Abstract
Chlorophyll loss is a major problem during green vegetable storage. However, the molecular mechanism is still unclear. In this study, a 21 days of storage experiments showed chlorophyll content was higher in light-stored Chinese cabbage (Brassica chinensis L.) leaves than those in dark-stored samples. Transcriptome analyses were performed on these samples to determine the effects of light. Among 311 differentially expressed genes (DEGs), early light-induced protein 1 (ELIP1) was identified as the main control gene for chlorophyll synthesis. Tissues and subcellular localization indicated that ELIP1 was localized in the nucleus. Motifs structure analyses, chromatin immunoprecipitation (ChIP) assays, luciferase reporter assays, and overexpression experiments demonstrated that ELIP1 regulated the expressions of genomes uncoupled 4 (GUN4), Glutamyl-tRNA reductase family protein (HEMA1), and Mg-protoporphyrin IX methyltransferase (CHLM) by binding to G-box-like motifs and affected chlorophyll biosynthesis during the storage of Chinese cabbage. It is a possible common tetrapyrrole biosynthetic pathway for chlorophylls, hemes, and bilin pigments in photosynthetic organisms. Our research also revealed that white light can be used as a regulatory factor to improve the storage ability and extent shelf life of Chinese cabbage.
Similar content being viewed by others
Introduction
Chinese cabbage (Brassica chinensis L.) belongs to cruciferous plants, which is one of the most important autumn vegetable varieties1,2,3. Chlorophyll content sometimes represents the storage quality of green vegetables4,5. Dark-adapted leaf senescence caused by chlorophyll loss is an essential problem during vegetable storage6. Some studies have revealed that light storage can prevent the leaf senescence and maintain the total chlorophyll content higher than dark storage7. However, the molecular mechanism is still unclear.
In green plants, chlorophyll biosynthesis starts with glutamyl-tRNA and ends with chlorophyll b synthesis8, which is controlled by multiple genes. For example, genomes uncoupled 4 (GUN4) is involved in the control of tetrapyrrole synthesis during the chlorophyll biosynthesis as a regulatory sub-unit of Mg-chelatase9, glutamyl-tRNA reductase family protein (HEMA1) is acutely dependent on phytochrome signaling and permitted the tetrapyrrole synthesis for the accumulation of chlorophyll10, and Mg-protoporphyrin IX methyltransferase (CHLM) plays a crucial role in chlorophyll biosynthesis and converts Mg-protoporphyrin IX to Mg-protoporphyrin IX monomethyl ester11,12,13,14. But recent research indicates that the chlorophyll a/b-binding (CAB) protein family is a crucial precursor signal during chlorophyll biosynthesis and metabolism15,16. At least 30 CAB family proteins have been found in Arabidopsis thaliana17,18, including photo-system protein19, one-helix protein20, and early light-induced proteins (ELIPs)21.
As a photoinduced protein, ELIPs broadly exists in various plants22. Chinese cabbage has two ELIPs alleles, namely ELIP1 and ELIP223. ELIP1 is nucleus-encoded protein that is synthesized on cytoplasmic ribosomes, then transfers to the chloroplast, and anchors to thylakoid membranes through three transmembrane domains22,23,24,25. ELIP1 can be expressed transiently in response to light in etiolated plants26. Under moderate light intensities conditions, ELIP1 only plays a slight role in the chlorophyll accumulation27. However, ELIP1 expression and chlorophyll accumulation dramatically decrease at low temperature and low light intensity28. In brief, the physiological function of ELIP1 may be related to the regulation of chlorophyll biosynthesis, but molecular mechanism on regulatory pathway has barely been reported, especially during the storage of green vegetable. In this study, we demonstrated some molecular mechanism on the light regulation of chlorophyll biosynthesis via ELIP1 during the storage of Chinese cabbage.
Results
Screening for chlorophyll regulatory genes during the storage of Chinese cabbage
After 21 days of storage, Chinese cabbage leaves stored under white light condition were still green, whereas those under dark condition lost most of their green pigment and become senescence (Fig. 1A). Chlorophyll content was higher in light-stored samples than those in dark-stored samples as revealed in Fig. 1B. The percentage of fresh weight loss of light-stored cabbage samples was also much lower than those of dark-stored samples (Fig. 1C). These results showed that light was very important to maintain the chlorophyll content and fresh weight during the storage of Chinese cabbage.
Transcriptome analyses were performed on Chinese cabbage samples to explore the molecular mechanisms. TS01 was fresh Chinese cabbage samples as control, TS02 samples were obtained on the 10th day of dark storage, and TS03 samples were obtained on the 20th day of dark storage. The results uncovered 3,687 DEGs at T02 versus T01 (Fig. 1D, Supplementary Fig. S1), 3,621 DEGs at T03 versus T02 (Fig. 1D, Supplementary Fig. S2), 2,472 DEGs at T02 versus T01 (Fig. 1D, Supplementary Fig. S3), and 311 common DEGs at T03 versus T02 versus T01 (Fig. 1D,E). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were applied for the analyses of these DEGs and revealed the genes involved in biological process, molecular function, and cellular component that were markedly altered in the storage of Chinese cabbage (Supplementary Figs. S1–S3). Clustering analysis suggested that more genes were down-regulated in dark-stored Chinese cabbage (Fig. 1E); therefore, light is a possible regulatory factor in the expression of these down-regulated genes. Among 311 DEGs, three CAB family members, namely, ELIP1, LHCAB1.1, and LHCAB2.1 may be closely related to the changes in chlorophyll content during the storage of Chinese cabbage (Fig. 1E, Supplementary Table S3).
Light regulation experiment
Transcriptome analysis revealed that light, as an important factor, had potential regulatory effects on chlorophyll content and gene expression. Further qRT-PCR results showed that ELIP1 was down-regulated in the dark but up-regulated under light exposure (Fig. 2A). The results of western blot indicated that ELIP1 signal was increased after light exposure, but was hardly detected after dark storage (Fig. 2B,C). Expression levels of LHCBA1.1 and LHCBA2.1 in light exposure had no substantial changes (Fig. 2D,E), but LHCBA2.1 was up-regulated in dark condition (Fig. 2D). We analyzed the expression of ELONGATED HYPOCOTYL 5 (HY5) and PHYTOCHROME INTERACTING FACTOR 3 (PIF3) and the results displayed elevated HY5 and decreased PIF3 in the dark (Supplementary Fig. S4A,B). This was consistent with the results of Cazzonelli et al.28. PIF3 and HY5 may be as a retrograde signal to regulating LHCBA2.1 expression to maintain the stability of chlorophyll content during dark storage in Chinese cabbage. Chlorophyll synthesis genes GUN4, HEMA1 and CHLM were prominently increased under light condition (Fig. 2F–H), but dramatically decrease under dark condition (Fig. 2F–H). In addition, we also found that the chlorophyll content increased significantly under light condition, but decreased slightly under dark condition after a 24-h light regulation experiment (Fig. 2I). These results were consistent with the expression trends of ELIP1 (Fig. 2A) and suggested that ELIP1 could be the control gene for chlorophyll biosynthesis during the storage of Chinese cabbage.
Tissue and subcellular localization analyses of ELIP1
We performed the tissue and subcellular localization with immunohistochemistry (IHC) and ELIP1-CFP fusion protein to investigate the localization of the ELIP1 protein in Chinese cabbage. The completely overlapped fluorescence signals of DAPI and IHC in tissue sections demonstrate that ELIP1 was localized near the nucleus (Fig. 2A–C). ELIP1-CFP fusion protein was also localized in the nucleus by comparing the overlapping areas of DAPI signal in the protoplast cells (Fig. 2B–D). In the 40 × field, ELIP1-CFP signal was showed in nucleus (white arrow) and cell wall of Chinese cabbage protoplasts (Fig. 2E–G). These findings indicated that ELIP1 was localized in the nucleus, which provided a molecular basis for its regulation on chlorophyll synthesis genes.
ELIP1 regulates GUN4, HEMA1, and CHLM via the G-box-like motifs
As a cis-acting DNA regulatory element, the G-box (CACGTG) can be bound to proteins known as G-box factors (GBFs) in a context-specific manner in plant genomes30. The motif structure analyses indicated that there were two typical G-box motifs in the promoter regions of ELIP1, nine G-box-like (CACGTN) motifs in the promoter regions of CHLM, one G-box-like motif in the promoter regions of GUN4, and one G-box-like motif in the promoter regions of HEMA1 (Fig. 4A). The binding relationship between ELIP1 and CHLM, GUN4, and HEMA1 was investigated by ChIP assays. The results revealed that ELIP1 could bind to G-box-like motifs in CHLM (− 1034, Fig. 4B,C), CHLM (− 1089, Fig. 4B,D), GUN4 (− 1586, Fig. 4B,E), and HEMA1 (− 168, Fig. 4B,F).
Subsequently, ELIP1 cDNA was cloned into the pCambia1301 vector. Promoter sequence of CHLM, GUN4, and HEMA1, including ELIP1 binding sites and 5′-untranslated region, was cloned into pGreenII 0800-Luc vectors and transformed into agrobacterium. The luciferase gene reporter assays were performed three days after agro-inoculation. The results demonstrated that luciferase activities mediated by G-box-like motifs on CHLM-Luc, GUN4-Luc, and HEMA1-Luc were activated by pCambia1301-ELIP1 (Fig. 4G–I). A construct was designed wherein the G-box-like motifs were deleted to further investigate their potential role. CHLM-mut-Luc, GUN4-mut-Luc, and HEMA1-mut-Luc lost luciferase activities when added to the luciferase system with pCambia1301-ELIP1 (Fig. 4G–I). In summary, G-box-like motifs played key roles on the transcription regulation of ELIP1 on CHLM, GUN4, and HEMA1.
ELIP1 overexpression increased the expression levels of CHLM, GUN4, and HEMA1 and the biosynthesis of chlorophyll
Positive A. tumefaciens EHA105 colonies containing pBI121–ELIP1 vectors were employed to infect Chinese cabbage leaves for ELIP1 overexpression to verify the chlorophyll changes caused by the downregulation of ELIP1 in dark-stored Chinese cabbage (Fig. 1A). The ELIP1 overexpression transgenic line (EO line) showed a higher level of ELIP1 expression, whereas the WT and control exhibited lower ELIP1 expression levels (Fig. 5A). Similarly, CHLM (Fig. 5B), GUN4 (Fig. 5C), and HEMA1 (Fig. 5D) all showed higher expression levels in the EO line. We also measured the chlorophyll content in the WT, control, and EO line. The results showed that the chlorophyll content was higher in the EO line than that in the control group (Fig. 5E). We also found that there was no significant difference on chlorophyll content between the WT and EO line, which may be due to the inevitable chlorophyll loss in the working solution during leaf infection in the EO line (Fig. 5E). These results demonstrated that the ELIP1 gene was successfully introduced into the Chinese cabbage plants and substantially increased the expression levels of CHLM, GUN4, and HEMA1, as well as chlorophyll biosynthesis.
Discussion
In this study, a 21 days of storage experiments showed Chinese cabbage leaves stored under white light were still green, whereas those under dark storage lost most of their green pigment and became senescence. The chlorophyll content was higher in light-stored Chinese cabbage leaves than those in dark-stored samples. Transcriptome analyses showed that ELIP1 may be the main control gene for chlorophyll synthesis during the storage of Chinese cabbage.
Light can improve vegetable quality at harvest and postharvest, but the molecular mechanism has not been revealed. In this study, we discussed some molecular mechanism on the light regulation of chlorophyll biosynthesis via ELIP1 during the storage of Chinese cabbage. ELIP1was closely related to chlorophyll biosynthesis and seed germination27,29. The results showed that chlorophyll content and ELIP1 expression in light-stored cabbage samples were much higher than those of dark-stored samples (Fig. 1A,B). CHLM, GUN4, and HEMA1 were upregulated under light exposure (Fig. 2F–H) and in the EO line (Fig. 5B–D). Combined with previous findings, our study revealed synergistic regulations between ELIP1, chlorophyll content, CHLM, GUN4, and HEMA1 during the storage of Chinese cabbage.
Like other eukaryotes, the expression of plant genes is regulated at the chromosome, transcription, post-transcription, translation, and post-translation levels, among which transcriptional regulation is of great importance8. The transcription regulation of plant genes plays its role through the interactions between regulatory factors and the specific sequences of promoter regions (cis-acting elements). Plants have various types of cis-acting elements, such as G-box31,32, E-box33, D-box34, and F-box35. Among them, G-box (CACGTG), a typical motif with six base palindrome sequences, is broadly distributed in plants and plays an essential role on the photoinduced regulation of plant genes36. Figure 4A revealed the presence of a large number of G-box and G-box-like motifs (CACGTN) in the promoter regions of ELIP1, CHLM, HEMA1, and GUN4. Transcriptional regulation by promoter-targeted protein needs to be undertaken in the nucleus to promote transcription activation or silencing37. Previous studies showed that mature ELIPs in pea was transferred to the chloroplast and anchored to thylakoid membranes through three transmembrane domains24. However, these studies did not suggest whether ELIP1 exists in the nucleus. Therefore, proving that ELIP1 is located in the nucleus of Chinese cabbage plants is important. In our study, the tissue and subcellular localization clearly showed that ELIP1 located in the nucleus of plant tissues and protoplasts in Chinese cabbage (Fig. 3). These findings provide a very strong evidence that ELIP1 could regulate downstream genes through interactions with cis-acting elements.
ChIP assays and luciferase experiments showed that ELIP1 regulated the luciferase activities of CHLM, GUN4, and HEMA1 by directly binding with G-box-like motifs (Fig. 4B–F). Figures 1, 2, and 5 showed that ELIP1, CHLM, GUN4, and HEMA1 were directly correlated to the chlorophyll content during the storage of Chinese cabbage. However, chlorophyll biosynthesis in higher plants contains a series of enzymatic reactions and a great number of genes. In fact, the motif structure analyses indicated that there was a typical G-box motif in the promoter regions of PROTOCHLOROPHYLLIDE OXIDOREDUCTASE C (POR C)28. We checked the expression levels of POR C under dark/light storage and found a decreased expression in dark and increased expression in light (Supplementary Fig. S4C). However, the results of luciferase and ChIP assay showed that ELIP1 could not drive the expression of POR C-luc and bind to the promoter region of POR C (Supplementary Fig. S4D–E), so we speculated that POR C was regulated by light but not by ELIP1. In addition, there is a POR gene (Bra010646) in Chinese cabbage, but the functional annotation is not clear, so we did not detect the expression of POR. Whether other genes were regulated by ELIP1 still needs further investigation. Previous studies have shown that photosynthetic organisms synthesize chlorophyll, heme, and bilin pigments via a common tetrapyrrole biosynthetic pathway11. Our study also provided a possible molecular mechanism for this common pathway (Fig. 5F).
Studies have shown that photosynthetic carbon assimilation can increase CO2 fixation and biomass yield in tobacco38. Moreover, due to the lack of photosynthesis during dark storage, Chinese cabbage leaves often rot and lost weight, which was also calculated in the fresh weight loss. Therefore, the percentage of fresh weight loss in light-stored cabbage samples was much lower than those of dark-stored samples (Fig. 1C). The results suggested that light can maintain the green and weight and had good freshness-keeping effects on Chinese cabbage (Fig. 1A–C). In short, this study suggests that light can regulate chlorophyll biosynthesis via ELIP1 and prolong the shelf life of Chinese cabbage.
In conclusion, our study provided some molecular mechanism about vegetable chlorophyll biosynthesis during storage. ELIP1 regulates CHLM, HEMA1, and GUN4 via the G-box-like motifs to affect chlorophyll biosynthesis during the storage of Chinese cabbage.
Materials and methods
Plant materials, storage conditions, and leaves sampling
Chinese cabbage (BeijingNew No. 3) samples came from Merris vegetable base in Qiqihar, Heilongjiang Province, China. “Beijing New No. 3” cabbage is the F1 of self-incompatibility lines 832,172 cabbage and 84,427 cabbage. “Beijing New No. 3” cabbage was selected and bred by the Vegetable Research Center of Beijing Academy of Agriculture and Forestry, Beijing, China. We have permission to collect the plant samples used in the study.
These samples for storage were kept in a storage cabinet with a temperature of 2–6 °C and a humidity of 60–70% RH. In addition to light intensity, dark storage condition was the same as light storage. The light intensity of light storage was about 1100–1700 lx, but the light intensity of dark storage was reduced to about 0–3 lx.
Multi-point sampling collection was selected for the leaves sampling. Each leaf had 1–2 sampling points, and a maximum of 2 g leaves were collected each point. The leaves were washed three times with double distilled water and dried with absorbent paper for further experiments. Leaves sampling needs to be repeated 3–6 times for each experiment.
Chlorophyll content and the percentage fresh weight loss
Chlorophyll content was measured with the acetone methods. First, about 2 g Chinese cabbage leaves were cut into small pieces, placed into a 50-ml centrifuge tube, added with 5 ml of 80% acetone, and ground with a grinding pestle. The supernatant was retained for further experimental use. Second, the supernatant was centrifuged at 10,000 r/min for 1 min. The supernatant (2 ml) was added with 18 ml of ethanol to prepare a 20 ml working fluid. Finally, the working fluid was analyzed at 645 and 663 nm with a 721 spectrophotometer. Chlorophyll content was calculated as the sum of chlorophyll a and chlorophyll b.
Twelve fresh Chinese Cabbage were selected, removed sediment and impurities, wiped clean, wrapped around fresh-keeping film, weighed (Initial mass), and put into the storage cabinet mentioned above. There were six cabbages in the light layer and six cabbage in the dark layer. Twenty-one days later, these cabbages were taken out, the rotten tissues were removed, and the final weigh was gotten (final mass). The percentage of fresh weight loss = ((initial mass − final mass)/initial mass)) × 100.
RNA quantification for transcriptome analysis
Total RNA was extracted from the Chinese cabbage leaves on the first day (TS01, fresh samples), the 10th day (TS02), and the 20th day (TS03) using Trizol (Invitrogen) for transcriptome sequencing according to the manufacturer's protocol. Each sample needed about 100 mg leaves. The sample was cut into small pieces, placed into a 2-ml centrifuge tube, added with 500 µl of Trizol, and ground with a grinding pestle. First-strand cDNAs were synthesized using random hexamer primers and M-MuLV reverse transcriptase (RNase H-), and second-strand cDNA was synthesized using DNA polymerase I and RNase H. Remaining overhangs was converted into blunt ends via exonuclease/polymerase activities. The 3′ ends of DNA fragments were adenylated, and NEBNext Adaptor with a hairpin loop structure was ligated to prepare for hybridization. The library fragments were purified with the AMPure XP system (Beckman Coulter) to select cDNA fragments with preferentially 240 bps in length. Then, 3 μl of USER enzyme was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before polymerase chain reaction (PCR). PCR was performed using a high-fidelity DNA polymerase, Universal PCR primers, and Index (X) primer. The PCR products were purified, and library quality was assessed on the Agilent Bioanalyzer 2100 system.
Deep sequencing-based transcriptome analysis
Clean reads were mapped to the Chinese cabbage reference genome (ftp://brassicadb.org/bra'chrome'v1.5/) by TopHat v2.0.13. Gene expression was calculated by Cufflinks. Gene expression levels were normalized by reads per kilobase of transcript per million mapped reads. Differentially expressed genes (DEGs) were identified by the DESeq R package (1.10.1), and the resulting p values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with adjusting p < 0.05 found by DESeq were assigned as differentially expressed.
Total RNA extraction for quantitative real-time PCR (qRT-PCR)
Total RNAs were extracted from 50 mg Chinese cabbage using TRIzol® reagent (Ambion). The sample was cut into small pieces, placed into a 1.5-ml centrifuge tube, added with 500 µl of Trizol, and ground with a grinding pestle. The purified total RNAs (2 µl) were reverse transcribed into cDNAs. qRT-PCR was performed with a Bio-Rad Laboratories instrument with the TB-Green detection system. Primers (Comate Bioscience Technology) used for qRT-PCR are listed in Supplementary Table S1. All results were normalized to the expression level of the housekeeping gene ACTIN2. qRT-PCR results are shown as a relative expression level calculated using the 2−ΔΔ CT method. p values were calculated with one-way ANOVA test.
Light regulation experiment
Fresh Chinese cabbage samples were separately stored in white light (1100–1700 lx) and dark (0–3 lx) for 24 h in the 4 °C storage cabinet. Then, about 2 g leaves were immediately harvested with light avoidance for qRT-PCR and chlorophyll content analysis. qRT-PCR was performed to analyze the gene expression under light and dark conditions. Fresh Chinese cabbage samples were used as the control and marked as Zero, the light-stored samples were marked as L, and the dark-stored samples were marked as D (Fig. 2).
Western blot
About 20 mg samples were collected, chopped, and ground with 200 μl of lysis buffer (Beyotime Biotechnology). The precipitates were mixed with sodium dodecyl sulfate (SDS)–polyacrylamide (PA) gel electrophoresis sample buffer in a ratio of 4:1, boiled for 3–5 min, and run on 12% SDS-PA gel. The western blot was conducted with anti-ELIP1 (1:3000) and anti-ACTIN-plant (1:800) rabbit polyclonal antibodies. The quantitative analysis of the Western blot strips was carried out by Image J (National Institutes of Health).
Tissue localization of ELIP1 with immunohistochemistry (IHC)
Chinese cabbage samples were fixed in 4% paraformaldehyde at 4 °C for 1–2 days, dehydrated with different concentrations of ethanol gradient elution, and made transparent with xylene solution. The samples were embedded with optimum cutting temperature compound and cut into 6–8 μm sections. The sections were baked under 80 °C for 30 min, then washed with PBS solution, permeated with 0.1% Triton X-100 for 10 min, and blocked with 3% BSA for 30 min. The sections were incubated with the primary antibody of polyclonal rabbit anti-ELIP1 under 4 °C for one night, then washed with PBS solution, and incubated with the secondary antibody, fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG, under 37 °C in the dark for 1 h. Finally, the sections were softly rinsed with PBS and stained with 4,6-diamidino-2-phenylindole (DAPI) for about 10 min. The stained upper epidermis of cabbage leaf were observed under a fluorescence microscope (Olympus, BX53).
Cloning and subcellular localization of ELIP1
ELIP1 cDNA (603 bp) was amplified by PCR and cloned into pBI121-NLS-CFP vector (MiaoLing Plasmid Sharing Platform) with XbaI and BamHI sites. The recombinant pBI121–NLS–CFP plasmids were introduced into the protoplast cells from the leaves of Chinese cabbage using PEG400 solution. The protoplast cells were cultivated on MS plates overnight at 25 °C (about 16 h) and observed under a fluorescence inverted microscope (Olympus, IX53).
Generation of ELIP1 overexpression line in Chinese cabbage
ELIP1 cDNA (603 bp) was joined to pBI121 vector with XbaI and BamHI sites. The recombinant vector pBI121-ELIP1 was transformed into Agrobacterium tumefaciens EHA105 using the freeze–thaw method. Positive A. tumefaciens EHA105 colonies containing PBI121-ELIP1 were employed for the transformation of cabbage leaves using the floral dip method (n = 110). Negative A. tumefaciens EHA105 colonies were used as the control (n = 110). “Beijing new No. 3” Chinese cabbage leaves were used as wild type (n = 110). In particular, these cabbage leaves were cut into circles by the puncher, and their diameter was about 6–8 mm.
qRT-PCR was performed with the RNA samples from the wild type (WT), control and ELIP1 overexpression transgenic line (EO line) cultured for 24 h with MS medium to assay the transient expression of ELIP1, CHLM, GUN4, and HEMA1. The chlorophyll content was measured using the acetone method as described above.
Chromatin immunoprecipitation (ChIP)
ChIP assays was performed according to the manufacturer’s protocol (56383 SimpleChIP® Plus Sonication ChIP Kit 4 and RT Reagents, Cell Signaling Technology). Briefly, 100–150 mg Chinese cabbage leaves were cut into small pieces and divided and cross-linked with 1% formaldehyde at room temperature for 30 min. The crosslinking was terminated by adding 100 μl of 10 × glycine, and then the sample was mixed and incubated on ice for 5 min, washed with the mixture of PBS and 200 × protease inhibitor cocktail (PIC, twice every 10 min), and centrifuged. The supernatant was removed, and the tissue was resuspended in 1 ml mixture of 1 × ChIP sonication cell lysis buffer and PIC. After lysate was sonicated and centrifuged, 50 μl supernatant was taken for chromatin digestion. 10 μl digested chromatin was used as the “Input”, and the rest was subjected to the following immunoprecipitation experiments. Normal rabbit IgG (Invitrogen) was used as the negative control. ChIP PCRs were performed using primers that flank the G-box-like motif sites and primers that do not flank the sites in the promoter regions of target genes as controls. Primers (Comate Bioscience Technology) for ChIP are listed in Supplementary Table S2. The quantitative analysis of the ChIP gel electrophoresis map was carried out by Image J (National Institutes of Health).
Luciferase assays
ELIP1 cDNA (603 bp) was amplified by PCR and cloned into pCambia1301vector. The 5′ promoter fragments of GUN4 (1672 bp), HEMA1 (450 bp), and CHLM (1313 bp) were separately cloned into the pGreenII 0800-Luc vectors. The mutagenized vectors were constructed by PCR-based site-directed mutagenesis. Restriction enzyme-treated PCR products were transformed into Escherichia coli. Positive clones were selected and verified by gene sequencing.
One microgram each of CHLM-Luc, GUN4-Luc, and HEMA1-Luc reporter vectors and 1 μg ELIP1 plasmid were transformed by agrobacterium-sensitive cells and injected into the 1-month-old tobacco blade. PRL-TK vector was added for control. Then, the same amount of injected tobacco blade was adopted, fully ground, and added with the cell lysate to divide the tobacco blade. Then, cells of the cracked tobacco blade were collected. One hundred milliliters each of the sample and luciferase reagent was added, and luciferase reporter assays were done using a dual-reporter assay system (Promega, E1980).
Data analyses
Results correspond to mean ± SD of the quantification of three independent experiments. Asterisks indicate a significant difference compared to control negative as analyzed by Dunnett, one-way ANOVA. (*p < 0.05; **p < 0.01; ***p < 0.001).
Statement
The study comply with local and national guidelines.
Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information files.
References
Kobayashi, Y. & Tanaka, K. Transcriptional regulation of tetrapyrrole biosynthetic genes explains abscisic acid-induced heme accumulation in the unicellular red alga Cyanidioschyzon merolae. Front. Plant Sci. https://doi.org/10.3389/fpls.2016.01300 (2016).
Hu, C. M., Hou, X. L. & Wang, M. Effects of low temperature on photosynthetic and fluorescent parameters of non-heading Chinese cabbage. Acta Bot. Boreali-Occidentalia Sin. 28, 2478–2484 (2008).
Wang, W. et al. Research progress of storage of Chinese cabbage. J. Anhui Agric. 45, 72–73 (2017).
Lin, T. T. et al. Genetic analysis of chlorophyll content using mixed major gene plus polygene inheritance model in non-heading Chinese cabbage. J. Nanjing Agric. Univ. 37, 34–40. https://doi.org/10.7685/j.issn.1000-2030.2014.05.006 (2014).
Hortensteiner, S. Chlorophyll degradation during senescence. Annu. Rev. Plant Biol. 57, 55–77. https://doi.org/10.1146/annurev.arplant.57.032905.105212 (2006).
Charoenchongsuk, N., Ikeda, K., Itai, A., Oikawa, A. & Murayama, H. Comparison of the expression of chlorophyll-degradation-related genes during ripening between stay-green and yellow-pear cultivars. Sci. Hortic. 181, 89–94. https://doi.org/10.1016/j.scienta.2014.10.005 (2015).
Noichinda, S., Bodhipadma, K., Mahamontri, C., Narongruk, T. & Ketsa, S. Light during storage prevents loss of ascorbic acid, and increases glucose and fructose levels in Chinese kale (Brassica oleracea var. alboglabra). Postharvest. Biol. Technol. 44, 312–315. https://doi.org/10.1016/j.postharvbio.2006.12.006 (2007).
Shi, D. Y., Liu, Z. X. & Jin, W. W. Biosynthesis, catabolism and related signal regulations of plant chlorophyll. Hereditas 31, 698–704. https://doi.org/10.3724/sp.j.1005.2009.00698 (2009).
Formighieri, C., Ceol, M., Bonente, G., Rochaix, J. D. & Bassi, R. Retrograde signaling and photoprotection in a gun4 mutant of Chlamydomonas reinhardtii. Mol. Plant 5, 1242–1262. https://doi.org/10.1093/mp/sss051 (2012).
Apitz, J., Schmied, J., Lehmann, M. J., Hedtke, B. & Grimm, B. GluTR2 complements a hema1 mutant lacking glutamyl-tRNA reductase 1, but is differently regulated at the post-translational level. Plant Cell Physiol. 55, 645–657. https://doi.org/10.1093/pcp/pcu016 (2014).
Vavilin, D. V. & Vermaas, W. F. J. Regulation of the tetrapyrrole biosynthetic pathway leading to heme and chlorophyll in plants and cyanobacteria. Physiol. Plant. 115, 9–24. https://doi.org/10.1034/j.1399-3054.2002.1150102.x (2002).
Huang, C., David, M. P., Cao, Z.-L. & Xiao, L.-T. Mutation of chloroplast CHLM contributes to down-regulation of multiple stress response genes in Arabidopsis. Plant Growth Regul. 91, 209–219 (2020).
Fan, L. L., Shi, J. Y., Lu, J. Y., Zuo, J. H., Gao, L. P., Wang, Q. Effects of storage methods on chlorophyll degradation in Chinese cabbage. J. Anhui Agric. Sci. 44(20), 86–89 (2016).
Wang, F., Wang, G., Li, X., Huang, J. & Zheng, J. Heredity, physiology and mapping of a chlorophyll content gene of rice (Oryza sativa L.). J. Plant Physiol. 165, 324–330. https://doi.org/10.1016/j.jplph.2006.11.006 (2008).
Alexander, L. et al. Nucleotide sequence of Cab-8, a new type I gene encoding a chlorophyll a/b-binding protein of LHC II in Pisum. Plant Mol. Biol. 17, 523–526. https://doi.org/10.1007/bf00040649 (1991).
Falconet, D., White, M. J., Fristensky, B. W., Dobres, M. S. & Thompson, W. F. Nucleotide sequence of Cab-215, a type II gene encoding a photosystem II chlorophyll a/b-binding protein in Pisum. Plant Mol. Biol. 17, 135–139. https://doi.org/10.1007/bf00036815 (1991).
Pietrzykowska, M. et al. The light-harvesting chlorophyll a/b binding proteins Lhcb1 and Lhcb2 play complementary roles during state transitions in Arabidopsis. Plant Cell 26, 3646–3660. https://doi.org/10.1105/tpc.114.127373 (2014).
Chandrasekar, S. & Shan, S. O. Anionic phospholipids and the Albino3 translocase activate signal recognition particle-receptor interaction during light-harvesting chlorophyll a/b-binding protein targeting. J. Biol. Chem. 292, 397–406. https://doi.org/10.1074/jbc.M116.752956 (2017).
Funk, C., Adamska, I., Green, B. R., Andersson, B. & Renger, G. The nuclear-encoded chlorophyll-binding photosystem II-S protein is stable in the absence of pigments. J. Biol. Chem. 270, 30141–30147. https://doi.org/10.1074/jbc.270.50.30141 (1995).
Andersson, U., Heddad, M. & Adamska, I. Light stress-induced one-helix protein of the chlorophyll a/b-binding family associated with photosystem I. Plant Physiol. 132, 811–820. https://doi.org/10.1104/pp.102.019281 (2003).
Heddad, M. & Adamska, I. Light stress-regulated two-helix proteins in Arabidopsis thaliana related to the chlorophyll a/b-binding gene family. Proc. Natl. Acad. Sci. U.S.A. 97, 3741–3746. https://doi.org/10.1073/pnas.050391397 (2000).
Grimm, B., Kruse, E. & Kloppstech, K. Transiently expressed early light-inducible thylakoid proteins share transmembrane domains with light-harvesting chlorophyll binding proteins. Plant Mol. Biol. 13, 583–593. https://doi.org/10.1007/bf00027318 (1989).
Harari-Steinberg, O., Ohad, I. & Chamovitz, D. A. Dissection of the light signal transduction pathways regulating the two early light-induced protein genes in arabidopsis. Plant Physiol. 127, 986–997. https://doi.org/10.1104/pp.010270 (2001).
Adamska, I. & Kloppstech, K. Evidence for an association of the early light-inducible protein (ELIP) of pea with photosystem II. Plant Mol. Biol. 16, 209–223. https://doi.org/10.1007/bf00020553 (1991).
Adamska, I., Kruse, E. & Kloppstech, K. Stable insertion of the early light-induced proteins into etioplast membranes requires chlorophyll a. J. Biol. Chem. 276, 8582–8587. https://doi.org/10.1074/jbc.M010447200 (2001).
Grimm, B. & Kloppstech, K. The early light-inducible proteins of barley. Characterization of two families of 2-h-specific nuclear-coded chloroplast proteins. Eur. J. Biochem. 167, 493–499. https://doi.org/10.1111/j.1432-1033.1987.tb13364.x (1987).
Rossini, S. et al. Suppression of both ELIP1 and ELIP2 in Arabidopsis does not affect tolerance to photoinhibition and photooxidative stress. Plant Physiol. 141, 1264–1273. https://doi.org/10.1104/pp.106.083055 (2006).
Cazzonelli, C. I. et al. A cis-carotene derived apocarotenoid regulates etioplast and chloroplast development. Elife 9, e45310 (2020).
Casazza, A. P., Rossini, S., Rosso, M. G. & Soave, C. Mutational and expression analysis of ELIP1 and ELIP2 in Arabidopsis thaliana. Plant Mol. Biol. 58, 41–51. https://doi.org/10.1007/s11103-005-4090-1 (2005).
Menkens, A. E., Schindler, U. & Cashmore, A. R. The G-box: A ubiquitous regulatory DNA element in plants bound by the GBF family of bZIP proteins. Trends Biochem. Sci. 20, 506–510. https://doi.org/10.1016/s0968-0004(00)89118-5 (1995).
Suekawa, M., Fujikawa, Y. & Esaka, M. Two G-box-like elements essential to high gene expression of SlAKR4B in tomato leaves. Biosci. Biotechnol. Biochem. 82, 425–432. https://doi.org/10.1080/09168451.2018.1429887 (2018).
Ezer, D. et al. The G-Box transcriptional regulatory code in Arabidopsis. Plant Physiol. 175, 628–640. https://doi.org/10.1104/pp.17.01086 (2017).
Liu, Y. J. et al. Arabidopsis AtbHLH112 regulates the expression of genes involved in abiotic stress tolerance by binding to their E-box and GCG-box motifs. New Phytol. 207, 692–709. https://doi.org/10.1111/nph.13387 (2015).
Guo, L. et al. The anaphase-promoting complex initiates zygote division in Arabidopsis through degradation of cyclin B1. Plant J. 86, 161–174. https://doi.org/10.1111/tpj.13158 (2016).
Lee, B. D. et al. The F-box protein FKF1 inhibits dimerization of COP1 in the control of photoperiodic flowering. Nat. Commun. 2259, 1–10. https://doi.org/10.1038/S41467-017-02476-2 (2017).
Sa, Q. L., Li, W. B. & Sun, Y. R. Transcriptional regulation of the G-box and G-box-binding proteins in plant gene expression. Plant Physiol. Mol. Biol. 39, 89–92 (2003).
Dillon, N. The impact of gene location in the nucleus on transcriptional regulation. Dev. Cell 15, 182–186 (2008).
Andrew, J. S. et al. Multigene manipulation of photosynthetic carbon assimilation increases CO2 fixation and biomass yield in tobacco. J. Exp. Bot. 66(13), 4075–4090 (2015).
Acknowledgements
This work was supported by the Natural Science Foundation of Heilongjiang Province (LC2019C066), the Special Fund for Plant Food Processing Technology from the Colleges and Universities Basic Scientific Research Project in Heilongjiang Province (YSTSXK201829, YSTSXK201807, and YSTSXK201876), the Fundamental Research Funds of Department of Education of Heilongjiang Province (135309371 and 135209371), University Nursing Program in Heilongjiang Province (UNPYSCT-2017157), and Qiqihar University Graduate Innovative Research Project (YJSCX2019079). We are thankful to the support of Professor Xiaolan Liu and Wei Sha.
Author information
Authors and Affiliations
Contributions
T.W., T.M., and W.W. provided essential ideas and technical supports for the study. T.W., S.T., and S.L. conducted the study. T.W., S.L., S.T., and W.W. performed statistical analysis. T.W. wrote the manuscript, and S.L., T.M., and W.W. revised the manuscript. All authors have read and approved the final manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Wang, T., Liu, S., Tian, S. et al. Light regulates chlorophyll biosynthesis via ELIP1 during the storage of Chinese cabbage. Sci Rep 12, 11098 (2022). https://doi.org/10.1038/s41598-022-15451-9
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-022-15451-9
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.