Abstract
Plant genomes contain numerous genes encoding chitinase‐like (CTL) proteins, which have a similar protein structure to chitinase belonging to the glycoside hydrolase (GH) family but lack the chitinolytic activity to cleave the β‐1,4‐glycosidic bond in chitins, polymers of N‐acetylglucosamine. CTL1 mutations found in rice and Arabidopsis have caused pleiotropic developmental defects, including altered cell wall composition and decreased abiotic stress tolerance, likely due to reduced cellulose content. In this study, we identified suppressor of hot2 1 (suh1) as a genetic suppressor of the ctl1 hot2‐1 mutation in Arabidopsis. The mutation in SUH1 restored almost all examined ctl1 hot2‐1 defects to nearly wild‐type levels or at least partially. SUH1 encodes a Golgi‐located type II membrane protein with glycosyltransferase (GT) activity, and its mutations lead to a reduction in cellulose content and hypersensitivity to cellulose biosynthesis inhibitors, although to a lesser extent than ctl1 hot2‐1 mutation. The SUH1 promoter fused with the GUS reporter gene exhibited GUS activity in interfascicular fibers and xylem in stems; meanwhile, the ctl1 hot2‐1 mutation significantly increased this activity. Our findings provide genetic and molecular evidence that the antagonistic activities of CTL1 and SUH1 play an essential role in assembling the cell wall in Arabidopsis.
Keywords: cell wall, chitinase‐like protein, Domain of Unknown Function 266 (DUF266), genetic suppression, glycosyltransferase, Golgi complex
1. INTRODUCTION
Chitinases (EC 3.2.1.14) comprise a subfamily of glycoside hydrolases (GHs) that catalyze the hydrolysis of β‐1,4 glycosidic bonds in amino polysaccharides such as chitin and chitooligosaccharides (Kesari et al., 2015). They are primarily grouped into six classes, I to VI. Classes III and V belong to the GH18 family, which exhibits a TIM (triosephosphate isomerase) fold consisting of a β/α barrel structure (Funkhouser & Aronson, 2007). The other classes belong to the GH19 chitinases, which are composed of an α‐helical structure in the catalytic region (Ohnuma et al., 2012). In addition to the well‐known role of chitinases in plant defense mechanisms against fungal pathogens, there have been several reports of chitinase‐like proteins (CTLs) participating in cell wall synthesis in Arabidopsis and rice, ultimately affecting their growth and development (Hauser et al., 1995; Hermans et al., 2010; Wu et al., 2012; Zhong et al., 2002). CTLs, which have a catalytic region composed of an α‐helical structure, are structurally characterized by the absence of a hevein domain, known as the chitin‐binding domain, and the chitinase activity motif (H‐E‐E‐T). Hence, these features allow them to be further categorized as class II, which belongs to the GH19 family (Grover, 2012). Although no chitinolytic activity was detected (Kwon et al., 2007; Wu et al., 2012), CTLs in Arabidopsis and Caribbean pine were reported to bind to glucan chains and arabinogalactan, respectively (Domon et al., 2000; Sánchez‐Rodríguez et al., 2012). These findings suggest that these interactions affect the cell wall assembly, which is composed mainly of complex polysaccharides.
Plant cell walls (CWs) can be classified into primary and secondary CWs, which differ in composition and function (Houston et al., 2016; Rui & Dinneny, 2019). The primary CW is relatively thin and flexible, consisting of cellulose microfibrils embedded in a matrix of various polysaccharides such as hemicelluloses, pectins, and glycoproteins. It determines cell shape and controls cell expansion during growth, contributes to the response to environmental stimuli, and allows for cell–cell communication. The secondary CW is thicker and more rigid than the primary CW. It is deposited on the inner side of the primary CW after cell expansion has ceased. The secondary CW mainly comprises cellulose microfibrils, hemicelluloses, and lignin, which is a polyphenolic compound that provides rigidity and waterproofing properties to the secondary CW. Consequently, depositing secondary CWs in interfascicular cells and xylem elements is crucial for providing structural support and facilitating water transport.
Various mutations affecting cell wall synthesis have resulted in abnormal growth and development, demonstrating the biological importance of CWs in plants. Cellulose is synthesized at the plasma membrane by a large membrane‐bound protein complex. The catalytic core comprises three cellulase synthase (CESA) types in the primary and secondary CWs (Endler & Persson, 2011). In Arabidopsis, the CESA complex, which contains CESA1 (RADIAL SWELLING1 [RSW1]), CESA3 (ISOXABEN RESISTANT1 [IXR1]), and CESA6 (PROCUSTE1[PRC1]) subunits, is mainly responsible for cellulose synthesis in the primary CW. Insertion of T‐DNA into CESA1 and CESA3 leads to lethal male gametophytes (Persson, Paredez, et al., 2007). However, a null mutation in CESA6 (cesa6 prc1‐1 ) exhibits mild phenotypes, such as reduced root and etiolated hypocotyl growth (Fagard et al., 2000). Genetic evidence shows that mutations in CESA4 (IRREGULAR XYLEM5 [IRX5]), CESA7 (IRX3), and CESA8 (IRX1), which are involved in cellulose synthesis in the secondary CW, cause a collapse in xylem development (Chen et al., 2005; Taylor et al., 1999, 2003). In addition, the small stature of many mutants defective in noncellulose polysaccharides such as pectin (Bouton et al., 2002; Liwanag et al., 2012) and hemicellulose (Xiao et al., 2016) provides genetic evidence of the physiological roles of the cell wall. Mutations in the Arabidopsis QUASIMODO1 (QUA1), which encodes a protein with amino acid sequence similarity to α‐1,4‐D‐galacturonosyltransferase that belongs to the glycosyltransferase 8 (GT8) family, cause a 25% reduction in pectin, leading to dwarfism and reduced cell adhesion (Bouton et al., 2002). The Arabidopsis mutations in IRX8 (encoding a protein similar to α‐1,4‐D‐galacturonosyltransferase, belonging to the GT8 family) exhibit a significant decrease in hemicellulose, such as homogalacturonan and xylan and display dwarfism and a reduction in secondary cell wall thickness (Persson, Caffall, et al., 2007). Moreover, rice brittle culm (bc) mutants with reduced mechanical internode strength also demonstrate the essential role cell walls perform in growth and development (Zhang & Zhou, 2011).
Despite numerous reports that CTLs are involved in cell wall assembly in Arabidopsis and rice, their lack of enzymatic activity and the structural complexity of their potential substrates—polysaccharides—further complicate the understanding of their functions. Therefore, to overcome these difficulties, we employed a genetic approach to further comprehend their function in cell wall assembly by isolating suppressor mutations that alleviate the defects caused by CTL1 mutations in Arabidopsis. Here, we report that the suppressor of hot2 1 (SUH1) gene encodes a Golgi‐localized type II membrane protein with glycosyltransferase activity, which is also potentially involved in glycan synthesis. Our findings provide genetic evidence that the interaction between CTL1 and SUH1, which have opposing enzymatic activities in glycan synthesis, plays a vital role in cell wall assembly in Arabidopsis.
2. MATERIALS AND METHODS
2.1. Plant growth conditions
Seeds were surface‐sterilized and grown on half‐strength (1/2) Murashige and Skoog (MS) media supplemented with 1% (w/v) sugar and .8% (w/v) agar, adjusted to pH 5.8 with KOH, either in total darkness or under long‐day conditions (16 h light/8 h dark, 22°C/18°C cycle under light density of 120 μmol m−2 s−1) in a growth chamber. Seedlings (light grown on 1/2 MS media) were transferred to the soil at 10 days old and grown under long‐day conditions in a growth chamber (light density of 120 μmol m−2 s−1). Unless stated otherwise, all mutants and transgenic lines used in this study were obtained from the Arabidopsis Columbia‐0 ecotype (Col‐0). We received the Arabidopsis thaliana SAIL_912_D02 T‐DNA insertion line (suh1‐4) with T‐DNA inserted in the first intron of At5g14550 from the Arabidopsis Information Resource (TAIR). Genomic DNA was extracted from individual plants. To confirm T‐DNA insertion, PCR amplification was performed using gene‐specific primers and the T‐DNA left border primer LBb1 (Table S2). The precise position of the T‐DNA insertion was determined by sequencing the PCR products using the T‐DNA left border primer LBb1.
2.2. EMS mutagenesis and isolation of the suppressor of ctl1 hot2‐1 mutation
Approximately 10,000 ctl1 hot2‐1 seeds (M1 seeds) were imbibed overnight in water at 4°C before incubation in 50 ml of .25% ethyl methane sulfonate (EMS) for 12 h. Subsequently, the EMS was washed several times with water. The M2 seeds were independently harvested from 100 trays, with each containing about 100 M1 plants. Since the dark‐grown ctl1 hot2‐1 seedlings display a short hypocotyl, about 120,000 M2 seeds were grown in the dark to identify three independent seedlings with a long hypocotyl. Then, it was confirmed that these three suppressors grown in the dark also showed a long hypocotyl in the successive M3 generation, and they were designated as suh (suppressor of hot2) mutants. To determine the inheritance characteristics of the suppressor mutation, three suh mutant plants were crossed with the parental ctl1 hot2‐1 plants, and all pairwise combinations of the three suh mutants were crossed to conduct allelism tests.
2.3. Map‐based cloning of SUH1 gene
To perform map‐based cloning of the suh1 mutation, we looked for a ctl1 mutant allele in the Landsberg erecta (Ler) ecotype by conducting a genetic screening of EMS‐mutagenized Ler seeds. Complementation testing and sequencing analysis revealed that a new ctl1 allele (Ler) harbors the same mutation as the ctl1 hot2‐1 (G881A).
To generate a mapping population, ctl1 hot2‐1 suh1‐1 mutant (Col‐0) was crossed with Ler‐derived ctl1 plants named ctl1 hot2‐3 . The resulting F1 plants were self‐pollinated, and DNA was extracted from individual F2 plants displaying the wild‐type phenotype of seedlings grown in the dark. PCR was performed using simple sequence length polymorphism (SSLP) markers (Bell & Ecker, 1994). The recombination frequency between the SUH1 locus and the SSLP makers was determined to identify the position of the SUH1 locus.
2.4. Semiquantitative RT‐PCR and cDNA synthesis
Total RNA was extracted from stems of Arabidopsis and rice using the Ribospin™ kit from GeneAll (https://geneall.com/). For RT‐PCR, 2–5 μg of total RNA was reverse transcribed using a Superscript First‐Strand Synthesis system (Invitrogen, Carlsbad, CA, USA). The quantitative RT‐PCR was performed using the suh1‐4 RT primers (Table S2) and Arabidopsis actin2 as an endogenous reference. PCR products were visualized by agarose gel electrophoresis using EtBr staining.
First‐strand cDNA was synthesized from 5 μg of total RNA using the PrimeScript™II cDNA Synthesis kit from Takara (https://takara.com/). Full‐length cDNAs of SUH1 and BC10 were amplified using the Taq LA DNA polymerase PCR kit from Takara (https://takara.com/) and cDNA‐specific primers (Table S2). The resulting fragments were cloned into the pGEM‐T easy vector and sequenced using an ABI 3730 automated sequencer (Applied Biosystems).
2.5. Construction of various vectors and plant transformation
The promoter fragment, located between −1060 and −1 in the SUH1 gene, was amplified (the A site in the ATG translation start codon was designated +1). The pSUH1::GUS construct was generated by replacing the 35S promoter in the pBI121 binary vector with a fragment containing the SUH1 promoter. To develop the constructs used in the complementation tests, the full‐length cDNAs of SUH1 and BC10 were placed under the control of the SUH1 promoter sequence (1060 bp) in the pBI121 binary vector. The resulting binary vectors (pSUH1::SUH1 and pSUH1::OsBC10) were introduced into the Agrobacterium tumefaciens strain GV3101 for Arabidopsis transformation using the floral dip method (Clough & Bent, 1998). Seedlings from the T1 generation were selected on half‐strength MS medium containing 30 μg/ml kanamycin. T3 homozygous progenies were isolated for each transgene, and three independent lines were selected for each construct for further examination.
2.6. Cell wall composition assay and histology
The Updegraff assay was applied to determine the cellulose content in the inflorescence stems (the bottom 5 cm of the stem) from 6‐week‐old wild‐type and mutant plants (Kumar & Turner, 2015). Samples were incubated in 70% ethanol at 70°C for 1 h, washed with 100% acetone, and dried overnight. After adding the acetic nitric agent (8:1:2, acetic acid:nitric acid:water), the samples were placed in a bath with boiling water for 30 min, precipitated by centrifugation at 14,000 rpm for 15 min, and incubated in 67% sulfuric acid in boiling water for 5 min. Finally, the anthrone–sulfuric acid colourimetric assay was performed to analyze the samples. The absorbance was measured at 620 nm using a spectrometer (UV‐1600, Shimadzu). The cellulose contents were expressed as the percentage of cellulose in the cell wall composition, with glucose standards.
Hand‐cut sections of inflorescence stems were incubated in 1% (w/v) phloroglucinol in 18% HCl, .01% (w/w) ruthenium red or 2 mg/ml β‐glycosyl Yariv agent in .1‐M NaCl to stain the lignin, pectin, and arabinogalactan proteins, respectively. After staining, the samples were washed with deionized water and observed using a Carl Zeiss AX10 microscope (Zeiss Corp, Germany).
To determine the shapes of the pith cells, the inflorescence stems of 6‐week‐old wild‐type and mutant plants were fixed in 2.5% glutaraldehyde at 4°C overnight. Tissues were gradually dehydrated in ethanol and embedded in Historesin (Leica Microsystems, France) according to the manufacturer's instructions. Transverse sections (5‐μm‐thick) were prepared using an ultramicrotome (LEICA, Germany), stained with 1% toluidine blue, and examined using a Carl Zeiss AX10 microscope (Zeiss Corp, Germany).
2.7. Treatment with cellulose biosynthesis inhibitors and abiotic stress
To determine their sensitivity to cellulose biosynthesis inhibitors (CBIs), sterilized Arabidopsis seeds were grown vertically on a half‐strength MS medium supplemented with the indicated concentrations of 2,6‐dichlorobenzonitrile (DCB) or isoxaben (ISX) in .5% dimethyl sulfoxide (DMSO). An equal volume of DMSO was used as a control. The primary root growth was measured after10 days.
To assess thermotolerance, seedlings were grown vertically in the dark for 2.5 days, subjected to heat stress at 45°C for 2 h, and further grown in the dark for 2.5 days. To apply salinity stress, 3‐day‐old seedlings grown vertically on a half‐strength MS medium were transferred to a medium with or without the indicated concentrations of NaCl. After 7 days of growth under long‐day conditions (16 h light/8 h dark), the primary root lengths of wild‐type and mutant plants were measured.
2.8. Histochemical assay for GUS activity
Histochemical analysis of β‐glucuronidase (GUS) activity was conducted as described previously (Jefferson et al., 1987). GUS enzyme activity in transgenic plants was determined by staining with 1 mg/ml X‐Gluc (Duchefa, the Netherlands) as the substrate. The GUS‐stained tissues in this report represent the typical results obtained in three independent transgenic lines.
2.9. Assay of C2GnT enzyme activity
The C2GnT assay was performed as described previously (Bierhuizen & Fukuda, 1992). To this end, SUH1 and BC10 were fused in‐frame with EGFP using the pEGFP‐N1 vector. The plasmids were transiently transfected into Chinese hamster ovary (CHO) cells. After 48 h, the cells were washed with phosphate‐buffered saline, and suspended in lysis buffer (10‐mM TrisCl at pH 8.0, .1‐mM EDTA, 5‐mM DTT, .9% NaCl, and 1% Triton X‐100). The cell lysate was centrifuged at 1000 g at 4°C for 10 min. Then, the supernatant was stored in aliquots of 250 μl at −80°C until further use. Protein concentration was determined using a Bio‐Rad protein assay with bovine serum albumin (BSA) as the standard. As the accepter, Galβ1 → 3GalNAcα1 → p‐nitrophenyl (Sigma) was employed for the C2GnT assay. Nontransfected and empty pEGFP‐N1‐transfected CHO cells were used as the negative control group. The reaction mixtures contained 50‐mM HEPES‐NaOH at pH 7.0, 1‐mM UDP‐[glucosamine‐U‐14C] GlcNAc (3.7 kBq, Amersham Pharmacia Biotech), 1‐mM acceptor oligosaccharide, .1‐M GlcNAc, and 5‐mM DTT. After incubating at 37°C for 3 h, the reaction products were adjusted to .25‐M ammonium formate at pH 4.0 and applied to a C18 reverse phase column (Alltech Associates Inc, IL, United States). After washing the column using the same solution, the product was eluted using 70% methanol. The radioactivity was measured using scintillation counting.
2.10. Tobacco transient expression and confocal microscopic analysis of SUH1–GFP fusion proteins
To investigate its subcellular location, SUH1 was fused in‐frame with the GFP‐encoding sequence of the pCAMBIA1300 vector. The ER marker mCherry‐HDEL and the Golgi marker MAN49‐mCherry were obtained from the Daegu Gyeongbuk Institute of Science and Technology (DGST). Transient transformations of Nicotiana benthamiana with A. tumefaciens strain (GV3101) containing the SUH1‐GFP construct and organelle markers were conducted. The Agrobacterium cells were incubated in an infiltration buffer (10‐mM MgCl2, 10‐mM MES at pH 5.9, and 150‐μM acetosyringone) at room temperature for 3 h. Before infiltration, the bacterial suspension was mixed with an equal volume of a bacterial suspension harboring pBin61‐P19 to co‐introduce the RNA‐silencing suppressor gene into the cells. Next, the mixture of Agrobacterium suspensions was infiltrated into the abaxial side of the second, third, and fourth leaves of 6‐week‐old N. benthamiana plants using a 5‐ml syringe without a needle and grown in a growth chamber at 25°C under long‐day conditions (16 h light/8 h dark). The agroinfiltrated plants were placed back into the same growth chamber, and their fluorescence was examined using a Carl Zeiss LSM 710 confocal scanning microscope.
3. RESULTS
3.1. SUH1 mutations restore the growth retardation of ctl1 hot2‐1 seedlings grown in the dark
To investigate the molecular mechanisms underlying CTL1‐mediated cell wall assembly in Arabidopsis, a genetic screen was conducted on over 120,000 M2 ethyl methane sulfonate (EMS)‐mutagenized ctl1 hot2‐1 seedlings. CTL1 mutations have been reported to inhibit Arabidopsis growth in both dark and light conditions. In particular, 5‐day‐old dark‐grown seedlings of ctl1 hot2‐1 exhibited a hypocotyl that is shorter than half that of the wild‐type seedlings. Therefore, identifying suppressor mutations that restore hypocotyl elongation in dark conditions enables mass screening of EMS‐mutagenized M2 seeds of ctl1 hot2‐1 in a Petri dish. Therefore, M2 seedlings were grown in dark conditions for 5 days, and seedlings whose hypocotyl elongated to wild‐type levels were selected. Finally, we isolated three independent suppressors that exhibited hypocotyl to wild‐type length in successive generations (Figure 1a,b). In addition, these suppressors also restored the abnormal morphology of light‐grown ctl1 hot2‐1 mutants, including the presence of numerous lateral branches and shorter stature of the aerial parts, to that of wild‐type plants (Figure 1c). Pair‐wise crosses of three suppressors demonstrated that the three mutations affected a single gene, leading to their designation as suh1‐1, suh1‐2, and suh1‐3 (Table S1). Their crosses with ctl1 hot2‐1 mutants also revealed that the restoration of hypocotyl elongation is inherited in a recessive manner.
3.2. Map‐based cloning of SUH1
The SUH1 locus was mapped to a 53‐kb segment on chromosome 5 (Figure 1d). Through sequencing analysis of suh1‐1 mutants, we identified a premature stop codon caused by a substitution of Arg at position 111 in the second exon of At5g14550, which consists of 11 exons and 10 introns (Figure 1e). Nonsense mutations were also detected in suh1‐2 and suh1‐3, where Gln (Cheng & Radhakrishnan, 2011) and Trp82 in the first exon of At5g14550 were replaced with stop codons, respectively (Figure 1d,e). Moreover, we identified a fourth mutant allele, suh1‐4, which carried T‐DNA in the first intron of At5g14550 (Figure 1d,e). No SUH1 transcripts were detected in suh1‐4 mutant plants (Figure S1A), and suh1‐4 was confirmed to restore the growth defects of ctl1 hot2‐1 mutants under both dark and light conditions. The introduction of SUH1 cDNA driven by its promoter in ctl1 hot2‐1 suh1‐1 mutants resulted in a reversion to the ctl1 hot2‐1 phenotype under both dark and light conditions (Figure S1B,C), providing further evidence that supports the mutation in SUH1 (At5g14550) is responsible for suppressing the ctl1 hot2‐1 phenotype.
Hématy et al. (2007) showed that mutations in THE1, which encodes a receptor‐like kinase, partially restore the short hypocotyl of etiolated cesa6 prc1‐1 carrying a null mutation in CESA6 (resulting in reduced cellulose levels) and pom1‐2 seedlings, which is another CTL1 allele in Arabidopsis. To examine whether suh1 can also restore the short hypocotyl of etiolated cesa6 prc1‐1 mutant, suh1‐4 cesa6 prc1‐1 double mutants were generated by crossing suh1‐4 and cesa6 prc1‐1 mutants. When grown in the dark, the appearance of suh1‐4 cesa6 prc1‐1 mutant seedlings was indistinguishable from the cesa6 prc1‐1 mutants (Figure S2), suggesting that suh1‐4 is unable to rescue the growth defects exhibited by cesa6 prc1‐1 mutants. This implies that SUH1 is involved in cell wall assembly associated with CTL1 rather than directly responding to the lack of a functional CESA6 in Arabidopsis.
3.3. SUH1 mutations restore multiple defects caused by ctl1 hot2‐1
To examine the effect of suh1 on the various defects linked to the CTL1 mutation, we compared the phenotypes of plants carrying four combinations of ctl1 hot2‐1 and suh1‐4. First, we confirmed that suh1‐4 restored the root growth of ctl1 hot2‐1 mutants to wild‐type levels (Figure 2a). As shown in Figure 2b, suh1‐4 suppressed the increase in root hair density in ctl1 hot2‐1 plants; the root hair density of ctl1 hot2‐1 suh1‐4 plants was indistinguishable from the wild‐type and suh1‐4 plants. We also compared the growth of wild‐type and mutant plants in a growth chamber under long‐day conditions for 6 weeks (Figure S3A). The suh1‐4 mutation clearly restored the semi‐dwarf and multiple branched defects exhibited by the ctl1 hot2‐1 mutants. However, the height of the ctl1 hot2‐1 suh1‐4 and suh1‐4 plants was slightly shorter, although not statistically significant, compared with the wild‐type plants (Figure S3B).
Next, we examined whether suh1 could restore changes in cell wall composition and the abnormal cell shape demonstrated by ctl1 hot2‐1 mutants. First, to determine lignin content, 5‐day‐old seedlings of wild‐type and mutant plants grown in the dark were treated with phloroglucinol‐HCl, which stains lignin red (Figure 2c). Unlike ctl1 hot2‐1 , which displayed dark‐red patches, no red patches were detected in the ctl1 hot2‐1 suh1‐4 and suh1‐4 mutant seedlings, similar to the wild‐type seedlings. Using the Updegraff assay (Kumar & Turner, 2015), we also found that the cellulose content in the ctl1 hot2‐1 mutants decreased to 66.9% of that in the wild‐type plants and that suh1‐4 restored the cellulose content in the ctl1 hot2‐1 mutants to 81.6% of that in the wild‐type plants (Figure 2d). In addition to these restorations, further histochemical staining analyses revealed that the deposition patterns of pectin (Figure S4A) and arabinogalactan proteins (AGPs; Figure S4B) in the inflorescence stems of the ctl1 hot2‐1 suh1‐4 mutants were indistinguishable from those in the suh1‐4 and wild‐type plants, unlike the ctl1 hot2‐1 plants, which displayed ectopic depositions. These histochemical staining analyses clearly show that the suh1‐4 mutation can recover their phenotype, although it is difficult to exclude the suh1‐4 effect on lignin, pectin, and AGP synthesis. Cross‐sections of the mature stems showed that suh1‐4 rescued the larger and irregularly shaped cells in the pith of the ctl1 hot2‐1 mutants to the wild‐type phenotype (Figure S4C). Moreover, we confirmed that suh1‐4 also restored the reduced tolerance to high temperature (Figure S5A,B) and salinity stress (Figure S5C,D) of ctl1 hot2‐1 plants. Interestingly, ctl1 hot2‐1 suh1‐4 and suh1‐4 exhibited partial recovery when placed in half‐strength MS media containing 50‐mM NaCl compared with wild‐type plants. Contrastingly, the tolerance to high temperature was restored almost to wild‐type levels. These findings indicate similarities between defects in suh1 and ctl1 hot2‐1 mutant plants, albeit to different degrees. Importantly, our results clearly demonstrate that the suh1 mutation restores almost all defects caused by the CTL1 mutation, at least partially or virtually, to wild‐type levels.
3.4. suh1 alters the response to cellulose biosynthesis inhibitors
The discovery that suh1‐4 affects cellulose synthesis prompted us to investigate the response of wild‐type and mutant plants to chemicals that inhibit cellulose synthesis. Thus, we examined root growth in seedlings grown for 10 days in the light on half‐strength MS media supplemented with cellulose biosynthesis inhibitors (CBIs), such as 2,6‐dichlorobenzonitrile (DCB) and isoxaben (ISX). The primary root growth of wild‐type plants on the medium with the lowest concentration of CBIs was more than 70% of the control plants grown on the medium without CBIs. In contrast, the primary roots of the ctl1 hot2‐1 mutants barely grew under the same conditions (Figure 3). As expected, the primary roots of the ctl1 hot2‐1 suh1‐4 mutants exhibited significant elongation at all inhibitor concentrations tested compared with the ctl1 hot2‐1 mutants, suggesting that suh1‐4 partially restores root growth retardation in ctl1 hot2‐1 mutants grown on half‐strength MS media containing CBIs. Furthermore, suh1‐4 exhibited root growth comparable to ctl1 hot2‐1 suh1‐4, which is shorter than the wild‐type plants at all concentrations of the CBIs. In addition, suh1‐4 was more sensitive to DCB than to ISX, which induces CESA internalization. However, DCB affects microtubule‐associated proteins (MAPs) that play a key role in vesicle transport (Wormit et al., 2012). Therefore, these results suggest that suh1 may be more sensitive to vesicle transport disruption rather than a lack of cellulose synthase due to CESA internalization.
3.5. SUH1 encodes a Golgi‐localized type II membrane protein containing the Domain of Unknown Function 266
The SUH1 cDNA has an open reading frame of 1134 nucleotides, which encodes a polypeptide of 377 amino acids with a predicted molecular mass of 44.6 kDa. Sequence analyses using Pfam (Finn et al., 2006) revealed an N‐terminal transmembrane domain (TM, amino acids 20–39) followed by a C‐terminus that contains the Domain of Unknown Function 266 (DUF266, amino acids 66–322). The multiple alignment of various DUF266‐containing proteins showed that SUH1 is most similar to the rice BC10 (Figure 4a). Bioinformatic analyses suggest that DUF266‐containing proteins have structural similarities to the leukocyte core‐2 β1, 6N‐acetylglucosaminyltransferase (C2GnT‐L; Figure 4b), which is a member of the glycosyltransferase 14 (GT14) family (Hansen et al., 2009). DUF266‐containing proteins are structurally related to the GT14 family and have been found in almost all plants (Ye et al., 2011). However, the study of Brittle Culm 10 (OsBC10), which encodes a type II membrane protein containing DUF266 in rice, was the first report on their functional characterization (Zhou et al., 2009). The catalytic region of human C2GnT contains three functional domains (Hansen et al., 2009; Figure 4b). The first domain is the Rossmann‐type nucleotide‐binding domain (amino acids 125–225 in C2GnT), followed by the substrate‐interaction domain (amino acids 286–345 in C2GnT), and the final domain binds to the diphosphate group in the nucleotide (amino acids 396–424 in C2GnT). We identified counterparts exhibiting high similarity to the three functional domains in C2GnT in the DUF266 proteins (Figure 4b). It is notable that the Glu320 and Lys401 residues in C2GnT, which are involved in catalysis and nucleotide binding, are also identified in DUF266 proteins. These residues are indicated by an asterisk and a circle, respectively (Figure 4b).
3.6. SUH1 is the Arabidopsis ortholog of the rice BC10 gene
OsBC10, which has a protein structure most similar to SUH1 (Figure 5a), is a Golgi‐localized type II membrane protein with about 1% of human C2GnT activity in Chinese hamster ovary (CHO) cells (Zhou et al., 2009). This finding led us to perform the following functional characterization. Before conducting functional analyses, we confirmed that the introduction of the SUH1‐GFP construct under the SUH1 promoter restored the ctl1 hot2‐1 suh1‐4 phenotype to that of the ctl1 hot2‐1 mutants (Figure S6). This result suggests that SUH1‐GFP retains the SUH1 biological functions. To determine the subcellular localization of SUH1, the SUH1‐GFP fusion protein was transiently co‐expressed with MAN49‐mCherry or mCherry‐HDEL in tobacco leaf epidermal cells. SUH1‐GFP displayed a punctate localization pattern (Figure 5b), which exactly overlapped with the known Golgi marker, MAN49‐mCherry, but not with the mCherry‐HDEL localized in the endoplasmic reticulum lumen (Nelson et al., 2007). These findings suggest that SUH1, similar to the rice OsBC10, encodes a Golgi‐localized protein containing a DUF266 domain.
Next, we performed the C2GnT assay using CHO cells as described previously (Bierhuizen & Fukuda, 1992). The plasmid carrying either the BC10‐GFP or SUH1‐GFP construct was transfected into CHO cells. As shown in Figure 5c, CHO cells expressing SUH1‐GFP displayed C2GnT activity at levels comparable to the cells transfected with BC10‐GFP. However, the C2GnT activity was barely detectable in nontransfected CHO cells, or CHO cells transfected with an empty vector. To further assess the potential of SUH1 as an ortholog of OsBC10, BC10 cDNA under the control of the SUH1 promoter (pSUH1::BC10) was introduced into ctl1 hot2‐1 suh1‐4 mutant plants (Figure S1B). As anticipated, the growth phenotype of ctl1 hot2‐1 suh1‐4 plants expressing OsBC10 was indistinguishable from the ctl1 hot2‐1 mutant plants under both dark (Figure 5d) and light conditions (Figure S1C). These findings demonstrate that SUH1, an Arabidopsis ortholog of OsBC10, encodes a Golgi‐localized type II membrane protein with glycosyltransferase activity.
3.7. SUH1 is predominantly expressed in tissues associated with secondary cell wall deposition
To investigate the SUH1 expression pattern, we generated transgenic wild‐type and ctl1 hot2‐1 mutant plants expressing the β‐glucuronidase (GUS) reporter gene driven by the 1060 bp SUH1 promoter region located upstream of the ATG start codon (pSUH1::GUS). In the wild‐type plants carrying the pSUH1::GUS construct, GUS activity was barely detectable in embryos, 5‐day‐old dark‐grown seedlings, and 10‐day‐old light‐grown seedlings; indeed, deposition of secondary cell walls was minimized during these early developmental stages (Figure 6a–c). However, a strong GUS activity was detected in the interfascicular fibers, xylem cells in the inflorescence stems (Figure 6d), and mature anthers (Figure 6e), where the secondary cell walls have been reported to be deposited (Taylor‐Teeples et al., 2015). Similar to the wild‐type plants, ctl1 hot2‐1 showed little pSUH1‐driven GUS activity in embryos (Figure 6f) and 10‐day‐old seedlings grown in the light (Figure 6g), where lignin is not deposited. However, in contrast to the wild‐type plants, ctl1 hot2‐1 exhibited strong GUS staining in etiolated seedlings (Figure 6h) and pith cells (Figure 6i), where ectopic lignin is deposited due to the ctl1 hot2‐1 mutation. An increase in GUS staining due to ctl1 hot2‐1 was also found in the flowers (Figure 6e,j). These results suggest that the SUH1 expression is associated with tissues that deposit the secondary cell wall and is enhanced by alterations in cell wall composition caused by the ctl1 hot2‐1 mutation.
4. DISCUSSION
Mutations in CTL1 cause several defects in Arabidopsis, including growth retardation and changes in cell wall composition, due to reduced cellulose contents (Hauser et al., 1995; Hong et al., 2003; Zhong et al., 2002). Our findings demonstrate the suh1‐mediated recovery of multiple defects caused by ctl1 hot2‐1 mutation, including growth and development, cell wall composition, and abiotic stresses. Notably, we showed that suh1‐4 affects the cellulose content (Figure 2d), response to salinity stress (Figure S5B), and root growth under CBI treatments (Figure 3), although to a lesser extent. These results suggest that suh1‐4 also displays similarities to defects in ctl1 hot2‐1 involving cell wall assembly in Arabidopsis. It has previously been posited that if a suppressor mutation displays a phenotype similar to the original mutation, the two gene products likely regulate the same step in a multistep pathway (Prelich, 1999). Therefore, considering the genetic relationship between CTL1 and SUH1, it is probable that these two genes are involved in the same process in a multistep pathway. Moreover, we revealed that SUH1 encodes a Golgi‐localized type II membrane protein with glycosyltransferase activity. These observations provide further insights into the cell wall synthesis mechanism mediated by CTL1 in Arabidopsis.
SUH1 was delivered to the Golgi complex similar to BC10 of rice (Figure 5b). At the same time, CTL1 was found in various organelles on the secretory pathway from the Golgi complex to the cell wall in Arabidopsis (Sánchez‐Rodríguez et al., 2012). Therefore, the assumption that CTL1 and SUH1 regulate the same step in a multistep pathway suggests that CTL1‐mediated processing possibly occurs in the Golgi complex, where the two proteins coexist, rather than in the apoplastic region where CTL1 is delivered alone. However, our results cannot exclude the possible role of apoplastic CTL1 suggested in a previous report (Sánchez‐Rodríguez et al., 2012). Interestingly, mutations in BC15/OsCTL1 in rice, the closest ortholog of AtCTL1, affect cell wall synthesis by reducing cellulose synthesis, while its product is also targeted to the Golgi complex (Wu et al., 2012). These results also make it extremely interesting to investigate whether the same genetic relationship between ctl1 hot2‐1 and suh1 in Arabidopsis would also be observed between bc10 and bc15 in rice.
Elucidating the substrates of CTL1 is important for understanding its molecular mechanism. Assuming that CTL1 and SUH1 regulate the same step in a multistep pathway, we consider understanding the possible substrates of SUH1 to be equally important. We showed that SUH1 contains DUF266, which is known to be related to the GT14 family (Figure 4b), and exhibits C2GnT activity (Figure 5c). In animal cells, C2GnT is implicated in the elongation of the core 2 O‐glycan branch of mucin, analogous to AGP glycans in plants (Basu et al., 2013; Cheng & Radhakrishnan, 2011). The side chain in the AGP glycans is also documented to be elongated by members of the GT14 family in Arabidopsis (Dilokpimol & Geshi, 2014; Knoch et al., 2013). Therefore, we suggest the possibility that SUH1 and CTL1 collaboratively regulate the glycosylation of AGPs in Arabidopsis, probably by promoting and blocking/degrading the same glycans of AGPs, respectively.
Finally, while AtCTL1 expression is relatively strong in almost all tissues (Hossain et al., 2010), SUH1 is predominantly expressed in interfascicular fibers and xylems with secondary cell walls (Figure 6a–e). In particular, ctl1 hot2‐1 causes increased expression of SUH1 along with lignin accumulation in etiolated hypocotyl (Figure 6h) and stem pith cells (Figure 6i), which are not observed in wild‐type plants (Figure 6c,d). These findings suggest that SUH1 transcripts are upregulated by an aberrant deposition of cell wall components caused by ctl1 hot2‐1 . Therefore, to further understand the roles of CTL1 in cell wall synthesis, it is essential to identify its endogenous substrates and elucidate how cell wall integrity affects the expression of SUH1. Overall, these results strongly reveal that suh1 mutations suppress ctl1 hot2‐1 ‐induced defects in Arabidopsis. Additionally, the genetic relationship between these two mutations suggests that CTL1 and SUH1 may regulate the same step in a multistep pathway, indicating that they may share substrates. Therefore, further characterizations of SUH1 are essential for a comprehensive understanding of cell wall synthesis in Arabidopsis.
AUTHOR CONTRIBUTIONS
Design and project management: Suk‐Whan Hong. Experiments and data analyses: Nguyen Thi Thuy, Hyun‐Jung Kim, and Suk‐Whan Hong. Writing and editing: Nguyen Thi Thuy, Hyun‐Jung Kim, and Suk‐Whan Hong.
CONFLICT OF INTEREST STATEMENT
The Authors did not report any conflict of interest.
CONFLICT OF INTEREST STATEMENT
The authors have no conflicts of interest to declare.
Supporting information
ACKNOWLEDMENTS
We thank ABRC for providing the T‐DNA insertional mutant for SUH1 (At5g14550, TAIR accession no. SAIL_912_D02) and the prc1‐1 (Col‐O) seeds. We also thank June M. Kwak (Daegu Gyeongbuk Institute of Science and Technology, Korea) for providing the ER marker gene mCherry‐HDEL and the Golgi marker gene MAN49‐mCherry. This work was supported by a grant (NRF‐2022R1A2C1002724) from the National Research Foundation of Korea.
Thuy, N. T. , Kim, H.‐J. , & Hong, S.‐W. (2024). Antagonistic functions of CTL1 and SUH1 mediate cell wall assembly in Arabidopsis . Plant Direct, 8(3), e580. 10.1002/pld3.580
DATA AVAILABILITY STATEMENT
All data are provided in the main text or Supporting Information.
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Data Availability Statement
All data are provided in the main text or Supporting Information.