Overexpression of 7-hydroxymethyl Chlorophyll a Reductase from Cucumber in Tobacco Accelerates Dark-Induced Chlorophyll Degradation

7-hydroxymethyl chlorophyll (Chl) a reductase (HCAR) plays critical roles in the Chl cycle and degradation during leaf senescence, however, its function in horticultural crops remains unknown. Here, we identified an HCAR gene (CsHCAR) from cucumber (Cucumis sativus L.) and investigated its roles in response to dark-induced Chl degradation. CsHCAR encoded 459 amino acids, which were orthologous to Arabidopsis HCAR, had the conserved domains, and localized in the chloroplast. Gene expression analysis showed that CsHCAR expression was the highest in senescent leaves and was responsive to different stresses and phytohormone treatments. Overexpression of CsHCAR in tobacco accelerated dark-induced Chl degradation through enhancing the expression of Chl catabolic genes. After 10 d of darkness treatment, the biomass of CsHCAR overexpression plants was reduced. Furthermore, the value of net photosynthetic rate, maximum quantum yield of photosystem II, and effective quantum yield of photosystem II in CsHCAR overexpression plants was significantly reduced in comparison to that in wild-type (WT) plants. The photosynthetic protein content, including Lhcb1, Lhcb2, Lhcb4, RbcS, and RbcL in CsHCAR overexpression plants exhibited a lower level as compared to that observed in WT plants. In addition, the expression of genes encoding these proteins in CsHCAR overexpression plants was significantly lower than that in WT plants. Moreover, CsHCAR overexpression plants inhibited the dark-induced accumulation of reactive oxygen species (ROS). These results indicate that CsHCAR affects the stability of photosynthetic proteins in chloroplasts, positively regulates Chl degradation, and plays an important role in maintaining ROS homeostasis in leaves.


Introduction
Chlorophyll (Chl) plays a central role in the photosynthetic system and is actively synthesized by glutamate during plant development and degraded into non-fluorescent Chl catabolites during senescence [1]. Chl intermediate molecules are also considered to be important signals of cellular processes, such as the cell cycle [2]. Excessive accumulation of Chl intermediate molecules may result in the production of large amounts of reactive oxygen species (ROS), leading to growth retardation and cell death [3]. As a result, Chl metabolism is strictly regulated by a variety of mechanisms, including gene expression, feedback inhibition, and protein stability [4].
The Chl cycle refers to the mutual transformation between Chl a and Chl b, which plays vital roles in the aging process [5]. In higher plants, Chl a is the degradable form of Chls [6]. The first step of Chl a degradation is catalyzed by magnesium (Mg)-dechelatase, encoded by Mendel's green cotyledon gene, STAY-GREEN (SGR), which catalyzes the conversion of Chl a to pheophytin a (Phetin a) [7]. The phytol chain of Phetin a is subsequently degradation in Chl-b-overproducing plants [22]. So far, the research on HCAR is mainly focused on Arabidopsis and rice, however, it has not been reported in horticultural plants.
To identify the HCAR gene, we used cucumber as a test material and found that cucumber's HCAR (CsHCAR) amino acid sequence is orthologous to the Arabidopsis; however, its physiological role remains unknown regarding dark-induced Chl degradation. In this study, we performed the functional analysis of CsHCAR and found that CsHCAR positively regulated dark-induced Chl degradation through increasing Chl catabolic genes (CCGs) expression.

Identification of Cucumber HCAR
For identification of the HCAR gene in cucumber, we used the amino acid sequence of Arabidopsis HCAR (AtHCAR) (AT1G04620) as the probe to perform BLAST research in the cucurbitaceae genome database. Only one putative HCAR sequence was identified, and was named as CsHCAR. Further analysis revealed that the CsHCAR gene was located on chromosome 3, and the length of coding DNA sequence (CDS) was 1380 bp, encoding 459 amino acids ( Table 1). The physicochemical analysis showed that the molecular weight (MW) and theoretical isoelectric point (pI) of CsHCAR was 51.19 KDa and 7.54, respectively (Table 1). Cucumber HCAR was identified from the cucurbitaceae genome database (http://cucurbitgenomics.org/ (accessed on 3 August 2021)) through BLAST research using the amino acid sequence of Arabidopsis HCAR (AT1G04620) as the probe. The protein MW and pI were analyzed using Protparam (http://web.expasy.org/protparam/ (accessed on 3 August 2021)). HCAR, 7-hydroxymethyl chlorophyll a reductase; Chr, chromosome; CDS, coding DNA sequence; bp, base pair; MW, molecular weight; pI, isoelectric point. In order to further investigate the evolutionary relationship between CsHCAR and other species, we searched the HCAR proteins of 35 species in the NCBI database (Table S1). After multiple sequence alignment of the 35 HCAR protein sequences using the Clustal W method in MEGA 5.0 software, a phylogenetic tree was constructed with the Neighborjoining method. The phylogenetic tree analysis showed that the HCAR proteins were divided into four groups, and the CsHCAR was orthology with those in cucurbitaceous plants ( Figure S1). Most of the plants had only one HCAR protein, however, a few plants had evolved two HCAR members ( Figure S1 and Table S1). Amino acid sequence alignment showed that the HCAR protein structure of cucumber, melon (Cucumis melo), Arabidopsis, tobacco (Nicotiana tabacum), rice, tomato (Solanum lycopersicum), and maize (Zea mays) was highly conserved and had the same domains, such as the cysteine residues and motif in FAD-containing proteins (Figure 1), indicating that they might have the same functions. The protein MW and pI were analyzed using Protparam (http://web.expasy.org/protparam/ (accessed on 3 August 2021)). HCAR, 7-hydroxymethyl chlorophyll a reductase; Chr, chromosome; CDS, coding DNA sequence; bp, base pair; MW, molecular weight; pI, isoelectric point.
In order to further investigate the evolutionary relationship between CsHCAR and other species, we searched the HCAR proteins of 35 species in the NCBI database (Table  S1). After multiple sequence alignment of the 35 HCAR protein sequences using the Clustal W method in MEGA 5.0 software, a phylogenetic tree was constructed with the Neighbor-joining method. The phylogenetic tree analysis showed that the HCAR proteins were divided into four groups, and the CsHCAR was orthology with those in cucurbitaceous plants ( Figure S1). Most of the plants had only one HCAR protein, however, a few plants had evolved two HCAR members ( Figure S1 and Table S1). Amino acid sequence alignment showed that the HCAR protein structure of cucumber, melon (Cucumis melo), Arabidopsis, tobacco (Nicotiana tabacum), rice, tomato (Solanum lycopersicum), and maize (Zea mays) was highly conserved and had the same domains, such as the cysteine residues and motif in FAD-containing proteins (Figure 1), indicating that they might have the same functions. Figure 1. Alignment of the amino acid sequences of HCAR proteins among seven species. Conserved residues are shaded with black. Filled triangles represent the cysteine residues that are predicted to coordinate the iron-sulfur cluster. Open triangles represent the conserved sequence motif in FAD-containing proteins (K×××××G×G). Cs, Cucumis sativus; Cm, Cucumis melo; At, Arabidopsis thaliana; Nt, Nicotiana tabacum; Sl, Solanum lycopersicum; Os, Oryza sativa; Zm, Zea mays.

Analysis of CsHCAR Expression Profiles and Subcellular Localization
In order to test the expression of CsHCAR in different tissues, cucumber roots, stems, young leaves, mature leaves, senescent leaves, fruits, sepals, and tendrils were used as materials for quantitative real-time PCR (qPCR) analysis. As shown in Figure 2A, CsHCAR Figure 1. Alignment of the amino acid sequences of HCAR proteins among seven species. Conserved residues are shaded with black. Filled triangles represent the cysteine residues that are predicted to coordinate the iron-sulfur cluster. Open triangles represent the conserved sequence motif in FAD-containing proteins (K×××××G×G). Cs, Cucumis sativus; Cm, Cucumis melo; At, Arabidopsis thaliana; Nt, Nicotiana tabacum; Sl, Solanum lycopersicum; Os, Oryza sativa; Zm, Zea mays.

Analysis of CsHCAR Expression Profiles and Subcellular Localization
In order to test the expression of CsHCAR in different tissues, cucumber roots, stems, young leaves, mature leaves, senescent leaves, fruits, sepals, and tendrils were used as materials for quantitative real-time PCR (qPCR) analysis. As shown in Figure 2A, CsHCAR expression was mainly concentrated in leaves, and also expressed in sepals, stems, fruits, and tendrils. The expression levels of CsHCAR in mature leaves and senescent leaves were 3.8-fold and 11.4-fold higher than those in young leaves, respectively (Figure 2A), and higher expression levels were observed in senescent leaves, indicating that CsHCAR might play an important regulatory role in the senescence process of cucumber plants. and tendrils. The expression levels of CsHCAR in mature leaves and senescent leaves were 3.8-fold and 11.4-fold higher than those in young leaves, respectively (Figure 2A), and higher expression levels were observed in senescent leaves, indicating that CsHCAR might play an important regulatory role in the senescence process of cucumber plants.
To determine the localization of CsHCAR in cells, Agrobacterium tumefaciens harboring the pFGC5941-CsHCAR-GFP fusion expression vector was infiltrated into Nicotiana benthamiana leaves, and the fluorescence of GFP was observed by laser confocal microscope. As shown in Figure 2B, the fluorescence of CsHCAR-GFP fusion protein was overlapped with the chloroplast auto-fluorescence signal, indicating that the CsHCAR protein was localized in the chloroplast.

Response of CsHCAR to Multiple Phytohormones and Abiotic Stresses
It has been demonstrated that environmental stresses and phytophormones mediate Chl degradation in plants [3,6]. Considering the critical role of HCAR in Chl breakdown, we analyzed the response of CsHCAR to cold, heat, dark, drought stress, abscisic acid (ABA), salicylic acid (SA), methyl jasmonate (MeJA), and gibberellin (GA3), respectively. Cold, heat stress, and SA treatment had similar effects on CsHCAR, and the expression level of CsHCAR was firstly decreased and then increased, and reached the highest level To determine the localization of CsHCAR in cells, Agrobacterium tumefaciens harboring the pFGC5941-CsHCAR-GFP fusion expression vector was infiltrated into Nicotiana benthamiana leaves, and the fluorescence of GFP was observed by laser confocal microscope. As shown in Figure 2B, the fluorescence of CsHCAR-GFP fusion protein was overlapped with the chloroplast auto-fluorescence signal, indicating that the CsHCAR protein was localized in the chloroplast.

Response of CsHCAR to Multiple Phytohormones and Abiotic Stresses
It has been demonstrated that environmental stresses and phytophormones mediate Chl degradation in plants [3,6]. Considering the critical role of HCAR in Chl breakdown, we analyzed the response of CsHCAR to cold, heat, dark, drought stress, abscisic acid (ABA), salicylic acid (SA), methyl jasmonate (MeJA), and gibberellin (GA 3 ), respectively. Cold, heat stress, and SA treatment had similar effects on CsHCAR, and the expression level of CsHCAR was firstly decreased and then increased, and reached the highest level at 48 h, which increased by approximately 8.5-fold, 5.1-fold, and 7.6-fold, respectively ( Figure 3). Under drought and darkness treatment, the expression of CsHCAR increased rapidly in a short time and reached the highest level at 12 h ( Figure 3). In the case of exogenous GA 3 treatment, CsHCAR expression was rapidly up-regulated within 3 h, recovered to the pre-treatment level at 6 h, and reached the highest level at 12 h, increasing by about 11.2 times compared with 0 h (Figure 3). Under ABA treatment, the expression of CsHCAR increased continuously and reached the highest level at 24 h, which increased by about 12.9 times (Figure 3). Under exogenous MeJA treatment, the expression of CsHCAR was firstly increased and then decreased, and reached the highest level at 6 h, which was about 7.6 times higher than that of the control plants ( Figure 3). Therefore, CsHCAR could be induced by different stresses and phytohormone treatments, suggesting that it might play a critical role in environmental stresses-and phytohormones-induced Chl degradation. at 48 h, which increased by approximately 8.5-fold, 5.1-fold, and 7.6-fold, respectively ( Figure 3). Under drought and darkness treatment, the expression of CsHCAR increased rapidly in a short time and reached the highest level at 12 h ( Figure 3). In the case of exogenous GA3 treatment, CsHCAR expression was rapidly up-regulated within 3 h, recovered to the pre-treatment level at 6 h, and reached the highest level at 12 h, increasing by about 11.2 times compared with 0 h (Figure 3). Under ABA treatment, the expression of CsHCAR increased continuously and reached the highest level at 24 h, which increased by about 12.9 times ( Figure 3). Under exogenous MeJA treatment, the expression of CsHCAR was firstly increased and then decreased, and reached the highest level at 6 h, which was about 7.6 times higher than that of the control plants ( Figure 3). Therefore, CsHCAR could be induced by different stresses and phytohormone treatments, suggesting that it might play a critical role in environmental stresses-and phytohormones-induced Chl degradation.

Overexpression of CsHCAR Promotes Chl Degradation
In order to investigate the function of CsHCAR, the Agrobacteria-mediated leaf plate method was used to perform genetic transformation in tobacco to obtain the transgenic plants that were overexpressing the CsHCAR. The rooting plants were screened by hygromycin, the DNA was extracted and verified by PCR, and two independent overexpression positive lines were identified ( Figure S2A). Immunoblotting analysis revealed that these two lines expressed high CsHCAR protein levels ( Figure S2B). Therefore, OE-1# and OE-2# lines were selected for subsequent experiments.
After darkness treatment for 10 d, the fresh weight (FW) and dry weight (DW) of CsH-CAR overexpression plants were significantly lower than that of the wild-type (WT) plants ( Figure 4A,B). After 10 d of darkness treatment, all of the plants showed different degrees of yellowing; however, the overexpression lines had wrinkle and more obvious chlorosis of the leaves ( Figure 4C). The total Chl content in WT plants decreased by 44.06%, while OE-1# and OE-2# lines decreased by 56.11% and 63.48%, respectively, in comparison to the control plants ( Figure 4D). To further verify the function of CsHCAR in Chl degradation, we detected the expression level of CCGs in WT and CsHCAR overexpression plants. Although darkness induced the expression of CCGs in WT and CsHCAR overexpression plants, their expression levels in CsHCAR overexpression plants were significantly higher than that in WT plants ( Figure 5). Therefore, overexpression of CsHCAR promoted dark-induced Chl degradation through upregulating the expression of CCGs.
Plants 2021, 10, 1820 6 of 16 results represent the mean ± SE (n=3). Means with the same letter did not significantly differ at p < 0.05 according to Tukey's test.

Overexpression of CsHCAR Promotes Chl Degradation
In order to investigate the function of CsHCAR, the Agrobacteria-mediated leaf plate method was used to perform genetic transformation in tobacco to obtain the transgenic plants that were overexpressing the CsHCAR. The rooting plants were screened by hygromycin, the DNA was extracted and verified by PCR, and two independent overexpression positive lines were identified ( Figure S2A). Immunoblotting analysis revealed that these two lines expressed high CsHCAR protein levels ( Figure S2B). Therefore, OE-1# and OE-2# lines were selected for subsequent experiments.
After darkness treatment for 10 d, the fresh weight (FW) and dry weight (DW) of CsHCAR overexpression plants were significantly lower than that of the wild-type (WT) plants ( Figure 4A,B). After 10 d of darkness treatment, all of the plants showed different degrees of yellowing; however, the overexpression lines had wrinkle and more obvious chlorosis of the leaves ( Figure 4C). The total Chl content in WT plants decreased by 44.06%, while OE-1# and OE-2# lines decreased by 56.11% and 63.48%, respectively, in comparison to the control plants ( Figure 4D). To further verify the function of CsHCAR in Chl degradation, we detected the expression level of CCGs in WT and CsHCAR overexpression plants. Although darkness induced the expression of CCGs in WT and CsHCAR overexpression plants, their expression levels in CsHCAR overexpression plants were significantly higher than that in WT plants ( Figure 5). Therefore, overexpression of CsHCAR promoted dark-induced Chl degradation through upregulating the expression of CCGs.   . Expression of chlorophyll degradation related genes in leaves of wild-type and CsHCAR overexpression tobacco seedlings. The results represent the mean ± SE (n = 3). Means with the same letter did not significantly differ at p < 0.05 according to Tukey's test. WT, wild-type; OE-1# and OE-2#, 2 independent transgenic tobacco overexpression of CsHCAR. DDI, day(s) of dark incubation; NYC1, non-yellow coloring1; NOL, NYC1-like; SGR1, stay-green1; PPH, pheophytinase; PAO, pheophorbide a oxygenase; RCCR, red chlorophyll catabolite reductase.

Overexpression of CsHCAR Affects Photosynthesis
In order to test the effect of CsHCAR on photosynthesis, we compared the net photosynthetic rate (Pn) of CsHCAR overexpression and WT plants. No significant difference was observed between WT and CsHCAR overexpression plants under normal growth conditions, while WT plants showed higher Pn under darkness stress ( Figure 6A). Darkness induced the decrease in the maximum quantum yield of photosystem II (Fv/Fm) and effective quantum yield of photosystem II [Y(II)] in all of the plants; however, the values of Fv/Fm and Y(II) in WT plants were significantly higher than those in CsHCAR overexpression plants at 10 d of darkness treatment ( Figure 6B-D). The dark-induced photosystem II damage was more serious in CsHCAR overexpression tobacco plants as a result of reduction in photosynthesis efficiency.

Overexpression of CsHCAR Affects Photosynthesis
In order to test the effect of CsHCAR on photosynthesis, we compared the net photosynthetic rate (Pn) of CsHCAR overexpression and WT plants. No significant difference was observed between WT and CsHCAR overexpression plants under normal growth conditions, while WT plants showed higher Pn under darkness stress ( Figure 6A). Darkness induced the decrease in the maximum quantum yield of photosystem II (Fv/Fm) and effective quantum yield of photosystem II [Y(II)] in all of the plants; however, the values of Fv/Fm and Y(II) in WT plants were significantly higher than those in CsHCAR overexpression plants at 10 d of darkness treatment ( Figure 6B-D). The dark-induced photosystem II damage was more serious in CsHCAR overexpression tobacco plants as a result of reduction in photosynthesis efficiency. To further investigate the role of CsHCAR on photosynthesis, the differences in the content of photosystem proteins were analyzed by immunoblotting. As shown in Figure  7A, the protein levels of Lhcb1, Lhcb2, Lhcb4, RbcS, and RbcL in WT plants were higher than those in CsHCAR overexpression plants under normal conditions. Although the protein abundances of Lhcb1, Lhcb2, RbcS, and RbcL in WT and CsHCAR overexpression plants were decreased under darkness treatment, the levels of these proteins in WT plants were higher than those in CsHCAR overexpression plants ( Figure 7A). The abundances of Lhcb4 in CsHCAR overexpression plants were also lower than those observed in WT plants after 10 d of darkness stress ( Figure 7A). Furthermore, the expression of genes encoding these proteins in the plants was restrained, however, their expression levels in WT plants were still higher than those in CsHCAR overexpression plants ( Figure 7B-F). These results indicated that the decline of photosynthesis efficiency in CsHCAR overexpression plants might result from the lower abundance of photosystem proteins and decreasing the expression of genes encoding these proteins. To further investigate the role of CsHCAR on photosynthesis, the differences in the content of photosystem proteins were analyzed by immunoblotting. As shown in Figure 7A, the protein levels of Lhcb1, Lhcb2, Lhcb4, RbcS, and RbcL in WT plants were higher than those in CsHCAR overexpression plants under normal conditions. Although the protein abundances of Lhcb1, Lhcb2, RbcS, and RbcL in WT and CsHCAR overexpression plants were decreased under darkness treatment, the levels of these proteins in WT plants were higher than those in CsHCAR overexpression plants ( Figure 7A). The abundances of Lhcb4 in CsHCAR overexpression plants were also lower than those observed in WT plants after 10 d of darkness stress ( Figure 7A). Furthermore, the expression of genes encoding these proteins in the plants was restrained, however, their expression levels in WT plants were still higher than those in CsHCAR overexpression plants ( Figure 7B-F). These results indicated that the decline of photosynthesis efficiency in CsHCAR overexpression plants might result from the lower abundance of photosystem proteins and decreasing the expression of genes encoding these proteins.

Overexpression of CsHCAR Reduces ROS Production
Cell death is often accompanied by an increase in ROS level [23]. Previous studies on HCAR are mainly focused on plant senescence, and there are few reports on the effect of HCAR on the content of ROS in leaves. Therefore, we compared the accumulation and production of H2O2 and O2 •− in leaves of WT and CsHCAR overexpression tobacco seedlings. 3,3′-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) histochemical staining results showed that H2O2 and O2 •− were accumulated in the leaves of all plants after 10 d of darkness stress, while ROS accumulation in leaves of WT plants was higher than that of CsHCAR overexpression seedlings ( Figure 8A,B). Furthermore, the content of H2O2 and O2 •− in CsHCAR overexpression was significantly lower than that in WT plants, which was consistent with the results of tissue staining ( Figure 8C,D). These results suggested

Overexpression of CsHCAR Reduces ROS Production
Cell death is often accompanied by an increase in ROS level [23]. Previous studies on HCAR are mainly focused on plant senescence, and there are few reports on the effect of HCAR on the content of ROS in leaves. Therefore, we compared the accumulation and production of H 2 O 2 and O 2 •− in leaves of WT and CsHCAR overexpression tobacco seedlings. 3,3 -diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) histochemical staining results showed that H 2 O 2 and O 2 •− were accumulated in the leaves of all plants after 10 d of darkness stress, while ROS accumulation in leaves of WT plants was higher than that of CsHCAR overexpression seedlings ( Figure 8A,B). Furthermore, the content of H 2 O 2 and O 2 •− in CsHCAR overexpression was significantly lower than that in WT plants, which was consistent with the results of tissue staining ( Figure 8C,D). These results suggested that overexpression of CsHCAR inhibited the accumulation of ROS, which might alleviate dark-induced cell death.
Plants 2021, 10, 1820 1 that overexpression of CsHCAR inhibited the accumulation of ROS, which might all dark-induced cell death.

Discussion
Chl is a potential molecule for the production of ROS, and it is converted into fluorescent Chl decomposition metabolites during aging [3,24]. Disorder of Chl me lism will lead to the accumulation of intermediate molecules, which will cause ne changes in plant leaves [1,25]. However, when Chl supply is limited, photosynthe tivity is reduced, leading to plant growth retardation [26]. Therefore, it is very impo to strictly control the synthesis and degradation of Chl during greening and senesc The Chl cycle plays an important role in maintaining the balance of Chl. This recip transformation pathway between Chl a and Chl b is called the Chl cycle, which is trolled by CBR and HCAR [2,12,14]. At present, studies on HCAR are mainly focus Arabidopsis and rice, and it has not been reported in horticultural plants. In this s we found that there was one HCAR gene in the cucumber genome, which was locat chromosome 3 and encoded 459 amino acids (Table 1). Amino acid sequence compa showed that the protein structure of HCAR in cucumber was highly conserved an the same domain as other species (Figure 1). The amino acid sequence of cucumber H was orthologous to that of Arabidopsis, suggesting that it may have a similar funct Arabidopsis. In Arabidopsis, HCAR catalyzes the conversion of 7-HMChl a to Chl HCAR gene mutants show delaying Chl degradation, while its overexpression plan celerate leaf yellowing in dark-induced senescence [14,18]. Furthermore, rice hcar m also displays persistent green phenotype in dark-induced and natural senescence Here, we found that CsHCAR was induced by darkness, and highly expressed in sene leaves (Figures 2A and 3). Furthermore, leaves of tobacco plants that were overexpre CsHCAR turned yellow, the Chl degradation rate was accelerated, and the CCGs significantly up-regulated during darkness treatment (Figures 4 and 5), indicating

Discussion
Chl is a potential molecule for the production of ROS, and it is converted into nonfluorescent Chl decomposition metabolites during aging [3,24]. Disorder of Chl metabolism will lead to the accumulation of intermediate molecules, which will cause necrotic changes in plant leaves [1,25]. However, when Chl supply is limited, photosynthetic activity is reduced, leading to plant growth retardation [26]. Therefore, it is very important to strictly control the synthesis and degradation of Chl during greening and senescence. The Chl cycle plays an important role in maintaining the balance of Chl. This reciprocal transformation pathway between Chl a and Chl b is called the Chl cycle, which is controlled by CBR and HCAR [2,12,14]. At present, studies on HCAR are mainly focused on Arabidopsis and rice, and it has not been reported in horticultural plants. In this study, we found that there was one HCAR gene in the cucumber genome, which was located on chromosome 3 and encoded 459 amino acids (Table 1). Amino acid sequence comparison showed that the protein structure of HCAR in cucumber was highly conserved and had the same domain as other species (Figure 1). The amino acid sequence of cucumber HCAR was orthologous to that of Arabidopsis, suggesting that it may have a similar function to Arabidopsis. In Arabidopsis, HCAR catalyzes the conversion of 7-HMChl a to Chl a, and HCAR gene mutants show delaying Chl degradation, while its overexpression plants accelerate leaf yellowing in dark-induced senescence [14,18]. Furthermore, rice hcar mutant also displays persistent green phenotype in dark-induced and natural senescence [21]. Here, we found that CsHCAR was induced by darkness, and highly expressed in senescent leaves (Figures 2A and 3). Furthermore, leaves of tobacco plants that were overexpressing CsHCAR turned yellow, the Chl degradation rate was accelerated, and the CCGs were significantly up-regulated during darkness treatment (Figures 4 and 5), indicating that up-regulating CsHCAR promotes Chl degradation and its function is similar to AtHCAR and OsHCAR.
Phytohormones play critical roles in regulating leaf senescence [24]. Environmental stresses accelerate leaf senescence, accompanied by the production of ABA, SA, and JA [27][28][29][30]. It has been demonstrated that ABA, SA, and JA positively regulate leaf senescence through activating the expression of CCGs [31][32][33]. Transcription factors in the downstream of these phytohormones, such as ABF3, ABF4, ABI5, MYC2, MYC3, and MYC4, directly bind to the promoters of NYC1, PPH, and PAO to trigger their expression, resulting in promoting Chl breakdown [32,34,35]. In this study, we found that CsHCAR was induced by foliar application of ABA, SA, and MeJA, suggesting that it might mediate these phytohormones-induced Chl degradation. In addition, HCAR physically interacts with CCEs and LHCII to form a complex, which plays critical roles in Chl degradation [18,21]. Crystal structure analysis reveals that AtHCAR has the potential to form trimers, which may be critical for its interaction with LHCII [36]. HCAR can form the dimer or trimer through interaction with LHCII and other CCEs, which might enhance its functions [18,21]. We found that the protein abundances of Lhcb1, Lhcb2, and Lhcb4 in CsHCAR overexpression tobacco plants were reduced and lower than those in WT plants under darkness stress ( Figure 7A). However, the stability of the photosystem protein in oshcar mutants was higher than that in WT plants [21]. These results indicated that the gene abundance of HCAR was proportional to the degradation rate of Chl and photosystem protein in senescent leaves.
It is reported that silencing or knockout of CCG, such as PAO and RCCR, exhibits an accelerated cell death phenotype via accumulation of excess phototoxic Chl intermediates [9,37]. HCAR may be involved in regulating cell death signaling by regulating the metabolic process of Chl degradation [25]. Cell death is often accompanied by an increase in ROS levels [23]. It was found that after 10 d of darkness stress, ROS accumulation in leaves of WT plants was significantly higher than that of CsHCAR overexpression tobacco seedlings (Figure 8). Athcar and oshcar mutants accumulate more ROS than WT plants, while their overexpression plants maintain lower levels of ROS [21]. Furthermore, knockout of HCAR promotes the accumulation of 7-HMChl a and Pheide a [14,21]. WT and hcar mutant protoplasts treated with 7-HMChl a or Pheide a both induce singlet oxygen production, however, the intensity in hcar mutant protoplasts is more obvious [21]. Thus, the accumulation of 7-HMChl a and Pheide a incudes the production of singlet oxygen to trigger cell death. During natural senescence and dark-induced senescence, overexpression of HCAR can alleviate the symptoms of non-apoptotic programmed death in plants with excessive accumulation of Chl b [22]. Therefore, these results suggested that CsHCAR might play an important role in the regulation of leaf cell death.

Plant Material and Growth Conditions
Cucumber (Cucumis sativus L. cv Jinchun No. 2) was used as the test material and the seeds were purchased from Tianjin Kernel Cucumber Research Institute (Tianjin, China) and used in our experiments. The uniformly germinated seeds were sown in plastic pots (10 cm × 7 cm × 8 cm) covered with a mixture of peat and vermiculite (2:1, v:v). The growth conditions were maintained as follows: 14/10 h light/dark cycle, 25/18 • C day/night, 75-80% relative humidity, and 300 µmol m −2 s −1 photosynthetic photon flux density (PPFD).
To analyze the possible effect of phytohormones and stress on CsHCAR expression, cucumber seedlings at the third leaves stage were treated with different phytohormones and imposed seedlings to the different abiotic stresses. For phytohormones treatment, 100 µM ABA, SA, MeJA, and GA 3 were sprayed on cucumber leaves, respectively. Cucumber plants were treated with 4 • C as cold stress, 42 • C as heat stress, 20% polyethylene glycol 6000 (PEG) as drought stress, and 48 h darkness as dark stress. The leaf samples were collected at various time points (0, 3, 6, 12, 24, and 48 h).
WT and transgenic tobacco seeds were sown on a plate containing Murashige & Skoog solid medium, and, after germination, seedlings were transferred into seedling substrate (10 cm × 7 cm × 8 cm). When the seedlings grew to 4 leaves, WT and transgenic tobacco seedlings with the same growth character were selected and cultured in darkness for 10 d. Seedlings grown with normal light were used as the control. After 10 d of darkness treatment, the seedlings were sampled and frozen with liquid nitrogen and then stored in the −80 • C.

Identification and Sequence Analysis of HCAR
To identify CsHCAR, BLAST, a search of the cucurbitaceae genome database (http: //cucurbitgenomics.org/ (accessed on 3 August 2021)) was performed with Arabidopsis HCAR amino acid sequence. The protein MW and pI were analyzed using Protparam (http://web.expasy.org/protparam/ (accessed on 3 August 2021)). The conservative structure of the protein domains was analyzed using Pfam database (http://pfam.xfam. org/ (accessed on 3 August 2021)). The phylogenetic tree of a set of HCAR protein sequences, searched in the NCBI database, was constructed using MEGA 5.0 software with Neighbor-joining method.

Total RNA Extraction and Gene Expression Analysis
Total RNA was extracted using RNA simple Total RNA Kit (Tiangen, Beijing, China) according to the manufacturer's instruction. The total RNA (1 µg) was reverse transcribed using the HiScript II Q RT SuperMix for qPCR (+gDNA wiper) Kit (Vazyme, Nanjing, China). qPCR assays were performed using the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster, CA, USA) with the ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China). The PCR program consisted of predenaturation at 95 • C for 5 min, followed by 40 cycles of 95 • C for 10 s, and 60 • C for 30 s. Specific primers (Table S2) were designed according to the CDS. The actin gene was used as an internal control. The relative gene expression was calculated based on the mean of three biological replications and were calculated using the 2 −∆∆CT method [38].

Subcellular Localization of HCAR
The full-length CDS of CsHCAR was amplified with specific primers (Table S3) and inserted into pFGC5941-GFP vector to generate a CsHCAR-GFP fusion expression vector, then transformed into the N. benthamiana leaves using A. tumefaciens strain GV3101. Control samples were transformed with an empty pFGC5941-GFP vector. After inoculation for 2 d, the GFP fluorescence was monitored under an LSM 800 confocal microscope (Zeiss, Oberkochen, Germany). All transient expression assays were repeated at least three times.

Plasmid Construction and Screening Transgenic Plants
To generate the CsHCAR overexpression construct, the full-length CDS was amplified with specific primers (Table S3) using cucumber cDNA as the template. The PCR product was inserted into the plant transformation vector pFGC1008-HA using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China). The constructed plasmid was transformed into A. tumefaciens strain EHA105, then transferred into NC89 tobacco plants as described previously [39]. Transgenic plants that were overexpressing the CsHCAR were identified by genomic PCR and immunoblotting. Two independent homozygous lines of the T 2 progeny (OE-1# and OE-2#, overexpression line 1 and 2) were used for further experiments.

Measurement of Chl Content
The Chl was extracted from the fifth leaves (0.2 g) and the samples were shredded and soaked in 25 mL ethanol in the darkness until completely whitened. Then, Chl concentrations were measured by spectrophotometry at 665 and 649 nm according to the method previously described [41].

Determination of Growth, Chl Fluorescence Parameters and Pn
For measurement of the FW and DW of tobacco seedlings, the plants were washed with distilled water and dried with absorbent paper after darkness stress for 10 d. The FW was measured by electronic balance (Sartorius, Goettingen, Germany). After that, the whole plants were enclosed in the envelopes and placed in an oven (Shanghai Yiheng Scientific Instrument Co., Ltd., Shanghai, China) at 105 • C for 15 min. Afterward, the oven temperature was adjusted to 75 • C to obtain a constant DW.
Tobacco plants were dark-adapted for 30 min to measure the Chl fluorescence parameters with the portable fluorometer (PAM-2100, Heinz Walz, Effeltrich, Germany) according to previously described method [42].
A portable photosynthetic apparatus LI-6400 (LI-COR, Lincoln, NE, USA) was used to measure the Pn of tobacco leaves, as in the method described by Zhang et al. [43]. For the measurement of data, the cuvette conditions were provided as PPFD of 800 µmol m −2 s −1 , relative humidity at 60-70%, temperature of 25 • C, and CO 2 concentration of 380 ± 10 µmol mol −1 .

Measurement of ROS Content and Histochemical Staining
The H 2 O 2 concentration in leaves was estimated by a method described previously [44]. The superoxide anion (O 2 •− ) production was determined as previously described [45]. Tobacco leaves treated with darkness for 0 d and 10 d were cut into 1.5 cm leaf discs for H 2 O 2 and O 2 •− staining. DAB staining method was used for H 2 O 2 histochemical staining [46]. After soaking tobacco leaf plates in 50 mM Tris-HCl (pH 3.8) solution containing 1 mg L −1 DAB, they were subjected to light for staining until the brown spots appeared on the leaves. O 2 •− histochemical staining was performed using NBT [46]. After soaking tobacco leaves in 25 mM HEPES (pH 7.8) solution containing 1 mg L −1 NBT, they were dyed in the dark for 3-5 h. Decolorization was carried out in boiling water bath with 95% ethanol until Chl was completely removed from the leaves.

Statistical Analysis
Experimental data were analyzed using the SPSS statistics software IBM SPSS statistics 20.0 (SPSS Inc., Chicago, IL, USA). Statistically significant differences among the treatments were determined using Tukey's test at p < 0.05. Three independent biological replicates were used for each determination.

Conclusions
In conclusion, we identified one HCAR gene in the cucumber genome, which was orthologous with other species, including Arabidopsis, melon, and rice. The gene expression levels of CsHCAR in mature leaves and senescent leaves were higher, indicating that it might play an important regulatory role in the senescence process of cucumber. Overexpression of CsHCAR promoted dark-induced Chl degradation through elevating the transcription of CCGs. The photosynthesis efficiency was also hampered in CsHCAR overexpression plants by the reduction of Fv/Fm and suppression the light-catching antenna protein content, as well as their encoding gene expression. Furthermore, the ROS accumulation in leaves of CsHCAR overexpression lines significantly decreased after 10 d of darkness treatment. These results indicated that CsHCAR affected the stability of chloroplast photosynthetic proteins, and positively regulated Chl degradation.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/plants10091820/s1, Figure S1: Phylogenetic tree of CsHCAR protein from cucumber and other species. Figure S2: Identification of the CsHCAR overexpression transgenic tobacco plants. Table S1: GenBank accession numbers of HCAR used to build phylogenetic tree.