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.