4. Discussion

The contents of E2H increased by 4.3 times during the colour transformation of jujube (Song, Bi, Chen, Wu, Lyu & Meng, 2019); E2H showed an exponential increase trend during the colour transformation of tomato (Wang, Baldwin, Luo, Zhao, Brecht & Bai, 2019), then decreased significantly after the colour transformation (Liu, Meng, Chen, Yin, Hu, Shao, Liu, Zhu, Ye & Wang, 2019). Coincidentally, the carotenoid accumulation and the E2H are both synthesized in the plastids (Joyard, Ferro, Masselon, Seigneurin-Berny, Salvi, Garin & Rolland, 2010). There are regulatory relationships between carotenoids and E2H.
Before chloroplasts IMS transformation, carotenoids synthesis has started in grana and stroma thylakoids and then packed into IMS (Schweiggert et al. , 2011, Sun et al. , 2018). In papaya, tubular elements (te) were not found until the carotenoids accumulation in colored ripening stages (Schweiggertet al. , 2011). The red crystal remnants (cr) appeared only when induced by lycopene accumulation in the cultured juice vesicles of Newhall oranges (Lu et al. , 2017). Therefore, the IMS of the chloroplasts are regulated by carotenoids (Sun & Li, 2020).
Based on the above evidences and our results, it is hypothesis that the E2H plays critical role in carotenoids activated IMS transformation in the chloroplasts during tomato colour transformation.
Exogenous E2H inhibited tomato colour development with a concentration-dependent manner (Figure 1A). It established the connection of exogenous E2H and the colour transformation. After E2H treatments in 72 h, green pericarps and red columellas illustrated that E2H can easily penetrate through connected tissues but locular gel physically or via signal transduction (Figure 1B). Instead of overall colour transformation from pink to red, patchy red areas were observed (Figure 1C). So E2H is more likely to suppress colour transformation through signal transduction than physical penetration.
Endogenous E2H increased during the colour transformation in jujube and tomato (Song et al. , 2019, Wang et al. , 2019), but why the exogenous E2H suppressed the colour transformation? It is indicated that exogenous E2H interfered the endogenous E2H functions during colour transformation. The exogenous E2H can be used as an interference tool to investigate the regulating mechanism among carotenoids accumulation (colour transformation), endogenous E2H and IMS transformation.
Ethylene initiates fruit ripening including colour changes. In the tomatoes pretreated or combine treated with the E2H, the colour development was not initiated by ET, whereas in the ET initiated tomatoes, the E2H cannot reverse the initiated colour change (Figure 2). The E2H signal transduction is suggested to function downstream of ET. Similarly, in cherry tomato, rapid increase of ET was observed in 7-15 day whereas the rapid increase of E2H was in 15-22 d (Wu, Tao, Ai, Luo, Mao, Ying & Li, 2018).
It is detected by TEM that the IMS of the chloroplasts (thylakoid membranes and envelope membranes) transformed to the characteristic structures of chromoplasts (tubular elements, plastoglobules and crystal remnants). These processes are conducted by membrane fusion or vesicles budding (Schweiggert et al. , 2011). According to the IMS transformation status (Figure 3I), four stages can be observed in the electron micrographs, including the chloroplast stage (S1) (Figure 3A), two transition stages (S3 and S4) (Figure 3B and C) and the chromoplast stage (S4) (Figure 3D and G). E2H accelerated the programed IMS transformation. The evidences included that (1) S1 and S2 plastids in the E2H group were not observed (Figure 3 E, F and H); (2) fewer pg and no cr was observed (Figure 3 E, F and J). E2H have been reported to change the fatty acids composition in the membrane and accelerated the release of free fatty acids (Patrignani, Iucci, Belletti, Gardini, Guerzoni & Lanciotti, 2008). The E2H biosynthesis process can also consume the free fatty acids released from the IMS (Beckett, Loreto, Velikova, Brunetti, Di Ferdinando, Tattini, Calfapietra & Farrant, 2012). Base on the mechanism, the IMS transformation can be started by E2H when the carotenoids are not sufficiently accumulated. As a result, the tomato colour transformation were inhibited (Figure 1A).
By analogy, the endogenous E2H may acted downstream of carotenoids as a signal molecular in starting the IMS transformation when the carotenoids contents reached a threshold during fruit ripening. Combining the results above (Figures 1, 2 and 3), a possible regulatory relationship was speculated: ethylene → carotenoids biosynthesis → E2H signaling → IMS transformation → more carotenoids accumulation.
The results of the pigments also support the above hypothesis (Figure 4). The a* value was used as the parameter indicating the colour development dynamics. The decrease of chlorophyll and the increase of lycopene are consistent with the results of a * value (Figure 4A-C). It is indicated that the chlorophyll and lycopene contents determine the colour of the tomatoes.
The low level E2H delayed the chlorophyll decline and lycopene increase, while the high level E2H completely prevented the changes of chlorophyll and lycopene contents (Figure 4A-C). A series of key proteins involved in lycopene biosynthesis, including phytoene synthase (PSY), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), ζ-carotene isomerase (Z-ISO), and carotenoid isomerase (CRTISO) are all located or function in the chloroplast IEM (Sun et al. , 2018, Zhou, McQuinn, Fei, Wolters, Van Eck, Brown, Giovannoni & Li, 2011). When E2H accelerated the IEM transformation, some of the key proteins above possibly lost the functions as the structural basis disappeared, therefore the lycopene biosynthesis was delayed or completely prevented. The delayed chlorophyll decline can be explained similarly. The chlorophyll catabolism-related enzymes, which located in the thylakoid membrane and the structural basis disappeared.
The lycopene β-cyclase (LycB) which located in the pg catalyzed the lycopene to synthesis β-carotene. It’s worth noting that the β-carotene contents in the E2H (2.5) and E2H (12.5) groups were much higher than the control at 6 h, and the E2H (12.5) showed more significant elevation than the E2H (2.5) (Figure 4D). The results can be explained as the E2H accelerated the transformation of the IEM to pg at 6 h, more LycB in the pg improved the synthesis of β-carotene, at the same time the pg in the control group were less formed (Fanciullino, Bidel & Urban, 2014). In 36-72 h, the decrease of β-carotene contents in both of the E2H group can be explained as less pg structures formed than the control (Figure 4D).
Thylakoid membranes and IEM in the chloroplasts are enriched in galactolipids, in the fatty acid parts of the galactolipids, linolenic acid contents are up to 95% in some plant species. In response to biotic or abiotic stimuli, fatty acids including linolenic acids released from the thylakoid membranes or IEM (Joyard et al. , 2010). Although there is no direct evidence for the release of linolenic acid from the thylakoid membranes or IEM during tomato colour transformation, the linolenic acid in tomato reached a minimum content in the turning and pink periods (Saini, Zamany & Keum, 2017).
In this study, the expression of lipoxygenase C gene (LOXC ) increased during colour transformation in the control group. E2H expedited the LOXC expression increase (Figure 5A). It is hypothesized that the release of linolenic acids may be accelerated by E2H because the LOXC is the key enzyme involve in the linolenic acids metabolism and produces hydroperoxyl-linolenoic acids. The expedited LOXC expression may explained the E2H accelerated IMS transformation because more linolenic acids were released and consumed by E2H treatments. Following that, the composition changes altered the IMS (Szilágyi, Selstam & Åkerlund, 2008).
The decomposition of linolenic acids also produces endogenous E2H, and the 13-hydroperoxide lyase (13-HPL) is the key enzyme involved. TheHPL expression was significantly decreased in the control group during colour transformation, and the exogenous E2H treatment further reduced the HPL expression (Figure 5B). Considering that HPL is localized in the plastid outer envelope membrane (OEM), it is less affected by the transformations in the plastid inner membranes (Joyard et al. , 2010). Therefore, it is speculated that exogenous E2H feedback regulate the HPLexpression, which may be a mechanism to maintain endogenous E2H balance.
The quick increase responses (6 h) of TA and SCC to E2H treatments can be explained as the stress response (Table 2). In the following time, similar contents in of TA and SCC in the control and the E2H groups were detected (Table 2). It is indicated that the quality of tomato was less affected by E2H which may have less correlation with the IMS of the plastids.
The ascorbic acids are produced in mitochondria and enters other organelles including chloroplasts to function as antioxidants and coenzymes. Ascorbate acid transporters are located in the envelope membrane and the thylakoid membrane (Miyaji, Kuromori, Takeuchi, Yamaji, Yokosho, Shimazawa, Sugimoto, Omote, Ma, Shinozaki & Moriyama, 2015). In this study, E2H accelerated the IEM and the thylakoid membrane transformation, the ascorbate acid transporters lost their structural basis, therefore ascorbic acids usage were inhibited. Therefore, the E2H increased the ascorbic acid content at 36-72h (Table 2).
The E2H treatments increased the MDA contents in part of the time points. In particular, the MDA contents in the E2H (2.5) group were higher than the E2H (12.5) at 18-36h. Based on the results, the effects of E2H on the IMS transformation may not base on the phytotoxicities, but the signaling activation (Table 2).
As shown in Figure 6, the mechanism of carotenoids activated inner membrane structures transformation in the chloroplasts during tomato fruit colour development were speculated. The E2H play critical role in this process. (I) the carotenoids are biosynthesis before IMS transform in the chloroplasts; (II) the carotenoids are packed into the IMS; (III) free linolenic acids are released from the IMS; (IV) LOXC decompose the free linolenic acids to produce hydroperoxy-linolenic acid; (V) HPL decompose the hydroperoxy-linolenic acid to produce E2Hs; (VI) The E2Hs are released inside or outside the plastid; (VII) The E2Hs altered the structure of the IMS; (VIII) the te, pg or cr structure in the chromoplasts form and accumulate more carotenoids; (IX) The E2Hs outside one plastid transfer to function in the neighbor plastids; (X) Excessive E2H feedback regulates the HPL enzyme to form a balanced level.