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<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Plant Physiol.</journal-id>
<journal-title>Frontiers in Plant Physiology</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Plant Physiol.</abbrev-journal-title>
<issn pub-type="epub">2813-821X</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="doi">10.3389/fphgy.2024.1372307</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Plant Physiology</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Mutations alter <italic>pll</italic> gene promoter from constitutive to inducible, coffering palmately lobed leaf trait of melon</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Yong</surname>
<given-names>LiangMin</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="author-notes" rid="fn003">
<sup>&#x2020;</sup>
</xref>
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</contrib>
<contrib contrib-type="author">
<name>
<surname>Dai</surname>
<given-names>Wenting</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="author-notes" rid="fn003">
<sup>&#x2020;</sup>
</xref>
<role content-type="https://credit.niso.org/contributor-roles/methodology/"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-review-editing/"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Zhang</surname>
<given-names>Xuan</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="author-notes" rid="fn003">
<sup>&#x2020;</sup>
</xref>
<role content-type="https://credit.niso.org/contributor-roles/formal-analysis/"/>
<role content-type="https://credit.niso.org/contributor-roles/software/"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-review-editing/"/>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Zhang</surname>
<given-names>Qiang</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="author-notes" rid="fn001">
<sup>*</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/2631809"/>
<role content-type="https://credit.niso.org/contributor-roles/conceptualization/"/>
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</contrib-group>
<aff id="aff1">
<sup>1</sup>
<institution>International Tourism Institute, Hainan College of Economics and Business</institution>, <addr-line>Haikou</addr-line>, <country>China</country>
</aff>
<aff id="aff2">
<sup>2</sup>
<institution>Institute of Processing &amp; Design of Agroproducts, Hainan Academy of Agricultural Science</institution>, <addr-line>Haikou</addr-line>, <country>China</country>
</aff>
<author-notes>
<fn fn-type="edited-by">
<p>Edited by: Yasuyuki Kawaharada, Iwate University, Japan</p>
</fn>
<fn fn-type="edited-by">
<p>Reviewed by: Seisuke Kimura, Kyoto Sangyo University, Japan</p>
<p>Murat Aycan, Niigata University, Japan</p>
</fn>
<fn fn-type="corresp" id="fn001">
<p>*Correspondence: Qiang Zhang, <email xlink:href="mailto:821861127@qq.com">821861127@qq.com</email>
</p>
</fn>
<fn fn-type="other" id="fn003">
<p>&#x2020;These authors share first authorship</p>
</fn>
</author-notes>
<pub-date pub-type="epub">
<day>29</day>
<month>04</month>
<year>2024</year>
</pub-date>
<pub-date pub-type="collection">
<year>2024</year>
</pub-date>
<volume>2</volume>
<elocation-id>1372307</elocation-id>
<history>
<date date-type="received">
<day>17</day>
<month>01</month>
<year>2024</year>
</date>
<date date-type="accepted">
<day>03</day>
<month>04</month>
<year>2024</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#xa9; 2024 Yong, Dai, Zhang and Zhang</copyright-statement>
<copyright-year>2024</copyright-year>
<copyright-holder>Yong, Dai, Zhang and Zhang</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</p>
</license>
</permissions>
<abstract>
<p>The leaves of the common melon are approximately round and uncut. A natural mutant of melon named <italic>bm7</italic>, which has palmately-lobed leaves. We found that <italic>bm7</italic> plants were more conducive to increasing planting density and achieving higher yields, and showed better disease resistance. Therefore, the germplasm resources of muskmelon split leaves have the potential of application. Previous study showed that the palmately-lobed leaf trait was controlled by a single recessive gene, <italic>pll</italic> (numbered MELO3C010784). By cloning and sequencing the <italic>pll</italic> genes of Jiashi (wide-type) and <italic>bm7</italic> (mutant) plants, we noticed that there was no mutation in this gene, but its expression level in <italic>bm7</italic> was far below than that in Jiashi. The silencing of <italic>pll</italic> gene with virus induced gene silencing (VIGS) confers palmately-lobed leaf trait of Jiashi plants. We found three mutations in the <italic>pll</italic> gene promoter of <italic>bm7</italic> plants, located at 373, 493, 506 bp upstream the translation initiation codon. Then, we had found the <italic>pll</italic> gene promoter activity of <italic>bm7</italic> was significantly lower than that of Jiashi. In addition, the expression level and promoter activity of <italic>pll</italic> gene in Jiashi plants were not affected by environmental factors, but in <italic>bm7</italic> plants, the expression level and promoter activity of <italic>pll</italic> gene, all decreased with the increase in light intensity and/or temperature. All the results indicated that the mutations convert the <italic>pll</italic> gene promoter from constitutive to inducible, and results in significantly reduced expression of <italic>pll</italic> gene, conferring the palmately-lobed leaf trait of melon.</p>
</abstract>
<kwd-group>
<kwd>muskmelon</kwd>
<kwd>lobed leaf trait</kwd>
<kwd>PLL gene</kwd>
<kwd>promoter</kwd>
<kwd>transient expression</kwd>
</kwd-group>
<counts>
<fig-count count="7"/>
<table-count count="0"/>
<equation-count count="0"/>
<ref-count count="35"/>
<page-count count="10"/>
<word-count count="4239"/>
</counts>
<custom-meta-wrap>
<custom-meta>
<meta-name>section-in-acceptance</meta-name>
<meta-value>Environmental Interactions</meta-value>
</custom-meta>
</custom-meta-wrap>
</article-meta>
</front>
<body>
<sec id="s1" sec-type="intro">
<label>1</label>
<title>Introduction</title>
<p>Leaf, an important organ associated with photosynthesis of plants, can convert solar energy into bioenergy, providing oxygen, food, fiber, biomass and other materials for human survival. In addition, plant leaf also has the ability to sense environmental changes, receive and transmit environmental signals (<xref ref-type="bibr" rid="B11">Edgerton, 2009</xref>). Changes in leaf morphology may affect plant physiological functions such as photosynthesis, transpiration and stress resistance, and eventually influencing plant growth and development. Therefore, leaf development has always been one of the major focuses of plant science (<xref ref-type="bibr" rid="B29">Tsukaya, 2013</xref>; <xref ref-type="bibr" rid="B2">Avramova et&#xa0;al., 2015</xref>). Research on leaf development at the genetic level began in the 1990s (<xref ref-type="bibr" rid="B26">Smith et&#xa0;al., 1992</xref>), and has revealed many genes associated with leaf morphogenesis. The leaves of common melons are approximately round and not distinctly lobed. However, we found a natural mutant of melon, named <italic>bm7</italic>, which has palmately lobed leaves.</p>
<p>Melon, watermelon, pumpkin, etc. are important cash crops of the cucurbitaceae family, and they all involve split leaf phase Genetic analysis showed that the split leaf traits were controlled by a single gene, and there were recessive differences, but there was no gene function report. The leaves of most watermelon varieties are deeply pinnatifid, however, a natural mutant with nonlobed leaf, Black Diamond, was first reported by Mohr in 1953 (<xref ref-type="bibr" rid="B24">Mohr, 1953</xref>), and this study showed that the nonlobed leaf trait was controlled by an incompletely-dominant gene <italic>nl</italic>. Another watermelon with nonlobed leaf, Banye No. 1, was discovered in 1983 (<xref ref-type="bibr" rid="B7">Chai and Wang, 1983</xref>), and it was reported that its leaf trait was controlled by a recessive gene. <xref ref-type="bibr" rid="B17">Huang (1988)</xref> developed a new watermelon cultivar with nonlobed leaf, Zhongkai No.1. Cui et&#xa0;al. further proved that this leaf trait was controlled by a recessive gene (<xref ref-type="bibr" rid="B8">Cui&#xa0;et&#xa0;al., 1996</xref>). Then, a tetraploid watermelon cultivar with nonlobed leaf QB-3 was derived from an introduced diploid cultivar in 1993 (<xref ref-type="bibr" rid="B34">Zhou, 1993</xref>). Another watermelon cultivar with nonlobed leaf Kaifeng No. 25 was generated in 2008 (<xref ref-type="bibr" rid="B20">Li et&#xa0;al., 2008</xref>). Two lobed leaf-linked genes <italic>lo-1</italic> and <italic>lo-2</italic> have been reported in pumpkin (<xref ref-type="bibr" rid="B10">Dyutin, 1980</xref>; <xref ref-type="bibr" rid="B16">Herrington and Brown, 2013</xref>). In melons, only Ganesan et&#xa0;al. reported that the schizoleaf trait of Maine Rock was controlled by a dominant single gene and named it L (<xref ref-type="bibr" rid="B13">Ganesan and Sambandam, 1985</xref>).</p>
<p>Arabidopsis thaliana plants with mutations in the genes that regulate gibberellin (GA) synthesis have lobed leaf margins. The lobed leaf trait in tomato plant disappears when exogenous gibberellin is applied (<xref ref-type="bibr" rid="B15">Hay et&#xa0;al., 2002</xref>). Decreasing GA signal or GA synthesis promotes the formation of lobed leaf margins, while application of exogenous GA or enhanced GA signal inhibits KNOX gene expression, resulting in decreased depth and number of leaf lobes (<xref ref-type="bibr" rid="B6">Byrne et&#xa0;al., 2000</xref>). The study of Zhang et&#xa0;al. suggested that low expression of <italic>BrLOM2</italic> gene may be the main reason causing lobed leaf trait in Chinese cabbage (<xref ref-type="bibr" rid="B33">Zhang et&#xa0;al., 2016</xref>). However, our previous work showed that the application of exogenous GA has no effect on leaf shape of the mutant <italic>bm7</italic>.</p>
<p>Our earlier studies have shown that the palmately lobed leaf trait of <italic>bm7</italic> is governed by a recessive gene <italic>pll</italic>. Then, using map-based cloning and SSR markers, this gene was located to a region of approximately 9 000 bp, which contains only one candidate gene (<xref ref-type="bibr" rid="B14">Gao et&#xa0;al., 2014</xref>). In melonomic database, <italic>pll</italic> gene is numbered cm010784, spans over 3 392 bp, and consists of seven exons and six introns. In addition, we also found that <italic>bm7</italic> plants had different leaf traits under different light intensity and temperature, which is different from the stable traits of most mutants. In this study, using a melon cultivar with nonlobed leaf Jiashi as a control, <italic>pll</italic> gene was cloned and sequenced, and its expression in both Jiashi and <italic>bm7</italic> plants grown under different light and temperature conditions was quantified and compared. We also found that the silencing of <italic>pll</italic> gene via virus-induced gene silencing (VIGS) conferred the palmately lobed leaf trait of Jiashi plants. After measuring <italic>pll</italic> gene promoter activity in both Jiashi and <italic>bm7</italic> plants, we found that the promoter activity and expression level of <italic>pll</italic> gene and leaf lobe depth of <italic>bm7</italic> were all influenced by light intensity and air temperature. These results provide a theoretical basis for further revealing the formation of palmately-lobed leaf trait in plants.</p>
</sec>
<sec id="s2">
<label>2</label>
<title>Materials and methods</title>
<sec id="s2_1">
<label>2.1</label>
<title>Plant material and treatments</title>
<p>Jiashi and <italic>bm7</italic> plants were provided by the College of Life Science and Technology, Xinjiang University, Urumqi, China. Melon seeds were sown in a greenhouse at 30&#xb0;C, and DNA sample used for gene cloning and vector construction was extracted from true leaves. RNA was extracted from 2-, 5-, 8-, 11-, 14- and 17-day-old true leaves for fluorescence quantitative PCR. Tobacco plants (Nicotiana NC89.) were used for transient expression analysis of transgenes.</p>
</sec>
<sec id="s2_2">
<label>2.2</label>
<title>Gene and promoter cloning and sequencing</title>
<p>Genomic DNA was extracted from plants with the Hi-DNAsecure Plant Kit (TIANGEN, China). The fragment (3 451 bp) including pll gene (3 392 bp) was amplified by polymerase chain reaction (PCR) using Long <italic>Taq</italic> DNA polymerase (TIANGEN, China), the forward primer 5&#x2019;-TTTCATTACCACAAAATTCTTTCAAT-3&#x2019; and the reverse primer 5&#x2019;-ATTGGGTTCTTGCAGCTAGGAAAAGA-3&#x2019;, and then, the gene DNA was cloned into the pEASY-T5 Zero Cloning vector (TRANS, China) for sequencing.</p>
<p>Based on the genomic sequence of pll (LOC-Cm010784), a 1 531-bp regulatory region upstream of the translation initiation codon was identified in the melonomics database. The region of the putative pll promoter was PCR amplified with the forward primer 5&#x2019;-<italic>TTGCCTGCAGG</italic>ACTGTTTCATACCCCACCCC-3&#x2019; and the reverse primer 5&#x2019;-<italic>CGCGGATCC</italic>CAAACCTCTTGGCTCCTCTTA-3&#x2019;. The cloned fragment was inserted into pEASY-T5 Zero Cloning vector (TRANS, China) for sequencing. &#x2018;<italic>TTGCCTGCAGG&#x2019;</italic> is the restriction enzyme and protected base of Sbf I, &#x2018;<italic>CGCGGATCC&#x2019;</italic> is the restriction enzyme and protected base of BamHI. The two restriction enzyme sequences were introduced into promoter fragment for the following experiment of pBI121 vector construction.</p>
</sec>
<sec id="s2_3">
<label>2.3</label>
<title>RNA extraction and real-time quantitative RT-PCR expression analysis</title>
<p>Total RNA samples were extracted from leaf material with the EasyPure Plant RNA Kit (TRANS, China) following the manufacturer&#x2019;s protocol. CDNA of interest was amplified by PCR using the forward primer 5&#x2019;-ATAACAATAATAGTTGGTT-3&#x2019; and the reverse primer 5&#x2019;-GAGTGATGATGAAGAGAGG-3&#x2019;, synthesized with the EasyScript First-Strand cDNA Synthesis SuperMix (TRANS China), to be used as the template for RT-PCR (<xref ref-type="bibr" rid="B35">Zienkiewicz et&#xa0;al., 2018</xref>). Quantitative RT-PCR reactions were performed in Bio-RAD MyiQTM Real-time PCR Detection System (Bio-Rad, USA) using the TransStart Top Green qPCR SuperMix (TRANS, China) according to the manufacturer&#x2019;s instructions, and the CmActin gene (MELO3C011913) was employed as the internal control for RT-PCR analysis. The primers of CmActin gene: the forward as 5&#x2019;-CCAAAGGCTGCAAGAATAGC-3&#x2019; and the reverse as 5&#x2019;- TTTGACCTTTGGGTGGGTAG-3&#x2019;. Amplification was carried out through initial denaturation at 94&#xb0;C for 2 min, followed by 38 cycles of denaturation at 94&#xb0;C for 3 min, annealing at 57&#xb0;C for 30 s, and elongation at 72&#xb0;C for 2 min. The PCR products from each amplification reaction were separated on 2.5% (w/v) agarose gels (<xref ref-type="bibr" rid="B5">Bi et&#xa0;al., 2017</xref>).</p>
</sec>
<sec id="s2_4">
<label>2.4</label>
<title>VIGS vector construction and gene silencing</title>
<p>Single-stranded cDNA was prepared from 1 mg of total RNA. A fragment of 405 bp from PLL corresponding to nucleotides 355 to 759 of the gene and including a region that was conserved in this family, the fragment was amplified by PCR using the following primers: 5&#x2019;-CTCAAGCTTACCCCCAAATTCTCTGCTTT-3&#x2019;(Hind III site was introduced) as forward and 5&#x2019;-CTGAAGCTTCAATGCCTGAAGTGTCCTCA-3&#x2019;(BamHI site was introduced) as reverse. The resulting PCR products were cloned into pEASY-T5 Zero Cloning vector (TRANS, China). The cDNAs of gene were excised from pEASY-T5 Zero by digestion and cloned into TRV2 cut with Hind III and BamHI (Roche Diagnostics) to obtain TRV: PLL. Agrobacterium strain containing TRV vectors was grown at 28&#xb0;C in YEB medium containing 10 mmol/L MES and 20 mmol/L acetosyringone with the appropriate antibiotics. After 24 h, Agrobacterium cells were harvested and resuspended in Agrobacterium infiltration buffer (10 mmol/L MgCl<sub>2</sub>, 10 mmol/L MES, pH 5.6, 150 mmol/L acetosyringone). Then the Agrobacterium suspension was injected into the stem of the plant with a syringe (<xref ref-type="bibr" rid="B19">Lentz et&#xa0;al., 2018</xref>; <xref ref-type="bibr" rid="B32">Yan et&#xa0;al., 2018</xref>).</p>
</sec>
<sec id="s2_5">
<label>2.5</label>
<title>Promoter vector construction</title>
<p>After sequencing the promoter fragment, the cloned fragment was inserted upstream of the GUS gene in the vector pBI121 using BamHI and SbfI (<xref ref-type="bibr" rid="B1">Avile&#x301;s-Arnaut and De&#x301; lano-Frier, 2012</xref>; <xref ref-type="bibr" rid="B27">Spangler et&#xa0;al., 2019</xref>).</p>
</sec>
<sec id="s2_6">
<label>2.6</label>
<title>Leaf disc vacuum infiltration</title>
<p>The Agrobacterium transformed to express the genes of interest were cultured in LB medium containing kanamycin (50mg/mL), and rifampicin (100mg/mL) overnight at 28&#xb0;C by shaking at 180 r/min. Bacterial concentrations were determined by measuring optical density (OD) at 600 nm.</p>
<p>Leaf discs were cut from Nicotiana NC89 leaves approximately 7-8 weeks after germination using a cork borer (8.5 mm). Poured the Agrobacterium suspension and leaf disc into a 20 ml syringe, pulled the plunger to create a small vacuum in the syringe and shook vigorously then released plunger rapidly. After infiltration, the well-infiltrated leaf discs were selected and incubated upper side down in Petri dishes with MS medium containing 0.8% agar under lighting programs (16 h of light: 8 h of dark) at 23&#xb0;C (<xref ref-type="bibr" rid="B23">Matsuo et&#xa0;al., 2016</xref>).</p>
</sec>
<sec id="s2_7">
<label>2.7</label>
<title>Histochemical GUS assay</title>
<p>For histochemical staining, the Agrobacterium infiltrated leaf discs were washed twice with 50 mM Tris-HCl buffer (pH 7.5) containing carbenicillin (500 mg/mL) and then twice with distilled water. The washed leaf discs were prefixed with chilled 90% (v/v) aqueous acetone and then washed with chilled water. They were then immersed in GUS staining solution consisting of 50 mM phosphate buffer (pH 7.2), 5 mM K<sub>3</sub>Fe(CN)<sub>6</sub>, 5 mM K<sub>4</sub>Fe(CN)<sub>6</sub>, 0.2%(v/v) Triton X-100, and 2 mM X-Gluc salt. They were then incubated overnight at 37&#xb0;C. Stained leaf discs were successively submerged in 25%, 50%, 70%, and 95% (v/v) ethanol to remove chlorophyll (<xref ref-type="bibr" rid="B9">Dai et&#xa0;al., 2017</xref>).</p>
</sec>
<sec id="s2_8">
<label>2.8</label>
<title>Quantitative GUS activity assay</title>
<p>The leaf blade to be tested was placed in a mortar, ground to a fine powder by adding liquid nitrogen, transferred to a sterile centrifuge tube. After extraction buffer was added, the sample was centrifuged at 12 000 r/min and 4&#xb0;C for 10 min to collect the supernatant (GUS protein extract). Then, 50 &#x3bc;l of the GUS protein extract was added to 450 &#x3bc;l of 4-MUG (2 mmol/L), incubated at 37&#xb0;C for 30 min, before fluorescence emission was measured, at 365 nm excitation and 455 nm emission. GUS activity was calculated according to the relative change in the product per unit time. Three biological replicates were used in this experiment (<xref ref-type="bibr" rid="B9">Dai et&#xa0;al., 2017</xref>).</p>
</sec>
<sec id="s2_9">
<label>2.9</label>
<title>Statistical analysis</title>
<p>The data were analyzed using the Statistical Analysis System (version 9.1, SAS Institute, Cary, NC, USA). Significant changes in gene expression were determined using Fisher&#x2019;s least significant differences (P &lt; 0.05).</p>
</sec>
</sec>
<sec id="s3" sec-type="results">
<label>3</label>
<title>Results</title>
<sec id="s3_1">
<label>3.1</label>
<title>No mutation sites were found in the <italic>pll</italic> gene of <italic>bm7</italic> plants, but there was a significant difference in <italic>pll</italic> gene expression between Jiashi and <italic>bm7</italic>
</title>
<p>Sequencing analysis showed that both the <italic>pll</italic> gene fragments cloned from Jiashi and <italic>bm7</italic> have the same sequences, which are consistent with the reference gene of melon. Since changes in gene expression can result in changes in phenotypical traits, we examined the expression of <italic>pll</italic> gene in both Jiashi and <italic>bm7</italic>. As shown in <xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1</bold>
</xref>, there was a significant difference in <italic>pll</italic> gene expression between Jiashi and <italic>bm7</italic>. In detail, the expression level of <italic>pll</italic> gene in Jiashi was approximately equal to that of <italic>CmActin</italic> gene, but was 25 times higher than <italic>pll</italic> gene expression level of in <italic>bm7</italic>.</p>
<fig id="f1" position="float">
<label>Figure&#xa0;1</label>
<caption>
<p>Expression of <italic>pll</italic> gene in Jiashi and <italic>bm7</italic>. Relative expression levels of <italic>pll</italic> gene in 2-, 5-, 8-, 11-, 14- and 17-day-old leaves of Jiashi and <italic>bm7</italic>. The macro comparison of <italic>pll</italic> gene expression was shown in <bold>(A)</bold>, and the quantitative expression analysis was shown in <bold>(B)</bold>. Diferent letters for each species denote signifcant diferences (<italic>p</italic> &#x2264; 0.05).</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fphgy-02-1372307-g001.tif"/>
</fig>
</sec>
<sec id="s3_2">
<label>3.2</label>
<title>Low expression of <italic>pll</italic> gene conferred the palmately lobed leaf character of melon</title>
<p>Although there was a significant difference in the expression of <italic>pll</italic> gene between Jiashi and <italic>bm7</italic>, it was still uncertain that the low expression of <italic>pll</italic> gene is the cause of palmately lobed leaf character. So, a 405-bp domain in the first exon of the <italic>pll</italic> gene of Jiashi plant was silenced by VIGS to observe the changes in leaf shape. The results showed that the leaves became palmately lobed after <italic>pll</italic> gene was silenced (<xref ref-type="fig" rid="f2">
<bold>Figures&#xa0;2A, B</bold>
</xref>), proving that low expression of <italic>pll</italic> gene in Jiashi plants is the cause of palmately lobed leaves.</p>
<fig id="f2" position="float">
<label>Figure&#xa0;2</label>
<caption>
<p>The <italic>pll</italic> gene of Jiashi was silenced by VIGS. <bold>(A)</bold> The leaf phenotype of Jiashi, pTRV2:00(negative control of empty vector), <italic>bm7</italic> and pTRV2-<italic>pll</italic> (<italic>pll</italic>-silencing) plants. <bold>(B)</bold> Relative expression of <italic>pll</italic> gene in Jiashi, pTRV2:00, <italic>bm7</italic> and <italic>pll</italic>-silencing Jiashi plants. The red arrow points to a leaf with a split shape, indicating that the <italic>pll</italic> gene had been successfully silenced.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fphgy-02-1372307-g002.tif"/>
</fig>
</sec>
<sec id="s3_3">
<label>3.3</label>
<title>The <italic>pll</italic> gene promoter of <italic>bm7</italic> had mutations at three sites, and the activity of <italic>pll</italic> gene promoter of Jishi and <italic>bm7</italic> was also significantly different</title>
<p>Promoters are important elements in the regulation of gene expression. Therefore, we sequenced and detected the activity of <italic>pll</italic> gene promoters of Jiashi and <italic>bm7</italic>. Sequencing of the cloned fragments revealed that there were three mutation sites at the 373 (T to C), 493 (C to T) and 506 bp (A to C) upstream the translation initiation codon of the <italic>pll</italic> gene in <italic>bm7</italic> plant (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3A</bold>
</xref>). The GUS activity was detected by <italic>Agrobacterium</italic>-mediated transient gene expression, and the GUS activity of Jiashi was significantly higher than that of <italic>bm7</italic> (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3B</bold>
</xref>). As shown in, the <italic>pll</italic> gene promoter activity of <italic>bm7</italic> was significantly lower than that of Jiashi (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3C</bold>
</xref>).</p>
<fig id="f3" position="float">
<label>Figure&#xa0;3</label>
<caption>
<p>Detection of <italic>pll</italic> gene promoter activity of Jiashi and <italic>bm7.</italic> <bold>(A)</bold> Mutation sites of <italic>pll</italic> gene promoters of <italic>bm7</italic>, the blue letters are Jiashi and the green letters are <italic>bm7</italic>, and TCT is the translation initiation codon of <italic>pll</italic> gene. <bold>(B)</bold> GUS activity in NC89 tobacco leaf discs. <bold>(C)</bold> GUS staining of transformed leaf discs.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fphgy-02-1372307-g003.tif"/>
</fig>
</sec>
<sec id="s3_4">
<label>3.4</label>
<title>Mutation sites -373 and -506 influence promoter activity greatly</title>
<p>We constructed PBI121 transient expression vectors carrying each of the following three full-length promoter fragments: L1 (containing mutation sites -373, -493 and -506), L2 (containing mutation sites -493 and -506) and L3 (containing mutation site -506), to analyze the effect of the three mutation sites on the activity of pll gene promoter. The results are shown in <xref ref-type="fig" rid="f4">
<bold>Figure&#xa0;4</bold>
</xref>. In bm7, L2 and L1 promoter fragments exhibited significant difference in activity, while L2 and L3 promoter fragments did not, indicating that the mutation site -373 has a greater influence than the mutation site -493 on promoter activity. In addition, the activity of L3 promoter fragment showed significant difference between Jiashi and bm7, indicating the mutation site -506 also has a great effect on promoter activity. Moreover, L1 fragment showed more significant difference in activity between Jiashi and bm7 than the other two fragments, indicating that the three mutation points have an additive effect on promoter activity, so simultaneous mutation of the three sites is able to further reduce activity of pll promoter.</p>
<fig id="f4" position="float">
<label>Figure&#xa0;4</label>
<caption>
<p>Quantitative GUS activity of different length promoter.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fphgy-02-1372307-g004.tif"/>
</fig>
</sec>
<sec id="s3_5">
<label>3.5</label>
<title>Expression of <italic>pll</italic> gene and leaf shape of <italic>bm7</italic> plants were influenced by light and temperature</title>
<p>We found that the pll gene expression and leaf shape of Jiashi did not change in different environments (<xref ref-type="fig" rid="f5">
<bold>Figures&#xa0;5A, C</bold>
</xref>). However, <italic>pll</italic> gene expression and leaf lobe depth of <italic>bm7</italic> varied significantly among different environments (<xref ref-type="fig" rid="f5">
<bold>Figures&#xa0;5B, D</bold>
</xref>). The expression level of <italic>pll</italic> gene in <italic>bm7</italic> plants exposed to intense sunlight and high temperature in outdoor environment was about two times lower than that in indoor environment, while <italic>pll</italic> gene in Jiashi plants showed no significant differences between the two environments (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5E</bold>
</xref>).</p>
<fig id="f5" position="float">
<label>Figure&#xa0;5</label>
<caption>
<p>
<italic>pll</italic> gene expression and leaf shape of Jiashi and <italic>bm7</italic> in outdoor and indoor environments. Leaf shape of Jiashi <bold>(A)</bold> and <italic>bm7</italic> <bold>(B)</bold> in outdoor environment; <bold>(C)</bold>&#xa0;Leaf shape of Jiashi <bold>(C)</bold> and <italic>bm7</italic> <bold>(D)</bold> in indoor environment; Expression of <italic>pll</italic> gene in Jiashi and <italic>bm7</italic> in outdoor and indoor environments <bold>(E)</bold>. The letters "a, b" are symbols that indicate a significant difference between the two.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fphgy-02-1372307-g005.tif"/>
</fig>
<p>We determined the expression of <italic>pll</italic> gene in the leaves of Jiashi and <italic>bm7</italic> under three different light intensity and temperature conditions. At 30&#xb0;C, the expression of <italic>pll</italic> gene of <italic>bm7</italic> plants gradually declined while the leaf lobe depth gradually increased, with light intensity increasing from 20 to 180 LUX (<xref ref-type="fig" rid="f6">
<bold>Figures&#xa0;6A, B</bold>
</xref>). At a light intensity of 50 LUX, the expression of <italic>pll</italic> gene of <italic>bm7</italic> plants gradually declined while the leaf lobe depth gradually increased, with temperature increasing from 20 to 40&#xb0;C (<xref ref-type="fig" rid="f6">
<bold>Figures&#xa0;6C, D</bold>
</xref>). These results indicated that the expression of <italic>pll</italic> gene in <italic>bm7</italic> was negatively regulated by temperature and/or light intensity, and the leaf lobe depth increased with the increase of light intensity and/or temperature. However, the <italic>pll</italic> gene expression and leaf shape of Jiashi plants were not affected by light and/or temperature.</p>
<fig id="f6" position="float">
<label>Figure&#xa0;6</label>
<caption>
<p>Expression of <italic>pll</italic> gene and leaf shape of Jiashi and <italic>bm7</italic> plants under different light and temperature conditions. <bold>(A)</bold> The leaf blade shape of Jiashi and <italic>bm7</italic> at light intensity of 20, 100 and 180 LUX and temperature of 30&#xb0;C; <bold>(B)</bold> The <italic>pll</italic> gene expression of Jiashi and <italic>bm7</italic> at light intensity of 20, 100 and 180 LUX and temperature of 30&#xb0;C; <bold>(C)</bold> the leaf blade shape of Jiashi and <italic>bm7</italic> at light intensity of 50 LUX and temperature of 20, 30, and 40&#xb0;C; <bold>(D)</bold> The <italic>pll</italic> gene expression of Jiashi and <italic>bm7</italic> at light intensity of 50 LUX and temperature of 20, 30, and 40&#xb0;C. The letters "a&#x2013;c" are symbols that indicate a significant difference between the three.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fphgy-02-1372307-g006.tif"/>
</fig>
</sec>
<sec id="s3_6">
<label>3.6</label>
<title>The <italic>pll</italic> gene promoter activity of <italic>bm7</italic> was affected by light and temperature</title>
<p>We measured the <italic>pll</italic> gene promoter activity under different light intensity and temperature conditions. GUS staining showed that similar to <italic>pll</italic> gene expression, the promoter activity of this gene in Jiashi plant was not affected by light intensity or temperature, but that in <italic>bm7</italic> plants gradually decreased with light intensity or temperature increasing (<xref ref-type="fig" rid="f7">
<bold>Figures&#xa0;7A&#x2013;D</bold>
</xref>).</p>
<fig id="f7" position="float">
<label>Figure&#xa0;7</label>
<caption>
<p>
<italic>pll</italic> gene promoter activity of Jiashi and <italic>bm7</italic> plants under different light and temperature conditions. <bold>(A)</bold> GUS activity of tobacco NC89 leaf discs at 20, 100 and 180 LUX; <bold>(B)</bold> GUS staining of transformed tobacco leaf discs after 20 days of culture at 20, 100 and 180 LUX; <bold>(C)</bold> GUS activity of tobacco NC89 leaf discs at 20, 30 and 40&#xb0;C; <bold>(D)</bold> GUS staining of transformed tobacco leaf discs after 20 days of culture at 20, 30 and 40&#xb0;C. The letters "a&#x2013;c" are symbols that indicate a significant difference between the three.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fphgy-02-1372307-g007.tif"/>
</fig>
</sec>
</sec>
<sec id="s4" sec-type="discussion">
<label>4</label>
<title>Discussion</title>
<p>As a unique germplasm resource, the mutant <italic>bm7</italic>, which has palmately lobed leaves, has the following potentials: (I) The leaves of bm7 are less overlapped with each other and occupy less space than those of other cultivars, so it is more tolerant to high-density planting to improve melon production per unit area. (II) Lobed leaves can improve plant&#x2019;s resistance to direct sunlight exposure (<xref ref-type="bibr" rid="B30">Vogel, 2009</xref>). Cotton cultivars that have moderately lobed leaves exhibit high photosynthetic efficiency (<xref ref-type="bibr" rid="B4">Barber, 2009</xref>). (III) Lobed leaves can also improve melon&#x2019;s resistance to disease to some extent. Field trials showed that lobed leaf mutant is less susceptible to powdery mildew than its wild-type sibling lines (without lobed leaf trait). (IV) Lobed leaf trait can be used as an indicator to identify the purity of F<sub>1</sub> generation seeds in hybrid-seed production.</p>
<p>By comparing with the published melon genome data, we found that the similarity between pll gene and ANT transcription factor is up to 89.3%. Studies have shown that ANT is involved in the early development of all tissues except roots, and maintains the ability of cells to proliferate continuously (<xref ref-type="bibr" rid="B18">Klucher et&#xa0;al., 1996</xref>; <xref ref-type="bibr" rid="B12">Elliott et al., 1996</xref>; <xref ref-type="bibr" rid="B31">Yamaguchi et&#xa0;al., 2015</xref>). Bandupriya et&#xa0;al. reported that ANT gene is involved in the formation of serrated leaf margins, and Arabidopsis thaliana plants that overexpress the ANT gene cloned from coconut have serrated leaf margins (<xref ref-type="bibr" rid="B3">Bandupriya and Dunwell, 2012</xref>), which is the only report that ANT is associated with split leaf development.</p>
<p>Gene mutation can cause changes in plant phenotypes. Mutations in the genes associated with gibberellin biosynthesis in <italic>Arabidopsis</italic> can lead to the formation of foliage leaf lobes, and leaf polymorphism in tomato can be eliminated by the use of exogenous gibberellins (<xref ref-type="bibr" rid="B15">Hay et&#xa0;al., 2002</xref>). However, in this study, we found no mutation in the coding sequence of the pll gene in mutant <italic>bm7</italic>.</p>
<p>Changes in gene expression levels can also lead to changes in plant phenotypes. By cloning leaf morphology- related gene BrLOM2 using RNA-Seq technology, Zhang et&#xa0;al. confirmed that the low expression of this gene might be the main reason for the formation leaf lobes (<xref ref-type="bibr" rid="B33">Zhang et&#xa0;al., 2016</xref>). Li et&#xa0;al. mapped the major QTL loci controlling the lobe depth at leaf margin of <italic>Brassica napus</italic> L., and found that the expression level of BrGA20OX3 in the leaves of the parental material Yellow Sarson C634 (early flowering stage, Indian rape) was only 1/6 of the normal level, suggesting that leaf lobes of the parent C634 might be caused by the low expression of BrGA20OX3 gene (<xref ref-type="bibr" rid="B21">Li et&#xa0;al., 2009</xref>). Therefore, we speculated that palmate lobes in leaf was probably a result of gene expression regulation. By measuring the expression of <italic>pll</italic> gene in Jiashi and <italic>bm7</italic>, we found that the expression level of <italic>pll</italic> gene in <italic>bm7</italic> was only about 1/25 of that in Jiashi.</p>
<p>Although the expression of <italic>pll</italic> gene in mutants is significantly lower than that in wide-type plants, but there may be multiple genes being expressed differently between plants. It was still unclear whether the low expression of <italic>pll</italic> gene is the cause of palmately-lobed leaves. So, in this study, <italic>pll</italic> gene was silenced by VIGS, an effective method for verifying gene function. The results showed that palmately lobed leaves were observed after <italic>pll</italic> gene was silenced in Jiashi plants, which was similar to the leaf trait of the mutant. The results confirmed that the low expression of <italic>pll</italic> gene is the cause of palmately lobed leaves of melon plants.</p>
<p>Promoter, an important component in the regulation of gene expression, can control the timing and level of gene expression. Constitutive promoters are not regulated by external environmental factors, and can drive gene expression continuously (<xref ref-type="bibr" rid="B28">Sun et&#xa0;al., 2018</xref>). Our results showed that environment had no significant effect on the expression of <italic>pll</italic> gene in Jiashi. Transient expression analysis suggested that the promoter of <italic>pll</italic> gene in Jiashi was constitutive, as its activity was not affected by light and temperature.</p>
<p>Inducible promoters can significantly increase or decrease the transcription level of a gene under certain physical or chemical signal stimulation. The expression of GUS gene in the roots and leaves of transgenic rice was greatly regulated via the treatment with MeJA and <italic>Xanthomonas oryzae</italic> (<xref ref-type="bibr" rid="B22">Li et&#xa0;al., 2018</xref>), indicating that MeJA and <italic>X. oryzae</italic> can induce the PRJA140 promoter. In addition, mutations in promoter or changes in DNA conformation can also affect gene expression and plant phenotypes. Alexandr Muterko et&#xa0;al. found that allelic variations at VRN-A1 and VRN-B1 could be identified based on DNA curvature and flexibility in the promoter region, and single-nucleotide mutations within VRN-box change DNA conformation, and influence the vernalization requirement and growth habit of wheat (<xref ref-type="bibr" rid="B25">Muterko et&#xa0;al., 2016</xref>). In the present study we found that there are three mutations in <italic>pll</italic> gene promoter of <italic>bm7</italic>, and this gene is highly expressed at high light intensity and/or low temperature. The transient expression analysis in tobacco indicated its promoter activity decreases with light intensity and/or temperature increasing, suggesting that <italic>pll</italic> gene promoter of <italic>bm7</italic> plants is a typical inducible promoter. Therefore, we can speculate that the mutations change the <italic>pll</italic> gene promoter from constitutive to inducible, resulting in the changes in <italic>pll</italic> gene expression, which ultimately leads to the formation of palmately-lobed leaf trait of melon.</p>
<p>A promoter is a region of DNA that initiates transcription of a particular gene, and it may contains a number of response elements. Mutations may cause changes in these response elements, or changes in the spatial structure of the DNA fragment. All these variations may affect the characteristics of the promoter. Therefore, the molecular mechanism by which the <italic>pll</italic> gene promoter is converted from constitutive to inducible still needs to be further studied.</p>
</sec>
<sec id="s5" sec-type="data-availability">
<title>Data availability statement</title>
<p>The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.</p>
</sec>
<sec id="s6" sec-type="author-contributions">
<title>Author contributions</title>
<p>LY: Methodology, Software, Writing &#x2013; review &amp; editing. WD: Methodology, Writing &#x2013; review &amp; editing. XZ: Formal analysis, Software, Writing &#x2013; review &amp; editing. QZ: Conceptualization, Data curation, Methodology, Resources, Supervision, Validation, Writing &#x2013; original draft.</p>
</sec>
</body>
<back>
<sec id="s7" sec-type="funding-information">
<title>Funding</title>
<p>The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was supported by the Hainan College of Economics and Business high-level talent introduction project (hnjmk2022303).</p>
</sec>
<ack>
<title>Acknowledgments</title>
<p>We thank Mr. Fan Wen Lin (College of Life Science and Technology, Xinjiang University) for Jiashi and bm7 melon seeds.</p>
</ack>
<sec id="s8" sec-type="COI-statement">
<title>Conflict of interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
<sec id="s9" sec-type="disclaimer">
<title>Publisher&#x2019;s note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
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