Plant Physiology| 湖南农业大学园艺学院邹学校院士团队刘峰教授等揭示CaBBX10调控辣椒色素合成

叶绿素和类胡萝卜素是直接影响辣椒（Capsicum annuum L.）果实经济价值的两种重要光合色素。然而，在辣椒果实发育过程中调控叶绿素和类胡萝卜素积累的协调机制仍不清楚。近日，湖南农业大学园艺学院邹学校院士团队刘峰教授等揭示CaBBX10调控辣椒色素合成，相关结果以The transcription factor CaBBX10 promotes chlorophyll and carotenoid pigment accumulation in Capsicum annuum fruit发表在Plant Physiology。

在这项研究中，辣椒B-box 10（CaBBX10）被发现是一个候选枢纽转录因子，在辣椒果实发育早期起双重作用。CaBBX10的病毒诱导基因沉默和过表达实验表明，该转录因子促进辣椒果实中叶绿素和类胡萝卜素的积累。进一步分析显示，CaBBX10直接结合到叶绿素和类胡萝卜素生物合成途径中镁螯合酶D亚基（CaCHLD）和番茄红素合酶（CaPSY1）的启动子，从而激活其表达。此外，生光形态建成因子CaCOP1被发现能够与CaBBX10发生物理相互作用并导致其降解。因此，CaBBX10可能作为连接叶绿素和类胡萝卜素生物合成与光信号传导的关键纽带。总之，本研究揭示了一个同时促进辣椒果实中叶绿素和类胡萝卜素积累的复杂转录调控机制。

湖南农业大学园艺学院邹学校院士团队博士后王瑾为第一作者，湖南农业大学刘峰教授、朱凡教授和熊程副教授为共同通讯作者。

Figure 1. Basic information of CaBBX10 gene. A) Expression analysis of the CaBBX family at different pepper fruit developmental stages. The lighter the color (closer to yellow) and the larger the circle, the higher the expression level; the darker the color (closer to blue) and the smaller the circle, the lower the expression level. B) Relative expression analysis of CaBBX10 in different tissues of pepper. C) Phylogenetic tree analysis of CaBBX10 protein and its homologs in other species, with 1,000 bootstraps by neighbor-joining (NJ) method, the scale bar represent branch length, substitution per site: 20% changes were observed between 2 sequence. D) Conserved domain analysis revealed 2 B-box domains represented by yellow rectangles. E) Gene structure analysis of CaBBX10 revealed exons (purple rectangles) and introns (black lines), with a 960-bp uninterrupted open Reading frame (ORF). F) Schematic of effector and reporter constructs used for transcriptional activation activity analysis. The LUC levels were normalized using the REN gene, which was regulated by the 35S promoter. GAL4DB, GAL4 DNA-binding domain; 5×GAL4-TATA, 5 GAL4-binding sites; REN, Renilla; LUC, luciferase. G) Relative LUC/REN ratio shows the transcriptional activation activity of CaBBX10, (one-way ANOVA, **P < 0.01, SD; n = 3). H) Subcellular localization analysis of CaBBX10 in Nicotiana benthamiana. Pepper eGFP-CaBBX10 and empty eGFP plasmids were transiently expressed in transgenic N. benthamiana (expressed with nucleus-located mCherry), and 35S::eGFP served as the positive control.

Figure 2. Virus-induced gene silencing (VIGS) of CaBBX10. A) Phenotypes of pepper fruits after silencing CaBBX10. The upper row is the phenotype of mature green (MG) fruit stage after silencing CaBBX10, and the bottom row is the phenotype of red ripe (RR) fruit stage after silencing CaBBX10. Image was digitally extracted for comparison. B) The relative expression of CaBBX10 in TRV2:00, TRV2:CaBBX10#1 and TRV2:CaBBX10#4 fruits. C) The total chlorophyll content in mature green fruits of TRV2:00, TRV2:CaBBX10#1 and TRV2:CaBBX10#4, FW: fresh weight of sample. D) The total carotenoid content in red ripe fruits of TRV2:00, TRV2:CaBBX10#1 and TRV2:CaBBX10#4. E) The relative content of lycopene, α-carotene, lutein, β-carotene, and violaxanthin in mature red fruits of TRV2:00, TRV2:CaBBX10#1 and TRV2:CaBBX10#4. F) The relative expression analysis of chlorophyll biosynthetic genes in the mature green fruits of TRV2:00, TRV2:CaBBX10#1 and TRV2:CaBBX10#4. G) The relative expression analysis of carotenoid biosynthetic genes in the red ripe fruits of TRV2:00, TRV2:CaBBX10#1 and TRV2:CaBBX10#4. All the data in this figure are expressed as mean ± SDs (n = 3), and statistical significance is denoted by *P < 0.1, **P < 0.01, ***P < 0.001 (one-way ANOVA).

Figure 3. The phenotypes of CaBBX10-OE and WT fruits. A) The phenotype of CaBBX10-OE (OE) and WT (Alisa Craig) fruits at 2 developmental stages (MG and RR), the ruler = 2 cm. B) The cross-section of CaBBX10-OE and WT fruits, the ruler = 2 cm. C) Analysis of the ultrastructure of chloroplasts in mature green fruit using transmission electron microscopy, ×10.0 K, the ruler = 1.0 μm. D) The total carotenoid content in the fruits of WT and CaBBX10-OE lines. E) The total chlorophyll content in the fruits of WT and CaBBX10-OE lines. F) The content of violaxanthin, lycopene, lutein, α-carotene, and β-carotene in the fruits of WT and CaBBX10-OE lines. G) The relative expression analysis of chlorophyll biosynthetic genes in the mature green fruits of CaBBX10-OE lines and WT lines. H) The relative expression analysis of carotenoid biosynthetic genes in the RR fruits of CaBBX10-OE lines and WT. All the data in this figure are expressed as mean ± SDs (n = 6), and statistical significance is denoted by *P < 0.1, **P < 0.01, ***P < 0.001 (one-way ANOVA).

Figure 4. Identification of potential DNA-binding sites and cis-elements bound by CaBBX10. A) Western blot detected nuclear protein of CaBBX10 by specific antibody, and Anti-H3(H3C15) as Actin for nuclear protein detection. B) Consensus sequence of cis-element bound by CaBBX10 was obtained from peak calling analysis on CUT&Tag data using HOMER. The cis-element is a G-box sequence motif. C) Genome-wide distribution of 28,232 DNA-binding peaks of CaBBX10. Distribution (intergenic regions, promoters, exons or introns) of peaks in various elements of the pepper reference genome. D) Chlorophyll biosynthetic pathway in pepper. E) Carotenoid biosynthetic pathway in pepper. F) KEGG (Kyoto encyclopedia of genes and genomes) pathway enrichment analysis on the target genes of CaBBX10. G) Gene Ontology (GO) enrichment analysis on the promoter of genes targeted by CaBBX10. p.adjust, adjusted P-value.

Figure 5. CaBBX10 binds to the CaCHLD promoter and activates its expression. A) Schematic showing CaCHLD promoter and electrophoretic mobility shift assay (EMSA) probes. Blue dots represent the cis-element, G-box. CHLD-wt is a wild-type probe synthesized based on the CaCHLD promoter sequence. CHLD-mt is a mutant probe, whose cis-element sequence was replaced with GGGGG, the red font indicates the specific binding site of CaBBX10. B) The binding of CaBBX10 to CaCHLD promoter was detected thought the yeast one-hybrid assay. The effector vector pGADT7-CaBBX10 was introduced into the yeast strain Y1HGold together with the reporter vector pAbAi-CaCHLD. The combination of reporter vector and empty vector pGADT7 was used as the negative control. The transformants were grown on a selective medium (SD/-Leu) supplemented with or without 80 ng/mL AbA. C) EMSA assay was used to detect the interaction between CaBBX10 and the G-box motif in the CaCHLD promoter. The core binding sequence is marked in bold. “+” and “−” denote presence and absence, respectively. D) Structure diagram for luciferase complementary imaging experiments. The effector vector pGreenII62SK-CaBBX10 and the reporter vector pGreenII0800-CaCHLD-Pro. E) The LUC/REN ratio indicates the binding of CaBBX10 to CaCHLD promoter. The LUC/REN ratio of empty effector and reporter-transformed N. benthamiana leaves was set to 1. LUC, firefly luciferase activity. REN, Renilla luciferase activity. Data were expressed as mean ± SD; n = 6. ***P < 0.001 (Student's t-test). F) Imaging of LUC activity of N. benthamiana leaf transiently infected by CaBBX10 and CaCHLD.

Figure 6. CaBBX10 binds to the CaPSY1 promoter and activates its expression. A) Schematic showing CaPSY1 promoter and EMSA probes. PSY1-wt is a wild-type probe synthesized based on the CaPSY1 promoter sequence. PSY1-mt is a mutant probe, whose cis-element sequence was replaced with GGGGG, respectively. The red font indicates the specific binding site of CaBBX10. B) The binding of CaBBX10 to CaPSY1 promoter was detected using the yeast one-hybrid assay. The transformants were grown on selective medium (SD/-Leu) supplemented with or without 75 ng/mL AbA. C) EMSA assay was used to detect the interaction between CaBBX10 and the G-box motif of CaPSY1 promoter. D) Structure diagram for luciferase complementary imaging experiments. Effector vector pGreenII62SK-CaBBX10. Reporter vector pGreenII0800-CaPSY1-Pro. E) The LUC/REN ratio indicates the binding of CaBBX10 to CaPSY1 promoter. Data were expressed as mean ± SD; n = 6. ***P < 0.001 (Student's t-test). F) Imaging of LUC activity of N. benthamiana leaf transiently infected by CaBBX10 and CaPSY1.

Figure 7. Verification of the interaction between CaBBX10 protein and CaCOP1. A)  In vivo detection of ubiquitinated CaBBX10. B) The interaction between CaBBX10 and CaCOP1 detected using the yeast two-hybrid system. The empty vectors PGBKT7, PGADT7, PGBKT7-T, and PGBKT7-lam were used as negative controls, and PGBKT7-T and PGBKT7-53 were co-transfected as positive controls. C) Co-immunoprecipitation (Co-IP) of CaBBX10 and CaCOP1. CaBBX10-FLAG was co-expressed with the control or CaCOP1-MYC in rice protoplasts. Total protein was extracted and incubated with anti-MYC magnetic beads. D) LUC complementary imaging was used to detect LUC signals from the interaction between CaBBX10 and CaCOP1. E) Relative expression of NbCOP1-silenced N. benthamiana plants. Data were expressed as mean ± SD; n = 6. ***P < 0.001 (Student's t-test). F) TRV2:NbCOP1-silenced N. benthamiana were used for CaBBX10-FLAG expression. The plants co-infected with TRV1 and empty TRV2 were used as controls. The accumulation of CaBBX10 was detected using immunoblotting with anti-FLAG antibody. Anti-actin was used as the loading control.