A MADS-Box Gene CiMADS43 Is Involved in Citrus Flowering and Leaf Development through Interaction with CiAGL9

Abstract

MADS-box genes are involved in various developmental processes including vegetative development, flower architecture, flowering, pollen formation, seed and fruit development. However, the function of most MADS-box genes and their regulation mechanism are still unclear in woody plants compared with model plants. In this study, a MADS-box gene (CiMADS43) was identified in citrus. Phylogenetic and sequence analysis showed that CiMADS43 is a GOA-like Bsister MADS-box gene. It was localized in the nucleus and as a transcriptional activator. Overexpression of CiMADS43 promoted early flowering and leaves curling in transgenic Arabidopsis. Besides, overexpression or knockout of CiMADS43 also showed leaf curl phenotype in citrus similar to that of CiMADS43 overexpressed in Arabidopsis. Protein-protein interaction found that a SEPALLATA (SEP)-like protein (CiAGL9) interacted with CiMADS43 protein. Interestingly, CiAGL9 also can bind to the CiMADS43 promoter and promote its transcription. Expression analysis also showed that these two genes were closely related to seasonal flowering and the development of the leaf in citrus. Our findings revealed the multifunctional roles of CiMADS43 in the vegetative and reproductive development of citrus. These results will facilitate our understanding of the evolution and molecular mechanisms of MADS-box genes in citrus.

Keywords: CiAGL9; CiMADS43; citrus; flowering; leaf.

Figure 1

Characterization of the CiMADS43. (a) Multiple sequence alignment of CiMADS43 protein and its homolog proteins. CiMADS43, Citrus sinensis (XP_006468501.1). PvDEFHL21, Pistacia vera (XP_031278613.1). VrFBPL24, Vitis riparia (XP_034698718.1). VvFBP24, Vitis vinifera (XP_010648959.1). PgFBP24, Punica granatum (XP_031400121.1). SoFBPL24, Syzygium oleosum (XP_030467505.1). AtABS, Arabidopsis thaliana (NP_001330404.1). AtGOA, Arabidopsis thaliana (NP_174399.2). (b) Phylogenetic analysis of MADS-box proteins from citrus and other plants. The gene ID of all MADS-box proteins were listed in Table S1. The red triangle indicates the citrus GORDITA (GOA)-like Bsister gene and the black triangle indicates the citrus ABS-like Bsister gene. (c) Subcellular localization of CiMADS43 in tobacco leaves. Green fluorescent protein (GFP) fused to the C-terminal region of CiMADS43, and the fusion protein was driven by 35S promoter. Red fluorescent protein (RFP) label was used as a nuclear marker driven by 35S promoter, and 35S::GFP was used as positive control. Scale bar = 5 0 µm. (d) Analysis of CiMADS43 transcription activation. CiMADS43 represents a full-length protein, and CiMADS43MIK was a truncated protein that the C-terminal region was removed, containing M domain, I domain and K domain. CiMADS43MI indicates the N-terminal region of CiMADS43 protein including M domain and I domain, while CiMADS43KC indicates the C-terminus of CiMADS43 protein including K domain and C domain.

Figure 2

Ectopic expression of CiMADS43 caused early flowering and leaf curling in transgenic Arabidopsis. (a) CiMADS43 transgenic Arabidopsis significantly earlier flowering than the control. Two transgenic lines (CiMADS43-OE5 and CiMADS43-OE7) were selected as representatives of 12 transgenic lines for evaluating MADS43 function. (b) Statistical analysis of the flowering days of the CiMADS43 transgenic Arabidopsis. (c) Statistical analysis of the number of rosette leaves when the CiMADS43 transgenic Arabidopsis and the control begin to flowering. (d) Two types of curling rosette leaf from CiMADS43 transgenic Arabidopsis. Type 1: all rosette leaves except cotyledons severely curly. Type 2: only some rosette leaves slightly curly. Arranged from left to right according to different stages of leaf development, with the cotyledons on the far left. (e,f) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of Arabidopsis flowering-related genes (AtFT and AtAP1) in the CiMADS43 transgenic Arabidopsis. (g,h) qRT-PCR analysis of Arabidopsis leaf development related genes (CLF and STM). Scale bar = 0.5 cm. Statistically significant is marked with asterisk(s) (* p < 0.05; ** p < 0.01; and *** p < 0.001; Student’s t-test).

Figure 3

Overexpression and knockout of CiMADS43 in lemon. (a) Wild type lemon. (b–d) The phenotype of three overexpressed citrus transgenic lines with CiMADS43. (e) qRT-PCR analysis of citrus leaf development related genes (CLF, STM, and FIE) in CiMADS43 overexpression lines. (f,g) Phenotypic analysis of CiMADS43 knockout lines by CRISPR/Cas9 system. (h) DNA sequencing analysis of gRNA target in CiMADS43 knockout lines. The red box indicates the position of the gRNA target. Double peaks appears due to the deletion of three nucleotides adjacent to the PAM (Protospacer Adjacent Motif) region. (i,j) The three-dimensional structure of CiMADS43 protein (i) and the residual protein which knocked out three bases by CRISPR/Cas9 system (g). The red arrow indicates the location of the change. (k) qRT-PCR analysis of citrus leaf development related genes (CLF, STM, and FIE) in CiMADS43 knockout lines. Scale bar = 0.2 cm. Statistically significant is marked with asterisk(s) (** p < 0.01 and *** p < 0.001, Student’s t-test).

Figure 4

The interaction between CiAGL9 and CiMADS43 protein. (a) Multiple sequence alignment of CiAGL9 protein and its homolog proteins. CiAGL9, Citrus sinensis (NP_001306995.1). AtAGL9, Arabidopsis (AAC00586.1). BnAGL9, Brassica napus (XP_022545908.1). MdAGL9, Malus domestica (XP_028952174.1). PtAGL9, Populus trichocarpa (XP_024453001.1). GmAGL9, Glycine max (NP_001242742.2). VvAGL9, Vitis vinifera (NP_001268114.1). (b) Phylogenetic analysis of AGL9 proteins of citrus and other plants. The protein ID of all AGL9 proteins were listed in Table S1. (c) Y2H assays between CiMADS43MIK and CiAGL9 proteins. (d) Pull-down assay of the interaction between CiMADS43 and CiAGL9. (e) Bimolecular fluorescence complementation (BiFC) analysis of protein interactions between CiMADS43 and CiAGL9 in tobacco leaf epidermis cells. CiMADS43-YN + CiAGL9-YC, coexpression of 35S::CiMADS43-nYFP and 35S::CiAGL9-cYFP. CiMADS43-YN + YC and CiAGL9-YC + YN were used as the negative control. Scale bar = 25 µM.

Figure 5

Expression patterns and promoter cis-elements analysis. (a,b) qRT-PCR analysis of CiMADS43 and CiAGL9 in different tissues. (c) The 2.0 kb CiMADS43 promoter was analyzed by the PlantCARE software. Boxes with different colors represent different elements. The sequences and cis-elements were listed in Table S3.

Figure 6

CiAGL9 binds to the CiMADS43 promoter. (a) Subcellular localization of CiAGL9 in tobacco leaves. GFP fused to the C-terminal region of CiAGL9, and the fusion protein was driven by 35S promoter. 4′,6-diamidino-2-phenylindole (DAPI) staining showed the location of the nucleus and 35S::GFP was used as the positive control. Scale bar = 25 µM. (b) Sequence of MADS-box protein binding site. (c) The interaction between the CiMADS43 promoter and CiAGL9 by yeast one-hybrid assay. pCiMADS43: 482 bp CiMADS43 promoter sequence was cloned into pAbAi vector. AD: pGADT7 empty vector. CiAGL9-AD: CiAGL9 fused with GAL4-AD. (d) Schematic diagrams of vectors used for the dual-luciferase (LUC) assay. (e) CiAGL9 activated the expression of CiMADS43. LUC reporter system was used in tobacco leaves. The relative LUC/REN were measured after 2 days of Agrobacterium infiltration. Statistically significant is marked with asterisk(s) (*** p < 0.001, Student’s t-test).

Figure 7

Ectopic expression of CiAGL9 causes slightly early flowering and leaf curling in transgenic Arabidopsis. (a) The phenotype of CiAGL9 transgenic Arabidopsis. Two transgenic lines (CiAGL9-OE6 and CiAGL9-OE17) were selected as representatives of 17 transgenic lines for evaluating CiAGL9 function. Scale bar = 0.5 cm. (b) Statistical analysis of flowering days of the transgenic Arabidopsis and the control. (c) Statistical analysis of rosette leaves number of the transgenic Arabidopsis and the control at flowering stage. (d,e) qRT-PCR analysis of Arabidopsis flowering-related genes (AtFT and AtAP1). (f,g) qRT-PCR analysis of Arabidopsis leaf development related genes (CLF and STM). Statistically significant was marked with asterisk(s) (** p < 0.01, and *** p < 0.001, Student’s t-test).

Figure 8

The expression patterns of CiMADS43 and CiAGL9 during buds and leaves development in citrus. (a) The seasonal expression changes of CiMADS43 and CiAGL9 in sweet orange buds. April is the flowering period of sweet orange. (b) The development process of sweet orange leaves was divided into six periods (S1–S6) from small to large. Scale bar = 1 cm. (c,d) qRT-PCR of CiMADS43 and CiAGL9 in the six periods of sweet orange leaf development.