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The Gene, Genomics, and Molecular Breeding in Cruciferae Plants (2nd Edition)

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Plant Sciences".

Deadline for manuscript submissions: 31 October 2024 | Viewed by 4477

Special Issue Editor

Prof. Dr. Maoteng Li

E-Mail Website
Guest Editor
Department of Biotechnology, Huazhong University of Science and Technology, Wuhan 430074, China
Interests: QTL mapping; gene cloning; gene editing and molecular breeding of Brassica napus
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Cruciferae plants include many important vegetables and oil crops, for example, Brassica rapa, B. napus, and Raphanus sativus. To increase the production (e.g., seed oil production of B. napus and yield of Chinese Cabbage), improve the quality (e.g., improve the polyunsaturated fatty acid and phytosterol in seeds), overcome disease resistance (e.g., Sclerotinia sclerotiorum and Clubroot disease), and overcome abiotic stress resistance (e.g., drought, salt, and cold) were the most important tasks at present.

The aim of this Special Issue was mainly to focus on the following: (1) The innovation of new germplasm by using traditional and modern biotechnology; (2) Genome sequencing and re-sequencing analysis of Cruciferae plants; (3) Genetic dissection (e.g., QTL and GWAS analysis) and molecular mechanisms analysis of important agronomy characteristics; (4) Candidate gene functional analysis (e.g., gene editing) and molecular breeding in Cruciferae plants. The research of other agronomic characteristics that were not mentioned above was also encouraged.

Prof. Dr. Maoteng Li
Guest Editor

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Keywords

  • Cruciferae plants
  • genome analysis
  • genetic dissection
  • gene function
  • molecular breeding

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Related Special Issue

Published Papers (7 papers)

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Research

19 pages, 5703 KiB  
Article
Physiological Parameters and Transcriptomic Levels Reveal the Response Mechanism of Maize to Deep Sowing and the Mechanism of Exogenous MeJA to Alleviate Deep Sowing Stress
by Fang Wang, Zhijin Feng, Xinyi Yang, Guangkuo Zhou and Yunling Peng
Int. J. Mol. Sci. 2024, 25(19), 10718; https://doi.org/10.3390/ijms251910718 - 5 Oct 2024
Viewed by 333
Abstract
Deep sowing, as a method to mitigate drought and preserve soil moisture and seedlings, can effectively mitigate the adverse effects of drought stress on seedling growth. The elongation of the hypocotyl plays an important role in the emergence of maize seeds from deep-sowing [...] Read more.
Deep sowing, as a method to mitigate drought and preserve soil moisture and seedlings, can effectively mitigate the adverse effects of drought stress on seedling growth. The elongation of the hypocotyl plays an important role in the emergence of maize seeds from deep-sowing stress. This study was designed to explore the function of exogenous methyl jasmonate (MeJA) in the growth of the maize mesocotyl and to examine its regulatory network. The results showed that the addition of a 1.5 μ mol L−1 MeJA treatment significantly increased the mesocotyl length (MES), mesocotyl and coleoptile length (MESCOL), and seedling length (SDL) of maize seedlings. Transcriptome analysis showed that exogenous MeJA can alleviate maize deep-sowing stress, and the differentially expressed genes (DEGs) mainly include ornithine decarboxylase, terpene synthase 7, ethylene responsive transcription factor 11, and so on. In addition, candidate genes that may regulate the length of maize hypocotyls were screened by Weighted Gene Co-expression Network Analysis (WGCNA). These genes may be involved in the growth of maize hypocotyls through transcriptional regulation, histones, ubiquitin protease, protein binding, and chlorophyll biosynthesis and play an important role in maize deep-sowing tolerance. Our research findings may provide a theoretical basis for determining the tolerance of maize to deep-sowing stress and the mechanism of exogenous hormone regulation of deep-sowing stress. Full article
(This article belongs to the Special Issue The Gene, Genomics, and Molecular Breeding in Cruciferae Plants (2nd Edition))
►▼ Show Figures

Figure 1

Figure 1
<p>Effects of exogenous MeJA on endogenous hormones of maize inbred line seedlings under deep-sowing stress. CK: distilled water treatment at 3 cm sowing depth; CM: 3 cm sowing depth with 1.5 μmol·L<sup>−1</sup> exogenous MeJA treatment; DS: distilled water treatment at 15 cm sowing depth; DM: 15 cm sowing depth with 1.5 μmol·L<sup>−1</sup> exogenous MeJA treatment. Different lowercase letters represent the same inbred line with significant differences under different treatments (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 2
<p>Longitudinal structure and cell length of mesocotyl cells of maize inbred lines treated with exogenous MeJA under deep-sowing stress. <b>A</b>: 3 cm sowing depth + distilled water treatment (Qi319); <b>B</b>: 3 cm sowing depth + MeJA treatment (Qi319); <b>C</b>: 15 cm sowing depth + distilled water treatment (Qi319); <b>D</b>: 15 cm sowing depth + MeJA treatment (Qi319); <b>E</b>: 3 cm sowing depth + distilled water treatment (Zi330); <b>F</b>: 3 cm seeding depth + MeJA treatment (Zi330); <b>G</b>: 15 cm sowing depth + distilled water treatment (Zi330); <b>H</b>: 15 cm seeding depth + MeJA treatment (Zi330); <b>I</b>: cell lengths of mesocotyls. CK: distilled water treatment at 3 cm sowing depth; CM: 3 cm sowing depth with 1.5 μmol·L<sup>−1</sup> exogenous MeJA treatment; DS: distilled water treatment at 15 cm sowing depth; DM: 15 cm sowing depth with 1.5 μmol·L<sup>−1</sup> exogenous MeJA treatment. Different lowercase letters represent the same inbred line with significant differences under different treatments (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 3
<p>Differential gene expression analysis. (<b>a</b>) Number distribution of down-regulated DEGs in different comparison groups. (<b>b</b>,<b>c</b>) Venn diagram analysis of the normal sowing depth and deep-sowing stress and the normal sowing depth and deep-sowing stress with exogenous MeJA applied. Qi_CK3: Qi319 under 3 cm sowing depth and distilled water treatment; Qi_CK15: Qi319 distilled water treatment at 15 cm sowing depth; Qi_T3: Qi319 under 3 cm sowing depth and MeJA treatment; Qi_T15: Qi319 under 15 cm sowing depth and MeJA treatment; Zi_CK3: distilled water treatment of Zi330 at 3 cm sowing depth; Zi_CK15: distilled water treatment of Zi330 at 15 cm sowing depth; Zi_T3: MeJA treatment of Zi330 at 3 cm sowing depth; Zi_T15: MeJA treatment of Zi330 at 15 cm sowing depth.</p> Full article ">Figure 4
<p>GO analysis of two inbred lines in different comparison groups. (<b>a</b>) GO analysis of Qi319 maize inbred line at normal sowing depth under deep-sowing stress. (<b>b</b>) GO analysis of Zi330 maize inbred line at normal sowing depth under deep-sowing stress. (<b>c</b>) GO analysis of Qi319 maize inbred line after adding exogenous MeJA under deep-sowing stress. (<b>d</b>) GO analysis of Zi330 maize inbred line after adding exogenous MeJA under deep-sowing stress. The treatments and abbreviations are the same as those given in <a href="#ijms-25-10718-f001" class="html-fig">Figure 1</a>.</p> Full article ">Figure 4 Cont.
<p>GO analysis of two inbred lines in different comparison groups. (<b>a</b>) GO analysis of Qi319 maize inbred line at normal sowing depth under deep-sowing stress. (<b>b</b>) GO analysis of Zi330 maize inbred line at normal sowing depth under deep-sowing stress. (<b>c</b>) GO analysis of Qi319 maize inbred line after adding exogenous MeJA under deep-sowing stress. (<b>d</b>) GO analysis of Zi330 maize inbred line after adding exogenous MeJA under deep-sowing stress. The treatments and abbreviations are the same as those given in <a href="#ijms-25-10718-f001" class="html-fig">Figure 1</a>.</p> Full article ">Figure 5
<p>Pathway enrichment analysis of the two inbred lines in different comparison groups. (<b>a</b>) Pathway enrichment analysis of maize inbred line Qi319 at a normal sowing depth under deep-sowing stress. (<b>b</b>) Pathway enrichment analysis of maize inbred line Zi330 at a normal sowing depth under deep-sowing stress. (<b>c</b>) Pathway enrichment analysis of maize inbred line Qi319 after adding exogenous MeJA under deep-sowing stress. (<b>d</b>) Pathway enrichment analysis of maize inbred line Zi330 adding exogenous MeJA under deep-sowing stress. The treatments and abbreviations are the same as those given in <a href="#ijms-25-10718-f001" class="html-fig">Figure 1</a>.</p> Full article ">Figure 6
<p>GO and KEGG analysis of expressed genes in different treatment groups. (<b>a</b>) GO analysis of two varieties under normal sowing and deep-sowing stress. (<b>b</b>) Pathway enrichment analysis of two cultivars under normal sowing and deep-sowing stress. (<b>c</b>) GO analysis of two varieties under deep-sowing stress after adding exogenous MeJA. (<b>d</b>) Pathway enrichment analysis of two cultivars under deep-sowing stress after adding exogenous MeJA.</p> Full article ">Figure 7
<p>Real-time quantitative PCR validation of significantly up-regulated differentially expressed genes between two varieties under deep-sowing stress treatment. (<b>a</b>) The expression changes in response to the QCK, QDS, ZCK, and ZDS treatments for each candidate gene as measured by qRT-PCR. (<b>b</b>) Scatter plot showing the changes in the expression (log fold changes) of selected genes based on RNA-seq via qRT-PCR. The red line in the figure represents RNA seq, and the blue dots represent qRT-PCR. QCK: distilled water treatment of Qi319 at 3 cm sowing depth; QDS: distilled water treatment of Qi319 at 15 cm sowing depth; ZCK: distilled water treatment of Zi330 at 3 cm sowing depth; ZDS: distilled water treatment of Zi330 at 15 cm sowing depth.</p> Full article ">Figure 8
<p>Gene cluster analysis and correlation analysis of phenotypes and modules. (<b>a</b>) Hierarchical clustering analysis of co-expression genes. Different colors represent all modules, with gray indicating genes that cannot be classified into any module by default. (<b>b</b>) Correlated heat maps between modules. A color block in the picture represents a numerical value. The redder the color, the higher the expression level, and the bluer the color, the lower the expression level. (<b>c</b>) Correlations between gene modules and phenotypes. Each tree diagram in the figure represents a module, each branch represents a gene, and the darker the color of each point (white → yellow → red), the stronger the connectivity between the two genes corresponding to the row and column. (<b>d</b>) Heat map of correlations between gene modules and phenotypes. The leftmost color block represents the module, and the rightmost color bar represents the correlation range. In the heatmap of the middle part, the darker the color, the higher the correlation, with red indicating positive correlation and blue indicating negative correlation. The numbers in each cell represent correlation and significance. MES: mesocotyl length; COL: coleoptile length; MESCOL: mesocotyl length and coleoptile length; MEWCOW: mesocotyl weight and coleoptile weight; RL: root length; RW: root fresh weight; SDL: seedling length; SDW: seedling fresh weight.</p> Full article ">Figure 8 Cont.
<p>Gene cluster analysis and correlation analysis of phenotypes and modules. (<b>a</b>) Hierarchical clustering analysis of co-expression genes. Different colors represent all modules, with gray indicating genes that cannot be classified into any module by default. (<b>b</b>) Correlated heat maps between modules. A color block in the picture represents a numerical value. The redder the color, the higher the expression level, and the bluer the color, the lower the expression level. (<b>c</b>) Correlations between gene modules and phenotypes. Each tree diagram in the figure represents a module, each branch represents a gene, and the darker the color of each point (white → yellow → red), the stronger the connectivity between the two genes corresponding to the row and column. (<b>d</b>) Heat map of correlations between gene modules and phenotypes. The leftmost color block represents the module, and the rightmost color bar represents the correlation range. In the heatmap of the middle part, the darker the color, the higher the correlation, with red indicating positive correlation and blue indicating negative correlation. The numbers in each cell represent correlation and significance. MES: mesocotyl length; COL: coleoptile length; MESCOL: mesocotyl length and coleoptile length; MEWCOW: mesocotyl weight and coleoptile weight; RL: root length; RW: root fresh weight; SDL: seedling length; SDW: seedling fresh weight.</p> Full article ">Figure 9
<p>Analysis of hub gene network interaction in phenotypic significant enrichment modules. (<b>a</b>) Network interaction analysis of hub genes in royalblue module. (<b>b</b>) Network interaction analysis of hub genes in bisque4 module. The color gradients of the dots represent high or low soft thresholds of connectivity, with a redder dot color representing a higher soft threshold of connectivity. The color gradients of the dots represent high or low soft thresholds of connectivity, with a redder dot color representing a higher soft threshold of connectivity.</p> Full article ">Figure 10
<p>Model of the molecular mechanisms underlying deep-sowing tolerance and MeJA mitigation of deep-sowing-stress-induced damage in maize.</p> Full article ">
15 pages, 4742 KiB  
Article
BnUC1 Is a Key Regulator of Epidermal Wax Biosynthesis and Lipid Transport in Brassica napus
by Fei Ni, Mao Yang, Jun Chen, Yifei Guo, Shubei Wan, Zisu Zhao, Sijie Yang, Lingna Kong, Pu Chu and Rongzhan Guan
Int. J. Mol. Sci. 2024, 25(17), 9533; https://doi.org/10.3390/ijms25179533 - 2 Sep 2024
Viewed by 419
Abstract
The bHLH (basic helix–loop–helix) transcription factor AtCFLAP2 regulates epidermal wax accumulation, but the underlying molecular mechanism remains unknown. We obtained BnUC1mut (BnaA05g18250D homologous to AtCFLAP2) from a Brassica napus mutant with up-curling leaves (Bnuc1) and epidermal wax deficiency [...] Read more.
The bHLH (basic helix–loop–helix) transcription factor AtCFLAP2 regulates epidermal wax accumulation, but the underlying molecular mechanism remains unknown. We obtained BnUC1mut (BnaA05g18250D homologous to AtCFLAP2) from a Brassica napus mutant with up-curling leaves (Bnuc1) and epidermal wax deficiency via map-based cloning. BnUC1mut contains a point mutation (N200S) in the conserved dimerization domain. Overexpressing BnUC1mut in ZS11 (Zhongshuang11) significantly decreased the leaf epidermal wax content, resulting in up-curled and glossy leaves. In contrast, knocking out BnUC1mut in ZS11-NIL (Zhongshuang11-near-isogenic line) restored the normal leaf phenotype (i.e., flat) and significantly increased the leaf epidermal wax content. The point mutation weakens the ability of BnUC1mut to bind to the promoters of VLCFA (very-long-chain fatty acids) synthesis-related genes, including KCS (β-ketoacyl coenzyme synthase) and LACS (long-chain acyl CoA synthetase), as well as lipid transport-related genes, including LTP (non-specific lipid transfer protein). The resulting sharp decrease in the transcription of genes affecting VLCFA biosynthesis and lipid transport disrupts the normal accumulation of leaf epidermal wax. Thus, BnUC1 influences epidermal wax formation by regulating the expression of LTP and genes associated with VLCFA biosynthesis. Our findings provide a foundation for future investigations on the mechanism mediating plant epidermal wax accumulation. Full article
(This article belongs to the Special Issue The Gene, Genomics, and Molecular Breeding in Cruciferae Plants (2nd Edition))
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Figure 1

Figure 1
<p>Cloning and expression pattern analysis of <span class="html-italic">BnUC1</span> from the mapping parents. (<b>A</b>) The point mutation (N200S) of BnUC1 in mapping parent ZS11 (BnUC1<sup>WT</sup>) and ZS11-NIL (BnUC1<sup>mut</sup>). The blue box represents conserved bHLH domain. (<b>B</b>) The gene expression pattern analysis for the <span class="html-italic">BnUC1</span> gene. Expression levels of <span class="html-italic">BnUC1</span> detected by qRT-PCR in various tissues, including roots, leaves, siliques, stems, cotyledons, buds, and flowers of ZS11 and ZS11-NIL. The <span class="html-italic">BnActin7</span> was used as the internal reference gene. * represents a significance level of less than 0.05, ** represents a significance level of less than 0.01.</p> Full article ">Figure 2
<p>Phenotypes and expression analysis of OE-<span class="html-italic">BnUC1<sup>mut</sup></span> lines. (<b>A</b>) Morphology comparison of ZS11 and OE-<span class="html-italic">BnUC1<sup>mut</sup></span> (OE-1, OE-5) lines at seedling stage. Bar = 5 cm. (<b>B</b>) Determine the expression level of <span class="html-italic">BnUC1</span> gene in OE-<span class="html-italic">BnUC1<sup>mut</sup></span> lines by qRT-PCR. Using <span class="html-italic">BnActin7</span> as the internal reference gene. Error bars indicate ± SD (<span class="html-italic">n</span> = 3). ** <span class="html-italic">p</span> &lt; 0.01. (<b>C</b>,<b>D</b>): SEM images of cuticle wax crystals on ZS11 (<b>C</b>) and OE-1 (<b>D</b>) line abaxial sides of leaves in <span class="html-italic">B. napus</span>. Bar = 10 μm.</p> Full article ">Figure 3
<p>The leaf cuticular wax comparison of ZS11 and OE-<span class="html-italic">BnUC1<sup>mut</sup></span> lines. (<b>A</b>) Total wax coverage and amount in leaf epidermis of ZS11 and OE-<span class="html-italic">BnUC1<sup>mut</sup></span> (OE−1 and OE−5) lines. (<b>B</b>) Amounts of epidermal wax in leaf epidermis of ZS11 and OE-<span class="html-italic">BnUC1<sup>mut</sup></span> (OE−1 and OE−5) lines. Cuticular wax samples were extracted from seven-week-old plants with chloroform and analyzed using GC-MS. Error bars indicate ± SD from three biological replicates (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 4
<p>Targets and phenotypes analysis of <span class="html-italic">BnUC1<sup>mut</sup></span> knockout lines. (<b>A</b>) The construct of <span class="html-italic">BnUC1<sup>mut</sup></span> CRISPR-Cas9 vector: a hygromycin resistance cassette consisting of the hygromycin phosphotransferase gene (<span class="html-italic">HPT</span>) driven by the cauliflower mosaic virus 35S promoter; a Cas9 expression cassette comprising the sequence encoding Cas9 driven by 35S promoter; and two sgRNAs (target1 and target2) driven by the U6 promoters from <span class="html-italic">Arabidopsis</span>. (<b>B</b>) Four CRISPR-Cas9-induced mutant alleles (named CR-1~4) detected by Sanger sequencing. PAM is indicated by a red underline, while nucleotide mutations are indicated by red letters. (<b>C</b>) Morphology of ZS11-NIL and <span class="html-italic">BnUC1<sup>mut</sup></span> knockout lines. Bars = 5 cm.</p> Full article ">Figure 5
<p>The comparison of leaf cuticular wax components between mapping parent ZS11-NIL and the <span class="html-italic">BnUC1<sup>mut</sup></span> knockout lines. (<b>A</b>) Total wax coverage and amount in leaf of ZS11-NIL and <span class="html-italic">BnUC1<sup>mut</sup></span> gene knockout (CR-1 and CR-3) lines. (<b>B</b>) Amounts of individual components in leaf of ZS11-NIL and <span class="html-italic">BnUC1<sup>mut</sup></span> gene knockout (CR−1 and CR−3) lines. Cuticular wax samples were extracted from eight-week-old plants with chloroform and analyzed using GC-MS. Error bars indicate ± SD from three biological replicates (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 6
<p>The <span class="html-italic">BnUC1<sup>mut</sup></span> regulates gene expression of long-chain fatty acid biosynthesis. Expression level of six VLCFA biosynthesis genes (<span class="html-italic">BnA01.CER2/BnaA01G0141100ZS</span>, BnA10.KCS2/BnaA10G0024400ZS, BnC02.KCS20/BnaC02G0385300ZS, BnC04.CER26/BnaC04G0357800ZS, BnC04.LACS1/BnaC04G0007500ZS, BnC02.CER3/BnaC02G0140500ZS) in <span class="html-italic">BnUC1<sup>mut</sup></span>-overexpressing lines (OE-1, OE-5, and ZS11) and the control ZS11 (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, <span class="html-italic">n</span> = 3).</p> Full article ">Figure 7
<p>The BnUC1s interact with <span class="html-italic">LACS1</span> and <span class="html-italic">KCS20</span> gene promoters. (<b>A</b>) Observe the growth status of yeast containing two plasmids in SD/-Leu and SD/-Leu+200 ng mL<sup>−1</sup> AbA (Aureobasidin A) media to determine the interactions between BnUC1<sup>WT</sup> and BnUC1<sup>mut</sup> with wax synthesis-related gene promoters, respectively. p53-AbAi+AD and pAbAi+AD were used as positive and negative controls, respectively. (<b>B</b>) The luciferase assay showed the binding between BnUC1s and wax synthesis-related gene promoters, respectively, in <span class="html-italic">N. benthamiana</span> leaves.</p> Full article ">Figure 8
<p>Expression analysis of <span class="html-italic">LTP</span> genes in <span class="html-italic">BnUC1<sup>mut</sup></span>-overexpressing and ZS11 lines. Expression level comparison of four <span class="html-italic">LTP</span> genes (<span class="html-italic">BnA03.LTP11</span>/<span class="html-italic">BnaA03G0536300ZS</span>, <span class="html-italic">BnC02.LTP1</span>/<span class="html-italic">BnaC02G0158700ZS</span>, <span class="html-italic">BnC02.LTP2</span>/<span class="html-italic">BnaC02G0159100ZS</span>, <span class="html-italic">BnC02.LTP3</span>/<span class="html-italic">BnaC02G054 0400ZS</span>) in <span class="html-italic">BnUC1<sup>mut</sup></span>-overexpressing lines (OE-1, OE-5, and ZS11) and the control ZS11. Statistical significance of the measurements was determined using Student’s <span class="html-italic">t</span> test (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, <span class="html-italic">n</span> = 3).</p> Full article ">Figure 9
<p>The BnUC1 binding to <span class="html-italic">LTP</span> gene promoters. (<b>A</b>) Observe the growth status of yeast containing two plasmids in SD/-Leu and SD/-Leu+200 ng/mL AbA (Aureobasidin A) media to determine the interactions between BnUC1<sup>WT</sup> and BnUC1<sup>mut</sup> with the <span class="html-italic">LTP</span> gene promoters, respectively. The p53−AbAi+AD and pAbAi+AD were used as positive and negative controls, respectively. (<b>B</b>) The luciferase assay showed the binding between BnUC1s and the <span class="html-italic">LTP</span> gene promoters, respectively, in <span class="html-italic">N. benthamiana</span> leaves.</p> Full article ">Figure 10
<p>BnUC1<sup>mut</sup> interacts with BnA03.LTP11, BnA05.LTP6, BnC04.LTP1, BnaA03.MYB57, BnaA07.ZFP22, and BnaA08.ZIP11. (<b>A</b>) The point-to-point validation of the protein interaction in yeast cell. The pGADT7-T/pGBKT7-Lam combination was used as the negative control, while the pGADT7-T/pGBKT7-53 combination was used as the positive control. (<b>B</b>) The validation of the protein interaction by BiFC assay in tobacco mesophyll cells. The YFP fluorescence and autofluorescence from chloroplasts are indicated in yellow and red, respectively. N, YFP N-terminal; C, YFP C-terminal. The empty vector C+N was used as the negative control. Bars = 20 μm.</p> Full article ">
14 pages, 3206 KiB  
Article
Mining Candidate Genes for Leaf Angle in Brassica napus L. by Combining QTL Mapping and RNA Sequencing Analysis
by Aoyi Peng, Shuyu Li, Yuwen Wang, Fengjie Cheng, Jun Chen, Xiaoxiao Zheng, Jie Xiong, Ge Ding, Bingchao Zhang, Wen Zhai, Laiqiang Song, Wenliang Wei and Lunlin Chen
Int. J. Mol. Sci. 2024, 25(17), 9325; https://doi.org/10.3390/ijms25179325 - 28 Aug 2024
Viewed by 518
Abstract
Leaf angle (LA) is an important trait of plant architecture, and individuals with narrow LA can better capture canopy light under high-density planting, which is beneficial for increasing the overall yield per unit area. To study the genetic basis and molecular regulation mechanism [...] Read more.
Leaf angle (LA) is an important trait of plant architecture, and individuals with narrow LA can better capture canopy light under high-density planting, which is beneficial for increasing the overall yield per unit area. To study the genetic basis and molecular regulation mechanism of leaf angle in rapeseed, we carried out a series of experiments. Quantitative trait loci (QTL) mapping was performed using the RIL population, and seven QTLs were identified. Transcriptome analysis showed that the cell wall formation/biogenesis processes and biosynthesis/metabolism of cell wall components were the most enrichment classes. Most differentially expressed genes (DEGs) involved in the synthesis of lignin, xylan, and cellulose showed down-regulated expression in narrow leaf material. Microscopic analysis suggested that the cell size affected by the cell wall in the junction area of the stem and petiole was the main factor in leaf petiole angle (LPA) differences. Combining QTL mapping and RNA sequencing, five promising candidate genes BnaA01G0125600ZS, BnaA01G0135700ZS, BnaA01G0154600ZS, BnaA10G0154200ZS, and BnaC03G0294200ZS were identified in rapeseed, and most of them were involved in cell wall biogenesis and the synthesis/metabolism of cell wall components. The results of QTL, transcriptome analysis, and cytological analysis were highly consistent, collectively revealing that genes related to cell wall function played a crucial role in regulating the LA trait in rapeseed. The study provides further insights into LA traits, and the discovery of new QTLs and candidate genes is highly beneficial for genetic improvement. Full article
(This article belongs to the Special Issue The Gene, Genomics, and Molecular Breeding in Cruciferae Plants (2nd Edition))
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Figure 1

Figure 1
<p>Leaf angle frequency distribution of RIL population in the two environments. (<b>A</b>) LPA frequency distribution of RIL population in Gansu. (<b>B</b>) LIA frequency distribution of RIL population in Gansu. (<b>C</b>) LPA frequency distribution of RIL population in Nanchang. (<b>D</b>) LIA frequency distribution of RIL population in Nanchang.</p> Full article ">Figure 2
<p>GO enrichment analysis of DEGs between R036 and R009.</p> Full article ">Figure 3
<p>Synthetic pathway of main components in cell wall. (<b>A</b>) DEG expression in lignin synthesis. (<b>B</b>) DEG expression in xylan and cellulose synthesis.</p> Full article ">Figure 3 Cont.
<p>Synthetic pathway of main components in cell wall. (<b>A</b>) DEG expression in lignin synthesis. (<b>B</b>) DEG expression in xylan and cellulose synthesis.</p> Full article ">Figure 4
<p>The anatomical structure of the junction between the main stem and leaf petiole. (<b>A</b>) The transverse section of the junction tissue of the R036 plant. Scale bars, 0.5 mm. (<b>B</b>) The transverse section of the junction tissue of the R009 plant. Scale bars, 0.5 mm. (<b>C</b>) The longitudinal section of the junction tissue of the R036 plants. Scale bars, 160 μm. (<b>D</b>) The longitudinal section of the junction tissue of the R009 plant. Scale bars, 160 μm. Abbreviations: EP: epidermis CO: cortex PI: pith VB: vascular bundle P: phloem VC: vascular cambium ABS: abaxial side; ADS: adaxial side.</p> Full article ">Figure 5
<p>Relative expression of candidate genes in the R036 and R009 by qPCR.</p> Full article ">Figure 6
<p>Potential regulatory model for LPA in <span class="html-italic">Brassica napus</span> L.</p> Full article ">
18 pages, 10338 KiB  
Article
Comparative Analysis of Transcriptomes Reveals Pathways and Verifies Candidate Genes for Clubroot Resistance in Brassica oleracea
by Fuquan Ce, Jiaqin Mei, Yu Zhao, Qinfei Li, Xuesong Ren, Hongyuan Song, Wei Qian and Jun Si
Int. J. Mol. Sci. 2024, 25(17), 9189; https://doi.org/10.3390/ijms25179189 - 24 Aug 2024
Viewed by 570
Abstract
Clubroot, a soil-borne disease caused by Plasmodiophora brassicae, is one of the most destructive diseases of Brassica oleracea all over the world. However, the mechanism of clubroot resistance remains unclear. In this research, transcriptome sequencing was conducted on root samples from both [...] Read more.
Clubroot, a soil-borne disease caused by Plasmodiophora brassicae, is one of the most destructive diseases of Brassica oleracea all over the world. However, the mechanism of clubroot resistance remains unclear. In this research, transcriptome sequencing was conducted on root samples from both resistant (R) and susceptible (S) B. oleracea plants infected by P. brassicae. Then the comparative analysis was carried out between the R and S samples at different time points during the infection stages to reveal clubroot resistance related pathways and candidate genes. Compared with 0 days after inoculation, a total of 4991 differential expressed genes were detected from the S pool, while only 2133 were found from the R pool. Gene function enrichment analysis found that the effector-triggered immunity played a major role in the R pool, while the pathogen-associated molecular pattern triggered immune response was stronger in the S pool. Simultaneously, candidate genes were identified through weighted gene co-expression network analysis, with Bol010786 (CNGC13) and Bol017921 (SD2-5) showing potential for conferring resistance to clubroot. The findings of this research provide valuable insights into the molecular mechanisms underlying clubroot resistance and present new avenues for further research aimed at enhancing the clubroot resistance of B. oleracea through breeding. Full article
(This article belongs to the Special Issue The Gene, Genomics, and Molecular Breeding in Cruciferae Plants (2nd Edition))
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<p>Transcriptomic response profiling in <span class="html-italic">B. oleracea</span> roots following inoculation with <span class="html-italic">P. brassicae</span>. (<b>A</b>) Histogram of number of up- or down-regulated DEGs. The red box represents the number of up-regulated genes; the green box represents the number of down-regulated genes. (<b>B</b>) Heatmap of all DEGs of R and S pools at different infection stages compared to 0 DAI. DAI, days after inoculation. R, resistant pool. S, susceptible pool.</p> Full article ">Figure 2
<p>GO enrichment analysis of the differentially expressed genes in R and S pools at different infection stages compared to 0 DAI. (<b>A</b>) The GO significant enrichment analysis of up-regulated genes. (<b>B</b>) The GO significant enrichment analysis of down-regulated genes. The <span class="html-italic">y</span>-axis corresponds to the enriched GO terms, while the <span class="html-italic">x</span>-axis represents the varying time points post-inoculation for both R and susceptible S pools. The size of each dot indicates the number of genes enriched for each term, and the color of each dot signifies the adjusted <span class="html-italic">p</span>-value, representing the significance of each term. DAI, days after inoculation. R, resistant pool. S, susceptible pool.</p> Full article ">Figure 3
<p>Significant enrichment of up-regulated (in red) and down-regulated (in green) DEGs in KEGG pathways across both pools. DAI, days after inoculation. R, resistant pool. S, susceptible pool.</p> Full article ">Figure 4
<p>The hub genes identified by the gene co-expression network. The network of “Red” module (<b>A</b>), “Black” module (<b>B</b>), “Brown” module (<b>C</b>), and “Dark Turquoise” module (<b>D</b>) revealed the hub genes colored by red.</p> Full article ">Figure 5
<p>Validation of candidate genes through qRT-PCR and identification of clubroot resistance. (<b>A</b>) The expression levels of the four genes measured by RNA sequencing and qRT-PCR. (<b>B</b>) Relative gene expression in WT and T-DNA mutant lines of four candidate genes in <span class="html-italic">A. thaliana</span> at 28 DAI by <span class="html-italic">P. brassicae</span>. (<b>C</b>) Disease index and percentages of WT and four mutant lines in the individual disease classes. Disease index = (1 × n1 + 2 × n2 + 3 × n3 + 4 × n4) × 100/4Nt, where n1–4 represents the number of plants in each severity class, and the total number of plants tested is denoted as Nt. (<b>D</b>) Hypocotyl width of WT and four mutants at 28 DAI by <span class="html-italic">P. brassicae.</span> (<b>E</b>) The <span class="html-italic">P. brassicae</span> biomass of root among WT and four mutants at 28 DAI by <span class="html-italic">P. brassicae.</span> (<b>F</b>) Root symptoms of WT and four mutants at 28 days after uninoculated and inoculated by <span class="html-italic">P. brassicae</span>. Treatments were replicated three times with 34–45 plants per replicate. White bar: 1 cm. The asterisk indicates a significant difference at <span class="html-italic">p</span>-value ≤ 0.01.</p> Full article ">
17 pages, 3971 KiB  
Article
Characteristics and Cytological Analysis of Several Novel Allopolyploids and Aneuploids between Brassica oleracea and Raphanus sativus
by Mingyang Hu, Shiting Fang, Bo Wei, Qi Hu, Mengxian Cai, Tuo Zeng, Lei Gu, Hongcheng Wang, Xuye Du, Bin Zhu and Jing Ou
Int. J. Mol. Sci. 2024, 25(15), 8368; https://doi.org/10.3390/ijms25158368 - 31 Jul 2024
Viewed by 621
Abstract
Polyploids are essential in plant evolution and species formation, providing a rich genetic reservoir and increasing species diversity. Complex polyploids with higher ploidy levels often have a dosage effect on the phenotype, which can be highly detrimental to gametes, making them rare. In [...] Read more.
Polyploids are essential in plant evolution and species formation, providing a rich genetic reservoir and increasing species diversity. Complex polyploids with higher ploidy levels often have a dosage effect on the phenotype, which can be highly detrimental to gametes, making them rare. In this study, offspring plants resulting from an autoallotetraploid (RRRC) derived from the interspecific hybridization between allotetraploid Raphanobrassica (RRCC, 2n = 36) and diploid radish (RR, 2n = 18) were obtained. Fluorescence in situ hybridization (FISH) using C-genome-specific repeats as probes revealed two main genome configurations in these offspring plants: RRRCC (2n = 43, 44, 45) and RRRRCC (2n = 54, 55), showing more complex genome configurations and higher ploidy levels compared to the parental plants. These offspring plants exhibited extensive variation in phenotypic characteristics, including leaf type and flower type and color, as well as seed and pollen fertility. Analysis of chromosome behavior showed that homoeologous chromosome pairing events are widely observed at the diakinesis stage in the pollen mother cells (PMCs) of these allopolyploids, with a range of 58.73% to 78.33%. Moreover, the unreduced C subgenome at meiosis anaphase II in PMCs was observed, which provides compelling evidence for the formation of complex allopolyploid offspring. These complex allopolyploids serve as valuable genetic resources for further analysis and contribute to our understanding of the mechanisms underlying the formation of complex allopolyploids. Full article
(This article belongs to the Special Issue The Gene, Genomics, and Molecular Breeding in Cruciferae Plants (2nd Edition))
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<p>FISH analyses of chromosome constitutions of the offspring plants. Blue signals indicate DAPI staining, and red signals labeled by BAC BoB014O06 probe indicate C subgenome chromosomes. (<b>A1</b>,<b>A2</b>) The mitotic cell of RRRCC with 27R genome chromosomes (blue) and 18C genome chromosomes (red). (<b>B1</b>,<b>B2</b>) The mitotic cell of RRRRCC with 36R genome chromosomes (blue) and 18C genome chromosomes (red). (<b>C1</b>,<b>C2</b>) The mitotic cell of RRRCC<sup>2</sup> with 27R genome chromosomes (blue) and 16C genome chromosomes (red). (<b>D1</b>,<b>D2</b>) The mitotic cell of RRRCC<sup>1</sup> with 27R genome chromosomes (blue) and 17C genome chromosomes (red). (<b>E1</b>,<b>E2</b>) The mitotic cell of RRRRCC<sup>1</sup> with 36R genome chromosomes (blue) and 19C genome chromosomes (red). Bar: 10 μm.</p> Full article ">Figure 2
<p>Characteristics of reproductive organ types of <span class="html-italic">R. sativus</span>, <span class="html-italic">B. oleracea</span>, and these offspring of RRRC. (<b>A</b>–<b>G</b>) The morphology of flowers of <span class="html-italic">R. sativus</span>, <span class="html-italic">B. oleracea,</span> RRRCC, RRRRCC, RRRCC<sup>2</sup>, RRRCC<sup>1</sup>, and RRRRCC<sup>1</sup>, respectively. The flower edges of RRRCC<sup>1</sup> and RRRRCC<sup>1</sup> are tinged with pale purple. (<b>H</b>) Siliques traits of <span class="html-italic">R. sativus</span>, <span class="html-italic">B. oleracea,</span> and these offspring plants. (<b>I</b>,<b>J</b>) Seeds traits of <span class="html-italic">R. sativus</span>, <span class="html-italic">B. oleracea</span>, RRRCC, RRRRCC, RRRCC<sup>1</sup>, and RRRRCC<sup>1</sup>, respectively. Bar: 1 cm. Shared letters a–e are significantly different detected by one-way analysis of variance (ANOVA), <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 3
<p>The integrated agronomic traits of <span class="html-italic">R. sativus</span>, <span class="html-italic">B. oleracea,</span> and the offspring of RRRC. (<b>A</b>–<b>E</b>) Phenotype of <span class="html-italic">R. sativus</span>, <span class="html-italic">B. oleracea,</span> RRRCC<sup>2</sup>, RRRCC<sup>1</sup>, RRRCC, RRRRCC, and RRRRCC<sup>1</sup>, respectively. (<b>H</b>) Leaves of <span class="html-italic">R. sativus</span>, <span class="html-italic">B. oleracea</span>, and the offspring of RRRC. Bar: 10 cm.</p> Full article ">Figure 4
<p>Pollen fertility analysis of <span class="html-italic">R. sativus</span>, <span class="html-italic">B. oleracea</span>, and the offspring of RRRC. (<b>A</b>–<b>C</b>) The pollen characteristics of <span class="html-italic">R. sativus</span>, RRRCC, and RRRRCC. The deep brown pollens indicate fertility, while the light brown ones indicate abnormal fertility. The red solid arrows indicate fertile pollen grains, and white solid arrows indicate sterile pollen grains. Bar: 100 µm. (<b>D</b>) Comparison of pollen fertility across parental species and above-mentioned hybrids. Shared letters a–f are extremely significantly different as detected by one-way analysis of variance (ANOVA), <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 5
<p>BAC-FISH analyses of chromosome behaviors of the offspring of RRRC at diakinesis. Blue signals indicate DAPI staining, and red signals from BAC BoB014O06 probe indicate C subgenome chromosomes. (<b>A1</b>,<b>A2</b>) Pollen mother cell (PMC) chromosomal configurations of RRRCC<sup>2</sup> with a C-R-R trivalent (white arrow). (<b>B1</b>,<b>B2</b>) PMC chromosomal configurations of RRRCC<sup>1</sup> with seven C-C bivalents and a C-C-R-R-R-C hexavalent (red hollow arrow). (<b>C1</b>,<b>C2</b>) PMC chromosomal configuration of RRRCC. One diakinesis with seven C-C bivalents and a C-C-C-C-R-R hexavalent (white hollow arrow). (<b>D1</b>,<b>D2</b>) PMC chromosomal configurations of RRRCC with six C-C bivalents, a C-C-C trivalent, a univalent, and a C-C-R-R quadrivalent. (<b>E1</b>,<b>E2</b>) PMC chromosomal configurations of RRRRCC. One diakinesis with seven C-C bivalents and a C-C-C-C-R pentavalent (red arrow). One diakinesis with seven C-C bivalents, a C-C-C trivalent, and a C-R-R-R-R-R hexavalent (yellow arrow). (<b>F1</b>,<b>F2</b>) An apparent R-R-R-R quadrivalent seems to be present (red solid arrow). PMC chromosomal configurations of RRRRCC<sup>1</sup>. (<b>G1</b>,<b>G2</b>) One diakinesis with seven C-C bivalents, a C-C-C trivalent, and a C-C-R-R-R-R hexavalent (yellow solid arrow). (<b>H1</b>,<b>H2</b>) One diakinesis with seven C-C bivalents, a C-C-C trivalent, a univalent, and a C-R-R trivalent (white arrow). Bar, 10 μm.</p> Full article ">Figure 6
<p>BAC-FISH analyses of chromosome behaviors of each offspring of RRRC at anaphase I (AI) and anaphase II (AII). The blue signal indicates DAPI staining, and red indicates labeled BAC BoB014O06. (<b>A1</b>,<b>A2</b>) PMC chromosomal configurations of RRRCC<sup>2</sup> separated as 19 (12R + 7C): 24 (15R + 9C). (<b>B1</b>,<b>B2</b>) PMC chromosomal configurations of RRRCC<sup>1</sup> separated as 25 (17R + 8C): 19 (10R + 9C). (<b>C1</b>,<b>C2</b>) PMC chromosomal configurations of RRRCC at AI. Chromosomes separated as 23 (14R + 9C): 22 (13R + 9C). (<b>D1</b>,<b>D2</b>) PMC chromosomal configurations of RRRRCC at AI. Chromosomes separated as 29 (20R + 9C): 25 (16R + 9C). (<b>E1</b>,<b>E2</b>) PMC chromosomal configurations of RRRRCC<sup>1</sup> at AI separated as 28 (18R + 10C): 27 (18R + 9C). Irregularity of chromosome behavior occurs at AI and AII. (<b>F1</b>,<b>F2</b>) The formation of chromosome bridge occurs at AI from meiotic cell of RRRCC<sup>1</sup> (white arrow). (<b>G1</b>,<b>G2</b>) Lagged chromosomes occur at AI from meiotic cell of RRRCC<sup>2</sup> (red arrow). (<b>H1</b>,<b>H2</b>) None of the C chromosomes separate to the cluster (red hollow arrow). Bar, 10 μm.</p> Full article ">Figure 7
<p>Imaginary representation of the origin of the autoallopolyploid offspring from RRRC hybrid. The diagrams with blue and red colors represent R and C genomes, respectively. The upper “1” and “2” indicate that the aneuploid plants have one or two chromosomes deviating from the chromosome number of euploid plants.</p> Full article ">
18 pages, 17205 KiB  
Article
Circadian Rhythm and Nitrogen Metabolism Participate in the Response of Boron Deficiency in the Root of Brassica napus
by Ling Liu, Xianjie Duan, Haoran Xu, Peiyu Zhao, Lei Shi, Fangsen Xu and Sheliang Wang
Int. J. Mol. Sci. 2024, 25(15), 8319; https://doi.org/10.3390/ijms25158319 - 30 Jul 2024
Viewed by 636
Abstract
Boron (B) deficiency has been shown to inhibit root cell growth and division. However, the precise mechanism underlying B deficiency-mediated root tip growth inhibition remains unclear. In this study, we investigated the role of BnaA3.NIP5;1, a gene encoding a boric acid channel, [...] Read more.
Boron (B) deficiency has been shown to inhibit root cell growth and division. However, the precise mechanism underlying B deficiency-mediated root tip growth inhibition remains unclear. In this study, we investigated the role of BnaA3.NIP5;1, a gene encoding a boric acid channel, in Brassica napus (B. napus). BnaA3.NIP5;1 is expressed in the lateral root cap and contributes to B acquisition in the root tip. Downregulation of BnaA3.NIP5;1 enhances B sensitivity in B. napus, resulting in reduced shoot biomass and impaired root tip development. Transcriptome analysis was conducted on root tips from wild-type B. napus (QY10) and BnaA3.NIP5;1 RNAi lines to assess the significance of B dynamics in meristematic cells during seedling growth. Differentially expressed genes (DEGs) were significantly enriched in plant circadian rhythm and nitrogen (N) metabolism pathways. Notably, the circadian-rhythm-related gene HY5 exhibited a similar B regulation pattern in Arabidopsis to that observed in B. napus. Furthermore, Arabidopsis mutants with disrupted circadian rhythm (hy5/cor27/toc1) displayed heightened sensitivity to low B compared to the wild type (Col-0). Consistent with expectations, B deficiency significantly disrupted N metabolism in B. napus roots, affecting nitrogen concentration, nitrate reductase enzyme activity, and glutamine synthesis. Interestingly, this disruption was exacerbated in BnaA3NIP5;1 RNAi lines. Overall, our findings highlight the critical role of B dynamics in root tip cells, impacting circadian rhythm and N metabolism, ultimately leading to retarded growth. This study provides novel insights into B regulation in root tip development and overall root growth in B. napus. Full article
(This article belongs to the Special Issue The Gene, Genomics, and Molecular Breeding in Cruciferae Plants (2nd Edition))
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<p>Downregulated expression of <span class="html-italic">BnaA3NIP5;1</span> enhances boron sensitivity. (<b>A</b>) The phenotypic characteristics and statistical analysis were conducted on primary root length and lateral root number in QY10 and NSQ lines cultured on plates for 10 days under 0.1 μM B. The study involved a minimum of three replicates. (<b>B</b>) Additionally, longitudinal sections (<b>C</b>–<b>E</b>) and diameter statistics (<b>F</b>) of the root tips from QY10 and NSQ lines in scenario (<b>A</b>) were examined (<span class="html-italic">n</span> ≥ 3). (<b>G</b>) Furthermore, the phenotypes of QY10 and NSQ lines were observed after 14 days of culture in nutrient solution under 0.25 μM B. (<b>H</b>–<b>J</b>) Microstructural analysis of the root tips from QY10 and NSQ lines (in scenario (<b>G</b>)) revealed the non-root-hair zone (NRHZ) length. (<b>K</b>) The NRHZ length was statistically analyzed (<span class="html-italic">n</span> ≥ 3), and the reported values represent means ± standard deviation. Letters denote significant differences between different treatments based on Duncan’s test (<span class="html-italic">p</span> &lt; 0.05). The abbreviation ‘NSQ’ refers to the <span class="html-italic">BnaA3NIP5;1</span> RNAi lines.</p> Full article ">Figure 2
<p>Differentially expressed genes (DEGs) among different groups. (<b>A</b>–<b>C</b>) We conducted principal component analysis (PCA), sample correlation analysis, and generated a gene coexpression Venn diagram using data from nine samples. These samples included NB_QY10 (normal boron QY10, 100 μM B), LB_QY110 (low-boron QY10, 0.25 μM B), and LB-NSQ (low-boron NSQ, 0.25 μM B), each with three biological replicates. (<b>D</b>) We quantified the differentially expressed genes (DEGs) between various groups. (<b>E</b>) Additionally, we created a Venn diagram to compare DEGs among LB_QY10 versus NB_QY10, LB_NSQ versus NB_QY10, and LB_NSQ versus LB_QY10. (<b>F</b>) Finally, we determined the count of upregulated and downregulated DEGs in the union of the DEGs identified in scenario (<b>E</b>).</p> Full article ">Figure 3
<p>qRT-PCR verification transcriptome data results of selected 10 DEGs. The gene expression profiles were assessed using RNA-seq (<b>left panel</b>) and qRT-PCR (<b>right panel</b>) in NB_QY10, LB_QY10, and LB_NSQ. The study involved three pools, each containing 30–50 root tips, and the reported values represent means ± SD.</p> Full article ">Figure 4
<p>KEGG functional enrichment analysis of DEGs in different groups. (<b>A</b>) KEGG enrichment analysis of DEGs in LB_QY10 vs. NB_QY10. (<b>B</b>) KEGG enrichment analysis of DEGs in LB_NSQ vs. NB_QY10. (<b>C</b>) KEGG enrichment analysis of DEGs in LB_NSQ vs. LB_QY10. (<b>D</b>) KEGG enrichment analysis of DEGs in LB_NSQ vs. LB_QY10 vs NB_QY10. (<b>E</b>) KEGG enrichment analysis of up-regulated DEGs in LB_NSQ vs. LB_QY10 vs NB_QY10. (<b>F</b>) KEGG enrichment analysis of down-regulated DEGs in LB_NSQ vs. LB_QY10 vs NB_QY10. The dot size and color correspond to the number of genes and the corrected <span class="html-italic">p</span>-value, respectively. The gene ratio represents the proportion of differentially expressed genes (DEGs) annotated within a specific pathway term relative to the total number of genes annotated in that term. A higher gene ratio signifies greater pathway intensity. The padj value, ranging from 0 to 1, reflects the corrected <span class="html-italic">p</span>-value, with lower values indicating stronger significance. Only the top 20 enriched pathways are displayed, and the red box highlights the pathways of particular interest.</p> Full article ">Figure 5
<p>Expression patterns of circadian rhythm pathway genes in <span class="html-italic">B. napus</span> and <span class="html-italic">Arabidopsis</span>. (<b>A</b>) The gene identity (ID), gene annotation (name), and differential expression multiples of circadian rhythm pathway genes were investigated under two conditions: LB_QY10 versus NB_QY10 and LB_NSQ versus NB_QY10 using RNA-seq. (<b>B</b>) The expression pattern of <span class="html-italic">AtHY5</span> in <span class="html-italic">Arabidopsis</span> Col-0 roots was examined after 12 days of growth on agar plates under normal (100 μM B) and low-boron (0.1 μM B) conditions. The study involved three pools, each containing 20 roots, (<span class="html-italic">n</span> = 3). and the reported values represent means ± standard deviation. Letters denote significant differences between different treatments based on Duncan’s test (<span class="html-italic">p</span> &lt; 0.05). Additionally, GUS staining was performed on 7-day-old <span class="html-italic">ProAtHY5: GUS</span> seedlings cultured on agar plates with normal boron ((<b>C</b>,<b>E</b>,<b>G</b>,<b>I</b>,<b>K</b>) (100 μM B)) and boron-deficient ((<b>D</b>,<b>F</b>,<b>H</b>,<b>J</b>,<b>L</b>) (0.1 μM B)) conditions.</p> Full article ">Figure 6
<p><span class="html-italic">hy5/cor27/toc1</span> mutants are more sensitive to boron deficiency than Col-0. (<b>A</b>) The phenotypes of wild-type (Col-0) and <span class="html-italic">hy5/cor27/toc1</span> mutant plants were observed after 12 days of growth on agar plates under both normal (100 μM B) and boron-deficient (0.1 μM B) conditions. (<b>B</b>) Microstructure images of the root tips from Col-0 and <span class="html-italic">hy5/cor27/toc1</span> mutants were captured under boron deficiency (0.1 μM B) conditions in scenario (<b>A</b>). The root tips were stained with propidium iodide (PI), and the upper row displays the view after PI staining, while the lower row shows the view under bright field. Arrows indicate the location of root hairs. (<b>C</b>) Statistical analysis was performed on primary root length, meristematic zone length, elongation zone length, and the number of lateral roots in Col-0 and <span class="html-italic">hy5/cor27/toc1</span> mutants from scenario (<b>A</b>) (<span class="html-italic">n</span> ≥ 10). The reported values represent means ± standard deviation. Letters denote significant differences between different treatments based on Duncan’s test (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 7
<p>Nitrogen metabolism participates in the response of <span class="html-italic">B. napus</span> roots to B deficiency. (<b>A</b>) The gene identity (ID), gene annotation (name), and differential expression multiples of nitrogen metabolism pathway genes were analyzed in RNA-seq data for LB_QY10 versus NB_QY10 and LB_NSQ versus NB_QY10. (<b>B</b>) A schematic diagram illustrates transcriptome changes in the main pathways of nitrate absorption and assimilation. Genes in green font indicate decreased expression during the low-boron response, while genes in red represent increased expression. (<b>C</b>–<b>F</b>) Nitrogen concentration, nitrate reductase (NR), glutamine synthase (GS), and glutamate dehydrogenase (GDH) were determined in roots of Y10 and NSQ lines cultured in nutrient solution for 14 days under 100 and 0.25 μM B. The values represent means ± SD (standard deviation), and letters indicate significant differences between different treatments based on Duncan’s test (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 8
<p>A working model illustrating the involvement of circadian rhythms and nitrogen metabolism in B deficiency-mediated root tip growth inhibition. Boron (B) deficiency activates the activity of circadian-rhythm-related transcription factors (<b>on the left</b>) and reduces the transcription level and enzyme activity of nitrogen metabolism-related genes (<b>on the right</b>, where green font represents decrease and red font represents increase). The gray box indicates cells, and the orange circle represents B.</p> Full article ">
15 pages, 8324 KiB  
Article
Overexpression of BnaA10.WRKY75 Decreases Cadmium and Salt Tolerance via Increasing ROS Accumulation in Arabidopsis and Brassica napus L.
by Xiaoke Ping, Qianjun Ye, Mei Yan, Jia Wang, Taiyuan Zhang, Sheng Chen, Kadambot H. M. Siddique, Wallace A. Cowling, Jiana Li and Liezhao Liu
Int. J. Mol. Sci. 2024, 25(14), 8002; https://doi.org/10.3390/ijms25148002 - 22 Jul 2024
Cited by 2 | Viewed by 876
Abstract
Soil is indispensable for agricultural production but has been seriously polluted by cadmium and salt in recent years. Many crops are suffering from this, including rapeseed, the third largest global oilseed crop. However, genes simultaneously related to both cadmium and salt stress have [...] Read more.
Soil is indispensable for agricultural production but has been seriously polluted by cadmium and salt in recent years. Many crops are suffering from this, including rapeseed, the third largest global oilseed crop. However, genes simultaneously related to both cadmium and salt stress have not been extensively reported yet. In this study, BnaA10.WRKY75 was screened from previous RNA-seq data related to cadmium and salt stress and further analyses including sequence comparison, GUS staining, transformation and qRT-PCR were conducted to confirm its function. GUS staining and qRT-PCR results indicated BnaA10.WRKY75 was induced by CdCl2 and NaCl treatment. Sequence analysis suggested BnaA10.WRKY75 belongs to Group IIc of the WRKY gene family and transient expression assay showed it was a nuclear localized transcription factor. BnaA10.WRKY75-overexpressing Arabidopsis and rapeseed plants accumulated more H2O2 and O2− and were more sensitive to CdCl2 and NaCl treatment compared with untransformed plants, which may be caused by the downregulation of BnaC03.CAT2. Our study reported that BnaA10.WRKY75 increases sensitivity to cadmium and salt stress by disrupting the balance of reactive oxygen species both in Arabidopsis and rapeseed. The results support the further understanding of the mechanisms underlying cadmium and salt tolerance and provide BnaA10.WRKY75 as a valuable gene for rapeseed abiotic stress breeding. Full article
(This article belongs to the Special Issue The Gene, Genomics, and Molecular Breeding in Cruciferae Plants (2nd Edition))
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Figure 1
<p>Response of <span class="html-italic">WRKY</span> transcription factors to cadmium stress. (<b>a</b>) Expression levels of 75 differently expressed <span class="html-italic">WRKY</span> transcription factors as revealed by RNA-seq. Expression levels were described by fold change and (Cd<sup>2+</sup> 0 h) was used as the control. Four <span class="html-italic">BnaWRKY75s</span> and <span class="html-italic">BnaA10.WRKY75</span> were indicated by line and star, respectively. (<b>b</b>) Expression levels of four <span class="html-italic">BnaWRKY75s</span> under cadmium stress. (<b>c</b>) GUS staining results of <span class="html-italic">Arabidopsis</span> transgenic plants expressing <span class="html-italic">pBnaA10.WRKY75::GUS</span>. White arrows indicate the difference in GUS signal between cadmium treated and untreated plants. Bars: 1 cm.</p> Full article ">Figure 2
<p>Protein sequences analysis and cis-element identification of four <span class="html-italic">BnaWRKY75s</span>. (<b>a</b>) Multiple sequences alignment. Two domains and representative amino acids were marked by line and star, respectively. (<b>b</b>) A phylogenetic tree including four BnaWRKY75s and 7 AtWRKY proteins from Group IIc. The green shading indicates the proteins that are closely related to BnaA10.WRKY75. (<b>c</b>) Genomic location of cis-element in <span class="html-italic">BnaWRKY75s</span> promoter.</p> Full article ">Figure 3
<p>Localization of BnaA10.WRKY75 in tobacco epidermal cells.</p> Full article ">Figure 4
<p>Expression of <span class="html-italic">BnaA10.WRKY75</span> in tissues detected by GUS staining: (<b>a</b>) 5 and (<b>b</b>) 14-day-old seedling; (<b>c</b>) 30-day-old leaf; (<b>d</b>) 10-day-old leaf; (<b>e</b>) stem; (<b>f</b>) flower; (<b>g</b>–<b>i</b>) silique at 1, 7 and 14 days after flowering. Bar: 1 cm.</p> Full article ">Figure 5
<p>The effects of cadmium stress on wild-type and <span class="html-italic">BnaA10.WRKY75</span>-overexpressing plants. (<b>a</b>,<b>b</b>) Root length performance of seedlings grown on MS medium with or without CdCl<sub>2</sub> added for three weeks. (<b>c</b>,<b>d</b>) Performance of leaves and (<b>e</b>) H<sub>2</sub>O<sub>2</sub> and O<sub>2</sub><sup>−</sup> accumulation of plants irrigated by 500 μM CdCl<sub>2</sub> solution for 7 d. White arrows in (<b>c</b>) indicate the difference in leaves between <span class="html-italic">BnaA10.WRKY75</span> overexpressing and Col-0 seedlings. (<b>f</b>,<b>g</b>) The performance of rapeseed seedlings irrigated by 1000 μM CdCl<sub>2</sub> solution for 10 d and white arrows indicate the difference in leaves between <span class="html-italic">BnaA10.WRKY75</span> overexpressing and J9709 seedlings. (<b>h</b>) DAB and NBT staining results of rapeseed plants irrigated by 1000 μM CdCl<sub>2</sub> solution. Values in (<b>b</b>) are the mean ± SD of three replications and differences in comparisons were revealed by student’s <span class="html-italic">t</span>-test. **, <span class="html-italic">p</span> &lt; 0.01; ***, <span class="html-italic">p</span> &lt; 0.001. Bars: (<b>a</b>,<b>c</b>–<b>e</b>,<b>h</b>) 1 cm; (<b>f</b>,<b>g</b>) 2 cm.</p> Full article ">Figure 6
<p>Response of <span class="html-italic">BnaWRKY75s</span> to stresses as obtained from BnIR database. (<b>a</b>,<b>b</b>) Expression patterns of <span class="html-italic">BnaWRKY75s</span> in leaves and roots, respectively. Gray shadings in (<b>a</b>,<b>b</b>) indicate significant upregulation of <span class="html-italic">BnaA10.WRKY75</span>.</p> Full article ">Figure 7
<p>Response of <span class="html-italic">BnaA10.WRKY75</span> to three types of abiotic stress. (<b>a</b>) qRT-PCR result in rapeseed cv. J9709; (<b>b</b>,<b>c</b>) GUS staining results of transgenic <span class="html-italic">Arabidopsis</span> plants expressing <span class="html-italic">pBnaA10.WRKY75::GUS</span> under control and 100 mM NaCl treatments. Values in (<b>a</b>) are the mean ± SD of three replications. Bars: (<b>b</b>,<b>c</b>) 1 cm.</p> Full article ">Figure 8
<p>The effects of salt stress on wild-type and <span class="html-italic">BnaA10.WRKY75</span>-overexpressing plants. (<b>a</b>) Comparison of plant performance under salt treatment. (<b>b</b>–<b>e</b>) Differences in fresh weight, chlorophyll, proline and MDA content between Col-0 and transgenic <span class="html-italic">Arabidopsis</span> plants. (<b>f</b>) DAB and NBT staining revealed H<sub>2</sub>O<sub>2</sub> and O<sub>2</sub><sup>−</sup> accumulation in leaves of <span class="html-italic">Arabidopsis</span> plants under salt treatment. (<b>g</b>) Performance of hydroponic rapeseed seedlings treated with salt solution for 10 d. (<b>h</b>) DAB and NBT staining revealed H<sub>2</sub>O<sub>2</sub> and O<sub>2</sub><sup>−</sup> accumulation in leaves of rapeseed plants under salt treatment. Values in (<b>b</b>–<b>e</b>) are the mean ± SD of three replications and differences in comparisons were revealed by student’s <span class="html-italic">t</span>-test. **, <span class="html-italic">p</span> &lt; 0.01. Bars: (<b>f</b>,<b>h</b>) 1 cm; (<b>a</b>,<b>g</b>) 2 cm.</p> Full article ">Figure 9
<p>BnaA10.WRKY75 regulates the expression of genes related to cadmium and salt stress. (<b>a</b>) <span class="html-italic">BnaC03.HMA4c</span>, (<b>b</b>,<b>c</b>) <span class="html-italic">AtSOS1</span>, (<b>d</b>) <span class="html-italic">BnaCAT2s</span>, (<b>e</b>) <span class="html-italic">AtCAT2</span> and (<b>f</b>) <span class="html-italic">BnaC03.CAT2</span>. Values in (<b>a</b>–<b>f</b>) are the mean ± SD of three replications and differences in comparisons were revealed by student’s <span class="html-italic">t</span>-test. *, <span class="html-italic">p</span> &lt; 0.05; **, <span class="html-italic">p</span> &lt; 0.01; ns, no significance.</p> Full article ">Figure 10
<p>The role and working frame of <span class="html-italic">BnaA10.WRKY75</span> in response to cadmium and salt stress. Green lines mean promotion and red lines mean inhibition. Solid and dashed lines represent determined and undetermined regulatory relationships, respectively. <span class="html-italic">BnaA10.WRKY75</span> was induced by cadmium and salt stress then repressed <span class="html-italic">BnaC03.CAT2</span>, which is responsible for ROS scavenging. <span class="html-italic">BnaA10.WRKY75</span> also promotes the expression of <span class="html-italic">BnaC03.HMA4c</span> and increases Cd<sup>2+</sup> transport.</p> Full article ">
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