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12 pages, 5675 KiB  
Article
Two Sugarcane Expansin Protein-Coding Genes Contribute to Stomatal Aperture Associated with Structural Resistance to Sugarcane Smut
by Zongling Liu, Zhuoxin Yu, Xiufang Li, Qin Cheng and Ru Li
J. Fungi 2024, 10(9), 631; https://doi.org/10.3390/jof10090631 - 3 Sep 2024
Viewed by 416
Abstract
Sporisorium scitamineum is a biotrophic fungus responsible for inducing sugarcane smut disease that results in significant reductions in sugarcane yield. Resistance mechanisms against sugarcane smut can be categorized into structural, biochemical, and physiological resistance. However, structural resistance has been relatively understudied. This study [...] Read more.
Sporisorium scitamineum is a biotrophic fungus responsible for inducing sugarcane smut disease that results in significant reductions in sugarcane yield. Resistance mechanisms against sugarcane smut can be categorized into structural, biochemical, and physiological resistance. However, structural resistance has been relatively understudied. This study found that sugarcane variety ZZ9 displayed structural resistance compared to variety GT42 when subjected to different inoculation methods for assessing resistance to smut disease. Furthermore, the stomatal aperture and density of smut-susceptible varieties (ROC22 and GT42) were significantly higher than those of smut-resistant varieties (ZZ1, ZZ6, and ZZ9). Notably, S. scitamineum was found to be capable of entering sugarcane through the stomata on buds. According to the RNA sequencing of the buds of GT42 and ZZ9, seven Expansin protein-encoding genes were identified, of which six were significantly upregulated in GT42. The two genes c111037.graph_c0 and c113583.graph_c0, belonging to the α-Expansin and β-Expansin families, respectively, were functionally characterized, revealing their role in increasing the stomatal aperture. Therefore, these two sugarcane Expansin protein-coding genes contribute to the stomatal aperture, implying their potential roles in structural resistance to sugarcane smut. Our findings deepen the understanding of the role of the stomata in structural resistance to sugarcane smut and highlight their potential in sugarcane breeding for disease resistance. Full article
(This article belongs to the Special Issue Genomics of Fungal Plant Pathogens, 3rd Edition)
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Figure 1

Figure 1
<p>Smut resistance evaluations were conducted on the smut-susceptible sugarcane variety GT42 and the smut-resistant variety ZZ9, following 100 d of either soaking or puncture inoculation with <span class="html-italic">Sporisorium scitamineum</span>. Sugarcane inoculated with H<sub>2</sub>O was considered the control. Red arrows indicate black whips. Bars = 30 cm.</p>
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<p>Observation and quantification of stomatal density, aperture, and area on sugarcane buds of different varieties. (<b>A</b>) Observation of stomatal density on sugarcane buds. Red circles indicate stomata on sugarcane buds. Bars = 200 μm. (<b>B</b>) Observation of stomata on sugarcane buds. Bars = 10 μm. (<b>C</b>) Statistical results of stomatal density, aperture, and area of outermost bud scales of sugarcane. Values followed by different letters are significantly different by Tukey’s test (<span class="html-italic">p</span> &lt; 0.05). Three biological replications were performed.</p>
Full article ">Figure 3
<p>Infection of <span class="html-italic">S. scitamineum</span> through the stomata on the outermost bud scale of sugarcane. (<b>A</b>–<b>D</b>) Observations of <span class="html-italic">S. scitamineum</span> infection through the stomata on the outermost bud scale of sugarcane. Arrows indicate the germ tube of germinated smut teliospores. Bars = 10 μm. (<b>E</b>) Statistics of <span class="html-italic">S. scitamineum</span> infection events in the stomata per cm<sup>2</sup>. Values followed by different letters are significantly different by Tukey’s test (<span class="html-italic">p</span> &lt; 0.05). Ten biological replications were performed.</p>
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<p>RNA-seq analysis of sugarcane GT42 and ZZ9 buds. (<b>A</b>) Volcano plot of differentially expressed genes (DEGs). There were 2769 upregulated and 3137 downregulated genes in GT42. (<b>B</b>) KEGG enrichment analysis of DEGs. (<b>C</b>) Expression profiles of <span class="html-italic">Expansin</span> genes in GT42 and ZZ9. (<b>D</b>) RT-qPCR analysis of four stomatal aperture-related gene expression levels in GT42 and ZZ9. GAPDH was used as internal standard. Three biological replications were conducted. * <span class="html-italic">p</span> &lt; 0.05.</p>
Full article ">Figure 5
<p>Sequence alignment analysis of c111037.graph_c0 and c113583.graph_c0. The Expansin protein sequences of <span class="html-italic">Oryza sativa</span> and <span class="html-italic">Arabidopsis thaliana</span> were used for analysis using MEGA 7. Black shades indicate that all proteins have the same amino acids. Grey shades indicate that some proteins have the same amino acids.</p>
Full article ">Figure 6
<p>Phylogenetic tree analysis of c111037.graph_c0 and c113583.graph_c0. c111037.graph_c0 and c113583.graph_c0 belong to α-Expansin and β-Expansin, respectively. The domains were predicted by the Batch CD-search Tool.</p>
Full article ">Figure 7
<p>The transient expression of c111037.graph_c0 and c113583.graph_c0 increased the stomatal aperture on <span class="html-italic">Nicotiana benthamiana</span> leaves. (<b>A</b>) Observations of the stomatal aperture after the transient expression of c111037.graph_c0 and c113583.graph_c0. 35S::GFP was considered the control. Yellow arrows indicate the stomatal aperture. Bars = 10 μm. (<b>B</b>) Statistics of the stomatal aperture after the transient expression of c111037.graph_c0 and c113583.graph_c0. The maximum distance of the stomatal opening was quantified using ImageJ v1.8.0 and taken as the stomatal aperture. Values followed by different letters are significantly different by Tukey’s test (<span class="html-italic">p</span> &lt; 0.05). Twelve biological replications were performed.</p>
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24 pages, 7903 KiB  
Article
Populus trichocarpa EXPA6 Facilitates Radial and Longitudinal Transport of Na+ under Salt Stress
by Zhe Liu, Kexin Yin, Ying Zhang, Caixia Yan, Ziyan Zhao, Jing Li, Yi Liu, Bing Feng, Rui Zhao, Jian Liu, Kaiyue Dong, Jun Yao, Nan Zhao, Xiaoyang Zhou and Shaoliang Chen
Int. J. Mol. Sci. 2024, 25(17), 9354; https://doi.org/10.3390/ijms25179354 - 29 Aug 2024
Viewed by 299
Abstract
Expansins are cell wall (CW) proteins that mediate the CW loosening and regulate salt tolerance in a positive or negative way. However, the role of Populus trichocarpa expansin A6 (PtEXPA6) in salt tolerance and the relevance to cell wall loosening is still unclear [...] Read more.
Expansins are cell wall (CW) proteins that mediate the CW loosening and regulate salt tolerance in a positive or negative way. However, the role of Populus trichocarpa expansin A6 (PtEXPA6) in salt tolerance and the relevance to cell wall loosening is still unclear in poplars. PtEXPA6 gene was transferred into the hybrid species, Populus alba × P. tremula var. glandulosa (84K) and Populus tremula × P. alba INRA ‘717-1B4’ (717-1B4). Under salt stress, the stem growth, gas exchange, chlorophyll fluorescence, activity and transcription of antioxidant enzymes, Na+ content, and Na+ flux of root xylem and petiole vascular bundle were investigated in wild-type and transgenic poplars. The correlation analysis and principal component analysis (PCA) were used to analyze the correlations among the characteristics and principal components. Our results show that the transcription of PtEXPA6 was downregulated upon a prolonged duration of salt stress (48 h) after a transient increase induced by NaCl (100 mM). The PtEXPA6-transgenic poplars of 84K and 717-1B4 showed a greater reduction (42–65%) in stem height and diameter growth after 15 days of NaCl treatment compared with wild-type (WT) poplars (11–41%). The Na+ accumulation in roots, stems, and leaves was 14–83% higher in the transgenic lines than in the WT. The Na+ buildup in the transgenic poplars affects photosynthesis; the activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT); and the transcription of PODa2, SOD [Cu-Zn], and CAT1. Transient flux kinetics showed that the Na+ efflux of root xylem and leaf petiole vascular bundle were 1.9–3.5-fold greater in the PtEXPA6-transgenic poplars than in the WT poplars. PtEXPA6 overexpression increased root contractility and extensibility by 33% and 32%, indicating that PtEXPA6 increased the CW loosening in the transgenic poplars of 84K and 717-1B4. Noteworthily, the PtEXPA6-promoted CW loosening was shown to facilitate Na+ efflux of root xylem and petiole vascular bundle in the transgenic poplars. We conclude that the overexpression of PtEXPA6 leads to CW loosening that facilitates the radial translocation of Na+ into the root xylem and the subsequent Na+ translocation from roots to leaves, resulting in an excessive Na+ accumulation and consequently, reducing salt tolerance in transgenic poplars. Therefore, the downregulation of PtEXPA6 in NaCl-treated Populus trichocarpa favors the maintenance of ionic and reactive oxygen species (ROS) homeostasis under long-term salt stress. Full article
(This article belongs to the Special Issue Plant Response to Abiotic Stress—3rd Edition)
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Figure 1

Figure 1
<p>Transcription profile of <span class="html-italic">PtEXPA6</span> in the leaves, stems, and roots of <span class="html-italic">Populus trichocarpa</span> during the period of salt stress. Uniform plants of <span class="html-italic">P. trichocarpa</span> were treated with NaCl saline (0 or 100 mM) for 48 h. The fine roots, stems, and upper leaves (3rd to 8th from shoot tip) were sampled at 0, 3, 6, 12, 24, and 48 h, respectively. For the RT-qPCR analysis, the primer sequences for <span class="html-italic">PtEXPA6</span> and the reference gene, <span class="html-italic">PtUBQ</span>, are shown in <a href="#app1-ijms-25-09354" class="html-app">Supplementary Table S1</a>. The data are means ± SD (<span class="html-italic">n</span> = 3), and the bars with asterisks indicate significant differences, **: <span class="html-italic">p</span> &lt; 0.01.</p>
Full article ">Figure 2
<p>Sequence and phylogenetic analysis of <span class="html-italic">Populus trichocarpa PtEXPA6</span>. (<b>A</b>) The multiple sequence alignment of EXPA and expansin family from <span class="html-italic">Populus</span> and other species. The black shading indicates identical amino acid residues, and the blue and pink shadings indicate conserved amino acids, respectively. The lower-case letters represent the same amino acids in different species. (<b>B</b>) The phylogenetic analysis of expansin from various species. <span class="html-italic">Populus euphratica</span> (Pe), <span class="html-italic">Populus trichocarpa</span> (Pt), <span class="html-italic">Populus tremula × Populus tremuloides</span> (Ptt), <span class="html-italic">Populus alba</span> (Pa), <span class="html-italic">Arabidopsis thaliana</span> (At), <span class="html-italic">Zea mays</span> (Zm), <span class="html-italic">Nicotiana tabacum</span> (Nt), and <span class="html-italic">Oryza sativa</span> (Os), <span class="html-italic">Glycine max</span> (Gm), <span class="html-italic">Salix viminalis</span> (Sv), <span class="html-italic">Morus notabilis</span> (Mn), <span class="html-italic">Rosa rugosa</span> (Rr), <span class="html-italic">Prunus persica</span> (Pp), <span class="html-italic">Pistacia vera</span> (Pv), <span class="html-italic">Ziziphus jujuba</span> (Zj), <span class="html-italic">Cucumis melo</span> (Cm), <span class="html-italic">Gossypium arboreum</span> (Ga), <span class="html-italic">Nicotiana sylvestris</span> (Ns), <span class="html-italic">Syzygium oleosum</span> (So), <span class="html-italic">Hibiscus syriacus</span> (Hs), <span class="html-italic">Pistacia vera</span> (Pv), and <span class="html-italic">Salix purpurea</span> (Sp). <a href="#app1-ijms-25-09354" class="html-app">Supplementary Table S2</a> lists the accession numbers of the EXPA orthologs. The blue, yellow, pink and green shadings indicate expansin orthologs of EXPA, EXLA, EXLB, and EXPB, respectively. The PtEXPA6 is labelled with star symbols in (<b>A</b>,<b>B</b>).</p>
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<p>Molecular verification of the transgenic lines overexpressing <span class="html-italic">P. trichocarpa PtEXPA6</span> in 84K and 717-1B4. (<b>A</b>) The PCR assay of the transgenic poplars. The primer sequences for <span class="html-italic">PtEXPA6</span> are shown in <a href="#app1-ijms-25-09354" class="html-app">Supplementary Table S1</a>. WT: negative control (wild type); CK: blank control; P: positive control. (<b>B</b>) The Western blot of the transgenic lines. The Western blot analysis performed with an anti-MYC-specific antibody for PtEXPA6.</p>
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<p>Phenotypic tests of the wild-type (WT) and <span class="html-italic">PtEXPA6</span>-overexpressing lines of 84K and 717-1B4 under long-term salt stress. The <span class="html-italic">PtEXPA6</span>-overexpressing lines of 84K (L11, L12, and L13) and 717-1B4 (L9, L15, and L16), and wild-type (WT) were exposed to NaCl with 0 or 100 mM for 15 days. The stem height and diameter of the no-salt control and salinized plants were measured after 15 days of the salt treatment. The relative growth of the stem height and diameter during the observation period are shown. (<b>A</b>) Representative images showing plant performance after the salt treatment. Scale bars = 5 cm. (<b>B</b>) Relative growth of the stem height. (<b>C</b>) Relative growth of the stem diameter. The data are means ± SD (<span class="html-italic">n</span> = 3), and the bars with different letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p>
Full article ">Figure 5
<p>Effect of NaCl on leaf gas exchange in the wild-type and <span class="html-italic">PtEXPA6</span>-overexpressing lines of 84K and 717-1B4. The <span class="html-italic">PtEXPA6</span>-overexpressing lines of 84K (L11, L12, and L13) and 717-1B4 (L9, L15, and L16), and wild-type (WT) were exposed to NaCl with 0 or 100 mM for 15 days. Leaf gas exchange, i.e., net photosynthetic rate, transpiration rate, and stomatal conductance were measured in the leaves of the no-salt control and salinized plants after 15 days of the salt treatment. (<b>A</b>) Net photosynthetic rate (Pn). (<b>B</b>) Transpiration rate (E). (<b>C</b>) Stomatal conductance (Cleaf). The data are means ± SD (<span class="html-italic">n</span> = 3), and the bars with different letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p>
Full article ">Figure 6
<p>Effect of NaCl on chlorophyll fluorescence in the wild-type and <span class="html-italic">PtEXPA6</span>-overexpressing lines of 84K and 717-1B4. The <span class="html-italic">PtEXPA6</span>-overexpressing lines of 84K (L11, L12, and L13) and 717-1B4 (L9, L15, and L16), and wild-type (WT) were exposed to NaCl with 0 or 100 mM for 15 days. Chlorophyll fluorescence, i.e., the relative electron transport rate, the actual photosynthetic quantum yield, and the maximum photochemical efficiency of PSII were measured in the leaves of the no-salt control and salinized plants after 15 days of the salt treatment. (<b>A</b>) The relative electron transport rate (ETR). (<b>B</b>) The actual photosynthetic quantum yield (YII). (<b>C</b>) The maximum photochemical efficiency of PSII (Fv/Fm). The data are means ± SD (<span class="html-italic">n</span> = 3), and the bars with different letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p>
Full article ">Figure 7
<p>Effect of NaCl on antioxidant enzyme activity and relative electrolyte leakage in the wild-type and <span class="html-italic">PtEXPA6</span>-overexpressing lines of 84K and 717-1B4. The <span class="html-italic">PtEXPA6</span>-overexpressing lines of 84K (L11, L12, and L13) and 717-1B4 (L9, L15, and L16), and wild-type (WT) were exposed to NaCl with 0 or 100 mM for 15 days. The activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and relative electrolyte leakage were measured in the leaves of the no-salt control and salinized plants after 15 days of the salt treatment. (<b>A</b>) POD activity. (<b>B</b>) SOD activity. (<b>C</b>) CAT activity. (<b>D</b>) Relative electrolyte leakage. The data are means ± SD (<span class="html-italic">n</span> = 3), and the bars with different letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Effect of NaCl on the transcription levels of antioxidant enzymes in the wild-type and <span class="html-italic">PtEXPA6</span>-overexpressing lines of 84K and 717-1B4. The <span class="html-italic">PtEXPA6</span>-overexpressing lines of 84K (L11, L12, and L13), 717-1B4 (L9, L15, and L16), and wild-type (WT) were exposed to NaCl with 0 or 100 mM for 15 days. The relative expression of the antioxidant enzyme genes such as peroxidase a2 (<span class="html-italic">PODa2</span>), superoxide dismutase [Cu-Zn] (<span class="html-italic">SOD [Cu-Zn]</span>), and catalase 1 (<span class="html-italic">CAT1</span>) were examined in the WT and <span class="html-italic">PtEXPA6</span>-overexpressing poplars after 15 days of the salt treatment. (<b>A</b>) <span class="html-italic">PODa2</span>. (<b>B</b>) <span class="html-italic">SOD [Cu-Zn]</span>. (<b>C</b>) <span class="html-italic">CAT1</span>. The primer sequences of <span class="html-italic">PODa2</span>, <span class="html-italic">SOD [Cu-Zn]</span>, and <span class="html-italic">CAT1</span> and the reference actin gene, <span class="html-italic">PtUBQ</span>, are shown in <a href="#app1-ijms-25-09354" class="html-app">Supplementary Table S1</a>. The data are means ± SD (<span class="html-italic">n</span> = 3), and the bars with different letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p>
Full article ">Figure 9
<p>Na<sup>+</sup> content in roots, stems, and leaves of wild-type and <span class="html-italic">PtEXPA6</span>-overexpressing lines of 84K and 717-1B4 under long-term salt stress. <span class="html-italic">PtEXPA6</span>-overexpressing lines of 84K (L11, L12, and L13) and 717-1B4 (L9, L15, and L16), and wild-type (WT) were exposed to NaCl with 0 or 100 mM for 15 days. Data are means ± SD (<span class="html-italic">n</span> = 3), and bars with different letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Comparative contractability and comparative extensibility of the intact root tip sites in the wild-type and <span class="html-italic">PtEXPA6</span>-overexpressing lines of 84K and 717-1B4. The comparative contractability of the <span class="html-italic">PtEXPA6</span>-overexpressing lines of 84K (L11, L12, and L13) and 717-1B4 (L9, L15, and L16), and wild-type (WT) was measured after the intact root tips were exposed to 300 mOsmol kg<sup>−1</sup> mannitol (−0.75 MPa). Then, comparative extensibility of the intact root tip was measured after a 0.10 MPa osmotic jump. The data are means ± SD (<span class="html-italic">n</span> = 3), and the bars with different letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Na<sup>+</sup> flux of the root xylem and the response to an osmotic jump in the wild-type and <span class="html-italic">PtEXPA6</span>-overexpressing lines of 84K and 717-1B4. After exposure to 200 mM NaCl for 4 h, the intact root tips of the <span class="html-italic">PtEXPA6</span>-trangenic lines of 84K (L11, L12, and L13) and 717-1B4 (L9, L15, and L16), and wild-type (WT) were exposed to 300 mOsmol kg<sup>−1</sup> mannitol (−0.75 MPa), followed by a 0.1 MPa osmotic jump. The net Na<sup>+</sup> flux of the root xylem was measured before and after the addition of mannitol and the subsequent 0.1 MPa osmotic jump. The data are means ± SD (<span class="html-italic">n</span> = 3), and the bars with different letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Na<sup>+</sup> flux of the petiole vascular bundle and the response to an osmotic jump in the wild-type and <span class="html-italic">PtEXPA6</span>-overexpressing lines of 84K and 717-1B4. After exposure to 200 mM NaCl for 4 h, the intact root tips of the <span class="html-italic">PtEXPA6</span>-trangenic lines of 84K (L11, L12, and L13) and 717-1B4 (L9, L15, and L16), and wild-type (WT) were exposed to 300 mOsmol kg<sup>−1</sup> mannitol (−0.75 MPa), followed by a 0.1 MPa osmotic jump. The net Na<sup>+</sup> flux of the petiole vascular bundle was measured before and after the addition of mannitol, and the subsequent 0.1 MPa osmotic jump. The data are means ± SD (<span class="html-italic">n</span> = 3), and the bars with different letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p>
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17 pages, 3964 KiB  
Article
Characterization of the Expansin Gene Promoters in Populus trichocarpa
by Junkang Zhang, Xiaoyu Li, Lei Wang, Longfeng Gong, Mengtian Li and Jichen Xu
Forests 2024, 15(9), 1485; https://doi.org/10.3390/f15091485 - 24 Aug 2024
Viewed by 393
Abstract
The expansin genes are commonly expressed in plant cells, and the encoded proteins influence plant growth and stress resistance by loosening the structure and increasing the flexibility of the cell wall. The objective of this study was to characterize expansin gene promoters in [...] Read more.
The expansin genes are commonly expressed in plant cells, and the encoded proteins influence plant growth and stress resistance by loosening the structure and increasing the flexibility of the cell wall. The objective of this study was to characterize expansin gene promoters in Populus trichocarpa to clarify the regulatory mechanisms underlying gene expression and evolution. Sequence alignments revealed that the similarity among 36 poplar expansin genes was greater for the coding sequences than for the promoter sequences, which suggested these promoter sequences evolved asynchronously. The bases flanking the start codon exhibited a usage bias, with sites +3, +4, and +5 biased toward GC, whereas the other sites were biased toward AT. The flanking sites were significantly correlated with gene expression, especially sites −10 and −17, in which C and G are the bases positively associated with gene expression. A total of 435 regulatory elements (61 types) were identified on the promoters of the poplar expansin genes; Skn-1 was the most common element in 23 promoters. Some expansin genes had more regulatory elements on their promoters (e.g., PtrEXPA4, PtrEXPA3, PtrEXPB3, and PtrEXPB1), whereas some others had less (e.g., PtrEXLA2, PtrEXLB1, and PtrEXPA23). Furthermore, 26 types of elements were involved in expansin gene expression, 25 of which positively affected expression in all analyzed samples. The exception was the endosperm expression-related element Skn-1, which negatively regulated expression in four tissues or treatments. Expression analysis showed that the expansin genes in Populus trichocarpa performed much differently under regular and abiotic stress conditions, which well matched the diversity of their promoter sequences. The results show that expansin genes play an important role in plant growth and development and stress resistance through expression adjustment. Full article
(This article belongs to the Special Issue Latest Progress in Research on Forest Tree Genomics)
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Figure 1

Figure 1
<p>Relative expression of the expansin genes in <span class="html-italic">Populus trichocarpa</span> under different stress treatments. Values represent the mean of three replicates ± SD. Different letters denote statistically significant differences resulting from Duncan’s test following a one-way ANOVA.</p>
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<p>Four bases frequency in the flanking sites of the initial codon ATG (−20~+6). (<b>A</b>). frequency of AT and GC; (<b>B</b>). frequency of AG and TC. The <span class="html-italic">X</span>-axis means flanking sites of the initial codon. The <span class="html-italic">Y</span>-axis means the percentage of the detected bases at each site.</p>
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<p>Phylogenetic trees based on the poplar expansin gene promoter sequences (left) and coding sequences (right) by the neighbor-joining algorithm, MEGA7. The Bootstrap test was used to verify phylogenetic tree reliability (replications = 1000).</p>
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<p>Distribution of the regulatory elements in 100 bp sequential interception segment along the poplar expansin gene promoters. The <span class="html-italic">X</span>-axis means the segments of the promoters started from the initial codon. The <span class="html-italic">Y</span>-axis means the number of regulatory elements in the relative segment. (<b>A</b>) Distribution of the <span class="html-italic">cis</span>-elements on the promoters; (<b>B</b>) distribution of the <span class="html-italic">cis</span>-elements on the promoter regions; (<b>C</b>) comparison of the <span class="html-italic">cis</span>-elements among the expansin genes. <span class="html-italic">PtrEXPA14</span>, <span class="html-italic">PtrEXPA15,</span> and <span class="html-italic">PtrEXPA3</span>.</p>
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<p>The gene expression in response to the start codon flanking sites. The circles represent the significant correlation (<span class="html-italic">p</span> = 0.05) between the expression level of the poplar expansin genes and the base usage frequency of the site on the promotor based on THE Pearson correlation coefficient calculation by SPSS 20.0. The relevant base was marked inside the circle. The red circle indicates a positive correlation between the site and the gene expression of the poplar expansin genes in the tissue, while the green circle indicates a negative correlation.</p>
Full article ">Figure 6
<p>The gene expression in response to the cis-elements on the promoters. The circles represent the significant correlation (<span class="html-italic">p</span> = 0.05) between the expression level of the poplar expansin genes and the number of the specific cis-element on the promotor based on the Pearson correlation coefficient calculation by SPSS 20.0. The red circle indicates a positive correlation between the element and the gene expression of the poplar expansin genes in the tissue, while the green circle indicates a negative correlation.</p>
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26 pages, 18602 KiB  
Article
Integration of Phenotypes, Phytohormones, and Transcriptomes to Elucidate the Mechanism Governing Early Physiological Abscission in Coconut Fruits (Cocos nucifera L.)
by Lilan Lu, Zhiguo Dong, Xinxing Yin, Siting Chen and Ambreen Mehvish
Forests 2024, 15(8), 1475; https://doi.org/10.3390/f15081475 - 22 Aug 2024
Viewed by 540
Abstract
The abscission of fruits has a significant impact on yield, which in turn has a corresponding effect on economic benefits. In order to better understand the molecular mechanism of early coconut fruit abscission, the morphological and structural characteristics, cell wall hydrolysis and oxidase [...] Read more.
The abscission of fruits has a significant impact on yield, which in turn has a corresponding effect on economic benefits. In order to better understand the molecular mechanism of early coconut fruit abscission, the morphological and structural characteristics, cell wall hydrolysis and oxidase activities, phytohormones, and transcriptomes were analyzed in the abscission zone (AZ) from early-abscised coconut fruits (AFs) and non-abscised coconut fruits (CFs). These results indicated that the weight and water content of AFs are significantly lower than those of CFs, and the color of AFs is a grayish dark red, with an abnormal AZ structure. Cellulase (CEL), polygalacturonase (PG), pectinesterase (PE), and peroxidase (POD) activities were significantly lower than those of CFs. The levels of auxin (IAA), gibberellin (GA), cytokinins (CKs), and brassinosteroid (BR) in AFs were significantly lower than those in CFs. However, the content of abscisic acid (ABA), ethylene (ETH), jasmonic acid (JA), and salicylic acid (SA) in AFs was significantly higher than in CFs. The transcriptome analysis results showed that 3601 DEGs were functionally annotated, with 1813 DEGs upregulated and 1788 DEGs downregulated. Among these DEGs, many genes were enriched in pathways such as plant hormone signal transduction, carbon metabolism, peroxisome, pentose and gluconate interconversion, MAPK signaling pathway—plant, and starch and sucrose metabolism. Regarding cell wall remodeling-related genes (PG, CEL, PE, POD, xyloglucan endoglucosidase/hydrogenase (XTH), expansin (EXP), endoglucanase, chitinase, and beta-galactosidase) and phytohormone-related genes (IAA, GA, CKs, BR, ABA, JA, SA, and ETH) were significantly differentially expressed in the AZ of AFs. Additionally, BHLH, ERF/AP2, WRKY, bZIP, and NAC transcription factors (TFs) were significantly differently expressed, reflecting their crucial role in regulating the abscission process. This study’s results revealed the molecular mechanism of early fruit abscission in coconuts. This provided a new reference point for further research on coconut organ development and abscission. Full article
(This article belongs to the Section Genetics and Molecular Biology)
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<p>Development of early-abscised coconut fruits (AFs) and non-abscised coconut fruits (CFs). (<b>a</b>) The anatomical structure of fruit morphology and abscission zone (AZ). (<b>b</b>) Fresh weight and water content of coconut fruits. The data represent the mean ± standard deviation (SD) of ten samples, and the significance of fresh weight and water content of coconut fruits between CFs and AFs was determined using Student’s <span class="html-italic">t</span>-tests. * significant at <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Enzyme activity and phytohormones in AZ of AFs and CFs. CEL, cellulase; PG, polygalacturonase; PE, pectinesterase; POD, peroxidase; IAA, auxin; GA, gibberellin; CK, cytokinin; BR, brassinosteroid; ABA, abscisic acid; ETH, ethylene; JA, jasmonic acid; SA, salicylic acid. The data represent the mean ± standard deviation (SD) of three biological replicates, and the significance of enzyme activity and plant hormone contents in AZ between CFs and AFs was determined using Student’s <span class="html-italic">t</span>-tests. * significant at <span class="html-italic">p</span> &lt; 0.05. ** significant at <span class="html-italic">p</span> &lt; 0.01.</p>
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<p>Differentially expressed genes (DEGs) enriched in the top 20 enriched GO terms in terms of biological processes, molecular functions, and cellular components in AFs and CFs.</p>
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<p>KEGG analysis from CF vs. AF group. (<b>a</b>) KEGG classification chart of DEGs. (<b>b</b>) KEGG enrichment bar chart of all DEGs. (<b>c</b>) KEGG enrichment bar chart of upregulated DEGs. (<b>d</b>) KEGG enrichment bar chart of downregulated DEGs.</p>
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<p>Expression of identified DEGs involved in the main KEGG enrichment pathways in CF vs. AF group.</p>
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<p>Expression of identified DEGs involved in cell wall modification in CF vs. AF group.</p>
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<p>Heatmap of relative changes in expression patterns of 8 phytohormone-related genes in AZ of CF vs. AF group. The color scales on each heatmap display their expression values.</p>
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<p>Transcription factor analysis in CF vs. AF group. (<b>a</b>) Expression of identified DEGs involved in transcription factors in CF vs. AF group. (<b>b</b>) Distribution of overexpression of the regulatory transcription factor family in CF vs. AF group.</p>
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<p>Verification of the expression of 11 coconut fruit abscission-related genes through qRT-PCR analysis. The bar chart represents the value of FPKM. The line graph represents qRT-PCR values. The error bar represents the standard deviation of three biological replicates (<b>a</b>–<b>k</b>). Correlation of expression changes observed through RNA-seq (<span class="html-italic">y</span>-axis) and qRT-PCR (<span class="html-italic">x</span>-axis) (<b>l</b>).</p>
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<p>A hypothetical model for coconut fruit abscission.</p>
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20 pages, 10553 KiB  
Article
A Study on the Functional Identification of Overexpressing Winter Wheat Expansin Gene TaEXPA7-B in Rice under Salt Stress
by Xue Wang, Jing Ma, Fumeng He, Linlin Wang, Tong Zhang, Dan Liu, Yongqing Xu, Fenglan Li and Xu Feng
Int. J. Mol. Sci. 2024, 25(14), 7707; https://doi.org/10.3390/ijms25147707 - 14 Jul 2024
Viewed by 616
Abstract
Expansin is a cell wall relaxant protein that is common in plants and directly or indirectly participates in the whole process of plant root growth, development and morphogenesis. A well-developed root system helps plants to better absorb water and nutrients from the soil [...] Read more.
Expansin is a cell wall relaxant protein that is common in plants and directly or indirectly participates in the whole process of plant root growth, development and morphogenesis. A well-developed root system helps plants to better absorb water and nutrients from the soil while effectively assisting them in resisting osmotic stress, such as salt stress. In this study, we observed and quantified the morphology of the roots of Arabidopsis overexpressing the TaEXPAs gene obtained by the research group in the early stage of development. We combined the bioinformatics analysis results relating to EXPA genes in five plants and identified TaEXPA7-B, a member of the EXPA family closely related to root development in winter wheat. Subcellular localization analysis of the TaEXPA7-B protein showed that it is located in the plant cell wall. In this study, the TaEXPA7-B gene was overexpressed in rice. The results showed that plant height, root length and the number of lateral roots of rice overexpressing the TaEXPA7-B gene were significantly higher than those of the wild type, and the expression of the TaEXPA7-B gene significantly promoted the growth of lateral root primordium and cortical cells. The plants were treated with 250 mM NaCl solution to simulate salt stress. The results showed that the accumulation of osmotic regulators, cell wall-related substances and the antioxidant enzyme activities of the overexpressed plants were higher than those of the wild type, and they had better salt tolerance. This paper discusses the effects of winter wheat expansins in plant root development and salt stress tolerance and provides a theoretical basis and relevant reference for screening high-quality expansin regulating root development and salt stress resistance in winter wheat and its application in crop molecular breeding. Full article
(This article belongs to the Section Molecular Genetics and Genomics)
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<p>Root hairs of overexpressed and wild-type <span class="html-italic">Arabidopsis</span>. (<b>A</b>) Observation on root hairs of overexpressed <span class="html-italic">TaEXPA4-A/B/D</span>, <span class="html-italic">TaEXPA7-A/B/D</span>, <span class="html-italic">TaEXPA8-B/D</span> and <span class="html-italic">TaEXPA19-A/D</span> genes and wild-type <span class="html-italic">Arabidopsis</span>; bar = 500 um. (<b>B</b>) Statistics on the number of root hairs of overexpressed <span class="html-italic">TaEXPA4-A/B/D</span>, <span class="html-italic">TaEXPA7-A/B/D</span>, <span class="html-italic">TaEXPA8-B/D</span> and <span class="html-italic">TaEXPA19-A/D</span> genes and wild-type <span class="html-italic">Arabidopsis</span>. The letters (a–e) represent significant differences according to Tukey’s test (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Root length of overexpressed and wild-type <span class="html-italic">Arabidopsis</span>. (<b>A</b>) Observation on root length of overexpressed <span class="html-italic">TaEXPA4-A/B/D</span>, <span class="html-italic">TaEXPA7-A/B/D</span>, <span class="html-italic">TaEXPA8-B/D</span> and <span class="html-italic">TaEXPA19-A/D</span> genes and wild-type <span class="html-italic">Arabidopsis</span>; bar = 1.5 cm. (<b>B</b>) Statistics on root length of overexpressed <span class="html-italic">TaEXPA4-A/B/D</span>, <span class="html-italic">TaEXPA7-A/B/D</span>, <span class="html-italic">TaEXPA8-B/D</span> and <span class="html-italic">TaEXPA19-A/D</span> genes and wild-type <span class="html-italic">Arabidopsis</span>. The letters (a–d represent significant differences according to Tukey’s test (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Lateral root of overexpressed and wild-type <span class="html-italic">Arabidopsis</span>. (<b>A</b>) Observation on lateral root of overexpressed <span class="html-italic">TaEXPA4-A/B/D</span>, <span class="html-italic">TaEXPA7-A/B/D</span>, <span class="html-italic">TaEXPA8-B/D</span> and <span class="html-italic">TaEXPA19-A/D</span> genes and wild-type <span class="html-italic">Arabidopsis</span>; bar = 1 mm. (<b>B</b>) Statistics on the number of lateral roots of overexpressed <span class="html-italic">TaEXPA4-A/B/D</span>, <span class="html-italic">TaEXPA7-A/B/D</span>, <span class="html-italic">TaEXPA8-B/D</span> and <span class="html-italic">TaEXPA19-A/D</span> genes and wild-type <span class="html-italic">Arabidopsis</span>. The letters (a–d) represent significant differences according to Tukey’s test (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Root cell size of overexpressed and wild-type <span class="html-italic">Arabidopsis</span>. (<b>A</b>) Observation on root cell size of overexpressed <span class="html-italic">TaEXPA4-A/B/D</span>, <span class="html-italic">TaEXPA7-A/B/D</span>, <span class="html-italic">TaEXPA8-B/D</span> and <span class="html-italic">TaEXPA19-A/D</span> genes and wild-type <span class="html-italic">Arabidopsis</span>; bar = 200 um. (<b>B</b>) Statistics on the number of root cell length of overexpressed <span class="html-italic">TaEXPA4-A/B/D</span>, <span class="html-italic">TaEXPA7-A/B/D</span>, <span class="html-italic">TaEXPA8-B/D</span> and <span class="html-italic">TaEXPA19-A/D</span> genes and wild-type <span class="html-italic">Arabidopsis</span>. (<b>C</b>) Statistics on the number of root cell width of overexpressed <span class="html-italic">TaEXPA4-A/B/D</span>, <span class="html-italic">TaEXPA7-A/B/D</span>, <span class="html-italic">TaEXPA8-B/D</span> and <span class="html-italic">TaEXPA19-A/D</span> genes and wild-type <span class="html-italic">Arabidopsis</span>. The letters (a–c) represent significant differences according to Tukey’s test (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Amino acid phylogenetic tree of EXPA family members in five plants constructed using the maximum likelihood algorithm via MEGA7.0 software with 1000 Bootstrap replicates. The red represents the <span class="html-italic">Physcomitrella patens</span>; purple represents <span class="html-italic">Selaginella moellendorffii</span>; green represents <span class="html-italic">Picea abies</span>; blue represents <span class="html-italic">Triticum aestivum</span>; yellow represents <span class="html-italic">Arabidopsis thaliana</span>.</p>
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<p>Promoter elements of the EXPA family genes in five plants. The number of promoters of EXPA family genes in five plants was counted to produce the heat map: (<b>A</b>) <span class="html-italic">Physcomitrella patens</span>, (<b>B</b>) <span class="html-italic">Selaginella moellendorffii</span>, (<b>C</b>) <span class="html-italic">Picea abies</span>, (<b>D</b>) <span class="html-italic">Triticum aestivum</span> and (<b>E</b>) <span class="html-italic">Arabidopsis thaliana</span>.</p>
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<p>Subcellular localization results of TaEXPA7-B protein. (<b>A</b>) TaEXPA7-B expression vector construction for onion inner epidermal cells subcellular localization test. P<sub>35S</sub> indicates CaMV35S promoter; eGFP indicates enhanced green fluorescent protein; and NOS indicates terminator. (<b>B</b>) TaEXPA7-B subcellular localization results; bar = 50 um.</p>
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<p>Identification of overexpressed rice. (<b>A</b>) Construction of TaEXPA7-B expression vector. P35S indicates CaMV35S promoter; NOS indicates terminator. (<b>B</b>) Identification of <span class="html-italic">TaEXPA7-B</span> in wild-type and overexpressed rice. (<b>C</b>) Identification of TaEXPA7-B protein in wild-type and overexpressed rice.</p>
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<p>External structure of rice overexpressing <span class="html-italic">TaEXPA7-B</span> gene. (<b>A</b>) Phenotypes of wild-type and overexpressed <span class="html-italic">TaEXPA7-B</span> gene rice at 15 days. (<b>B</b>) Measurements of the plant height of wild-type and overexpressed rice. (<b>C</b>) The plant length of wild-type and overexpressed rice. (<b>D</b>) The number of roots of wild-type and overexpressed rice. * indicates significant differences in <span class="html-italic">t</span>-tests (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01).</p>
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<p>Observation and quantitative statistics of anatomical structure of wild-type and overexpressed <span class="html-italic">TaEXPA7-B</span> gene rice. (<b>A</b>) The observation of microstructure between wild-type and overexpressed <span class="html-italic">TaEXPA7-B</span> gene rice; a, b, c and d are the longitudinal cutting of rice roots; e, f, g and h are the cross-sections of rice roots. (<b>B</b>) Measurements of the cell height of rice root cortex. (<b>C</b>) The cell length of rice root cortex. (<b>D</b>) The cell width of rice root cortex. (<b>E</b>) The layers of rice root cortex. (<b>F</b>) The number of lateral root primordia. * indicates significant differences in <span class="html-italic">t</span>-tests (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01).</p>
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<p>Tolerance of overexpressed <span class="html-italic">TaEXPA7-B</span> rice to salt stress. (<b>A</b>) Phenotype of rice overexpressing <span class="html-italic">TaEXPA7-B</span> gene under salt stress. (<b>B</b>) The wilting rate of rice leaves under salt stress. * indicates significant differences in <span class="html-italic">t</span>-tests (* <span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Changes in related substances content in overexpressed rice under salt stress. (<b>A</b>) Soluble sugar content. (<b>B</b>) Soluble protein content. (<b>C</b>) Proline content. (<b>D</b>) Lignin content. (<b>E</b>) Cellulose content. (<b>F</b>) Hemicellulose content. (<b>G</b>) Activity of SOD. (<b>H</b>) Activity of POD. (<b>I</b>) Activity of CAT. * indicates significant differences in <span class="html-italic">t</span>-tests (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01).</p>
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22 pages, 5364 KiB  
Article
Unraveling the lncRNA-miRNA-mRNA Regulatory Network Involved in Poplar Coma Development through High-Throughput Sequencing
by Zihe Song, Chenghao Zhang, Guotao Song, Hang Wei, Wenlin Xu, Huixin Pan, Changjun Ding, Meng Xu and Yan Zhen
Int. J. Mol. Sci. 2024, 25(13), 7403; https://doi.org/10.3390/ijms25137403 - 5 Jul 2024
Viewed by 658
Abstract
Poplar coma, the fluff-like appendages of seeds originating from the differentiated surface cells of the placenta and funicle, aids in the long-distance dispersal of seeds in the spring. However, it also poses hazards to human safety and causes pollution in the surrounding environment. [...] Read more.
Poplar coma, the fluff-like appendages of seeds originating from the differentiated surface cells of the placenta and funicle, aids in the long-distance dispersal of seeds in the spring. However, it also poses hazards to human safety and causes pollution in the surrounding environment. Unraveling the regulatory mechanisms governing the initiation and development of coma is essential for addressing this issue comprehensively. In this study, strand-specific RNA-seq was conducted at three distinct stages of coma development, revealing 1888 lncRNAs and 52,810 mRNAs. The expression profiles of lncRNAs and mRNAs during coma development were analyzed. Subsequently, potential target genes of lncRNAs were predicted through co-localization and co-expression analyses. Integrating various types of sequencing data, lncRNA-miRNA-TF regulatory networks related to the initiation of coma were constructed. Utilizing identified differentially expressed genes encoding kinesin and actin, lncRNA-miRNA-mRNA regulatory networks associated with the construction and arrangement of the coma cytoskeleton were established. Additionally, relying on differentially expressed genes encoding cellulose synthase, sucrose synthase, and expansin, lncRNA-miRNA-mRNA regulatory networks related to coma cell wall synthesis and remodeling were developed. This study not only enhances the comprehension of lncRNA but also provides novel insights into the molecular mechanisms governing the initiation and development of poplar coma. Full article
(This article belongs to the Collection Advances in Molecular Plant Sciences)
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<p>Distribution of lncRNAs on chromosomes. The outermost bar chart represents the quantity of different types of lncRNA on each chromosome; the blue curve represents the density of lncRNA in different regions of each chromosome; the red curve indicates miRNA-lncRNA regulatory relationships predicted by the psRNAtarget online website (with an expectation &lt; 3).</p>
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<p>Comparative features of different types of lncRNA. (<b>A</b>). Comparison of the expression levels of different types of lncRNAs in three developmental stages of poplar coma. (<b>B</b>). Comparison of the lengths between different types of lncRNA. (<b>C</b>). Comparison of the number of exons between different types of lncRNA. (<b>D</b>). Comparison of ORF lengths among different types of lncRNA.</p>
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<p>Expression patterns of lncRNAs during different developmental stages of poplar coma. (<b>A</b>). Volcano plot of DELs and DEGs in the PvsW comparison group. (<b>B</b>). Volcano plot of DELs and DEGs in the YvsP comparison group. (<b>C</b>). Volcano plot of DELs and DEGs in the YvsW comparison group. The upper diagram represents lncRNA, while the lower diagram represents mRNA, red dots represent significantly upregulated genes, and green dots represent significantly downregulated genes. (<b>D</b>). Time series analysis of DELs. (<b>E</b>). Time series analysis of DEGs.</p>
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<p>Sequencing data expression identified by RT-qPCR. The broken line shows the relative expression levels obtained by RT-qPCR (left <span class="html-italic">y</span>-axis). The bar graph shows reads FPKM values obtained by sRNA-seq (right <span class="html-italic">y</span>-axis). For the results obtained using sRNA-seq and RT-qPCR, the expression levels at each developmental stage were normalized to the mean expression levels across the three biological replicates.</p>
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<p>The lncRNA-miRNA-mRNA regulatory network associated with the cytoskeleton organization of coma. (<b>A</b>). The lncRNA-miRNA-mRNA regulatory network constituted by kinesin-encoded transcripts. (<b>B</b>). The lncRNA-miRNA-mRNA regulatory network constituted by actin-encoded transcripts. The blue circle represents mRNA, the red square represents miRNA, and the green triangle represents lncRNA.</p>
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<p>The lncRNA-miRNA-mRNA regulatory network associated with cell wall synthesis and remodeling in coma. (<b>A</b>). The lncRNA-miRNA-mRNA regulatory network is constituted by cellulase synthase-encoded transcripts. (<b>B</b>). The lncRNA-miRNA-mRNA regulatory network is constituted by sucrose synthase-encoded transcripts. (<b>C</b>). The lncRNA-miRNA-mRNA regulatory network is constituted by expansin-encoded transcripts.</p>
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<p>lncRNA-miRNA-bHLH regulatory networks. The blue circle represents mRNA, the red square represents miRNA, and the green triangle represents lncRNA.</p>
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<p>Regulatory mechanism model at different developmental stages of poplar coma.</p>
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17 pages, 5811 KiB  
Article
Overexpression of Wild Soybean Expansin Gene GsEXLB14 Enhanced the Tolerance of Transgenic Soybean Hairy Roots to Salt and Drought Stresses
by Linlin Wang, Tong Zhang, Cuiting Li, Changjun Zhou, Bing Liu, Yaokun Wu, Fumeng He, Yongqing Xu, Fenglan Li and Xu Feng
Plants 2024, 13(12), 1656; https://doi.org/10.3390/plants13121656 - 14 Jun 2024
Viewed by 663
Abstract
As a type of cell-wall-relaxing protein that is widely present in plants, expansins have been shown to actively participate in the regulation of plant growth and responses to environmental stress. Wild soybeans have long existed in the wild environment and possess abundant resistance [...] Read more.
As a type of cell-wall-relaxing protein that is widely present in plants, expansins have been shown to actively participate in the regulation of plant growth and responses to environmental stress. Wild soybeans have long existed in the wild environment and possess abundant resistance gene resources, which hold significant value for the improvement of cultivated soybean germplasm. In our previous study, we found that the wild soybean expansin gene GsEXLB14 is specifically transcribed in roots, and its transcription level significantly increases under salt and drought stress. To further identify the function of GsEXLB14, in this study, we cloned the CDS sequence of this gene. The transcription pattern of GsEXLB14 in the roots of wild soybean under salt and drought stress was analyzed by qRT-PCR. Using an Agrobacterium rhizogenes-mediated genetic transformation, we obtained soybean hairy roots overexpressing GsEXLB14. Under 150 mM NaCl- and 100 mM mannitol-simulated drought stress, the relative growth values of the number, length, and weight of transgenic soybean hairy roots were significantly higher than those of the control group. We obtained the transcriptomes of transgenic and wild-type soybean hairy roots under normal growth conditions and under salt and drought stress through RNA sequencing. A transcriptomic analysis showed that the transcription of genes encoding expansins (EXPB family), peroxidase, H+-transporting ATPase, and other genes was significantly upregulated in transgenic hairy roots under salt stress. Under drought stress, the transcription of expansin (EXPB/LB family) genes increased in transgenic hairy roots. In addition, the transcription of genes encoding peroxidases, calcium/calmodulin-dependent protein kinases, and dehydration-responsive proteins increased significantly. The results of qRT-PCR also confirmed that the transcription pattern of the above genes was consistent with the transcriptome. The differences in the transcript levels of the above genes may be the potential reason for the strong tolerance of soybean hairy roots overexpressing the GsEXLB14 gene under salt and drought stress. In conclusion, the expansin GsEXLB14 can be used as a valuable candidate gene for the molecular breeding of soybeans. Full article
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<p>Amino acid phylogenetic tree of GsEXLB14 and soybean expansin family members.</p>
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<p>Transcriptional patterns of <span class="html-italic">GsEXLB14</span> in wild soybean roots under salt and drought stresses. Note: The experiment was conducted using a relative quantitative method, and the relative expression level represents the fold in the transcription level of <span class="html-italic">GsEXLB14</span> gene relative to that at 0 h after treatment. Different lowercase letters indicate that the transcript level of <span class="html-italic">GsEXLB14</span> under same treatment condition was significantly different between different treatment times (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Construction of overexpression vector of <span class="html-italic">GsEXLB14</span> and RT-PCR detection results of transgenic soybean hairy roots. Note: (<b>A</b>) Schematic diagram of overexpression vector construction. (<b>B</b>) RT-PCR detection results of transgenic soybean hairy roots. K599 represents the soybean hairy roots induced by the empty bacteria (without any exogenous expression vector).</p>
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<p>Phenotype of soybean hairy roots overexpressing <span class="html-italic">GsEXLB14</span> under normal, salt, and drought stress conditions (Bar = 2 cm).</p>
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<p>Statistical results of growth indicators of soybean hairy roots overexpressing <span class="html-italic">GsEXLB14</span> gene under salt and drought stress. Note: Relative growth indicates the change in different phenotypes of soybean hairy roots after 7 d under different treatments relative to the original state. (<b>A</b>) Relative increase in the number of hairy roots. (<b>B</b>) Relative growth of total length of hairy roots. (<b>C</b>) Relative increase in total weight of hairy roots. (* indicates that, under the same treatment condition, there was a significant difference between transgenic group and K599 control group, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, n = 30.)</p>
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<p>Clustering heatmap of differential genes among treatment groups in the transcriptome.</p>
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<p>KEGG classification information of differentially transcribed genes in overexpressing <span class="html-italic">GsEXLB14</span> and control group.</p>
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<p>Number of upregulated transcription genes involved in plant environmental stress response and abiotic stress resistance regulation between the treatments groups (left group of “vs” was used as control). Note: Data were derived from the transcriptome, with FPKM value ≥ 5 and transcriptional upregulation fold ≥5 as thresholds. (<b>A</b>) CK vs. CK-Salt. (<b>B</b>) GsEXLB14 vs. GsEXLB14-Salt. (<b>C</b>) CK-Salt vs. GsEXLB14-Salt. (<b>D</b>) CK vs. CK-Drought. (<b>E</b>) GsEXLB14 vs. GsEXLB14-Drought. (<b>F</b>) CK-Drought vs. GsEXLB14-Drought.</p>
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<p>qRT-PCR results of differentially transcribed genes between control group and overexpressing <span class="html-italic">GsEXLB14</span> group under salt and drought stress. Note: The experiment was conducted using a relative quantitative method, and the relative expression level represents the fold in the transcription level of gene relative to CK. (<b>A</b>) Salt stress. (B) Drought stress. (* indicates that, under the same treatment, the transcriptional level of candidate gene was significantly different between overexpression group and control group, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01.)</p>
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17 pages, 5565 KiB  
Article
Genome-Wide Identification, Phylogenetic and Expression Analysis of Expansin Gene Family in Medicago sativa L.
by Yajing Li, Yangyang Zhang, Jing Cui, Xue Wang, Mingna Li, Lili Zhang and Junmei Kang
Int. J. Mol. Sci. 2024, 25(9), 4700; https://doi.org/10.3390/ijms25094700 - 25 Apr 2024
Viewed by 1059
Abstract
Expansins, a class of cell-wall-loosening proteins that regulate plant growth and stress resistance, have been studied in a variety of plant species. However, little is known about the Expansins present in alfalfa (Medicago sativa L.) due to the complexity of its tetraploidy. [...] Read more.
Expansins, a class of cell-wall-loosening proteins that regulate plant growth and stress resistance, have been studied in a variety of plant species. However, little is known about the Expansins present in alfalfa (Medicago sativa L.) due to the complexity of its tetraploidy. Based on the alfalfa (cultivar “XinjiangDaye”) reference genome, we identified 168 Expansin members (MsEXPs). Phylogenetic analysis showed that MsEXPs consist of four subfamilies: MsEXPAs (123), MsEXPBs (25), MsEXLAs (2), and MsEXLBs (18). MsEXPAs, which account for 73.2% of MsEXPs, and are divided into twelve groups (EXPA-I–EXPA-XII). Of these, EXPA-XI members are specific to Medicago trunctula and alfalfa. Gene composition analysis revealed that the members of each individual subfamily shared a similar structure. Interestingly, about 56.3% of the cis-acting elements were predicted to be associated with abiotic stress, and the majority were MYB- and MYC-binding motifs, accounting for 33.9% and 36.0%, respectively. Our short-term treatment (≤24 h) with NaCl (200 mM) or PEG (polyethylene glycol, 15%) showed that the transcriptional levels of 12 MsEXPs in seedlings were significantly altered at the tested time point(s), indicating that MsEXPs are osmotic-responsive. These findings imply the potential functions of MsEXPs in alfalfa adaptation to high salinity and/or drought. Future studies on MsEXP expression profiles under long-term (>24 h) stress treatment would provide valuable information on their involvement in the response of alfalfa to abiotic stress. Full article
(This article belongs to the Section Molecular Genetics and Genomics)
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<p>Phylogenetic tree of Expansin proteins. The unrooted phylogenetic tree was constructed using all Expansin proteins from alfalfa, <span class="html-italic">Medicago trunctula</span>, and <span class="html-italic">Arabidopsis thaliana</span> using the MEGA7.0 neighbor-joining (NJ) method with a bootstrap of 1000. Clades in green, blue, tangerine, and purple branches refer to the EXPA, EXPB, EXLA, and EXLB subfamilies, respectively. EXPA is divided into 12 subgroups (EXPAI–XII) and the subgroups are alternately marked in dark or light green. The tangerine triangles, green dots, and blue stars represent EXPs from alfalfa, <span class="html-italic">M. truncatula</span>, and <span class="html-italic">Arabidopsis</span>, respectively.</p>
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<p>Chromosomal distribution of <span class="html-italic">MsEXPs</span>. Chromosome distributions of <span class="html-italic">MsEXPs</span> were visualized through Tbtools software v1.108 based on the physical location of each gene. The green vertical bars represent the chromosomes of alfalfa and are numbered on the top; 50,568–50,573 and 50,581 represent unplaced genomic scaffolds. Chromosome size is indicated by relative length. The scale (Mb) of chromosome length is displayed on the left. A total of 157 <span class="html-italic">MsEXPs</span> were mapped onto the 30 chromosomes of alfalfa, while another 11 <span class="html-italic">MsEXPs</span> were located on the unplaced genomic scaffolds. The tandem duplicated gene pairs are connected with red lines.</p>
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<p>Phylogenetic tree, structural analysis, and motif distribution of the MsEXP family. (<b>A</b>) The phylogenetic tree was constructed based on the full-length sequences of MsEXPs and divided into four groups. Different subgroups are highlighted in different colors. (<b>B</b>) Exon–intron structures of <span class="html-italic">MsEXPs</span>. Introns and exons are represented by lines and boxes, respectively. (<b>C</b>) Analysis of MsEXP motifs is based on MEME tools. Different motifs are shown in colored boxes, as indicated in the legend.</p>
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<p>Collinearity analysis of <span class="html-italic">MsEXPs</span>. The identification syntenic relationship in <span class="html-italic">MsEXPs</span> is performed using TBtools software v1.108 (E-value ≤ 1 × 10<sup>−5</sup>). The gray lines show the syntenic regions in alfalfa genome. The genes pairs in segmental duplication events are marked with red lines.</p>
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<p>Synteny analysis of <span class="html-italic">Expansin</span> genes between alfalfa and three model plant species. The chromosome labels are located above or below the corresponding chromosome. Gray lines in the background indicate collinear blocks within alfalfa and the indicated plant, whereas the red lines highlight syntenic <span class="html-italic">Expansin</span> gene pairs.</p>
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<p>A heatmap showing the counts of <span class="html-italic">cis</span>-acting elements in the promoters of <span class="html-italic">MsEXPs</span>. The <span class="html-italic">MsEXPs</span> are grouped according to the phylogenetic results, with the green, blue, tangerine, and purple vertical lines referring to the EXPA, EXPB, EXLA, and EXLB phylogenetic groups, respectively. EXPAI–XII labels are alternately marked with dark green or green. The digit in the box represents the number of <span class="html-italic">cis</span>-acting elements (at the top).</p>
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<p>Expression levels of <span class="html-italic">MsEXPs</span> under salt stress. Four-week-old alfalfa seedlings were treated. “CK” represents 1/2 Hoagland solution. “NaCl” represents salt treatment using 1/2 Hoagland solution with 200 mM NaCl. The error bars indicate the standard errors of three biological replicates. Lowercase letters indicate significant differences at <span class="html-italic">p</span> &lt; 0.05 according to ANOVA.</p>
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<p>Expression levels of <span class="html-italic">MsEXPs</span> under drought stress. Four-week-old alfalfa seedlings were treated. “CK” represents 1/2 Hoagland solution. “PEG” represents drought treatment using 1/2 Hoagland solution with 15% PEG (polyethylene glycol). The error bars indicate the standard errors of three biological replicates. Lowercase letters indicate significant differences at <span class="html-italic">p</span> &lt; 0.05 according to ANOVA.</p>
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15 pages, 11465 KiB  
Article
Multi-Omics Analysis Reveals the Distinct Features of Metabolism Pathways Supporting the Fruit Size and Color Variation of Giant Pumpkin
by Wenhao Xia, Chen Chen, Siying Jin, Huimin Chang, Xianjun Ding, Qinyi Fan, Zhiping Zhang, Bing Hua, Minmin Miao and Jiexia Liu
Int. J. Mol. Sci. 2024, 25(7), 3864; https://doi.org/10.3390/ijms25073864 - 29 Mar 2024
Viewed by 1204
Abstract
Pumpkin (Cucurbita maxima) is an important vegetable crop of the Cucurbitaceae plant family. The fruits of pumpkin are often used as directly edible food or raw material for a number of processed foods. In nature, mature pumpkin fruits differ in size, [...] Read more.
Pumpkin (Cucurbita maxima) is an important vegetable crop of the Cucurbitaceae plant family. The fruits of pumpkin are often used as directly edible food or raw material for a number of processed foods. In nature, mature pumpkin fruits differ in size, shape, and color. The Atlantic Giant (AG) cultivar has the world’s largest fruits and is described as the giant pumpkin. AG is well-known for its large and bright-colored fruits with high ornamental and economic value. At present, there are insufficient studies that have focused on the formation factors of the AG cultivar. To address these knowledge gaps, we performed comparative transcriptome, proteome, and metabolome analysis of fruits from the AG cultivar and a pumpkin with relatively small fruit (Hubbard). The results indicate that up-regulation of gene-encoded expansins contributed to fruit cell expansion, and the increased presence of photoassimilates (stachyose and D-glucose) and jasmonic acid (JA) accumulation worked together in terms of the formation of large fruit in the AG cultivar. Notably, perhaps due to the rapid transport of photoassimilates, abundant stachyose that was not converted into glucose in time was detected in giant pumpkin fruits, implying that a unique mode of assimilate unloading is in existence in the AG cultivar. The potential molecular regulatory network of photoassimilate metabolism closely related to pumpkin fruit expansion was also investigated, finding that three MYB transcription factors, namely CmaCh02G015900, CmaCh01G018100, and CmaCh06G011110, may be involved in metabolic regulation. In addition, neoxanthin (a type of carotenoid) exhibited decreased accumulation that was attributed to the down-regulation of carotenoid biosynthesis genes in AG fruits, which may lead to pigmentation differences between the two pumpkin cultivars. Our current work will provide new insights into the potential formation factors of giant pumpkins for further systematic elucidation. Full article
(This article belongs to the Section Molecular Biology)
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<p>The phenotype of two pumpkin cultivars: Atlantic Giant and Hubbard fruits.</p>
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<p>Annotation and pathway analysis of DEGs between AG and Hubbard fruits. (<b>a</b>) GO classification of all annotated genes and DEGs. (<b>b</b>,<b>c</b>) Scatter plot of the most enriched KEGG pathways of down-regulated (<b>b</b>) and up-regulated (<b>c</b>) DEGs.</p>
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<p>The expression heatmap of DEGs in several KEGG pathways related to phenotypic differentiation between Atlantic Giant and Hubbard fruits. The expression profile was determined based on log<sub>2</sub>(FPKM) in RNA-seq data for each gene. A1~3 and H1~3 represent AG and Hubbard fruits, respectively.</p>
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<p>Expression profile and annotation of DEPs identified from a comparison between Atlantic Giant and Hubbard fruits. The expression profile was determined based on log<sub>2</sub>(protein expression) in proteomic data for each protein. Red triangles indicate the protein annotations that we noticed. A1~3 and H1~3 represent AG and Hubbard fruits, respectively.</p>
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<p>The relative content of metabolites in pumpkin fruits generated from metabolomics data. * represents the differential metabolites between AG and Hubbard fruits. ‘ns’ represents no difference.</p>
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<p>Identification of MYB transcription factors and assimilation metabolism-related genes that exhibited similar expression patterns. (<b>a</b>) Heatmap of selected MYB TFs and assimilation metabolism-related genes. The red arrows represent the MYB TFs with potential regulatory functions. (<b>b</b>) <span class="html-italic">Cis</span>-acting element analysis in the promoters of eight assimilation metabolism-related genes. A1~3 and H1~3 represent AG and Hubbard fruits, respectively.</p>
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<p>The expression levels of assimilation metabolism-related genes detected by the RT-qPCR assay. Data are expressed as the means ± standard deviation (SD) of three technical replicates.</p>
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<p>A comprehensive scheme summarizing changes in the transcription of various genes, protein expression, and metabolites from small-fruited pumpkin to giant pumpkin. Single marks and three interlinked marks represent down-regulation and up-regulation, respectively.</p>
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16 pages, 5943 KiB  
Article
Mycorrhizal Symbiosis Enhances P Uptake and Indole-3-Acetic Acid Accumulation to Improve Root Morphology in Different Citrus Genotypes
by Chun-Yan Liu, Xiao-Niu Guo, Feng-Jun Dai and Qiang-Sheng Wu
Horticulturae 2024, 10(4), 339; https://doi.org/10.3390/horticulturae10040339 - 29 Mar 2024
Cited by 1 | Viewed by 2981
Abstract
Arbuscular mycorrhizal fungi (AMF) are known to enhance plant growth via stimulation of root system development. However, the extent of their effects and underlying mechanisms across different citrus genotypes remain to be fully elucidated. This study investigates the impact of Funneliformis mosseae ( [...] Read more.
Arbuscular mycorrhizal fungi (AMF) are known to enhance plant growth via stimulation of root system development. However, the extent of their effects and underlying mechanisms across different citrus genotypes remain to be fully elucidated. This study investigates the impact of Funneliformis mosseae (F. mosseae) inoculation on plant growth performance, root morphology, phosphorus (P), and indole-3-acetic acid (IAA) concentrations, as well as the expression of related synthesis and transporter genes in three citrus genotypes: red tangerine (Citrus tangerine ex. Tanaka), kumquat (Fortunella margarita L. Swingle), and fragrant citrus (Citrus junos Sieb. ex. Tanaka). Following 12 weeks of inoculation, significant improvements were observed in plant height, shoot and root biomass, total root length, average root diameter, second-order lateral root development, root hair density, and root hair length across all genotypes. Additionally, F. mosseae inoculation significantly increased root P and IAA concentrations in the three citrus genotypes. Notably, phosphatase activity was enhanced in F. margarita but reduced in C. tangerine and C. junos following inoculation. Gene expression analysis revealed a universal upregulation of the P transporter gene PT5, whereas expressions of the auxin synthesis gene YUC2, transporter gene LAX2, and phosphatase gene PAP1 were commonly downregulated. Specific to genotypes, expressions of YUC5, LAX5, PIN2, PIN3, PIN6, and expansin genes EXPA2 and EXPA4 were significantly upregulated in C. tangerine but downregulated in F. margarita and C. junos. Principal component analysis and correlation assessments highlighted a strong positive association between P concentration, P and auxin synthesis, and transporter gene expressions with most root morphology traits, except for root average diameter. Conversely, IAA content and phosphatase activities were negatively correlated with these root traits. These findings suggest that F. mosseae colonization notably enhances plant growth and root system architecture in citrus genotypes via modifications in P transport and IAA accumulation, indicating a complex interplay between mycorrhizal symbiosis and host plant physiology. Full article
(This article belongs to the Special Issue Citrus Plant Growth and Fruit Quality)
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<p>Root mycorrhizal colonization of three citrus genotype plants by <span class="html-italic">F. mosseae</span>. Root AM colonization in <span class="html-italic">C. tangerine</span> (<b>a</b>), <span class="html-italic">F. margarita</span> (<b>b</b>) and <span class="html-italic">C. junos</span> (<b>c</b>), and external hyphae in <span class="html-italic">C. tangerine</span> (<b>d</b>), <span class="html-italic">F. margarita</span> (<b>e</b>) and <span class="html-italic">C. junos</span> (<b>f</b>).</p>
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<p>Effect of <span class="html-italic">F. mosseae</span> on root hair development of three citrus genotypes. (<b>a</b>) Root hair density; (<b>b</b>) root hair length; (<b>c</b>) root hair diameter; and root hair morphology of non-AMF <span class="html-italic">C. tangerine</span> (<b>d</b>), AMF <span class="html-italic">C. tangerine</span> (<b>e</b>), non-AMF <span class="html-italic">F. margarita</span> (<b>f</b>), AMF <span class="html-italic">F. margarita</span> (<b>g</b>), non-AMF <span class="html-italic">C. junos</span> (<b>h</b>), and AMF <span class="html-italic">C. junos</span> (<b>i</b>). The different letters (a, b, c, d, and e) indicate significant differences (<span class="html-italic">p</span> ≤ 0.05) in the different AMF treatment treatments.</p>
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<p>Effect of <span class="html-italic">F. mosseae</span> on root P concentration (<b>A</b>) and root phosphatase activity (<b>B</b>) of three citrus genotype plants. The different letters (a, b, c, and d) indicate significant differences (<span class="html-italic">p</span> ≤ 0.05) in the different AMF treatment treatments.</p>
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<p>Effect of AMF <span class="html-italic">F. mosseae</span> on root IAA concentration of three citrus genotype plants. The different letters (a, b, c, d and e) indicate significant differences (<span class="html-italic">p</span> ≤ 0.05) in the different AMF treatment treatments.</p>
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<p>Effect of <span class="html-italic">F. mosseae</span> on the relative expression of root expansin genes of three citrus genotype plants. The different letters (a, b, c, and d) indicate significant differences (<span class="html-italic">p</span> ≤ 0.05) in the different AMF treatment treatments.</p>
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<p>Effect of <span class="html-italic">F. mosseae</span> on the relative expression of P transporter and phosphatase genes in roots of three citrus genotype plants. The different letters (a, b, c, and d) indicate significant differences (<span class="html-italic">p</span> ≤ 0.05) in the different AMF treatment treatments.</p>
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<p>Effect of <span class="html-italic">F. mosseae</span> on relative expression of root IAA synthesis and carrier genes of three citrus genotype plants. The different letters (a, b, c, and d) indicate significant differences (<span class="html-italic">p</span> ≤ 0.05) in the different AMF treatment treatments.</p>
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<p>Principal component analysis (PCA) from all six treatments and 30 variables, including RSA, P, and auxin transportation, assessed in different citrus genotypes inoculated with or without <span class="html-italic">F. mosseae.</span> PC1: variation between varieties; PC2: variation between inoculation and non-inoculation with <span class="html-italic">F. mosseae</span>.</p>
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<p>Pearson correlations between root system architecture parameters and P and auxin transport in different citrus genotypes for non-inoculated treatments (<b>a</b>) or inoculated with the AMF <span class="html-italic">F. mosseae</span> (<b>b</b>). Red and blue colors indicate positive and negative relationships, respectively. Circle sizes indicate the correlation strength. *, <span class="html-italic">p</span> &lt; 0.05, means significant; **, <span class="html-italic">p</span> &lt; 0.01, means extremely significant.</p>
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14 pages, 12833 KiB  
Article
Exploring the PpEXPs Family in Peach: Insights into Their Role in Fruit Texture Development through Identification and Transcriptional Analysis
by Yakun Guo, Conghao Song, Fan Gao, Yixin Zhi, Xianbo Zheng, Xiaobei Wang, Haipeng Zhang, Nan Hou, Jun Cheng, Wei Wang, Langlang Zhang, Xia Ye, Jidong Li, Bin Tan, Xiaodong Lian and Jiancan Feng
Horticulturae 2024, 10(4), 332; https://doi.org/10.3390/horticulturae10040332 - 28 Mar 2024
Viewed by 860
Abstract
Expansins (EXPs) loosen plant cell walls and are involved in diverse developmental processes through modifying cell-walls; however, little is known about the role of PpEXPs in peach fruit. In this study, 26 PpEXP genes were identified in the peach genome and grouped into [...] Read more.
Expansins (EXPs) loosen plant cell walls and are involved in diverse developmental processes through modifying cell-walls; however, little is known about the role of PpEXPs in peach fruit. In this study, 26 PpEXP genes were identified in the peach genome and grouped into four subfamilies, with 20 PpEXPAs, three PpEXPBs, one PpEXPLA and two PpEXPLBs. The 26 PpEXPs were mapped on eight chromosomes. The primary mode of gene duplication of the PpEXPs was dispersed gene duplication (DSD, 50%). Notably, cis-elements involved in light responsiveness and MeJA-responsiveness were detected in the promoter regions of all PpEXPs, while ethylene responsive elements were observed in 12 PpEXPs. Transcript profiling of PpEXPs in the peach fruit varieties of MF (melting), NMF (non-melting) and SH (stony hard) at different stages showed that PpEXPs displayed distinct expression patterns. Among the 26 PpEXPs, 15 PpEXPs were expressed in the fruit. Combining the expressing patterns of PpEXPs in fruits with different flesh textures, PpEXPA7, PpEXPA13 and PpEXPA15 were selected as candidate genes, as they were highly consistent with the patterns of previous reported key genes (PpPGM, PpPGF and PpYUC11) in regard to peach fruit texture. The genes with different expression patterns between MF and NMF were divided into 16 modules, of which one module, with pink and midnightblue, negatively correlated with the phenotype of fruit firmness and was identified as PpEXPA1 and PpEXPA7, while the other module was identified as PpERF in the pink module, which might potentially effect fruit texture development by regulating PpEXPs. These results provide a foundation for the functional characterization of PpEXPs in peach. Full article
(This article belongs to the Special Issue Advances in Developmental Biology in Tree Fruit and Nut Crops)
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<p>Phylogenetic analysis showed that EXPs in <span class="html-italic">Arabidopsis thaliana</span> (yellow round), rice (<span class="html-italic">Oryza sativa</span>, green triangle), Peach (<span class="html-italic">Prunus persica</span>, red star). A: EPXA; B: EXPB; LA:EXPLA; LB: EXPLB.</p>
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<p>Gene structure and motif analysis of PpEXPs. (<b>A</b>): Phylogenetic relationships of PpEXPs; (<b>B</b>): Gene structure of PpEXPs. Yellow box, turquoise box and black line represent exon, UTR and intron, respectively; (<b>C</b>): Conserved motif of PpEXPs. Different color boxes represent different motifs, noted in upper-right.</p>
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<p>Chromosome distribution and gene duplication of PpEXPs. The genes with the same color represented a type of gene duplication: red, DSD; blue, TD; green, WGD. Syntenic pairs were linked with lines, with colors representing different pairs.</p>
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<p>The <span class="html-italic">cis</span>-elements in the promoters of PpEXPs. (<b>A</b>): The different types of <span class="html-italic">cis</span>-elements, with different colors, were shown in the promoter region of each PpEXP. (<b>B</b>): The number of each <span class="html-italic">cis</span>-element in the promoter regions.</p>
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<p>The expression pattern of PpEXPs in melting, non-melting and stony hard peaches. (<b>A</b>): The expression pattern of PpEXPs in melting and non-melting. (<b>B</b>): The expression pattern of PpEXPs in melting and non-melting NMF−S1−4: S1-S4 stages in non-melting peaches CN14; MF−S1−4: S1−S4 stages in melting peaches HSM; SH−S3−4III: S3−S4III in stony hard peaches CN16; MF−S3−4III: S3−S4III in melting peaches CN13.</p>
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<p>WGCNA of expressed genes at four stages of HSM and CN fruit. (<b>A</b>): Hierarchical cluster tree of WGCNA analysis (R<sup>2</sup> = 0.85). (<b>B</b>): Module–trait correlation analysis. Sixteen modules were labeled with different colors. The numbers in each row represent the correlation of every module, the numbers in parentheses indicate the <span class="html-italic">p</span>-values of correlations, and the gene numbers were displayed in the right of each module.</p>
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14 pages, 2256 KiB  
Article
Identification of Candidate Expansin Genes Associated with Seed Weight in Pomegranate (Punica granatum L.)
by Chunyan Liu, Haoyu Zhao, Jiyu Li, Zhen Cao, Bo Deng, Xin Liu and Gaihua Qin
Genes 2024, 15(2), 212; https://doi.org/10.3390/genes15020212 - 6 Feb 2024
Cited by 1 | Viewed by 994
Abstract
Seed weight is an important target trait in pomegranate breeding and culture. Expansins act by loosening plant cell walls and cellulosic materials, permitting turgor-driven cell enlargement. However, the role of expansin genes (EXPs) in pomegranate seed weight remains elusive. A total [...] Read more.
Seed weight is an important target trait in pomegranate breeding and culture. Expansins act by loosening plant cell walls and cellulosic materials, permitting turgor-driven cell enlargement. However, the role of expansin genes (EXPs) in pomegranate seed weight remains elusive. A total of 29 PgrEXPs were identified in the ‘Dabenzi’ genome. These genes were classified into four subfamilies and 14 subgroups, including 22 PgrEXPAs, 5 PgrEXPBs, 1 PgrEXPLA, and 1 PgrEXPLB. Transcriptome analysis of PgrEXPs in different tissues (root, leaf, flower, peel, and seed testa) in ‘Dabenzi’, and the seed testa of the hard-seeded pomegranate cultivar ‘Dabenzi’ and soft-seeded cultivar ‘Tunisia’ at three development stages showed that three PgrEXPs (PgrEXPA11, PgrEXPA22, PgrEXPA6) were highly expressed throughout seed development, especially in the sarcotesta. SNP/Indel markers of these PgrEXPs were developed and used to genotype 101 pomegranate accessions. The association of polymorphic PgrEXPs with seed weight-related traits (100-seed weight, 100-kernel weight, 100-sarcotesta weight, and the percentage of 100-sarcotesta to 100-seed weight) were analyzed. PgrEXP22 was significantly associated with 100-seed weight and 100-sarcotesta weight and is a likely candidate for regulating seed weight and sarcotesta development in particular. This study provides an effective tool for the genetic improvement of seed weight in pomegranate breeding programs. Full article
(This article belongs to the Section Plant Genetics and Genomics)
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<p>Phylogenetic analysis of the expansin genes in <span class="html-italic">Arabidopsis thaliana</span> (<span class="html-italic">AtEXP</span>), <span class="html-italic">Z. jujuba</span>. (<span class="html-italic">ZjEXP</span>), <span class="html-italic">E. grandis</span> (<span class="html-italic">EgrEXP</span>), <span class="html-italic">P. granatum</span> (<span class="html-italic">PgrEXP</span>). The subgroups are marked by different color bars.</p>
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<p>Collinearity analysis of expansin genes in pomegranate. Red lines indicate the collinearity of expansin genes among chromosomes in pomegranate; gray lines in the background indicate all the collinear blocks among different chromosomes.</p>
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<p>Protein domain architecture and gene structure of PgrEXPs. The Leaf figure shows the conserved motif composition of PgrEXP proteins. The motifs (numbered 1–10) are displayed in different colored boxes. The right figure shows the exon–intron structure of PgrEXPs. The green boxes indicate untranslated regions (UTR), yellow boxes indicate exons, and black lines indicate introns.</p>
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<p>Expression profiles of the <span class="html-italic">PgrEXPs</span> in different tissues and varieties of pomegranate. (<b>A</b>): The transcript data of <span class="html-italic">PgrEXPs</span> in the root, flowers, and leaf as well as the peel, sarcotesta, and mesotesta at three development stages (50, 95, and 140 days after pollination) were processed with log2 normalization based on FPKM values. “OSC” = sarcotesta, “ISC” = mesotesta. (<b>B</b>): The transcript data of <span class="html-italic">PgrEXPs</span> in the mesotesta and sarcotesta of hard-seeded cultivar ‘Dabenzi’ and soft-seeded cultivar ‘Tunisia’ at three development stages (50, 95, and 140 days after pollination) were processed with log2 normalization based on FPKM values. “D” = ‘Dabenzi’; “T” = ‘Tunisia’, “O” = sarcotesta, and “I” = mesotesta. The subsequent number represents the days after pollination.</p>
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<p>Distribution of seed weight-related parameters for mature fruits of pomegranate accessions. (<b>A</b>) 100-seed weight; (<b>B</b>) 100-Kernel weight; (<b>C</b>) 100-sarcotesta weight; (<b>D</b>) PSW.</p>
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18 pages, 12009 KiB  
Article
Genome-Wide Identification of Expansins in Rubus chingii and Profiling Analysis during Fruit Ripening and Softening
by Zhen Chen, Danwei Shen, Yujie Shi, Yiquan Chen, Honglian He, Junfeng Jiang, Fan Wang, Jingyong Jiang, Xiaoyan Wang, Xiaobai Li and Wei Zeng
Plants 2024, 13(3), 431; https://doi.org/10.3390/plants13030431 - 1 Feb 2024
Cited by 1 | Viewed by 1147
Abstract
Improving fruit size or weight, firmness, and shelf life is a major target for horticultural crop breeding. It is associated with the depolymerization and rearrangement of cell components, including pectin, hemicellulose, cellulose, and other structural (glyco)proteins. Expansins are structural proteins to loosen plant [...] Read more.
Improving fruit size or weight, firmness, and shelf life is a major target for horticultural crop breeding. It is associated with the depolymerization and rearrangement of cell components, including pectin, hemicellulose, cellulose, and other structural (glyco)proteins. Expansins are structural proteins to loosen plant cell wall polysaccharides in a pH-dependent manner and play pivotal roles in the process of fruit development, ripening, and softening. Rubus chingii Hu, a unique Chinese red raspberry, is a prestigious pharmaceutical and nutraceutical dual-function food with great economic value. Thirty-three RchEXPs were predicted by genome-wide identification in this study, containing twenty-seven α-expansins (EXPAs), three β-expansins (EXPBs), one expansin-like A (EXPLA), and two expansin-like B (EXPLBs). Subsequently, molecular characteristics, gene structure and motif compositions, phylogenetic relationships, chromosomal location, collinearity, and regulatory elements were further profiled. Furthermore, transcriptome sequencing (RNA-seq) and real-time quantitative PCR assays of fruits from different developmental stages and lineages showed that the group of RchEXPA5, RchEXPA7, and RchEXPA15 were synergistically involved in fruit expanding and ripening, while another group of RchEXPA6 and RchEXPA26 might be essential for fruit ripening and softening. They were regulated by both abscisic acid and ethylene and were collinear with phylogenetic relationships in the same group. Our new findings laid the molecular foundation for improving the fruit texture and shelf life of R. chingii medicinal and edible fruit. Full article
(This article belongs to the Section Horticultural Science and Ornamental Plants)
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<p>Phylogenetic tree of expansins from <span class="html-italic">Rubus chingii</span>, <span class="html-italic">Fragaria vesca</span>, <span class="html-italic">Rubus idaeus,</span> and <span class="html-italic">R. occidentalis.</span> EXPA, EXPB, EXPLA, and EXPLB subfamilies were presented in light orange, yellow, blue, and green. “★”indicates the same expression tendency during fruit development stages which was highest at the red stage, while “▲” indicates that these gene expressions were initiated in earlier stages of fruit.</p>
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<p>Phylogenetic relationships, gene structures of exon/intron, and motif compositions of expansins in <span class="html-italic">Rubus chingii</span>.</p>
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<p>Chromosomal location of expansin encoding genes in <span class="html-italic">R. chingii.</span> The red dot means the tandem duplication events, including <span class="html-italic">RchEXPA8</span>–<span class="html-italic">10, RchEXPA16</span>–<span class="html-italic">19</span>, <span class="html-italic">RchEXPA20</span>–<span class="html-italic">21,</span> and <span class="html-italic">RchEXPA22</span>–<span class="html-italic">24</span>.</p>
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<p>Synteny analyses of <span class="html-italic">R. chingii</span> and <span class="html-italic">F. vesa</span> genomes. (<b>a</b>) Synteny analysis of <span class="html-italic">R. chingii</span> genome. The gene density was displayed in the form of heat maps and lines. Syntenic blocks were linked by gray lines, and syntenic relationships of <span class="html-italic">EXP</span> members were highlighted by red color. (<b>b</b>) Syntenic relationships of <span class="html-italic">R. chingii</span> and <span class="html-italic">F. vesa</span> genomes. Syntenic <span class="html-italic">EXP</span> gene pairs between <span class="html-italic">R. chingii</span> and <span class="html-italic">F. vesa</span> were highlighted by red color.</p>
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<p>Predicted cis-acting elements and protein–protein interaction networks of expansins in <span class="html-italic">R. chingii</span>. (<b>a</b>) Predicted cis-acting elements and their numbers in the promoters of expansin genes in <span class="html-italic">R. chingii</span>. (<b>b</b>) Construction of protein–protein interaction networks of expansins and other proteins in <span class="html-italic">R. chingii</span>.</p>
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<p>Expression profile of <span class="html-italic">RchEXPs</span> during fruit ripening and softening. (<b>a</b>) <span class="html-italic">RchEXPs</span> expression levels at the four representative stages, BGI, GY, YO, and Re, by RNA-seq. (<b>b</b>) <span class="html-italic">RchEXPs</span> expression levels at the red stages of the different degrees of ripeness by RNA-seq. (<b>c</b>–<b>f</b>) qPCR of <span class="html-italic">RchEXPA6</span>, <span class="html-italic">RchEXPA26</span>, <span class="html-italic">RchEXPA5,</span> and <span class="html-italic">RchEXPA15</span>. Different lower cases mean the significant difference at <span class="html-italic">p</span> &lt; 0.05 by a one-way analysis of variance with the LSD method.</p>
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<p>Biomass and real-time quantitative PCR of <span class="html-italic">RchEXPs</span> in red fruit of different <span class="html-italic">R. chingii</span> lineages. (<b>a</b>) Fresh weight (Fw); (<b>b</b>) fruit firmness; (<b>c</b>–<b>f</b>) qPCR of <span class="html-italic">RchEXPA6</span>, <span class="html-italic">RchEXPA26</span>, <span class="html-italic">RchEXPA7,</span> and <span class="html-italic">RchEXPA15.</span> Different lower cases mean a significant difference at <span class="html-italic">p</span> &lt; 0.05 by a one-way analysis of variance with the LSD method. (<b>g</b>) Pearson correlation analysis of <span class="html-italic">RchEXPs</span> and fruit fresh weight and firmness based on the qPCR data of red fruits from different lineages. “*” means a significant difference at <span class="html-italic">p</span> ≤ 0.05.</p>
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<p>Biomass and real-time quantitative PCR of <span class="html-italic">RchEXPs</span> in red fruit of different <span class="html-italic">R. chingii</span> lineages. (<b>a</b>) Fresh weight (Fw); (<b>b</b>) fruit firmness; (<b>c</b>–<b>f</b>) qPCR of <span class="html-italic">RchEXPA6</span>, <span class="html-italic">RchEXPA26</span>, <span class="html-italic">RchEXPA7,</span> and <span class="html-italic">RchEXPA15.</span> Different lower cases mean a significant difference at <span class="html-italic">p</span> &lt; 0.05 by a one-way analysis of variance with the LSD method. (<b>g</b>) Pearson correlation analysis of <span class="html-italic">RchEXPs</span> and fruit fresh weight and firmness based on the qPCR data of red fruits from different lineages. “*” means a significant difference at <span class="html-italic">p</span> ≤ 0.05.</p>
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14 pages, 4169 KiB  
Article
Comparative Proteomics Analysis of Primulina serrulata Leaves Reveals New Insight into the Formation of White Veins
by Quan-Li Dou, Da-Jun Xie, Tan Deng, Mo-Fang Chen, Zheng-Min Qian, Shuang-Shuang Wang and Ren-Bo Zhang
Horticulturae 2024, 10(1), 19; https://doi.org/10.3390/horticulturae10010019 - 23 Dec 2023
Viewed by 910
Abstract
Primulina serrulata is a valuable ornamental herb with rosette leaves and vibrant flowers. Some leaves of this species exhibit a bright and distinct white color along the upper veins, enhancing their ornamental value, while others are less white or entirely green. This variation [...] Read more.
Primulina serrulata is a valuable ornamental herb with rosette leaves and vibrant flowers. Some leaves of this species exhibit a bright and distinct white color along the upper veins, enhancing their ornamental value, while others are less white or entirely green. This variation is observed in adult leaves from natural habitats and among young leaves from seedlings grown in the laboratory. TMT-labeled proteomics technology was used to study the protein-level biogenesis of white-veined (WV) P. serrulata leaves. Our objective was to offer novel insight into the breeding of WV plants. Chlorophyll (Chl) content was significantly lower in the WV group than in the control group. Out of 6261 proteins identified, a mere 69 met the criteria for differentially expressed proteins (DEPs) after stringent screening for subsequent analyses. Among these DEPs, there were 44 proteins that exhibited downregulation and 25 that were upregulated in the WV plants. Some DEPs associated with chloroplasts and Chl biosynthesis were downregulated, leading to the absence of green coloration. Concurrently, Gene Ontology enrichment analysis further emphasized an insufficiency of magnesium, the key element in Chl biosynthesis. Many DEPs associated with abiotic or biotic stressors were downregulated, suggesting an overall weakening of stress resistance with certain compensatory mechanisms. Similarly, many DEPs related to modifying biomacromolecules were downregulated, possibly affected by the decrease in proteins involved in photosynthesis and stress resistance. Some DEPs containing iron were upregulated, indicating that iron is mainly used to synthesize heme and ferritin rather than Chl. Additionally, several DEPs related to sulfur or sulfate were upregulated, suggesting strengthened respiration. Expansin-A4 and pectinesterase were upregulated, coinciding with the emergence of a rough and bright surface in the white area of leaves, indicative of the elongation and gelation processes in the cell walls. These findings provide new insight for future studies to explore the mechanism of color formation in WV leaves. Full article
(This article belongs to the Special Issue Physiological and Molecular Biology Research on Ornamental Flower)
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<p>Variation in the ratio of white veins in <span class="html-italic">Primulina serrulata</span> leaves. (<b>A</b>) Bright and distinct white veins in the natural habitat; (<b>B</b>) few, indistinct white veins in the natural habitat; (<b>C</b>) compared seedling leaves grown in the laboratory.</p>
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<p>Chl <span class="html-italic">a</span> and Chl <span class="html-italic">b</span> contents in the WV and control groups.</p>
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<p>Gene Ontology annotation of the total proteins. (<b>a</b>) Bar chart of Biological Process categories; (<b>b</b>) Bar chart of Cellular Component categories; (<b>c</b>) Bar chart of Molecular Function categories.</p>
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<p>Volcano plot of the DEPs between the WV plants and the control. Red: upregulated DEPs; blue: downregulated DEPs; grey: no significant difference.</p>
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<p>Bar chart of the GO-BP enrichment results.</p>
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<p>Bar chart of GO-CC enrichment analysis.</p>
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<p>Bar chart of the GO-MF enrichment results.</p>
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<p>Bubble chart of the KEGG enrichment results.</p>
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<p>Protein interaction network in <span class="html-italic">Primulina serrulata</span> leaves. Different colored lines between the proteins indicate ofdifferent interactions.</p>
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<p>Top view of the white vein of <span class="html-italic">Primulina serrulata</span>. (<b>A</b>) White area; (<b>B</b>) green area.</p>
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20 pages, 4456 KiB  
Article
Molecular Cloning, In Silico Analysis, and Characterization of a Novel Cellulose Microfibril Swelling Gene Isolated from Bacillus sp. Strain AY8
by Md. Azizul Haque, Dhirendra Nath Barman, Aminur Rahman, Md. Shohorab Hossain, Sibdas Ghosh, Most. Aynun Nahar, Mst. Nur-E-Nazmun Nahar, Joyanta K. Saha, Kye Man Cho and Han Dae Yun
Microorganisms 2023, 11(12), 2857; https://doi.org/10.3390/microorganisms11122857 - 24 Nov 2023
Viewed by 1305
Abstract
A novel cellulose microfibril swelling (Cms) gene of Bacillus sp. AY8 was successfully cloned and sequenced using a set of primers designed based on the conserved region of the gene from the genomic database. The molecular cloning of the Cms gene revealed that [...] Read more.
A novel cellulose microfibril swelling (Cms) gene of Bacillus sp. AY8 was successfully cloned and sequenced using a set of primers designed based on the conserved region of the gene from the genomic database. The molecular cloning of the Cms gene revealed that the gene consisted of 679 bp sequences encoding 225 amino acids. Further in silico analysis unveiled that the Cms gene contained the NlpC/P60 conserved region that exhibited a homology of 98% with the NlpC/P60 family proteins found in both the strains, Burkholderialata sp. and Burkholderia vietnamiensis. The recombinant Cms enzyme had a significant impact on the reduction of crystallinity indices (CrI) of various substrates including a 3%, a 3.97%, a 4.66%, and a substantial 14.07% for filter paper, defatted cotton fiber, avicel, and alpha cellulose, respectively. Additionally, notable changes in the spectral features were observed among the substrates treated with recombinant Cms enzymes compared to the untreated control. Specifically, there was a decrease in band intensities within the spectral regions of 3000–3450 cm−1, 2900 cm−1, 1429 cm−1, and 1371 cm−1 for the treated filter paper, cotton fiber, avicel, and alpha cellulose, respectively. Furthermore, the recombinant Cms enzyme exhibited a maximum cellulose swelling activity at a pH of 7.0 along with a temperature of 40 °C. The molecular docking data revealed that ligand molecules, such as cellobiose, dextrin, maltose 1-phosphate, and feruloyated xyloglucan, effectively bonded to the active site of the Cms enzyme. The molecular dynamics simulations of the Cms enzyme displayed stable interactions with cellobiose and dextrin molecules up to 100 ns. It is noteworthy to mention that the conserved region of the Cms enzyme did not match with those of the bioadditives like expansins and swollenin proteins. This study is the initial report of a bacterial cellulose microfibril swellase enzyme, which could potentially serve as an additive to enhance biofuel production by releasing fermentable sugars from cellulose. Full article
(This article belongs to the Section Microbial Biotechnology)
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<p>Cloning strategies of Cms gene from <span class="html-italic">Bacillus</span> sp. strain AY8.</p>
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<p>Agarose gel (1%) electrophoresis of (<b>A</b>) #2684F, #2685R products using. S/M, size mark; Lane 1, #2684F + #2685R (0.2 kb); (<b>B</b>) #2709F + #2708R products. S/M, size mark; Lane 1, #2709F + #2708R (0.678 kb).</p>
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<p>Nucleotide and deduced amino acid sequences of Cms gene of <span class="html-italic">Bacillus</span> sp. AY8. The stop codon is indicated by a bar, while the signal peptide is depicted by underline.</p>
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<p>Expression of Cms gene of <span class="html-italic">Bacillus</span> sp. strain AY8. (<b>A</b>) Products Cms gene expression on SDS-PAGE; (<b>B</b>) pH; and (<b>C</b>) heat stabilities of Cms protein on Congo red adsorption enhancement on cotton cellulose, respectively [<a href="#B14-microorganisms-11-02857" class="html-bibr">14</a>].</p>
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<p>Phylogenetic relationships of <span class="html-italic">Bacillus</span> sp. strain AY8 Cms enzyme (blue) and other closely related proteins. Proteins highlighted with green color are cellulose swelling proteins involved in cellulose H-bonds disruption, whereas purple color are very similar proteins (unpublished) like Cms.</p>
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<p>(<b>A</b>). Structure of Cms–cellobiose docking complex. (<b>B</b>). Structure of Cms–dextrin docking complex. (<b>C</b>). Structure of Cms–feruloyatedxyloglycan complex.</p>
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<p>Molecular dynamic simulations of Cms proteins with cellobiose and dextrin complexes (<b>A</b>) Root mean square deviation (RMSD) of Cms–cellobiose and Cms–dextrin complexes, (<b>B</b>) root mean square fluctuation (RMSD) of Cms–cellobiose and Cms–dextrin complexes, (<b>C</b>) radius of Gyration (Rg) of Cms–cellobioase and Cms–dextrin complexes.</p>
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<p>FTIR spectra of untreated and recombinant Cms enzyme-treated (<b>A</b>) filter paper, (<b>B</b>) cotton fiber, (<b>C</b>) avicel, (<b>D</b>) alpha cellulose. Samples of untreated or treated with recombinant Cms enzyme in Tris-HCl buffer (pH 7) were incubated at 37 °C in water bath for 24 h.</p>
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<p>XRD spectra of untreated and recombinant Cms enzyme-treated (<b>A</b>) filter paper, (<b>B</b>) cotton fiber, (<b>C</b>) avicel, (<b>D</b>) alpha cellulose. Samples of untreated or treated with recombinant Cms enzyme in Tris-HCl buffer (pH 7) were incubated at 37 °C in water bath for 24 h.</p>
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