Citrus sinensis MYB Transcription Factor CsMYB85 Induce Fruit Juice Sac Lignification Through Interaction With Other CsMYB Transcription Factors

Abstract

Varieties of Citrus are commercially important fruits that are cultivated worldwide and are valued for being highly nutritious and having an appealing flavor. Lignification of citrus fruit juice sacs is a serious physiological disorder that occurs during postharvest storage, for which the underlying transcriptional regulatory mechanisms remain unclear. In this study, we identified and isolated a candidate MYB transcription factor, CsMYB85, that is involved in the regulation of lignin biosynthesis in Citrus sinensis, which has homologs in Arabidopsis and other plants. We found that during juice sac lignification, CsMYB85 expression levels increase significantly, and therefore, suspected that this gene may control lignin biosynthesis during the lignification process. Our results indicated that CsMYB85 binds the CsMYB330 promoter, regulates its expression, and interacts with CsMYB308 in transgenic yeast and tobacco. A transient expression assay indicated that Cs4CL1 expression levels and lignin content significantly increased in fruit juice sacs overexpressing CsMYB85. At4CL1 expression levels and lignin content were also significantly increased in Arabidopsis overexpressing CsMYB85. We accordingly present convincing evidence for the participation of the CsMYB85 transcription factor in fruit juice sac lignification, and thereby provide new insights into the transcriptional regulation of this process in citrus fruits.

Keywords: Citrus sinensis; CsMYB85; juice sacs; lignification; postharvest.

FIGURE 1

CsMYB85 sequence and phylogenetic analysis. (A) Alignment of the conserved R2R3 domains in CsMYB85 with those in AtMYB42, AtMYB85, AtMYB58, AtMYB63, AtMYB4, CsMYB330, and CsMYB308. (B) Phylogenetic tree for CsMYB85 and MYB transcription factors from other plants: Populus trichocarpa, Arabidopsis thaliana, Zea mays, and Oryza sativa. The accession numbers of the transcription factor proteins are as follows: PtMYB46 (XP_024465368.1), ZmMYB109 (NP_001241859.1), OsMYB83 (XP_015619488.1), AtMYB46 (AT5G12870), AtMYB83 (At3g08500), CsMYB85 (XP_006477265.1), AtMYB42 (AT4G12350), AtMYB85 (AT4G22680), AtMYB20 (AT4G66230), CsMYB330 (NC_023049), AtMYB63 (AT1G79180), AtMYB58 (AT1G16490), ZmMYB1 (P20024.1), CsMYB308 (NC_023053), AtMYB32 (AT4G34990), AtMYB4 (AT4G38620), ZmMYB31 (NP_001105949), AtMYB75 (AT1G56650), AtMYB61 (AT1G09540), AtMYB103 (AT1G63910), and AtMYB26 (AT3G13890) The phylogenetic tree was constructed using DNAMAN v. 6.0.

FIGURE 2

Relative expression of the CsMYB85 gene at different stages of citrus fruit granulation. CsMYB85 expression was determined by real-time qRT-PCR and normalized to the CsActin reference gene. Means were obtained from four biological replicates. Error bars represent standard errors and were determined using Duncan’s multiple range test (α = 0.05) in SAS software (SAS Institute, Cary, NC, United States). The ‘a–d’ above each column indicates P < 0.05 and the same letter indicates that the difference is not significant.

FIGURE 3

Subcellular localization and transcriptional activation of CsMYB85. (A) Subcellular localization of CsMYB85 in tobacco leaf cells. 35S:GFP was localized in both the nucleus and cytoplasm of tobacco leaf cells. In contrast, CsMYB330:GFP was observed only in the nucleus. 4’,6-Diamidino-2-phenylindole (DAPI) signals were localized only in the nucleus. Merged images show GFP and DAPI colocalization. Bar = 20 μm. (B) Transcriptional activation of CsMYB85 in yeast cells. The full-length coding sequence of CsMYB85 was inserted into the pGBKT7 (BD) vector. The pGADT7 (AD) vector and either BD-CsMYB85 or BD were transformed into cells of the Y2HGold yeast strain. Yeast cells containing AD and BD vectors were used as negative controls. The yeast was grown on SD media lacking –Leu/–Trp (–L/–T) or –Leu/–Trp/–His (–L/–T/–H) for 3 days at 30°C. Results were obtained from three independent transformation experiments.

FIGURE 4

Interactions between CsMYB85 and the promoters of lignin biosynthesis-related genes. (A) Interactions of CsMYB85 with the promoters of CsMYB330, CsMYB308, Cs4CL1, Cs4CL2, Cs4CCoAOMT1, CsHCT, CsPAL1, and CsPAL2 were identified using Y1H assays. The promoters were inserted into pHIS2 vectors. Empty AD and constructed pHIS2 vectors were transfected into yeast Y187 cells, which were grown on SD/–L/–T/–H dropout medium containing various concentrations of 3-amino-1,2,4-triazole (3-AT) to suppress background histidine leakiness. Numbers in the lower-right corner of the images indicate the optimal 3-AT concentrations. Results were obtained from three independent transformation experiments. (B) Activation of the CsMYB330 promoter by CsMYB85 was assayed transiently in Nicotiana benthamiana leaves using an effector and reporter system. The effector and reporter constructs are shown in the schematic diagram. The reporter vector contained LUC normalization and GUS reporter genes driven by the CaMV35S and CsMYB330 promoters, respectively. The effector vector contained a CsMYB85 gene under control of the CaMV35S promoter. T-R, terminator; Boxes, various DNA sequences. (C) Transcriptional activity of CsMYB85 was analyzed using a GUS reporter gene driven by the CsMYB330 promoter. The GUS/LUC ratio in leaves transformed with the empty vector (control) harboring the CsMYB330 promoter was set to 1. Error bars represent standard errors. Means were obtained from five biological replicates. Student’s t-test: ^∗∗P < 0.01.

FIGURE 5

Interaction between CsMYB85 and CsMYB308 determined using a yeast two-hybrid assay. Analyses of CsMYB85 and CsMYB308 interactions in yeast cells. The CsMYB85 and CsMYB308 coding regions were cloned into the AD and BD vectors, respectively. AD-T containing BD-53 and AD containing BD were used as positive and negative controls, respectively. Yeast clones were grown on SD media lacking –Leu/–Trp or –Leu/–Trp/–His for 3 days at 30°C. Results were obtained from three independent transformation experiments.

FIGURE 6

Interaction between CsMYB85 and CsMYB308 determined using a BiFC assay. CsMYB85 and CsMYB308 were fused with C-terminal yellow fluorescent protein (YFP^C) and N-terminal YFP (YFP^N), respectively, and driven by the CaMV35S promoter. The vectors were introduced into Nicotiana benthamiana leaves by agroinfiltration. YFP fluorescent signals were imaged under a confocal microscope 72 h after infiltration. Panels from left to right: YFP signal (YFP), 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining signal, and merged YFP and DAPI signals. Empty vectors (YFP^C and YFP^N) and the aforementioned vectors were co-expressed in epidermal cells as a control. Bar = 50 μm.

FIGURE 7

Lignin content and gene expression analyses in transiently overexpressing juice sacs. (A) Expression levels of the CsMYB85 gene in transiently overexpressing juice sac tissues were determined using real-time qRT-PCR. Empty SK vector was used as a control. The CsMYB85 expression level was set to 1 in the cintrol. Expression of the CsActin reference gene (NW_006256915.1) was used to normalize the CsMYB85 expression level. Error bars represent the standard error of four biological replicates. Student’s t-test: ^∗∗P < 0.01. (B) Transcription levels of CsMYB330, CsMYB308, and Cs4CL1 (NW_006257196.1) in transiently overexpressing juice sac tissues determined by real-time qRT-PCR and normalized to the CsActin reference gene. Expression levels of CsMYB330, CsMYB308, and Cs4CL1 in the SK control were set to 1. Error bars represent the standard error of four biological replicates. Student’s t-test: ^∗∗P < 0.01. (C) Lignin content in juice sac tissues. Means represent the averages of three replicate lignin content determinations in transiently overexpressing juice sac tissues. Error bars represent standard errors. Student’s t-test: ^∗∗P < 0.01.

FIGURE 8

CsMYB85 overexpression increased lignin content in Arabidopsis thaliana. (A) Six-week-old seedlings of wild-type A. thaliana (WT, left), MYB85-OE#1 (CsMYB85 overexpression line 1, center), and MYB85-OE#2 (CsMYB85 overexpression line 2, right). Note the smaller leaves of MYB85-OE#1 and MYB85-OE#2 plants relative to those of the WT. (B) Expression levels of At4CL1 (AT1G51680), AtMYB58, and AtMYB4 were increased in CsMYB85-OE#1 and MYB85-OE#2 plants compared with those in WT plants. At4CL1, AtMYB58, and AtMYB4 expression levels in the WT were set to 1. AtActin was used as an internal reference gene. Error bars represent the standard errors of four biological replicates. Student’s t-test: ^∗∗P < 0.01. (C) Lignin content in WT, CsMYB85-OE#1, and MYB85-OE#2 plants. Data are the averages of four biological replicates in WT, CsMYB85-OE#1, and MYB85-OE#2 plants. Error bars represent standard errors. Student’s t-test: ^∗∗P < 0.01.

FIGURE 9

The brief model for regulatory network of Citrus sinensis fruit juice sacs lignification. The MYB transcription factors can activate or inhibit the expression of lignin biosynthesis related genes in Citrus sinensis fruit juice sacs. The Cs4CL1 gene directly involved in lignin biosynthesis. The transcription factors, CsMYB330 and CsMYB330, regulated expression of Cs4CL1 gene, directly. The CsMYB85 transcription factor can interactive with CsMYB308 and regulated expression of CsMYB330 gene, and indirectly regulated expression of Cs4CL1 gene.