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Article

Altered Photoprotective Mechanisms and Pigment Synthesis in Torreya grandis with Leaf Color Mutations: An Integrated Transcriptome and Photosynthesis Analysis

by
Yujia Chen
1,2,
Lei Wang
1,
Jing Zhang
1,
Yilu Chen
1 and
Songheng Jin
1,2,*
1
Jiyang College, Zhejiang A&F University, Zhuji 311800, China
2
School of Forestry and Biotechnology, Zhejiang A&F University, Hangzhou 311300, China
*
Author to whom correspondence should be addressed.
Horticulturae 2024, 10(11), 1211; https://doi.org/10.3390/horticulturae10111211
Submission received: 20 October 2024 / Revised: 14 November 2024 / Accepted: 15 November 2024 / Published: 17 November 2024
(This article belongs to the Special Issue Advances in Developmental Biology in Tree Fruit and Nut Crops)
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Abstract

:
Torreya grandis is a widely cultivated fruit species in China that is valued for its significant economic and agricultural importance. The molecular mechanisms underlying pigment formation and photosynthetic performance in Torreya leaf color mutants remain to be fully elucidated. In this study, we performed transcriptome sequencing and measured photosynthetic performance indicators to compare mutant and normal green leaves. The research results indicate that the identified Torreya mutant differs from previously reported mutants, exhibiting a weakened photoprotection mechanism and a significant reduction in carotenoid content of approximately 33%. Photosynthetic indicators, including the potential maximum photosynthetic capacity (Fv/Fm) and electron transport efficiency (Ψo, φEo), decreased significantly by 32%, 52%, and 49%, respectively. While the quantum yield for energy dissipation (φDo) increased by 31%, this increase was not statistically significant, which may further reduce PSII activity. A transcriptome analysis revealed that the up-regulation of chlorophyll degradation-related genes—HCAR and NOL—accelerates chlorophyll breakdown in the Torreya mutant. The down-regulation of carotenoid biosynthesis genes, such as LCY1 and ZEP, is strongly associated with compromised photoprotective mechanisms and the reduced stability of Photosystem II. Additionally, the reduced expression of the photoprotective gene psbS weakened the mutant’s tolerance to photoinhibition, increasing its susceptibility to photodamage. These changes in gene expression accelerate chlorophyll degradation and reduce carotenoid synthesis, which may be the primary cause of the yellowing in Torreya. Meanwhile, the weakening of photoprotective mechanisms further impairs photosynthetic efficiency, limiting the growth and adaptability of the mutants. This study emphasizes the crucial roles of photosynthetic pigments and photosystem structures in regulating the yellowing phenotype and the environmental adaptability of Torreya. It also provides important insights into the genetic regulation of leaf color in relation to photosynthesis and breeding.

1. Introduction

Renowned for its economic value, Torreya grandis is a highly prized tree species. Its popularity stems from its versatility, with applications in various fields such as food, medicine, timber, oil production, and ecological restoration [1]. Leaf color mutation is a frequently observed dominant phenotypic trait in higher plants. Previous research has identified a yellow-leafed Torreya mutant in Zhejiang Province [2]. Recently, we identified a novel yellow-leafed mutant of Torreya in the natural environment of Zhuji County, Zhejiang Province. Different mutants may display variations in photosynthetic performance. The photosynthesis of plants mainly occurs in leaves, so leaf color is an important phenotypic characteristic that affects plant traits. Hence, various leaf color mutants provide the optimal materials for studying the regulation of a plant’s photosynthetic performance, photosynthetic pigment biosynthesis, and chlorophyll structure development in relation to the cultivation of efficient photosynthetic plants [2,3,4,5].
Some leaf color mutants have become valuable genetic resources and are widely used in genetic breeding studies due to their unique traits. For example, the formation of red leaf mutants is often attributed to anthocyanin accumulation—a phenomenon that has been extensively studied [6]. In contrast, the mechanisms underlying yellow leaf mutant development in woody plants are significantly more complex and hold substantial research value, particularly in economically important tree species. In contrast, the formation mechanism of yellow leaves in native plants, especially in economic tree species, is more complex and remains insufficiently studied, offering substantial research value. The molecular mechanisms underlying leaf yellowing can be classified into several categories, such as changes in the expression levels of genes related to chlorophyll synthesis or degradation, changes in the expression of genes related to chloroplast development and function, changes in the expression of genes in pathways related to photosynthesis, and mutations affecting genes related to carotenoid biosynthesis [2,7,8]. Changes in the expression levels of genes associated with chlorophyll synthesis and degradation, genes related to chloroplast development and function, genes involved in photosynthesis, and genes linked to carotenoid biosynthesis constitute the molecular mechanisms that lead to the yellowing phenotype of leaves. Thus far, the inclusion of a comprehensive analysis of photosynthetic performance to explore the mechanisms of the yellowing of leaves in woody plants has been limited to a few species.
Chloroplasts contain a variety of pigments, of which chlorophyll and carotenoids are particularly prominent. Carotenoids function as antenna pigments in photosynthesis, capturing light energy and facilitating its transfer to chlorophyll, as well as functioning to protect chlorophyll from light damage [9]. Chlorophyll forms a photosynthetic complex by binding to specific proteins that capture light energy and facilitate electron transport to reaction centers [10]. The electron transport in photosynthesis occurs sequentially through three major pigment–protein complexes that are situated on the thylakoid membrane—Photosystem II (PSII), cytochrome b6f (cytb6f), and Photosystem I (PSI) [11]. Chlorophyll fluorescence has become an indispensable tool for studying plant photosynthesis; it enables the rapid, accurate, and non-invasive detection of plant photosystems and photosynthetic electron transport processes that contain delayed chlorophyll a fluorescence (DF) and fast chlorophyll a fluorescence (PF) [12,13,14]. PF kinetics has emerged as a critical tool for studying the structural stability of PSII, offering a comprehensive understanding of the energy flow among its various components [15]. By introducing JIP test parameters, it is possible to effectively characterize the changes in energy flux within the photosynthetic electron transport chain, enabling a quantitative assessment of photosynthetic efficiency [16]. DF reveals the charge recombination reaction in PSII, which is markedly influenced by the energy state of the thylakoid membrane [17,18]. Plant survival is dependent on the occurrence of photosynthesis within chloroplasts, which furnish the vital materials and energy required for plant activities. Thus, investigating the photosynthetic mechanisms and regulatory pathways of mutant yellow leaves represents a significant area of focus in biological research.
Next-generation sequencing (NGS) technology has been widely applied to elucidate the molecular mechanisms underlying the formation of plant leaf color mutants. This technology is essential for identifying key genes and exploring biological processes at the transcriptional level [4,5,8,19].
The objective of this study was to compare the differences in photosynthetic performance and molecular mechanisms between the Torreya mutant and its wild type, with a focus on elucidating how leaf color regulation affects photosynthetic efficiency. These findings provide theoretical foundations for a deeper understanding of the genetic regulatory mechanisms of plant photosynthesis and contribute to the cultivation of plants with enhanced photosynthetic performance.

2. Materials and Methods

2.1. Plant Material

Torreya seedlings were sourced from the Chinese Torreya Museum, with one-year-old Torreya scions grafted onto one-year-old Torreya Fort. ex Lindl. rootstocks. The experimental materials were obtained from normal plants and natural mutants displaying yellow leaves, all cultivated under the same environmental conditions. The rootstock was trimmed approximately 5–6 cm above the root collar before being grafted onto the scion. Each grafted tree was individually cultivated in liter plastic pots filled with 7 kg of loam. These were watered to saturation when the soil dried naturally and were fertilized monthly to maintain nutrient levels. While wild-type plants display dark green leaves, the mutant features yellow leaves. Reduced pigmentation makes the veins more visible, adding contrast against the deeper leaf background (Figure 1). In August 2023, three replicates of green wild-type Torreya and yellow Torreya natural mutant leaf tissues were randomly selected as research materials.

2.2. Measurement of Photosynthetic Pigment

The chlorophyll (Chl) and total carotenoid contents were assessed using the ethanol extraction method, while calculations were carried out following Lichetenthaler’s methodology [20].

2.3. Measurements of Chlorophyll a Fluorescence

Using a Multi-Function Plant Efficiency Analyzer M-PEA (Hansatech, Norfolk, UK), the simultaneous determination of the kinetic profiles of PF and DF was carried out following 30 min of the leaves being subjected to complete dark adaptation. The measurement method was detailed by Strasser et al. [21]. The M-PEA emitted red light at an intensity of 5000 μmol·m−2·s−1 and a wavelength of 627 ± 10 nm, and the light–dark conversion began after 300 μs of exposure. The PF light reflection signal was recorded under illumination, and the DF signal was recorded in the dark [21]. Based on the methodologies described in prior studies, the PF and DF curves were generated [21,22].
The parameters that could be directly obtained in the kinetics curve included Fo at 20 μs, the maximum fluorescence intensity Fm, FJ at 3 ms, and FI at 30 ms. The standardization calculated from the O-P phase is represented by the following equation: Vt = (Ft − Fo)/(Fm − Fo). The JIP test parameters are defined as follows: the potential maximum photosynthetic capacity (Fv/Fm), the ratio of exciton-driven electron transfer captured by the active reaction center of PSII (Ψo), the quantum yield of electron transport (φEo), the quantum yield for the reduction of the end electron acceptors on the PSI acceptor side (φRo), the probability with which an electron from the intersystem electron carriers moves to reduce the end electron acceptors on the PSI acceptor side (δRo), and the quantum efficiency of energy dissipation (φDo). The specific fluxes (per active reaction center in PSII) quantify the absorbed energy flux (ABS/RC), the trapped energy flux (TRo/RC), the electron transport flux (ETo/RC), the electron flux reducing the end electron acceptors at the PSI acceptor side (REo/RC), and the energy dissipation flux (DIo/RC). The phenomenological fluxes (per cross-sectional area unit) include the flux absorbed (ABS/CSm), the flux trapped (TRo/CSm), the flux dissipated as heat (DIo/CSm), the flux transferred through electron transport (ETo/CSm), and the reduction in the specific electron flux to the terminal electron acceptor of PSI (REo/CSm).

2.4. Transcriptome Sequencing

All gathered leaf samples were promptly frozen in liquid nitrogen and maintained at −80 °C for later use. Total RNA from the samples was extracted using the TRIzol reagent (Invitrogen, Waltham, MA, USA). After performing RNA extraction, assessing RNA integrity, and constructing the DNA library, sequencing was conducted using NGS technology on the Illumina platform. This process was facilitated by Huanran Biotechnology Co., Ltd., (Hangzhou, China). Prior to the bioinformatics analysis, the sequencing data underwent filtering to eliminate low-quality reads and those containing adaptor sequences.
Clean reads derived from the raw data were mapped to the Torreya reference genome (https://doi.org/10.6084/m9.figshare.21089869) (accessed on 12 December 2023) using HISAT v2.0.4 software. A differential expression pattern analysis was performed using DESeq [23]. Differentially expressed genes (DEGs) were identified based on the criteria of |log2FoldChange| > 1 and Padj < 0.05. Gene function annotation was conducted using multiple public databases, including NCBI non-redundant (NR), Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), Swiss-Prot, and the Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups (eggNOGs).

2.5. Validation by RT-qPCR of Some DEGs

Total RNA was used as a template for cDNA synthesis, utilizing a HiScript III All-in-One RT SuperMix Perfect for qPCR (Vazyme, Nanjing, China) according to the manufacturer’s instructions. The gene-specific primer sequences were designed by PrimerPremier 5.0 software (Table S1). The gene Actin from Torreya was used as the internal reference gene. qRT-PCR was conducted using a Taq Pro Universal SYBR qPCR Master Mix Kit (Vazyme, Nanjing, China) on a LightCycler® 480 II (Roche Diagnostics, Mannheim, Germany). The cycling program consisted of an initial denaturation step at 95 °C for 30 s, followed by 40 cycles comprising 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 10 s. Gene expression levels were quantified using the 2−ΔΔCt method.

2.6. Statistical Analysis

Physiological data were analyzed using a one-way analysis of variance (ANOVA) with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA). Results are expressed as mean ± standard deviation (SD). When p < 0.05, there was a statistically significant difference, which is denoted by different letters.

3. Results

3.1. Determination of Photosynthetic Pigment

Table 1 demonstrates that the contents of Chl a and b in the mutant significantly decreased by 59% and 55%, respectively, while carotenoid levels also decreased substantially by 33%. Similarly, the ratio of Chl a to b displayed a comparablea trend.

3.2. Assessment of Fluorescence Kinetic Parameters

The PF curve of the mutant leaves exhibited significant changes, including a marked decrease in the maximum fluorescence (Fm). Additionally, the transitions between the J-I and I-p phases disappeared, and the J-step approached the p level (Figure 2A). To facilitate a comparison, the PF curve was standardized from step O (20 μs) to step p (300 ms) (Figure 2B). In the mutant, Vt was elevated as a whole, with VJ and VI showing particularly significant increases. At the same time, the I2 peak was not obvious, and the DF decay between the I1 and I2 peaks nearly disappeared; this observation aligns with the OJIP transient phase of PF (Figure 2C). Additionally, the decay kinetics of DF at the I1 peak showed a significant decrease in the mutant (Figure 2D).
There was a significant reduction in the parameters Ψo, φEo, φRo, δRo, and Fv/Fm. Conversely, φDo increased in the mutant leaves, though with minimal variations in magnitude (Table 2).
The energy flux absorption is illustrated in Figure 3. In the mutant leaves, ETo/RC and REo/RC were lower, while DIo/RC increased significantly. In the mutant, the phenomenological energy fluxes, including ABS/CSm, TRo/CSm, ETo/CSm, and REo/CSm, were significantly reduced. Only DIo/CSm increased slightly, although this change was not significant (Figure 3).

3.3. Transcriptomic Analysis of Mutant and Wild-Type Torreya

Transcriptome sequencing was conducted on both wild-type and mutant samples; a total of 300,334,628 raw reads were generated. After removing low-quality sequences, filtering adapters, and eliminating ambiguous reads, we obtained 294,982,568 clean reads. In the filtered RNA data, the average Q20 and Q30 scores exceeded 97.69% and 95.85%, respectively, with the GC content ranging from 43.55% to 43.72%. The raw sequence data and assemblies were deposited in the NCBI BioProject under the accession number PRJNA1150738. Subsequently, the clean read sequences were aligned with the published Torreya genome, revealing that over 94.97% of the sequences successfully mapped the reference genome (Table S2). The Pearson correlation coefficient (R2) among the biological replicates exceeded 0.77. The above results indicate a good within-group reproducibility of the samples (Figure S1).
A total of 47,089 genes were annotated using the NR, eggNOG, Swiss-Pro, KEGG, and GO databases to provide annotation information for transcripts. The highest annotation success rate was achieved with the NR database (82.55%) and the lowest was achieved with the KEGG database (32.09%) (Table S3).

3.4. Functional Annotation and Classification of the DEGs

In this study, differentially expressed genes (DEGs) were identified using the criteria |log2FoldChange| > 1 and a corrected Padj value < 0.05. A total of 4607 DEGs were detected between wild-type and mutant samples, with 2433 being up-regulated and 2174 being down-regulated. The overall distribution of mRNA is illustrated in a volcano plot (Figure 4A). Hierarchical clustering of the DEGs was performed, with leaves of the same type being clustered together. This clustering suggests that genes within the same cluster may have similar biological functions. Additionally, the abundance of DEGs was visually represented using FPKM values and color coding (Figure 4B). GO annotation of these DEGs was conducted based on the categories of Cellular Components (CCs), Molecular Functions (MFs), and Biological Processes (BPs) to evaluate their potential functions. The results indicated that genes associated with the ‘cell periphery’ and the ‘plasma membrane’ were the most highly enriched; these genes were classified under the CC category (Figure 4A). Following this, ‘response to stimulus’ was the most enriched category within Biological Processes (BPs). In the Molecular Function (MF) category, ‘glucosyltransferase activity’ exhibited the highest level of enrichment (Figure 5A). Based on the KEGG analysis, a total of 1235 DEGs were identified and enriched in 119 pathways. The top 20 enrichment pathways, based on the false discovery rate, are shown in Figure 4B. The most significantly enriched KEGG pathways included ‘plant–pathogen interaction’, ‘amino sugar and nucleotide sugar metabolism’, and ‘galactose metabolism’ (Figure 5B).

3.5. Identification of the DEGs Associated with Leaf Coloration

Using the KEGG database, we analyzed the metabolic pathways directly associated with leaf color, specifically focusing on carotenoid biosynthesis, porphyrin metabolism, and photosynthesis. We identified the DEGs annotated within these pathways. In this study, five DEGs were identified as being the core genes involved in encoding carotenoid biosynthetic enzymes.
These included carotenoid cleavage dioxygenase 4 (CCD4), zeaxanthin epoxidase (ZEP), 9-cis-epoxy carotenoid dioxygenase (NCED1), and lycopene beta-cyclase (LCY1). CCD4 and NCED1 are involved in carotenoid degradation, whereas ZEP and LCY1 are involved in carotenoid synthesis. The expression levels of CCD4 genes (TG3g00961 and TG3g00950) showed different degrees of regulation, with some genes being up-regulated and some being down-regulated compared to the wild type. In contrast, the expression levels of the other three genes—NCED1 (TG3g00946), ZEP (TG3g01751), and LCY1 (TG5g03735)—were uniformly down-regulated in the mutant (Figure 6A).
Four DEGs were identified within the photosynthesis pathway, encoding enzymes associated with photosynthesis—PSI P700 chlorophyll a apoprotein A2 (psaB), ATP synthase subunit alpha (atpA), PSI P700 chlorophyll a apoprotein A1 (psaA), and PSII 22 kDa protein (psbS). The expression levels of psaB (TG11g00862), psaA (TG8g03353), and atpA (TG5g03088) were significantly higher in the mutant compared to the wild type. Conversely, the expression of psbS (TG4g01167) was significantly down-regulated in the mutant relative to the wild type (Figure 6C).
In the porphyrin metabolic pathway, we concentrated on analyzing the coding genes of the key enzymes involved in chlorophyll metabolism. A total of six DEGs were identified, with each encoding an enzyme involved in chlorophyll metabolism. These enzymes included chlorophyllide a oxygenase (CAO), Chl b reductase (NOL), 7-hydroxymethyl chlorophyll a reductase (HCAR), uroporphyrinogen decarboxylase (hemE1), chlorophyllase-2 (CLH2), and light-independent protochlorophyllide reductase (chlN) (Figure 6B).
Among these genes, chlN, CAO, and hemE1 are involved in chlorophyll synthesis, while HCAR, NOL, and CLH2 are associated with chlorophyll degradation. The expression levels of CAO (TG8g02191), NOL (TG9g00978), and HCAR (TG11g01233) were significantly up-regulated in the mutant compared to the wild type. Conversely, the expression levels of chlN (TG7g03643), hemE1 (TG7g00721), and CLH2 (TG8g02048) were down-regulated in the mutant relative to the wild type (Figure 5B). The expression levels of these genes are further detailed in Figure 6C.

3.6. Validation of Transcription Data Accuracy

Several DEGs associated with carotenoid biosynthesis, photosynthesis, and porphyrin metabolism pathways were validated using qRT-PCR to further assess the confidence of the RNA-seq data. The DEGs from qRT-PCR are consistent with the expression patterns observed in the RNA-seq data, indicating that the RNA-seq data were highly accurate (Figure 7).

4. Discussion

4.1. Photosynthesis Performance in the Mutant Torreya

Previous research has demonstrated that Chl-deficient mutants consistently display reduced levels of chlorophyll and carotenoids. The concentration and composition of these pigments are closely linked to the development of yellowing leaves in plants [4,5]. The changes in photosynthetic pigment content observed in a previous mutant of Torreya differ from those found in this research, as the earlier study reported an increase in carotenoid content [2]. We observed a significant reduction in chlorophyll and carotenoid content in the mutant leaves of Torreya, which altered the normal leaf coloration and resulted in a Chl-deficient phenotype (Table 1). In this study, the induction kinetics of PF and DF were analyzed in both wild-type and mutant leaves. This method, which involves simultaneous measurements, provides complementary information on electron transport through optical coupling [22]. The increase in PF kinetics from the lowest point (O) to the highest point (p) primarily indicates alterations in the original photochemical processes of PSII [21,24]. The results indicated that the p point in the mutant Torreya was significantly lower than that in the wild type (Figure 2A). Previous studies suggest that the reduction in fluorescence at p may result from several different factors, i.e., the denaturation of chlorophyll–protein complexes [25], the inhibition of the PSI acceptor side [26], a reduction in the number of active reaction centers available to support electron transport to PSI [27], changes in the properties of PSII electron acceptors [28], and the development of radiation-free dissipation of the chlorophyll excited state within PSII antennas [29]. From the standardized Vt curve obtained after the O-p phase, it can be observed that the J and I steps were elevated in the mutant compared to the wild type, while the O and P steps remained unchanged (Figure 2B). The apparent enhancement of VJ and VI reflected the kinetic inhibition of the electron transport chain in the subsequent processes. The significant enhancement of VJ and VI indicated kinetic inhibition in the electron transfer chain during later stages. The observed inhibition may be related to a decreased electron transfer efficiency between QA and QB on the acceptor side of PSII, as well as a limited reoxidation of plastoquinol (PQH2) [30,31]. DF is directly generated from the reaction centers of PSII, making it a reliable indicator of PSII function [32]. The kinetic values of DF were altered by the reduced electron transfer rate in PSII (Figure 2C,D). This reduction is manifested as a decrease in DF yield, which subsequently affects charge recombination and the regeneration of antenna pigments [21]. The loss of DF observed between peaks I1 and I2 was associated with the absence of the J-I process in the PF curve. The significant reduction in I1 indicated a potential decrease in P680, which could result in decreased electron transfer efficiency between the donor and acceptor sides of PSII [28]. Additionally, changes in I2 may have been correlated with the prolonged reopening of PSII reaction centers [33], which is consistent with the PF results mentioned above. Fv/Fm is regarded as a crucial indicator for assessing the extent of photoinhibition in plants [16]. Previous studies have indicated that the Fv/Fm in green leaves is significantly higher than that in mutant leaves [2], which is consistent with the results of this study. The elevated ABS/RC values observed in the mutant samples compared to the wild-type samples are primarily due to the accelerated degradation of pigments within PSII [34]. Furthermore, the significant increase in the ABS/RC and TRo/RC values is believed to result from the enlargement of the antenna complex relative to the size of P680 [35]. The down-regulation of Ψo and φRo has been observed in the mutant, which is consistent with previous findings [2]. The significantly lower φEo in the mutant suggests the utilization of trapped photons for electron transfer from QA to QB, and beyond QB, in the electron transport chain [16].
The variations in φRo and δRo reflect changes in PSI electron transfer efficiency, with their decreased values in mutant leaves being interpreted as an impairment of PSI activity. DIo/RC and φDo are not significantly enhanced. Moreover, the rise in DIo/CSm values indicates the intensified thermal inactivation of RCs, which consequently lowers the values of REo/CSm and ETo/CSm [36]. The chlorophyll and carotenoid content in the mutants was significantly reduced, leading to impaired photosynthetic efficiency, which was evidenced by decreased photosynthetic electron transfer efficiency and intensified photoinhibition. Unlike previous studies that reported enhanced PSI activity in Torreya mutants [2], the mutant in this study lacked additional photoprotective mechanisms. This was reflected not only in the reduced photosynthetic efficiency, but also in the decreased stability and tolerance of the photosystem. Such inefficiency and instability may place the mutant plants at a disadvantage in terms of growth and development, potentially impairing their adaptability and competitiveness in natural environments.

4.2. Integrated Analysis of DEGs in Carotenoid, Chlorophyll, and Photosynthesis Pathways

The molecular mechanisms underlying yellow leaf formation vary among different plant species. The yellowing of plants is typically attributed to a deficiency in chlorophyll, which is the primary pigment that is responsible for imparting green coloration to leaves [11]. To date, numerous mutants with defects in chlorophyll biosynthesis and degradation have been discovered among different plant species, including rice [37], Arabidopsis [38], and pakchoi [39]. In this study, we applied a combined criterion of |log2FoldChange| > 1 and a corrected Padj value < 0.05 to identify DEGs associated with porphyrin metabolism in both wild-type and mutant samples. Among the selected DEGs, NOL and HCAR, which are involved in chlorophyll degradation, exhibited up-regulation, whereas CLH2 showed down-regulation. The expression level of the chlN gene, which is associated with the chlorophyll biosynthesis pathway, was down-regulated. Six enzymes involved in chlorophyll metabolism have been identified in both Arabidopsis and rice, including HCAR and NOL, which were annotated in this study [37,40]. Chlorophyll degradation plays a crucial role in the disassembly of PSII and the reduction of chlorophyll–protein complexes, which directly impacts the efficiency of photosynthesis [41]. In a study on tobacco plants overexpressing cucumber HCAR, accelerated chlorophyll degradation was observed, which was accompanied by a reduction in photosynthetic proteins, ultimately leading to a decline in quantum yield and net photosynthetic rate [42]. In Arabidopsis, plants overexpressing HCAR exhibited accelerated leaf yellowing [38]. The up-regulation of the ZjNOL gene, cloned from Zoysia japonica, can impair the structure and function of the photosystem, thereby reducing photosynthetic efficiency [43]. These findings suggest that the up-regulation of NOL and HCAR may reduce the photosynthetic capacity of Torreya mutants by accelerating chlorophyll degradation and impairing the photosystem, thereby limiting resource accumulation, growth, and development. The expression of CLH2 was inversely correlated with the yellowing of broccoli [44]. However, in our study, the expression level of the CLH2 gene decreased, which typically leads to chlorophyll accumulation or preservation, suggesting that CLH2 may have a more complex regulatory role in the color modulation of Torreya leaves. The reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) is a critical step in the chlorophyll biosynthesis pathway; this process is mediated by the light-dependent protochlorophyllide oxidoreductase (LPOR) and the dark-active Pchlide oxidoreductase (DPOR) [45]. The dark treatment of Norway spruce resulted in etiolation, which was attributed to the inhibition of the transcription of genes encoding DPOR [46]. The chlorophyll gene chlN, which encodes DPOR, was down-regulated in the mutant of Torreya. The alterations in these gene expression levels may serve as the primary factor contributing to the reduction in the chlorophyll content in leaves, subsequently facilitating the development of yellowing phenotypes. At the same time, the DEGs associated with the carotenoid metabolic pathway in the Torreya mutant also exhibited changes in expression. The LCY1 gene encodes a carotenoid cyclase involved in the conversion of lycopene to α-carotene [47]. This process is crucial for a plant’s photosynthetic efficiency, particularly in enhancing leaf photoprotection and antioxidant responses [48]. The overexpression of Ntβ-LCY1 in tobacco has been proven to result in a strong accumulation of carotenoids [49]. The expression level of the LCY1 gene identified in this study was significantly lower in the mutant compared to that in the wild-type. Carotenoids not only capture light energy as photosynthetic pigments, but also regulate the stability of PSII in the light protection mechanism [50]. We speculate that in the yellowing mutant of Torreya, the down-regulation of the LCY1 gene may reduce carotenoid synthesis, resulting in a decreased photosynthetic capacity and yellowing. Zeaxanthin epoxidase (ZEP) serves as a crucial enzyme in the xanthophyll cycle, catalyzing the downstream reactions within the carotenoid biosynthesis pathway [4,47]. The xanthophyll cycle mechanism has been shown to protect plants from photodamage [49], particularly in PSII [51], where excess light energy is dissipated primarily through thermal dissipation and photochemical quenching mechanisms [52]. In addition, the accumulation of zeaxanthin plays a crucial role in mitigating light stress, thereby supporting normal plant growth [11]. Previous studies have demonstrated that the elevated abundance of zeaxanthin in striped leaves forms an adaptive protective mechanism, which enables rice plants to alleviate the negative impact of surrounding white leaf stripes on overall plant growth and development; this mechanism is primarily driven by the up-regulation of OsZEP expression [53]. The down-regulation of ZEP expression in the mutant leaves of Torreya may impair the function of the xanthophyll cycle, making the leaves more susceptible to photo-oxidative damage, thereby compromising the stability of PSII. Abscisic acid (ABA) synthesis is mainly achieved by the oxidative cleavage of carotenoids, and 9-cis-epoxycarotenoid dioxygenase (NCED1) is a key regulatory gene in ABA biosynthesis [54,55]. In the Torreya mutant, the expression levels of the NCED1 and ZEP genes were found to be down-regulated, which aligns with the findings observed in the poplar golden leaf mutant [4].
The expression of the psbB and psbD genes was blocked in the yellow leaves of common wheat, and a further chloroplast ultrastructure analysis suggested that the formation of yellow leaves was closely associated with an abnormal chloroplast structure [56]. According to the KEGG database, it was revealed that a total of 37 unigenes were identified within the photosynthesis pathway of the mutant yellow leaves of ginkgo [5]. In the mutant, only the psbS gene was down-regulated in the photosynthetic pathway compared to that in the wild type. The level of psbS plays a regulatory role in cyclic electron flow, and psbS absence increases susceptibility to damage to PSI and PSII [57]. Thus, the Torreya mutants examined in this study exhibited significant deficiencies in their ability to cope with chlorophyll deficiency and the decline in PSII linear electron transport [2]. Related studies have shown that transgenic tobacco lines overexpressing SaPsbS exhibit higher Fv/Fm values [58], while the photochemical efficiency of Photosystem II is significantly reduced in Arabidopsis mutants lacking PsbS [59]. This finding aligns with the chlorophyll fluorescence results obtained in this study. Studies have shown that Chl-deficient mutants attempt to increase non-photochemical quenching (NPQ), which enhances heat dissipation and improves the photoprotection provided by the xanthophyll cycle pool [60]. The PsbS protein, an essential component of the PSII core complex, has a critical function in NPQ [61,62], while ZEP serves a critical function in the lutein cycle [19]. The expression levels of ZEP and psbS were significantly down-regulated in Torreya mutants, suggesting a deficiency in photoprotection mechanisms. This may increase the mutant’s susceptibility to light-induced damage, leading to reduced photosystem efficiency.
In order to further advance the understanding of the pigment formation and photosynthetic efficiency in Torreya mutants, several future directions of study could be pursued. CRISPR technology could complement the findings of this study on chlorophyll and carotenoid biosynthesis. Recent studies have shown that CRISPR/Cas9 can be used to knock out or overexpress genes in plant systems, enabling the targeted modifications of the pathways involved in pigment synthesis. For example, in the case of carotenoid biosynthesis, CRISPR has been used to successfully knock out genes like lycopene ε-cyclase (LCYE) in Chlamydomonas reinhardtii, which led to altered carotenoid profiles, enhancing the production of valuable pigments like astaxanthin [63]. This technology can be employed for the selective knockout or overexpression of annotated genes, such as HCAR, NOL, and ZEP, in the context of this study. By generating precise genetic modifications, we can explore the molecular mechanisms behind the observed phenotype and their impact on the plant’s photosynthetic performance. Additionally, combining CRISPR with transcriptomic analyses could enable the identification of novel regulators or signaling pathways that influence pigment biosynthesis, opening up new avenues for crop improvement strategies under changing environmental conditions.

5. Conclusions

In this study, the content of Chl and carotenoids was found to be reduced in Torreya mutants, which correlated with impaired photosynthetic activity compared to the wild type. This impairment led to a significant decrease in parameters associated with electron transfer (ETo/CSm, ETo/RC, and φEo). The genes involved in chlorophyll biosynthesis (chlN, hemE1, and CLH2) were down-regulated; those associated with carotenoid biosynthesis (NCED1, LCY1, and ZEP) also exhibited a downward trend. In contrast, genes related to chlorophyll degradation (NOL and HCAR) showed up-regulated expression. These findings highlight significant differences in the regulation and alteration of photosynthetic pigment biosynthesis among the mutant varieties of Torreya. The weakening of photoprotective mechanisms results in a marked decrease in electron transfer, which may restrict the growth and competitiveness of these mutants in natural environments. Overall, this study provides valuable insights for further investigation into leaf color changes and their molecular mechanisms in other plant species, while also offering important information for the cultivation and breeding of Torreya.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae10111211/s1, Figure S1: Pearson’s correlation coefficients between two biological replicates in the six samples. Table S1: The gene-specific primer sequences used in qRT-PCR. Table S2: Statistics of sequencing data quality from Torreya grandis samples. Table S3: Reference genome information.

Author Contributions

Conceptualization, Y.C. (Yujia Chen), S.J. and L.W.; methodology, Y.C. (Yujia Chen) and J.Z.; software, Y.C. (Yilu Chen) and J.Z.; validation, Y.C. (Yujia Chen) and J.Z.; formal analysis, Y.C. (Yujia Chen); resources, S.J.; writing—original draft preparation, Y.C. (Yujia Chen); writing—review and editing, Y.C. (Yujia Chen), L.W. and S.J.; funding acquisition, S.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Key Research and Development Project (2019YFE0118900), the National Natural Science Foundation of China (31971641), and the Zhejiang Provincial Team Technology Commissioner Project (Horticulture Team in Wencheng).

Data Availability Statement

Raw sequence data and assemblies were deposited under NCBI BioProject PRJNA1150738.

Conflicts of Interest

The authors declare no competing interests.

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Figure 1. Leaf appearance of wild type and mutant Torreya.
Figure 1. Leaf appearance of wild type and mutant Torreya.
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Figure 2. (A) Prompt chlorophyll a fluorescence (PF). (B) Normalized curve; Vt = [(Ft − FO)/(FM − Fo)], J reflects early electron transport blockage; I reflects the size of the PQ pool and the efficiency of electron flow; P represents the maximum PSII photochemical efficiency. (C) Delayed chlorophyll a fluorescence (DF). I1 represents the redox state of QA and PSII functionality; I2 represents the reduction of the PQ pool and the efficiency of electron transfer; D2 represents the charge separation stability and recombination dynamics. (D) The decay kinetics of DF at the characteristic maxima I1 (7 ms). Each curve represents the mean value derived from three replicate measurements.
Figure 2. (A) Prompt chlorophyll a fluorescence (PF). (B) Normalized curve; Vt = [(Ft − FO)/(FM − Fo)], J reflects early electron transport blockage; I reflects the size of the PQ pool and the efficiency of electron flow; P represents the maximum PSII photochemical efficiency. (C) Delayed chlorophyll a fluorescence (DF). I1 represents the redox state of QA and PSII functionality; I2 represents the reduction of the PQ pool and the efficiency of electron transfer; D2 represents the charge separation stability and recombination dynamics. (D) The decay kinetics of DF at the characteristic maxima I1 (7 ms). Each curve represents the mean value derived from three replicate measurements.
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Figure 3. Radar plot of energy fluxes in mutant and wild type Torreya. The radar plot reflected specific activity values at individual PSII reaction centers (RCs) and cross-sections (CSs). Each data point represents the mean value derived from three replicate measurements.
Figure 3. Radar plot of energy fluxes in mutant and wild type Torreya. The radar plot reflected specific activity values at individual PSII reaction centers (RCs) and cross-sections (CSs). Each data point represents the mean value derived from three replicate measurements.
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Figure 4. Differentially expressed genes (DEGs) in different leaves of Torreya. (A) DEGs were displayed in the form of a volcano plot. The red dots denote up-regulated genes, while the blue dots denote down-regulated genes. (B) Hierarchical cluster analysis of DEGs. Each row represents a gene, with red indicating a more pronounced up-regulation and green indicating a more pronounced down-regulation.
Figure 4. Differentially expressed genes (DEGs) in different leaves of Torreya. (A) DEGs were displayed in the form of a volcano plot. The red dots denote up-regulated genes, while the blue dots denote down-regulated genes. (B) Hierarchical cluster analysis of DEGs. Each row represents a gene, with red indicating a more pronounced up-regulation and green indicating a more pronounced down-regulation.
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Figure 5. Functional annotation of DEGs. (A) GO classification of differentially expressed genes—the top 30 enriched GO terms. (B) KEGG enrichment of differentially expressed genes; the larger the bubble, the more DEGs that are enriched.
Figure 5. Functional annotation of DEGs. (A) GO classification of differentially expressed genes—the top 30 enriched GO terms. (B) KEGG enrichment of differentially expressed genes; the larger the bubble, the more DEGs that are enriched.
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Figure 6. Regulation of gene expression and metabolic pathways associated with leaf color at the transcriptional level. (A) Analysis of differentially expressed genes related to chlorophyll biosynthesis and degradation pathways. (B) Differential expressions of genes related to carotenoid biosynthesis and degradation pathways. (C) Transcriptome data (FPKM) were used for heat mapping.
Figure 6. Regulation of gene expression and metabolic pathways associated with leaf color at the transcriptional level. (A) Analysis of differentially expressed genes related to chlorophyll biosynthesis and degradation pathways. (B) Differential expressions of genes related to carotenoid biosynthesis and degradation pathways. (C) Transcriptome data (FPKM) were used for heat mapping.
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Figure 7. The expression levels of DEGs in wild-type and mutant Torreya. (A) Gene ID: TG8G02048 (CHL2). (B) Gene ID: TG5G03735 (LCY1). (C) Gene ID: TG3G00961 (CCD4). (D) Gene ID: TG9G00978 (NOL). (E) Gene ID: TG7G03643 (chlN). (F) Gene ID: TG3G0175 (ZEP). (G) Gene ID: TG8G003353 (psaA). (H) Gene ID: TG4G00167 (psbS). (I) Gene ID: TG11G01233 (HCAR). (J) Gene ID: TG3G00946 (NECD1). Each error bar represents the SD calculated from three biological replicates, each of which includes three technical replicates.
Figure 7. The expression levels of DEGs in wild-type and mutant Torreya. (A) Gene ID: TG8G02048 (CHL2). (B) Gene ID: TG5G03735 (LCY1). (C) Gene ID: TG3G00961 (CCD4). (D) Gene ID: TG9G00978 (NOL). (E) Gene ID: TG7G03643 (chlN). (F) Gene ID: TG3G0175 (ZEP). (G) Gene ID: TG8G003353 (psaA). (H) Gene ID: TG4G00167 (psbS). (I) Gene ID: TG11G01233 (HCAR). (J) Gene ID: TG3G00946 (NECD1). Each error bar represents the SD calculated from three biological replicates, each of which includes three technical replicates.
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Table 1. Comparison of pigment concentrations between mutant and wild-type Torreya leaves.
Table 1. Comparison of pigment concentrations between mutant and wild-type Torreya leaves.
Torreya grandisChl (a + b) (mg/g FW)Chl a (mg/g FW)Chl b (mg/g FW)Chl a/b (mg/g FW)Carotenoid (mg/g FW)
Wild type0.42 ± 0.01 a0.32 ± 0.02 a0.09 ± 0.01 a3.47 ± 0.08 a0.12 ± 0.00 a
Mutant016 ± 0.00 b0.12 ± 0.01 b0.04 ± 0.01 b2.82 ± 0.20 b0.08 ± 0.01 b
The values are presented as mean ± SD (n = 3); significant differences are indicated by different letters; p < 0.05.
Table 2. JIP test parameters in the leaves of mutant and wild-type Torreya grandis.
Table 2. JIP test parameters in the leaves of mutant and wild-type Torreya grandis.
Torreya grandisFv/FmφEoφRoφDoδRoΨo
Wild type0.84 ± 0.01 a0.70 ± 0.02 a0.26 ± 0.01 a0.16 ± 0.01 b0.38 ± 0.01 a0.83 ± 0.02 a
Mutant0.57 ± 0.12 b0.36 ± 0.16 b0.18 ± 0.03 b0.21 ± 0.14 b0.27 ± 0.05 b0.40 ± 0.06 b
Fv/Fm: the potential maximum photosynthetic capacity; Ψo: the ratio of exciton-driven electron transfer captured by the active reaction center of PSII at t = 0; φEo: quantum efficiency of electron transfer at t = 0; φRo: quantum yield for the reduction of the terminal electron acceptors on the PSI acceptor side; φDo: quantum yield for energy dissipation at t = 0; δRo: quantum yield for the reduction of the terminal electron acceptors on the PSI acceptor side. The values are presented as mean ± SD (n = 3); significant differences are indicated by different letters; p < 0.05.
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Chen, Y.; Wang, L.; Zhang, J.; Chen, Y.; Jin, S. Altered Photoprotective Mechanisms and Pigment Synthesis in Torreya grandis with Leaf Color Mutations: An Integrated Transcriptome and Photosynthesis Analysis. Horticulturae 2024, 10, 1211. https://doi.org/10.3390/horticulturae10111211

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Chen Y, Wang L, Zhang J, Chen Y, Jin S. Altered Photoprotective Mechanisms and Pigment Synthesis in Torreya grandis with Leaf Color Mutations: An Integrated Transcriptome and Photosynthesis Analysis. Horticulturae. 2024; 10(11):1211. https://doi.org/10.3390/horticulturae10111211

Chicago/Turabian Style

Chen, Yujia, Lei Wang, Jing Zhang, Yilu Chen, and Songheng Jin. 2024. "Altered Photoprotective Mechanisms and Pigment Synthesis in Torreya grandis with Leaf Color Mutations: An Integrated Transcriptome and Photosynthesis Analysis" Horticulturae 10, no. 11: 1211. https://doi.org/10.3390/horticulturae10111211

APA Style

Chen, Y., Wang, L., Zhang, J., Chen, Y., & Jin, S. (2024). Altered Photoprotective Mechanisms and Pigment Synthesis in Torreya grandis with Leaf Color Mutations: An Integrated Transcriptome and Photosynthesis Analysis. Horticulturae, 10(11), 1211. https://doi.org/10.3390/horticulturae10111211

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