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25 pages, 4496 KiB  
Article
FaNPR3 Members of the NPR1-like Gene Family Negatively Modulate Strawberry Fruit Resistance against Colletotrichum acutatum
by Victoria Súnico, José Javier Higuera, Francisco Amil-Ruiz, Isabel Arjona-Girona, Carlos J. López-Herrera, Juan Muñoz-Blanco, Ana María Maldonado-Alconada and José L. Caballero
Plants 2024, 13(16), 2261; https://doi.org/10.3390/plants13162261 - 14 Aug 2024
Viewed by 495
Abstract
Strawberry fruit is highly appreciated worldwide for its organoleptic and healthy properties. However, this plant is attacked by many pathogenic fungi, which significantly affect fruit production and quality at pre- and post-harvest stages, making chemical applications the most effective but undesirable strategy to [...] Read more.
Strawberry fruit is highly appreciated worldwide for its organoleptic and healthy properties. However, this plant is attacked by many pathogenic fungi, which significantly affect fruit production and quality at pre- and post-harvest stages, making chemical applications the most effective but undesirable strategy to control diseases that has been found so far. Alternatively, genetic manipulation, employing plant key genes involved in defense, such as members of the NPR-like gene family, has been successful in many crops to improve resistance. The identification and use of the endogenous counterpart genes in the plant of interest (as it is the case of strawberry) is desirable as it would increase the favorable outcome and requires prior knowledge of their defense-related function. Using RNAi technology in strawberry, transient silencing of Fragaria ananassa NPR3 members in fruit significantly reduced tissue damage after Colletotrichum acutatum infection, whereas the ectopic expression of either FaNPR3.1 or FaNPR3.2 did not have an apparent effect. Furthermore, the ectopic expression of FaNPR3.2 in Arabidopsis thaliana double-mutant npr3npr4 reverted the disease resistance phenotype to Pseudomonas syringe to wild-type levels. Therefore, the results revealed that members of the strawberry FaNPR3 clade negatively regulate the defense response to pathogens, as do their Arabidopsis AtNPR3/AtNPR4 orthologs. Also, evidence was found showing that FaNPR3 members act in strawberry (F. ananassa) as positive regulators of WRKY genes, FaWRKY19 and FaWRKY24; additionally, in Arabidopsis, FaNPR3.2 negatively regulates its orthologous genes AtNPR3/AtNPR4. We report for the first time the functional characterization of FaNPR3 members in F. ananassa, which provides a relevant molecular basis for the improvement of resistance in this species through new breeding technologies. Full article
(This article belongs to the Section Plant Genetics, Genomics and Biotechnology)
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Figure 1

Figure 1
<p>Expression pattern of <span class="html-italic">FaNPR3</span> genes in strawberry fruit after agroinfiltration with pFRN::FaNPR3<span class="html-italic">all</span>.RNAi construct. (<b>A</b>) Gene expression of <span class="html-italic">FaNPR3.1</span>. (<b>B</b>) Gene expression of <span class="html-italic">FaNPR3.2</span>. (<b>C</b>) Gene expression of <span class="html-italic">FaNPR3.3</span>. qRT-PCR analysis was accomplished in agroinfiltrated strawberry fruit before (0 and 2 days) and after (3, 5, and 7 days) <span class="html-italic">C. acutatum</span> inoculation (<span class="html-italic">Ca</span>). The time scale refers to days after agroinfiltration (dai). For all the genes, the arrow and horizontal line indicate the time of <span class="html-italic">Ca</span> inoculation. The black and grey columns show pFRN (control) and pFRN::FaNPR3<span class="html-italic">all</span>.RNAi (silencing) agroinfiltrations, respectively. Data from all time points are referred to data in time zero, represented as 1 (white column). Bars, mean ± standard error. Note the different scales in the relative-expression-level axis. Statistical significance was determined by one-way ANOVA. Letters indicate significant differences (<span class="html-italic">p</span>  &lt;  0.05) in HSD Tukey’s post hoc test.</p>
Full article ">Figure 2
<p>Expression pattern of <span class="html-italic">FaNPR3</span> genes in strawberry fruit after agroinfiltration with pB7GWIWG2::FaNPR32.RNAi. (<b>A</b>) Gene expression of <span class="html-italic">FaNPR3.1</span>. (<b>B</b>) Gene expression of <span class="html-italic">FaNPR3.2</span>. (<b>C</b>) Gene expression of <span class="html-italic">FaNPR3.3</span>. qRT-PCR analysis was accomplished in agroinfiltrated strawberry fruit before (0 and 2 days) and after (3, 5, and 7 days) <span class="html-italic">C. acutatum</span> inoculation (<span class="html-italic">Ca</span>). The time scale refers to days after agroinfiltration (dai). For all the genes, the arrow and the horizontal line indicate the time of <span class="html-italic">Ca</span> inoculation. The black and grey columns show pB7GWIWG2 and pB7GWIWG2::FaNPR32.RNAi agroinfiltrations, respectively. Data from all time points are referred to data in time zero, represented as 1 (white column). Bars, mean ± standard error. Note the different scales in the relative-expression-level axis. Statistical significance was determined by one-way ANOVA. Letters indicate significant differences (<span class="html-italic">p</span>  &lt;  0.05) in HSD Tukey’s post hoc test.</p>
Full article ">Figure 3
<p>Silencing effect of pFRN::FaNPR3<span class="html-italic">all</span>.RNAi and ectopic overexpression of <span class="html-italic">FaNPR3.1</span> in strawberry fruit after <span class="html-italic">C. acutatum</span> infection. (<b>A</b>,<b>B</b>) Upper panels: external surface disease symptoms on the two agroinfiltrated opposite halves of the same fruit, after silencing and overexpression, respectively. (<b>A</b>,<b>B</b>) Lower panels: internal tissue damage of the same fruit shown in the corresponding upper panels. pFRN::FaNPR3<span class="html-italic">all</span>.RNAi and pFRN, silencing construct and its corresponding empty vector, as control. pK7::FaNPR31.OE and pK7, overexpression construct and its corresponding empty vector, as control. A relevant fruit is shown for each condition, as an example. (<b>C</b>) Statistical analysis of internal tissue damage ratio of the two opposite halves of the same fruit, according to the 1 to 5 severity scale; striped and plain blue bars, pFRN/pFRN and pFRN/pFRN::FaNPR31-RNAi agroinfiltrated values, respectively; striped and plain red bars, pK7/pK7 and pK7/pK7::FaNPR31-OE agroinfiltrated values, respectively. Data correspond to mean ± SD. Within each bar, means with different letters are significantly different by LSD test at <span class="html-italic">p</span> &lt; 0.05. A ratio value of 1 indicates no differences between the opposite halves of the same fruit.</p>
Full article ">Figure 4
<p>Silencing effect of pB7GWIWG2::FaNPR32.RNAi and ectopic overexpression of <span class="html-italic">FaNPR3.2</span> in strawberry fruit after <span class="html-italic">C. acutatum</span> infection. (<b>A</b>,<b>B</b>) Upper panels: external surface disease symptoms on the two agroinfiltrated opposite halves of the same fruit, after silencing and overexpression, respectively. (<b>A</b>,<b>B</b>) Lower panels: internal tissue damage of the same fruit shown in the corresponding upper panels. pB7GWIWG2::FaNPR32.RNAi and pB7GWIWG2, silencing construct and its corresponding empty vector, as control. pB7WG2::FaNPR32.OE and pB7WG2, overexpression construct and its corresponding empty vector, as control. A relevant fruit is shown for each condition, as an example. (<b>C</b>) Statistical analysis of internal tissue damage ratio of the two opposite halves of the same fruit, according to the 1 to 5 severity scale; striped and plain blue bars, pB7GWIWG2/pB7GWIWG2 and pB7GWIWG2/pB7GWIWG2::FaNPR32-RNAi agroinfiltrated values, respectively; striped and plain red bars, pB7WG2/pB7WG2 and pB7WG2/pB7WG2::FaNPR32-OE agroinfiltrated values, respectively. Data correspond to mean ± SD. Within each bar, means with different letters are significantly different by LSD test at <span class="html-italic">p</span> &lt; 0.05. A ratio value of 1 indicates no differences between opposite halves of the same fruit.</p>
Full article ">Figure 5
<p>Silencing effect of <span class="html-italic">FaNPR3</span> members on the expression of <span class="html-italic">FaWRKY19</span> and <span class="html-italic">FaWRKY24</span> genes in strawberry fruit after <span class="html-italic">C. acutatum</span> infection. (<b>A</b>) Silencing effect of pFRN::FaNPR3<span class="html-italic">all</span>.RNAi. (<b>B</b>) Silencing effect pB7GWIWG2::FaNPR32.RNAi. Black columns, the expression of <span class="html-italic">FaWRKY19</span> and <span class="html-italic">FaWRKY24</span> genes in half fruit agroinfiltrated with pFRN (<b>A</b>) or pB7GWIWG2 (<b>B</b>) control vectors. Grey columns, the expression of <span class="html-italic">FaWRKY19</span> and <span class="html-italic">FaWRKY24</span> genes in half fruit agroinfiltrated with pFRN::FaNPR3<span class="html-italic">all</span>.RNAi (<b>A</b>) or pB7GWIWG2::FaNPR32.RNAi (<b>B</b>). qRT-PCR analysis was accomplished in agroinfiltrated strawberry fruit before (0 and 2 days) and after (3, 5, and 7 days) <span class="html-italic">C. acutatum</span> inoculation (<span class="html-italic">Ca</span>). The time scale refers to days after agroinfiltration (dai). Arrow and horizontal line indicate the time of <span class="html-italic">Ca</span> inoculation. Data from all time points are referred to data in time zero, represented as 1 (white column). Bars, mean ± standard error. Note the different scales in the relative-expression-level axis. Statistical significance was determined by one-way ANOVA. Letters indicate significant differences (<span class="html-italic">p</span>  &lt;  0.05) in HSD Tukey’s post hoc test.</p>
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<p>Characterization of Arabidopsis lines. (<b>A</b>) <span class="html-italic">FaNPR3.2</span> expression in Arabidopsis WT-, <span class="html-italic">npr3npr4</span> mutant-, and <span class="html-italic">FaNPR3.2</span>-overexpressing lines <span class="html-italic">npr3npr4</span>::FaNPR32 and WT::FaNPR32 (black, grey, white and striped bars, respectively). Transcript accumulation was monitored by qRT-PCR in non-infected plants as described in the <a href="#sec4-plants-13-02261" class="html-sec">Section 4</a>. Expression levels were normalized with respect to the internal control <span class="html-italic">ACTIN2</span> and displayed relatively to the threshold value of the wild-type (no expression of <span class="html-italic">FaNPR3.2</span>) that was given a value of 1 for convenience. Note that no amplification of <span class="html-italic">FaNPR3.2</span> strawberry gene is detected in neither WT nor double mutant <span class="html-italic">npr3npr4</span> using the specific primers (<a href="#app1-plants-13-02261" class="html-app">Supplementary Materials Table S1</a>). Bars represent the mean levels of transcript quantified from three independent biological experiments (±SD). (<b>B</b>–<b>D</b>) Disease resistance phenotype of Arabidopsis lines upon <span class="html-italic">Pst</span> inoculation. (<b>B</b>) Symptoms development on leaves 3, 5, and 7 days post-inoculation (dpi) (10<sup>6</sup> CFU mL<sup>−1</sup>); (<b>C</b>) trypan blue staining for the detection of cell death 1 dpi (10<sup>5</sup> CFU mL<sup>−1</sup>); (<b>D</b>) in planta bacterial growth monitored 3, 5, and 7 days post-inoculation (10<sup>6</sup> CFU mL<sup>−1</sup>). CFU, colony-forming units. Statistically significant differences are labelled by letters (one-way ANOVA, Tukey’s multiple comparisons test, <span class="html-italic">p</span> &lt; 0.05). <span class="html-italic">Pst</span> was pressure infiltrated into fully expanded mature leaves of 4–5-week-old Arabidopsis plants. As control, leaves were infiltrated with 10 mM MgCl<sub>2</sub>. The whole experiment was performed three times with similar results.</p>
Full article ">Figure 7
<p>Expression of the defense-related genes <span class="html-italic">PR1</span>, <span class="html-italic">PR2</span>, and <span class="html-italic">PR5</span> in Arabidopsis lines. Relative expression level was monitored by qRT-PCR in control (<b>A</b>) and infected plants 1 and 2 days post-inoculation (dpi) with <span class="html-italic">Pst</span> (10<sup>6</sup> CFU mL<sup>−1</sup>) (<b>B</b>), in WT, WT::FaNPR32, double mutant <span class="html-italic">npr3npr4</span>, and <span class="html-italic">npr3npr4</span>::FaNPR32 (black, grey, white and striped bars, respectively). Expression levels were normalized with respect to the internal control <span class="html-italic">ACTIN2</span> and displayed relative to the expression in mock-treated wild-type samples (<b>A</b>) or to the expression in mock-treated samples of each line (<b>B</b>) that were given a value of 1. Bars refer to mean ± standard error. Note the different scales in the relative-expression-level axis. Statistical significance was determined by one-way ANOVA. Letters indicate significant differences (<span class="html-italic">p</span>  &lt;  0.05) in HSD Tukey’s post hoc test.</p>
Full article ">Figure 8
<p>Expression of the endogenous <span class="html-italic">AtNPR1</span>, <span class="html-italic">AtNPR3</span>, and <span class="html-italic">AtNPR4</span> genes in Arabidopsis. Relative expression level was monitored by qRT-PCR in control (<b>A</b>) and infected plants 1 and 2 days post-inoculation (dpi) with <span class="html-italic">Pst</span> (10<sup>6</sup> CFU mL<sup>−1</sup>) (<b>B</b>), in WT, WT::FaNPR32, double mutant <span class="html-italic">npr3npr4</span>, and <span class="html-italic">npr3npr4</span>::FaNPR32 (black, grey, white and striped bars, respectively). Expression levels were normalized with respect to the internal control <span class="html-italic">ACTIN2</span> and displayed relative to the expression in mock-treated wild-type samples (<b>A</b>) or to the expression in mock-treated samples of each line (<b>B</b>) that were given a value of 1. Bars refer to mean ± standard error. Note the different scales in the relative-expression-level axis. Statistical significance was determined by one-way ANOVA. Letters indicate significant differences (<span class="html-italic">p</span>  &lt;  0.05) in HSD Tukey’s post hoc test. Note that the absence of transcript for <span class="html-italic">AtNPR3</span> and <span class="html-italic">AtNPR4</span> in the overexpressing lines in the double mutant <span class="html-italic">npr3npr4</span> background. It proves that Arabidopsis primers do not amplify strawberry orthologous genes.</p>
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20 pages, 3868 KiB  
Article
Genome-Wide Analysis of the WRKY Transcription Factor Family in Roses and Their Putative Role in Defence Signalling in the Rose–Blackspot Interaction
by Helena Sophia Domes and Thomas Debener
Plants 2024, 13(8), 1066; https://doi.org/10.3390/plants13081066 - 10 Apr 2024
Viewed by 1115
Abstract
WRKY transcription factors are important players in plant regulatory networks, where they control and integrate various physiological processes and responses to biotic and abiotic stresses. Here, we analysed six rose genomes of 5 different species (Rosa chinensis, R. multiflora, R. [...] Read more.
WRKY transcription factors are important players in plant regulatory networks, where they control and integrate various physiological processes and responses to biotic and abiotic stresses. Here, we analysed six rose genomes of 5 different species (Rosa chinensis, R. multiflora, R. roxburghii, R. sterilis, and R. rugosa) and extracted a set of 68 putative WRKY genes, extending a previously published set of 58 WRKY sequences based on the R. chinensis genome. Analysis of the promoter regions revealed numerous motifs related to induction by abiotic and, in some cases, biotic stressors. Transcriptomic data from leaves of two rose genotypes inoculated with the hemibiotrophic rose black spot fungus Diplocarpon rosae revealed the upregulation of 18 and downregulation of 9 of these WRKY genes after contact with the fungus. Notably, the resistant genotype exhibited the regulation of 25 of these genes (16 upregulated and 9 downregulated), while the susceptible genotype exhibited the regulation of 20 genes (15 upregulated and 5 downregulated). A detailed RT–qPCR analysis of RcWRKY37, an orthologue of AtWRKY75 and FaWRKY1, revealed induction patterns similar to those of the pathogenesis-related (PR) genes induced in salicylic acid (SA)-dependent defence pathways in black spot inoculation experiments. However, the overexpression of RcWRKY37 in rose petals did not induce the expression of any of the PR genes upon contact with black spot. However, wounding significantly induced the expression of RcWRKY37, while heat, cold, or drought did not have a significant effect. This study provides the first evidence for the role of RcWRKY37 in rose signalling cascades and highlights the differences between RcWRKY37 and AtWRKY75. These results improve our understanding of the regulatory function of WRKY transcription factors in plant responses to stress factors. Additionally, they provide foundational data for further studies. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Plant Defense against Fungal Pathogens)
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Figure 1

Figure 1
<p>Positions of the <span class="html-italic">RcWRKYs</span> on chromosomes according to the <span class="html-italic">R. chinensis</span> genome of Hibrand Saint-Oyant et al. [<a href="#B22-plants-13-01066" class="html-bibr">22</a>]. Two sequences located only on the genome of Raymond et al. [<a href="#B17-plants-13-01066" class="html-bibr">17</a>] are highlighted in red.</p>
Full article ">Figure 2
<p>A phylogenetic tree of <span class="html-italic">A. thaliana</span> and <span class="html-italic">R. chinensis</span> WRKY genes. The tree was inferred using the maximum likelihood method and JTT matrix-based mode. The tree with the highest log likelihood (−138,959.49) is shown. The percentage of trees in which the associated sequences clustered together is illustrated by the triangles on the branches. This analysis revealed 149 WRKY amino acid sequences, of which 81 were from <span class="html-italic">Arabidopsis</span> and 68 were from <span class="html-italic">R. chinensis</span>. There were a total of 2992 positions in the final dataset. Evolutionary analyses were conducted in MEGA X, and the graphic was created with iTOL. Three <span class="html-italic">R. chinensis</span> sequences are marked in red because they were mapped to Group I but had only one WRKY domain.</p>
Full article ">Figure 3
<p>The transcriptional changes (log2-fold change) of 27 <span class="html-italic">WRKY</span> genes in rose leaves infected with <span class="html-italic">D. rosae</span> were compared to those in mock-inoculated leaves at 24 and 72 h post inoculation (hpi). The susceptible genotype ‘Pariser Charme’ (PC) and the <span class="html-italic">D. rosae</span>-resistant genotype 91/100-5 were used. Only the 27 <span class="html-italic">WRKY</span> genes that were differentially expressed between the inoculated leaves and the mock-inoculated leaves are shown and the gene <span class="html-italic">RcWRKY37</span>, described in more detail later, is marked with an * (<span class="html-italic">n</span> = 3).</p>
Full article ">Figure 4
<p>Promoter analysis of the region 1500 bp upstream of each gene on the plus strand.</p>
Full article ">Figure 5
<p>Promoter analysis of the region 1500 bp upstream of each gene on the minus strand.</p>
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<p>Changes in the expression of <span class="html-italic">RcWRKY37</span> and defence-related genes in ‘Pariser Charme’ (PC) and PC::<span class="html-italic">Rdr1</span> [<a href="#B31-plants-13-01066" class="html-bibr">31</a>] leaves inoculated with <span class="html-italic">D. rosae</span>. Changes in expression were calculated for each time point relative to mock-inoculated leaves of the respective genotypes. PC is susceptible and PC::<span class="html-italic">Rdr1</span> is resistant to <span class="html-italic">D. rosae</span>. The following genes were analysed as additional defence-related genes: <span class="html-italic">EDS 1</span> &amp; <span class="html-italic">5</span>, <span class="html-italic">ERF113</span>, <span class="html-italic">PAD4</span>, <span class="html-italic">PR1</span>, <span class="html-italic">PR4</span>, and <span class="html-italic">PR6</span>. The samples were taken at 0, 24, 48, and 72 h post inoculation (hpi). Stars indicate significant differences between the two genotypes at the indicated points in time. The lines represent the fold change mean values, and the boxplots indicate the distribution of biological replicates (<span class="html-italic">n</span> = 3).</p>
Full article ">Figure 7
<p>Gene expression of various defence-related genes after transient 35s:<span class="html-italic">WRKY37</span> overexpression relative to PC petals infiltrated with an empty vector (GFP). The data were obtained from three independent infiltration experiments with three biological replicates each. The gene names indicate their function in pathways related to ethylene (ET with <span class="html-italic">EIN3</span> and <span class="html-italic">ERF1</span>), jasmonic acid (JA with <span class="html-italic">PDF1-2</span> and <span class="html-italic">MYC2</span>), or salicylic acid (SA with <span class="html-italic">EDS1</span>, <span class="html-italic">PAD4</span>, <span class="html-italic">PR1</span>, <span class="html-italic">PR4</span>, <span class="html-italic">PR6</span>, and <span class="html-italic">SAG101</span>).</p>
Full article ">Figure 8
<p>The expression levels of <span class="html-italic">RcPR1</span>, <span class="html-italic">RcPR4</span>, and <span class="html-italic">RcWRKY37</span> in PC leaves were measured in response to abiotic stress and compared to those in the control treatment. Significant changes in regulation with at least a 3-fold change are marked with an asterisk.</p>
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12 pages, 2674 KiB  
Article
Physiological and Transcriptomic Analyses Revealed That Humic Acids Improve Low-Temperature Stress Tolerance in Zucchini (Cucurbita pepo L.) Seedlings
by Haiping Li, Fanrong Kong, Tingting Tang, Yalan Luo, Haoran Gao, Jin Xu, Guoming Xing and Lingzhi Li
Plants 2023, 12(3), 548; https://doi.org/10.3390/plants12030548 - 25 Jan 2023
Cited by 6 | Viewed by 2067
Abstract
Zucchini (Cucurbita pepo L.) is one of the main vegetable crops grown under protected cultivation in northern China. Low-temperature (LT) stress severely inhibits the growth of zucchini seedlings, resulting in reductions in yield and quality. Here, using three kinds of different humic [...] Read more.
Zucchini (Cucurbita pepo L.) is one of the main vegetable crops grown under protected cultivation in northern China. Low-temperature (LT) stress severely inhibits the growth of zucchini seedlings, resulting in reductions in yield and quality. Here, using three kinds of different humic acids, including coal-based humic acid (CHA), fulvic acid (FA), and biochemical humic acid (BHA), we investigated the effects of humic acids against LT stress (5 °C) in zucchini seedlings. Treatment with all three kinds of humic acids improves LT stress tolerance by decreasing oxidative damage through increases in antioxidative enzyme activities and the contents of soluble sugar and proline in zucchini seedlings, especially after BHA application. Comparative transcriptomic analysis revealed that a total of 17 differentially expressed genes (DEGs) were commonly induced in the leaves of FA-, CHA-, and BHA-treated zucchini seedlings under LT stress, including calmodulin, ethylene-responsive transcription factors (TFs), peroxidases, and 10 TFs, including two NAC and seven WRKY genes. Altogether, these results indicated that supplementation with humic acids reprograms plant metabolism and modulates the expression of genes involved in ROS scavenging, phytohormone metabolism, or signaling pathways, finally improving LT stress tolerance in zucchini seedlings. Full article
(This article belongs to the Section Plant Response to Abiotic Stress and Climate Change)
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Figure 1

Figure 1
<p>Effects of humic acid treatment on the (<b>A)</b> reactive oxygen contents, (<b>B</b>) SOD activity, (<b>C</b>) POD activity, (<b>D</b>) soluble sugar contents, (<b>E</b>) proline contents, and (<b>F</b>) total chlorophyll contents in zucchini leaves under LT stress (<span class="html-italic">p</span> &lt; 0.05). LT, 5 °C. CHA, 5 °C + 0.05% coal-based humic acid. FA, 5 °C+ 0.05% fulvic acid. BHA, 5 °C + 0.05% biochemical humic acid. Error bars represented the ± SEs (<span class="html-italic">p</span> &lt; 0.05). Different letters indicate significantly different values at <span class="html-italic">p</span> &lt; 0.05, according to Tukey’s test.</p>
Full article ">Figure 2
<p>Transcriptomic analysis to determine the differentially expressed genes (DEGs) in the leaves of coal-based humic acid (CHA)-, fulvic acid (FA)-, and biochemical humic acid (BHA)-treated zucchini seedlings under LT stress. (<b>A</b>) Hierarchical clustering analysis of the transcriptome data. (<b>B</b>–<b>E</b>) Volcano map of DEGs. The scale represents log<sub>10</sub> (FPKM+1). (<b>B</b>) LT/CK comparison. (<b>C</b>) CHA/LT comparison. (<b>D</b>) FA/LT comparison. (<b>E</b>) BHA/LT comparison. The log<sub>2</sub>-fold change (FC) ≥ 1 or ≤ −1 and FDR &lt; 0.05. LT, 5 °C. CHA, 5 °C + 0.05% coal-based humic acid. FA, 5 °C + 0.05% fulvic acid. BHA, 5 °C + 0.05% biochemical humic acid.</p>
Full article ">Figure 3
<p>GO (<b>A</b>) and KEGG (<b>B</b>) pathway analyses. LT, 5 °C. CHA, 5 °C + 0.05% coal-based humic acid. FA, 5 °C+ 0.05% fulvic acid. BHA, 5 °C + 0.05% biochemical humic acid.</p>
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<p>Correlation analysis between the 14 common differentially expressed TFs in FA/LT, CHA/LT, and BHA/LT comparisons and the physiological indexes. CC, chlorophyll content. POD, peroxidase. SSC, soluble sugar contents. SOD, superoxide dismutase. PRO, proline. ROS, reactive oxygen species. MP, plasma membrane permeability.</p>
Full article ">Figure 5
<p>Weighted gene coexpression network analysis of DEGs in zucchini leaves. (<b>A</b>) Hierarchical clustering tree and heatmap of genes were constructed by correlation coefficients between genes. Different branches of the clustering tree represent different gene modules, and different colors represent different modules. (<b>B</b>) Correlation between the physio-biochemical indexes and each module. Module names are displayed on the Y-axis, and the physio-biochemical indexes are displayed on the X-axis. The depth of the color indicates the degree of correlation (red, positive correlation; blue, negative correlation). CC, chlorophyll content. PRO, proline. MP, membrane-lipid peroxidation damage. SSC, soluble sugar contents. (<b>C,D</b>) The circle diagram shows the predicted regulatory network for the yellow module (<b>C</b>) and blue module (<b>D</b>).</p>
Full article ">
15 pages, 8724 KiB  
Article
Importance of FaWRKY71 in Strawberry (Fragaria × ananassa) Fruit Ripening
by Maolan Yue, Leiyu Jiang, Nating Zhang, Lianxi Zhang, Yongqiang Liu, Yan Wang, Mengyao Li, Yuanxiu Lin, Yunting Zhang, Yong Zhang, Ya Luo, Xiaorong Wang, Qing Chen and Haoru Tang
Int. J. Mol. Sci. 2022, 23(20), 12483; https://doi.org/10.3390/ijms232012483 - 18 Oct 2022
Cited by 20 | Viewed by 2237
Abstract
WRKY transcription factors play a nonnegligible role in plant growth and development, but little is known about the involvement of WRKY transcription factors in the regulation of fruit ripening. In this study, FaWRKY71 was identified to be closely related to fruit maturation in [...] Read more.
WRKY transcription factors play a nonnegligible role in plant growth and development, but little is known about the involvement of WRKY transcription factors in the regulation of fruit ripening. In this study, FaWRKY71 was identified to be closely related to fruit maturation in octoploid strawberry. FaWRKY71 protein localized in the nucleus and responded to cold, salt, low phosphate, ABA, and light quality in strawberry seedlings. The temporal and spatial pattern expression analysis indicated that FaWRKY71 was expressed in all the detected tissues, especially in the full red fruits. In addition, FaWRKY71 gave rise to the accumulation of anthocyanin content by promoting the expression of structural genes FaF3’H, FaLAR, FaANR, and transport factors FaTT19 and FaTT12 in the flavonoid pathway, and softening the texture of strawberry via up-regulating the abundance of FaPG19 and FaPG21. Furthermore, FaWRKY71 was a positive regulator that mediated resistance against reactive oxygen species by enhancing the enzyme activities of SOD, POD, and CAT, reducing the amount of MDA. Altogether, this study provides new and comprehensive insight into the regulatory mechanisms facilitating fruit ripening in strawberry. Full article
(This article belongs to the Special Issue Advances in Research for Fruit Crop Breeding and Genetics)
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<p>Sequence analysis and subcellular localization of FaWRKY71. (<b>a</b>) Alignment of the WRKY motif and C2H2 zinc finger structure of FaWRKY71 proteins with it′ s homologous in other species. The black box indicates the WRKY domain and C2H2 motif. (<b>b</b>) Phylogenetic analysis of FaWRKY71. The bold font stands for FaWRKY71. FvWRKY71 (<span class="html-italic">Fragaria vesca</span>): XP_004303874.1; RcWRKY71 (<span class="html-italic">Rosa chinensis</span>): XP_024182610.1; PaWRKY71 (<span class="html-italic">Prunus avium</span>): XP_021811687.1; PdWRKY28-like (<span class="html-italic">Prunus dulcis</span>): XP_034208919.1; PpWRKY71 <span class="html-italic">(Prunus persica</span>): XP_007217821.1; CsWRKY28 (<span class="html-italic">Citrus sinensis</span>): KAH9770763.1; PtWRKY71 (<span class="html-italic">Populus trichocarpa</span>): XP_002306743.1; MdWRKY71 (<span class="html-italic">Malus domestica</span>): XP_008383508.2; CsWRKY71 (<span class="html-italic">Camellia sinensis</span>): XP_028100462.1; VvWRKY71 (<span class="html-italic">Vitis vinifera</span>): XP_002272089.1; AtWRKY71 (<span class="html-italic">Arabidopsis thaliana</span>): NP_174279.1. (<b>c</b>) Subcellular localization of FaWRKY71 protein. Scale bars represent 20 µm.</p>
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<p>Expression characteristics of <span class="html-italic">FaWRKY71</span>. (<b>a</b>) Diagrams about different tissues of ‘Benihoppe’. Scale bars represent 10 mm. (<b>b</b>) Spatial and temporal expression of <span class="html-italic">FaWRKY71</span> in different tissues and different developmental stages of strawberries. Multiple comparisons were tested using Turkey’s test and significant differences (<span class="html-italic">p</span> &lt; 0.05) were indicated by different lowercase letters. (<b>c</b>) Effects of different light qualities on <span class="html-italic">FaWRKY71</span> gene expression in strawberry full red fruits. W (White light); B (Blue light); W: B = 1:1 (White light: Blue light = 1:1); W: R = 1:1 (White light: Blue light = 1:1). (<b>d</b>) Effects of different light qualities on <span class="html-italic">FaWRKY71</span> gene expression in tissue culture seedlings of strawberry. Error bars were SEs for three replicates, and statistical significance was determined with the white light by Student’s t-test (**, <span class="html-italic">p</span> &lt; 0.01; *, <span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Expression patterns of <span class="html-italic">FaWRKY71</span> under different abiotic stresses. Multiple comparisons were tested using Turkey’s test and significant differences (<span class="html-italic">p</span> &lt; 0.05) were indicated by different lowercase letters.</p>
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<p><span class="html-italic">FaWRKY71</span> increased the accumulation of anthocyanin and lignin and contributed to fruit softening. (<b>a</b>) The phenotype of overexpressed <span class="html-italic">FaWRKY71</span> in ‘Xiaobai’ fruit. 35SN (empty vector) was as control. Scale bars represent 10 mm. (<b>b</b>) Relative expression level of <span class="html-italic">FaWRKY71</span> and anthocyanin content in it′ s overexpressed samples. Error bars were SEs for three replicates, and statistical significance was determined by Student’s t-test (**, <span class="html-italic">p</span> &lt; 0.01). (<b>c</b>) qRT-PCR analysis of key structural genes and transport factors in the flavonoid biosynthesis pathway. <span class="html-italic">Phenylalanine ammonia lyase</span> (<span class="html-italic">PAL</span>); <span class="html-italic">cinnamate 4-hydroxylase</span> (<span class="html-italic">C4H</span>); <span class="html-italic">chalcone synthase</span> (<span class="html-italic">CHS</span>); <span class="html-italic">flavonoid 3</span><span class="html-italic">′-hydroxylase</span> (<span class="html-italic">F3</span><span class="html-italic">′H</span>); <span class="html-italic">leucoanthocyanidin reductase</span> (<span class="html-italic">LAR</span>); <span class="html-italic">anthocyanidin reductase</span> (<span class="html-italic">ANR</span>); <span class="html-italic">TRANSPARENT TESTA 19</span> (<span class="html-italic">TT19</span>); <span class="html-italic">TRANSPARENT TESTA 12</span> (<span class="html-italic">TT12</span>); <span class="html-italic">H<sup>+</sup>-ATPase 10</span> (<span class="html-italic">AHA10</span>). (<b>d</b>) qRT-PCR analysis of key structural genes of lignin pathway. <span class="html-italic">shikimate O-hydroxycinnamoyl transferase</span> (<span class="html-italic">HCT</span>); <span class="html-italic">coumarate 3-hydroxylase</span> (<span class="html-italic">C3H</span>); <span class="html-italic">cinnamyl alcohol dehydrogenase</span> (<span class="html-italic">CAD</span>); <span class="html-italic">caffeoyl CoA O-methyl transferase</span> (<span class="html-italic">CCoAOMT</span>); <span class="html-italic">cinnamoyl CoA reductase</span> (<span class="html-italic">CCR</span>); <span class="html-italic">caffeic acid O-methyltransferase</span> (<span class="html-italic">COMT</span>); <span class="html-italic">ferulate 5-hydroxylase</span> (<span class="html-italic">F5H</span>); <span class="html-italic">peroxidase</span> (<span class="html-italic">POD</span>). (<b>e</b>) Safranin-stained section of strawberry fruit. The lignified cell walls were dyed red. (<b>f</b>–<b>h</b>) The expression levels of <span class="html-italic">ARF</span>, <span class="html-italic">NCED</span>, <span class="html-italic">PG</span>, and <span class="html-italic">PL</span> in <span class="html-italic">FaWRKY71</span> overexpressed samples.</p>
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<p>The activity of antioxidant enzymes in <span class="html-italic">FaWRKY71</span> overexpressed samples and Regulatory model of FaWRKY71 in strawberry. (<b>a</b>) Enzymes activity of SOD, POD, CAT and the content of MDA. Error bars were SEs for three replicates, and statistical significance was determined by Student’s t-test (**, <span class="html-italic">p</span> &lt; 0.01; *, <span class="html-italic">p</span> &lt; 0.05). (<b>b</b>). Relative expression of <span class="html-italic">MYBs</span> in <span class="html-italic">FaWRKY71</span> overexpressed samples. (<b>c</b>) A model indicating how FaWRKY71 regulates strawberry ripening.</p>
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26 pages, 7233 KiB  
Article
A Comprehensive Study of the WRKY Transcription Factor Family in Strawberry
by José Garrido-Gala, José-Javier Higuera, Antonio Rodríguez-Franco, Juan Muñoz-Blanco, Francisco Amil-Ruiz and José L. Caballero
Plants 2022, 11(12), 1585; https://doi.org/10.3390/plants11121585 - 15 Jun 2022
Cited by 8 | Viewed by 2184
Abstract
WRKY transcription factors play critical roles in plant growth and development or stress responses. Using up-to-date genomic data, a total of 64 and 257 WRKY genes have been identified in the diploid woodland strawberry, Fragaria vesca, and the more complex allo-octoploid commercial [...] Read more.
WRKY transcription factors play critical roles in plant growth and development or stress responses. Using up-to-date genomic data, a total of 64 and 257 WRKY genes have been identified in the diploid woodland strawberry, Fragaria vesca, and the more complex allo-octoploid commercial strawberry, Fragaria × ananassa cv. Camarosa, respectively. The completeness of the new genomes and annotations has enabled us to perform a more detailed evolutionary and functional study of the strawberry WRKY family members, particularly in the case of the cultivated hybrid, in which homoeologous and paralogous FaWRKY genes have been characterized. Analysis of the available expression profiles has revealed that many strawberry WRKY genes show preferential or tissue-specific expression. Furthermore, significant differential expression of several FaWRKY genes has been clearly detected in fruit receptacles and achenes during the ripening process and pathogen challenged, supporting a precise functional role of these strawberry genes in such processes. Further, an extensive analysis of predicted development, stress and hormone-responsive cis-acting elements in the strawberry WRKY family is shown. Our results provide a deeper and more comprehensive knowledge of the WRKY gene family in strawberry. Full article
(This article belongs to the Section Plant Genetics, Genomics and Biotechnology)
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<p>Protein domains (PFAM v32.0) found in FvWRKY proteins. Only the largest splice forms are represented. See <a href="#app1-plants-11-01585" class="html-app">Table S2</a> for more details.</p>
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<p>Chromosome mapping of the <span class="html-italic">Fragaria vesca WRKY</span> gene family. Segmental duplicated gene pairs (syntenic paralogs) share the same colors. Tandemly duplicated gene clusters are colored in pink, and underlined genes are collinear with genes outside that syntenic block. Ruler size is in Megabases.</p>
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<p>GEvo analyses comparing microsynteny between genomic regions from <span class="html-italic">A. thaliana</span> and <span class="html-italic">F. vesca</span>. (<b>A</b>) Genomic regions containing the genes <span class="html-italic">FvWRKY60</span>, <span class="html-italic">FvWRKY61</span> and <span class="html-italic">AtWRKY70</span> with red blocks and connectors show high-scoring sequence pairs between both sequences. The region shown in B is framed. (<b>B</b>) Homology details among <span class="html-italic">FvWRKY60</span>, <span class="html-italic">FvWRKY61</span> (black arrows) and <span class="html-italic">AtWRKY70</span>. A short, non-<span class="html-italic">WRKY</span> gene (<span class="html-italic">FvH4_7g26100</span>, red arrow) was identified as part of the <span class="html-italic">FvWRKY59-60-61</span> tandem (see text for details).</p>
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<p>Chromosome mapping of the <span class="html-italic">Fragaria</span> × <span class="html-italic">ananassa WRKY</span> gene family within each subgenome. Segmental duplicated gene pairs (syntenic paralogs) share the same colors. Non-syntenic gene duplicates are underlined. Tandemly duplicated gene clusters are colored in orange. Ruler size is in Megabases.</p>
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<p>Phylogenetic analysis of <span class="html-italic">Fragaria vesca</span> (Fv), <span class="html-italic">Arabidopsis thaliana</span> (At) and <span class="html-italic">Vitis vinifera</span> (Vv) WRKY proteins. WRKY proteins are clustered into Groups I + IIc, IIa + IIb, IId + IIe and III. R protein-WRKY from Fv and At are clustered within their respective groups. The tree was inferred using the Neighbor-Joining method (1000 bootstrap replicates) and drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. All positions with less than 95% site coverage were eliminated. Connecting lines represent shared synteny between Fv-Vv (red) and Fv-At (blue).</p>
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<p>GO functional annotation plot comparing the number and percentage of FvWRKY and FaWRKY proteins sharing the same functions. The <span class="html-italic">y</span>-axis represents the number (right) and the percentage (left) of WRKY genes in <span class="html-italic">Fragaria</span> × <span class="html-italic">ananassa</span> (green) and in <span class="html-italic">Fragaria vesca</span> (blue) for each GO category (<span class="html-italic">x</span>-axis).</p>
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<p>Phylogenetic tree of the <span class="html-italic">AtWRKY55</span> and <span class="html-italic">AtWRKY54/70</span> orthologous genes (red and blue branches, respectively) in strawberry (Fv), soybean (Glyma), walnut (WALNUT), apple (MD), grapevine (Vv) and <span class="html-italic">Amborella</span> (<span class="html-italic">AmTr</span>). Strawberry orthologs to <span class="html-italic">AtWRKY54/70</span> paralogs underwent tandem duplications after the gamma hexaploidization and independently to the more recent WGD events in other species. The evolutionary history was inferred using the Maximum Likelihood method (100 bootstraps) with optimized parameters (TN + G + I). The tree is drawn to scale, with lengths of branches representing the number of substitutions per site. Analyses were conducted in MEGA7.</p>
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<p>Phylogenetic tree of the <span class="html-italic">FvWRKY</span> family showing expression values during fruit ripening (Ruegen receptacle tissue at 15 and at 22 DPA and Yellow Wonder receptacle tissue at 15 and at 22 DPA: Yw-15D, Yw-22D, Rg-15D and Rg-22D, respectively) and roots (Root, collected from 7-week-old plants grown in aerated hydroponic culture, and Root_P, after 2 days of inoculation with <span class="html-italic">Phytophthora cactorum</span>). <span class="html-italic">FvWRKY</span> paralogs are shown in red (see <a href="#plants-11-01585-t001" class="html-table">Table 1</a>. Color scale represents the expression level as log-transformed TPM (Transcripts per Million) values.</p>
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<p>(<b>A</b>) Heatmap of differentially expressed <span class="html-italic">FaWRKY</span> genes during strawberry fruit ripening stages in receptacle (White_R, Turning_R, and Red_R; left side) and achene (White_A, Turning_A, and Red_A; right side) tissues. Changes in gene expression, with respect to green fruit tissues, are represented as log2 fold change if absolute values were &gt;1 (padj &lt; 0.01), otherwise, they were colored in grey. (<b>B</b>) Venn diagram showing those genes which were differentially expressed in receptacle and achene only, or in both.</p>
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<p>Heatmap of differentially expressed <span class="html-italic">FaWRKY</span> genes in response to <span class="html-italic">C. fructicola</span> infection in leaves. Changes of gene expression in infected strawberry (Inf_24h, Inf_72h, Inf_96h; 24, 72 and 96 h after spore inoculation, respectively) vs. mock-inoculated strawberry are represented as log2 fold change if absolute values were &gt;1 (padj &lt; 0.01), in a color scale from lowest (blue) to highest (red). Otherwise, they were colored grey.</p>
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19 pages, 3890 KiB  
Article
Impact of Chitosan, Sucrose, Glucose, and Fructose on the Postharvest Decay, Quality, Enzyme Activity, and Defense-Related Gene Expression of Strawberries
by Yuanhua Wang, Zhiming Yan, Weihua Tang, Qing Zhang, Bei Lu, Qiong Li and Geng Zhang
Horticulturae 2021, 7(12), 518; https://doi.org/10.3390/horticulturae7120518 - 24 Nov 2021
Cited by 10 | Viewed by 4228
Abstract
Strawberry is one of the most highly consumed fruits worldwide, but is extremely perishable. This study systematically compared the effects of chitosan, sucrose, glucose, and fructose immersion on the physiology and disease development in harvested strawberries. After storage at 15 °C for 9 [...] Read more.
Strawberry is one of the most highly consumed fruits worldwide, but is extremely perishable. This study systematically compared the effects of chitosan, sucrose, glucose, and fructose immersion on the physiology and disease development in harvested strawberries. After storage at 15 °C for 9 days, all sugar treatment groups had significantly higher total soluble solids and total anthocyanin content than those of the control group. All sugar treatment groups inhibited malondialdehyde accumulation. At the end of the storage, chitosan, glucose, and fructose maintained higher superoxide dismutase activity and chitosan maintained higher catalase activity. The chitosan and glucose groups had lowest fruit decay index, followed by sucrose and fructose groups. The fruit firmness and luster were better maintained in the glucose group. Furthermore, genes related to sucrose metabolism (e.g., FaSUS1 and FaSUS2), titratable acidity accumulation (e.g., FaMDH1, FaMDH2, FaCS1, and FaCS2), disease resistance (e.g., FaPGIP1, FaWRKY1, and FaWRKY33) and to anabolic jasmonic acid and abscisic acid pathways (e.g., FaJAZ1, FaJAZ2, FaOPR3, FaNCED1, and FaNCED2) were regulated to varying degrees, suggesting that chitosan and glucose participate in plants’ immune signaling networks and regulate disease resistance in fruit through hormone pathways. The findings provide new insights into the physiological regulation of harvested strawberries. Full article
(This article belongs to the Section Postharvest Biology, Quality, Safety, and Technology)
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<p>Experimental process flow chart. TSS, total soluble solids; TA, titratable acidity; TAC, total anthocyanin content; MDA, malondialdehyde; SOD, superoxide dismutase; CAT, catalase.</p>
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<p>Incidence grade and descriptor for strawberry (<span class="html-italic">Fragaria × ananassa</span> Duch.) fruit.</p>
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<p>Effects of different sugar treatments on the (<b>A</b>) visual quality and (<b>B</b>) decay index of strawberry fruits stored at 15 ± 2 °C for 11 d. Data are mean of three replicates. Different letters on top of bars in the same storage time indicate a significant difference (Tukey’s HSD test, <span class="html-italic">p</span> &lt; 0.05). CK represents that strawberry fruits were immersed with water before storage; 1, 3, 5, 7 g/L CTS represents that strawberry fruits were immersed with 1, 3, 5, 7 g/L chitosan before storage, respectively; 1, 3, 5, 7 g/L Suc represents that strawberry fruits were immersed with 1, 3, 5, 7 g/L sucrose before storage, respectively; 5, 10, 15, 20 g/L Glc represents that strawberry fruits were immersed with 5, 10, 15, 20 g/L glucose before storage, respectively; 5, 10, 15, 20 g/L Fru represents that strawberry fruits were immersed with 5, 10, 15, 20 g/L fructose before storage, respectively; the same below.</p>
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<p>Effects of chitosan (3 g/L), sucrose (5 g/L), glucose (15 g/L), and fructose (15 g/L) on the (<b>A</b>) decay index and (<b>B</b>) commodity rate of strawberry fruits stored at 15 ± 2 °C for 9 d. CK represents that strawberry fruits were immersed with water before storage. Data are mean of three replicates. Different letters on top of bars in the same storage time indicate a significant difference (Tukey’s HSD test, <span class="html-italic">p</span> &lt; 0.05). CTS, chitosan; Suc, sucrose; Glc, glucose; Fru, fructose.</p>
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<p>Sensory evaluation of fresh strawberry fruits at the 0th day and strawberry fruits treated with 3 g/L chitosan, 5 g/L sucrose, 15 g/L glucose, and 15 g/L fructose, respectively, at the 9th day, stored at 15 ± 2 °C. CK represents that strawberry fruits were immersed with water before storage. The information about sensory indicator and standard for evaluation can be found in <a href="#app1-horticulturae-07-00518" class="html-app">supplementary Table S1</a>. Data are mean of scores given by twenty panelists. CTS, chitosan; Suc, sucrose; Glc, glucose; Fru, fructose.</p>
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<p>Effects of chitosan (3 g/L), sucrose (5 g/L), glucose (15 g/L), and fructose (15 g/L) on the (<b>A</b>) titratable acid, (<b>B</b>) total soluble solids, and (<b>C</b>) total anthocyanin content of strawberry fruits stored at 15 ± 2 °C for 9 d. CK represents that strawberry fruits were immersed with water before storage. Data are mean of three replicates. Different letters in the same storage time indicate a significant difference (Tukey’s HSD test, <span class="html-italic">p</span> &lt; 0.05). CTS, chitosan; Suc, sucrose; Glc, glucose; Fru, fructose.</p>
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<p>Effects of chitosan (3 g/L), sucrose (5 g/L), glucose (15 g/L), and fructose (15 g/L) on the (<b>A</b>) malondialdehyde content, (<b>B</b>) SOD activity, and (<b>C</b>) CAT activity of strawberry fruits stored at 15 ± 2 °C for 9 d. CK represents that strawberry fruits were immersed with water before storage. Data are mean of three replicates. Different letters in the same storage time indicate a significant difference (Tukey’s HSD test, <span class="html-italic">p</span> &lt; 0.05). CTS, chitosan; Suc, sucrose; Glc, glucose; Fru, fructose; SOD, superoxide dismutase; CAT, catalase.</p>
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<p>Effects of chitosan (3 g/L), sucrose (5 g/L), glucose (15 g/L), and fructose (15 g/L) on transcription of genes related to fruit quality and disease resistance. The fruit treated with distilled water was used as the control (CK), relative gene expressions in strawberries treated with 3 g/L chitosan, 5 g/L sucrose, 15 g/L glucose, and 15 g/L fructose were calculated. The expressions in the chitosan, sucrose, glucose, and fructose groups were then each divided by that in the control, and the results were used to create a heat map. CTS, chitosan; Suc, sucrose; Glc, glucose; Fru, fructose.</p>
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14 pages, 4565 KiB  
Article
A Transcriptomic Analysis of Gene Expression in Chieh-Qua in Response to Fusaric Acid Stress
by Min Wang, Wenrui Liu, Jinqiang Yan, Piaoyun Sun, Feng Chen, Biao Jiang, Dasen Xie, Yu’e Lin, Qingwu Peng and Xiaoming He
Horticulturae 2021, 7(4), 88; https://doi.org/10.3390/horticulturae7040088 - 19 Apr 2021
Cited by 3 | Viewed by 2686
Abstract
Fusarium wilt results in undesirable effects on the quality and production of chieh-qua (Benincasa hispida Cogn. var. Chieh-qua How). Fusaric acid (FA), a secondary metabolite of biotin produced by pathogens of genus Fusarium, induced resistant responses in chieh-qua; however, the physiological and [...] Read more.
Fusarium wilt results in undesirable effects on the quality and production of chieh-qua (Benincasa hispida Cogn. var. Chieh-qua How). Fusaric acid (FA), a secondary metabolite of biotin produced by pathogens of genus Fusarium, induced resistant responses in chieh-qua; however, the physiological and molecular mechanism(s) of FA resistance remains largely unknown. In our study, ‘A39’ (FA-resistant cultivar) exhibited decreased malondialdehyde (MDA) content and increased superoxide dismutase (SOD) enzyme activity when exposed to FA compared with ‘H5’ (FA-susceptible cultivar). More apoptosis cells existed in ‘H5’ than ‘A39’ after 2 days of FA treatment. RNA-seq results revealed that a total of 2968 and 3931 differentially expressed genes (DEGs) were detected under normal conditions (1562 up-regulated and 1406 down-regulated) and FA treatment (2243 up-regulated and 1688 down-regulated), respectively. Interestingly, DEGs associated with pathogen-related protein and ethylene (ET) biosynthesis and signal pathways were most significantly changed during FA stress. Notably, several crucial genes encoding pathogenesis-related protein (CL4451.Contig2, CL2175.Contig4), peroxidase (Unigene49615 and CL11695.Contig2), and ET-responsive transcription factors (TFs) (CL9320.Contig1, CL9849.Contig3, CL6826.Contig2, CL919. Contig6, and CL518.Contig7) were specifically induced after FA treatment. Collectively, the study provides molecular data for isolating candidate genes involved in FA resistance, especially ET related genes in chieh-qua. Full article
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<p>Phenotype observation of ‘A39’ and ‘H5’ under FA stress. (<b>A</b>) Seedlings of ‘A39’ and ‘H5’ under normal condition. (<b>B</b>,<b>C</b>) Seedlings of ‘A39’ and ‘H5’ at the second day after FA stress. (<b>D</b>,<b>E</b>) MDA content and SOD activity before treatment and after 2 days of FA stress. Data is presented as the mean ± standard deviation (n = 9). ** Student’s <span class="html-italic">t</span>-test at <span class="html-italic">p</span> &lt; 0.01.</p>
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<p>TUNEL assay of ‘A39’ and ‘H5’ from seedling leaves. (<b>A</b>) Apoptosis detection of the control plant under normal condition. Bars = 200 μm. (<b>B</b>) ‘A39’ and (<b>C</b>) ‘H5’ after 1 day of FA treatment. Bars = 200 μm. (<b>D</b>) ‘A39’ and (<b>E</b>) ‘H5’ after 2 days of FA treatment. Bars =400 μm. The Green Fluorescence indicates the signal for apoptosis. The Merged represents the combination of PI staining and Green Fluorescence.</p>
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<p>Principal components analysis (PCA) of ‘A39’ and ‘H5’ (n = 12). (<b>A</b>) Two-dimensional chart produced with the clean data from the samples. (<b>B</b>) Three-dimensional chart produced with the clean data from the samples.</p>
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<p>DEGs comparison in leaves of ‘A39’ and ‘H5’ under normal conditions (<b>A</b>) and 2 days of FA stress (<b>B</b>) Red, green, and blue correspond to up-regulated, unaltered, and down-regulated gene expression, respectively.</p>
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<p>The top 20 significantly enriched KEGG pathways in the seedlings. (<b>A</b>) Normal conditions: ‘A39’_N-vs. ‘H5’_N. (<b>B</b>) FA stress: ‘A39’_F-vs. ‘H5’_F. The y-axis represents different KEGG pathways and the x-axis represents enrichment factors. Larger circles indicate more DEGs in the pathway and higher Q-value is indicated by green color.</p>
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<p>Relative expression of genes related to pathogen-related protein. Data is presented as the mean ± standard deviation (n = 9). Student’s <span class="html-italic">t</span> test: ** <span class="html-italic">p</span> ≤ 0.01.</p>
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<p>Relative expression of genes related to plant hormones. Data is presented as the mean ± standard deviation (n = 9). Student’s <span class="html-italic">t</span> test: ** <span class="html-italic">p</span> ≤ 0.01.</p>
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<p>A model for the mechanism of FA resistance in chieh-qua. Under FA stress, ‘A39’ exhibited enhanced resistance compared with ‘H5’ due to induction of genes encoding pathogen-related protein and ET biosynthesis and signal transduction. Also, increased expression of peroxidase and SOD activity would improve ROS scavenging ability, reducing stress effects.</p>
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17 pages, 2504 KiB  
Article
Strawberry FaWRKY25 Transcription Factor Negatively Regulated the Resistance of Strawberry Fruits to Botrytis cinerea
by Sizhen Jia, Yuanhua Wang, Geng Zhang, Zhiming Yan and Qingsheng Cai
Genes 2021, 12(1), 56; https://doi.org/10.3390/genes12010056 - 31 Dec 2020
Cited by 19 | Viewed by 3415
Abstract
WRKY genes and jasmonic acid (JA) play a crucial role in plants’ responses against biotic and abiotic stress. However, the regulating mechanism of WRKY genes on strawberry fruits’ resistance against Botrytis cinerea is largely unknown, and few studies have been performed on their [...] Read more.
WRKY genes and jasmonic acid (JA) play a crucial role in plants’ responses against biotic and abiotic stress. However, the regulating mechanism of WRKY genes on strawberry fruits’ resistance against Botrytis cinerea is largely unknown, and few studies have been performed on their effect on the JA-mediated defense mechanism against B. cinerea. This study explored the effect of FaWRKY25 on the JA-mediated strawberry resistance against B. cinerea. Results showed that the JA content decreased significantly as the fruits matured, whereas the FaWRKY25 expression rose substantially, which led to heightened susceptibility to B. cinerea and in strawberries. External JA treatment significantly increased the JA content in strawberries and reduced the FaWRKY25 expression, thereby enhancing the fruits’ resistance against B. cinerea. FaWRKY25 overexpression significantly lowered the fruits’ resistance against B. cinerea, whereas FaWRKY25 silencing significantly increased resistance. Moreover, FaWRKY25 overexpression significantly lowered the JA content, whereas FaWRKY25 silencing significantly increased it. FaWRKY25 expression level substantially affects the expression levels of genes related to JA biosynthesis and metabolism, other members of the WRKY family, and defense genes. Accordingly, FaWRKY25 plays a crucial role in regulating strawberries’ resistance against B. cinerea and may negatively regulate their JA-mediated resistance mechanism against B. cinerea. Full article
(This article belongs to the Section Plant Genetics and Genomics)
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Figure 1

Figure 1
<p>Phylogenetic analysis of WRKY25 protein. (<b>A</b>) Phylogenic analysis of WRKY25 homologs from various species. Species name abbreviations are follows: At, <span class="html-italic">Arabidopsis</span>; Br, <span class="html-italic">Brassica rapa</span>; Rs, <span class="html-italic">Raphanus sativus</span>; Bn, <span class="html-italic">Brassica napus</span>; Gh, <span class="html-italic">Gossypium hirsutum</span>; Gm, <span class="html-italic">Glycine max</span>; Cs, <span class="html-italic">Camelina sativa</span>; Zm, <span class="html-italic">Zea mays</span>; Ls, <span class="html-italic">Lactuca sativa</span>; Ca, <span class="html-italic">Capsicum annuum</span>; Cp, <span class="html-italic">Carica papaya</span>; Ptr, <span class="html-italic">Populus trichocarpa</span>. (<b>B</b>) Sequence alignment of the deduced amino acid sequences of WRKY25. The gene accession numbers are found in <a href="#app1-genes-12-00056" class="html-app">Table S2</a>.</p>
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<p>Variation of <span class="html-italic">FaWRKY25</span> gene expression and jasmonic acid (JA) content in strawberry fruits. (<b>A</b>) Phenotypes showing different developmental stages of strawberry fruit: SG (15–20 days after flowering), MG (20–25 days after flowering), BG (25–30 days after flowering), Wh (30–35 days after flowering), Tu (35–38 days after flowering), HF (38–40 days after flowering), and Re (40–45 days after flowering). (<b>B</b>) Changes in jasmonic acid levels throughout fruit development. (<b>C</b>) Changes in <span class="html-italic">FaWRKY25</span> gene expression levels throughout fruit development. (<b>D</b>) <span class="html-italic">FaWRKY25</span> relative expression in red fruit under different treatments. (<b>E</b>) Phenotypic effects of JA on red fruits after <span class="html-italic">Botrytis cinerea</span> inoculation. Control, fruits treated with sterile water; <span class="html-italic">B. cinerea</span>, fruits inoculated with <span class="html-italic">Botrytis cinerea</span>; JA, fruits treated with 250 µM JA; JA+<span class="html-italic">B. cinerea</span>, fruits inoculated with <span class="html-italic">Botrytis cinerea</span> after being treated with 250 µM JA. As for MeJA treatments, the fruits were inoculated with <span class="html-italic">B. cinerea</span> right after being treated with different concentrations of MeJA for 2 h. The experiment was repeated three times, and 30 fruits were used in each batch for each treatment.</p>
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<p>Effect of <span class="html-italic">FaWRKY25</span>-OE (-overexpression) and -RNAi (-ribonucleic acid interference) on the levels of <span class="html-italic">FaWRKY25</span> transcript and JA. (<b>A</b>) Expression of green fluorescent protein (eGFP) after <span class="html-italic">Agrobacterium tumefaciens</span> injection. (<b>B</b>) Phenotypes of <span class="html-italic">FaWRKY25</span>-OE and <span class="html-italic">FaWRKY25</span>-RNAi strawberry fruits after <span class="html-italic">Agrobacterium tumefaciens</span> injection and <span class="html-italic">Botrytis cinerea</span> inoculation. DABI denotes days after <span class="html-italic">Botrytis cinerea</span> inoculation, and DAI denotes days after <span class="html-italic">Agrobacterium tumefaciens</span> injection. (<b>C</b>) Effect of <span class="html-italic">FaWRKY25</span>-OE and -RNAi on <span class="html-italic">FaWRKY25</span> relative expression 5 days after <span class="html-italic">Agrobacterium tumefaciens</span> injection (DAI-5). (<b>D</b>) Effect of <span class="html-italic">FaWRKY25</span>-OE and -RNAi on jasmonic acid levels 5 days after <span class="html-italic">Agrobacterium tumefaciens</span> injection (DAI-5). (<b>E</b>) Comparison of the incidence rate in <span class="html-italic">FaWRKY25</span>-OE and <span class="html-italic">FaWRKY25</span>-RNAi strawberry fruits after <span class="html-italic">Botrytis cinerea</span> inoculation at DABI-6. Values are means ± SD of three biological replicates. Asterisks above the columns denote a significant difference at * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01 levels according to Student’s <span class="html-italic">t</span>-test.</p>
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<p>Effects of <span class="html-italic">FaWRKY25</span>-OE and -RNAi on transcription of resistance-related genes. OE and RNAi denote overexpression and silencing expression of the <span class="html-italic">FaWRKY25</span> gene. With the wild strawberries without bacterial injection as the control group, the relative gene expression in the empty vectors (EV-OE or EV-As), <span class="html-italic">FaWRKY25</span>-OE, and <span class="html-italic">FaWRKY25</span>-RNAi groups was calculated. The expression in the <span class="html-italic">FaWRKY25</span>-OE and <span class="html-italic">FaWRKY25</span>-RNAi groups was than divided by that in the empty vector group, and the results were used to create a heat map.</p>
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