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Article

CfCHLM, from Cryptomeria fortunei, Promotes Chlorophyll Synthesis and Improves Tolerance to Abiotic Stresses in Transgenic Arabidopsis thaliana

by
Guangqian Wei
1,2,†,
Yingting Zhang
1,2,†,
Ye Yang
1,2,
Junjie Yang
1,2 and
Jin Xu
1,2,*
1
State Key Laboratory of Tree Genetics and Breeding, College of Forestry, Nanjing Forestry University, Nanjing 210037, China
2
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Forests 2024, 15(4), 628; https://doi.org/10.3390/f15040628
Submission received: 22 February 2024 / Revised: 20 March 2024 / Accepted: 27 March 2024 / Published: 29 March 2024
(This article belongs to the Section Genetics and Molecular Biology)
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Abstract

:
Mg-protoporphyrin IX methyltransferase (CHLM) is essential for the synthesis of chlorophyll (chl). However, no CHLM gene has been reported in Chinese cedar (Cryptomeria fortunei). Here, we cloned the CHLM gene from C. fortunei, and the full-length CfCHLM sequence was 1609 bp, with a 1077 bp ORF region encoding a protein 358 amino acids long. A homologous comparison analysis showed that CfCHLM was highly evolutionarily conserved among different plant species. A phylogenetic tree was drawn using CHLM proteins from ten angiosperms and three gymnosperms, and CfCHLM was found to be most closely related to the TcCHLM protein of Chinese yew (Taxus chinensis). The CfCHLM is located in chloroplasts and does not exhibit self-activation. The expression of CfCHLM was highest in the needles and was downregulated under abiotic stress, i.e., cold, heat, drought, or salt stress. Under cold, heat, drought, and salt abiotic stresses, CfCHLM transgenic A. thaliana showed higher chl fluorescence parameters, elevated chl levels, increased net photosynthetic rate (Pn), and enhanced antioxidant enzyme activities. Conversely, it showed a lower stomatal conductance (Gs), a reduced transpiration rate (Tr), and decreased malondialdehyde (MDA) levels compared to the wild type (WT). In summary, the CfCHLM gene augments chloroplast function, photosynthetic capacity, and stress resistance in plants. This study provides a reference for future research on the growth and development of C. fortunei.

1. Introduction

Photosynthesis in green plants is fundamental to the survival and development of almost all life on Earth [1]. Chlorophyll (chl) is an important substance for photosynthesis in plants, and its function is to collect and transmit light energy and drive electron transfer to reaction centers for energy conversion [2,3]. Chl biosynthesis, including from glutamate (Glu) to chl b, is catalyzed by fifteen enzymes encoded by twenty-seven genes [4,5,6]. First, Glu is catalytically converted to 5-aminolevulinicacid (ALA) with an incomplete carbon ring structure by enzymes such as glutamyl-tRNA synthetase (GluRS) and glutamyl-tRNA reductase (GluTR). Then, two molecules of ALA are used to synthesize bilirubinogen containing a pyrrole ring, and four bilirubinogen units polymerize to form protoporphyrin IX (Proto IX). Proto IX is a watershed in the tetrapyrrole biosynthetic pathway, including the iron branch, catalyzed by ferrous chelatase to synthesize ferrous heme and phytochrome, and the magnesium branch for the synthesis of chl. Mg-protoporphyrin IX (Mg-Proto IX) is obtained via magnesium protoporphyrin IX chelatase (Mg-chelatase) and then forms Mg-protoporphyrin IX monomethyl ester (MgPME) under the catalytic action of Mg-protoporphyrin IX methyltransferase (CHLM). It is then acted upon by Mg-protoporphyrin IX monomethyl ester cyclase (MgPEC), protochlorophyllide oxidoreductase (POR), chl synthase (ChlG), and a series of other enzymes to finally form chl a and chl b.
Chl metabolism causes changes in the leaf color of plants. Specifically, the absence or depletion of genes involved in chl biosynthesis leads to a decrease in chl content, resulting in mutant phenotypes such as albinism [7] and yellowing [8] in plant leaves. For example, gene mutations related to chl synthesis cause barley (Hordeum vulgare) to show albinism [9], and mutation in MgPEC causes a yellow and blotchy phenotype in rice (Oryza sativa) [10]. The process of chl synthesis involves multistep enzymatic reactions, but the catalytic mechanism or regulatory mechanism of some of these enzyme-encoding genes is poorly understood, such as CHLM.
CHLM is essential for chl synthesis and chloroplast development in plants [11]. It catalyzes methyl transfer from S-adenosyl methionine (AdoMet) to MgPIX to form MgPME and S-adenosyl homocysteine (AdoHcy) [12] and is involved in regulating the activity of Mg-chelatase [6]. In addition, the CHLM gene is necessary for the formation of chl-containing protein complexes [13]. CHLM genes have been mutated in many plants, such as Arabidopsis (Arabidopsis thaliana) [14], tobacco (Nicotiana tabacum) [15], and rice [16]. For example, rice chlm mutation resulted in a decrease in CHLM enzyme activity and impaired chl synthesis, resulting in yellow-green leaves [16]. A. thaliana plants with a T-DNA insertion mutation in chlm accumulated excessive amounts of Mg-Proto IX and exhibited albinism [13]. A. thaliana chlm-4 mutant plants accumulated more superoxide anion radicals (O2−) and fewer photosystem II proteins, and chlm-4 plants also exhibited a hypersensitive response to salt stress by downregulating the expression of stress-responsive CBF family genes during seed germination [17]. These results suggest that the CHLM gene is essential for plant growth and photosynthesis. However, no studies on the CHLM gene of Chinese cedar (Cryptomeria fortunei) have been reported to date.
C. fortunei is an evergreen species and has broad application prospects due to its strong adaptability, excellent materials, and rapid growth. The research on C. fortunei is mainly about its economic value, abiotic stress response, and timber properties. In a previous study, we identified the CHLM gene based on two different phenotypes of C. fortunei (needles are yellowish-brown in autumn and winter, returning to green in spring in asexual line #3 and are evergreen in asexual line #X1). In this study, we cloned the CHLM gene from C. fortunei and explored the function of the CfCHLM gene by sequence analysis, phylogenetic analysis, subcellular localization analysis, transcriptional activity assay, functional validation of transgenic A. thaliana, and expression pattern analysis.

2. Materials and Methods

2.1. Cultivation and Condition Setting of Plant Materials

C. fortunei clones from our laboratory were planted at the Baima Experimental Base of Nanjing Forestry University (119°10′48″ E, 31°37′12″ N), and cuttings of these C. fortunei clones were grown in the Garden Experimental Teaching Center of Nanjing Forestry University (118°49′12″ E, 32°04′48″ N). Young needles were collected from well-growing cutting seedlings for RNA extraction.
The seeds of A. thaliana or N. benthamiana were sterilized with 70% alcohol and 5% H2O2, and the sterilized seeds were spread evenly on Murashige and Skoog (MS) solid medium and placed upside-down in a refrigerator at 4 °C for 3 d. Then, they were placed in an incubator with a photosynthetic photon flux density of 120 μmol m−2 s−1, a photoperiod of 16 h light/8 h dark, a constant temperature of 23 °C, and a relative humidity of 70%. Two-true-leaf seedlings were transplanted into pots after a 7 d culture, and the seedlings were then placed in the incubator, grown further under the above conditions, and watered once every three days.

2.2. Cloning of a CHLM cDNA Sequence

Total RNA was extracted from the harvested needles using the FastPure Universal Plant Total RNA Isolation Kit (Nanjing Vazyme Biotech Co., Ltd., Nanjing, China). RNA integrity and concentration were examined by 1% agarose gel electrophoresis and NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA), respectively. Reverse transcription was then performed using the HiScript® III 1st Strand cDNA Synthesis Kit Nanjing Vazyme Biotech Co., Ltd., Nanjing, China).
We used the AtCHLM (AT4G25080) sequence of A. thaliana to screen CfCHLM homologous sequences in the local C. fortunei BLAST database in our laboratory. The cloning, primer design, sequencing, and splicing of the intermediate fragment and the 5′/3′RACE fragment were carried out according to the description of Zhang et al. [18], and, finally, the full length of the CfCHLM gene was obtained.

2.3. Phylogenetic and Protein Property Analysis of CfCHLM

To understand the phylogenetic and protein property analysis of the CfCHLM sequence, we used the ORFfinder program to predict the open reading frame (ORF). The molecular weight (MW), theoretical isoelectric point (pI), and amino acid composition of the CfCHLM protein were predicted and calculated by ExPASy ProtParam. The secondary structure of the CfCHLM amino acid sequences was predicted by SOPMA. The tertiary structure of the CfCHLM protein was predicted based on the AlphaFold DB model (gene: A9NUI2_PICSI) of Sitka spruce (Pinus sitchensis) using the Swiss-Model Online software (https://swissmodel.expasy.org/).
The amino acid homologous sequences of CfCHLM were collected using the BLAST tool on the NCBI website, and these screened CHLM amino acid sequences were aligned using the DNAMAN8.0 software. Then, we constructed a phylogenetic tree using the MEGA 11 software.

2.4. Subcellular Localization and Transcriptional Activity Assay of CfCHLM

The ORF of CfCHLM was amplified and introduced into the XbaI/BamHI cloning sites of the pBI121-green fluorescent protein (GFP) vector or the EcoRI/BamHI cloning sites of the pGBKT7 vector. The pBI121-GFP (35S::GFP) vector and recombinant plasmid (35S::CfCHLM-GFP) were transformed into Agrobacterium tumefaciens strain GV3101 (Shanghai Weidi Biotech Co., Shanghai, China) and then injected into the 28 d N. benthamiana leaves. The GFPs were detected by a laser scanning confocal microscope (CarlZeiss LSM710) (Carl Zeiss Jena, Oberkochen, Germany). The yeast expression vectors pGBKT7 (negative control CK), pGBKT7-CfCHLM, and pCL1 (positive control CK) were transformed into AH109 (Shanghai Weidi Biotech Co., Ltd., Shanghai, China) yeast cells. The yeast cells were spread on SD/-Trp, SD/-Ade/-His/-Trp, and SD/-Ade/-His/-Trp/X-α-gal plates (Coolaber) and cultured, inverted, in an incubator at 29 °C.

2.5. A. thaliana Transformation

With the cDNA as the template, the pBI121 vector was double-digested at the XbaI and BamHI sites, and then the recombinant expression vector was constructed using the ClonExpress® II One Step Cloning Kit (Nanjing Vazyme Biotech Co., Ltd., Nanjing, China). The expression vector was transferred into WT A. thaliana by floral infection [19]. Then, the transgenic seeds were screened using an MS medium supplemented with 50 mg L−1 of kanamycin and transplanted into soil after 10 d of culture in an incubator with a photosynthetic photon flux density of 120 μmol m−2 s−1, a photoperiod of 16 h light/8 h dark, a constant temperature of 23 °C, and a relative humidity of 70%. The transgenic seedlings were continuously screened and cultured to the T2-generation following this method. We collected leaves of T2-generation and WT seedlings and then extracted DNA using the DNAsecure Plant Kit (TianGen Biotech Co., Ltd., Beijing, China). To test the success of the transgene, we performed gel electrophoresis using the collected DNA, and both negative (without DNA) and positive (with subcloned target gene) controls were established (Figure S1).
Total RNA was extracted from leaves of T2-generation and WT seedlings using the FastPure Universal Plant Total RNA Isolation Kit (Nanjing Vazyme Biotech Co., Ltd., Nanjing, China). Reverse transcription was then performed using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Nanjing Vazyme Biotech Co., Ltd., Nanjing, China). ChamQ SYBR qPCR Master Mix (Low ROX Premixed) (Nanjing Vazyme Biotech Co., Ltd., Nanjing, China) was used for qRT-PCR. The expression of the CfCHLM gene in six transgenic A. thaliana lines was determined (Figure S2), and the two lines with high expression levels (CfCHLM-L3 and CfCHLM-L6) were screened for the subsequent studies.

2.6. Setting of Abiotic Stress Conditions in A. thaliana

Twenty-five-day-old WT and T3-generation CfCHLM transgenic A. thaliana plants (60 plants each) were cold-adapted at 4 °C for 24 h and then transferred to −6 °C cold stress for 8 h or were placed in a 40 °C incubator for 6 h of heat stress [18].
Twenty-five-day-old WT and T3-generation CfCHLM transgenic A. thaliana plants (60 plants each) were irrigated with a PEG-6000 solution (10% or 15%) or irrigated with a NaCl solution (300 mM or 400 mM) to simulate drought or salt stress. Each pot of A. thaliana was irrigated with 50 mL of solution once every two days, for three times.

2.7. Detection of Plant Phenotypic and Physiological Indicators

Plant phenotypes were observed at the end of each group of treatments, and the average number of leaves in each line under the different stress treatments was recorded. The plants were shaded in darkness for 20 min in advance, and then fresh leaves were harvested without damage, and the maximum photochemical quantum yield of PSII (Fv/Fm), effective photochemical quantum yield of PSII (Y(II)), and coefficient of photochemical fluorescence quenching (qP) values [20,21] were measured using a dual-channel PAM-100 fluorometer (Walz, Effeltrich, Baden-Wuertenberg, Germany). Setting the light intensity to 120 μmol m−2 s−1 at room temperature, the net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr) of the leaves were measured using a CIRAS-3 (PP Systems Co., Ltd., Amesbury, MA, USA) photosynthesis meter.
We measured the chl levels using the method of Lichtenthaler and Wellburn [22]. Fresh plant leaves (0.2 g) were ground with a small amount of quartz sand and calcium carbonate, and chloroplast pigments were extracted with 96% ethanol. The levels of chloroplast pigments could be determined in this manner due to the different wavelengths of the maximum absorption peaks of chl a and chl b in 96% ethanol. The plant leaves (0.2 g) were placed in a 2 mL grinding tube, quickly frozen in liquid nitrogen, and ground into a powder using a high-throughput tissue lyzer (Ningbo Xinzhi Biotech Co., Ltd., Ningbo, China). Then, 1.6 mL of 50 mM precooled phosphate buffer (PBS, pH = 7.8) was added to the powder, and the mixture was subjected to vortex oscillation, which caused the phosphate buffer to come into full contact with the powder. The mixture was then centrifuged at 4000 rpm for 10 min at 4 °C, and the supernatant was diluted to 2 mL. This supernatant was the enzyme solution and was used to assay SOD, POD, and CAT activities and MDA content. SOD activity was determined using the photoreduction method with nitro-blue tetrazolium [23]. POD activity was determined by the o-methoxy-phenol method, and CAT activity was determined by the UV absorption method [24]. In addition, the reaction of MDA with 2-thiobarbituric acid (TBA) was used to determine the MDA content [25].

2.8. The Expression Pattern of CfCHLM

Different tissues (needles, roots, stems, seeds, and cones) of C. fortunei were collected under normal growth conditions (25/23 °C (day/night)). C. fortunei was treated with cold (4 °C), heat (42 °C), drought (15% PEG-6000), and salt (200 mM NaCl) stress [18]. We designed specific quantitative primers for the CfCHLM sequence (Table S1), and, under these abiotic stresses, needles of C. fortunei were collected over different periods (0, 2, 6, 12, and 48 h), and CfCHLM expression was measured by qRT-PCR. CYP [26] of C. fortunei were used as the internal reference genes, and the expression level of CfCHLM was calculated by the 2−ΔΔCt method [27].

2.9. Data Statistics and Analysis

Data analysis was conducted using IBM SPSS Statistics 25.0 (IBM, Chicago, IL, USA), and Duncan’s tests were used to evaluate significant differences at p ≤ 0.05.

3. Results

3.1. Full-Length and Phylogenetic Analysis of the CfCHLM Gene

The full-length CfCHLM sequence was 1609 bp with a 1077 bp ORF region (GenBank ID: OQ867966), and this ORF region encoded a protein 358 amino acids long. The amino acid sequences encoded by the CfCHLM gene of C. fortunei were compared using the NCBI online software Blast (BLAST+ 2.15.0), from which nine sequences with a high similarity were selected and imported into DNAMAN for comparison. The amino acid sequences of CHLM are relatively conserved among species (Figure 1A), and our homologous comparison showed that these amino acid sequences shared 71.97% of similarity. All CHLM proteins contain the typical motifs of S-adenosylmethionine-dependent methyltransferases (within the red rectangular box).
In addition, we selected nine angiosperm and two gymnosperm species containing the CHLM proteins and constructed a phylogenetic tree (Figure 1B) using MEGA11. The phylogenetic tree showed that the distribution of the CHLM proteins was different in angiosperms and gymnosperms, and, among them, CfCHLM was closely related to TcCHLM in the gymnosperm Chinese yew (Taxus chinensis).

3.2. Physicochemical Properties Analysis of CfCHLM Protein

The molecular weight of the CfCHLM protein was 38.925 kDa, and the pI was 6.770. The CfCHLM protein was predominantly composed of Ala (13.100%), followed by Leu (8.900%) and Ser (8.400%) (Table S2). We also analyzed the structural features of the CfCHLM protein, and the secondary structure of the CfCHLM protein mainly consisted of alpha helices (50.840%), random coils (30.450%), extended strands (10.340%), and beta turns (8.380%) (Figure 1D; Table S3). The tertiary structure of the CfCHLM protein mainly consisted of alpha helices and random coils (Figure 1C).

3.3. Subcellular Localization and Transcriptional Activity of CfCHLM

The fluorescence signal of pBI121-GFP appeared in the cell membrane, nucleus, and cytoplasm, whereas the fluorescence signal of 35S::CfCHLM-GFP partially overlapped with the chloroplasts (Figure 2A).
The pGBKT7-CfCHLM yeast cells grew normally on the SD/-Trp medium, failed to grow on the SD/-Ade/-His/-Trp medium, or turned blue on the SD/-Ade/-His/-Trp/X-α-gal medium (Figure 2B), showing that CfCHLM has no self-activation ability.

3.4. Expression Pattern of CfCHLM

The expression levels of CfCHLM in different tissues was significantly (p ≤ 0.05) different, with the highest expression in needles, followed by cones and stems, in which the expression levels were 14.764% and 12.638% of those of the needles, respectively, and the expression levels of roots and seeds were 7.444% and 6.693% of the needles’ levels, respectively (Figure 3A).
Under cold stress, the expression of CfCHLM decreased significantly (p ≤ 0.05) from 3 h to 6 h and decreased to 0.435 of CK at 6 h, with no significant difference between 6 h and 48 h (Figure 3B). Under heat stress, the expression of CfCHLM showed a significant (p ≤ 0.05) downward trend before 12 h and decreased to 0.058 of CK at 12 h, and there was no significant difference in terms of its expression after 12 h (Figure 3C). Under drought and salt stress, the expression of CfCHLM decreased significantly (p ≤ 0.05) over time, reaching the lowest at 48 h, with values 0.195 and 0.299 of CK, respectively (Figure 3D–E).

3.5. Expression Level Assay of CfCHLM in Transgenic A. thaliana Lines

We screened a total of six T2-generation CfCHLM transgenic A. thaliana lines (CfCHLM-L2, CfCHLM-L3, CfCHLM-L4, CfCHLM-L5, CfCHLM-L6, and CfCHLM-L9) (Figure S1). The expression levels of the CfCHLM gene in the six transgenic A. thaliana lines were calculated, using A. thaliana Actin as the internal reference gene, and the expression level of CfCHLM-L2 was 1. CfCHLM-L3 exhibited the highest expression, followed by CfCHLM-L6 (Figure S2). Therefore, these two lines were selected for our experiment.

3.6. Response of CfCHLM Gene to Cold or Heat Stress

After 8 h of treatment at −6 °C, WT and CfCHLM transgenic A. thaliana showed leaf shrinkage (Figure 4A–F) and a significant (p ≤ 0.05) decrease in the number of leaves compared to CK (Figure 4J). But leaf chlorosis and damage were more severe in WT compared to transgenic A. thaliana (Figure 4D–F). Under cold treatment, the values of Fv/Fm, Y(II), and qP were 0.424, 0.272, and 0.586 for WT, 0.663, 0.459, and 0.688 for CfCHLM-L3, and 0.652, 0.478, and 0.730 for CfCHLM-L6, respectively (Figure 4K–M). The Fv/Fm, Y(II), and qP values were significantly (p ≤ 0.05) greater in both transgenic A. thaliana lines than in the WT. Similarly, most of the indicators of the chl levels (chl a, chl b, and chl a + b), photosynthetic parameters (Pn, Gs and Tr), and antioxidant enzyme (SOD, POD, and CAT) activities were higher in transgenic A. thaliana than in WT (Figure 4N–V). The results suggested that the transgenic lines may be better tolerant to cold stress than the WT.
The chl fluorescence parameters (Fv/Fm, Y(II), and qP), chl levels (chl a, chl b, and chl a + b), activities of antioxidant enzymes (SOD, POD, and CAT), and MDA content of transgenic A. thaliana were non-significantly changed compared to those of the WT after treating each line at 40 °C for 8 h (Figure 4K–P,T–W). However, it is noteworthy that, under the heat treatment, the Pn, Gs, and Tr values were 5.900, 281.750, and 2.750 for WT; 2.967, 173, and 1.950 for CfCHLM-L3; and 3.330, 136, and 1.900 for CfCHLM-L6, respectively (Figure 4Q–S). The data showed that the photosynthetic parameters (Pn, Gs, and Tr) were significantly (p ≤ 0.05) smaller in both transgenic A. thaliana lines than in the WT (p ≤ 0.05). The results also showed that, in response to heat stress, transgenic A. thaliana and WT behaved differently, mainly in terms of their Pn, Gs, and Tr.

3.7. Response of CfCHLM Gene to Drought Stress

The WT and transgenic plants were treated with 10% and 15% PEG-6000 solutions, respectively. Under the 10% PEG treatment, most of the indicators of A. thaliana were non-significantly different compared to CK, while, under the 15% PEG treatment, most of the indicators of A. thaliana were significantly different (p ≤ 0.05) compared to CK. Under the PEG treatments, the chl fluorescence parameters (Fv/Fm, Y(II), and qP) were non-significantly different in transgenic A. thaliana compared to those of WT (Figure 5K–M), and the chl levels (chl a, chl b, and chl a + b), Pn, and antioxidant enzyme activities (SOD, POD, and CAT) were relatively high compared to those of WT (Figure 5N–Q,T–V). The GS, Tr, and MDA levels were relatively low compared to those of the WT (Figure 5R–S,W). These results suggest that transgenic A. thaliana may have a better drought tolerance than the WT.

3.8. Response of CfCHLM Gene to Salt Stress

To investigate the response of CfCHLM to salt stress, we treated the WT and transgenic lines with NaCl solutions with concentrations of 300 mM and 400 mM, respectively. Under salt stress, A. thaliana was subject to yellowing and wilting (Figure 6A–I), while transgenic A. thaliana showed less yellowing and wilting than the WT (Figure 6D–I). Under salt stress, the chl fluorescence parameters (Fv/Fm, Y(II), and qP) of transgenic A. thaliana were non-significantly different compared to those of the WT (Figure 6K–M); the chl levels (chl a, chl b, and chl a + b), Pn, and SOD activity were relatively high compared to those of the WT (Figure 6N–Q,T); and the Gs, Tr, and MDA content were relatively low compared to those of the WT (Figure 6R–S,W). These results indicate that salt stress causes some damage to A. thaliana plants but relatively little damage to transgenic plants and that transgenic plants may be more salt-tolerant than WT plants.

4. Discussion

4.1. Functional Analysis and Expression Pattern of CfCHLM

We found that the CfCHLM protein showed a high similarity with CHLM proteins from the other species (Figure 1A). The phylogenetic analysis showed that the CHLM proteins of gymnosperms and angiosperms clustered separately; among them, CfCHLM was most closely related to TcCHLM (Figure 1B). Our prediction analysis showed that the CfCHLM protein is predominantly composed of alpha helices and random coils (Figure 1C), which is similar to the structural composition of the A. thaliana CHLM protein [17]. Our subcellular localization results showed that CfCHLM is located in chloroplasts (Figure 2A), and it has been reported that CHLM is located in both envelope and thylakoid chloroplast membranes [14], which is similar to what was found in our study. Therefore, we speculated that the CfCHLM protein may bind to the chloroplast membrane and promote chl synthesis.
The expression levels of CfCHLM varied in different tissues, with the highest expression found in the needles and the lowest in the roots and seeds (Figure 3A). In rice, the gene encoding the CHLM protein, YGL18, has the highest expression in the leaves and the lowest expression in the roots [16], and, even though the expression of this gene in seeds has not been detected, the tissues with the highest and lowest expression levels in rice are similar to those identified in our results. In addition, abiotic stress had an effect on the gene expression levels (Figure 3B–E). In this study, the expression of CfCHLM in C. fortunei was shown to be downregulated under abiotic stress, and the downregulation of the CHLM gene may have inhibited the synthesis of chl. Many studies have shown that the synthesis of chl is inhibited under various abiotic stresses [28,29,30,31], which demonstrates the downregulation of the CfCHLM gene in response to stress.

4.2. The Function of the CfCHLM Gene in Photosynthesis

In our study, the chl levels of plants decreased under abiotic stress, but the chl levels of CfCHLM transgenic A. thaliana were higher than those of WT (Figure 4N–P, Figure 5N–P and Figure 6N–P). Many studies have shown that the chl content of plants can be affected by abiotic stresses. For example, salt stress affects the chl content of cowpeas (Vigna unguiculata) [32]; drought stress causes a decrease in the chl content in rice [33]; and cold stress specifically inhibits chl biosynthesis in young pakchoi (Brassica rapa ssp. chinensis) leaves [31]. The above studies are similar to our results, demonstrating that the CfCHLM gene promotes chl synthesis in A. thaliana.
Chl fluorescence parameters reflect the photochemical processes and efficiency of plants. Fv/Fm is a reliable indicator of photoinhibition—the lower the Fv/Fm is, the higher the degree of photoinhibition—and this indicator can be used to identify plant resistance to stress. Y(II) can reflect the actual photosynthetic efficiency of a plant, and qP can reflect the electron transfer activity of the PSII reaction center [20,34]. Plants produce large amounts of reactive oxygen species (ROS) when subjected to abiotic stress, and ROS promote the senescence of plant leaves [35], which reduces a plant’s ability to photosynthesize. The mechanism of photosynthesis involves various components, including photosystem activity, the electron transport system, the CO2 transport level, and so on [36]. Chl fluorescence parameters are closely related to photosynthesis in plants and can reflect the photosynthetic capacity of plants [37]. Therefore, when plants are subjected to stress in a manner which results in a decrease in their photosynthetic capacity, their chl fluorescence parameters will also decrease accordingly. In our study, the Fv/Fm, Y(II), and qP values of A. thaliana showed a decreasing trend under the stress treatments compared to CK (Figure 4K–M, Figure 5K–M and Figure 6K–M). Arminian et al. [38] found that the chl fluorescence parameters of canola (Brassica napus L.) leaves decreased during cold. Li et al. [39] found that the Fv/Fm and qP of two different varieties of cucumber (Jinchun No.4, Zhongnong No.12) under high temperatures both decreased. Hou et al. [40] found that hypertonic salt stress reduced the Fv/Fm and qP values in A. thaliana. These studies showed that chl fluorescence parameters decrease under stress, similarly to our results, but, in our study, the Fv/Fm, Y(II), and qP values of transgenic A. thaliana were higher under each stress than those of the WT (Figure 4K–M, Figure 5K–M and Figure 6K–M), demonstrating that the CfCHLM gene can enhance photosynthesis in A. thaliana.
Pn, Gs, and Tr can also be used as important indicators of plant chloroplast function. In response to external stress, plants lower their Gs to reduce water loss, forming a self-protection mechanism. Many studies have shown that Pn is positively correlated with Gs in plants under normal growth conditions, but, under stress conditions, influenced by other factors, Pn is not as well correlated with Gs [41,42,43]. Zhang et al. [44] found that the Pn, Gs, and Tr of wild apricot (Prunus armeniaca L. var. ansu) decreased with decreasing soil moisture. Ying et al. [45] found that the Pn, Gs, and Tr values of happy tree (Camptotheca acuminata) decreased with increasing levels of water stress. In this study, the A. thaliana lines (WT and transgenic lines) showed a decreasing trend for Pn, Gs, and Tr under the stress treatments compared to CK (Figure 4Q–S, Figure 5Q–S and Figure 6Q–S). Under heat stress, drought stress, and salt stress, the Gs and Tr of transgenic A. thaliana were lower than those of WT (Figure 4R–S, Figure 5R–S and Figure 6R–S), demonstrating that transgenic A. thaliana was more capable of self-protection than WT. Notably, both Gs and Tr decreased under 300 mM salt stress but rebounded under 400 mM salt stress in the A. thaliana lines (WT and transgenic lines) (Figure 6R–S). We hypothesized that, under 300 mM salt stress, the A. thaliana plants adapted to the stress and, thus, reduced their Gs, which led to a reduction in Tr and Pn, but, under 400 mM salt stress, cell damage led to an increase in Gs and Tr but a reduction in Pn. These results indicated that CfCHLM enhanced chloroplast function in A. thaliana.

4.3. The Function of the CfCHLM Gene in the Response to Abiotic Stress

A phenotype is the most intuitive manifestation of plants under stress. In our study, A. thaliana leaves showed different degrees of damage under various stresses (Figure 4A–I, Figure 5A–I and Figure 6A–I). Notably, in our study, A. thaliana under the 10% PEG treatment was greener than CK (Figure 5D–F). Ma et al. [46] found that PEG pretreatment increased plants’ chl content, chl fluorescence, and photosynthetic parameters, promoted rice seedling growth, and regulated the tolerance of rice seedlings to drought. We, therefore, hypothesized that, because 10% PEG was a moderate concentration or the treatment period was short, this treatment had a positive effect instead of acting as a stress. This could be improved in the future by increasing the concentration or extending the treatment time.
SOD, POD, and CAT are important antioxidant enzymes that scavenge ROS in plants. SOD is the first line of defense against plant antioxidants, scavenging excess superoxide anions from cells, while POD and CAT scavenge disproportionate amounts of H2O2 into water and oxygen molecules [47]. The MDA content can be used as an indicator of oxidative damage to plant cell membranes [48]. Our results indicated that the SOD, POD, and CAT activities and MDA content showed an increasing trend in A. thaliana under abiotic stress, but, compared to WT, CfCHLM transgenic A. thaliana generally possessed a higher antioxidant enzyme activity and a lower MDA content (Figure 4T–W, Figure 5T–W and Figure 6T–W). Zhang et al. [49] found that the antioxidant enzyme activities and MDA content of Limonium sinense Kuntze increased with increasing time under NaCl stress. Wang et al. [50] found that cold stress increased antioxidant enzyme activities and MDA concentrations in maize (Zea mays) seedlings. Khanna-Chopra and Chauhan [51] found that, under heat stress, antioxidant enzyme activities were higher in the heat-tolerant wheat (Triticum aestivum) line Hindi62 than in the heat-sensitive wheat line PBW343, and this increased antioxidant capacity contributed to an improved heat tolerance. Huang et al. [17] found that mutant plants of A. thaliana chlm-4 accumulated excessive ROS and had a defective enzyme system for scavenging ROS. Our results were similar to those of these reports, showing that transgenic A. thaliana had a higher antioxidant capacity and could effectively reduce membrane lipid peroxidation in leaves, demonstrating that the CfCHLM gene enhances plant stress tolerance.
In summary, we hypothesized that the CfCHLM gene may enhance the photosynthetic function of plants mainly by promoting the synthesis of chl, thus increasing the resistance of plants to stress.

5. Conclusions

We first obtained and characterized the CfCHLM gene from C. fortunei and verified its response to abiotic stresses. CfCHLM was expressed at different levels in various tissues and downregulated under abiotic stress (cold, heat, drought, or salt). Compared with WT plants, transgenic A. thaliana showed higher chl fluorescence parameters, chl levels, Pn values, and antioxidant enzyme activities and lower Gs values, Tr values, and MDA contents. This demonstrates that the CfCHLM gene is essential to chl synthesis and photosynthesis in A. thaliana and enhances the tolerance of A. thaliana to abiotic stresses. Thus, the CfCHLM gene might serve as a potential candidate for improving photosynthesis and abiotic stress tolerance in plants. These findings provide a basis for future in-depth studies on the role played by the CfCHLM gene in the growth and development of C. fortunei.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/f15040628/s1: Table S1: Primer sequences used in this study; Table S2: Amino acid composition of the CHLM protein in C. fortunei; Table S3: Composition of the secondary structure of the CHLM protein in C. fortunei; Figure S1: PCR detection of CfCHLM transgenic A. thaliana; and Figure S2: CfCHLM gene expression of WT A. thaliana and six transgenic A. thaliana lines.

Author Contributions

Conceptualization, G.W., Y.Z., Y.Y., J.Y. and J.X.; methodology, G.W. and Y.Z.; software, G.W.; validation, G.W., Y.Z., Y.Y., J.Y. and J.X.; formal analysis, G.W.; investigation, G.W.; resources, J.X.; data curation, G.W.; writing—original draft preparation, G.W. and Y.Z.; writing—review and editing, J.X. and Y.Z.; visualization, J.X.; supervision, J.X.; project administration, J.X.; funding acquisition, J.X. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Seed Industry Innovation and Industrialization Project of the Fujian province (ZYCX-LY-202101) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Abbreviations

CAT: catalase; cDNA: complementary DNA; chl: chlorophyll; CHLM: Mg-protoporphyrin IX methyltransferase; CK: control; Fv/Fm: the maximum quantum yield of photosystem II (PSII); GFP: green fluorescent protein; Gs: stomatal conductance; MDA: malondialdehyde; ORF: open reading frame; Pn: net photosynthetic rate; POD: peroxidase; qP: photochemical quenching; qRT-PCR: quantitative real-time polymerase chain reaction; RT: room temperature; ROS: reactive oxygen species; SOD: superoxide dismutase; Tr: transpiration rate; WT: wild type; and Y(II): the effective quantum yield of PSII.

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Figure 1. Sequence alignment, phylogenetic analysis, and structural composition of CfCHLM protein. (A) Multi-sequence alignment of CHLM proteins, different colors represent the level of base conservation (yellow > pink > blue). (B) Phylogenetic analysis of CHLM proteins: AtCHLG (Arabidopsis thaliana, NP_190750.1); AtCHLM (Arabidopsis thaliana, NP_001328344.1); AtCRD1 (Arabidopsis thaliana, NP_001190112.1); CasCHLM (Camellia sinensis, AVP39682.1); CisCHLM (Citrus sinensi, XP_006485173.1); GhCHLM (Gossypium hirsutum, XP_040973278.1); GmCHLM (Glycine max, XP_003532350.3); NsCHLM (Nicotiana sylvestris, XP_009773258.1); PbCHLM (Pyrus bretschneideri, XP_048445884.1); PsCHLM (Picea sitchensis, ABK21950.1); PtCHLM (Populus trichocarpa, XP_002318168.2); QsCHLM (Quercus suber, XP_023887941.1); SsCHLM (Salix suchowensis, KAG5234333.1); and TcCHLM (Taxus chinensis, KAH9300938.1). (C) Tertiary structure analysis of the CfCHLM protein. (D) Secondary structure analysis of the CfCHLM protein; long blue lines and short red/green/pink lines show predicted alpha helices and extended strands/beta turns/random coils, respectively.
Figure 1. Sequence alignment, phylogenetic analysis, and structural composition of CfCHLM protein. (A) Multi-sequence alignment of CHLM proteins, different colors represent the level of base conservation (yellow > pink > blue). (B) Phylogenetic analysis of CHLM proteins: AtCHLG (Arabidopsis thaliana, NP_190750.1); AtCHLM (Arabidopsis thaliana, NP_001328344.1); AtCRD1 (Arabidopsis thaliana, NP_001190112.1); CasCHLM (Camellia sinensis, AVP39682.1); CisCHLM (Citrus sinensi, XP_006485173.1); GhCHLM (Gossypium hirsutum, XP_040973278.1); GmCHLM (Glycine max, XP_003532350.3); NsCHLM (Nicotiana sylvestris, XP_009773258.1); PbCHLM (Pyrus bretschneideri, XP_048445884.1); PsCHLM (Picea sitchensis, ABK21950.1); PtCHLM (Populus trichocarpa, XP_002318168.2); QsCHLM (Quercus suber, XP_023887941.1); SsCHLM (Salix suchowensis, KAG5234333.1); and TcCHLM (Taxus chinensis, KAH9300938.1). (C) Tertiary structure analysis of the CfCHLM protein. (D) Secondary structure analysis of the CfCHLM protein; long blue lines and short red/green/pink lines show predicted alpha helices and extended strands/beta turns/random coils, respectively.
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Figure 2. Subcellular localization and transcriptional activity assay of CfCHLM. (A) Subcellular localization of pBI121-GFP and 35S::CfCHLM-GFP. Bars = 20 μm. (B) Growth of pCL1, pGBKT7 and pGBKT7-CfCHLM yeast cells on SD/-Trp, SD/-Ade/-His/-Trp, and SD/-Ade/-His/-Trp/X-α-gal plates.
Figure 2. Subcellular localization and transcriptional activity assay of CfCHLM. (A) Subcellular localization of pBI121-GFP and 35S::CfCHLM-GFP. Bars = 20 μm. (B) Growth of pCL1, pGBKT7 and pGBKT7-CfCHLM yeast cells on SD/-Trp, SD/-Ade/-His/-Trp, and SD/-Ade/-His/-Trp/X-α-gal plates.
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Figure 3. Expression patterns of CfCHLM gene in different C. fortunei tissues and abiotic stresses. (A) Expression patterns of CfCHLM in different tissues. (B) CfCHLM expression under 4 °C cold stress. (C) CfCHLM expression under 42 °C heat stress. (D) CfCHLM expression under 15% PEG-6000. (E) CfCHLM expression under 200 mM NaCl. Each bar represents the mean ± standard deviation (n = 3), and different lowercase letters above each bar indicate significant differences (p ≤ 0.05).
Figure 3. Expression patterns of CfCHLM gene in different C. fortunei tissues and abiotic stresses. (A) Expression patterns of CfCHLM in different tissues. (B) CfCHLM expression under 4 °C cold stress. (C) CfCHLM expression under 42 °C heat stress. (D) CfCHLM expression under 15% PEG-6000. (E) CfCHLM expression under 200 mM NaCl. Each bar represents the mean ± standard deviation (n = 3), and different lowercase letters above each bar indicate significant differences (p ≤ 0.05).
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Figure 4. Analysis of phenotypic and physiological indicators of A. thaliana under cold/heat stress. (A–C) Untreated WT, CfCHLM-L3, and CfCHLM-L6 lines. (D–F) Cold treatment. (G–I) Heat treatment; bars = 1 cm. (J–W) Mean number of leaves per plant (J); Fv/Fm values (K); Y(II) values (L); and qP values (M); chl a content (N); chl b content (O); chl a + b (P); Pn values (Q); Gs values (R); Tr values (S); SOD activity (T); POD activity (U); CAT activity (V); and MDA content (W). Each bar represents the mean ± standard deviation (n = 3), and different lowercase letters above each bar indicate significant differences (p ≤ 0.05).
Figure 4. Analysis of phenotypic and physiological indicators of A. thaliana under cold/heat stress. (A–C) Untreated WT, CfCHLM-L3, and CfCHLM-L6 lines. (D–F) Cold treatment. (G–I) Heat treatment; bars = 1 cm. (J–W) Mean number of leaves per plant (J); Fv/Fm values (K); Y(II) values (L); and qP values (M); chl a content (N); chl b content (O); chl a + b (P); Pn values (Q); Gs values (R); Tr values (S); SOD activity (T); POD activity (U); CAT activity (V); and MDA content (W). Each bar represents the mean ± standard deviation (n = 3), and different lowercase letters above each bar indicate significant differences (p ≤ 0.05).
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Figure 5. Analysis of phenotypic and physiological indicators of A. thaliana under drought stress. (A–C) Untreated WT, CfCHLM-L3, and CfCHLM-L6 lines. (D–F) 10% PEG treatment. (G–I) 15% PEG treatment; bars = 1 cm. (J–W) Mean number of leaves per plant (J); Fv/Fm values (K); Y(II) values (L); qP values (M); chl a content (N); chl b content (O); chl a + b (P); Pn values (Q); Gs values (R); Tr values (S); SOD activity (T); POD activity (U); CAT activity (V); and MDA content (W). Each bar represents the mean ± standard deviation (n = 3), and different lowercase letters above each bar indicate significant differences (p ≤ 0.05).
Figure 5. Analysis of phenotypic and physiological indicators of A. thaliana under drought stress. (A–C) Untreated WT, CfCHLM-L3, and CfCHLM-L6 lines. (D–F) 10% PEG treatment. (G–I) 15% PEG treatment; bars = 1 cm. (J–W) Mean number of leaves per plant (J); Fv/Fm values (K); Y(II) values (L); qP values (M); chl a content (N); chl b content (O); chl a + b (P); Pn values (Q); Gs values (R); Tr values (S); SOD activity (T); POD activity (U); CAT activity (V); and MDA content (W). Each bar represents the mean ± standard deviation (n = 3), and different lowercase letters above each bar indicate significant differences (p ≤ 0.05).
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Figure 6. Analysis of phenotypic and physiological indicators of A. thaliana under salt stress. (A–C) Untreated WT, CfCHLM-L3, and CfCHLM-L6 lines. (D–F) 300 mM NaCl treatment. (G–I) 400 mM NaCl treatment; bars = 1 cm. (J–W) Mean number of leaves per plant (J); Fv/Fm values (K); Y(II) values (L); qP values (M); chl a content (N); chl b content (O); chl a + b (P); Pn values (Q); Gs values (R); Tr values (S); SOD activity (T); POD activity (U); CAT activity (V); and MDA content (W). Each bar represents the mean ± standard deviation (n = 3), and different lowercase letters above each bar indicate significant differences (p ≤ 0.05).
Figure 6. Analysis of phenotypic and physiological indicators of A. thaliana under salt stress. (A–C) Untreated WT, CfCHLM-L3, and CfCHLM-L6 lines. (D–F) 300 mM NaCl treatment. (G–I) 400 mM NaCl treatment; bars = 1 cm. (J–W) Mean number of leaves per plant (J); Fv/Fm values (K); Y(II) values (L); qP values (M); chl a content (N); chl b content (O); chl a + b (P); Pn values (Q); Gs values (R); Tr values (S); SOD activity (T); POD activity (U); CAT activity (V); and MDA content (W). Each bar represents the mean ± standard deviation (n = 3), and different lowercase letters above each bar indicate significant differences (p ≤ 0.05).
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Wei, G.; Zhang, Y.; Yang, Y.; Yang, J.; Xu, J. CfCHLM, from Cryptomeria fortunei, Promotes Chlorophyll Synthesis and Improves Tolerance to Abiotic Stresses in Transgenic Arabidopsis thaliana. Forests 2024, 15, 628. https://doi.org/10.3390/f15040628

AMA Style

Wei G, Zhang Y, Yang Y, Yang J, Xu J. CfCHLM, from Cryptomeria fortunei, Promotes Chlorophyll Synthesis and Improves Tolerance to Abiotic Stresses in Transgenic Arabidopsis thaliana. Forests. 2024; 15(4):628. https://doi.org/10.3390/f15040628

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Wei, Guangqian, Yingting Zhang, Ye Yang, Junjie Yang, and Jin Xu. 2024. "CfCHLM, from Cryptomeria fortunei, Promotes Chlorophyll Synthesis and Improves Tolerance to Abiotic Stresses in Transgenic Arabidopsis thaliana" Forests 15, no. 4: 628. https://doi.org/10.3390/f15040628

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