Genome encode analyses reveal the basis of convergent evolution of fleshy fruit ripening

Altres ajuts: Generalitat de Catalunya/CERCA ProgrammeFleshy fruits using ethylene to regulate ripening have developed multiple times in the history of angiosperms, presenting a clear case of convergent evolution whose molecular basis remains largely unknown. Analysis of the fruitENCODE data consisting of 361 transcriptome, 71 accessible chromatin, 147 histone and 45 DNA methylation profiles reveals three types of transcriptional feedback circuits controlling ethylene-dependent fruit ripening. These circuits are evolved from senescence or floral organ identity pathways in the ancestral angiosperms either by neofunctionalisation or repurposing pre-existing genes. The epigenome, H3K27me3 in particular, has played a conserved role in restricting ripening genes and their orthologues in dry and ethylene-independent fleshy fruits. Our findings suggest that evolution of ripening is constrained by limited hormone molecules and genetic and epigenetic materials, and whole-genome duplications have provided opportunities for plants to successfully circumvent these limitations

Genome-Scale Computational Identification and Characterization of UTR Introns in <i>Atalantia buxifolia</i>

Accumulated evidence has shown that CDS introns (CIs) play important roles in regulating gene expression. However, research on UTR introns (UIs) is limited. In this study, UIs (including 5′UTR and 3′UTR introns (5UIs and 3UIs)) were identified from the Atalantia buxifolia genome. The length and nucleotide distribution characteristics of both 5UIs and 3UIs and the distributions of cis-acting elements and transcription factor binding sites (TFBSs) in 5UIs were investigated. Moreover, PageMan enrichment analysis was applied to show the possible roles of transcripts containing UIs (UI-Ts). In total, 1077 5UIs and 866 3UIs were identified from 897 5UI-Ts and 670 3UI-Ts, respectively. Among them, 765 (85.28%) 5UI-Ts and 527 (78.66%) 3UI-Ts contained only one UI, and 94 (6.38%) UI-Ts contained both 5UI and 3UI. The UI density was lower than that of CDS introns, but their mean and median intron sizes were ~2 times those of the CDS introns. The A. buxifolia 5UIs were rich in gene-expression-enhancement-related elements and contained many TFBSs for BBR-BPC, MIKC_MADS, AP2 and Dof TFs, indicating that 5UIs play a role in regulating or enhancing the expression of downstream genes. Enrichment analysis revealed that UI-Ts involved in ‘not assigned’ and ‘RNA’ pathways were significantly enriched. Noteworthily, 119 (85.61%) of the 3UI-Ts were genes encoding pentatricopeptide (PPR) repeat-containing proteins. These results will be helpful for the future study of the regulatory roles of UIs in A. buxifolia

A New Leaf Blight Disease Caused by <i>Alternaria jacinthicola</i> on Banana in China

A leaf blight disease with an incidence level of about 50% was found on Robusta banana in Guangdong province of China in September 2020. The early symptom appeared as pale gray to black brown, irregular, small, necrotic lesions mainly on the top 3–5 leaves. Severely infected leaves were withered and necrotic. Two representative fungus strains, strain L1 and strain L2, were isolated from affected banana leaves, and morphological and molecular identification analysis confirmed that the two fungi were both Alternaria jacinthicola. Many Alternaria species have been reported to cause wilting, decay, leaf blight and leaf spots on plants and lead to serious economic losses in their production, including A. alternata, causing leaf blight and leaf sport diseases on banana. The Koch’s postulates of A. jacinthicola causing the leaf blight disease was further fulfilled, which confirmed that it is the causal agent of this disease. To our knowledge, this is the first report of A. jacinthicola causing leaf blight on banana in China

Effect of ABA concentration on seed germination and root growth in WT and <i>RhNAC3</i> overexpressing <i>A. thaliana</i> plants.

<p><b>A,</b> Seed germination phenotypes. The homozygous T3 seeds of <i>RhNAC3</i>-overexpressing lines (OE#3, OE#6 and OE#12), wild type (WT) and vector plants were plated on MS supplemented with 0, 0.2 or 0.4 µM ABA. Images were obtained 7 days after planting. <b>B,</b> Seed germination rates. The germination rates were measured 7 days after planting. Error bars represent standard error (<i>n</i> = 3). **: <i>P</i><0.01, *: <i>P</i><0.05, <i>t</i> test. <b>C,</b> Root growth phenotypes. Five-day-old seedlings of WT, vector only and three <i>RhNAC3</i>-overexpressing <i>A. thaliana</i> lines (OE#3, #6 and #12) were transferred to MS plates supplemented with 0, 5, 10 and 30 µM ABA. Root phenotypes were visualized 10 days after planting. <b>D,</b> Primary root length analysis. **: P<0.01, *: P<0.05, <i>t</i> test. <b>E,</b> Lateral root number analysis. Both primary root length and lateral root number were measured after 10 days of growth. Three independent experiments were performed using 15 plants in each experiment in D and E. Error bars represent standard error (<i>n</i> = 3).</p

Stomatal aperture of the <i>RhNAC3</i> overexpressing <i>A. thaliana</i> plants in response to ABA and drought treatments.

<p><b>A,</b> Stomatal aperture in response to ABA. Mature leaves from three-week old wild type (WT), vector control and independent <i>RhNAC3</i>-overexpressing plants (OE#3, OE#6 and OE#12) were treated with a stomatal opening solution for 2 h (0 µM) and incubated with 10 µM ABA for 2 h (10 µM). Stomata on the abaxial surfaces were imaged by light microscopy. Stomatal aperture (the ratio of width to length) was quantified using at least 20 guard cells from each sample. <i>Bar</i> 10 µm. <b>B,</b> Stomatal aperture of <i>RhNAC3</i> overexpressing lines in response to drought stress. Three-week old seedlings of WT, vector control and independent <i>RhNAC3</i>-overexpressing plants (OE#3, OE#6 and OE#12) were subjected to 10 days without water. Plants grown under normal well watered conditions were used as a control. The leaves were harvested and the stomata on the leaf abaxial surfaces were immediately photographed. Stomatal apertures were then quantified (n = 20). <i>Bar</i> 10 µm. **: <i>P</i><0.01, *: <i>P</i><0.05, <i>t</i> test.</p

ABA-related gene expression in <i>RhNAC3</i>-silenced rose petals.

<p><b>A</b>, The putative ABA signaling and downstream rose genes from the ABA-signaling pathway in rose. a, The clone ID from the rose transcriptome database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109415#pone.0109415-Dai1" target="_blank">[7]</a>. b, Description of the <i>A. thaliana</i> homolog given by The Arabidopsis Information Resource (TAIR, <a href="http://www.arabidopsis.org" target="_blank">http://www.arabidopsis.org</a>). <b>B</b>, qRT-PCR analysis of <i>RhNAC3</i>-silenced rose petals. The rose cDNAs of TRV and <i>RhNAC3</i>-silenced (TRV-<i>RhNAC3</i>) petals were described in our previous report <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109415#pone.0109415-Jiang1" target="_blank">[26]</a>. Data represent the fold change of each gene by TRV-<i>RhNAC3</i> relative to the TRV control. <i>RhUbi1</i> was used as the internal control. Error bars indicate SE (<i>n</i> = 3). <b>C</b>, Sequences and positions of putative RhNAC3 binding elements used for the EMSA. Probes were derived from the regulatory sequence of three selected ABA-related rose genes. Underlined letters indicate the core sequences of putative NAC protein-binding sites. The sense strands of oligonucleotide probes corresponding to the predicted RhNAC3 binding sites are shown. <b>D</b>, DNA-binding specificity for RhNAC3 with the probes indicated in <b>C</b>. The arrows indicate the positions of protein/DNA complexes and the free probes, respectively. Purified protein (2 µg) was incubated with 0.2 pmol of biotin probe. <b>E</b>, DNA-binding specificity for RhNAC3 with <i>RU03861</i>. The <i>RU03861</i> (P3) probe incubated with GST was used as a control, and a 10, 100, and 1000 fold excess of the unlabeled P3 was used for competitive binding.</p

Analysis of putative NAC binding <i>cis</i>-elements in the promoter regions of genes downstream from <i>RhNAC3</i> action.

<p>Analysis of putative NAC binding <i>cis</i>-elements in the promoter regions of genes downstream from <i>RhNAC3</i> action.</p

The Rose (<i>Rosa hybrida</i>) NAC Transcription Factor 3 Gene, <i>RhNAC3</i>, Involved in ABA Signaling Pathway Both in Rose and <i>Arabidopsis</i>

<div><p>Plant transcription factors involved in stress responses are generally classified by their involvement in either the abscisic acid (ABA)-dependent or the ABA-independent regulatory pathways. A stress-associated NAC gene from rose (<i>Rosa hybrida</i>), <i>RhNAC3</i>, was previously found to increase dehydration tolerance in both rose and <i>Arabidopsis</i>. However, the regulatory mechanism involved in RhNAC3 action is still not fully understood. In this study, we isolated and analyzed the upstream regulatory sequence of <i>RhNAC3</i> and found many stress-related <i>cis</i>-elements to be present in the promoter, with five ABA-responsive element (ABRE) motifs being of particular interest. Characterization of <i>Arabidopsis thaliana</i> plants transformed with the putative <i>RhNAC3</i> promoter sequence fused to the β-glucuronidase (GUS) reporter gene revealed that <i>RhNAC3</i> is expressed at high basal levels in leaf guard cells and in vascular tissues. Moreover, the ABRE motifs in the <i>RhNAC3</i> promoter were observed to have a cumulative effect on the transcriptional activity of this gene both in the presence and absence of exogenous ABA. Overexpression of <i>RhNAC3</i> in <i>A. thaliana</i> resulted in ABA hypersensitivity during seed germination and promoted leaf closure after ABA or drought treatments. Additionally, the expression of 11 ABA-responsive genes was induced to a greater degree by dehydration in the transgenic plants overexpressing <i>RhNAC3</i> than control lines transformed with the vector alone. Further analysis revealed that all these genes contain NAC binding <i>cis</i>-elements in their promoter regions, and RhNAC3 was found to partially bind to these putative NAC recognition sites. We further found that of 219 <i>A. thaliana</i> genes previously shown by microarray analysis to be regulated by heterologous overexpression <i>RhNAC3,</i> 85 are responsive to ABA. In rose, the expression of genes downstream of the ABA-signaling pathways was also repressed in <i>RhNAC3</i>-silenced petals. Taken together, we propose that the rose RhNAC3 protein could mediate ABA signaling both in rose and in <i>A. thaliana</i>.</p></div

Deletion analysis and ABA dose dependent response of <i>RhNAC3</i> promoter activity.

<p><b>A,</b> Assays of GUS activity in <i>A. thaliana</i> protoplasts containing RhNAC3 promoter deletion and ABRE mutation constructs. The numbers on top represent the positions of the ABRE <i>cis</i>-elements and mutations of ABREs in the <i>RhNAC3</i> promoter region. Relative GUS activity in transient expression experiments using five different constructs (N0, mN0, N1, mN1 and N2) and the vector control (NC) is shown at the bottom. GUS activity was determined after 24 h of incubation. Error bars represent standard error (<i>n</i> = 3). **: <i>P</i><0.01, *: <i>P</i><0.05, <i>t</i> test. <b>B,</b> Effects of exogenous ABA on GUS activity in <i>A. thaliana</i> protoplasts containing <i>RhNAC3</i> promoter deletion constructs. The truncated <i>RhNAC3</i> promoter constructs (N0, N1 and N2) and vector control (NC) were transformed into <i>A. thaliana</i> protoplasts, which were then exposed to 0 and 10 µM exogenous ABA. GUS activity in protoplast extracts was measured after 24 h of incubation with ABA. Error bars represent standard error (<i>n</i> = 5). <b>C,</b> Histochemical analysis of <i>RhNAC3</i> promoter::GUS expression in response to ABA. 9-day-old transgenic seedlings were grown on MS medium only or MS medium plus ABA (transferred to MS medium plus 100 µM ABA for 4 days) before being subjected to histochemical GUS staining. Scale bar = 1 mm.</p