Characterization of Cellulose synthase-like D (CSLD) family revealed the involvement of PtrCslD5 in root hair formation in Populus trichocarpa

Cellulose synthase-like D (CSLD) family was characterized for their expression and functions in Populus trichocarpa. Ten members, PtrCslD1-10, were identified in the P. trichocarpa genome, and they belong to 4 clades by phylogenetic tree analysis. qRT-PCR and promoter:GUS assays in Arabidopsis and P. trichocarpa displayed divergent expression patterns of these 10 PtrCSLD genes in root hairs, root tips, leaves, vascular tissues, xylem and flowers. Among PtrCslD2, PtrCslD4, PtrCslD5, PtrCslD6, and PtrCslD8 that all exhibited expression in root hairs, only PtrCslD5 could restore the root hairless phenotype of the atcsld3 mutant, demonstrating that PtrCslD5 is the functional ortholog of AtCslD3 for root hair formation. Our results suggest more possible functions for other PtrCslD genes in poplar.

Yin et al. developed atcsld2/csld3, atcsld2/csld5, atcsld3/csld5 and atcsld2/csld3/csld5 double and triple Arabidopsis knockout mutants. All mutants were dwarf and displayed severe necrosis, indicating the collaborative effects among AtCslD2, AtCslDd3 and AtCslD5 33 . The expression of AtCslD2 is AtCslD3-dependent, and the defects in the atcsld3 mutant were partially compensated for by AtCslD2 overexpression 14 . Two other CSLD genes, PdCslD5 and PdCslD6, were complementation remedies for defects and abnormalities of atcsld3 mutants, which proved that the aforementioned genes are functional orthologs of the AtCslD3 34 .
AtCslD1 and AtCslD4 are responsible for cellulose deposition in cell walls to avoid ectopic pollen tubes and pollen grains 35 . AtCslD3 is crucial for the tensile strength of root hair tip cells by deposition of cellulose, and atcsld3 mutants were unable to maintain homeostasis, and terminated bulge elongation at early stage was observed 14,36 . Stunted root and shoot growth, a decreased concentration of homoglacturonan and xylans, and an elevated concentration of the cellulose synthase inhibitor isoxaben were observed in the atcsld5 mutants 15 . The rice oscsld1 mutant had normal root hair initiation, but displayed stunted root hair growth, swelling and kinking, showing that OsCslD1 is a functional ortholog of AtCslD3/KOJAK/RHD7 and functioning in root hair elongation 30 . Retarded growth and arrested cell division due to lack of cellulose deposition in culm and root tips of rice nd1 mutants (OsCslD4) was observed 37 .
Root and root hair growth have already been explored in maize, rice, cotton and Arabidopsis 14,[37][38][39] . In this study, we identified 10 CSLD genes (PtrCslD1-10) in P. trichocarpa and investigated their possible functions. We studied their expression pattern by qRT-PCR and promoter::GUS staining, and their involvement in root hair formation was investigated by complementation in the Arabidopsis atcsld3 mutant. We demonstrate the functions of PtrCslD5 in root hair formation and provide preliminary evidence of the involvement of CSLD members in xylem formation.
On phyletic lineage, gene pair PtrCslD1 and PtrCslD2 shared the same clade I with ZmCslD4, OsCslD4, AtCslD5, GaCslD5, GhCslD5, and GrCslD5 (Fig. 1). In this clade, functions of OsCslD4 and AtCslD5 were studied, and both mutants displayed defective root hairs 15,37 . In clade II, PtrCslD3 and PtrCslD4 shared the same lineage with AtCslD6, GrCslD6, GaCslD6, GhCslD6; none of those were identified for their functions. PtrCslD5 and PtrCslD6 belong to gene pair, sharing high similarity with AtCslD2, AtCslD3, OsCslD1, OsCslD2, GrCslD2, GaCslD2, GhCslD2, NaCslD3, ZmCslD1 and ZmCslD2 in clade III. Among these 9 genes, AtCslD2, AtCslD3, OsCslD1 are required for root hair morphogenesis, and ZmCslD1 is essential for cell division of rapidly growing tissues 14,29,30,43,47 . Gene pair PtrCslD7 and PtrCslD8 and gene pair PtrCslD9 and PtrCslD10 belong to clade IV. PtrCslD7 and PtrCslD8 are closely related to AtCslD4, and PtrCslD9 and PtrCslD10 are closely related to AtCslD1. Mutation of both AtCslD1 and AtCslD4 caused abnormal flowers, pollen tubes, and pollen grains 35 . Based on the phylogenetic tree analysis, PtrCslD1, PtrCslD2, PtrCslD5 and PtrCslD6 may function in root hair formation, and PtrCslD7, PtrCslD8, PtrCslD9 and PtrCslD10 may participate in flower and pollen tube development. expression patterns of CSLD members in P. trichocarpa plants. Understanding the gene expression pattern can give some clue as to their possible functions. We used quantitatively RT-PCR to examine the expression patterns of these CSLD genes in P. trichocapra. Their absolute transcript abundance in young roots, mature roots, young stem, leaves, xylem, phloem, and apex were determined. Considering the high nucleotide similarity between gene pairs, we designed specific primers to distinguish the gene pairs.
PtrCslD1 and PtrCslD2 displayed similar expression patterns, with high expression levels in young roots, mature roots, young stems, and phloem, while with relatively low levels in xylem and apex, and the lowest level in leaves. However, PtrCslD1 and PtrCslD2 displayed inverse expression levels between young and mature roots ( Fig. 2A,B). A comparatively higher transcript abundance of PtCslD3, 4, 5 and 6 was detected in roots than in young stems, xylem and phloem, and apex had low transcript abundance ( Fig. 2C-F). A high expression level in roots was observed for PtrCslD7 and PtrCslD8. PtrCslD7 was highly expressed in both young and mature roots, while PtCslD8 was detected only for its expression in young roots (Fig. 2G,H). PtrCslD9 and PtrCslD10 were expressed in all tissues, but absolute transcript abundance of PtrCslD10 was very low in these tissues (Fig. 2I,J). Although similar expression patterns between each gene pair were generally observed, in some tissues the gene pair exhibited variable expression levels. For example, high transcript abundance was detected in mature roots for PtrCslD7 but not for PtrCslD8.
Expression analysis by promoter::GUS staining. We used the β-glucuronidase (GUS) gene driven by these 10 CSLD gene promoters to provide more detailed information about their gene expression patterns. About 2.3-3.4 kb of the promoter regions upstream start codon were amplified and the promoter:GUS was transformed into Arabidopsis. GUS signals were stained in roots, leaves, xylem and flowers (petal and style) in both pPtrCslD1:GUS and pPtrCslD2:GUS transgenic Arabidopsis (Fig. 3A and leaf vascular tissues in both transgenics, but GUS signals were observed only in root hairs of pPtrCslD2:GUS transgenics ( Fig. 3A,B). PtrCslD4 promoter-driven GUS signals were detected in various tissues, including root hairs, vascular tissues of leaves and petals, vascular bundles of stems, and pollen grains (Fig. 3D). Compared to the PtrCslD4 promoter, the PtrCslD3 promoter only gave weak GUS signals in phloem and vascular tissue of leaves ( Fig. 3C). GUS staining showed both PtrCslD5 and PtrCslD8 promoters were activated in root hairs (Fig. 3E,F), and PtrCslD5 promoter-driven GUS expression was also detected in pollen sac (Fig. 3E). The observed GUS signals in Arabidopsis root hairs were consistent with the high transcript abundance determined by qRT-PCR (Fig. 2H). In pPtrCslD9:GUS transgenic Arabidopsis, GUS signals were detected only in pollen grains (Fig. 3G). We did not observe any GUS signals in the transgenic Arabidopsis of pPtrCslD6:GUS and pPtrCslD7:GUS. pPtrCslD2:GUS in Arabidopsis had GUS signals in root hairs, but pPtrCslD2:GUS did not, indicating PtrCslD2 might express in root hairs of P. trichocarpa. To confirm the expression of PtrCslD2 in root hairs of P. trichocarpa, we transformed pPtrCslD2:GUS into P. trichocarpa. GUS staining in P. trichocarpa showed strong signals in developing xylem, root hairs, and root tips ( Fig. 4A-C), consistent with the GUS staining in Arabidopsis. Both PtrCslD6 and PtrCslD7 promoters did not give GUS signals in Arabidopsis. We selected the PtrCslD6 promoter to test its ability in P. trichocarpa. GUS was stained in xylem, root hairs and root tips in pPtrCslD6:GUS transgenic poplar ( Fig. 4D-F).
Complementation to the atcsld3 mutant. In the above promoter:GUS experiments, we observed GUS signals in root hairs in several transgenics. The promoters of PtrCslD2, PtrCslD4, PtrCslD5, and PtrCslD8 could drive GUS expression in Arabidopsis (Fig. 3). Transformation of PtrCslD2 and PtrCslD6 promoter-driven GUS in P. trichocarpa gave GUS signals in root hairs. To identify the functions of these five CSLD genes in root hairs, we overexpressed these genes in the root hair mutant atcsld3. Absolute transcript abundance of transgenes in the complementation Arabidopsis was determined by qRT-PCR to confirm their expression (Fig. 5). Numerous root hairs were observed in wildtype Arabidopsis (Fig. 6A). In the atcsld3 mutant, root hairs were hardly seen (Fig. 6B). Complementation of atcsld3 with PtrCslD2, PtrCslD4 and PtrCslD8 gave only a few short root hairs (Fig. 6C-E). Compared with PtrCslD2, PtrCslD4 and PtrCslD8, overexpression of PtrCslD6 in atcsld3 produced more and longer root hairs, but the root hair number was much fewer and root hairs were shorter than in wildtype (Fig. 6F). PtrCslD5 complemented mutants appeared with bunches of root hairs (Fig. 6G), and the root hair length was the same as in the wildtype, indicating that PtrCslD5 is the functional ortholog of AtCslD3.

Discussion
Root hairs, tubular appendages of trichoblast cells of rhizodermis are integral for plant growth, soil anchorage, water and mineral adsorption, symbiotic interface for mychorhizae and nitrogen-fixing bacteria. Arabidopsis thaliana root epidermis cells are being used as a model to study cell growth and function 1,2,14,48-50 . Rapid axillary mitotic division of root hairs requires proper cellulose deposition 1,14 . Cellulose synthase A (CesA) family members are responsible for cellulose biosynthesis, while cellulose synthesis like D (CSLD) family members are involved in cellulose deposition in both primary and secondary cell walls 5 . Disruption of cellulose deposition would affect root hair formation. In Arabidopsis, several CSLD members, including AtCslD2, AtCslD3 and AtCslD5, have been characterized for their functions in root hair formation 33 . The root hairless phenotype was observed in the atcsld3 mutant 31 , and mutant characterization showed that AtCslD3 is functioning in the initiation of root hair formation 33 . AtCslD2 is functioning at a later stage of root hair development, and the atcsld2 mutant had abnormal root hairs, with many rupturing late in development 43 . AtCslD5 has functions redundant with AtCslD2 and AtCslD3 33 . In our studies on CSLD members in P. trichocarpa, only PtrCslD5 could restore the root hairless phenotype of atcsld3, indicating that PtrCslD5 is the functional ortholog of AtCslD3. However, PtrCslD6 shared 96% amino acid sequence identity with PtrCslD5 but had very little complementation with atcsld3. In another study, both PdCslD5 and PdCslD6 from P. deltoids could rescue the root hairless phenotype in the atcsld3 mutation 34 . The difference on the complementation to atcsld3 between PtrCslD6 and the other three CSLD genes (PtrCslD5, PdCslD5 and PdCslD6) indicates that some key amino acids may be changed in PtrCslD6, reducing its function in root hair formation. PtrCslD6 and PdCslD6 share a 99% amino acid identity, with two amino acid difference (valine vesus isoleucine, and glycine versus serine). It will be interesting to further investigate the roles of these two amino acids in root hair formation. Based on the GUS staining in promoter:GUS transgenic Arabidopsis and poplar, PtrCslD2, PtrCslD4, PtrCslD6, and PtrCslD8 may also function in root hair formation. It is interesting that the GUS expression driven by PtrCslD5 and PtrCslD8 promoters were at the same places, root hair and epidermis of root hair zone, but PtrCslD8 did not restore the phenotype of atcsld3. GUS staining shows that PtrCslD2 promoter activity is induced at an early stage of root hair development, while PtrCslD4 promoter activity is induced at a later stage of root hair development, suggesting PtrCslD2 and PtrCslD4 may function at different stages of root hair development. The functions of PtrCslD2, PtrCslD4, PtrCslD6, and PtrCslD8 genes in root hair formation and whether they are functioning cooperatively with PtrCslD5 need further studies, such as complementation to atcsld2 and atcsld5 mutants or knockout poplar mutant generation. In Arabidopsis and other species, CSLD members function not only in root hair formation but also in other tissues, such as vascular tissues and pollen 35 . Strong GUS staining of PtrCslD1 and PtrCslD2 promoters was observed in vascular tissue of leaves and vascular bundles of stems (Fig. 3A,B), and comparatively light GUS signals in vascular tissue were detected for PtrCslD3 and PtrCslD4 promoters (Fig. 3C,D). Phylogenetic tree analysis shows PtrCslD1 and PtrCslD2 are in the clade with AtCslD5 and OsCslD4, and PtrCslD3 and PtrCslD4 are in the same clade with AtCslD6. Triple mutant csld2/csld3/csld5 had asymmetric loops and discontinuous vascular elements, showing that AtCslD5, a gene important for root hair formation, is also functioning in vascular tissues 33 . OsCslD4 is expressed in the apex of many organs with rapid growth, and its mutation had many effects, such as inhibited plant growth, thin culms, small grains etc 37 . In clade IV, AtCslD1 and AtCslD4 are closely related to gene pair PtrCslD9/10 and gene pair PtrCslD7/8. Both AtCslD1 and AtCslD4 are important for pollen tube growth 35 . Combining the gene expression patterns and functional characterizations of these 10 PtrCSLD genes and other CSLD genes in other species, we found that some CSLD genes are functioning in multiple tissues, such as root hairs, pollen tubes and vascular tissues. For example, the qRT-PCR and promoter:GUS staining experiment (Figs 2 and 4) showed that PtrCslD2 was expressed in various tissues, including root hairs, root tips, and xylem. The expression is in accordance with AtCslD5 expression in Arabidopsis 15 . The expression of PtrCslD6 was observed in root hairs, root tip, and xylem in P. trichocarpa, indicating its roles in the root hairs, root tip and xylem. The expression pattern and predicted function of PtrCslD6 are in accordance with the functions of AtCslD2, AtCslD3 and AtCslD5 in root hairs, xylem and tip growth 33 . Although PtrCslD2 and PtrCslD6 promoters also drove GUS signals in the P. trichocarpa cambium that is lacking in Arabidopsis stems, we assume the expression in cambium is in accordance with the expression of AtCslD2, AtCslD3 and AtCslD5 in tip tissue 51 (Fig. 3H).
These results indicate that the CSLD genes share a certain level of conservation between Arabidopsis and poplar, and the CSLD genes in P. trichocarpa may play roles in the same tissues of root hair, vascular tissue and pollen tube, as in Arabidopsis. We also observed difference between Arabidopsis and poplar related to CSLD gene functions and regulation. The activities of the promoters of PtrCslD1 to PtrCslD9 were studied in Arabidopsis through promoter:GUS experiments. Surprisingly, no GUS staining was observed for PtrCslD6 and PtrCslD7 promoters. However, the PtrCslD6 promoter was active in P. trichocarpa, with staining in root hairs, root tips, cambium, and xylem (Fig. 4). This difference indicates that the upstream regulator(s) of PtrCslD6 between Arabidopsis and poplar may be different. PtrCslD1 and PtrCslD2 transcripts were detected at a very low level in leaves in P. trichocarpa ( Fig. 2A,B), but both promoters gave strong signals in Arabidopsis leaves (Fig. 3A,B), indicating that the promoters are activated differently between Arabidopsis and poplar. Besides, the expression level of PtrCslD1 in young roots was higher than that in mature roots, but PtrCslD2 displayed an opposite expression pattern in young roots and mature roots. The occurrence of the different expression patterns between PtrCslD1 and PtrCslD2 in roots may be formed after chromosome duplication.

Quantitative reverse transcription and PCR (qRT-PCR).
For the PtrCslD gene expression pattern analysis, leaves, shoot apices, young stems of 1-3 internodes, xylem, phloem, young roots and mature roots were collected from six-month-old trees and put in liquid nitrogen immediately. Total RNA was extracted using the CTAB method 53 . For the qRT-PCR analysis of PtrCslD genes in the atcsld3 mutant, the total RNA was extracted from the roots using an RNeasy Plant Mini Kit (Qiagen). The reverse transcription of RNA to cDNA and quantitative  polymerase chain reaction (PCR) were carried out as described previously 52 . The primers used in the qRT-PCR are listed in Supplemental Table S1.
Mutant complementation. The total RNA isolated from the xylem for qRT-PCR analysis was reverse-transcribed to cDNA using an Omniscript RT kit (Qiagen). Using the cDNA as templates, the full-length cDNAs of PtrCslD2, PtrCslD4, PtrCslD5, PtrCslD6 and PtrCslD8 were amplified with designed primers (Supplemental Table S1). The PCR fragments were inserted into pBI121 to replace the GUS gene, generat-