber development. We also investigated the regulatory network of miRNAs and corresponding targets in fiber initiation and elongation, and secondary wall formation. Our Gene Ontology-based term classification and KEGG-based pathway enrichment analyses showed that the miRNA targets covered 220 biological processes, 67 molecular functions, 45 cellular components, and 10 KEGG pathways. Three of ten KEGG pathways were involved in lignan synthesis, cell elongation, and fatty acid biosynthesis, all of which have important roles in fiber development. Overall, our study shows the potential regulatory roles of miRNAs in cotton fiber development and the importance of miRNAs in regulating different cell types. This is helpful to design miRNA-based biotechnology for improving fiber quality and yield.

Functional Characterization of Populus PsnSHN2 in Coordinated Regulation of Secondary Wall Components in Tobacco

Wood formation is a biological process during which the most abundant lignocellulosic biomass on earth is produced. Although a number of transcription factors have been linked to the regulation of wood formation process, none of them has been demonstrated to be a higher hierarchical regulator that coordinately regulates secondary wall biosynthesis genes. Here, we identified a Populus gene, PsnSHN2, a counterpart of the Arabidopsis AP2/ERF type transcription factor, SHINE2. PsnSHN2 is predominantly expressed in xylem tissues and acted evidently as a high hierarchical transcriptional activator. Overexpression of PsnSHN2 in tobacco significantly altered the expression of both transcription factors and biosynthesis genes involved in secondary wall formation, leading to the thickened secondary walls and the changed cell wall composition. The most significant changes occurred in the contents of cellulose and hemicellulose that increased 37% and 28%, respectively, whereas the content of lignin that decreased 34%. Furthermore, PsnSHN2 activated or repressed the promoter activities of transcription factors involved in secondary wall biosynthesis and bound to five cis-acting elements enriched in the promoter regions of these transcription factors. Taken together, our results suggest PsnSHN2 coordinately regulate secondary wall formation through selective up/down-regulation of its downstream transcription factors that control secondary wall formation.

Downregulation of RWA genes in hybrid aspen affects xylan acetylation and wood saccharification

High acetylation of angiosperm wood hinders its conversion to sugars by glycoside hydrolases, subsequent ethanol fermentation and (hence) its use for biofuel production. We studied the REDUCED WALL ACETYLATION (RWA) gene family of the hardwood model Populus to evaluate its potential for improving saccharification. The family has two clades, AB and CD, containing two genes each. All four genes are expressed in developing wood but only RWA-A and -B are activated by master switches of the secondary cell wall PtNST1 and PtMYB21. Histochemical analysis of promoter::GUS lines in hybrid aspen (Populus tremula×tremuloides) showed activation of RWA-A and -B promoters in the secondary wall formation zone, while RWA-C and -D promoter activity was diffuse. Ectopic downregulation of either clade reduced wood xylan and xyloglucan acetylation. Suppressing both clades simultaneously using the wood-specific promoter reduced wood acetylation by 25% and decreased acetylation at position 2 of Xylp in the dimethyl sulfoxide-extracted xylan. This did not affect plant growth but decreased xylose and increased glucose contents in the noncellulosic monosaccharide fraction, and increased glucose and xylose yields of wood enzymatic hydrolysis without pretreatment. Both RWA clades regulate wood xylan acetylation in aspen and are promising targets to improve wood saccharification.

Control of secondary cell wall patterning involves xylan deacetylation by a GDSL esterase.

O-acetylation, a ubiquitous modification of cell wall polymers, has striking impacts on plant growth and biomass utilization and needs to be tightly controlled. However, the mechanisms that underpin the control of cell wall acetylation remain elusive. Here, we show a rice brittle leaf sheath1 (bs1) mutant, which contains a lesion in a Golgi-localized GDSL esterase that deacetylates the prominent hemicellulose xylan. Cell wall composition, detailed xylan structure characterization and enzyme kinetics and activity assays on acetylated sugars and xylooligosaccharides demonstrate that BS1 is an esterase that cleaves acetyl moieties from the xylan backbone at O-2 and O-3 positions of xylopyranosyl residues. BS1 thus plays an important role in the maintenance of proper acetylation level on the xylan backbone, which is crucial for secondary wall formation and patterning. Our findings outline a mechanism for how plants modulate wall acetylation and endow a plethora of uncharacterized GDSL esterases with surmisable activities.

Activation tagging of Arabidopsis POLYGALACTURONASE INVOLVED IN EXPANSION2 promotes hypocotyl elongation, leaf expansion, stem lignification, mechanical stiffening, and lodging.

Pectin is the most abundant component of primary cell walls in eudicot plants. The modification and degradation of pectin affects multiple processes during plant development, including cell expansion, organ initiation, and cell separation. However, the extent to which pectin degradation by polygalacturonases affects stem development and secondary wall formation remains unclear. Using an activation tag screen, we identified a transgenic Arabidopsis thaliana line with longer etiolated hypocotyls, which overexpresses a gene encoding a polygalacturonase. We designated this gene as POLYGALACTURONASE INVOLVED IN EXPANSION2 (PGX2), and the corresponding activation tagged line as PGX2AT . PGX2 is widely expressed in young seedlings and in roots, stems, leaves, flowers, and siliques of adult plants. PGX2-GFP localizes to the cell wall, and PGX2AT plants show higher total polygalacturonase activity and smaller pectin molecular masses than wild-type controls, supporting a function for this protein in apoplastic pectin degradation. A heterologously expressed, truncated version of PGX2 also displays polygalacturonase activity invitro. Like previously identified PGX1AT plants, PGX2AT plants have longer hypocotyls and larger rosette leaves, but they also uniquely display early flowering, earlier stem lignification, and lodging stems with enhanced mechanical stiffness that is possibly due to decreased stem thickness. Together, these results indicate that PGX2 both functions in cell expansion and influences secondary wall formation, providing a possible link between these two developmental processes.

PtoMYB156 is involved in negative regulation of phenylpropanoid metabolism and secondary cell wall biosynthesis during wood formation in poplar

Some R2R3 MYB transcription factors have been shown to be major regulators of phenylpropanoid biosynthetic pathway and impact secondary wall formation in plants. In this study, we describe the functional characterization of PtoMYB156, encoding a R2R3-MYB transcription factor, from Populus tomentosa. Expression pattern analysis showed that PtoMYB156 is widely expressed in all tissues examined, but predominantly in leaves and developing wood cells. PtoMYB156 localized to the nucleus and acted as a transcriptional repressor. Overexpression of PtoMYB156 in poplar repressed phenylpropanoid biosynthetic genes, leading to a reduction in the amounts of total phenolic and flavonoid compounds. Transgenic plants overexpressing PtoMYB156 also displayed a dramatic decrease in secondary wall thicknesses of xylem fibers and the content of cellulose, lignin and xylose compared with wild-type plants. Transcript accumulation of secondary wall biosynthetic genes was down-regulated by PtoMYB156 overexpression. Transcriptional activation assays revealed that PtoMYB156 was able to repress the promoter activities of poplar CESA17, C4H2 and GT43B. By contrast, knockout of PtoMYB156 by CRISPR/Cas9 in poplar resulted in ectopic deposition of lignin, xylan and cellulose during secondary cell wall formation. Taken together, these results show that PtoMYB156 may repress phenylpropanoid biosynthesis and negatively regulate secondary cell wall formation in poplar.

A Transcriptional and Metabolic Framework for Secondary Wall Formation in Arabidopsis.

Plant cell walls are essential for plant growth and development. The cell walls are traditionally divided into primary walls, which surround growing cells, and secondary walls, which provide structural support to certain cell types and promote their functions. While much information is available about the enzymes and components that contribute to the production of these two types of walls, much less is known about the transition from primary to secondary wall synthesis. To address this question, we made use of a transcription factor system in Arabidopsis (Arabidopsis thaliana) in which an overexpressed master secondary wall-inducing transcription factor, VASCULAR-RELATED NAC DOMAIN PROTEIN7, can be redirected into the nucleus by the addition of dexamethasone. We established the time frame during which primary wall synthesis changed into secondary wall production in dexamethasone-treated seedlings and measured transcript and metabolite abundance at eight time points after induction. Using cluster- and network-based analyses, we integrated the data sets to explore coordination between transcripts, metabolites, and the combination of the two across the time points. We provide the raw data as well as a range of network-based analyses. These data reveal links between hormone signaling and metabolic processes during the formation of secondary walls and provide a framework toward a deeper understanding of how primary walls transition into secondary walls.

Chemistry of developing bordered-pit rims in balsam-fir trees

Rims of bordered pits form on the primary walls of radially enlarged cambial derivatives prior to the onset of general secondary-wall formation. A recent report (Botany, 2014, 92(7): 495–511) raised the possibility that the chemical composition of the rim might be different from that of the secondary wall. To investigate this, early-stage nonfluorescent and late-stage autofluorescent rims were separated from cambial derivatives of Abies balsamea (L.) Mill. and purified to homogeneity by density-gradient centrifugation. Solid state nuclear magnetic resonance spectroscopy, Raman microspectroscopy, combined gas chromatography – mass spectroscopy, enzyme digestion, and chemical resilience data support the interpretation that cellulose alone is the microfibrillar polysaccharide of nonfluorescent early-stage rims. A lignin is additionally present in late-stage rims, and it evidently bonds with cellulose because rims are extraordinarily resistant to hydrolysis by either enzymes or strong acid.

Living on the Edge: Contrasted Wood-Formation Dynamics in Fagus sylvatica and Pinus sylvestris under Mediterranean Conditions.

Wood formation in European beech (Fagus sylvatica L.) and Scots pine (Pinus sylvestris L.) was intra-annually monitored to examine plastic responses of the xylem phenology according to altitude in one of the southernmost areas of their distribution range, i.e., in the Moncayo Natural Park, Spain. The monitoring was done from 2011 to 2013 at 1180 and 1580 m a.s.l., corresponding to the lower and upper limits of European beech forest in this region. Microcores containing phloem, cambium and xylem were collected biweekly from twenty-four trees from the beginning of March to the end of November to assess the different phases of wood formation. The samples were prepared for light microscopy to observe the following phenological phases: onset and end of cell production, onset and end of secondary wall formation in xylem cells and onset of cell maturation. The temporal dynamics of wood formation widely differed among years, altitudes and tree species. For Fagus sylvatica, the onset of cambial activity varied between the first week of May and the third week of June. Cambial activity then slowed down and stopped in summer, resulting in a length of growing season of 48–75 days. In contrast, the growing season for P. sylvestris started earlier and cambium remained active in autumn, leading to a period of activity varying from 139-170 days. The intra-annual wood-formation pattern is site and species-specific. Comparison with other studies shows a clear latitudinal trend in the duration of wood formation, positive for Fagus sylvatica and negative for P. sylvestris.

PtrWRKY19, a novel WRKY transcription factor, contributes to the regulation of pith secondary wall formation in Populus trichocarpa

WRKY proteins are one of the largest transcription factor families in higher plants and play diverse roles in various biological processes. Previous studies have shown that some WRKY members act as negative regulators of secondary cell wall formation in pith parenchyma cells. However, the regulatory mechanism of pith secondary wall formation in tree species remains largely unknown. In this study, PtrWRKY19 encoding a homolog of Arabidopsis WRKY12 was isolated from Populus trichocarpa. PtrWRKY19 was expressed in all tissues tested, with highest expression in stems, especially in pith. PtrWRKY19 was located in the nucleus and functioned as a transcriptional repressor. Ectopic expression of PtrWRKY19 in an atwrky12 mutant successfully rescued the phenotype in pith cell walls caused by the defect of AtWRKY12, suggesting that PtrWRKY19 had conserved functions for homologous AtWRKY12. Overexpression of PtrWRKY19 in poplar plants led to a significant increase in the number of pith parenchyma cells. qRT-PCR analysis showed that lignin biosynthesis-related genes were repressed in transgenic plants. In transcient reporter assays, PtrWRKY19 was identified to repress transcription from the PtoC4H2 promoter containing the conserved W-box elements. These results indicated that PtrWRKY19 may function as a negative regulator of pith secondary wall formation in poplar.

Proteomic Analysis of Microtubule Interacting Proteins over the Course of Xylem Tracheary Element Formation in Arabidopsis

Plant vascular cells, or tracheary elements (TEs), rely on circumferential secondary cell wall thickenings to maintain sap flow. The patterns in which TE thickenings are organized vary according to the underlying microtubule bundles that guide wall deposition. To identify microtubule interacting proteins present at defined stages of TE differentiation, we exploited the synchronous differentiation of TEs in Arabidopsis thaliana suspension cultures. Quantitative proteomic analysis of microtubule pull-downs, using ratiometric (14)N/(15)N labeling, revealed 605 proteins exhibiting differential accumulation during TE differentiation. Microtubule interacting proteins associated with membrane trafficking, protein synthesis, DNA/RNA binding, and signal transduction peaked during secondary cell wall formation, while proteins associated with stress peaked when approaching TE cell death. In particular, CELLULOSE SYNTHASE-INTERACTING PROTEIN1, already associated with primary wall synthesis, was enriched during secondary cell wall formation. RNAi knockdown of genes encoding several of the identified proteins showed that secondary wall formation depends on the coordinated presence of microtubule interacting proteins with nonoverlapping functions: cell wall thickness, cell wall homogeneity, and the pattern and cortical location of the wall are dependent on different proteins. Altogether, proteins linking microtubules to a range of metabolic compartments vary specifically during TE differentiation and regulate different aspects of wall patterning.