This result demonstrated that SlARF6A targets the promoters of CAB, RbcS, and SlGLK1 genes and positively regulates chlorophyll accumulation, chloroplast development and photosynthesis.Motif analysis showed that the SAMS1 promoter contains the conserved ARF binding site, the TGTCTC box. The transient expression assays showed that the LUC/REN ratios were significantly decreased compared with that of the control, suggesting that SlARF6A negatively regulates the expression of SAMS1 genes . ChIP-qPCR was carried out to confirm the binding of SlARF6A with the SAMS1 promoter in vivo, and the results showed that the promoter sequences containing the TGTCTC of SAMS1 were significantly enriched compared with those with the negative control anti-IgG . The direct binding of SlARF6A protein to the SAMS1 promoter was further verified by EMSA. The results indicated that the SlARF6A protein directly bound to the TGTCTC motif in the SAMS1 promoter . Taken together, SlARF6A can target the SAMS1 promoter and negatively regulate the expression of SAMS1 genes. The data demonstrate that SlARF6A plays an important role in ethylene production and fruit ripening.In this study, we functionally characterized the transcription factor SlARF6A in tomato. However, there are two very similar SlARF6 genes in the tomato genome, namely, SlARF6A and SlARF6B. We also examined the function of SlARF6B using genetic approaches and found no obvious phenotypes in the transgenic RNAi and over expression tomato plants . This may be related to the fact that SlARF6B lacks the AUX/IAA domain in the C-terminus of the protein .Previous studies reported that chlorophyll accumulation increased in Arabidopsis roots when they were detached from shoots,lettuce vertical farming which was repressed by auxin treatment. Mutant analyses showed that auxin inhibits the accumulation of chlorophyll through the function of IAA14, ARF7, and ARF19 in Arabidopsis.
In tomato, SlARF4 plays an important role as an inhibitor in chlorophyll biosynthesis and sugar accumulation via transcriptional inhibition of SlGLK1 expression in tomato. In this study, over expression of SlARF6A resulted in enhanced chlorophyll accumulation and chloroplast development, whereas down regulation of SlARF6A decreased chlorophyll accumulation and chloroplast number in tomato . These results demonstrate that SlARF6A positively regulates chlorophyll accumulation and chloroplast number in tomato. Our study also showed that SlARF6A directly targeted the SlGLK1 promoter and positively regulated SlGLK1 expression . Nguyen et al. reported that over expression of SlGLK1 and SlGLK2 produced dark-green fruits and increased chlorophyll accumulation and chloroplast development. The fact that the phenotypes of SlGLK1 over expression plants resembled those described in the OE-SlARF6A plants further suggests that SlARF6A positively regulates SlGLK1 to improve chlorophyll accumulation and chloroplast development in tomato leaves and fruits. Although SlGLK1 and SlGLK2 have similar functions, SlGLK1 functions largely in leaves, while SlGLK2 functions in fruits. However, SlGLK2 does not account for the chlorophyll phenotypes in OE and RNAi-SlARF6A plants because the ‘Micro-Tom’ variety possesses two null alleles of SlGLK2. In our study, down regulation of SlARF6A reduced SlGLK1 expression and chlorophyll accumulation, whereas over expression of SlARF6A increased SlGLK1 expression and chlorophyll accumulation in leaves and fruits of tomato plants . The data demonstrate that SlGLK1 may be involved in chlorophyll accumulation not only in tomato leaves but also in fruits. Further study is needed to elucidate the important role of SlGLK1 in tomato fruit using CRISPR/Cas9 technologies. The chlorophyll a/b-binding proteins are the apoproteins of the light-harvesting complex of photosystem II . CABs are normally complexed with xanthophylls and chlorophyll, functioning as the antennacomplex, and are involved in photosynthetic electron transport. Meng et al. reported that SlBEL11 directly acted on the promoter of CABs to suppress their transcription. Silencing of SlBEL11 increased the expression of CAB genes, resulting in enhanced chlorophyll accumulation and stability in thylakoid membranes of chloroplasts in green tomato fruit.
In our study, SlARF6A targeted the promoter of CABs, which positively regulated chlorophyll accumulation, chloroplast development and photosynthesis in tomato . Our data further demonstrate important roles of CABs in chloroplast activity and photosynthesis in tomato. Rubisco, a key enzyme in the fixation of CO2, is the ratelimiting factor in the photosynthesis pathway under conditions of saturating light and atmospheric CO2. The RbcL and RbcS genes encode two subunits that form the Rubisco enzyme. The RbcL and RbcS genes are localized to the chloroplasts and to the nucleus, respectively. Our study showed that over expression of SlARF6A increased the expression of the RbcS gene. Moreover, SlARF6A directly targeted the RbcS promoter and positively regulated RbcS expression . In addition, SlARF6A positively affected photosynthesis in the fruits and leaves of tomato plants . Our study demonstrates that SlARF6A has important roles in photosynthesis via the direct regulation of the RbcS gene in tomato. Interestingly, RNA-Seq data showed that the expression levels of SlARF4 and SlARF10 genes were not altered in RNAi-SlARF6A and OE-SlARF6A plants, suggesting that SlARF6A may act on chlorophyll accumulation independently of SlARF4 and SlARF10. However, studies indicate that ARFs must form dimers on palindromic TGTCTC AuxREs to form a stable complex, leading to the possibility that SlARF6A, SlARF4 and SlARF10 could form dimers with each other to regulate chlorophyll metabolism. Further study could focus on the interactions among SlARF6A, SlARF4, and SlARF10 to comprehensively elucidate the effects of the transcriptional regulation of ARFs on chlorophyll accumulation in tomato.Down regulation of SlARF4 increased the photosynthesis rate and enhanced the accumulation of starch, glucose and fructose in tomato fruits. In this study, the increased chlorophyll accumulation and photosynthesis rate in OESlARF6A plants resulted in the increased contents of starch and soluble sugars in fruits . Starch is a dominant factor in the nutrients and flavor of fruits. AGPase catalyzes the first regulatory step in starch synthesis, converting glucose-1-phosphate and ATP into ADP-glucose. This critical catalytic reaction is also a limiting step during starch biosynthesis in potato tubers. Knockdown of SlARF4 increases the expression of AGPase genes and starch content. In this study, SlARF6A was positively correlated with the expression of AGPase genes , suggesting the important role of AGPase genes in starch biosynthesis in tomato. However, the EMSA failed to detect any binding between SlARF6A and the promoters of AGPase genes, even though auxin-responsive motifs were detected in the promoters of AGPase S1 and AGPase S2 genes.
Evidence suggests that sucrose induces the expression of AGPase genes in leaves and fruits in tomato. In this study, over expression of SlARF6A led to increased sucrose content in tomato fruits, while the RNAi-SlARF6A fruits displayed decreased sucrose accumulation . The altered accumulation of starch in OE-SlARF6A and RNAi-SlARF6A lines may be explained by the altered expression of AGPase genes influenced by sucrose in tomato. Over expression of SlARF6A also resulted in increased glucose and fructose content, vertical grow shelf which was likely due to the increased starch content degraded into increased contents of soluble sugars in tomato fruits. Our results are consistent with the notion that incipient starch content determines soluble sugars in the process of fruit development. Our study also provides a valuable method to improve the nutritional value of tomato fruits via regulation of SlARF6A expression.The tomato ARF2A gene was reported to positively regulate fruit ripening. Over expression of ARF2A in tomato resulted in blotchy ripening, and silencing of ARF2A led to retarded fruit ripening. Overexpression of ARF2A in tomato promoted early production of ethylene and expression of ethylene biosynthesis and receptor genes. In this study, SlARF6A negatively regulated fruit ripening and ethylene biosynthesis in tomato fruit . S-adenosyl-L-methionine , synthesized by SAM synthetase from ATP and methionine, is a substrate for ethylene biosynthesis . SAM is converted to ACC by the ACS enzyme, and ACC is then converted to ethylene by ACO. The level of SAM is tightly controlled to integrate developmental signals into the hormonal control of plant development. In Arabidopsis, over expression of SAMS1 increases the SAM and ethylene levels, whereas sam1/2 mutants show the opposite phenotype in seedlings. Similarly, in tomato plants, over expression of SAMS1 results in higher concentrations of ACC and ethylene compared with those in WT plants. These data indicate the important role of the SAMS1 gene in ethylene biosynthesis in plants. In this study, SlARF6A directly targeted the SAMS1 promoter and negatively regulated SAMS1 expression . The regulatory mechanism by which SlARF6A affects fruit ripening and ethylene production in tomato fruits can be explained by the interaction between SlARF6A and the SAMS1 promoter. It is interesting that ethylene and auxin interact with each other to control some plant developmental processes. For example, ethylene controls root growth through regulation of auxin biosynthesis, transport and signaling, while the formation of hypocotyl apical hooks is also regulated in a similar fashion in Arabidopsis. In tomato, knockdown of IAA3 results in both auxin and ethylene phenotypes, suggesting that IAA3 might be the molecular connection between ethylene and auxin. Liu et al. reported that the ethylene response factor SlERFB3 integrated ethylene and auxin signaling through direct regulation of the Aux/IAA27 gene in tomato. Our results indicate that SlARF6A negatively regulates ethylene biosynthesis and that the interaction of SlARF6A and SAMS1 represents an important integrative hub mediating ethylene-auxin cross-talk in tomato. In summary, our results demonstrate that SlARF6A regulates chlorophyll level and chloroplast development by directly binding to the promoters of the SlGLK1, CAB1, and CAB2 genes. SlARF6A also directly targets the RbcS gene promoter, activating RbcS expression and increasing the photosynthetic rate. The increased chlorophyll accumulation and chloroplast activity improve photosynthesis, resulting in the increased accumulation of starch and soluble sugars in tomato. In addition, SlARF6A can act directly on the promoter of SAMS1 and negatively regulate its expression, thereby influencing ethylene production and fruit ripening.
The present study provides new insight into the link between auxin signaling, chloroplast activity, and ethylene biosynthesis during tomato fruit development. Our data also provide an effective way to improve fruit nutrition of horticulture crops via regulation of chlorophyll accumulation and photosynthetic activity.For chlorophyll content determination, the fruits at different developmental stages and leaf tissues were collected and examined based on the methods described by Powell et al.. To determine chlorophyll auto- fluorescence, pericarp was peeled off tomato fruits and observed with a TCS SP2 laser confocal microscope . For transmission electron microscopy, pericarp tissues were examined with an FEI Tecnai T12 twin transmission electron microscope according to the method described by Nguyen et al.. For measurements of photosynthesis rates, the green mature fruits and leaves were measured via a PAM-2500 pulse-amplitude modulation fluorometer . The chlorophyll fluorescence parameter was measured based on the method described by Maury et al..For sugar extraction, 1 g of fruit tissue was collected and ground under liquid nitrogen. Subsequently, 10 mL of 80% ethanol was used for extraction three times at 80℃ for 30 min. After centrifugation, samples were completely evaporated under vacuum and then dissolved in 4 mL of distilled water. Using the dissolved samples, HPLC was carried out to determine the content of sucrose, fructose and glucose. Starch content determination was performed using fruit pellets. Four milliliters of 0.2 M KOH was used to dissolve the pellet by incubating the sample in a boiling water bath for 30 min. Then, 1.48 mL of 1 M acetic acid with 7 units of amyloglucosidase was employed to hydrolyze each sample for 45 min. Finally, 10 mL of distilled water was adopted to dissolve the sample, and then the dissolved sample was used for starch content measurement. For metabolite measurement, HPLC analysis was conducted using an Agilent 1260 Series liquid chromatograph system with a vacuum degasser, an autosampler, a binary pump, and a diode array detector controlled by Agilent ChemStation software. A precolumn and a Waters XBridge Amide column were used for analysis. The separation was performed via an isocratic solvent system with solvent A and solvent B , while the mobile phase was maintained at 75% B for 15 min for elution. The column temperature was maintained at 38 °C, and the solvent flow rate was 0.6 mL/ min. Meanwhile, the injection volume was 10 μL for each sample. With a drift tube temperature at 80 °C, the detection system for HPLC was an ELSD 2000, and air was used as the carrier gas with a flow rate of 2.2 L/min. Finally, the contents of glucose, fructose, sucrose and starch in tomato fruits were determined based on the methods described by Geigenberger et al..