BEL1-LIKE HOMEODOMAIN 11 regulated chloroplast development and chlorophyll synthesis in tomato fruit

Authors: MENG, LANHUAN - China Agricultural University; FAN, ZHONGQI - South China Agricultural University; QIANG, ZHANG - Chongqing University; WANG, CUICUI - China Agricultural University; GAO, YING - China Agricultural University; DENG, YIKANG - China Agricultural University; ZHU, BENZHONG - China Agricultural University; ZHU, HONGLIANG - China Agricultural University; CHEN, JIANYE - South China Agricultural University; SHAN, WEI - South China Agricultural University; YIN, XUNREN - Zhejiang University; ZONG, SILIN - The Chinese University Of Hong Kong (CUHK); GRIERSON, DONALD - University Of Nottingham; Jiang, Cai-Zhong; LUO, YUNBO - China Agricultural University; FU, DA-QI - China Agricultural University

Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/5/2018
Publication Date: 4/16/2018
Citation: Meng, L., Fan, Z., Qiang, Z., Wang, C., Gao, Y., Deng, Y., Zhu, B., Zhu, H., Chen, J., Shan, W., Yin, X., Zong, S., Grierson, D., Jiang, C., Luo, Y., Fu, D. 2018. BEL1-LIKE HOMEODOMAIN 11 regulated chloroplast development and chlorophyll synthesis in tomato fruit. Plant Journal. 94(6):1126-1140. https://doi.org/10.1111/tpj.13924.
DOI: https://doi.org/10.1111/tpj.13924

Interpretive Summary: Chloroplasts are essential for converting light energy into chemical energy by photosynthesis and for the synthesis and metabolism of sugars and other compounds that provide resources for plant development and human health. Chloroplasts also play an important photosynthetic role in green fruits and other tissues, in addition to leaves, and ultimately contribute to fruit growth, development, and quality by increasing their photosynthetic capacity, leading to starch accumulation (Klee, 2010). Unlike those in leaves, fruit chloroplasts are often converted into colored chromoplasts, a process involving degradation of chlorophyll and accumulation of lycopene and ß-carotene during tomato fruit ripening. Chloroplast development and chlorophyll synthesis in plant leaves have been well studied, and key structural genes for chlorophyll synthesis have been identified. Overexpression or silencing of Chl biosynthesis genes can significantly affect Chl levels in plants, thereby altering their photosynthetic ability and fruit quality. The accumulation of Chl in fruits is influenced by many factors. Chloroplast and chromoplast development can also be influenced by biosynthetic processes. But the factors regulating these processes are poorly understood. Overexpression of Golden like 2 (SlGLK2) enhances Chl level by increasing plastid number and size and generates dark-green fruits. Fruits of high-pigment 1 and 2 (hp-1 and hp-2) mutants have more chloroplasts and increased the size of plastid compartments, resulting in fruits with higher accumulation of chlorophyll and carotenoids compared to control fruits. Overexpression of SlAPRR2-LIKE results in increased size and number of plastids and higher concentrations of Chl and carotenoids in transgenic tomato fruits. A dominant gain-of-function mutation of knotted 2 (TKN2) displays similar chloroplast development to the SlGLK2 overexpression lines in the fruits. Although these TFs appear to play an important role in the control of chloroplast development in tomato fruit, the mechanism that regulates chloroplast number and size is still largely unknown. On the other hand, plant hormones also regulate the development of chloroplasts. Down-regulation of an auxin-response-factor (DR12) produces dark green tomato fruits. It has been shown that an ethylene response factor, CitERF13, was involved in degradation of chlorophyll during de-greening in citrus fruits. Thus, Chl metabolism in fruits is also regulated by a series of hormone-related TFs. Genes that delay fruit ripening generally inhibit the degradation of Chl during fruit ripening. Therefore, screening ripening-associated genes is an effective way to study Chl synthesis and degradation in fruits. In order to identify additional TFs with putative roles in fruit development and ripening, we performed functional characterization of seven Homeobox-containing TFs that were highly expressed in the fruits during tomato fruit ripening using virus-induced gene silencing (VIGS). Silencing one of these TFs, BEL1-LIKE HOMEODOMAIN 11 (SlBEL11), led to high accumulation of Chl in immature tomato fruits. The enhanced Chl accumulation in the fruits was verified using RNA interference (RNAi) in tomato. Further experiments demonstrated that SlBEL11 negatively regulated the expression of genes involved in Chl synthesis chloroplast development in unripe tomato fruit. Taken together, our findings demonstrate that SlBEL11 plays an important role in controlling chloroplast development and chlorophyll synthesis in tomato fruit. Our results expand our understanding of the regulatory network that controls Chl metabolism and plastid development during tomato fruit ripening.

Technical Abstract: Chloroplast development and chlorophyll content and metabolism in unripe tomato contribute to the growth and development of the fruit, and also the ripe fruit quality, but the mechanism is poorly understood. In this work, seven homeobox-containing transcription factors (TFs) with specific ripening-associated expression patterns were studied by virus-induced gene silencing (VIGS) technology. We found that inhibiting the expression of BEL1-LIKE HOMEODOMAIN11 (SlBEL11, Solyc11g068950.1.1) significantly increased the chlorophyll content in tomato unripe fruit, and this phenotype was confirmed by RNAi-SlBEL11 in transgenic tomatoes. RNA-seq of RNAi-SlBEL11 fruit at the mature green showed that 48 genes involved in chlorophyll biosynthesis, photosynthesis and chloroplast development were significantly up-regulated compared to wild type, including 5 key genes involved in the chlorophyll biosynthesis pathway, and 19 chlorophyll-binding a/b proteins genes (CAB) and other genes involved in chloroplast development. Genomic global scanning combined with RNA-seq differential gene analysis showed that 22 of these 48 genes could be the direct target genes of SlBEL11 protein, including 3 chlorophyll biosynthesis genes such as POR (Protochlorophyllide reductase), CHLH (Magnesium chelatase H subunit), CAO (Chlorophyllide a oxygenase), 6 CAB (chlorophyll-binding a/b proteins) genes, TKN2 (knotted 2; Solyc02g081120) and APRR2-like related to chloroplast development. EMSA and ChIP-qPCR assays verified that SlBEL11 protein directly interacted with the conserved promoter regions of TKN2, CAB (Solyc02g071010) and POR (Solyc12g013710), which are involved in the chloroplast development and chlorophyll accumulation. Taken together, our findings demonstrate that SlBEL11 plays an important role in controlling chloroplast development and chlorophyll synthesis in tomato fruit.