A total of 9 mature ‘Lane late’ navel orange (Citrus sinensis Osbeck) trees (10-year-old) with red tangerine (Citrus tangerina Hort) as rootstock from an orchard located in the Three Gorges Reservoir area (E 110°41’, N 30°54’) in Zigui County were selected for sampling in this study, and fruits were collected at 350 days after flowering according to our previous study (Wu et al., 2020). The experiments were conducted with three biological replicates, and 15 fruits collected from 3 trees in different directions were mixed together as one biological replicate. The juice sacs were isolated from the fruits, immediately frozen with liquid nitrogen, and stored at -80 °C for RNA extraction. For low temperature treatment, the juice sacs isolated from normal ‘Lane late’ navel orange fruits were placed on a plate and covered with sterilized gauze to keep moist. Then, the plates with juice sacs were transported into an incubator at 4 °C for low temperature treatment. The samples were collected at 0 h, 3 h, 9 h, 12 h, and 24 h, and 30 individual juice sacs were collected as one biological replicate. Each sample had three biological replicates, and all the juice sacs were immediately frozen with liquid nitrogen and stored in -80 °C for RNA extraction.

To explore the phylogenetic relationship of CsMYB15 and other MYB family transcription factors, a multiple sequence alignment of the amino acid sequences of CsMYB15 and other 32 MYB proteins was performed using MUSCLE (www.ebi.ac.uk/Tools/msa/muscle/ ) with default parameters (Edgar, 2004). The phylogenetic tree was constructed using the maximum likelihood method of MEGA X software with 1000 bootstraps (Kumar et al., 2018). The final phylogenetic tree was visualized and polished with the Interactive Tree of Life software (iTOL, version 5, https://itol.embl.de/) (Letunic and Bork, 2016).

Total RNA was extracted from the juice sacs using TRIzol™ reagent according to the manufacturer’s instructions (Thermo Scientific). RNA quality was assessed by agarose gel electrophoresis, and 5X All-In-One RT MasterMix with AccuRT (Applied Biological Materials) kit was used for ss/dsDNA digestion and cDNA synthesis. Quantitative RT-PCR was performed on a QuantStudio 7 Flex system (Thermo Scientific, USA) according to the manufacturer’s instruction with EvaGreen 2X qPCR MasterMix kit (Applied Biological Materials, Canada). Elongation factor 1 (Ef1, Cs8g16990) gene was utilized as an internal reference gene (Li et al., 2022), and the relative expression was calculated with 2^-ΔΔCt method. Student’s t-test was performed to determine statistical significance, and ** indicated significant differences at the level of P < 0.01. The primers used for qRT-PCR were listed in Supplementary Table S1 .

The cDNA synthesized from the total RNA extracted from juice sacs of ‘Lane late’ navel orange (Citrus sinensis Osbeck) was used to amplify the full-length coding sequence (CDS) of CsMYB15 using the following primers: Forward 5’ -ATGATGGGGAGGGCTCC- 3’ and Reverse 5’ -GGAAATGGTAATGTTAATGAGTCTGCC-3’. PCR amplification was performed using the Phanta Max Super-Fidelity DNA Polymerase (Vazyme, Nanjing, China) following the manufacturer’s instructions. The PCR product was purified and cloned into pTOPO vector for sequencing. The coding sequence of CsMYB15 was deposited in Genebank with the accession number of OQ985393. The 2000 bp region upstream the start codon ATG of CsMYB15 was cloned as the promoter sequence from the total DNA extracted from the leaf of ‘Lane late’ navel orange. The promoter of CsMYB15 was further analyzed using the online software of plantPan 3.0 (http://plantpan.itps.ncku.edu.tw/) and plantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The promoter sequence of CsMYB15 was provided in Supplementary Table S2 .

The CDS of CsMYB15 without stop codon was ligated with GFP (green fluorescent protein), and then cloned into pICH86988 vector containing cauliflower mosaic virus 35S promoter to construct 35S:CsMYB15:GFP vector by Golden Gate method (Engler et al., 2014). Subsequently, the recombinant 35S:CsMYB15:GFP vector was transformed into Agrobacterium tumefaciens (strain GV3101 with pSoup-p19), and co-expressed with a nucleolus marker 35S:FIB2:mCherry in tobacco leaves (Chang et al., 2016) according to a previous study (Sparkes et al., 2006). The subcellular localization of CsMYB15 was observed via a confocal laser-scanning microscope (TCS SP6, Leica, Germany).

A yeast system was utilized to examine the transcriptional activity of CsMYB15 according to a previously described method (Ma et al., 2009). The coding sequences of CsMYB15 were cloned into the bait vector pGBKT7 by homologous recombination method using HiFi DNA Assembly Master Mix (NEB, Ipswich, USA) according to the manufacturer’s protocol. The CsMYB15-pGBKT7 + pGADT7-AD, pGBKT7-lam + pGADT7-T (negative control), and pGBKT7-53 + pGADT7-T (positive control) were transformed into Y2H strain yeast independently. After being cultured on SD/-Trp medium at 30 °C for 3 days, single colonies were selected and further amplified. Subsequently, the positive transformants were grown on SD/-Leu/-Trp and SD/-Leu/-Trp/-His/-Ade media, and the growth of all the transformants were observed after 3 days. Additionally, the galactosidase assay was performed by adding X-α-Gal to the medium according to a previously described method (Ma et al., 2009).

The CDS of CsMYB15 was cloned into pAML4 vector (containing cauliflower mosaic virus 35S promoter-driven GFP reporter module) together with CaMV35S promoter and nos terminator through Golden Gate method (Engler et al., 2014). The recombinant plasmid was transformed into Agrobacterium tumefaciens (strain GV3101) for further transformation. For juice sac transient expression, juice sacs were separated from ‘Lane late’ navel orange (Citrus sinensis Osbeck) fruits, and CsMYB15 was transiently over-expressed in juice sacs, as described in previous study (Shi et al., 2019). The transformed juice sacs were carefully collected at 6 days post infection. For kumquat (F. crassifolia Swingle) fruit transient expression, kumquat fruits at the green ripening stage (120-180 days after flowering) were selected for transformation. These fruits were infiltrated with A. tumefaciens carrying the CsMYB15 expression vector by using a sterile 1 ml hypodermic syringe according to the method described previously (Gong et al., 2021a). The infiltrated fruit sections were screened with GFP fluorescence and sampled at 5 days post infection by using a fluorescence microscope (Olympus, Japan). For citrus calli stable transformation, the healthy and plump calli of navel orange (Citrus sinensis Osbeck) were selected, and the transformation was performed according to a previously described method (Shi et al., 2020). The positive calli were selected by GFP fluorescence using a fluorescence microscope (Olympus, Japan), and then validated by PCR amplification.

Half of the juice sac, kumquat fruit, and citrus callus samples were immediately frozen with liquid nitrogen and stored at −80°C for RNA extraction and qRT-PCR analysis. The remaining samples were stoved in a bake oven for lignin content determination.

The lignin contents of the juice sacs, kumquat fruits, and citrus calli were measured by using a lignin assay kit (Solarbio, China) according to the manufacturer’s instruction (Yang et al., 2022), and each sample has three biological replicates.

Yeast one-hybrid assay was conducted as described previously (Gong et al., 2021b). The full-length CDS of CsMYB15 and promoter sequences of Cs4CL1, Cs4CL2, CsC3H, CsCCR2, CsPOD2, and CsPOD3 were cloned into pGADT7 and pAbAi vectors to produce prey construct of pGADT7:CsMYB15 and bait constructs of pAbAi:promoters, respectively. Then, the bait constructs were transformed into the yeast strain Y1H Gold to produce reporter strains, and their autoactivation was determined on SD/-Ura+ aureobasidin A (AbA) plates. The prey construct was introduced into the reporter strains, and pGADT7 empty vector was utilized as a negative control. The transformed yeast cells were cultured on SD/-Ura/-Leu/AbA medium for Y1H assay to examine the interaction between CsMYB15 and the promoters of Cs4CL1, Cs4CL2, CsC3H, CsCCR2, CsPOD2 and CsPOD3. The primers used for constructing Y1H vectors were listed in Supplementary Table S1 . The promoter sequences of Cs4CL1, Cs4CL2, CsC3H, CsCCR2, CsPOD2 and CsPOD3 were deposited in Genebank with the accession number of OQ985394 to OQ985399.

The electrophoretic mobility shift assay (EMSA) was conducted as described previously with minor modifications (Shi et al., 2020). Briefly, the MBP-CsMYB15 protein was expressed and purified, as described previously (Lu et al., 2018). The 5’ FAM-labeled oligonucleotide probes were directly synthesized and labeled by the Tianyi Huiyuan Company (Wuhan, China). The same oligonucleotides without labels were utilized as competitors. Probe information was provided in Supplementary Table S1 .

Dual luciferase transcriptional activity assay was performed using Nicotiana benthamiana leaves according to a previous report (Lu et al., 2018). Specifically, the the promoter sequence of Cs4CL2 was inserted into the upstream of LUC coding sequence in pGreen0800-LUC vector to produce reporter construct. The full-length CDS of CsMYB15 was cloned into pAML4 over-expression vector to produce 35S::CsMYB15 effector construct. The effector construct and reporter construct were respectively transformed into A. tumefaciens (strain GV3101 with pSoup-p19), and co-expressed in tobacco leaves according to a previously reported method (Hellens et al., 2005). The LUC activity was measured according to the method reported by Lu et al. (2018). All the primers were listed in Supplementary Table S1 .

The coding sequence of CsMYB15 was identified and cloned from ‘Lane late’ navel orange (Citrus sinensis Osbeck) to investigate its biological function. The coding sequence of CsMYB15 was 804 bp in length, and this coding sequence encoded a 268-amino acid protein. As shown in Figure 1A , the open reading frame of CsMYB15 was constructed with three exons. Then, the coding sequence of CsMYB15 was aligned with that of 8 other MYB genes with high homology. CsMYB15 protein was found to contain four conserved domains, including one R2-domain, one R3-domain, and two SG2 domains, indicating that CsMYB15 was an typical SG2-Type R2R3-MYB protein ( Figure 1B ).

To explore the phylogenetic relationship of CsMYB15, a phylogenetic tree was established with the amino acid sequences of 32 MYB proteins using the maximum likelihood method of MEGA. As shown in Figure 2 , the MYB genes were divided into 5 clades, including Clade I, Clade II, Clade III, Clade IV and Clade V, and the CsMYB15 was highly homologous to MYB15-like genes from Arabidopsis thaliana, Vitis vinifera, Solanum lycopersicum, Fragaria vesca, Prunus persica, and Malus domestica in Clade I. It was worth noting that CsMYB15 and AtMYB15 were clustered into a subclade with the closest genetic relationship and the highest homology. Further blast result revealed that CsMYB15 was the orthologous gene of cold tolerance-related AtMYB15.

To further investigate the role of CsMYB15 in low temperature-induced juice sac granulation, the expression of CsMYB15 under different degrees of low temperature-induced juice sac granulation was assessed by qRT-PCR. As shown in Figure 3A , the expression of CsMYB15 was significantly induced by juice sac granulation, and the induction of CsMYB15 was gradually increased with the increasing granulation degrees, indicating that CsMYB15 might be involved in the regulation network during juice sac granulation process. Interestingly, we also found that the expression of CsMYB15 was significantly induced under low temperature treatment ( Figure 3B ). Although the expression of CsMYB15 was increased first and then decreased under low temperature treatment, it was relatively higher at all treatment time points than at 0 h, suggesting a significant induction of CsMYB15 under low temperature stress. Further, we amplified the promoter sequence of CsMYB15 from ‘lane late’ navel orange, and analyzed the cis-elements involved in the promoter sequence. As shown in Figure 3C , the potential regulatory cis-elements were predicted, including low temperature responsive element, MYC recognition site, which have been predicted to be involved in low temperature responsiveness in many cases. We also predicted the light responsiveness elements, Box 4, GT-1, and G Box, and hormone responsiveness elements from the promoter of CsMYB15, indicating that the expression level of CsMYB15 might also been regulated by light and plant hormones. Additionally, qRT-PCR revealed that the expression level of 6 lignin biosynthesis genes were up-regulated under low temperature treatment ( Figure 3D ). Expect for the expression level of CsPAL2 was only induced at 3h, the expression levels of Cs4CL1, Cs4CL2, CsCCR1, CsCCR2, and CsPOD2 were increased first and then decreased, and the expression levels were relatively higher at all treatment time points than at 0 h, which was in line with the expression level of CsMYB15. In a word, these results indicating that CsMYB15 was induced by both juice sac granulation and low temperature treatment.

To explore the subcellular localization of CsMYB15 protein, CsMYB15-GFP fusion construct in pICH86988 vector was transiently co-expressed with a nucleolus marker (FIB2:mCherry) in tobacco leaves. As shown in Figure 4A , the green fluorescence of CsMYB15:GFP was localized to the nucleus, including both nucleolus and nucleoplasm. The GFP fluorescence of nucleolus was overlapped with the red fluorescence of FIB2:mCherry (a nucleolus marker), and the nucleolus was surrounded by the GFP fluorescence of nucleoplasm. A yeast system was adopted for assessing the transcriptional activity of CsMYB15. As shown in Figure 4B , the positive control and pGBKT7-CsMYB15 strains grew well on both SD/-Leu-Trp and SD/-Leu-Trp-His-Ade media, and these two strains turned blue on the medium containing X-α-gal, whereas the negative control strain failed to grow on SD/-Leu-Trp-His-Ade medium, indicating that CsMYB15 had transcriptional activation activity in yeast. Taken together, the above results suggested that CsMYB15 was a nuclear-localized protein with transcriptional activation activity.

The coding sequence of CsMYB15 was cloned from ‘Lane late’ navel orange, and individual transgenic experiments were conducted to explore the biological function of CsMYB15. Firstly, CsMYB15 was transiently expressed in the juice sacs isolated from ‘Lane late’ navel orange fruits. As shown in Figure 5B , qRT-PCR revealed that the expression level of CsMYB15 was up-regulated in the juice sac over-expressing CsMYB15. Comparing with that of wild type, the morphology of juice sacs transiently expressing CsMYB15 was slim, deformed, and granulated ( Figure 5A ). The lignin content in juice sacs over-expressing CsMYB15 was significantly higher than that in wide type ( Figure 5C ). These results were further verified by the transient expression of CsMYB15 in ‘Huapi’ kumquat (Fortunella crassifolia Swingle) fruits ( Figure S1 ).

Then, CsMYB15 was over-expressed in citrus calli through Agrobacterium-mediated stable transformation system. Two independent transgenic lines were selected through GFP fluorescence ( Figure 5D ). The expression of CsMYB15 in representative lines was validated by qRT-PCR ( Figure 5E ). As expected, the lignin content was significantly higher in the transgenic citrus calli over-expressing CsMYB15 than in wide type calli ( Figure 5F ). Taken together, these results indicated that CsMYB15 positively regulated lignin biosynthesis in citrus.

We speculated that CsMYB15 could directly bind to the promoters of lignin biosynthesis genes, thus affecting their expression, eventually regulating the lignin biosynthesis. Thus, 2000 bp sequences upstream the initiation codon were cloned as the promoters of Cs4CL1, Cs4CL2, CsC3H, CsCCR2, CsPOD2 and CsPOD3, and the Yeast one-hybrid assay (Y1H) was conducted to verify the interaction between CsMYB15 and the promoters of these genes. As shown in Figure 6A , clone CsMYB15 + proCs4CL2 was grown well on the SD-leu + 75 ng/μL AbAi (aureobasidin A) medium, indicating that CsMYB15 could bind to the promoter of Cs4CL2. However, CsMYB15 did not interact with the promoters of Cs4CL1 ( Figure S2 ), it did not interact with those of CsC3H, CsCCR2, CsPOD2 and CsPOD3, either (data not shown).

We predicted that CsMYB15 might bind to the MYB recognition sites (MRS, TGGTTG/A) on the Cs4CL2 promoter. To verify our prediction, the electrophoretic mobility shift assay (EMSA) was performed using purified MBP-CsMYB15 protein with a FAM-labeled Cs4CL2 promoter fragment (−51 to −89 bp region) used as a probe. As shown in Figure 6B , a shifted band was observed after the probe was incubated with MBP-MYB15, but this band became weaker under the actions of the corresponding competitors in a dosage-dependent manner. These results further demonstrated that CsMYB15 directly bound to the MRS cis-element of Cs4CL2 promoter in vitro.

In addition, a dual luciferase transcriptional activity assay (LUC) was performed in N. benthamiana leaf to reveal how CsMYB15 regulated the expression of Cs4CL2. The LUC: renillia (REN) ratio of leaf co-expressing CsMYB15 and Cs4CL2 promoter was significantly higher than that of the negative control, indicating that CsMYB15 acted as transcriptional activators of Cs4CL2 by directly interacting with the MRS elements ( Figures 6C, D ). Further, we detected the expression patterns of Cs4CL2 in the transgenic juice sac and citrus callus lines using qRT-PCR. As shown in Figures 6E, F , the expression level of Cs4CL2 was significantly higher in the transgenic plant lines over-expressing CsMYB15 than the wide type, which was further proof of the activation of CsMYB15 transcription factor on the Cs4CL2.