Advance in mechanism of plant leaf colour mutation

As a common mutation trait in plants, leaf colour mutation is related to the degree of chlorophyll and anthocyanin changes and the destruction of chloroplast structure. This study summarizes the latest research progress in leaf colour mutation mechanism, including the metabolic basis of plant leaf colour mutation, leaf colour mutation caused by gene mutation in the chlorophyll metabolism pathway, leaf colour mutation caused by blocked chloroplast development, leaf colour mutation controlled by key transcription factors and noncoding RNAs, leaf colour mutation caused by environmental factors, and leaf colour mutation due to the involvement of the mevalonate pathway. These results will lay a theoretical foundation for leaf colour development, leaf colour improvement, and molecular breeding for leaf colour among tree species.


Introduction
Leaf is an important organ for photosynthesis and gas exchange in plants (Meng et al., 2018). As a key visible feature of leaves, leaf colour is a reliable marker for plant breeding (Akhter et al., 2018). The leaves of most plants in the normal growth period are green, which results from chlorophyll (Chl) enrichment (Yang et al., 2015). With the increasing demand for a living environment, the number of green-leaved tree species cannot meet the needs of landscape engineering. To improve the ornamental effect of landscape plants, trees with colourful-leaves are widely used in gardening, e.g., Cercis canadensis with purple leaf (Roberts et al., 2015), Lagerstroemia indica with yellow leaf , and Prunus cerasifera with purple leaf (Gu et al., 2015).
The leaf colours of these trees are all caused by mutations. As a common mutation trait in plants, leaf colour mutation is a vital source of germplasm resources for colourful leaf plants. Previous studies have shown that 208 leaf colour mutants exist in Oryza sativa (Deng et al., 2014). Leaf mutants of Cucumis sativus (Ding et al., 2019), Glycine max , Ginkgo biloba (Liu et al., 2016a;Li et al., 2018a), Triticum aestivum (Rong et al., 2018), and Anthurium andraeanum (Wang et al., 2018a) also exist. Most of these mutants have 3 (Zhang et al., 2017b). In Brassica oleracea, the content of anthocyanin in purple leaves is 2.5 times that in ordinary green leaves .
Leaf colour mutation is a complex physiological process involving the effects of various substances. Considering the accuracy of mass spectrometry in identifying compounds, metabolome has become an important technique in plant research. A large number of compounds involved in plant leaf colour mutation have been identified through metabolome. Li et al. (2019a) compared the content of secondary metabolites in albino leaves of Camellia sinensis to that of normal green leaves through metabolic screening. Further analysis showed that the contents of total amino acids, L-theanine, and glutamic acid increased significantly, whereas the contents of alkaloid, catechin, and polyphenols decreased significantly. These substances contributed to formation of albino leaves in C. sinensis. Similarly, a decrease in total amino acids and L-theanine of 'ZH2', a leaf colour mutant of C. sinensis, was also observed . By comparing the metabolites in purple and green leaves of Tetrastigma hemsleyanum, the purple leaves were found to have accumulated a larger number of anthocyanins and flavone-glycosides than green leaves. Moreover, the contents of pelargonidin and dihydrokaempferol in purple leaves were significantly higher than in green leaves, indicating that these substances contribute to the purple colour of T. hemsleyanum leaves (Yan et al., 2020).
In addition to the effect of pigments on leaf colour formation, starch and sugar also affect leaf colour changes. During leaf development in Acer saccharum, the concentration of starch, glucose, and fructose were positively correlated with the expression of leaf colour, and the red colour of leaves were significantly affected by the content of sucrose and fructose (Schaberg et al., 2003). Murakami et al. (2008) found that girding in A. saccharum can significantly increase the content of sugar in leaves and accelerate the accumulation of anthocyanin. A comparative analysis of the metabolites of the three types of albino leaves in C. sinensis showed that the content of sugar (mainly sorbitol and erythrose) in albino leaves was significantly higher than in green leaves . Flavones and flavonols are important parts of flavonoids in plants, and the changes in their content also affect the expression of plant leaf colour (Martens et al., 2010). In G. biloba, the accumulation of flavonols and flavones promotes the expression of yellow leaves (Shi et al., 2012). However, for Camellia nitidissima, flavonols are the main component of golden leaves (Zhou et al., 2013a). The abovementioned studies showed that leaf colour mutation involves the interaction of multiple compounds. Moreover, the content and morphological changes of these compounds constitute a tight regulatory network for leaf colour mutation.

Leaf Colour Mutation Caused by Gene Mutation of Chlorophyll Metabolism
Chl is a major component of green leaves. Since the Chl biosynthesis pathway was first reported by Beale, a large number of genes related to Chl biosynthesis have been identified (Beale et al., 2005;Deng et al., 2014). In Arabidopsis thaliana, the synthesis of Chl starts from glutamyl-tRNA, and Chl finally forms through the action of 15 enzymes encoded by 27 genes (Meier et al., 2011). If any step in this process is hindered, then leaf colour mutation may occur. Previous studies showed that the mutation of genes related to Chl synthesis, such as CHLI/CHLD/CHLH, HemA, CHLG, CAO, and DVR, is one of the common sources of leaf colour mutation ( Figure 1). The three subunits coded by CHLI/CHLD/CHLH are the functional basis of Mg 2+ chelatase, which is a key protein complex for Chl synthesis, and the lack of any subunit destroys Chl synthesis (Hansson et al., 2002). In O. sativa, varied yellow-green leaf mutants are the results of the gene mutation of CHLI/CHLD/CHLH, such as chlorina-1, chlorina-9, chlorina-2, ygl3, ygl7, and ygl98 (Jung et al., 2003;Zhang et al., 2006;Sun et al., 2011;Tian et al., 2013;Deng et al., 2014). Interestingly, the mutation in different subunits also causes a variety of different mutant phenotypes. For example, the gene mutation of OsCHLD in mutant chlorina-1 led to the yellow-green leaf phenotype at the seedling stage, whereas the gene mutation of OsCHLI in mutant ell led to the yellow leaf phenotype at the seedling stage. However, the seedlings died after the trefoil stage (Zhang et al., 2006(Zhang et al., , 2015. Studies on mutant ygl1 found that YGL1 encodes Chl synthase 4 (CHLG), thereby causing leaf colour mutation (Wu et al., 2007). Similarly, the mutant ygl3 showed a yellowgreen phenotype, reduced plant height, and decreased grain yield (Zhang et al., 2006). Furthermore, the 9-bp deletion in the OsDVR sequence caused leaf colour mutation in mutant 824ys (Wang et al., 2010). OsCAO1 and OsCAO2 encode chlorophyllide an oxygenase, which catalyses the conversion of Chl a to Chl b (Figure1). Moreover, OsCAO1 was induced by light, whereas OsCAO2 was expressed in the dark, and the OsCAO knockout mutation led to the expression of leaf color mutation (Lee et al., 2005). HEMA gene encodes glutamyl-tRNA reductase (GluTR), which is a key catalytic enzyme for Chl synthesis. HEMA gene is regulated by light, and the expression of HEMA antisense RNA inhibits the formation of δ-aminolevulinic acid (ALA), thereby leading to the expression of Chl in A. thaliana (Kumar and Soll, 2000).
Chl and heme are two types of tetrapyrrole with a similar structure. They share a pathway from ALA to protoporphyrin IX (Figure 1) (Weller et al., 1996). Heme is necessary for photosynthesis and respiration.
However, excessive heme accumulation inhibits the activity of glutamyl-tRNA reductase and the synthesis of ALA, thereby affecting Chl biosynthesis (Terry et al., 1999). Many leaf colour mutants caused by abnormal heme metabolism have been identified, including A. thaliana (Xie et al., 2012), O. sativa (Xu et al., 2012;Li et al., 2014), Pisum sativum (Linley et al., 2006), Zea mays (Shi et al., 2013), and Brassica pekinensis (Zhang et al., 2020). Studies on the yellow leaf colour mutant pylm showed that the single-base mutation of recessive nuclear genes (PY1 and PY2), results in the dysfunction of heme oxygenase-1 (HO-1) . The accumulation of excessive heme in leaves activates the feedback inhibition of Chl synthesis (Weller et al., 1996), leading to the expression of the yellow leaf phenotype. This mutation mechanism was similar to that in the in rice mutant yellow-green leaf 2 (Chen et al., 2013). Furthermore, the functional defects of HO1 increase heme levels and cause the abnormal development of chloroplast thylakoids. For example, the HO1 mutation in the maize mutant elm1 showed decreased thylakoid basal accumulation, declined HEMA activity, and reduced Chl content (Shi et al., 2013). Studies in rice also showed that HO1 defective mutation affects thylakoid development (Li et al., 2014). Moreover, genes in the Chl degradation pathway, such as NYC1, NOL, and SGR, are important sources of leaf colour mutation (Ren et al., 2007(Ren et al., , 2010; Barry et al., 2008;Borovsky and Paran, 2008;Horie et al., 2009;Wang et al., 2018b).

Leaf Colour Mutation Caused by Destroyed Chloroplast Structure
Chloroplast, as the synthesis site of Chl and carotenoid, is important for the formation of plant leaf colour. Transmission electron microscopy (TEM) analysis of the ultrastructure of leaf colour mutant varieties showed that most of the leaf colour mutants showed a destroyed chloroplast structure, degraded thylakoid lamella, and dissolved thylakoid granule (Gao et al., 2020;Du et al., 2020). The yellow leaves of B. pekinensis expressed the inhibited development of chloroplast, and showed immature starch grains. Furthermore, the chloroplast had no complete granule and clear thylakoid membrane, which blocked Chl synthesis (Xie et al., 2018). In rice albino leaves, the chloroplast structure is destroyed. The chloroplast is filled with a large number of oval vesicles and has no thylakoid basal accumulation (Qiu et al., 2018). These studies indicated that chloroplast development defect is the important cause of leaf colour mutation in plants.
Chloroplasts in higher plants are developed from proplastids, which fold into vesicles and then develop into thylakoid lamella . A complete chloroplast usually consists of chloroplast membrane, thylakoid, and stroma. The number, size, morphology, and distribution of chloroplasts directly affect leaf colour. Therefore, the presence of dysfunctional chloroplasts always accompanies the lack of green colour in leaves (Yang et al., 2015). Several genes related to chloroplast development and chloroplast division have been identified. Their functions in leaf colour formation have been clarified through a previous study on a variety of leaf colour mutants. Golden2-like (GLK) transcription factor (TF) is a vital member of the GARP family in plants. GLK is reportedly involved in multiple biological processes and plays an important role in chloroplast development (Powell et al., 2012). The homologous genes of GLK have been identified from 5 various plants, such as A. thaliana , Z. mays (Rossini et al., 2001), and birch (Gang et al., 2019). Moreover, most GLK families include two members, i.e., GLK1 and GLK2. Through the functional analysis of the GLK gene in A. thaliana and rice, the GLK gene was shown wo exhibit functional redundancy (Nguyen et al., 2014;Wang et al., 2013a). Moreover, a transgenic functional verification experiment showed that only glk1glk2 double mutants expressed the virescent phenotype, and any overexpression of a GLK gene can restore the green phenotype of leaves (Fitter et al., 2002). In the birch mutant yl, a 40 kb deletion of the BpGLK gene on chromosome 2 caused the destruction of the chloroplast structure, blocked Chl synthesis, and resulted in leaf color mutation (Gang et al., 2019). In addition, the ectopic expression of GLKs increased the number of chloroplasts in the roots of rice and A. thaliana (Kobayashi et al., 2012(Kobayashi et al., , 2013. The blue box represents the key genes of MEP pathway related to leaf colour mutation, while the key genes of chlorophyll metabolism pathway involved in leaf colour mutation are marked with yellow box, and the red dotted "T" represents inhibition or hindrance. G3P, Glyceraldehyde 3-phosphate; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; IspD, 4-diphosphocytidyl-2-C-methyl-Derythritol synthase; IspE, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; IspF, 2-C-methyl-D-erythritol 2,4cyclodiphosphate synthase; IspG, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase; IspH, 1-hydroxy-2methyl-2-(E)-butenyl 4-diphosphate reductase; IPP, isopentenyl pyrophosphate; IDI, isopentenyl diphosphate isomerase; DMAPP, dimethylallyl pyrophosphate; GPP, geranyl diphosphate; GGPP, geranylgeranyl pyrophosphate; HemA, glutamyl-tRNA reductase; ALA, δ-aminolevulinic acid; CPO, coproporphyrinogen oxidative decarboxylase; Protogen IX, protoporphyrinogen IX; PPOX, protoporphyrinogen oxidase; Proto IX, protoporphyrin IX; CHLI, Mg chelatase I subunit; CHLD, Mg chelatase D subunit; CHLH, Mg chelatase H subunit; Mg-Proto IX, Mgprotoporphyrin IX; DV Pchlide, divinyl protochlorophyllide; DVR, divinyl reductase; PORA/B/C, NADPH: protochlorophyllide oxidoreductase; Chlide a, chlorophyllide a; Chlide b, chlorophyllide b; DV chlide a, divinyl chlorophyllide a; CAO, chlorophyllide a oxygenase; CHLG, chlorophyll synthase; NYC1, non-yellow colouring 1; NOL, NYC1-like; SGR, stay green gene; HO, heme oxygenase-1. Plastid ribosomal proteins (PRPs) are involved in the assembly of chloroplast structure and have great significance in chloroplast division and formation (Zhang et al., 2016). PRPs are highly conserved in chloroplasts and are indispensable for chloroplast development (Tiller and Bock, 2014). Lacking PRPs, the maize mutant lem1 and hcf60 both exhibited a lethal phenotype, whereas the tobacco mutant prps18 showed chloroplast development defects (Schultes et al., 2000;Ma and Dooner, 2004;Rogalski et al., 2006). The PRPs in A. thaliana are involved in many biological processes, such as leaf development, photosynthesis, and lowtemperature response (Zhang et al., 2016). In rice, many PRPs mutants, such as asl1, asl2, and al1, cannot develop into fully functional chloroplasts, leading to the development of albino leaves (Gong et al., 2013;Lin et al., 2015;Zhao et al., 2016). PRPs are necessary for chloroplast development under low temperatures (Song et al., 2014;Wang et al., 2017a). In the rice mutant wgl2, a single-base mutation (G to T) in the PRP gene results in defects in chloroplast development. Then, the leaves showed an albino phenotype and reduced contents of Chl and carotenoid (Qiu et al., 2018). In addition, other genes related to chloroplast development have been identified in previous studies, such as V1, V2, V3, St1, GRY79, and YCL1. These genes have a function similar to that of AtGLK indirectly regulate the function of chloroplasts (Kusumi et al., 2011;Sugimoto et al., 2007;Yoo et al., 2009;Wan et al., 2015;Zhou et al., 2013b). Furthermore, YL1 and WP1 in rice are indispensable for early chloroplast development (Chen et al., 2016;Wang et al., 2016a). Studies have shown that the members of Accumulation and Replication of Chloroplast (ARC) gene family cooperate with FtsZ protein to regulate the division of chloroplasts (Osteryoung and Nunnari, 2003;Maple and Moller, 2007). In this family, ARC3, ARC5, and ARC6 are key regulators for chloroplast development (Gao et al., 2003;Shimada et al., 2004;Vitha et al., 2003).

Key Transcription Factor and Non-coding RNAs Regulate Leaf Colour Mutation
In the process of plant pigment synthesis, many coding RNAs are involved. For example, structural genes encode various enzymes in the pigment synthesis pathway, which directly determine the accumulation or degradation of pigments. TFs regulate pigment synthesis by binding to cis-acting elements in their target gene promoters to induce or inhibit the expression of structural genes (Wang et al., 2016b;Kim et al., 2017). In the anthocyanin synthesis pathway, MYB TFs can combine with bHLH and WD40 proteins to form the MBW protein complex. The MBW protein complex is the core component of anthocyanin synthesis regulation that can directly control the key enzymes of anthocyanin synthesis pathway, such as ANS, DFR, and F3′H (Tohge et al., 2005;Gonzalez et al., 2008) (Figure 2).
In addition to the role of coding RNAs, many non-coding RNAs (ncRNAs) participate in the regulation of pigment synthesis (Li et al., 2019b). Moreover, several lncRNAs and miRNAs related to pigment synthesis have been identified along with genes mainly involved in the anthocyanin synthesis pathway Wu et al., 2019) (Figure 2). Through the study of non-coding RNAs in C. sinensis (Jeyaraj et al., 2017), csn-miRn27, csn-miRn49, csn-miRn56, and csn-miRn23 were found to be co-targeted to the F3′5′H gene and participated in the synthesis of anthocyanin and in the accumulation of flavonoids. Furthermore, the study confirmed that csn-miRn70 and csn-miRn30 target F3H and UFGT genes, respectively, to jointly regulate the accumulation of anthocyanin in new leaves (Jeyaraj et al., 2017). Previous studies have shown that miR156 interferes with the function of the MBW protein complex by targeting SPL9 and inhibits the synthesis of anthocyanin (Gou et al., 2011). This mechanism has been verified in a variety of plants (Liu et al., 2017;He et al., 2019). It is worth mentioning that miR156-SPL is also involved in plant stress response (Wang et al., 2013b;Stief et al., 2014), synthesis of secondary metabolites (Ye et al., 2020), and floral organ development (Wang et al., 2009b).
By comparing the gene expression profiles of different varieties of roses, five miRNAs (miR171, miR166i, miR159e, miR845, and miR396e) were found to be enriched only in white flowers of rose, suggesting that these miRNAs may negatively regulate the expressions of downstream genes. Thus, the accumulation of carotenoids or anthocyanins is hindered, resulting in the development of white flower in rose (Kim et al., 2012). The analysis of miRNAs in Malus pumila showed that the R2R3-MYB TF gene involved in anthocyanin synthesis is the target gene of miR858 (Xia et al., 2012). Most MYBs are common target genes of miR828 and miR858, indicating that miR828 and miR858 play a vital role in anthocyanin synthesis (Guan et al., 2014). Interestingly, Wang et al. (2016b) found that miR858a positively regulates anthocyanin synthesis by inhibiting the expression of MYBL2. Mutant dg is a dark green mutant in A. andraeanum, whose leaves are thicker than the wild-type and whose petioles have turned red. The back of the leaf veins of the mutant changed from green to red, because of the enhanced pigment synthesis due to the expression of the mutant dg (Xu et al., 2006;Yang et al., 2015). Jiang et al. (2018) identified 10 differentially expressed miRNAs through a comparative analysis of the miRNA sequencing results of the dg mutant and the wild-type. Aa-miR408 was significantly upregulated in the dg mutant, suggesting that Aa-miR408 may be closed to the colour mutation of the dg mutant. Recently, Wu et al. (2020) screened a total of eight up-regulated miRNAs from the yellow leaf mutant of G. biloba. Among them, the novel 158_mature is involved the synthesis of lutein through the regulation of the expression of the target gma-miR2118a-3p gene. Moreover, three miRNAs (novel 151_mature, ptc-miR396e-3p, and aly-miR156a-5p) were the key regulators of leaf colour mutation (Wu et al., 2020).
LncRNAs are small RNA molecules with lengths greater than 200nt and no protein-coding ability (Laurent et al., 2015). LncRNAs are widely distributed in plants, and many have been identified in A. thaliana (Liu et al., 2012a), Z. mays (Lv et al., 2016), Salvia miltiorrhiza , and Populus euphratica (Liu et al., 2018). For the synthesis of pigments also involves the regulation of multiple lncRNAs (Wu et al., 2019). Previous studies showed that lncRNAs perform their functions by interacting with miRNAs .
Two differentially expressed lncRNAs (LNC1 and LNC2) were screened through the transcriptome analysis of Hippophae rhamnoides fruits at different maturation stages. Transient expression experiments verified that LNC1 positively regulates SPL9 expression by interacting with miR156 and promotes anthocyanin synthesis by facilitating the stability of the MBW protein complex. On the contrary, LNC2 interacts with miR828, and affects the expression of MYB114 to regulate anthocyanin synthesis . However, for tomato, the accumulation of lycopene was significantly reduced in the lncRNA1459 mutant, leading to the delay in fruit ripening (Li et al., 2018c).

Leaf Colour Mutation Caused by Environmental Factor
The mechanism of plant leaf colour mutation is extremely complex. It is regulated by internal genes and affected by the external environment, which includes temperature and light. Temperature is critical to the formation of leaf colour in plants. In C. sinensis, the appearance of albino buds is controlled by temperature, and the synthesis of Chl a and b is inhibited under low temperature (≤15 ℃), leading to albino buds. However, when the albino buds were cultured at a high temperature (≥15 ℃), the process of Chl synthesis was restored, and the leaves turned green (Du et al., 2008). Mutants that exhibit different leaf colour changes at various temperatures are known as temperature-sensitive leaf colour mutants. Previous studies have identified temperature-sensitive leaf colour mutants in plants, such as O. sativa (Huang et al., 2011), B. oleracea (Zhou et al., 2013c), Z. mays (Pasini et al., 2005), and T. aestivum (Liu et al., 2012b). Studies on wheat mutant fa85 showed that with the extension of low temperature treatment time, the aboveground leaves of fa85 completely bleared and gradually turned green increasing temperature (Liu et al., 2012b). Results of comparisons between mutant fa85 and its wild-type Aibian showed that the ultrastructure and molecular genetic characteristics of fa85 are affected by low temperature treatment. Meanwhile, proteomics analysis indicated the presence of significant differences in the expression patterns of chloroplast protein between fa85 and its parent Aibian at low temperatures (Hou et al., 2009).
Temperature regulates the synthesis and accumulation of pigments by affecting gene expression, thereby controlling the features of leaf colour. For example, the Chl-deficient leaf in rice at low temperatures is caused by a mutation of the NUS1 gene (Kusumi et al., 2011). In tomato, the WV gene, which controls the yellowing phenotype, is sensitive to low temperature. Therefore, the leaves expressed an albino phenotype at low temperature (Gao et al., 2019). The type and content of pigments of the temperature sensitive mutant mt of Commelina purpurea changed under different temperature conditions. At low temperature, the anthocyanin content in the leaves reached its peak, the Chl and carotenoid contents were significantly reduced. Thus, the leaves expressed a pink phenotype. At room temperature (25 ℃), no significant difference was found between the mutant mt and the wild-type. The anthocyanin content decreased, whereas Chl content increased. Further experiments suggested that the expressions of structural genes (such as PAL, CHS, CHI, F3'H, F3'5'H, DFR, ANS, UFGT, and OMT) related to anthocyanin synthesis were induced at low temperatures, leading to the excessive accumulation of cyanidin, pelargonidin, delphinidin, and petunidin. Thus, the leaves presented a pink phenotype. Meanwhile, the chloroplast in mutant mt was replaced by leucoplast at low temperature, and this mutant could not accumulate Chl (Liu et al., 2016b). A few temperature-sensitive leaf colour mutants exhibited multiple leaf colour changes at different temperatures. For example, the leaves of mutant tsc1 showed albino, virescent, and green phenotypes at 23.0 ℃, 26.0 ℃, and 30.0 ℃, respectively (Dong et al., 2001).
Besides temperature, light also regulates the phenotype of leaves (Biswal et al., 2012). Studies showed that the expression of golden leaf in plants is affected by environmental light intensity. Under high light conditions, the leaf colour turned golden, whereas in low light, the leaf colour was yellow-green due to the increase in Chl content (Hu et al., 2007). Light can promote the differentiation of non-photosynthetic plastids into fully functional chloroplasts, thereby affecting the development of chloroplasts and the expression of genes 9 related to Chl synthesis (Su et al., 2012). Guo et al. (2013) found that the light regulates the expression of the CPO gene, which encodes an enzyme that catalyses the oxidative decarboxylation of Coprogen III to ProtoIX, resulting in light-dependent yellow leaves of tobacco and A. thaliana. In contrast, in Hordeum vulgare, high light leads to slow growth of mutant nyb and turns its leaves yellow (Yuan et al., 2010). The leaf colour of mutant gl1 in L. indica is regulated by light intensity (Wang et al., 2017b). The leaf colour of CPO deletion mutant line2 in A. thaliana is influenced by day-length. The leaves are yellow-green under long-day conditions, and leaves are yellow under short-day conditions (Ishikawa et al., 2001).

Leaf Colour Mutation Regulated by the Mevalonate Pathway
Terpenoids are the most abundant secondary metabolites in organisms (Sacchettini and Poulter, 1997). Terpenoids, also known as isoprene compounds, participate in various plant life activities, such as photosynthesis (Chl and carotenoids), growth (phytosterols) and development (GA and ABA), and plant defence responses (Phillips et al., 2008). As one of the pathways involved in the synthesis of terpenoids, the mevalonate pathway (MEP) pathway is catalyzed by multiple enzymes, and its final synthesis products are IPP and DMAPP (Samad et al., 2019). IsPF (MDS), the fifth synthetase in the MEP pathway, catalyses the cyclization reaction of CDP-MEP to generate ME-cPP. The IsPF gene is also involved in the regulation of leaf colour in plants (You et al., 2020). In the rice yellow-green leaf mutant 505ys, the IsPF gene has a base substitution (C to T), thereby changing the encoded amino acid. Moreover, the overexpression of the wildtype OsIsPF gene in the mutant can restore the phenotype of mutant 505ys, proving that the IsPF gene is the cause of leaf colour variations in mutant 505ys (Huang et al., 2018). qRT-PCR results of key genes in the Chl synthesis pathway of rice mutant indicated that YGL gene expression in the mutant 505ys significantly declined, suggesting the existence of a positive regulation between the OsIspF and YGL gene (Huang et al., 2018).
In A. thaliana, IspF T-DNA insertion mutant and IspF RNAi mutant showed albino phenotypes with extremely low Chl and carotenoid contents (1% and 2% of the contents in the wild-type, respectively). Further ultrastructure analysis results showed that chloroplast development was inhibited in the mutant, and thylakoids were replaced by numerous vesicles (Hsieh and Goodman, 2006). Therefore, the mutant 505ys possibly did not appear with the albino phenotype, because single-base mutation could not completely replace the function of the IspF gene, thereby further verifying the key role of the IspF gene in the development of plant leaf color. Similarly, the IspE gene on chromosome 1 of the rice mutant gry340 has base substitutions, resulting in the yellow-green leaf phenotype . Through the studies on A. thaliana and tobacco, IspD, IspH, IspG, DXS, DXR, and IspE genes were verified to have functions similar to those of the IspF gene. These genes are at the key cores of plant leaf mutation, pigment reduction, and thylakoid structure destruction (Mandel et al., 1996;Estevez et al., 2000;Budziszewski et al., 2001;Gutierrez et al., 2004;Guevara et al., 2005;Hsieh and Goodman, 2006;Xing et al., 2010;Hsieh et al., 2008;Ahn and Pai, 2008). During the ripening process of tomatoes, the transcription level of the DXS gene significantly increases, and a large amount of carotenoids accumulates, further promoting the colouring of tomato fruits at the ripening stage (Lois et al., 2000). Zhang et al. (2019) found that the albino leaves of the maize mutant scd was caused by a mutation of the IspH (HDS) gene in the MEP pathway. Moreover, the decreased activity of the key enzymes of the MEP pathway indirectly affects the accumulation of downstream products, such as carotenoid and Chl.

Conclusions
As a visible mutant, leaf colour mutant is an ideal material that can be used for plasmid development and photosynthesis. Also, leaf colour mutant has an important research value. Leaf colour mutation in higher plants is mostly related to the content changes of Chl and anthocyanin. The regulation mechanism of leaf colour mutation is extremely complicated. It involves the enzymes of pigment synthesis and is affected by chloroplast structure, the regulation of TFs, small RNAs, the interaction between plants and external environment, and the regulation of the plant secondary metabolite synthesis pathway. Although many studies have been conducted on leaf colour mutants, most of them reported the role of key Chl synthesis genes and chloroplast structure. Moreover, most of the studies used big data joint analysis methods, such as transcriptome, proteome, and metabolome. Few studies have been conducted on the upstream regulatory mechanism of the vital genes related to leaf colour mutation, such as the functions of miRNA and lncRNA in leaf colour mutation, which needs to be further analysed. At present, most miRNA and lncRNA studies on plant colour regulation focus on fruit and flower colour. Few studies on small RNA include leaf colour formation. Furthermore, the development of leaf colour involves the interaction of nuclear coding and chloroplast genes. However, studies on the plastid-nuclear reverse signal pathway have been slow, and the regulation process, and regulation molecular mechanisms are still unclear. Therefore, follow-up research works should focus on these two centres and maximize the advanced means of molecular biology to further analyse the regulation mechanism of leaf colour mutation, which would serve as the theoretical foundation for the improvement of leaf colour varieties of more plants.
Authors' Contributions M.Y.F. and F.X. designed and wrote the manuscript; S.Y.C., W.W.Z., J.R.Z., and L.W. collected and analysed the data; Z.X.C. and Z.B.L. revised the manuscript. All authors read and approved the final manuscript Hou DY, Xu H, Du GY, Lin JT, Duan M, Guo AG (2009 Tian X, Ling Y, Fang L, Du P, Sang X, Zhao F, ... He G (2013). Gene cloning and functional analysis of yellow green leaf3 (ygl3) gene during the whole-plant growth stage in rice. Genes & Genomics 35 (1)