Cellulose Deficiency Is Enhanced on Hyper Accumulation of Sucrose by a H⁺-Coupled Sucrose Symporter^,

The cellulose deficiency of the shv3svl1 mutant is enhanced by the uptake of exogenous sucrose, which results in carbon redirection from cellulose to starch biosynthesis.

Abstract

In order to understand factors controlling the synthesis and deposition of cellulose, we have studied the Arabidopsis (Arabidopsis thaliana) double mutant shaven3 shaven3-like1 (shv3svl1), which was shown previously to exhibit a marked cellulose deficiency. We discovered that exogenous sucrose (Suc) in growth medium greatly enhances the reduction in hypocotyl elongation and cellulose content of shv3svl1. This effect was specific to Suc and was not observed with other sugars or osmoticum. Live-cell imaging of fluorescently labeled cellulose synthase complexes revealed a slowing of cellulose synthase complexes in shv3svl1 compared with the wild type that is enhanced in a Suc-conditional manner. Solid-state nuclear magnetic resonance confirmed a cellulose deficiency of shv3svl1 but indicated that cellulose crystallinity was unaffected in the mutant. A genetic suppressor screen identified mutants of the plasma membrane Suc/H⁺ symporter SUC1, indicating that the accumulation of Suc underlies the Suc-dependent enhancement of shv3svl1 phenotypes. While other cellulose-deficient mutants were not specifically sensitive to exogenous Suc, the feronia (fer) receptor kinase mutant partially phenocopied shv3svl1 and exhibited a similar Suc-conditional cellulose defect. We demonstrate that shv3svl1, like fer, exhibits a hyperpolarized plasma membrane H⁺ gradient that likely underlies the enhanced accumulation of Suc via Suc/H⁺ symporters. Enhanced intracellular Suc abundance appears to favor the partitioning of carbon to starch rather than cellulose in both mutants. We conclude that SHV3-like proteins may be involved in signaling during cell expansion that coordinates proton pumping and cellulose synthesis.

Cellulose, a crystalline assembly of β-1,4-glucan chains, endows the plant cell wall with mechanical strength that selectively resists turgor pressure in order to define plant form at all scales. During the growth of primary organs, cellulose microfibrils are deposited transverse to the axis of cell expansion in order to restrict radial expansion. Thus, cellulose-deficient mutants typically exhibit radial swelling and reduced elongation of rapidly expanding cells in roots and hypocotyls (Somerville, 2006). Cellulose is synthesized from UDP-Glc at the plasma membrane (PM) by a large cellulose synthase complex (CSC) composed of cellulose synthase (CESA) catalytic subunits and several accessory proteins. The catalytic subunits are encoded by a family of 10 CESA genes in Arabidopsis (Arabidopsis thaliana). CESA1 and CESA3 encode nonredundant and essential components of the primary cell wall CSC, while CESA2, CESA5, CESA6, and CESA9 encode partially redundant components (Kumar et al., 2016). CESA10 is highly similar to CESA1 but, thus far, appears to be dispensable (Griffiths et al., 2015). A distinct CSC containing CESA4, CESA7, and CESA8 forms the cellulose of secondary cell walls (Kumar et al., 2016). Several accessory proteins have been shown to be bona fide components of the primary cell wall CSC as well: CELLULOSE SYNTHASE INTERACTIVE genes (CSI1 and CSI3) and COMPANION OF CELLULOSE SYNTHASE genes (CC1 and CC2) encode proteins involved in the association of CSCs with microtubules (Li et al., 2012; Lei et al., 2013; Endler et al., 2015). A PM-anchored endoglucanase encoded by KORRIGAN also is associated with the primary cell wall CSC and required for cellulose synthesis (Vain et al., 2014).

Cellulose synthesis is thought to be highly regulated (Wang et al., 2016). A major mechanism of this regulation appears to be the phosphorylation of CESA and accessory protein subunits of the CSC. Although regulatory kinases remain unidentified, CESA and CSI1 phosphopeptides have been detected by proteomic methods (Nühse et al., 2004; Taylor, 2007; Boex-Fontvieille et al., 2014). Moreover, phosphomimetic mutations of CESA1 (Chen et al., 2010), CESA3 (Chen et al., 2016), and CESA5 (Bischoff et al., 2011) result in altered CSC dynamics in living cells. The synthesis or channeling of UDP-Glc to CSCs also may play a role in the regulation of cellulose synthesis. Sucrose synthase (SuSy) catalyzes the reversible conversion of UDP and Suc into UDP-Glc and Fru and is thought to be a major source of UDP-Glc for cellulose synthesis in plants (Ruan, 2014). SuSy localization appears to be dynamically regulated by phosphorylation, peptide S-thiolation, and its oligomerization state, leading to an association with the cytoplasm, actin cytoskeleton, or membranes (Röhrig et al., 2004; Hardin et al., 2006; Duncan and Huber, 2007). The association of SuSy with the PM is thought to favor the channeling of UDP-Glc to CSCs, although this has not been demonstrated experimentally (Ruan, 2014). More generally, the in vivo role of SuSy in cellulose synthesis remains ambiguous. On the one hand, overexpression of SuSy in poplar (Populus spp.) results in increased cellulose accumulation in wood (Coleman et al., 2009). However, in Arabidopsis, quadruple SuSy mutants exhibit wild-type growth, although the amount of remaining SuSy activity in these plants is controversial (Barratt et al., 2009; Baroja-Fernández et al., 2012).

Cellulose biosynthesis is also regulated at the level of transcription, and the expression of genes encoding factors involved in cellulose biosynthesis is highly coordinated. This has allowed a guilt-by-association approach to reverse genetics, wherein uncharacterized genes coexpressed with known cellulose biosynthetic genes have been shown to be involved in the deposition of cellulose (Persson et al., 2005). One such gene that is highly coexpressed with genes encoding subunits of the primary cell wall CSC is SHAVEN3 (SHV3), named for its involvement in root hair formation (Jones et al., 2006; Hayashi et al., 2008). SHV3 encodes a glycosylphosphatidylinositol (GPI)-anchored protein with weak similarity to glycerophosphodiester phosphodiesterase (GDPD) and phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes. It is part of a six-member gene family in Arabidopsis that includes five SHV3-like genes (SVL1–SVL5; Hayashi et al., 2008). Hayashi et al. (2008) constructed double mutants of SHV3 and each of its homologs and showed that the double mutant shv3svl1 exhibited approximately 50% decreases in cellulose and hypocotyl length of dark-grown seedlings and only minor alterations in other cell wall polymers.

Prompted by the results of Hayashi et al. (2008), we sought to further understand the molecular basis of SHV3-like proteins’ involvement in cellulose biosynthesis in Arabidopsis. Unexpectedly, we discovered that the cellulose deficiency of shv3svl1 is highly conditional on the presence of exogenous Suc in the growth medium. We show that this phenomenon is due to a hyperpolarized PM H⁺ gradient and enhanced Suc uptake via the Suc/H⁺ symporter SUC1. In these conditions, the excess intracellular accumulation of Suc favors the synthesis of starch rather than cellulose. We conclude that SHV3-like proteins may be involved in coordinating cellulose synthesis with H⁺ secretion and wall loosening during cell expansion.

RESULTS

The Dark-Grown Seedling Phenotypes of shv3svl1 Are Enhanced by 30 mm or Greater Exogenous Suc

When we obtained shv3svl1 seeds and grew them using our standard conditions for skotomorphic growth, we observed only a minor deficiency in hypocotyl elongation, in contrast with the dramatic phenotype reported by Hayashi et al. (2008). As our growth medium contained no Suc and the previous work utilized medium with 60 mm Suc (2% [w/v] Suc), we examined the effect of exogenous Suc on hypocotyl growth in the wild type and shv3svl1. We observed a striking shortening of shv3svl1 relative to the wild type in the presence of 60 mm Suc, although hypocotyl elongation of the wild type also was inhibited to a lesser extent (Fig. 1A). We further investigated the effect of varying concentrations of Suc on hypocotyl elongation in the wild type and shv3svl1 and observed inhibition of hypocotyl elongation for both genotypes at Suc concentrations of 30 mm or greater, with shv3svl1 exhibiting enhanced Suc sensitivity relative to the wild type that does not increase at Suc concentrations of 60 mm or greater (Fig. 1B).

Figure 1.

Suc conditional phenotypes of shv3svl1. A, Effect of 60 mm exogenous Suc on dark-grown shv3svl1 seedlings. B, Quantification of seedling hypocotyl length on various concentrations of exogenous Suc (means ± se; n = 19–29). C, Lugol’s iodine staining of starch granules in seedlings. All seedlings were grown in the dark for 5 d on one-half-strength Murashige and Skoog (MS) medium with or without Suc as indicated. Asterisks indicate statistically significant differences compared with the wild type (WT) for the respective growth medium (Student’s t test, P < 0.05).

In order to determine whether the difference in the observed hypocotyl elongation phenotype was related to cell wall structure, we analyzed the cellulose content of shv3svl1 and wild-type seedlings grown in the dark in the presence or absence of 60 mm Suc. In the absence of exogenous Suc, shv3svl1 exhibited a 7% reduction in cellulose relative to the wild type (Table I). The addition of 60 mm Suc in the medium led to a more substantial decrease in cellulose content (48% reduction relative to the wild type), consistent with the results reported by Hayashi et al. (2008). The marked decrease in cellulose content of shv3svl1 grown in the presence of Suc is accompanied by an increase in starch accumulation (Table I). In fact, we observed approximately 4 times more starch accumulation than Hayashi et al. (2008), possibly because we analyzed older seedlings. The substantial accumulation of starch granules throughout the elongating hypocotyl also was observed by Lugol’s iodine staining (Fig. 1C). On the other hand, little starch accumulated in either genotype when seedlings were grown on medium without Suc (Table I; Fig. 1C).

Table I. Cell wall and starch composition of shv3svl1.

All values are expressed as µg mg⁻¹ alcohol-insoluble residue (AIR). Asterisks indicate statistically significant differences compared with the wild type for the respective growth medium (Student’s t test, *, P < 0.05). The data are means of three biological replicates ± sd.

Parameter	0 mm Suc		60 mm Suc
	Wild Type	shv3svl1	Wild Type	shv3svl1
Cellulose	122 ± 2	113 ± 3*	147 ± 6	76 ± 10*
Starch	17 ± 3	12 ± 1*	75 ± 7	260 ± 10*
Fuc	3.33 ± 0.05	3.13 ± 0.09*	4.47 ± 0.20	3.12 ± 0.05*
Rha	19.2 ± 0.2	18.1 ± 0.3*	15.0 ± 0.4	10.1 ± 0.4*
Ara	29 ± 1	32 ± 2	30 ± 1	26 ± 1*
Gal	64 ± 4	61 ± 2	66 ± 3	52 ± 2*
Xyl	27 ± 1	25 ± 1	33 ± 1	23 ± 1*
Man	5.7 ± 0.1	5.8 ± 0.2	6.5 ± 0.7	5.1 ± 0.1*
GalA	83 ± 4	73 ± 1*	70 ± 3	49 ± 2*
GluA	5.27 ± 0.05	4.66 ± 0.16*	3.53 ± 0.02	2.72 ± 0.17*

Aside from the glucans cellulose and starch, we assayed monosaccharides associated with hemicellulose and pectin of the cell wall (Table I). In the absences of exogenous Suc, only Fuc, Rha, GalA, and GlcA were statistically different in shv3svl1 than in the wild type, suggesting minor deficiencies in pectin, particularly rhamnogalacturonan I and II structures in the mutant. A concentration of 60 mm exogenous Suc resulted in significant decreases of all matrix monosaccharides. This is largely explained by the substantial contribution of starch to the AIR mass in the mutant (26%, w/w). When we recalculated values for cell wall composition that were normalized to AIR with the mass of starch subtracted, we found that alterations of only Fuc, Rha, Xyl, and GalA were of statistical significance (Supplemental Table S1). Taken together, these results indicate relatively minor alterations in cell wall composition in the absence of exogenous Suc. In the presence of exogenous Suc, the cellulose deficiency of shv3svl1 is greatly enhanced without substantial additional alteration of other cell wall polysaccharides.

shv3svl1 Is Specifically Sensitive to High Concentrations of Exogenous Suc and Not Other Sugars

In order to test the specificity of shv3svl1’s response to exogenous sugars, we grew shv3svl1 and wild-type seedlings in the dark on one-half-strength MS medium containing 60 mm sorbitol (Sorb), 60 mm Glc, 60 mm Fru, a mixture of 30 mm Fru and 30 mm Glc, and 60 mm Suc. The wild type exhibited reduced elongation of dark-grown hypocotyls with exogenous Suc, while the effects of other sugars were not significant (Fig. 2A). This result is consistent with a previous report that systematically investigated the effect of various carbon sources on the growth of wild-type Arabidopsis (Stevenson and Harrington, 2009). However, we observed that shv3svl1 exhibited a more dramatic response to Suc, with no other sugars inhibiting hypocotyl elongation and Fru slightly increasing hypocotyl elongation (Fig. 2B).

Figure 2.

Effects of various exogenous sugars on hypocotyl elongation in the dark. A, Wild-type (WT) seedlings (means ± se; n = 33–55). B, shv3svl1 seedlings (n = 36–70). C, cesa6 seedlings (n = 28–33) D, cob seedlings (n = 26–45). All seedlings were grown for 5 d on one-half-strength MS medium supplemented with sugars as indicated. For each graph, columns not sharing a common letter differ (P < 0.05) by one-way ANOVA and Tukey’s multiple comparison test.

To examine whether this Suc sensitivity syndrome was typical of cellulose-deficient mutants, we repeated this experiment with mutants of CESA6 (Fagard et al., 2000) and COBRA (COB; Ko et al., 2006), two well-characterized genes encoding a subunit of CESA and a GPI-anchored protein involved in cellulose biosynthesis, respectively (Fig. 2, C and D). In both cases, hypocotyl elongation was inhibited by exogenous Glc and Suc to the same extent, in contrast to what we observed with shv3svl1. Moreover, the extent of elongation inhibition by Suc was less severe than observed for shv3svl1.

A potential explanation for Suc-specific sugar sensitivity in our assay is that Suc is readily hydrolyzed to Fru and Glc by the action of invertases present in the cell wall, cytoplasm, and vacuole of plants (Ruan, 2014). As a result, a given concentration of Suc has the potential to contribute twice as much carbon or osmolarity compared with medium with the same molarity of a monosaccharide. To control for this possibility, we conducted additional experiments comparing the growth of the wild type, shv3svl1, cesa6, and cob on medium supplemented with 60 mm Suc or a mixture of 60 mm Glc and 60 mm Fru (Supplemental Fig. S1). Wild-type hypocotyl elongation was inhibited to a slightly greater extent by a mixture of 60 mm Glc and 60 mm Fru than 60 mm Suc, although an osmotic control with 120 mm Sorb was equally inhibitory (Supplemental Fig. S1A). In the case of shv3svl1, 60 mm Suc inhibited growth to a greater extent than the mixture of 60 mm Glc and 60 mm Fru or 120 mm Sorb (Supplemental Fig. S1B), indicating that Suc-specific inhibition in shv3svl1 is not due to invertase activity, as described above. While cesa6 exhibited the same trend as shv3svl1 (Supplemental Fig. S1C), cob exhibited equal sensitivity to 60 mm Suc and the 60 mm Glc/60 mm Fru mixture (Supplemental Fig. S1D).

Slower CSCs Synthesize Cellulose with Unaltered Crystallinity in shv3svl1

The rate of cellulose biosynthesis in living cells can be monitored by tracking the dynamics of fluorescently tagged CSCs using spinning-disk confocal microscopy. The movement of CSCs in the PM is due to the elongation of cellulose microfibrils, and their speed is proportional to the rate of UDP-Glc consumption and glucan synthesis occurring at each CSC (Paredez et al., 2006). To further characterize SHV3-like proteins’ involvement in cellulose biosynthesis, we generated transgenic plants expressing GFP-tagged CESA3 in the shv3svl1 background and examined CSC dynamics in hypocotyl epidermal cells of seedlings grown for 3 d in the dark in the presence or absence of 60 mm Suc (Fig. 3; Supplemental Movie S1).

Figure 3.

Live-cell imaging of CSC dynamics. A, B, D, and E, Representative images of time series collected from plants grown for 3 d in the dark on medium containing no Suc (A and B) or 60 mm Suc (D and E) showing single frames, time projections, and kymographs from plants expressing CesA3-GFP in the wild-type (WT; A and D) and shv3svl1 (B and E) backgrounds. The line used to calculate each kymograph is indicated with a yellow dashed line. C and F, Histograms of CESA particle speeds from CesA3-GFP expressed in wild-type and shv3svl1 plants grown on medium with no Suc (C) or 60 mm Suc (F).

Regardless of the presence of Suc in growth medium, shv3svl1 exhibited the typical array of linear tracks of CESA particles that was also observed in wild-type plants (Fig. 3 A, B, D, and E). Kymograph analysis of these tracks indicated that the particles moved in the bidirectional constant velocities that are typical of CSCs embedded in the PM. In order to quantify CSC speed, we used computational particle tracking to determine the speeds of thousands of CSCs (Table II). In the absence of exogenous Suc, shv3svl1 exhibited a minor decrease in CSC speeds relative to the wild type (Fig. 3C; Table II). The presence of 60 mm Suc in the growth medium resulted in a reduction of CSC speed in the wild type (Fig. 3F; Table II). We note that this is in contrast to what we observed previously with plants grown on medium with lower concentrations of Suc (30 mm; 1%, w/v), where CSC speeds were not affected in the wild-type background (Vellosillo et al., 2015). This suggests that Suc-dependent effects on CSC activity may occur at a threshold intracellular concentration that occurs when exogenous Suc concentration exceeds approximately 30 mm, similar to the reported threshold for Suc-dependent translational repression of ATB2/bZIP11 (Rook et al., 1998). In shv3svl1 plants grown on medium containing 60 mm Suc, we observed a further reduction in CSC speeds (Fig. 3F; Table II). Thus, while the average CSC speed of shv3svl1 was reduced by 8% relative to the wild type in the absence of Suc, with 60 mm Suc, shv3svl1 had CSCs with an average speed 21% slower than the wild type (Table II). In plants grown on Suc, the particle speed distribution is narrower (Fig. 3, F compared with C). We speculate that this may reflect improved tracking of particles in the flatter and swollen cells of Suc-grown plants. Since net cellulose synthesis is related to the number of particles as well as their speed, we calculated the density of particles observed in each time series. We did not observe any significant difference in particle density between the genotypes or growth media (Table II).

Table II. Quantification of CSC particle speed and density.

Growth Medium	Genotype	Average Speeda	Relative Speedb	Particle Densityc	No. of Particles
		nm min−1	%	µm−2
0 mm Suc	Wild type	283 ± 1 a	100	0.49 ± 0.04 e	9,699
	shv3svl1	260 ± 2 b	92	0.41 ± 0.05 e	7,359
60 mm Suc	Wild type	201 ± 1 c	100	0.51 ± 0.05 e	7,582
	shv3svl1	159 ± 1 d	79	0.54 ± 0.02 e	17,758

^aMeans ± se for n particles analyzed as indicated. All genotype/medium combinations were statistically distinct from each other (P < 0.001) by two-way ANOVA and Bonferroni’s multiple comparison test, as indicated by letters a to d. For each analysis, nine to 15 unique cells were imaged from three to five individual plants.  ^bAverage speed relative to the wild type of the respective growth medium.  ^cMean number of CSC particles tracked from nine to 15 cells ± se. All genotype/medium combinations were not significantly different (P > 0.05) by two-way ANOVA and Bonferroni’s multiple comparison test, as indicated by the letter e.

To probe the consequences of the shv3svl1 mutations on cellulose structure, we prepared uniformly ¹³C-labeled cell wall material from dark-grown seedlings and subjected it to ¹³C magic-angle-spinning solid-state NMR spectroscopy. A general reduction in signals attributed to cellulose glucans corroborated the reduction in cellulose amount seen previously (Fig. 4A). Comparison of the peaks from 88.5 to 91.5 ppm and 84 to 86 ppm yields a ratio of interior C4 of Glc (iC4) to surface C4 (sC4) of Glc (Fig. 4B). The iC4-sC4 ratio is analogous to crystallinity and has been reported to be altered in several cellulose-deficient mutants (Harris et al., 2012; Sorek et al., 2015). In our experiment, the wild type was found to have an iC4:sC4 ratio of 0.65 and shv3svl1 was found to have an iC4:sC4 ratio of 0.68, indicating that microfibril structure, in terms of this metric, is essentially unaltered in the shv3svl1 mutant (Fig. 4C).

Figure 4.

Quantitative ¹³C direct polarization magic-angle-spinning solid-state NMR of the wild type (WT) and shv3svl1. A, Relative intensity spectrum of the wild type and shv3svl1 with peak assignments indicated. B, Detail of the 78- to 94-ppm region from A highlighting iC4 and sC4 signals used for calculation of the interior-surface glucan ratio. C, Quantitative integration and calculation of the interior-surface glucan ratio.

Taken together, the live-cell imaging of CSCs and solid-state NMR indicate a reduced rate of cellulose synthesis without any apparent aberrant deposition or structure of the microfibrils in shv3svl1.

Mutation of a Suc/H⁺ Symporter Suppresses the Suc Sensitivity of shv3svl1

To identify additional genes connected to SHV3’s and SVL1’s role in cellulose biosynthesis and hypocotyl elongation, we conducted a genetic suppressor screen of shv3svl1. Bulked shv3svl1 seeds were mutagenized by treatment with ethyl methanesulfonate (EMS), and M2 progeny were screened in the dark for the restoration of hypocotyl elongation on one-half-strength MS medium containing 60 mm Suc. Five suppressors of shv3svl1 (sss1–sss5) were isolated from independent pools of M2 seeds, and their heritability was confirmed in the M3 generation (Fig. 5A). None of the suppressors that we recovered rescued the root hair phenotype of shv3svl1. In order to map the mutation underlying the suppression of the hypocotyl elongation defect of shv3svl1, shv3svl1sss1 was backcrossed to shv3svl1 and approximately 150 bulked segregants of the BCF2 generation were genotyped by Illumina deep sequencing at approximately 40× coverage. The frequency distribution of EMS-induced single-nucleotide polymorphisms (SNPs) indicated a peak centered at the bottom of chromosome 1 (Fig. 5B). Within this region, the two highest frequency SNPs predicted to result in an amino acid change were within At1g74180, encoding a receptor-like protein of unknown function (RLP14), and At1g71880, encoding a PM-localized Suc/H⁺ symporter (SUC1).

Figure 5.

Identification and mapping of suppressors of shv3svl1. A, Seedlings of the five shv3svl1 suppressors grown for 5 d on one-half-strength MS medium + 60 mm Suc in the dark, with the wild type (WT), shv3svl1, and suc1-2 for comparison. B, SNP frequency of BCF2 bulked segregants as a function of chromosome (Chr.) position. Blue symbols indicate nongenic or synonymous SNPs, and red symbols indicate those predicted to result in amino acid changes in protein-coding genes. The circle indicates the two highest frequency SNPs that are predicted to result in amino acid changes, as discussed in the text. C, Structure of the SUC1 gene with mutant alleles that suppress shv3svl1 indicated as well as the suc1-2 transposon insertion allele. Below the gene model is the predicted transmembrane structure of SUC1 (Omasits et al., 2014) with point mutations indicated.

The mutation in SUC1 was shown to be the causative SNP in the suppressors by sequencing of the SUC1 gene from the other independently isolated suppressors. In all cases, unique nonsynonymous mutations were detected, indicating that the suc1 mutation suppressed the hypocotyl elongation defect of shv3svl1 in the presence of Suc (Fig. 5C). Notably, all of the identified amino acid changes in SUC1 were located within or adjacent to predicted transmembrane helices (Fig. 5C). Since we suspected that the sss1 allele might be hypomorphic based on the hypocotyl elongation phenotype (Fig. 5A), we decided to include in our future analyses shv3svl1sss2, as it has a premature stop codon in SUC1 that is likely a null allele.

Further analysis of shv3svl1sss1 and shv3svl1sss2 grown on the same panel of exogenous sugars that we used previously revealed that both alleles suppressed the Suc-specific shortening of hypocotyls seen in shv3svl1, although the sss1 allele appears to be weaker in this regard (Fig. 6A; compare Fig. 2B). We also examined the sensitivity of the suc1-2 mutant in the absence of the shv3svl1 mutations. Notably, suc1-2 abolishes the less severe inhibition of hypocotyl elongation characteristic of the wild type grown on 60 mm Suc (Supplemental Fig. S2; compare Fig. 2A).

Figure 6.

Characterization of suppressors of shv3svl1. A, Hypocotyl lengths of shv3svl1sss1 and shv3svl1sss2 5-d-old seedlings grown in the dark (means ± se; n = 16–31). Asterisks indicate a statistically significant difference (****, P < 0.0001) between the genotypes for the given treatment as determined by two-way ANOVA followed by Bonferroni’s multiple comparison test; ns indicates no significant difference (P > 0.05) by the same test. B and C, Cellulose (B) and starch (C) contents of 5-d-old dark-grown seedlings grown on the indicated media. Asterisks indicate statistically significant differences compared with the wild type (WT) for the respective growth medium (Student’s t test, *, P < 0.05).

Next, we analyzed the cellulose content of the suppressors in comparison with the wild type, shv3svl1, and the suc1-2 single mutant grown on medium ± 60 mm Suc (Fig. 6B). The minor cellulose deficiency of shv3svl1 in the absence of Suc was not suppressed in either shv3svl1sss1 or shv3svl1sss2. However, the more severe Suc-conditional cellulose deficiency was partially rescued in shv3svl1sss1 and nearly completely rescued in shv3svl1sss2. The suc1-2 single mutant exhibited wild-type levels of cellulose in both conditions.

Since the cellulose deficiency of shv3svl1 in the presence of exogenous Suc is associated with the accumulation of starch, we expected that this effect also would be mitigated by suc1 mutation. The accumulation of starch in plants grown on Suc was partially suppressed in shv3svl1sss1 and more substantially in shv3svl1sss2 (Fig. 6C). The suc1-2 single mutant accumulated 63% less starch than the wild type on Suc-containing medium. Starch accumulation in all genotypes was low without exogenous Suc, and no pattern in its accumulation with regard to genotype was observed (Fig. 6C).

shv3svl1 Partially Phenocopies feronia

In the course of our work on shv3svl1, reports on the feronia (fer) mutant attracted our interest, since FER, like SHV3, is coexpressed with primary cell wall CESA genes (Ruprecht et al., 2014) and several phenotypes are shared between fer and shv3svl1. Both mutants exhibit collapsed root hairs and short hypocotyls, and the mature rosette leaves have misformed trichomes (Hayashi et al., 2008; Deslauriers and Larsen, 2010; Duan et al., 2010). Recently, it was reported that fer exhibits Suc-conditional ectopic starch accumulation and shortening of hypocotyls in the dark (Yang et al., 2015). Prompted by these reports, we further compared the phenotypes of fer and shv3svl1.

We first confirmed that the hypocotyl elongation defect of fer was enhanced by Suc (Fig. 7A). Like shv3svl1, fer seedlings also exhibit enhanced accumulation of anthocyanins in the presence of 60 mm Suc (Fig. 7A). We subjected fer seedlings to the same panel of exogenous sugars that we used previously and observed that hypocotyl elongation in fer was inhibited to the greatest extent by exogenous Suc (Fig. 7B). However, inhibition of fer hypocotyl elongation by medium containing Glc also was observed (Fig. 7B), and repeating the experiment with a mixture of 60 mm Glc and 60 mm Fru indicated equivalent inhibition to 60 mm Suc, suggesting that fer exhibits a less-specific sensitivity to Suc than we observed in shv3svl1 (Supplemental Fig. S3).

Figure 7.

The fer mutant partially phenocopies shv3svl1. A, Five-day-old dark-grown fer seedlings compared with the wild type (WT) and shv3svl1. B, Hypocotyl lengths of 5-d-old seedlings grown on one-half-strength MS medium or medium supplemented with the indicated sugars (means ± se; n = 24–29). Columns not sharing a common letter differ (P < 0.05) by one-way ANOVA and Tukey’s multiple comparison test. C and D, Cellulose (C) and starch (D) contents of wild-type and fer 5-d-old dark-grown seedlings grown on the indicated media. Asterisks indicate statistically significant differences compared with the wild type for the respective growth medium (Student’s t test, *, P < 0.05). The data in C and D are means of three biological replicates ± sd. E, Effect of RALF treatment on hypocotyl length (means ± se; n = 11–13). Asterisks indicate statistically significant differences (****, P < 0.0001) between the treated and untreated groups as determined by two-way ANOVA followed by Bonferroni’s multiple comparison test; ns indicates no significant difference (P > 0.05) by the same test.

Next, we analyzed the cellulose content of fer and observed that, like shv3svl1, fer exhibits a cellulose deficiency that is enhanced by exogenous Suc (Fig. 7C). Exogenous Suc also induced an accumulation of starch in the fer mutant to a similar degree to that observed in shv3svl1 (Fig. 7D). The minor starch accumulation in medium without Suc also was greater in fer than in the wild type (Fig. 7D). In contrast to shv3svl1, the majority of the matrix monosaccharides of fer were altered relative to the wild type in both conditions, suggesting alterations of both xyloglucan and pectin components of the cell wall in fer in addition to cellulose (Supplemental Table S2).

In order to address whether shv3svl1 was involved in the perception of the RALF peptide ligand of FER, we tested the sensitivity of shv3svl1 to the RALF peptide. Previously, it was demonstrated that fer is insensitive to the growth inhibition caused by RALF treatment (Haruta et al., 2014), so if SHV3 and SVL1 function in the same pathway as FER, we expected to see a reduction in RALF sensitivity in shv3svl1. However, shv3svl1 exhibited a similar response to RALF treatment as the wild type, indicating that, despite the phenotypic similarity of the two mutants, SHV3 and SVL1 are not involved in RALF perception and are not likely to participate directly in FER signaling (Fig. 7E).

A Hyperpolarized PM H⁺ Gradient Is Associated with an Enhanced Suc Accumulation of shv3svl1 and fer

A major physiological phenotype of fer is a hyperpolarization of the PM H⁺ gradient, presumably due to misregulation of the PM H⁺/ATPase AHA2 (Haruta et al., 2014). We suspected that hyperpolarization of the PM H⁺ gradient could lead to an excess accumulation of Suc in fer via the protonmotive force-driven activity of PM H⁺/Suc symporters. If shv3svl1 exhibits a similar enhancement of proton-pumping activity, this could explain the similar Suc-conditional alterations in starch and cellulose biosynthesis that we observed in both mutants (Fig. 7, C and D). To test whether this might be true, dark-grown seedlings were incubated for 16 h in an unbuffered bathing medium and pH was assayed using a fluorescent indicator. Indeed, in these conditions, shv3svl1 exhibited significantly greater acidification of the bathing medium than the wild type, consistent with a hyperpolarization of the PM (Fig. 8A). The strong suppressor allele shv3svl1sss2 exhibited acidification of the bathing medium to the same extent as the wild type (Fig. 8A).

Figure 8.

shv3svl1 and fer exhibit PM H⁺ hyperpolarization and enhanced accumulation of exogenous Suc. A, The pH of bathing media after 16 h of incubation of seedlings of the indicated genotypes (means ± se; n = 4). Bars not sharing a common letter differ (P < 0.05) by one-way ANOVA and Tukey’s multiple comparison test. B, Soluble Glc, Fru, and Suc assayed from 3-d-old seedlings (means ± se; n = 3). Statistically significant differences for Suc relative to the wild type (WT) by two-way ANOVA followed by Tukey’s multiple comparison test are shown by asterisks (*, P < 0.05 and ***, P < 0.001). FW, Fresh weight. C to H, Uptake of the fluorescent glucoside esculin in 4-d-old wild-type (C), shv3svl1 (D), suc1-2 (E), shv3svl1sss1 (F), shv3svl1sss2 (G), and fer (H) seedlings. Controls incubated with solutions containing dimethyl sulfoxide are shown in Supplemental Figure S4.

To further establish that exogenous Suc is taken up from the medium to a greater extent in fer and shv3svl1, we assayed soluble Suc, Fru, and Glc in seedlings (Fig. 8B). Both shv3svl1 and fer exhibited significantly greater amounts of Suc than the wild type. This was also the case for the weak suppressor allele shv3svl1sss1. On the other hand, the strong suppressor allele shv3svl1sss2 exhibited a reduced accumulation of soluble Suc, indicating that the increased accumulation of Suc in shv3svl1 was dependent on SUC1. To examine the activity of Suc transporters in vivo, we utilized the fluorescent Suc analog esculin. Esculin is a coumarin β-glucoside that is readily transported by type I Suc transporters like SUC1 (Sivitz et al., 2007). Previously, it was used as a fluorescent probe to visualize phloem loading and transport in planta (Knoblauch et al., 2015) and heterologous Suc transporter activity in Saccharomyces cerevisiae (Gora et al., 2012). After 30 min of incubation with 1 mm esculin, we observed blue fluorescent signal accumulating in roots of wild-type plants, indicating intracellular accumulation of esculin (Fig. 8C). The shv3svl1 mutant exhibited a similar intensity of fluorescence in roots but intense fluorescence throughout the hypocotyl, indicating enhanced uptake of esculin in the elongating hypocotyl (Fig. 8D). The suc1 null mutant exhibited a reduction in root-localized fluorescence relative to the wild type (Fig. 8E), and shv3svl1sss1 and shv3svl1sss2 had reduced hypocotyl-localized esculin accumulation compared with shv3svl1 (Fig. 8, F and G).

The fer mutant also exhibited strong esculin signal in the hypocotyl that was comparable with that observed in shv3svl1, indicating that Suc uptake activity is similarly enhanced in this mutant (Fig. 8H). Notably, both shv3svl1 and fer exhibited enhanced autofluorescence in control experiments without esculin that is likely due to ectopic lignification in these mutants (Supplemental Fig. S4). The intensity of this fluorescence, however, was negligible compared with what we observed during esculin uptake. Taken together, these results indicate that both shv3svl1 and fer exhibit a hyperpolarized PM H⁺ gradient that is associated with enhanced accumulation of exogenous Suc. In the case of shv3svl1, we demonstrate that this is dependent on the activity of SUC1. We suspect that suc1 also might suppress the Suc-conditional phenotypes of fer. However, generating a suc1fer double mutant to test this hypothesis proved prohibitively difficult due to suc1 and fer both exhibiting approximately 90% reduced transmission via the male and female gametophytes, respectively (Huck et al., 2003; Sivitz et al., 2008).

DISCUSSION

We discovered that the cellulose deficiency of shv3svl1 is conditional on the uptake of exogenous Suc from growth medium and that this is suppressed by mutation of SUC1. SUC1 encodes a PM-localized Suc/H⁺ symporter (Sauer and Stolz, 1994) that is expressed in pollen as well as young seedlings (Sivitz et al., 2008). The suc1 mutant is deficient in the accumulation of anthocyanins in response to growth on medium containing 90 mm (3%, w/v) Suc (Sivitz et al., 2008). In our work, shv3svl1 grown on medium containing 60 mm (2%, w/v) Suc exhibited anthocyanin accumulation while the wild type did not, and this phenotype was suppressed by mutation of suc1 (Fig. 5A). This indicates that shv3svl1 exhibits a more general enhancement of sensitivity to Suc not restricted to its cellulose deficiency, and this is due to increased intracellular accumulation of Suc (Fig. 8). Combined with the observation that 60 mm exogenous Suc reduces the average speed of CSCs in the wild type (Fig. 3; Table II), we interpret our results to indicate that SHV3 and SVL1 exert their effect on cellulose synthesis in part by an indirect mechanism that negatively impacts Suc uptake via PM Suc/H⁺ symporters and PM H⁺/ATPase activity. The intracellular concentration of Suc and/or downstream metabolites then affects carbon partitioning between cellulose and starch. SHV3 and SVL1 also may have an effect on CSC activity independent of Suc availability, as decreased cellulose and CSC velocities were observed in shv3svl1 in the absence of exogenous Suc (Table II; Fig. 3C).

Posttranslational regulation of several proteins involved in these processes in response to Suc is suggested by phosphoproteomic experiments (Niittylä et al., 2007; Wu et al., 2013). Within 3 min of Suc treatment, Niittylä et al. (2007) observed changes in the abundance of phosphopeptides from SUC1 and the H⁺/ATPase proteins AHA1 and AHA2. Suc up-regulation of H⁺/ATPase activity is consistent with our results showing that the suc1 mutation reduces medium acidification (Fig. 8A). Niittylä et al. (2007) also identified SUCROSE-INDUCED RECEPTOR KINASE1 (SIRK1), which is rapidly phosphorylated in response to Suc. In further work, putative downstream targets of SIRK1 were identified. Intriguingly, enhanced phosphorylation of CESA3 in response to Suc was identified in plants overexpressing SIRK1 (Wu et al., 2013). A potential connection between SHV3-like proteins and SIRK1-related signaling is suggested by coexpression analysis of SVL1. While SHV3 is coexpressed with primary cell wall CSC components (Persson et al., 2005), SVL1 is coexpressed with several receptor-like kinases, including SIRK1 (Supplemental Fig. S5; Obayashi et al., 2007). Another potential connection between Suc perception and cellulose synthesis is suggested by the work of Fujimoto et al. (2015). In this case, a shift from medium containing 30 mm Suc to medium without Suc was used in order to induce an increased internalization of CSCs in seedlings. While the authors attributed this effect to the hypotonic nature of their treatment, this could reflect Suc-induced relocalization of CSCs, potentially as a regulatory mechanism (Wang et al., 2016). On the other hand, our live-cell imaging, which did not involve a medium shift, did not show any apparent difference in CSC particle density related to growth on Suc (Fig. 3; Table II), and the Suc-conditional enhancement of cellulose deficiency in shv3svl1 can be interpreted largely in terms of the observed reduction in CSC velocities in these mutants (Fig. 3; Table II). However, one limitation of CSC live-cell imaging is that only epidermal cells are accessible to spinning-disk confocal microscopy and cellulose synthesis may occur at different rates in other cells. As we observed CSC dynamics in a single cell type at a single time with this technique, this may explain why we did not observe reduced cellulose accumulation in the wild type grown on Suc (Table I) despite a reduction in average CSC speed (Fig. 3; Table II).

We discovered that the Suc sensitivity syndrome of shv3svl1 also was exhibited by the unrelated mutant fer. In this mutant, we observed Suc-conditional enhancement of starch biosynthesis and cellulose deficiency at a Suc concentration of 60 mm (Fig. 7, C and D). A previous report noted that fer exhibits an accumulation of starch when grown on medium containing Suc (Yang et al., 2015). The authors attributed this to an interaction between the kinase domain of FER and glyceraldehyde-3-phosphate dehydrogenase, an enzyme that would indirectly affect starch synthesis through its role in glycolysis (Yang et al., 2015). The fact that fer hyperaccumulates exogenous Suc due to a hyperpolarized PM H⁺ gradient (Fig. 8) is likely to have a much larger effect on starch accumulation in these conditions. On the other hand, we speculate that, in the absence of exogenous Suc, the hypothesis of Yang et al. (2015) may be relevant, as we did observe higher levels of starch in fer in these conditions where energy is expected to be more limiting (Fig. 7D).

Previous reports have noted the induction of starch accumulation due to genetic or pharmacological inhibition of cellulose synthesis. Peng et al. (2000) observed modest starch accumulation in three temperature-sensitive cellulose-deficient radial swelling mutants, although our results cannot be directly compared due to differences in growth conditions. In more recent work, treatment of wild-type seedlings with the cellulose biosynthesis inhibitor isoxaben resulted in transient starch accumulation, but after 12 h of isoxaben treatment, starch returned to basal levels (Wormit et al., 2012). Notably, Wormit et al. (2012) did not observe an enhanced uptake of Suc from medium in response to isoxaben treatment, in contrast to what we observed in the shv3svl1 mutant. Thus, we do not believe that the enhanced Suc uptake and dramatic starch accumulation that we observed in shv3svl1 are generic results of cellulose deficiency.

What is the biochemical activity of SHV3-like proteins? SHV3-like proteins possess two tandem domains with sequence similarity to both GDPD and PI-PLC enzymes. However, SHV3 and its homologs lack several conserved catalytic residues of either of these enzymes, suggesting that they may lack such activity. Indeed, Hayashi et al. (2008) were unable to demonstrate GDPD activity of recombinant SHV3 expressed in Escherichia coli. We addressed two potential limitations of their assay by expressing SHV3 in the eukaryotic host Pichia pastoris and assaying for GDPD as well as PI-PLC activity at a range of pH conditions, including the acidic conditions expected of the periplasmic space where SHV3 is localized. Nevertheless, we were unable to demonstrate either GDPD or PI-PLC activity catalyzed by recombinant SHV3 (Supplemental Fig. S6). It appears likely that, despite the similarity to GDPD and PI-PLC, SHV3-like proteins have evolved distinct activities or do not possess any enzymatic activity at all.

Recently, it was shown that another GPI-anchored protein, LORELEI-LIKE-GPI-ANCHORED PROTEIN1 (LLG1), interacts with and acts as a chaperone or coreceptor of FER (Li et al., 2015). An attractive hypothesis is that SHV3-like proteins may play a similar role in complex with a different receptor kinase involved in signaling to coordinate cellulose biosynthesis and the PM H⁺ gradient during cell expansion. Since we did not observe a reduction in susceptibility to the RALF peptide in shv3svl1 (Fig. 7E), as seen in fer and llg1 mutants (Haruta et al., 2014; Li et al., 2015), we do not propose that SHV3-like proteins function in this signaling pathway but rather share several phenotypes that are associated with the misregulation of PM H⁺/ATPase activity. Future work will require the identification of interacting proteins and/or ligands of SHV3-like proteins. Toward this end, SIRK1 and the other receptor-like kinases coexpressed with SVL1 are promising candidates (Supplemental Fig. S5).

It is not surprising that suc1 failed to suppress the root hair defect of shv3svl1, as the root hair phenotype of shv3svl1 is not Suc conditional and SUC1 is not expressed in root hairs (Sivitz et al., 2007). However, based on our results, we speculate that the root hair defect of shv3 (and shv3svl1) may be due to the misregulation of PM H⁺/ATPase activity, as we observed in shv3svl1 whole seedlings (Fig. 8A). Monshausen et al. (2007) demonstrated that root hair growth involves oscillations in tip-localized extracellular pH that are thought to mediate localized cell wall loosening via the action of expansins and cell wall-modifying enzymes. We propose that aberrant pH-dependent cell wall loosening could result in root hair bursting in shv3. Hayashi et al. (2008) reported that the root hair defect in shv3 is partially suppressed by the application of exogenous borate. In the context of our hypothesis here, cell wall stiffening due to increased borate cross-linking of rhamnogalacturonan II could compensate for excessive pH-dependent cell wall loosening that otherwise results in cell wall failure and burst root hairs. Notably, borate did not rescue the hypocotyl defect of shv3svl1 grown on Suc (Hayashi et al., 2008), which would be consistent with further weakening of the cell wall due to Suc-conditional cellulose deficiency. We also suspect that the swelling of guard cells and the collapse of trichomes observed by Hayashi et al. (2008) could be explained in part by such ectopic cell wall loosening in combination with cellulose deficiency in shv3svl1.

While our work sheds some light on the role of SHV3-like proteins in cellulose synthesis, it also provides a cautionary note regarding the inclusion of high concentrations of Suc in plant growth media when working with mutants with disrupted PM H⁺ gradients. We found that, in both shv3svl1 and fer, the enhanced uptake of Suc from medium dramatically enhances the phenotypes of these mutants. In rapidly expanding cells of the dark-grown hypocotyl, induced cellulose deficiency resulted in radial swelling and reduced cell elongation. In the case of fer, this effect could mask phenotypes associated with FER’s role in the regulation of cell expansion in response to the RALF peptide (Haruta et al., 2014), mechanostimulation (Shih et al., 2014), or phytohormones (Deslauriers and Larsen, 2010; Yu et al., 2012). On the other hand, we still observed more subtle cellulose deficiency in fer and shv3svl1 in the absence of Suc, indicating that the underlying cellulose deficiency in both cases warrants further investigation aimed at understanding the role of these proteins in coordinating cellulose biosynthesis with H⁺ secretion during cell expansion.

MATERIALS AND METHODS

Plant Material

Arabidopsis (Arabidopsis thaliana) plants were grown at 22°C on one-half-strength MS medium consisting of 0.215% (w/v) Murashige and Skoog Basal Salts (Caisson Laboratories), 2.5 mm MES (pH 5.7), and 0.8% (w/v) agar, supplemented with sugars as indicated. Seeds were sterilized with household bleach, washed with water, and stratified for 5 d at 4°C in the dark. For skotomorphic growth, seeds were plated and exposed to ambient light for 3 h to induce germination. Plates were then wrapped in three layers of aluminum foil and transferred to the growth chamber. The specific mutant alleles used are indicated in Table III; all mutants were in the Columbia-0 (Col-0) background, which was used as the wild-type control in all experiments. Seedling images were obtained using a Perfection V600 Photo flatbed scanner (Epson). Quantification of hypocotyl length was based on tracing of images using ImageJ.

Table III. Arabidopsis genotypes used in this study.

Genotype	Alleles	Mutation Type	Reference
shv3svl1	shv3-2 svl1-1	Null insertions	Hayashi et al. (2008)
suc1	suc1-2	Null insertion	Sivitz et al. (2008)
cob	cob-6	Hypomorphic insertion	Ko et al. (2006)
cesa6	prc1-1	Null point	Fagard et al. (2000)
fer	fer-4	Null insertion	Duan et al. (2010)

Cell Wall Monosaccharide and Cellulose Quantification

Seedlings were flash frozen in liquid nitrogen and lyophilized. Dried material was beaten with three steel balls in an MM 400 ball mill (Retsch) twice for 2 min at 25 Hz. Balls were removed, and the powdered material was then washed twice with 70% (v/v) ethanol, three times with 1:1 (v/v) chloroform:methanol, and once with acetone. After each wash step, material was recovered by centrifugation at 20,000g for 10 min. The AIR is the final material after air drying overnight. For simultaneous quantification of matrix monosaccharides and cellulose, we used an adaptation of the one-step/two-step hydrolysis protocol described by Bauer and Ibáñez (2014). From each AIR sample, paired aliquots of approximately 1 mg were accurately weighed into a 2-mL Sarstedt tube. To one tube, 50 µL of 72% (w/v) sulfuric acid was added, and the tube was vortexed at room temperature for 1 h. The acid was then diluted by the addition of 1,400 µL of water. Material in the second tube was resuspended in 1,400 µL of water and 50 µL of 72% (w/v) sulfuric acid without any concentrated acid pretreatment step. Both tubes were then sealed and heated to 121°C for 1 h in an autoclave. Samples were cooled and diluted 100-fold with water before analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) on a Dionex ICS-4000 system (Thermo Fisher). All samples were analyzed on a CarboPac PA-20 column (Thermo Fisher) eluted with 2 mm KOH in order to resolve and quantify Fuc, Gal, Glc, Xyl, and Man. Samples without concentrated sulfuric acid pretreatment were further analyzed on a CarboPac PA-20 column eluted with 18 mm KOH in order to resolve and quantify Rha and Ara. These samples were also analyzed on a CarboPac PA-200 column eluted with 100 mm NaOH and a 10-min 50 to 200 mm sodium acetate gradient in order to resolve and quantify GalA and GlcA. A recovery standard consisting of a mixture of all nine quantified sugars was subjected to the same hydrolysis conditions, and correction factors were calculated for each sugar based on comparison with an untreated standard mix. The calculation of cellulose, based on a comparison of Glc recovered from the two hydrolysis regimes, was as described by Bauer and Ibáñez (2014).

Starch Quantification and Visualization

Starch was quantified using an adaptation of the thermostable α-amylase/amyloglucosidase-based Total Starch Assay kit (Megazyme). Briefly, approximately 1 mg of AIR material was accurately weighed into a 2-mL Sarstedt tube and wetted with 10 µL of 70% (v/v) ethanol. To this, 3 units of thermostable α-amylase was added in a solution of 150 µL of 100 mm sodium acetate and 5 mm CaCl₂, pH 5. Tubes were heated for 6 min at 95°C with constant vortexing. After cooling to room temperature, 3.3 units of amyloglucosidase was added, and the samples were incubated for 30 min at 50°C with constant vortexing. To stop the reaction, 840 µL of 0.5 m acetic acid was added. Samples were diluted 100-fold with water, and Glc was quantified by HPAEC-PAD as described for cell wall monosaccharides.

For visualization of starch granules, seedlings were cleared by washing for 30 min in 70% (v/v) ethanol followed by rinsing in water. Seedlings were then mounted in Lugol’s solution (Sigma; no. 62650) diluted 10-fold with water and examined with a 5×/0.15 air objective on a Leica DM5000B microscope using bright-field illumination.

Live-Cell Imaging of CSCs

Wild-type (Col-0) and shv3svl1 plants were transformed with a construct encoding GFP-tagged CesA3 using Agrobacterium tumefaciens-mediated transformation as described previously (Desprez et al., 2007). T2 plants grown for 3 d on the indicated media were analyzed by spinning-disk confocal microscopy, and CSC dynamics were analyzed using ImageJ and Imaris (BitPlane) software as described previously (Vellosillo et al., 2015). CSC particle density is reported as the number of spots detected and tracked in Imaris from each 170-s time series divided by the area of each cell with visible CSC particles. Seedlings were mounted in liquid one-half-strength MS medium containing the same Suc concentration used to grow the seedlings.

¹³C Solid-State NMR of Cell Walls

Liquid cultures of seedlings were grown in shaking flasks for 14 d in the dark in one-half-strength MS medium supplemented with 0.5% (w/v) uniformly ¹³C-labeled Glc as described previously (Dick-Pérez et al., 2011). Quantitative ¹³C direct polarization magic-angle-spinning solid-state NMR spectroscopy was performed as described by Sorek et al. (2015). Because of the technical limitation imposed by the necessity of uniform ¹³C labeling, we could not compare the structure of cellulose from seedlings grown in the absence of exogenous sugars by this method.

Suppressor Screen and Genetic Mapping

Bulked shv3svl1 seeds were mutagenized by treatment with EMS as described by Kim et al. (2006). M2 seeds were harvested in pools derived from selfing of approximately 100 M1 individuals. To screen for suppressor mutants, approximately 2,000 M2 seeds from each pool were sterilized with household bleach, stratified for 5 d, and sown in square petri dishes by embedding in cooled one-half-strength MS medium containing 60 mm Suc and 0.6% (w/v) agar. The initial screen was performed in this manner, since we found that growth through low-concentration agar rather than on the surface reduced variability in hypocotyl length in the mutant, particularly at high seedling densities. After 3 h in the light, plates were wrapped in aluminum foil and grown for 5 d at 22°C. Candidate suppressors were selected based on enhanced hypocotyl elongation in the dark and were transferred to soil and 16-h-light/8-h-dark growth conditions. To confirm the heritability of suppressor mutations, the M3 generation seedlings were rescreened in the dark. In total, 80 pools were screened, representing the descendants of approximately 8,000 M1 individuals.

To map the mutation underlying the suppression of shv3svl1, shv3svl1sss1 was backcrossed to shv3svl1 and the BCF2 progeny were collected and screened as before. Approximately 150 individuals with long dark-grown hypocotyls were pooled. Genomic DNA was extracted from the pooled segregants and from unmutagenized shv3svl1 using the DNeasy Plant Mini kit (Qiagen) following the manufacturer’s instructions. Barcoded libraries were prepared with an average fragment size of 500 bp by the University of California, Berkeley, Functional Genomics Laboratory. Both libraries were sequenced in 100-bp paired-end mode on a single lane of a HiSeq 2000 (Illumina) by the University of California, Berkeley, Vincent J. Coates Genomics Sequencing Laboratory. Reads were deposited in the National Center for Biotechnology Information Sequence Read Archive with accession number SRP072211.

The resulting reads from each library were aligned to the Arabidopsis Col-0 reference genome (The Arabidopsis Information Resource 9) using CLC Genomics Workbench 7 (Qiagen) with default settings and autodetection of paired distances. SNPs were called using the basic variant detection method, and preexisting SNPs present in shv3svl1 relative to the reference genome were filtered from those of the mapping population. Plotting allele frequency of the 866 remaining SNPs as a function of chromosome position revealed a peak at the bottom of chromosome 1. To confirm that the SUC1 SNP was causative, a fragment of genomic DNA spanning the SUC1 coding sequence was amplified from each suppressor by PCR using primers SUC1-P1 and SUC1-P2, and the resulting fragments were Sanger sequenced using primers SUC1-P3, SUC1-P4, SUC1-P5, and SUC1-P6 (Supplemental Table S3).

RALF Sensitivity

Recombinant AtRALFL1 with a His tag was expressed and purified from Escherichia coli as described previously (Morato do Canto et al., 2014). Individual seeds were placed in wells of 12-well plates containing 1 mL of one-half-strength MS medium without Suc. Plates were wrapped in foil and shaken gently for 2 d at 22°C. With green light illumination, 10 µL of a 200 µm RALF solution was added to each well for RALF treatment or 10 µL of water was added as the mock control. Plates were again wrapped in foil and grown for 3 d at 22°C with gentle shaking. Liquid was removed from each well, and seedlings were imaged as described above.

Bathing Medium Acidification

To assay H⁺ secretion, we monitored the pH of bathing medium containing fluorescein dextran as a pH indicator based on the protocol of Haruta et al. (2010) with some modifications. Briefly, unbuffered one-quarter-strength MS medium containing 60 mm Suc and 30 µg mL⁻¹ fluorescein dextran (Sigma; FD10S) was adjusted to pH 6.6. Seedlings were grown for 5 d in the dark on one-half-strength MS plates with 60 mm Suc. Under green light, five seedlings were transferred to each well of a 12-well plate containing 1 mL of the bathing medium. The plate was wrapped in foil and shaken gently for 16 h at 22°C. To measure the pH of each well, 200 µL was transferred to an opaque 96-well plate in triplicate, and fluorescence at 525 nm with excitation at 485 nm was recorded using a Paradigm plate reader (Beckman Coulter). The pH was calculated from a standard curve of bathing medium adjusted to pH 5.6 to 6.6.

Soluble Sugars

Seedlings were grown for 3 d in the dark, rinsed with water, and blotted dry with a paper towel. Pools of seedlings were put into a 2-mL Sarstedt tube containing five glass beads, and the fresh weight was recorded. Flash-frozen samples were homogenized by shaking in an MM 400 ball mill (Retsch) twice for 2 min at 25 Hz. To the frozen powdered samples, 400 µL of 80% (v/v) ethanol was added, and the sealed tubes were heated with constant vortexing for 20 min at 80°C. After cooling, samples were centrifuged at 20,000g for 10 min, and the supernatant was recovered. Extraction with ethanol was repeated three times, and the pooled extracts were dried by vacuum centrifugation. Dried residue was resuspended in 1 mL of water, and 0.5 mL of chloroform was added. Samples were vortexed and centrifuged at 20,000g for 1 min. The aqueous phase was recovered, and Glc, Fru, and Suc were quantified by HPAEC-PAD as described previously using a PA-20 column eluted with 30 mm KOH.

Esculin Uptake

Plants were grown on plates for 4 d in the dark on one-half-strength MS medium with 60 mm Suc. Seedlings were transferred to one-half-strength MS liquid medium with 60 mm Suc supplemented with 1 mm esculin (Sigma; E8250) for 30 min. Seedlings were then rinsed and mounted on glass slides in one-half-strength MS liquid medium without esculin. The samples were illuminated with 360-nm wavelength UV light and examined with a Leica MZ16 F microscope.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers SRP072211.

Supplemental Data

The following supplemental materials are available.

• Supplemental Figure S1. Comparison of the growth of seedlings on media containing 60 mm Suc, 60 mm Glc and 60 mm Fru, and 120 mm Sorb.

• Supplemental Figure S2. Effects of various exogenous sugars on the hypocotyl elongation of suc1 in the dark.

• Supplemental Figure S3. Growth of fer seedlings on media containing 60 mm Suc, 60 mm Glc and 60 mm Fru, and 120 mm Sorb.

• Supplemental Figure S4. Dimethyl sulfoxide controls for esculin uptake experiments.

• Supplemental Figure S5. SVL1 coexpression network.

• Supplemental Figure S6. Assays of recombinant SHV3 enzyme activity.

• Supplemental Table S1. Neutral sugar and uronic acid composition of shv3svl1 correcting for the mass of starch.

• Supplemental Table S2. Neutral sugar and uronic acid composition of fer.

• Supplemental Table S3. Primers used in this study.

• Supplemental Movie S1. Live-cell imaging of CSC dynamics.

Glossary

PM

plasma membrane

CSC

cellulose synthase complex

GPI

glycosylphosphotidylinositol

AIR

alcohol-insoluble residue

Sorb

sorbitol

MS

Murashige and Skoog

EMS

ethyl methanesulfonate

SNP

single-nucleotide polymorphism

Col-0

Columbia-0

HPAEC-PAD

high-performance anion-exchange chromatography with pulsed amperometric detection