Variability in CitXET expression and XET activity in Citrus cultivar Huangguogan seedlings with differed degrees of etiolation

Considering the known effects of xyloglucan endotransglycosylase (XET) on plant growth and development, we aimed to determine whether XETs help to regulate the growth and elongation of Huangguogan shoots and roots. We confirmed a possible role for XET during seedling etiolation. Our results revealed that the roots of etiolated seedlings (H-E) were longer than those of green seedlings (H-G). However, shoot length exhibited the opposite pattern. We also observed positive and negative effects on the xyloglucan-degrading activity of XET in the root sub-apical region and shoots of etiolated Huangguogan seedling, respectively. There was a significant down-regulation in CitXET expression in the etiolated shoots at 15 days after seed germination. On the contrary, it was significantly increased in the root sub-apical region of etiolated and multicolored seedlings at 15 days after seed germination. The XET coding sequence (i.e., CitXET) was cloned from Huangguogan seedlings using gene-specific primers. The encoded amino acid sequence was predicted by using bioinformatics-based methods. The 990-bp CitXET gene was highly homologous to other XET genes. The CitXET protein was predicted to contain 319 amino acids, with a molecular mass of 37.45 kDa and an isoelectric point of 9.05. The predicted molecular formula was C1724H2548N448O466S14, and the resulting protein included only one transmembrane structure. The CitXET secondary structure consisted of four main structures (i.e., 21% α-helix, 30.72% extended strand, 9.09% β-turn, and 39.18% random coil). Analyses involving the NCBI Conserved Domains Database (NCBI-CDD), InterPro, and ScanProsite revealed that CitXET was a member of the glycosyl hydrolase family 16 (GH16), and included the DEIDFEFLG motif. Our results indicate that the differed degrees of etiolation influenced the CitXET expression pattern and XET activity in Huangguogan seedlings. The differential changes in XET activity and CitXET expression levels in Huangguogan seedlings may influence the regulation of root and shoot development, and may be important for seedling etiolation.

Introduction and roots. Another objective was to elucidate the XET gene expression pattern and function in etiolated seedlings. Thus, we identified and isolated the Huangguogan XET gene and completed bioinformatics-based analyses. We herein describe the growth of Huangguogan seedlings with differed degree of etiolation, and discuss its role during the elongation of the roots and shoots of etiolated seedlings. Our findings may be useful for characterizing the function of CitXET in Huangguogan root and shoot development during the process of etiolation.

Plant materials
Huangguogan seeds were obtained from the Institute of Pomology and Olericulture, Sichuan Agricultural University, China. The seeds were presoaked in water for 4 h, incubated at 25 ± 1˚C for 3 days, and then transferred to pots filled with vermiculite and perlite (1:1, v/v). The pots were placed in a growth chamber set at 25 ± 1˚C and 50-60% relative humidity. The seedlings were exposed to a 12-h light/12-h dark photoperiod, and watered every 2 days. The etiolated (H-E), multicolored (H-M), and green (H-G) seedlings were harvested at 5, 10, 15, and 20 days after seed germination (i.e., emergence of the radicle through the seed coat). The collected samples were immediately frozen in liquid nitrogen and stored at −80˚C.

Root and shoot dry weight and length
Eight H-E, H-M, and H-G Huangguogan seedlings were collected at 20 days after germinating, and then divided into shoots and roots. The root and shoot length was measured using a vernier caliper. The shoots and roots were dried at 70˚C for 24 h. The root and shoot dry weight was measured by an electronic balance.

Enzyme extraction
For estimating enzyme activities, total proteins were extracted from the shoots and root subapical regions of the H-E, H-M, and H-G seedlings at different time points (i.e., 5, 10, 15, and 20 days after seed germination) as previously described [19,41].

Cloning and sequence analysis of CitXET
Total RNA was extracted from Huangguogan as previously described [42]. First-strand cDNA was synthesized with the HiScript 1st Strand cDNA Synthesis Kit (Vazyme Biotech Co., Ltd, Nanjing, China). The extracted RNA was treated with DNase I (Invitrogen) to eliminate contaminating genomic DNA, and then stored at −20˚C. The PCR amplification of the target sequence was completed in a 25-μL solution that included 12.5 μL 2× Taq Master Mix (Vazyme Biotech Co., Ltd, Nanjing, China), 1 μL forward and reverse gene-specific primers, 1 μL cDNA template, and double-distilled H 2 O up to 25 μL. The PCR program was as follows: 94˚C for 3 min; 35 cycles of 94˚C for 30 s, 55˚C for 30 s, and 72˚C for 90 s; 72˚C for 5 min. The amplified target fragment was analyzed by agarose gel electrophoresis, and then purified using the Agarose Gel DNA Recovery Kit (TIANGEN Biotech Co., Ltd, Beijing, China). The target fragment was incorporated into the pMD19-T vector, which was then inserted into Escherichia coli DH5α cells and sequenced.

Bioinformatics analysis
The BlastN online tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to analyze the homology between CitXET and the other plant XET genes. The amino acid sequence encoded by CitXET was determined using the DNAMAN program. The amino acid composition, isoelectric point and molecular mass of the CitXET protein were calculated with the ExPASy ProtParam tool (http://web.expasy.org/protparam/). Additionally, we analyzed the protein transmembrane region with the TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/ TMHMM/), while the protein signal peptide was predicted using SignalP 4.1. Furthermore, SOPMA, ClustalX 1.83, BioEdit, and MEGA 7.0.12 program were used to compare the amino acid sequences and construct a phylogenetic tree. ESPript 3.0 was used for multiple sequence alignment and homology modeling. The secondary and tertiary protein structures were predicted using RCSB PDB and SWISS-MODEL [43].

Statistical analysis
The data was analyzed using Duncan's multiple range test in the XLSTAT program (version 2010) (P = 0.05 level of significance).

Huangguogan seedling growth
The dry matter content as well as the root and shoot lengths of Huangguogan seedlings were measured at 20 days after seed germination (Fig 1). The dry weight, shoot length, and root-toshoot ratio of H-E seedlings were significantly lower than those of H-G seedlings. However, the opposite trend was observed for root length (Table 1). These results suggest that the H-E seedling roots and shoots grow faster and slower than those of the H-G seedlings, respectively.

Identification and isolation of the CitXET gene
The cDNA produced by reverse transcription was used as the template for PCR amplifications. The 990-bp amplicons observed during agarose gel electrophoresis was consistent with the expected fragment size (S1 Fig). The results of the sequencing by GENEWIZ Biotechnology Co., Ltd. indicated that the amplified DNA fragment consisted of 990 bp. A comparison with other sequences using the Blastn and GenBank online tools revealed that CitXET was 99% homologous to the corresponding Citrus sinensis gene. Additionally, CitXET was 82%, 81%, 80%, and 82% homologous to sequences of V. vinifera (AY043237.1), M. domestica (EU494960.1), P. pyrifolia (EU432411.1), and Glycine max (NM_001253317.2), respectively. These results confirmed that the cloned sequence represented the Huangguogan XET gene.
We observed a slight but significant down-regulation in CitXET expression in the H-E shoots (log2 fold change = −2.15) at 15 days after seed germination (Fig 2a). For the other time points (i.e., 5, 10, and 20 days after seed germination), there was a small but consistent decrease in the CitXET expression levels of H-E and H-M seedlings (log2 fold change between −2 and −1). However, this decrease was considered insignificant. In the root sub-apical region (Fig  2b), a small but consistent increase in the CitXET expression levels of H-E and H-M seedlings was observed at 15 days after seed germination. The log2 fold change values for the CitXET expression levels in H-E and H-M seedlings were about 2.49 and 2.28 (i.e., up-regulated), respectively. At 10 days after seed germination, a significant up-regulation in CitXET expression was detected in H-E seedlings (log2 fold change = 2.20).
The xyloglucan-degrading activity of XET during Huangguogan seedling etiolation was estimated using an iodine based detection of xyloglucans. Compared with H-G, a significant  (P < 0.05) decrease in extractable enzyme activity of H-E was evidenced in the shoots for 5, 10, 15, and 20 days after seed germination. In shoots (Fig 3a), the basal levels for H-E (0.17 U/mg) and H-M (0.27 U/mg) seedlings were observed on day 5. The XET activities of root sub-apical region were slightly more active (>1.50 U/mg) than that of shoots. Gradual increases in activity were observed for H-E (0.29-0.33 U/mg), H-M (0.30-0.35 U/mg), and H-G (0.34-0.42 U/ mg) seedlings between days 10 and 20 (Fig 3a). Maximum activity was recorded on day 15 in shoots and root sub-apical region. In shoots, XET activity of H-G seedlings was the most active, followed by that of H-M seedlings and H-E seedlings. But it was the opposite to the root sub-apical region, the XET activity of H-E seedlings was significantly (P < 0.05) higher than that of H-G seedlings (Fig 3b).

Analysis of the CitXET sequence
A 990-bp amplicon was generated using CitXET-specific primers. The encoded amino acid sequence was determined using the DNAMAN program. A subsequent search using the NCBI ORF finder and NCBI Protein-Blast algorithm revealed that CitXET containing a 960-bp coding region encoded a protein, which consisted of 319 amino acids (Fig 4). , with an isoelectric point of 9.05. The most common amino acid was phenylalanine (29, 9.1%), followed by aspartic acid (22, 6.9%), glycine (22, 6.9%), and lysine (21, 6.6%). The least common amino acids were methionine (8, 2.5%), cysteine (6, 1.9%), and histidine (6, 1.9%). The instability index was calculated as 44.12. Additionally, the grand average of hydropathicity value was −0.398. We submitted the full-length CitXET sequence to the GenBank database using the BankIt tool (Accession number: KY576851).

Secondary and tertiary protein structures of CitXET
An analysis of the CitXET protein secondary structure using SOPMA revealed the enzyme consists of four main structures (i.e., 21% α-helix, 30.72% extended strand, 9.09% β-turn, and 39.18% random curl) (Fig 5). The deduced amino acid sequence was compared with the XET protein sequences from other plants. Additionally, ESPript was used for homology modeling (Fig 6). The CitXET sequence and secondary structures were highly homologous to those of other plants. To investigate the evolutionary relationships between CitXET and the XETs of other plant species, we constructed a phylogenetic tree using the protein sequences for known plant XET sequences in the GenBank database (Fig 7). The phylogenetic tree was divided into two evolutionary branches. TaXTH1 (Triticum aestivum, AAT94293.1) and ZaXTH1 (Zea mays, AAC49011.1) clustered into one evolutionary branch (IV), while genes from the dicotyledonous plants were grouped together to form another main branch. CitXET, GaXET (Gossypium arboreum, KHG12145.1), and PtXET (Populus trichocarpa, XP-002297895.1) clustered together in branch I, suggesting these were the most closely related proteins. Using the NCBI Conserved Domains Database (NCBI-CDD), InterPro, and ScanProsite, we determined that CitXET carried the GH16 domain. The CitXET protein sequence also contained the consensus signature motif conserved among GH16 proteins (i.e., -EIDFEFLGNRT-). We used the NPS@ web server and ProScan to predict the active site of the Huangguogan XET protein. The results indicated that CitXET consisted of one GH16 active site, one N-glycosylation site, two protein kinase C phosphorylation sites, one casein kinase II phosphorylation site, one tyrosine kinase phosphorylation site, four N-myristoylation sites, and one amidation site ( Table 2). The TMHMM server predicted that the CitXET protein had only one transmembrane structure (Fig 8). Using SignalP to identify the signal peptide revealed that the CitXET protein likely lacked a signal peptide.
The SWISS-MODEL server was used to predict the tertiary structure of CitXET based on known crystal structures of homologous proteins. The model was refined to a resolution of 1.8 Å, oligo-state was monomer, and coverage was 0.84. According to the prediction of the tertiary structure of CitXET, there were two ligands, BGC-BGC-BGC-XYS: SUGAR (4-MER) and XYS-GAL: SUGAR (2-MER), respectively. The active site residues E136, Q149, N151, E161, R163, D225, W226, and G230 were included in BGC-BGC-BGC-XYS, and D159, E161, R163, Y297, and R305 in XYS-GAL. Results regarding tertiary structure indicated that the CitXET protein was similar to other family GH16 enzymes with β-jellyroll-type structure (Fig 9a), especially PttXET16A (PDB ID: 1UMZ_A). The highest scoring (Seq identity: 91.08) and validated model for CitXET that exhibited the greatest amino acid sequence identity with the crystal structure was the protein of Populus tremula PttXET16A (Fig 9b). However, a notable structural feature arised because of an insertion of 41 residued at the N-terminus of CitXET, forming α-helix and β-sheet in the molecule (Figs 5, 9a and 9b). QMEAN analysis was also used to evaluate and validate the model, the QMEAN4 score was 0.11 (between 0 and 1), all atoms (−1.21), C-beta interactions (−1.10), solvation (−1.20) and torsion (0.61), which showed a good quality of the model (Fig 9c).

Discussion
The XTHs catalyze reactions affect cell wall xyloglucans and xylans [9]. Additionally, XET activity is an important part of an ancient machinery that regulates cell wall modifications, and is common among all major groups of green plants [10], including all vascular plants [44]. To the best of our knowledge, there is limited information regarding the effects of XET on the elongation of plant roots and shoots, especially during seedling etiolation. Specific XET activity was detected in Huangguogan seedlings. Furthermore, we revealed a correlation between root and shoot elongation and changes in XET activity. We observed that XET activity was specific to elongation, which is consistent with the results of a previous study on liverworts [45].
Our data regarding XET activity and CitXET temporal expression patterns during etiolation indicated that there was a gradual increase in CitXET gene expression, especially between days 10 and 20, which coincided with the period when the seedling roots and shoots were rapidly growing. These findings confirmed that CitXET affected the etiolation of Huangguogan seedlings. Additionally, we detected relatively low and high CitXET gene expression levels in the shoots and root sub-apical regions of etiolated seedlings, respectively. The XET activities of root sub-apical region were higher than those of shoots during seedling etiolation. These implied that the higher CitXET expression and XET activity, the longer roots and shoots length ( Table 1, Figs 2 and 3).
The XETs are members of the GH16 family, and are encoded by multigene families. Generally, the XTHs can be divided into three or four subgroups, and those belonging to classes I, II,  and IIIB exhibit XET activity [36,46]. Several members of the XET gene family have been cloned and identified in many fruit trees. For example, three litchi, three longan, and four pear XET genes are available in the GenBank database. In this study, we used a homologous cloning method to isolate the complete coding sequence of the Huangguogan XET gene (i.e., CitXET), which encodes 319 amino acids (Fig 4). Based on comparisons with XET sequences from other plant species as well as the constructed phylogenetic tree, we determined that the N-terminal of CitXET was highly conservative. Additionally, there was a high degree of homology among the XET sequences from various plant species. The CitXET active site was identical to the functional site (-DEIDFEFLG-) of most GH16 XTHs (Fig 4), suggesting the catalytic domain was highly conservative. Similar results were reported by Nishitani et al. [18] and Henrissat et al. [47].  Table 2) was highly homologous to sequences from other known XETs (Fig 6). CitXET, GaXET (G. arboreum, KHG12145.1), and PtXET (P. trichocarpa, XP-002297895.1) were clustered together in branch I, implying a close relationship among these genes (Fig 7).

Conclusions
Our results indicate that the degree of etiolation affects the XET activity and CitXET expression patterns of Huangguogan seedlings. Furthermore, CitXET is vital for root and shoot growth in etiolated seedlings. The 960-bp CitXET coding sequence encodes a protein consisting of 319 amino acids. CitXET belongs to GH16, based on analyses using the NCBI-CDD, InterPro, and ScanProsite, and the protein has only one transmembrane structure. Our data regarding the XET-related activity and expression patterns in etiolated Huangguogan seedlings may be relevant to future studies on the root and shoot elongation of etiolated seedling. These studies should focus on biochemical and structural characterizations. A more thorough understanding of the effects of CitXET expression patterns and XET activities on root and shoot development may expand our knowledge regarding the role of XTHs during seedling etiolation.