SlARF10, an auxin response factor, is involved in chlorophyll and sugar accumulation during tomato fruit development

Journal of Experimental Botany, Vol. 69, No. 22 pp. 5507–5518, 2018 doi:10.1093/jxb/ery328 Advance Access publication 13 September 2018 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) RESEARCH PAPER SlARF10, an auxin response factor, is involved in chlorophyll and sugar accumulation during tomato fruit development Yujin Yuan1,†, Lihua Mei1,†, Mengbo Wu1,†, Wei Wei2, Wei Shan2, Zehao Gong1, Qian Zhang3, Fengqing Yang3, Fang Yan1, Qiang Zhang1, Yingqing Luo1, Xin Xu1, Wenfa Zhang1, Mingjun Miao4, Wangjin Lu2, Zhengguo Li1 and Wei Deng1,* School of Life Science, Chongqing University, Chongqing 400044, China State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and Vegetables, College of Horticulture, South China Agricultural University, Guangzhou 510642, China 3 School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044, China 4 Horticulture Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu 610066, China 2 * Correspondence: These authors contributed equally to this work. † Received 12 February 2018; Editorial decision 5 September 2018; Accepted 11 September 2018 Editor: Fabrizio Costa, Fondazione Edmund Mach, Italy Abstract The photosynthesis of green tomatoes contributes to fruit growth and carbon economy. The tomato auxin response factor 10 (SlARF10) belongs to the ARF family and is located in nucleus. In this study, we found that SlARF10 was highly expressed in green fruit. Overexpression of SlARF10 in fruit produced a dark-green phenotype whilst knockdown by RNAi produced a light-green phenotype. Autofluorescence and chlorophyll content analyses confirmed the phenotypes, which indicated that SlARF10 plays an important role in chlorophyll accumulation. Overexpression of SlARF10 positively affected photosynthesis in both leaves and fruit. Furthermore, SlARF10-overexpression lines displayed improved accumulation of starch, fructose, and sucrose in fruit, whilst SlARF10-RNAi lines showed decreased accumulation of starch and sucrose. Regulation of SlARF10 expression altered the expression of AGPase starch biosynthesis genes. SlARF10 positively regulated the expression of SlGLK1, POR, CBP1, and CBP2, which are related to chlorophyll metabolism and regulation. Electrophoretic mobility shift assays confirmed that SlARF10 directly targets to the SlGLK1 promoter. Our results thus indicate that SlARF10 is involved in chlorophyll accumulation by transcriptional activation of SlGLK1 expression in tomato fruit, and provide insights into the link between auxin signaling, chloroplast activity, and sugar metabolism during tomato fruit development. Keywords: ARF10, auxin, chlorophyll, fruit, starch, sugar, tomato. Introduction Tomato (Solanum lycopersicum), a nutrient-rich multicarpellar berry with strong adaptability and high yield, has become the world’s second largest vegetable crop (Tanksley, 2004). It has also become the research model species for fleshy fruits, due Abbreviations: ARF, auxin response factor; B3, B3 DNA-binding domain; CBP1, chlorophyll binding protein 1; CBP2, chlorophyll binding protein 2; DDB1, UV-DAMAGED DNA-BINDING PROTEIN 1; GLK, GOLDEN2-LIKE; qRT-PCR, quantitative real-time PCR; WT, wild-type. © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1 5508 | Yuan et al. of auxin-responsive genes (Ren et al., 2011). Most ARF proteins contain an N-terminal DNA-binding domain (B3), which is involved in transcription of auxin-response genes, a middle region acting as an activation domain or repression domain, and a C-terminal dimerization domain that requires the formation of heterodimers or homodimers (Zouine et al., 2014). An increasing number of studies have demonstrated that ARFs play important roles in many developmental processes in tomato (Krogan et al., 2012; Wang et al., 2012; Guan et al., 2013; Ckurshumova et al., 2014; Liu et al., 2014a, 2014b; Zhang et al., 2015). SlARF7 acts as a negative regulator of fruit set and development in tomato (De Jong et al., 2009). ARF6 and ARF8 have important roles in controlling flower growth and development (Liu et al., 2014a). SlARF9 is required for the regulation of cell division during early tomato fruit development (De Jong et al., 2015), SlARF3 is involved in the formation of epidermal cells and trichomes (Zhang et al., 2015), and SlARF4 controls the accumulation of chlorophyll and starch in the fruit (Jones et al., 2002; Sagar et al., 2013). The influence of SlARF4 on fruit chlorophyll accumulation seems to be mediated through the transcriptional up-regulation of SlGLK1 in the fruit (Sagar et al., 2013). Hendelman et al. (2012) reported that SlARF10 is posttranscriptionally regulated by Sl-miR160, and constitutive expression of mSlARF10 (Sl-miR160a-resistant version) produced narrow leaflet blades, sepals, and petals, and abnormally shaped fruit in tomato. Repression of SlARF10 expression by Sl-miR160 is essential for auxin-mediated blade outgrowth and early fruit development (Hendelman et al., 2012). In the present study, the functions of SlARF10 were studied in the development of tomato fruit, and it was found to be involved in chlorophyll and sugar accumulation. This study expands our understanding of the functions of ARFs during the development of tomato fruit, and provides new insights into the regulation mechanisms of chlorophyll and sugar accumulation. Materials and methods Plant material and growth conditions Tomato (Solanum lycopersicum L. cv. Micro Tom) plants were grown in a culture chamber under a 16/8 h light/dark photoperiod at 25 ± 2/18 ± 2 °C and 80% relative humidity. Sequence analysis BLAST analysis was performed at the website http://www.ncbi.nlm. nih.gov/blast/. Protein domains were identified using the Pfam program (http://pfam.xfam.org). Alignment of protein sequences was performed with ClustalX version 2.1. Expression patterns and qRT-PCR analysis Expression patterns were analysed online according to the tomato gene expression database (http://tomexpress.toulouse.inra.fr/). The pericarp of fruit was used for total RNA isolation. Total RNA was extracted using an RNeasy Plant Mini Kit (Qiagen). Quantitative real-time (qRT)-PCR was carried out as previously described by Deng et al. (2012). The relative transcript abundance was monitored on a CFX96 real-time PCR detection system (Bio-Rad) using All-in-One™ qPCR Mix (GeneCopoeia). The relative expression for each gene of interest was calculated using the ΔΔCt values with ubiquit (...truncated)