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The synthesis of xyloglucan, an abundant plant cell wall polysaccharide, requires CSLC function

Significance Plant cells have a polysaccharide-based wall that maintains their structural and functional integrity and determines their shape. Reorganization of wall components is required to allow growth and differentiation. One matrix polysaccharide that is postulated to play an important role in this reorganization is xyloglucan (XyG). While the structure of XyG is well understood, its biosynthesis is not. Through genetic studies with Arabidopsis CSLC genes, we demonstrate that they are responsible for the synthesis of the XyG glucan backbone. A quintuple cslc mutant is able to grow and develop normally but lacks detectable XyG. These results raise important questions regarding cell wall structure and its reorganization during growth. The series of cslc mutants will be valuable tools for investigating these questions.


Transcriptomics and qRT-PCR analyses
RNA was extracted using plant RNA extraction kit (www.mn-net.com). After RNA isolation, total RNA from 7-day old etiolated hypocotyls of wild type and cslc456 and cslc456812 mutant were sent to the Research Technology Support Facility at Michigan State University for generating RNAseq libraries and sequencing the libraries using Illumina platform (paired 125 bp). The RNAseq reads were analyzed with CLC Genomics Workbench version 8.5 (www.qiagenbioinformatics.com/products/clc-genomics-workbench/). Adapter sequence and lowquality regions were removed using the Trim Sequences tool (quality limit: 0.05). Trimmed reads were mapped onto the Arabidopsis thaliana reference genome (TAIR10) and alignments over gene regions were quantified using the RNA-Seq Analysis tool (length fraction: 0.8, similarity fraction: 0.8). The resulting read mappings and RPKM normalized expression values were imported into a custom database for further analysis. Total read counts for each gene were analyzed using DESeq2 (1).
For qRT-PCR or RT-PCR of CSLC family genes, 1 µg of total RNA was used to investigate expression of CSLC genes during Arabidopsis development. qRT-PCR and RT-PCR was performed using CSLC gene-specific primers (Table S5) as described previously (2). Ubiquitin was used to normalize the expression level of CSLC genes. Two to three biological replicates were tested, and a similar pattern of CSLC expression was observed among biological replicates.

Root hair measurements and pollination assays.
For root hair measurements, seedlings were grown for 1 week on plates containing ½ MS with 1% sucrose containing 0.7% agar at 16 h/ 8 h light/ dark (120 µmol/m 2 /s) at 22 °C. The images of root hairs from seedlings were obtained using Zeiss-Axio imager (www.zeiss.com). Images were analysed using ImageJ software to measure the length of root hairs.
Pollination assays were performed as described previously (3), except that floral buds were processed for microscopy exactly 1 hour after pollination. For each assay, wild-type stigmas were saturated with pollen from wild type or each of the various mutant plants. Pollen tubes were visualized using a Nikon A1 laser scanning confocal microscope by exciting with a 405-nm laser and collecting emission at 525 nm with the green band-pass filter channel. In order to both increase contrast between the pistil tissue and the pollen tubes and emphasize gradations in signal intensities, the "Rainbow Dark" LUT setting was applied to the captured images using NIS-Elements AR software. Pollen tubes that had penetrated the style of each wild-type pistil (n = 6 pistils for each pollen genotype) were counted, and treatments (mutants) that were statistically significantly different from the wild-type control were determined by Dunnett's test (P < 0.05) using Prism software.

Glycan array
Glycan array was performed as described previously (4)

Driselase digestion of alcohol-insoluble residue (AIR), and HPAEC and LC/ Qtof MS analyses
The driselase enzyme mix (www.sigmaaldrich.com) was partially purified as previously reported (7). AIR was prepared using 7-day-old etiolated hypocotyls as described (5). Approximately 1.3 mg of AIR obtained from the etiolated hypocotyls were digested with 300 µl of 0.03% of driselase enzyme mix (25 mM NaAc, pH 5.0) at 37 °C for 48 hours, and the reaction was stopped by heating the samples at 95 °C for 5 min. For HPAEC analysis, maltose was added as an internal standard for IP quantification. The digest was analysed using a modified procedure (8). The carbohydrates were separated using a Carbo Pac PA200 column with a linear gradient of 2-10 mM NaOH for 5 min, followed by 10-355 mM NaOH for 20 min. Eluting compounds were detected by pulsed amperometric detection (PAD). For LC/MS analyses, 233 µl of methanol was added to the driselase digest (100 µl) and incubated in 4 °C for 20 min. After centrifugation at 11k x g for 10 min, the supernatant containing digested sugars were dried and resuspended in 50% acetonitrile containing 2 µg/ml of 13 C-labelled sucrose (monitoring matrix effect) as an internal standard for quantification. IP quantification was done using a Waters Xevo G2-XS Qtof mass spectrometer interfaced with a Waters Acquity UPLC. Samples were injected onto a Waters Acquity BEH-Amide UPLC column (2.1x100 mm) and a 20-min gradient was run as follows: initial conditions were 5% solvent A (10 mM ammonium acetate in water), 95% B (acetonitrile) and held for 1 min, followed by a ramp to 35% A/ 65% B at 14 min, hold at 35% A until 16 min, return to 5% A/ 95% B at 16.01 min and hold until 20 min. Flow rate was 0.3 ml/min and column temperature was 40 ºC. Data were acquired using electrospray ionization in negative-ion mode with a target enhancement function (m/z 325). Peak areas for IP (M-H) and 13 C sucrose were processed using Quanlynx (Masslynx software).

Complementation of the cslc456812 quintuple mutant
To generate Gateway technology-compatible vector containing the Arabidopsis CSLC4 promoter, amplification of the CSLC4 promoter (1719 bp) was performed using CLSC4 promoter-specific primers. The amplified product was introduced in the pEarlygate100 vector using the Infusion system (www.takarabio.com) (9). Full-length coding sequences of CSLC family genes with Nterminal T7 tags were synthesized from Thermo-Fisher (www.thermofisher.com). The resulting synthesized genes in the pDONOR221 plasmid were used to generate the final constructs containing CSLC4pro::T7-CSLC4, 5,6,8,12 in the pEarlygate100 plasmid backbone.
Arabidopsis transformation by vacuum infiltration was performed to create complementation lines of the cslc456812 quintuple mutants as previously reported (8). The transformed seedlings were screened by observing root hair growth restoration. Due to difficulty of screening successful complementation lines by CSLC8 and CSLC12 with this method, we created 35Spro::T7-CSLC8 or CSLC12 in pGWB2 via the LR reaction in order to screen transformants using antibiotics (hygromycin). The CSLC8 or CSLC12 transformed seedlings were screened on ½ Murashige and

Fig. S2. Expression pattern of CSLC genes in Arabidopsis
The expression data of CSLCs was obtained from Arabidopsis eFP Browser (http://bar.utoronto.ca/efp2/Arabidopsis/Arabidopsis_eFPBrowser2.html). Mature pollen was combined with seed development to facilitate display of the data. The expression level of each CSLC gene was investigated using RNA from wild-type Col-0, and relative expression level of each CSLC gene was normalized using the expression level of CSLC4, n = 3, ± SD.  Asterisks indicate statistically significant differences from Col-0 (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by student's t-test).

Fig. S6. Root-hair lengths, and pollen-tube numbers from pollination assays, of various cell wall mutants.
(A) Lengths of root hairs from 1-week-old seedlings (n > 40, ± SD). One-way ANOVA (Tukey's test, P < 0.05) was used to determine statistical differences of root-hair length among genotypes. Each statistical group was labeled with lower-case letters (a, b, and c). (B) Numbers of pollen tubes that have penetrated the styles of Col-0 pistils, 1 hour after pollination with pollen from each of the indicated genotypes (n = 6, ± SEM). Asterisks denote treatments (mutants) that are statistically significantly different from the Col-0 control (Dunnett's test, *P < 0.05).

Fig. S7. Isoprimeverose profiling of wall preparations from Col-0 and Arabidopsis cslc and xxt1 xxt2 mutants using HPAEC-PAD
The AIR material obtained from the etiolated hypocotyls of Arabidopsis wild type and different mutants were digested with driselase. The digested material was analysed by HPAEC. The IP present in the digest was quantified using maltose as an internal standard.

Fig. S8. Glycosidic linkage analysis of compounds eluting at the retention time of IP.
Driselase-digested AIR was subjected to HPAEC (see Fig. 4, Fig. S7) and the compounds eluting at the retention time of IP (approximately 10 min) were collected. To account for the significant reduction in IP abundance in the xxt1 xxt2 and cslc456812 mutants (see Fig. 4, Fig. S7), 10 times the amount of driselase digest of the corresponding AIR material was subjected to HPAEC analysis and collected. The collected material was subjected to methylation analysis followed by acid hydrolysis, reduction, and acetylation to yield partially methylated alditol acetates. These were analysed by gas chromatography (panel A) fitted with an electron impact quadrupole detector (GC-MS). An IP standard subjected to this procedure (separation by HPAEC, collection and analysis by GC-MS) results in two peaks that based on their ion fragmentation patterns represent terminal xylose (T-Xylose), and 6-linked glucose (6-Glucose). These two peaks, at 12.3 minutes and 17.6 minutes, respectively, were also visible in the driselase-digested AIR from Col-0. They gave fragmentation patterns characteristic of terminal xylose and 6-linked glucose (panel B). However, similar peaks were not seen in the GC traces of the cslc quintuple mutant and the xxt1 xxt2 mutant (panel A) and the characteristic fragmentation pattern of these two glycosylmoieties could not be detected in the mass spectra of the material eluting at the appropriate retention times (panels C and D), even though 10 times the material was used for analysis.

Fig. S11. Verification of complementation lines of the cslc456812 mutant using RT-PCR.
Extracted RNA from each complementation line was used for RT-PCR using CSLC-specific primers. UBQ10 primers were used for the loading control. As positive and negative controls, RNA from Col-0 and the cslc quintuple mutant was used to show the specificity of primer sets used in this experiment. Significance was based on student's t-test (* P < 0.05 -yellow shade, ** P < 0.01-orange shade). a Glycosyl residues are expressed as percentage of the total glycosyl peak areas. Averages and standard deviations of the biological replicates (n = 3) are presented. Significance was based on student's t-test (* P < 0.05, yellow; ** P < 0.01, orange). b Non-plant cell wall component due to contamination of the agar growth medium.