CELLULOSE SYNTHASE9 serves a nonredundant role in secondary cell wall synthesis in Arabidopsis epidermal testa cells.

Herein, we sought to explore the contribution of cellulose biosynthesis to the shape and morphogenesis of hexagonal seed coat cells in Arabidopsis (Arabidopsis thaliana). Consistent with seed preferential expression of CELLULOSE SYNTHASE9 (CESA9), null mutations in CESA9 caused no change in cellulose content in leaves or stems, but caused a 25% reduction in seeds. Compositional studies of cesa9 seeds uncovered substantial proportional increases in cell wall neutral sugars and in several monomers of cell wall-associated polyesters. Despite these metabolic compensations, cesa9 seeds were permeable to tetrazolium salt, implying that cellulose biosynthesis, via CESA9, is required for correct barrier function of the seed coat. A syndrome of depleted radial wall, altered seed coat cell size, shape, and internal angle uniformity was quantified using scanning electron micrographs in cesa9 epidermal cells. By contrast, morphological defects were absent in cesa9 embryos, visually inspected from torpedo to bent cotyledon, consistent with no reduction in postgermination radical or hypocotyl elongation. These data implied that CESA9 was seed coat specific or functionally redundant in other tissues. Assessment of sections from glutaraldehyde fixed wild-type and cesa9 mature seeds supported results of scanning electron micrographs and quantitatively showed depletion of secondary cell wall synthesis in the radial cell wall. Herein, we show a nonredundant role for CESA9 in secondary cell wall biosynthesis in radial cell walls of epidermal seed coats and document its importance for cell morphogenesis and barrier function of the seed coat.

Perhaps one of the most important reasons for the successful radiation of land plants into the many diverse and extreme environments of our planet can be found in the evolution of seeds (Lidgard and Crane, 1988;Knapp et al., 2005). At the heart of this evolutionary step, from spore-mediated reproduction to seed-mediated reproduction (Holsinger, 2000), is the mechanistic structure of the seed. In a simple model, the seed is categorized into 3 components, the embryo, the endosperm and the seed coat (testa) (Fahn, 1990). With respect to the angiosperm testa, this portion of the seed consists of several layers of specialized tissues that are maternally inherited and differentiate from cells of the ovule integuments following fertilization (Vaughan and Whitehouse, 1971;Corner, 1976;Sagasser et al., 2002). Comprising the outermost cell layers of the seed, the testa is uniquely positioned at the interface between the embryo and the external environment and thus has evolved as a dynamic and specialized structure capable of protecting the embryo from environmental insults such as desiccation, mechanical stress, pathogen attack and UV damage (Windsor et al., 2000;Haughn and Chaudhury, 2005). For instance, there are numerous dispersal mechanisms that, whether mediated by animals, wind, or water, all require specific adaptations of the seed coat (Howe and Smallwood, 1982). The testa cells also play a major role in maintaining the dehydrated dormant state of the embryo until appropriate conditions exist (Windsor et al., 2000). A good example of the highly specialized role of testa cells is found in the epidermal seed coat layer of cotton (Gossypium hirsutum L.) from which the economically important cotton trichome or fiber is produced (Kim and Triplett, 2001). The visual appearance of the testa is also frequently used for taxonomic descriptions and to distinguish between closely related plant species (Rodin and Kapil, 1969;Chuang and Heckard, 1972).
Seed development in Arabidopsis thaliana (fruiting body are dehiscent siliques), has received substantial scientific scrutiny, and seed coats have been cytologically (Beeckman et al., 2000;Western et al., 2000;Windsor et al., 2000) and genetically (Reiser and Fisher, 1993;Klucher et al., 1996;Western et al., 2001Western et al., , 2004Dean et al., 2007;Arsovski et al., 2009ab) studied. The epidermal cells of the testa in A. thaliana form a hexameric matrix. Each epidermal testa cell also has a central columella mucilage pocket (Western et al., 2000(Western et al., , 2001. Subsequently, a thick secondary cell wall is deposited along the radial boundary wall and the outer tangential wall forming the volcano shaped columella. Upon imbibition, the pectinaceuous mucilage swells, ruptures the primary radial wall and extrudes from the seed coat and germination begins (Gutterman and Shemtov, 1996;Western et al., 2000;Western et al., 2001). The formation of the secondary cell wall includes reinforcement of the radial wall that assists in forming the unique hexagonal shapes of testa epidermal cells. This radial wall must inherently possess substantial biomechanical strength. The major load-bearing constituent of higher plant cell walls is cellulose, a polymer of 1,4-β-D-glucose residues (Brown et al., 1996). However, the molecular underpinning of seed coat reinforcement remains poorly characterized, particularly with respect to the contribution of cellulose biosynthesis.
The overarching goal of the current study sought to explore cellulose deposition in the uniquely hexagonally shaped epidermal seed coat (testa) cells. In this study, a reverse genetic approach in A. thaliana was used to show that CESA9, which was previously proposed to be a redundant component of cellulose biosynthesis, is central to the formation of the secondary wall in this cell type. With no change observed in embryogenesis, these results inferred intriguing cell type specificity for cellulose biosynthesis in the seed.

Gene expression analysis for CESA9
CESA9 encodes a 1088 amino acid protein and comprises 12 introns and 13 exons (Richmond, 2000). Gene expression (mRNA transcript abundance) of CESA9 was interrogated using GENEINVESTIGATOR expression profiling tool (Zimmerman et al., 2004). CESA9 gene expression was highest during fruit development, specifically, after stage 3 of seed development. CESA9 expression increased and peaked between stage 5 and stage 9 of seed development (data not presented, see GENEINVESTIGATOR output). Expression was low in rapidly elongating tissue such as hypocotyls or roots.
Consistent with these data, coexpression analysis (www.atted.bio.titech.ac.jp Obayashi et al., 2009) using CESA9 as bait did not reveal coexpression with any other primary or secondary cell wall genes (Fig. S1). Contrastingly, genes associated with both primary and secondary cell wall cellulose biosynthesis have previously been shown to cluster tightly together (Brown et al., 2005;Persson et al., 2005). For example, coexpression analysis performed using CESA3 as bait, identified CESA1, CESA2, CESA6, COBRA and KORRIGAN all following a tight transcriptional coexpression pattern (Fig. S1), consistent with Persson et al. (2005). Alternatively, transcripts that are coexpressed with CESA9 included (At3g25160) an ER lumen protein retaining receptor family protein, (At4g16160) ATOEP16-S protein, (At2g38905) a hydrophobic protein responsive to low temperature and salt, (At3g18570, At2g25890) two independent glycine-rich proteins/oleosins, (At3g14950) thioredoxin-like 2, (At1g48470) glutamine synthase and (At1g04920) sucrose phosphate synthase. These transcripts have no published association with cellulose biosynthesis. The presence in this cluster of oleosins, which are known to be seed-specific oil-body proteins, show that gene co-expression may be due only to seed specific transcripts and thus be unrelated to cell wall biosynthesis.

Isolation of T-DNA mutants for CESA9
Gene expression analyses showed that CESA9 was expressed during fruit development.
However, whether CESA9 was expressed in the embryo (Beeckman et al., 2002) or the seed coat was unclear. To address this and explore the role of CESA9 in seed physiology, a reverse genetic approach was taken and two alleles for CESA9 were CESA9 (cesa9-1; At2g21770) and in neither case was the phenotype of the seed examined and reported. Plants homozygous for cesa9-1 and cesa9-2 alleles were viable and displayed no growth feature differences in the mature plant, consistent with previous reports (Persson et al., 2007) (Fig. 1A). More specifically, when grown at 22º C, the growth habit of the cesa9 seedlings and mature plants showed no substantial radial swelling or dwarfing phenotypes as was expected for a cellulose deficient mutant (Sugimoto et al., 2001).

Phenotypic analysis of cesa9 plants
Quantitative (Fig. 1A) and visual inspection (Fig. 1B) revealed that cesa9 seeds were smaller in size than wild-type seed. Specifically, seed weight were 1.57 mg.100 seed -1 (± 6) in the wild-type relative to 1.23 mg.100 seed -1 (± 6) cesa9-1 and 1.20 mg.100 seed -1 (± 10) cesa9-2 (Fig. 1A). Since cesa9 displayed a dramatic seed size phenotype, we asked whether morphogenesis was affected during embryogenesis. Embryos were dissected from cesa9 and wild-type ovules, and the embryonic stages of development were compared. The early stages of cesa9 mutant development from globular to heart stage ( Fig. S3) exhibited no visible phenotypes relative to wild-type, which was consistent to mature embryo stage (not presented). No differences were observed in elongating seedlings (light or dark grown) and rosette leaf size between wild-type and the cesa9-1 and cesa9-2 alleles. Altered seed coat cell morphology was observed when performing differential interference contrast (DIC) microscopy (Normorski optics)( Fig.   2A) and incomplete clearing of testa pigment by the Hoyer's solution revealed that the ultrastructure of cesa9 was different to wild-type cells. In particular, the cell shape of cesa9 appeared less uniform than that of wild-type seed coats ( Fig. 2A).
To gain further insight into the defective cell shape phenotype, examination by scanning electron microscopy (SEM) was performed on cesa9-1 and cesa9-2 (Fig. 2B). Epidermal cell morphology was severely distorted among null cesa9 seeds relative to the uniform cell shape in wild-type seeds (Fig. 2B). In mature cesa9 seed imaged by SEM the radial cell wall was either thinner or not observable at all beneath the outer tangential cell wall draping over the columella ( Fig. 2C and Fig. 2D) suggesting that collapse in radial cell walls led to an indistinguishable border between neighboring cells and the appearance of cell fusion. Highly irregular shape was also observable in cesa9 by SEM relative to uniform cell hexagonal shapes in wild-type. Hence, loss of uniform cell shape in the mature seed was established in cesa9.

Quantitative SEM study of ablated epidermal cell shape and morphogenesis in cesa9 seed
To further distinguish altered shape parameters in the cesa9 seed coat epidermal cells, we quantitatively analyzed cell area (Fig. 3A), the area of columella ( μm 2 (standard error of 12.8 μm 2 ) relative to 717 μm 2 (standard error of 13.6 μm 2 ) in cesa9 (P>0.05 Wilcoxan Ranked Signed Test, Fig. 3), demonstrating a smaller overall cell size among epidermal testa cells in cesa9. In contrast to total cell area, columella area was measurably greater in the cesa9 (n= 300; 140 μm 2 (standard error of 2.4 μm 2 ) relative to 114 μm 2 in wild-type (n= 300; standard error of 2.2 μm 2 ) (Fig. 3B). Cell area/columella area was therefore greater in the wild-type testa cells and had a ratio of 7.28 whereas the ratio for cesa9 was 5.32 (Fig. 3C). However, the number did not provide a sufficient measure of the obvious non-uniformity seen in the cesa9 cells relative to wild-type. In an effort to obtain such an assessment, we determined the internal angle between the sides of the hexagonally shaped wild-type cells with those of cesa9 ( Fig. 3D). In wild-type cells, the average internal angle was 119.4° measured over 30 different seeds and composed of 1086 independent angle measurements. Not surprisingly, given the geometric constraints, the internal angles in cesa9 cells also averaged out to 118.5° (n=1080) however, the range of angles observed in the cesa9 was substantially greater than wild-type; for instance, the minimum internal angle was 43° and 64° respectively and the maximum internal angle was 257° and 162° respectively (Fig. 3D). Internal angles ranging from 43° to 257° are consistent with careful visual inspection that showed highly irregular and inconsistent shapes occurring between neighboring cells in cesa9 ( Fig. 3E  the mutant radial wall, although variable, appeared smaller than in wild type (Fig. 4B).
To quantify these changes, the radial wall height and width were measured from a total of 10 cells for each genotype. The results (Fig. 4C) indicate significant differences in the height of the radial walls when compared to wild type cells. Although a decrease in radial wall width was also observed the differences were not significant (Fig. 4D).

Histological and compositional studies of cesa9 seed
The relative amounts of acid insoluble glucose (relative estimate of crystalline cellulose) were examined in seed, stems and leaves of cesa9-1 and wild-type (Fig. 5A). Total acid insoluble glucose of the cesa9 mutant was on average 94 mg.g -1 (± 21) versus 126 mg.g -1 (± 4) in the wild-type seed, representing a 25.4% reduction. Further measurements indicated no change in acid insoluble glucose content in stems or leaves, consistent with no difference in radical and hypocotyl emergence and rosette leaf morphology (Fig. 1C). Staining with Calcofluor, a fluorescent stain for beta-glycans including cellulose (Windsor et al., 2000;Willats et al., 2001;Macquet et al., 2007), allowed the observation of the mucilage strands extending laterally from the seed coat epidermis (Fig. 5B), consistent with previous reports (Macquet et al., 2007). The main difference evident from Calcofluor staining was that cesa9-1 mucilage strands arose from the imbibed seed at slightly irregular angles and were more diffuse in its distribution relative to wild-type (Fig. 5B). To further explore biosynthetic feedback into other cell wall polysaccharides, such as that identified by Burton et al. (2000) we examined the content of arabinose, xylose, mannose, glucose, rhamnose and galactose in mature seed.
When seeds were incubated in a solution of tetrazolium salts (Debeaujon et al., 2001), cesa9 seeds were found to be more sensitive to salt uptake than wild-type seed (Fig. 6 inset). To determine whether the defect in cellulose synthesis resulted in cell wall associated polyesters in the seed coat, we analyzed the composition of lipid polyester monomers arising from whole seeds of the cesa9 mutant compared with wild-type seed.

DISCUSSION
The process of testa cell development in A. thaliana has been elegantly dissected microscopically (Beeckman et al., 2000;Western et al., 2000Western et al., , 2001 Haughn and Chaudhury 2005) revealing a complex process whereby mucilage is secreted and sequestered in the apoplast between the primary cell wall and the plasma membrane at the junction of the radial and outer tangential cell walls. Concurrent to mucilage production, the cytoplasm and starch granules of testa cells are shaped into a column in the center of the cell (Western et al., 2000). Following mucilage secretion, an elaborate secondary cell wall is produced around the cytoplasmic column forming the columella, and along the radial cell wall (Western et al., 2000;Windsor et al., 2000). During the final step of seed maturation, 'the cell dehydrates leaving the columella and radial walls visible as the epidermal plateau and reticulations visible on the seed coat' (Windsor et al., 2000). Analysis of developing seed by both SEM and sectioning showed that the 1 1 radial wall was present during early developmental stages in cesa9 (Fig. 4) but failed to show secondary cell wall thickening relative to wild-type during the latter stages of development (Fig. 4). For instance, careful examination of cross sectioned seed coat cells stained with toluidine blue showed that the secondary cell wall of wild-type cells extended up the entire radial wall whereas in cesa9 the secondary radial wall was less developed. Quantitative analysis of the height of radial walls in cross-sectioned tissue clearly demonstrated a significant reduction in radial wall height. Therefore, it is likely that after cesa9 seed matures and dehydrates, the unreinforced radial cell walls partially collapse due to a lack of cesa9 dependent cellulose biosynthesis resulting in the appearance that neighboring cells lack radial walls visualized by SEM (Fig. 2). A series of surface defects in cell shape and morphology were quantifiable in the mature cesa9 seed coat cells, such as cell area, columella area and internal angle uniformity (Fig. 3).
Given that the function of cellulose biosynthesis is to provide rigidity to the cell wall, one plausible explanation for the changes in cell morphogenesis is that the constraint of internal turgor pressure is disturbed. This may explain the collapsed cell boundary phenotype, irregular internal angles and altered cell and columella areas ( Fig. 2 and Fig.   3). Such a scenario has previously been documented for other tissue types (Burton et al., 2002;Desprez et al., 2002).
Previous studies have shown that where cellulose biosynthesis is inhibited in either primary cell wall or secondary cell wall xylem thickening, compensation by other cell wall polymers, such as hemicellulose attempt to overcome structural weakness (Turner and Somerville, 1997;Burton et al., 2000;Cano-Delgado et al., 2003;Bosca et al., 2006;Taylor, 2008). Evidence for proportional increase in cell wall polymers other than cellulose were also observed in cesa9 seed on a total cell wall basis (Fig. 5C). Noncellulosic cell wall polysaccharides (neutral sugars) and aliphatic monomers and some polyesters (Fig. 6) such as 18:1 DCA, 18:2 DCA, 18:1 and 18:2 fatty acids, ferulate and sinapate were proportionally more abundant in cesa9 relative to wild-type. Despite increased biosynthesis of non-cellulosic cell wall polymers, the uptake of tetrazolium salts could not be prevented (Fig. 6 inset) suggesting that the role of the seed coat in osmoprotection (boundary function) was compromised. Furthermore, the upregulation in non-cellulosic cell wall polymers could not rescue cell shape and morphogenesis supporting a non-redundant role for regulated cellulose biosynthesis, via CESA9, in these tissues. Given the paucity of cell type specificity for CESA subunits, results presented herein for CESA9 provide important evidence for specific requirements of secondary cell wall biosynthesis in epidermal seed coat radial cell walls that are distinct from secondary xylem thickening and primary cell wall cellulose synthesis. A non-redundant role for CESA9 in secondary cell wall thickening in seed coat cells (Fig. 4-6)  Conceptually, if the hexameric model holds true for seed coat secondary cell wall synthesis, CESA9 must occupy one stoichiometric location in the CESA complex during radial cell wall thickening. Because seed coat cell shape and morphogenesis phenotypes are distorted in cesa9 alleles, there is no evidence to suggest that a different subunit can fulfill its role. A reason for this scenario may be based on the specific cellular requirements of the testa cell layer (Haughn and Chaudhury, 2005), which may not offer the same cellular cues required to compensate for the loss of cesa9, such as upregulation of CESA2, CESA5, CESA6 or CESA10 as may occur in elongating tissues (Persson et al., 2007). Alternatively, encoded within CESA9 may be subtle differences in amino acid composition that facilitate some of the unique features of cellulose deposition in this hexagonally shaped cell type. Indeed, the contribution of structural macromolecules to highly specialized cell types remains a poorly understood area of plant cell biology and beckons further exploration.

Plant Material and Growth Conditions
All Arabidopsis lines used in this study were of the Columbia-0 ecotype. Seeds were surface sterilized using 30% bleach solution and stratified for 3 days in 0.15% agar at 4ºC. For phenotypic analysis and growth assays, plants were exposed to light for 1 h

Identification of T-DNA Insertions in CESA9
Polymerization chain reaction (PCR) confirmed homozygous alleles carrying exonal T-DNA insertion were as follows: cesa9-1 SALK_107750C (Persson et al., 2007, Harris et al., 2009, cesa9-2 (SALK_046455)(genotyping presented in Fig. S3). These alleles were sourced from The Arabidopsis Biological Resource Center (ABRC) as a fee for service product and identified through The Arabidopsis Information Resource (TAIR) and genotyped homozygous lines re-deposited as a public resource.

Staging of Floral Age
For developmental studies performed using light microscopy, pollination was defined as the time in days when the flower was beginning to open (days postanthesis; DPA). At this stage the stamen are beginning to grow over the gynoecium and the pollen is released (anthesis). Petals can be seen extending beyond the tops of the sepals.
Flowers at anthesis were marked using water soluble paint, and a different color was used for each day of marking. By contrast, SEM and Normarski optics used a different staging regime. Gyneoecium protrusion was defined as the point when the flower was fully open and the elongating gynoecium was clearly elongated beyond the petals and stamen. At this stage, the flower was tagged and each silique posterior to this event counted.

Resin Embedding for Bright Field Microscopy
Siliques staged at 4, 7, and 10 days post anthesis (DPA) were removed from the plant and dissected using a razor blade. The seeds were removed and the seed coat was punctured by either an insect pin, or a razor blade. High pressure freezing, freeze substitution, and resin embedding were performed according to Western et al. (2000).
Samples were loaded onto copper hats (Ted Pella) containing 1-hexadecene and frozen under high pressure using a Bal-Tec HPM 010 high-pressure freezer (RMC Products).
Copper hats were then transferred to frozen cryovials containing freeze substitution medium consisting of 2% (w/v) osmium tetroxide in acetone with 8% (v/v) dimethoxypropane. Freeze substitution was performed for 6 days at Spurr's epoxy resin (Canemco) (Spurr, 1969). Alternatively for the analysis of mature seeds, samples were fixed in 3% glutaraldehyde in 0.1M KH 2 PO 4 buffer pH 7.0 or FAA
Dehydration, embedding and sectioning were performed as described by Western et al. (2000). Sections were photographed using an Axioskop 2 microscope (Carl Zeiss) and Q Capture pro imaging software (Q imaging). Resin embedded samples were then thick sectioned (0.5mm) and stained with 1% (w/v) toluidine blue O in 1X (w/v) sodium borate (pH 11). Sections were examined by light microscopy and evaluated for intact seed coat cells and proper developmental stage based on the morphological criteria described by Western et al. (2000). Intact seed coat cells with a complete columella and radial wall were quantitatively assessed for height and width of the wall. The height of the wall was measured from the bottom of the mucilage pocket to the top of the wall. Width was measured for each radial wall. A total of 10 walls were measured for each genotype.
These measurements were made on the 3% glutaraldehyde fixed samples, because it enabled visual clarity of where the radial wall began and ended. Significance of comparisons was established based on a student's t-test with a two tailed analysis.

Analysis of seed lipid polyesters
Experimental procedures to extract soluble lipids and analyze polyesters of the cell wall residue were previously described in Molina et al. (2006). In brief, for each replicate, 50 to 100 mg mature seeds of wild-type and cesa9 were grinded, delipidated and the dried 1 5 residue was depolymerized using methanolysis. Released monomers were acetylated or sylilated and then separated, identified, and quantified by GC-MS using a splitless injection. Mass spectroscopy was performed using electron impact ionization, and peak quantification achieved on the basis of their total ion current. For addition details on GC-MS analysis see Molina et al. (2006).

Seed cell wall preparation and analysis
Cellulose content was measured colorimetrically and neutral sugar composition was determined by gas chromatography by using 500 mg (dry weight) of ball-milled material as described (Blakeney et al., 1983;Harris et al., 2009;Stork et al., 2009). Sugars from mature seed samples were prepared by sequential washing (five times) with 70% ethanol for 45 min at 70°C followed by five sequential acetone washes at room temperature for 2 min each. The main goal was to remove starch from the seed.   Analysis of columella area relative to the cell size. Columella area was approximately 14% of the wild type cell area, and 20% of the cesa9 mutant. D) Analysis of the internal angles between the cell sides (n=1080). Statistical significance of distribution shifts was calculated by using the Wilcoxon rank sum test for cesa9 relative to wild-type (P < 0.01). Wild-type and cesa9 cell area (P>0.001), wild-type and cesa9 columella area (P>0.008), wild-type and cesa9 cell area/columella area (P>0.001), wild-type and cesa9 internal angle (P>0.5). E-F) Provides an example of morphological variation between wild-type and cesa9 epidermal testa cells (scale bars = 30 μm). Fig. 4. Analysis of the structure and development of wild type and cesa9 seed coats. A) Epidermal cell morphology of wild type and cesa9 toluidine blue-stained sections of cryrofixed 4 DPA, 7 DPA, 10 DPA seeds. Arrows on 10 DPA images indicate the location of the radial wall where secondary cell wall synthesis is occuring (scale bar = 10 mm, all images same magnification). B) Epidermal cell morphology of wild type and cesa9 toluidine blue-stained sections of aqueous (3% v/v) glutaraldehyde fixed mature seeds (scale bar = 10 mm). C) Average height of the radial wall (RW) of wild type and cesa9 seed coat cells (mm). D) Average width of the radial wall (RW) of wild type and cesa9 seed coat cells (mm): Error bars are standard error from the mean. The * in C indicated significant difference from wild type based on one-way ANOVA at P>0.05 (student T-test).

Fig. 5. Composition of wild-type and cesa9 seed cell walls A) Acid insoluble
crystalline cellulose content of various tissues was determined colorometrically for mature seed, leaves and stems. Error bars are standard errors three technical replicates from 3 independent batches of seed as biological replicates. B) Calcofluor staining and subsequent illumination with UV light (scale bar = 250 μm). C) Gas chromatography analysis of cell wall neutral sugars. Error bars are standard error of 3 replicates. D) Mucilage weights determined for wild-type and cesa9 seed, error bars are standard error of 3 replicates. E) Ruthenium red stained seed visualized by light microscopy (scale bar = 200 μm).

Fig. 6. Polyester and aliphatic monomer composition for wild-type and cesa9
seed. GC and GC-MS analysis of mature cesa9 seed assessed lipid polyester monomers from seeds of wild-type and cesa9 plants. The insoluble dry residue obtained after grinding and delipidation of tissues with organic solvents was depolymerized by acid-catalyzed methanolysis and aliphatic and aromatic monomers released were analyzed by gas chromatography-mass spectroscopy. Error bars are the standard error of 4 replicates. Black bar is wild-type and red bar is cesa9. DCAs, Dicarboxylic acids; FAs, fatty acids; PAs, primary alcohols; br., branched. Inset documents tetrazolium salt uptake into the cesa9 seed relative to wild-type (scale bar = 250 μm).