Predicted CitXET active sites.

<p>Predicted CitXET active sites.</p

Features of the CitXET molecular structure.

<p><b>a</b>: CitXET tertiary structures, N- to C-terminals are color-coded blue to red; <b>b</b>: Model-Template Alignment of CitXET and PttXET16A; <b>c</b>: QMEAN analysis.</p

Deduced <i>CitXET</i> gene and encoded amino acid sequences.

<p>Underlined EIDFEFLGNRT was the conserved consensus signature motif of glycosyl hydrolase family 16 protein. The functional site (-DEIDFEFLG-) of most XTHs in family GH16 was highlighted in red color.</p

<i>CitXET</i> expression profiles of the etiolated (H-E) and multicolored (H-M) <i>Huangguogan</i> seedlings at different time points (i.e., 5, 10, 15, and 20 days after seed germination).

<p><b>a</b>: shoots; <b>b</b>: root sub-apical region. The <i>CitXET</i> expression levels are provided as the transcript inhibition levels (log2 fold change) relative to the values for the green <i>Huangguogan</i> seedlings. Data are presented as the mean ± standard deviation of three independent replicates (<i>n</i> = 3).</p

Predicted CitXET secondary structures.

<p>Blue line, α-helix; Red line, extended chain; Green line, β-sheet; Purple line, random curl.</p

Figures of <i>Huangguogan</i> seedlings in 20 days after seed germination.

<p>H-E, etiolated seedlings. H-M, multicoloured seedlings. H-G, green seedlings.</p

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

<div><p>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 <i>Huangguogan</i> 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 <i>Huangguogan</i> seedling, respectively. There was a significant down-regulation in <i>CitXET</i> 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., <i>CitXET</i>) was cloned from <i>Huangguogan</i> seedlings using gene-specific primers. The encoded amino acid sequence was predicted by using bioinformatics-based methods. The 990-bp <i>CitXET</i> gene was highly homologous to other <i>XET</i> 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 <i>CitXET</i> expression pattern and XET activity in <i>Huangguogan</i> seedlings. The differential changes in XET activity and <i>CitXET</i> expression levels in <i>Huangguogan</i> seedlings may influence the regulation of root and shoot development, and may be important for seedling etiolation.</p></div

Multiple sequence alignment and homology modeling of XETs from <i>Huangguogan</i> and other plant species.

<p>Multiple alignment analysis of CitXET protein sequence was generated with the protein sequences of other known plant <i>XET</i> sequences from the NCBI database (<a href="https://www.ncbi.nlm.nih.gov/" target="_blank">https://www.ncbi.nlm.nih.gov/</a>). <i>GaXET</i> (<i>Gossypium arboretum</i>, KHG12145.1), <i>PtXET</i> (<i>Populus trichocarpa</i>, XP-002297895.1), <i>AdXET</i> (<i>Actinidia deliciosa</i>, AAC09388.1), <i>VvXET2</i> (<i>Vitis vinifera</i>, AAK81881.1), <i>SIXET</i> (<i>Solanum lycopersicum</i>, BAA03923.1), <i>FcXTH1</i> (<i>Fragaria chiloensis</i>, ADE42488.1), <i>RbXET</i> (<i>Rosa x borboniana</i>, ABB86296.1), <i>PpXET</i> (<i>Pyrus pyrifolia</i>, ACA02823.1), <i>PcXET1</i> (<i>Pyrus communis</i>, BAC58038.1), <i>MdXET1</i> (<i>Malus domestica</i>, AAN07897.1), <i>GmXET</i> (<i>Glycine max</i>, BAA03922.1), <i>PsEXGT1</i> (<i>Pisum sativum</i>, BAA34946.1), <i>AcXET2</i> (<i>Annona cherimola</i>, ACK36946.1), <i>AcXET1</i> (<i>Annona cherimola</i>, ACK36945.1), <i>MdXET2</i> (<i>Malus domestica</i>, AAN07898.1), <i>LcXET3</i> (<i>Litchi chinensis</i>, ABK30789.1), <i>VvXET1</i> (<i>Vitis vinifera</i>, AAK81880.1), <i>AtXET</i> (<i>Arabidopsis thaliana</i>, CAA63553.1), <i>BjXTH3</i> (<i>Brassica juncea</i>, AEX07607.1), <i>TaXTH1</i> (<i>Triticum aestivum</i>, AAT94293.1) and <i>ZaXTH1</i> (<i>Zea mays</i>, AAC49011.1).</p

XET activity in excised segments of the etiolated (H-E), multicolored (H-M), and green (H-G) <i>Huangguogan</i> seedlings at different time points (i.e., 5, 10, 15, and 20 days after seed germination).

<p><b>a</b>: shoots; <b>b</b>: root sub-apical region. Total proteins were extracted from seedlings, and XET activity was estimated using xyloglucan polymer as the specific substrate (extracted from tamarind flour). The data are presented as the mean of six biological replicates ± standard error. Duncan’s multiple range tests were used to establish statistical significance. Different letters indicate significantly different values (at <i>P</i> = 0.05 level).</p

Predicted <i>Cit</i>XET protein transmembrane structures.

<p>Predicted <i>Cit</i>XET protein transmembrane structures.</p