LCD1 mutation caused earlier leaf senescence, whereas LCD1 overexpression significantly delayed leaf senescence compared with the wild type in 10-week tomato seedlings. Moreover, LCD1 overexpression was found to delay dark-induced senescence in detached tomato leaves, and the lcd1 mutant showed accelerated senescence.
1. Introduction
Leaf senescence represents the final stage of leaf development, which is a genetically controlled process
[1]. As leaves age, the decomposition of chloroplast is initiated, accompanied by the catabolism of macromolecules including nucleic acids, proteins, and lipids. The decomposed nutrients then transfer to other developing organs, such as young leaves and growing fruit
[2]. Chloroplasts constitute approximately 70% of the total proteins in green leaves and chlorophyll degradation causes the first visible signs of leaf senescence
[3]. Thus, the coordinated degradation of chlorophyll is crucial for the breakdown of chloroplasts. Terrestrial plants utilize two types of chlorophyll species (i.e., chlorophyll a and chlorophyll b) for photosynthesis
[4]. Chlorophyll b has to be converted to chlorophyll a before it can be processed into the degradation pathway and NON-YELLOW COLORING 1 (NYC1) catalyzes the reduction of chlorophyll b to 7-hydroxymethyl chlorophyll a
[5]. Chlorophyll a is further decomposed to pheophytin a by Mg-dechelatase NON-YELLOWINGs/STAY-GREENs (NYEs/SGRs)
[6]. Pheophytin a is then hydrolyzed by the pheophytinase PPH to generate pheophorbide a which is further catalyzed by oxygenase PAO to produce a red chlorophyll catabolite (RCC)
[7]. Moreover, hundreds of
senescence-associated genes (
SAGs), whose transcripts increase as leaves age
[8[8][9],
9], are also involved in the regulation of leaf senescence.
Plant hormones are major players influencing each stage of leaf senescence. For instance, ethylene, abscisic acid (ABA), and so on promote leaf senescence, while cytokinins (CKs), gibberellic acid (GA), and so on delay leaf senescence
[10,11,12][10][11][12]. Furthermore, leaf senescence-linked events are often associated with the pronounced accumulation of reactive oxygen species (ROS)
[13]. Among them, H
2O
2 is a well-defined inducer of leaf senescence. Recently, it was reported that transcription factor NAC075 delays leaf senescence by deterring ROS accumulation through directly binding the promoter of the antioxidant enzyme gene
catalase 2 (
CAT) in
Arabidopsis [14]. Hydrogen sulfide (H
2S) is an important gasotransmitter in both animals and plants
[15]. H
2S has not only been implicated in seed germination and root development, but can also enhance plant tolerance to various stresses such as heavy metals, drought, salinity, and cold by enhancing the antioxidant system
[16,17,18][16][17][18]. In addition, H
2S can extend the shelf life of bananas, grapes, strawberries, tomatoes, and so on
[19,20,21,22][19][20][21][22]. The underlining mechanism of H
2S in alleviating postharvest senescence may involve the activation of the antioxidant system, the inhibition of ethylene synthesis and the signaling pathway, etc.
H
2S is endogenously produced in a precise and regulated manner. Cysteine degradation by cysteine desulfhydrases (CDes) to the formation of sulfide, ammonia, and pyruvate was believed to be an important source of H
2S
[23]. Plant cells contain different CDes localized in the cytoplasm, plastids, and mitochondria
[23]. DES1, an O-acetylserine(thiol)lyase homolog with L-cysteine desulfhydrase activity, regulates cysteine homeostasis in
Arabidopsis [24]. Recently it was reported that the
des1 mutant was more sensitive to drought stress and displayed accelerated leaf senescence, while the leaves of
OE-DES1 contained adequate chlorophyll levels accompanied by significantly increased drought resistance, suggesting the role of DES1 in regulating leaf senescence
[25].
2. Role of LCD1 in Regulating Tomato Leaf Senescence
To elucidate the possible involvement of LCD1 in regulating leaf senescence, two previously reported tomato lines,
lcd1-7 and
lcd1-9, with mutations near the PAM sequence were used as
lcd1 mutants, and two lines (hereafter called
LCD1-oe and
LCD1-oe1) with an increased expression of
LCD1 under the control of the CaMV
35S promoter were also used. The overexpression efficacy of five
LCD1 overexpression lines was verified by RT-qPCR as shown in
Figure S1 and the lines
LCD1-oe and
LCD1-oe1, which showed a higher
LCD1 expression, were used in the present study. To confirm the role of LCD1 in catalyzing H
2S production, the H
2S producing rates were determined in leaves of
lcd1 and
LCD1-oe lines. The data in
Figure 1B suggest that
lcd1 leaves had a lower H
2S producing rate compared with the wild type, while
LCD1 overexpression induced a significantly higher level of the H
2S producing rate. Besides, H
2S production was also evaluated by lead acetate H
2S detection strips, and the results showed that
lcd1 leaves produced less H
2S and
LCD1 overexpression produced more H
2S (
Figure 1C). After 10 weeks of growth, the
LCD1 mutation caused earlier leaf senescence compared with the wild type. In contrast,
LCD1 overexpression significantly delayed leaf senescence (
Figure 1D).
Figure 1. Phenotypic characterization of lcd1 mutants and LCD-oe (over-expression) tomatoes. (A) Phenotype of 10-week-old wild-type (WT), lcd1-7, lcd1-9, LCD1-oe, and LCD1-oe1 plants. (B) H2S producing rate in the mature leaves from wild type, lcd1, and LCD1-oe lines of 10 weeks growth. (C) H2S production from the mature leaves of 10-week-old wild type, lcd1, and LCD1-oe lines detected by lead acetate H2S detection strips (Sigma-Aldrich). (D) The leaves of different tomato lines in (A) were detached and photographed. Data are means of three biological replicates ± standard deviation (SD). The symbols * and ** stand for p < 0.05 and p < 0.01 as determined by the Student’s t-test, respectively.
3. LCD1 Participates in Dark-Induced Senescence
Leaf senescence is an important phenomenon in the growth and development of plant leaves, and darkness is widely used as a tool to induce senescence in detached leaves. To study the role of LCD1 in dark-induced senescence, the mature leaves without visible senescence of 6-week-old wild type,
lcd1 mutant, and
LCD1-oe were stored in darkness for 8 days. As shown in
Figure 2A,
lcd1 showed the obvious syndrome of the leaf yellowing phenotype after 5 and 8 days in dark stress, whereas
LCD1 overexpression still maintained the green phenotype. To study the kinetics of tomato leaf H
2S production during senescence, H
2S production in the leaves at different developmental stages—young leaves (YL), mature leaves (ML), senescent leaves (SL), and late senescent leaves (LS)—was evaluated and the H
2S detection strips showed browning with senescence, suggesting H
2S production increased during leaf senescence (
Figure S2). Moreover, H
2S production in leaves of wild-type (WT),
lcd1, and
LCD1-oe tomatoes were also determined during dark-induced senescence (
Figure 2B). Generally, an increasing trend of H
2S production was observed in all samples during storage, while
LCD1-oe leaves showed a higher H
2S production compared with the wild-type control. In addition, the
lcd1 mutant produced a significantly lower level of H
2S compared with the wild type. Therefore, it can be concluded that
LCD1 deletion caused a lower H
2S release and the attenuated H
2S release may cause an accelerated senescence in the
lcd1 mutant. Overall, the present results indicate that
LCD1 plays a negative role in leaf senescence in both developmental and dark-induced senescence.
Figure 2. (A) Dark-induced senescence symptoms in detached leaves of 6-week-old wild-type (WT), lcd1, and LCD1-oe tomatoes for up to 8 days. (B) H2S producing rate in detached leaves of 6-week-old wild-type (WT), lcd1, and LCD1-oe tomatoes stored in darkness for up to 8 days. Different letters above the columns stand for significant difference between two values (p < 0.05) at the same time point.
4. Effect of LCD1 on Dark-Triggered Chlorophyll Degradation and Reactive Oxygen Species Accumulation in Detached Tomato Leaves
Chlorophyll degradation is the one of the most significant changes during leaf senescence; thus, chlorophyll contents were determined in wild-type,
lcd1 mutant, and
LCD1-oe leaves during dark-induced senescence. As shown in
Figure 3A, the content of total chlorophyll in the wild type decreased gradually during storage in darkness for 8 days, whereas the content of chlorophyll in the
lcd1 mutant showed an obvious decrease on days 5 and 8 under darkness, and the value on day 8 was about 32.6% of the initial value on day 0. In contrast,
LCD1 overexpression maintained a relatively higher chlorophyll content compared with the wild type and the
lcd1 mutant on days 5 and 8 under darkness. After 8 days in darkness, the chlorophyll content in
LCD1 overexpression decreased to 84.6% of the initial value, suggesting the role of
LCD1 in delaying dark-induced senescence. As shown in
Figure 3B, there were minor changes in the chlorophyll a content between different groups during storage. Moreover, only a slight decrease in chlorophyll a was observed during dark-induced senescence, except for a significant decline found in
lcd1 on day 8.
Figure 3C shows the change pattern of chlorophyll b content in wild type,
lcd1 mutant, and
LCD1-oe during dark-induced senescence. With the increase of storage days, the chlorophyll b content decreased in each group. At day 0, chlorophyll b content in
lcd1 leaves was about 53.4% of that in the
LCD1-oe group, and decreased to 21.3% on day 8 compared with the value on day 0. Furthermore, the ratio of chlorophyll a/b was also evaluated in dark-stored detached leaves of wild-type,
lcd1, and
LCD1-oe tomatoes for 0, 2, 5, and 8 days. As shown in
Figure 3D, the ratio of chlorophyll a/b in WT and
lcd1 mutant leaves increased during storage, while
LCD1 deletion caused the highest ratio compared with other groups. In contrast, the ratio of chlorophyll a/b in
LCD1 overexpression almost remained unchanged. The above results indicate that the
lcd1 mutation accelerated dark-induced leaf senescence and
LCD1 overexpression delayed leaf yellowing and chlorophyll degradation.
Figure 3. Changes in the contents of (A) total chlorophyll, (B) chlorophyll a, (C) chlorophyll b, and (D) the ratio of chlorophyll a/b in dark-stored detached leaves of 6-week-old wild-type (WT), lcd1, and LCD1-oe tomatoes for 0, 2, 5, and 8 days. Data are means of three biological replicates ± standard deviation (SD). Different letters above the columns stand for significant difference between two values (p < 0.05) at the same time point.
Leaf senescence is usually associated with the excessive accumulation of ROS; therefore, the levels of H
2O
2 and malondialdehyde (MDA) were monitored in wild-type,
lcd1 mutant, and
LCD1-oe leaves during dark-induced senescence. As shown in
Figure 4A, there was no significant difference in H
2O
2 content between the different groups on day 0. During the dark-induced senescence, the H
2O
2 content in each group showed an increasing trend, of which the
lcd1 group increased the fastest, followed by the wild-type and
LCD1-oe group. However, H
2O
2 content in the
LCD1-oe group increased slowly compared with other groups. As shown in
Figure 4B, the change of MDA content among the groups also showed a similar trend to H
2O
2. The content of MDA in
lcd1 leaves during storage was the highest compared with other groups, and the lowest MDA content was observed in
LCD1-oe leaves. Therefore, it can be concluded that the overexpression of
LCD1 could reduce the accumulation of ROS and MDA in leaves under dark-triggered senescence.
Figure 4. Changes in the contents of (A) H2O2 and (B) malondialdehyde (MDA) in dark-stored detached leaves of 6-week-old wild-type (WT), lcd1, and LCD1-oe tomatoes for 0, 2, 5, and 8 days. Data are means of three biological replicates ± standard deviation (SD). Different letters above the columns stand for significant difference between two values (p < 0.05) at the same time point.
5. Effect of LCD1 on the Expressions of Genes Related to Chlorophyll Degradation in Detached Tomato Leaves
Chlorophyll degradation marks the senescence stage of leaves. In order to explore the molecular mechanism of the differences in chlorophyll content of
lcd1,
LCD1-oe, and wild-type leaves during dark-induced senescence, the expression levels of key genes
NYC1,
PAO,
PPH, and
SGR1 in the chlorophyll degradation pathway were analyzed by RT-qPCR. The present data showed
NYC1 was transcriptionally induced during dark-induced senescence in all groups (
Figure 5A). In accordance with the early senescence phenotype of the
lcd1 mutant and late senescence in
LCD1-oe leaves, the expression of
NYC1 was significantly higher in the
lcd1 mutant and was less expressed in
LCD1-oe leaves during dark storage. Three other genes—
PAO (
Figure 5B),
PPH (
Figure 5C), and
SGR1 (
Figure 5D)—were also analyzed at the transcriptional level in wild-type,
lcd1 mutant, and
LCD1-oe leaves during dark-induced senescence and similar changes to that of the
NYC1 expression were observed. The higher expression of
PAO,
PPH, and
SGR1 in
lcd1 and lower expression in
LCD1-oe again supported the role of
LCD1 in delaying leaf senescence. The results suggest that LCD1 may delay the chlorophyll degradation by down-regulating the transcription of key genes in the chlorophyll degradation pathway.
Figure 5. Changes in the gene expressions of chlorophyll degradation related genes: (A) NYC1, (B) PAO, (C) PPH, and (D) SGR1 in detached leaves of 6-week-old wild-type (WT), lcd1, and LCD1-oe tomatoes stored in darkness for 0, 2, 5, and 8 days. Data are means of three biological replicates ± standard deviation (SD). Different letters above the columns stand for significant difference between two values (p < 0.05) at the same time point.
6. Effect of LCD1 on the Expressions of SAGs in Detached Tomato Leaves
To further analyze the senescence-alleviating role of LCD1, we conducted an RT-qPCR analysis to evaluate the expression patterns of senescence-associated genes (
SAGs) in
lcd1,
LCD1-oe, and wild-type leaves during dark-induced senescence. As shown in
Figure 6,
SAG12,
SAG15, and
SAG113 were transcriptionally induced during dark-induced senescence. Compared with
SAG15 and
SAG113,
SAG12 showed more fold changes during leaf senescence, which was 109.6 times in the wild type on day 8 compared with day 0 (
Figure 6A). In accordance with the early senescence phenotype of the
lcd1 mutant and late senescence in
LCD1-oe leaves, the expression of the three
SAGs was significantly higher in the
lcd1 mutant and was less expressed in
LCD1-oe leaves during dark storage, especially on day 8.
Figure 6. Changes in the gene expressions of senescence-related genes: (A) SAG12, (B) SAG15, and (C) SAG113 in detached leaves of 6-week-old wild-type (WT), lcd1, and LCD1-oe tomatoes stored in darkness for 0, 2, 5, and 8 days. Data are means of three biological replicates ± standard deviation (SD). Different letters above the columns stand for significant difference between two values (p < 0.05) at the same time point.