TKN2 and TKN4, two Class I KNOTTED1-LIKE HOMEOBOX proteins, act as transcriptional activators of SlGLK2 and APRR2-LIKE genes to promote chloroplast development in tomato fruits. BEL1-LIKE HOMEODOMAIN11 also plays an important role in chlorophyll synthesis and chloroplast development in tomato fruits. The ripening of tomato is mainly regulated by the ethylene pathway and many transcription factors. In the ethylene biosynthetic pathway, S-adenosylmethionine synthetase catalyzes the reaction of ATP and methionine to form S-adenosyl-L-methionine. 1-Aminocyclopropane-1-carboxylic acid synthase and ACC oxidase catalyze the conversion of SAM to ACC and of ACC to ethylene, respectively. The MADS box gene RIPENING INHIBITOR controls the early phase of ripening and ethylene production via transcriptional regulation of ACSs and ACOs. The other ripening regulators affecting ethylene production also include the NAC transcription factor NOR, the SQUAMOSA PROMOTER BINDING protein CNR, the ethylene response factor ERF B3, the AP2/ERF member AP2a, and several MADS box proteins, such as TDR4/SlFUL1, SlFUL2, SlMADS1, TAGL1, and TAG. Auxin is an important phytohormone involved in flower fertilization, fruit setting, fruit initiation and development. Auxin is also essential in the regulation of cell division and expansion, controlling final fruit size. Auxin modulates plant development through transcriptional regulation of auxin-responsive genes, which is primarily mediated by two gene families: the short-lived nuclear protein Aux/IAA family and auxin response factors. Most ARFs have an N-terminal DNAbinding domain required for transcriptional regulation of auxin response genes, a middle region functioning as a repression domain or activation domain ,grow lights for cannabis and a C-terminal dimerization domain involved in the formation of homodimers or heterodimers.
ARFs can act as either an activator or a repressor of the transcription of auxin-responsive genes. Numerous studies have indicated that ARFs are involved in many tomato developmental processes. SlARF4 negatively regulates chlorophyll accumulation and starch biosynthesis in tomato fruit. Our previous study showed that SlARF10 positively regulated chlorophyll accumulation via direct activation of the expression of SlGLK1. Down regulation of ARF6 and ARF8 by over expression of Arabidopsis microRNA167 results in the failure of pollen germination on the stigma surface and/or growth through the style in tomato. However, the function of SlARF6 in the regulation of fruit development is still not well understood. In this study, SlARF6A was found to be involved in photosynthesis, sugar accumulation and fruit development in tomato. Our data demonstrate that SlARF6A plays an important role in the regulation of fruit quality and development.The SlARF6A gene has an open reading frame of 2608 bp encoding a putative protein of 869 amino acids. Amino acid sequence analysis revealed that, like SlARF7 and SlARF8, which have typical conserved ARF domains, SlARF6A protein also contained B3-DNA, ARF, and AUX/IAA binding domains . A phylogenetic tree was constructed to gain insight into the phylogenetic relationship among ARF proteins in Arabidopsis and tomato. ARFs were divided into four major classes: I, II, III, and VI. SlARF6A along with SlARF6B and AtARF6 were grouped into subclass IIa and are closely related to AtARF8 and SlARF8 , indicating possible functional similarity among them. To determine the expression pattern of SlARF6A in planta, a transcriptional fusion was constructed between the SlARF6A promoter and the GUS reporter gene. GUS staining in the transgenic tomato plants was detected in leaves, stems, buds, flowers, and fruits at different developmental stages, an indication of the ubiquitous expression of SlARF6A in all tissues tested. The GUS staining was weak in the early fruits at 2 and 4 days post anthesis but became strong at 8, 30 and 45 DPA , suggesting possible roles of SlARF6A in the development of tomato fruits.
To examine its subcellular localization in plants, SlARF6A was fused to GFP and transferred into tobacco protoplasts.A GAL4-responsive reporter system in yeast was employed to reveal the transcriptional activity of SlARF6A. SlARF6A was fused to GAL4-BD to form a pGBKT7-SlARF6A fusion plasmid and subsequently transformed into yeast. Yeast transformants harboring the pGBKT7-SlARF6A construct grew well in the medium lacking Trp, His, and Ade , while the yeasts transformed with pGBKT7 vector alone could not . Assessing transcriptional activity revealed that SlARF6A is a transcriptional activator.To elucidate the physiological significance of the SlARF6A gene in fruit development, upregulated and down regulated transgenic lines corresponding to independent transformation events were generated in tomato plants. qRT-PCR was used to evaluate the expression level of SlARF6A in all transgenic lines. Compared with the level in the wild type , the expression level of SlARF6A was decreased in RNAi 2 and 6 plants but increased in OE-4 and 6 plants . It is noteworthy that altered SlARF6A expression led to a dramatic change in chlorophyll accumulation in transgenic lines. Compared with WT plants, the OE-SlARF6A plants had dark-green fruits at the green fruit stage, whereas the RNAi-SlARF6A plants had light-green fruits . The impact of altered SlARF6A expression on chlorophyll accumulation was analyzed by measuring the chlorophyll content in fruits and leaves. The SlARF6A over expression lines possessed greater accumulation of chlorophyll in the fruits at immature green, mature green, breaker, and orange stages, whereas the RNAi lines had lower chlorophyll accumulation in the fruits at immature green and mature green stages than the WT plants . In leaves, the upregulated and down regulated SlARF6A transgenic lines possessed higher and lower chlorophyll levels, respectively, than the WT plants . Then, chlorophyll autofluorescence in the pericarp was detected using confocal laser scanning microscopy.
OE-SlARF6A plants had stronger chlorophyll auto- fluorescence, while the RNAi-SlARF6A lines had weaker chlorophyll autofluorescence in both epicarp and endocarp tissues compared with that of the WT plants . Then, the chloroplasts were observed using a transmission electron microscope . The growth of individual chloroplasts in OE-SlARF6A fruits was obviously promoted, with a significant increase in size and length . However, the number of chloroplasts per cell in OE-SlARF6A fruits was the same as that in the WT plants. For the RNAi-SlARF6A lines,grow cannabis the number of chloroplasts per cell was obviously decreased, but the size of individual chloroplasts was not changed .The dark-green phenotype and associated increased chlorophyll content may potentially lead to enhanced photosynthetic performance in tomato plants. The photosynthetic performance in leaves and fruits of SlARF6A transgenic lines was measured. In both leaves and green fruits, the photochemical potential was elevated in OE-SlARF6A lines, whereas the value was decreased in RNAi-SlARF6A plants compared with the WT plants . The effective photochemical quantum yield of PSII in OE-SlARF6A lines was higher than that of the WT plants in both leaves and fruits, while the values for RNAiSlARF6A plants were lower than that for the WT plants in both leaves and fruits . Thus, the SlARF6A gene positively affects photosynthesis in the fruits and leaves of tomato plants. Sugars are the major products of photosynthesis, so it is essential to evaluate whether the altered chlorophyll level and photosynthetic performance in SlARF6A plants result in altered sugar accumulation. As shown in Fig. 4e, starch levels decreased rapidly throughout fruit development in the transgenic and WT plants. The starch content in OESlARF6A fruits was much higher than that in WT fruits at green fruit stages, whereas the starch content in RNAiSlARF6A fruits was much lower than that in the WT fruits at green stages . These data demonstrated that the SlARF6A gene positively affects starch accumulation during green fruit development. It is well established that starch degradation is the dominant source of soluble sugars in fruits. The contents of fructose, glucose and sucrose were analyzed in SlARF6A transgenic plants. The levels of glucose, fructose and sucrose were significantly higher in the OE-SlARF6A fruits than in the WT fruits, particularly at the orange and red fruit stages . Compared with the WT fruits, the RNAi-SlARF6A fruits exhibited obviously decreased contents in glucose, fructose and sucrose . Our data indicated that the SlARF6A gene positively affects the levels of glucose, fructose and sucrose during fruit development.The SlARF6A transgenic plants also exhibited different ripening of fruits than the WT plants. Down regulation of SlARF6A accelerated fruit ripening, with the breaker stage occurring 5 days sooner than that in the WT plants , while over expression delayed the breaker stage by 5 days compared with that of the WT plants . The assessment of color change via measurement of the evolution of hue angle values further confirmed the difference between the SlARF6A transgenic lines and WT plants throughout the ripening process . The ethylene production was measured using a GC method. When compared with that of the WT plants, the ethylene production of RNAi-SlARF6A plants showed a dramatic induction of ~2-fold and 4-fold at the breaker stage and remained at high levels for 2 and 3 days after the breaker stage, while that of over expressed lines was inhibited at the breaker stage and remained at low levels for 5 days after the breaker stage compared with the levels in the WT plants .To investigate the molecular mechanism of chlorophyll accumulation, photosynthesis and fruit ripening in SlARF6A transgenic plants, RNA-sequencing was conducted to analyze the differentially expressed genes in OE-SlARF6A and RNAi-SlARF6A plants. Under the criterion of a false discovery rate < 0.05, 591 upregulated and 508 down regulated DEGs were identified in 4 DPA ovaries of RNAi-SlARF6A plants, and 254 upregulated and 424 down regulated DEGs were identified in 35 DPA fruits of OE-SlARF6A plants .
GO function and pathway enrichment analyses showed that knockdown of SlARF6A affected multiple metabolic pathways, including those of porphyrin and chlorophyll metabolism, photosynthesis, photosynthesisantenna proteins, carbon fixation, starch and sucrose metabolism, fructose and mannose metabolism, and plant hormone signal transduction . Over expression of SlARF6A also affected metabolic pathways, including those of photosynthesis, photosynthesisantenna proteins, carbon fixation, starch and sucrose metabolism, fructose and mannose metabolism, and plant hormone signal transduction . The expression of two genes encoding chlorophyll A/B binding protein was induced in OE-SlARF6A plants. The expression of a gene encoding ribulose bisphosphate carboxylase small chain was upregulated in OE-SlARF6A plants. Moreover, the expression of a gene encoding SAM synthetase 1 , which is involved in ethylene biosynthesis, was induced in RNAi-SlARF6A plants. Analysis of the RNA-Seq data also showed that among tomato ARF family genes, only SlARF6A was down regulated in RNAi-SlARF6A plants, indicating the specific knockdown of SlARF6A by the RNAi method. To validate the RNA-Seq results, 11 DEGs in RNAi-SlARF6A plants and 8 DEGs in OE-SlARF6A plants were selected for qRT-PCR analysis, and the results were in accordance with the data from RNA-Seq , which showed that the results from the RNA-Seq were reproducible and reliable.Analysis of the promoter sequences in the CAB and RbcS genes revealed conserved ARF binding sites and TGTCTC boxes. In addition, the chlorophyll phenotypes of SlARF6A over expression fruits were similar to those in SlGLK over expressing lines, and the SlGLK1 promoter contained two TGTCTC motifs. qRT-PCR identified that SlARF6A over expression induced the expression of SlGLK1 and SlGLK2, and knockdown of SlARF6A decreased the expression levels of SlGLK1 and SlGLK2 in fruits and leaves . Dual-luciferase reporter transient expression assays were conducted to examine whether SlARF6A could directly activate or suppress the expression of CAB, RbcS, and SlGLK1 genes. Tobacco leaves were cotransformed with LUC reporter vectors driven by the promoters of CAB, RbcS and SlGLK1 genes together with effector vectors carrying the CaMV35S promoter-driven SlARF6A gene. The results showed that LUC/REN ratios were significantly increased compared with those in the control . The binding of SlARF6A with the promoters in vivo was verified by ChIPqPCR analysis. As expected, the promoter sequences containing a motif of TGTCTC in the CAB, RbcS and SlGLK1 genes were significantly enriched with antiSlARF6A compared with the negative control anti-IgG . Furthermore, the direct binding of SlARF6A protein to the promoters of CAB, RbcS, and SlGLK1 was verified by an electrophoretic mobility shift assay . We generated a recombinant glutathione S-transferase fusion protein with truncated SlARF6A . The purified GST-tSlARF6A fusion protein bound to biotin-labeled probes containing the TGTCTC motif from the promoters of CAB, RbcS, and SlGLK1 and caused a mobility shift. When unlabeled promoter fragments were used as competitors, the mobility shift was efficiently abrogated in a dose dependent manner . In addition, as a negative control, the mobility shift was also abolished when biotin labeled probes were incubated with GST only .