Light regulation of the biosynthesis of phenolics, terpenoids, and alkaloids in plants

Biosynthesis of specialized metabolites (SM), including phenolics, terpenoids, and alkaloids, is stimulated by many environmental factors including light. In recent years, significant progress has been made in understanding the regulatory mechanisms involved in light-stimulated SM biosynthesis at the transcriptional, posttranscriptional, and posttranslational levels of regulation. While several excellent recent reviews have primarily focused on the impacts of general environmental factors, including light, on biosynthesis of an individual class of SM, here we highlight the regulation of three major SM biosynthesis pathways by light-responsive gene expression, microRNA regulation, and posttranslational modification of regulatory proteins. In addition, we present our future perspectives on this topic.

HY5 is a key regulator of light-induced anthocyanin biosynthesis in various plant species, and it acts downstream of multiple photoreceptors, such as PHY, CRY, and UVR8, in the signal transduction pathways 37 .In the presence of light, the activated photoreceptors inhibit COP1/SPA activity by distinct regulatory mechanisms, leading to the stabilization of HY5 38 .HY5 activates the expression of anthocyanin pathway genes, such as CHS, DFR, and LDOX, as well as regulatory genes (such as MYBs) by directly binding to the G-box or ACE (ACGT-rich elements) motifs in the promoters of the target genes.In Arabidopsis, HY5 regulates anthocyanin biosynthesis directly by binding to the promoters of pathway genes and indirectly by activating the expression of PAP1 (MYB75) 39 and the MYB-like domain TF MYBD 40 .HY5 binds to the MYBD and PAP1 promoter to induce expression in response to light.MYBD or PAP1 overexpression induces the expression of anthocyanin pathway genes such as DFR and ANS.MYBD represses the activity of MYBL2, a negative regulator of anthocyanin biosynthesis, resulting in the promotion of anthocyanin biosynthesis 40 (Fig. 2b).Similar to Arabidopsis, in apple (Malus domestica), MdHY5 stimulates anthocyanin biosynthesis by inducing the expression of MdMYB10 and MdMYBDL1, and HY5 directly bind to the G-box motifs in these MYB promoters 41,42 .In apple, MdMYB1/10 are PAP1-like MYBs and components of the apple MBW complex, and MYBDL1 is closely related to Arabidopsis MYBD.MdHY5 and MdMYBDL1 inhibit the expression of MdMYB16 and MdMYB308, which are repressors of anthocyanin accumulation.Additionally, MdMYB308 interacts with MdbHLH3 and MdbHLH33, components of the apple MBW complex.MdMYB308-MdbHLH3/33 interaction likely affects MBW complex formation and influences anthocyanin accumulation in response to light 42 (Fig. 2c).Exposure to UV-B induces anthocyanin accumulation in apple.Recent studies identified two UV-B responsive WRKY TFs, MdWRKY71-L and MdWRKY72, that promote anthocyanin accumulation in apple by inducing the expression of MdHY5 and MdMYB1 43,44 .In blood orange (Citrus x sinensis), HY5 regulates the expression of the MYB TF Ruby to promote anthocyanin accumulation 45 .In peach (Prunus persica), PpHY5 regulates the expression of biosynthetic genes, such as PpCHS1, PpCHS2, PpDFR1, and regulatory gene PpMYB10.1 to induce anthocyanin accumulation 46 .In red-skinned pears (Pyrus pyrifolia), PyHY5 directly binds to the G-box element in the promoters of PyMYB10 and PyWD40, thereby enhancing their expression and resulting in increased anthocyanin accumulation 47 .SlHY5 regulates bluelight-induced anthocyanin accumulation by directly binding to the CHS and DFR promoters in tomato 37 .
In Arabidopsis, PIF3 positively, while PIF4 and PIF5 negatively, regulate anthocyanin biosynthesis in response to different light conditions (Fig. 2b).HY5 acts synergistically or antagonistically with PIFs to regulate different biological processes.Overexpression of PIF3 (PIF3-OE) increases anthocyanin levels under far-red light, while pif3 mutants exhibit reduced anthocyanin accumulation.However, the anthocyanin level in the hy5 mutant overexpressing PIF3 (PIF3-OEhy5) is similar to that in the hy5 single mutant, suggesting that the positive effects of PIF3 require a functional HY5.Indeed, PIF3 and HY5 activate the same set of genes in the anthocyanin biosynthetic pathway, and PIF3-mediated activation is dependent on HY5.However, PIF3 and HY5 do not compete for the same binding motif.PIF3 binds to the G/E-box, while HY5 binds to ACE motifs in the promoters of anthocyanin pathway genes 11 .In Arabidopsis, red light induces anthocyanin accumulation, which is enhanced in pif4 and pif5 mutants but suppressed in PIF4-OE and PIF5-OE plants.Consistent with anthocyanin phenotype, the expression of genes encoding anthocyanin pathway enzymes and regulators, such as CHS, F3′H, DFR, LDOX, PAP1, and TT8, is increased in pif4 and pif5 mutants but decreased in PIF4 and PIF5-OE plants.Additionally, PIF4 and PIF5 repress red lightinduced promoter activities of DFR and F3'H, and PIF5 binds to the G-box motif in the DFR promoter to repress its expression.These findings collectively suggest that PIF4 and PIF5 act as negative regulators of red light-induced anthocyanin accumulation 48 .A recent study demonstrated that PIF4 binds to the G-box motif in the PAP1 promoter to repress its expression and negatively regulates anthocyanin biosynthesis in response to white light 49 .Anthocyanin accumulation is also suppressed in response to other abiotic factors such as salt and phytohormone BA (6-benzylaminopurine).It has been demonstrated that PIF4 interacts with the R2R3 MYB TF PAP1 and competes with the bHLH TF TT8 for binding to PAP1, thus inhibiting the formation of the MBW complex.This mechanism is likely involved in PIF4 and PIF5 inhibition of anthocyanin accumulation in red light 50 .The function of HY5 in PIF4/5-mediated anthocyanin biosynthesis is not explored in these studies.PIFs also negatively regulate anthocyanin accumulation in other plants such as apple and pear.PIF8 positively regulates anthocyanin biosynthesis in pear skin and calli by activating the expression of CHS 51 .In apple, MdPIF7 is a negative regulator, while MdHY5 and MdBBX23 are positive regulators of anthocyanin biosynthesis.MdHY5 is regulated by MdBBX23.MdPIF7 interacts with MdBBX23 and attenuates the transcriptional activation of MdBBX23 on MdHY5, thereby repressing anthocyanin accumulation.In apple MdbZIP44 and MdERF38 interact with MdMYB1 to regulate anthocyanin biosynthesis.MdPIF7 interacts with MdERF38 and MdbZIP44 and weakens the formation of the MdERF38/MdbZIP44-MdMYB1 complex to affect anthocyanin accumulation (Fig. 2c) 52 .
The B-box (BBX) proteins regulate anthocyanin biosynthesis in response to light either independently or by interacting with HY5 53,54 .In Arabidopsis, anthocyanin biosynthesis is positively regulated by BBX21/22/23 18,55 but generally negatively regulated by BBX24/25 56 (Fig. 2b).In apple (Malus domestica), while a group of light-inducible BBX proteins (BBX1/20/21/22/33) promote anthocyanin biosynthesis by regulating the expression of HY5 and MYB1/10 57,58 , MdBBX37 inhibits anthocyanin biosynthesis through two different mechanisms (Fig. 2c).MdBBX37 interacts with MdMYB1 and MdMYB9, preventing them from binding to the promoters of the anthocyanin pathway genes.Alternatively, MdBBX37 binds to the MdHY5 promoter to repress its expression 59 .Similar to apple, a number of BBX proteins regulate anthocyanin accumulation in pear (Pyrus pyrifolia).PpBBX16 and PpBBX18 interact with PpHY5 to activate the expression of PpMYB10 and anthocyanin pathway genes to regulate anthocyanin accumulation.PpBBX21, on the other hand, acts as a negative regulator by inhibiting the formation of the PpBBX18-PpHY5 complex, thus repressing anthocyanin biosynthesis 60,61 .In strawberry (Fragaria x ananassa), FaBBX22 interacts with FaHY5 to enhance anthocyanin accumulation, and the interaction upregulates the expression of anthocyanin biosynthetic genes such as FaPAL, FaANS, FaF30H, and FaUFGT1, as well as the transporter FaRAP, in a lightdependent manner 62 .In summary, light-induced anthocyanin biosynthesis is regulated by a well-conserved group of TFs, i.e.PIFs, HY5, BBX, and MYBs in Arabidopsis and their homologs in other plant species, such as apple, pear, and strawberry.In addition, other regulators, such as WRKYs and the AP2/ERFs, have been identified in apple as the regulators of anthocyanin biosynthesis.
MicroRNAs (miRNAs) are a class of endogenous small RNAs, typically 21 or 22 nucleotides in length, found in both animals and plants 63 .They are important mediators of posttranscriptional gene regulation that fine-tune many developmental and metabolic processes in plants.MiRNAs are known to regulate biosynthesis of SM, including anthocyanins in Arabidopsis and other plant species 28,29,[64][65][66] , terpenoid indole alkaloids (TIAs) in Catharanthus roseus 26 , and sesquiterpenes in Arabidopsis 67 .In this context, we will specifically discuss the influence of light on miRNA regulation of anthocyanin biosynthesis in plants.
In Arabidopsis, the expression of miR858a, a positive regulator of anthocyanin biosynthesis, is induced by light.Light-induced expression of miR858a is HY5-dependent, and HY5 binds to the miR858a promoter to regulate its expression.Overexpression of miR858a partially complements the long hypocotyl phenotype of the hy5 mutant and rescues the anthocyanin levels in the mutant.Additionally, overexpression of miR858a in wild-type Arabidopsis significantly induces expression anthocyanin pathway genes, such as DFR, ANS, TT8, GL3, and increases anthocyanin accumulation 68 .miR858a and HY5 regulate the activity of MYBL2, a negative regulator of anthocyanin, by translational inhibition and chromatin modification.Overall, multiple regulatory mechanisms involving miRNA858a and HY5 regulate the light-induced anthocyanin accumulation in Arabidopsis.In apple, light induces anthocyanin accumulation but reduces lignin level.A recent study has demonstrated that the inverse correlation between anthocyanin and lignin accumulation is mediated by the light-induced miRNA7125 69 .RNAseq and miRNAseq analyses, combined with experimental data, suggest that cinnamoyl-coenzyme A reductase gene (CCR), a key enzyme in the lignin pathway, is the target of miRNA7125.Additionally, both the activator MdMYB1 and the repressor MdMYB16 of anthocyanin biosynthesis bind to the miRNA7125 promoter to regulate its expression.The interplay among MdMYB1/MYB16, CCR, and miRNA7125 maintains the homeostasis between anthocyanin and lignin in response to light 69 .In red Chinese sand pear (Pyrus spp.), light-induced anthocyanin accumulation is regulated by the PyPIF5-PymiR156a-PySPL9-PyMYB114/ MYB10 module.PyMYB114 and PyMYB10 are components of the MBW complex that positively regulates anthocyanin biosynthesis.In contrast, pySPL9 and PyPIF5 negatively regulate anthocyanin biosynthesis.PySPL9 is a target of PymiR156, and it interacts with PyMYB114/MYB10, inhibiting the MBW complex formation and affecting anthocyanin accumulation.PyPIF5 regulates the PymiR156 expression by binding to the G-box motif in the promoter.Light induces the expression of PymiR156 but reduces that of PyPIF5.The light-induced upregulation of miRNA156 results in the degradation of PySPL9, leading to higher anthocyanin accumulation.In addition, overexpression of miR156 induces anthocyanin accumulation in pear fruit skin.Collectively, these studies underscore the importance of miRNAs in light-induced anthocyanin accumulation (Fig. 3a) 70 .
Light plays a crucial role in plant adaptation to changing environments and is perceived by photoreceptors that transduce different wavelengths of light into physiological responses through intracellular signaling pathways 71 .The protein kinases (PKs) are major components of various signaling pathways including light.PKs regulate many biological processes in plants, including growth and development, phytohormone signaling, and responses to abiotic and biotic factors.PKs are activated in response to various signals and phosphorylate diverse substrates, including TFs and enzymatic proteins, to induce appropriate responses.In Arabidopsis, PAP1 is a component of the MBW complex that positively regulates anthocyanin biosynthesis.The mitogen-activated protein kinase 4 (MAPK4) interacts with PAP1 to increase its stability, which is essential for anthocyanin accumulation in response to light 72 .In apple, MPK4 has been reported to mediate the phosphorylation of MYB1 to enhance light-induced anthocyanin accumulation 73 .Analysis of apple fruit phosphoproteome in response to light identified HY5 to be differentially phosphorylated.Protein-protein interaction and phosphorylation assays revealed that MPK6 phosphorylates HY5 and its activity is increased by light, which is crucial for increased anthocyanin accumulation 74 .Apart from MPKs, other groups of kinases also regulate anthocyanin biosynthesis.In Arabidopsis, the Sucrose non-fermenting Related Kinase 1 (SnRK1) represses anthocyanin biosynthesis at both transcriptional and post-translational levels.SnRK1 inhibits the expression of PAP1, triggering the dissociation of the MBW complex and the degradation of PAP1 (Fig. 3b) 75 .
In Arabidopsis, anthocyanin accumulation is induced by light and suppressed in the dark.COP1 and SPA are components of the E3 ubiquitin ligase complex, and are involved in the dark suppression of anthocyanin accumulation.E3 ubiquitin ligases are components of the 26 S proteasomal degradation machinery.The cop1 mutant accumulates anthocyanin in the dark when a functional PAP1 is present.Protein-protein interaction studies indicated that PAP1 and PAP2 interact with COP1 and SPA1, and PAP1 and PAP2 proteins are degraded in the dark but stable under light.In addition, PAP2 degradation is dependent on the 26 S proteasome and COP1 76 .Similarly, in apple, MdMYB1, a homolog of PAP1 and positive regulator of anthocyanin, is accumulated under light but degraded in the dark.MdCOPs interact with MdMYB1 and are necessary for the MYB1 degradation in the dark 77 .
Regulation of stilbene biosynthesis by light.Stilbenes, particularly trans-Resveratrol (t-Res), are phenolic compounds found in various plant species such as grapes (Vitis vinifera).t-Res has gained significant attention due to its roles in plant defense mechanisms and its potential health-promoting properties.Stilbenes are derived from the general phenylpropanoid pathway, and stilbene synthase (STS) catalyzes the conversion of three units of malonyl-CoA and one unit of p-coumaroyl CoA into t-Res in a single step.Res accumulation is induced by UV light (UV-B and UV-C).UV-C is more effective than UV-B in inducing stilbenes, including Res, in grapes.The regulation of Res in response to UV has been extensively studied in grape vines.R2R3 MYB (VvMYB14 and VvMYB30) and WRKY (WRKY8) TFs are involved in the regulation of Res biosynthesis in response to UV irradiation.VvMYB14 and WRKY8 are activators, whereas MYB30 is a repressor of Res biosynthesis.UV-B and UV-C irradiation induce the expression of the grape STS15/21, VvMYB14, and VvWRKY8 genes, but repress VvMYB30, suggesting that an activator-repressor module controls the accumulation of Res in response to UV 78 .

Regulation of carotenoid and artemisinin biosynthesis by light.
Terpenoids are synthesized from the five-carbon precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) 79 .They are classified based on the number of fivecarbon units in their skeletons, ranging from hemi-(C5) to tetra-(C40) terpenoids.Terpene synthase (TPS) is the key enzyme responsible for synthesizing the basic backbone structures.In nature, terpenoids serve various biological functions 80 , for instance, carotenoids function as photosynthetic pigments, while certain terpenoids emit volatile floral scents to attract the pollinators.Many terpenoids are defense molecules against pathogens.In addition, various terpenoids possess therapeutic properties and have been used in the treatment of various diseases.For example, artemisinin, produced by Artemisia annua (sweet wormwood), is a well-known effective antimalarial drug.Light plays a crucial role in regulating biosynthesis and accumulation of terpenoids in many plant species [81][82][83][84] .Here, we highlight the roles of light on in regulation of the biosynthesis of two terpenoids, carotenoids and artemisinin.
Carotenoids are tetra-terpenoids widely distributed in plants and are responsible for the red, orange, or yellow colors observed in flowers, fruits, and roots 85 (Fig. 1).In addition to their roles in pigmentation, carotenoids serve as essential photosynthetic pigments in chloroplasts.The primary function of carotenoid is to protect the photosynthetic apparatus from photooxidative damage caused by excessive illumination 85,86 .The biosynthesis of carotenoids is intricately regulated by light and is closely associated with the development of chloroplasts 87,88 .Carotenoids biosynthesis starts with the formation of phytoene, which is produced from two molecules of geranylgeranyl pyrophosphate (GGPP) catalyzed by phytoene synthase (PSY) 85 .Phytoene undergoes a series of enzymatic steps, including desaturation, cyclization, and hydroxylation, resulting in the production of carotenoids with a wide range of colors (Fig. 4).Among the biosynthetic enzymes involved in carotenoid biosynthesis, PSY is considered a rate-limiting enzyme and its gene expression is regulated by light.Recent evidence suggests that the major light signaling factors, including PIFs, HY5, BBXs, COP1, and DET1, regulate the expression of genes involved in carotenoid biosynthesis and accumulation 88 .PIFs play a negative regulatory role in carotenoid biosynthesis by repressing the expression of PSY.The PIF-PSY regulatory module has been observed in many plant species, including Arabidopsis 89 , tomato 90 , and grape berry 91 .PIFs directly repress PSY in Arabidopsis and tomato fruit.In contrast, HY5 positively regulates carotenoid biosynthesis in both Arabidopsis 12 and tomato 92,93 .In tomato fruit, HY5 directly regulates multiple carotenoid biosynthetic genes, including PSY, Z-ISO (15-cis-zeta-carotene isomerase), CrtISO1 (carotene isomerase 1), LCYB (lycopene beta-cyclase), LCYE (lycopene epsilon-cyclase), and VDE (violaxanthin de-epoxidase), indicating a broader regulatory role for HY5 on the carotenoid pathway compared to PIFs.Both PIF1 and HY5 directly bind to the G-box in the PSY promoter in Arabidopsis, and these two proteins exhibit antagonistic effects on PSY expression, highlighting their opposing roles in carotenoid biosynthesis 12 .BBX proteins have also been implicated as regulators of carotenoid biosynthesis in tomato 19 and pepper 94 .However, their regulatory roles appear to be limited to specific carotenoid biosynthetic genes, such as PSY in tomato and CCS (capsanthin-capsorubin synthase) in pepper.Silencing of COP1 or DET1 significantly enhanced the accumulation of carotenoids in tomato 92,95 , suggesting the negative roles of both light-regulated E3 ubiquitin ligases in carotenoid metabolism (Fig. 4a).
The regulation of carotenoids in response to light involves not only the core light signaling regulators, but also other TFs that play important roles in various plant species.In papaya (Carica papaya), two bHLH TFs, CpbHLH1 and CpbHLH2, are induced by light during fruit ripening and activate carotenoid biosynthetic genes 96 .Similarly, during kumquat (Fortunella crassifolia) fruit ripening, a NAC (NAM/ATAF/CUC2) family TF gene, FcrNAC22, is upregulated by light, triggering the expression of carotenoids pathway genes 97 .However, the relationships of these bHLH and NAC TFs with the core light signaling components need further investigation and verification.
The development of GSTs in A. annua leaves is crucial for the accumulation of artemisinin.Recent research has shed light on the regulatory mechanisms governing GST development and initiation, particularly involving the AaCOP1 protein 104 .In the darkness, AaCOP1 interacts with and facilitates the degradation of AaSEP1, a positive regulator of GST initiation.The AaSEP1-AaMYB16-AaHD1 regulatory complex regulates GST initiation 104,105 .AaHD1 directly activates the expression of AaGSW2, an activator of GST initiation 106 .AaMYB16 enhances the activation of AaHD1 on the AaGSW2 promoter through their interaction, and the interaction between AaSEP1 and AaMYB16 further strengthens this activation process.AaCOP1 not only regulates the protein level of AaSEP1 but also influences the transcript level of AaSEP1 in response to light.The specific mechanism through which light induces the transcription of AaSEP1 remains unknown 104 .
Regulation of the biosynthesis of alkaloids and nitrogencontaining metabolites by light.Alkaloids, which are nitrogencontaining compounds, have been studied extensively for their functions in plant defense against pathogens and insects.Different types of alkaloids and nitrogen-containing metabolites, such as terpenoid indole alkaloids (TIA) in Catharanthus roseus (Madagascar periwinkle) 107 , steroidal glycoalkaloids (SGA) in Solanum lycopersicum (tomato) [108][109][110] , and glucosinolates (GSL) in Arabidopsis 111 , have been the focus of research for their roles in plant defense and benefits in human health.While the biosynthesis and regulation of these SM by the phytohormone jasmonate acid (JA) have been a major focus of many studies 112 , recent research has highlighted the regulatory roles of light in the biosynthesis of GSLs 113 , alkaloids including vindoline in C. roseus 114 , camptothecin in Camptotheca acuminata (Chinese Happy Tree) 115 , and SGAs in tomato (Fig. 1) 116 .
C. roseus is the source of more than 130 TIAs including the anticancer drugs vincristine and vinblastine 117 .TIA biosynthesis is a highly complex process involving more than 30 enzymes and 20 regulatory proteins.The biosynthesis of TIAs starts with the condensation of tryptamine, derived from the indole branch of the pathway, and secologanin, derived from the iridoid branch, to form strictosidine, which is the universal precursor for all TIAs, including ajmalicine, serpentine, tabersonine, catharanthine, and vindoline (Fig. 5) 118,119 .Vincristine and vinblastine are derived from the condensation of vindoline and catharanthine.Vindoline is preferentially accumulated in the aerial parts of the plant and is induced by light in a phytochromedependent manner 120 .The biosynthesis of vindoline from tabersonine involves seven enzymatic steps 121 , and the expression of five of the seven genes, including T16H2 (tabersonine 16hydroxylase), T3O (tabersonine 3-oxidase), T3R (tabersonine 3reductase), D4H (desacetoxyvindoline-4-hydroxylase), and DAT (deacetylvindoline 4-Oacetyltransferase) is induced by light 114 .A recent study has revealed that the core components of the light signaling influence vindoline biosynthesis in C. roseus.CrPIF1 is a negative regulator of vindoline biosynthesis while the GATAtype zinc finger family TF, CrGATA1, acts as a positive regulator 114 .Specifically, CrPIF1 represses the expression of CrGATA1, T16H2, and DAT, whereas CrGATA1 activates the expression of the five vindoline biosynthetic genes that are responsive to light 114 .It has been suggested that the CrPIF1 suppresses vindoline biosynthesis in the dark, and the degradation of CrPIF1 upon exposure to light triggers the induction of vindoline biosynthesis by CrGATA1 (Fig. 5a) 114 .
Camptothecin is a TIA produced in C. acuminata and has shown antitumor activity against cancers in breast, ovarian, colon, lung, and stomach 122,123 .Camptothecin is accumulated in all tissues with the shoot apex and young leaf accumulating the highest levels 124 .In C. acuminata, camptothecin biosynthesis begins with the condensation of tryptamine and secologanic acid to produce strictosidinic acid (Fig. 5).The subsequent steps in camptothecin biosynthesis have not been fully elucidated 124 .
Although it has been known for some time that camptothecin production is negatively regulated by light, the regulatory mechanism remained unknown 125 .A recent research has revealed that light suppresses camptothecin biosynthesis by affecting the expression of four crucial camptothecin biosynthesis genes, CaTDC1, CaG8O, CaCYC1, and Ca7-DLS 115 .A bZIP TF, CaLMF (Light-Mediated camptothecin Biosynthesis Factor), has been identified as a regulator of the light-induced suppression of camptothecin biosynthesis.Light induces the expression of CaLMF, which represses the expression of four camptothecin biosynthetic genes (Fig. 5a) 115 .Although CaLMF shares significant sequence similarity with AtHY5, its functional role as a HY5-like protein requires further investigation.
Solanaceous species, such as tomato and potato, produce SGAs, which are toxic alkaloids involved in defense against biotic stress.In tomato, the major SGA is α-tomatine, which is highly accumulated in immature fruits to provide protection against pathogens and predators 108 .SGAs are derived from cholesterol through a series of enzymatic reactions, including hydroxylation, oxidation, transamination, and glycosylation.The enzymes catalyzing these steps are well characterized and known as GLYCOALKALOID METABO-LISM enzymes (GAMEs) (Fig. 5) 109,110 .SGA biosynthesis is light inducible as most of the GAMEs are upregulated in response to light exposure 126 .The core light signaling components, including the activator SlHY5 116,126 and the repressor SlPIF3 126 , regulate SGA biosynthesis.Specifically, SlHY5 directly activates the genes encoding GAME1, GAME4, and GAME17, while SlPIF3 acts as a repressor (Fig. 5a) 126 .These findings suggest that the tomato SGAs biosynthesis is tightly regulated by the core light signaling components.
GSLs are a diverse group of nitrogen and sulpfur-containing SM found in the Brassicaceae family plants including Arabidopsis 111,127 .GSLs play important roles in plant defense against various pests and diseases and have significant medicinal values 127,128 .They can be converted by intestinal bacteria into isothiocyanates, which have anti-inflammatory, anti-cancer, antiobesity, and neuroprotective properties 129 .Recent research has shed light on the molecular mechanisms that govern the regulation of two major types of GSLs, aliphatic and indolic GSL, which are derived from the amino acid methionine and tryptophan, respectively (Fig. 5).The effects of light on GSL biosynthesis have been studied in many plant species 113,130,131 .The indolic and aliphatic GSL biosynthetic pathways can be divided into three independent stages, i.e. chain elongation of the amino acids, construction of the GSL structure, and secondary modifications of the amino acid side chain 132 .Light upregulates six GSL biosynthetic genes, including three involved in the biosynthesis of aliphatic GSL (CYP79F1, MAM-L, and SOT17), two in indolic GSL (CYP79B2 and SOT16), and one associated with both types of GSL (SOT18) 113 .The expression of GSL pathway genes is regulated by a group of MYB TFs in Arabidopsis.The biosynthesis of aliphatic GSL is positively regulated by MYB28, MYB29, and MYB76, while that of indolic GSL is regulated by MYB34, MYB51, and MYB122 133,134 .The MYB genes associated with aliphatic GSL are induced by light and repressed in dark, while the expression patterns of those associated with indolic GSL vary in response to light 113 .Expression of MYB51 is repressed in the dark but not induced by light, whereas that of MYB34 is induced by light but not affected in the dark.MYB122 is induced in the dark but does not respond to light.Light regulation of GSL is partially dependent on HY5, which positively regulates CYP79F1, CYP79B2, and SOT18.Expression of all three aliphatic GSL-associated MYB28, MYB29, and MYB76 are strongly induced by light in the hy5 mutant compared to wild-type Arabidopsis, whereas that of indolic GSL-associated MYB34 and MYB122 are repressed, suggesting that HY5 may act as a repressor of aliphatic GSL and an activator of indolic GSL.Interestingly, although the aliphatic MYBs are strongly induced in the hy5 mutant, the degree of upregulation of the pathway genes are not significant, and, therefore, the hierarchy of MYBs and HY5 still needs to be established 113 .In addition, the BBX family TF AtBBX29 activates GSL biosynthesis in response to light, possibly acting through MYB34 and MYB51 135 .COP1, the negative regulator of light signaling pathway, plays a positive role in regulating GLS biosynthesis 134 .
In cop1 mutant lines, the reduced accumulation of both types of GSLs is consistent with downregulation of the aliphatic GSL biosynthetic gene CYP83A1 and the indolic GSL biosynthetic genes CYP79B3 and CYP83B1, as well as MYB34.However, the expression of MYB51 and MYB122 is upregulated in cop1 mutant lines.In contrast to the positive regulator HY5, some activators of GSL biosynthesis are negatively regulated by light, with COP1 playing a positive role in this regulation (Fig. 5b).These findings highlight the complexity of the regulatory network governing GSL biosynthesis in response to light.Conclusion and Future Perspective.The biosynthesis of SM is influenced by many environmental factors including light (Fig. 1).However, this process is energy-intensive, and excessive accumulation of certain SM can be toxic to cells.Consequently, plants have evolved intricate regulatory mechanisms to prevent overactivation and fine-tune SM biosynthesis.Accumulating evidence suggests that the regulations of most SM biosynthetic pathways are conserved across plant species.One example of such conservation is observed in the regulation of anthocyanin biosynthesis, which is controlled by the MBW complex, in a wide range of plants, including the model plant Arabidopsis.Another instance is the regulation of terpene biosynthesis, where the bHLH TF MYC2 or its orthologues govern the biosynthetic process from the eudicot Arabidopsis to the primitive land plant, Marchantia polymorpha 136 .Furthermore, this conserved nature extends to the biosynthesis of structurally diverse SM.For example, the biosynthesis of TIAs in C. roseus, SGA in tomato and potato, and nicotine in tobacco are regulated MYC2 and a group of phylogenetically related JA-responsive AP2/ERFs (Apetala 2/Ethylene Responsive Factors) [137][138][139][140][141] .The conserved nature of the regulatory mechanism is also apparent in the light signaling pathway, in which the core light signaling pathway TFs PIFs, HY5, and BBX regulate the biosynthesis of diverse groups of SM, including anthocyanins, terpenes, and TIAs in various plant species.Nonetheless, some regulatory factors seem to be speciesspecific, e.g., some TFs involved in anthocyanin biosynthesis are found in apples but not Arabidopsis (Fig. 2b, c), suggesting that some plant species have evolved unique responses to light.Light and JA are important regulators of many SM.However, the relationship between JA and light signaling pathways and their combined effects on the regulation of SM biosynthesis have been relatively less studied.A previous study has shown that JAinduced artemisinin biosynthesis is dependent on light.Subsequent transcriptomic analysis of JA and light-treated A. annua seedlings has identified several candidate TFs potentially involved in the regulation of artemisinin biosynthesis 142 .Recently, a light and JA-inducible WRKY TF, AaWRKY9 that acts downstream of AaHY5, has been identified as a positive regulator of artemisinin biosynthesis 100 .Similarly, AaMYB108, identified from light and JA-treated transcriptomes, is a positive regulator of artemisinin biosynthesis 103 .Future studies will likely identify factors involved in the light and JA-dependent regulation of other plant SM.
While recent years have seen significant progress in understanding photo-regulation of SM biosynthesis, some existing questions are still left unanswered.Compared to transcriptional regulation, light-mediated posttranscriptional and posttranslational regulation of SM are insufficiently studied.Identification and functional characterization of additional protein kinases and miRNAs related to SM biosynthesis will further advance our knowledge on regulatory mechanisms.Follow-up questions would thus concern why such multilayered regulatory mechanisms are necessary.Phosphorylation of the MBW components by different light-responsive kinases can lead to stabilization or disassociation of the complex (Fig. 3).How then do plants balance such countereffects by kinases in response to light?In addition, many TFs are not only regulated by phosphorylation but also de-phosphorylation by protein phosphatases (PPs).The involvement of phosphatases in the photo-regulation of SM biosynthesis is currently poorly investigated.Furthermore, literature on other posttranslational modifications, such as SUMOylation, related to photo-regulation of SM biosynthesis is limited.
Phosphorylation-dephosphorylation by PKs and PPs, respectively, is an ancient regulatory mechanism that influences many biological processes in plants and animals 143 .Although several PKs that phosphorylate PIFs have been identified, related information on PP is limited 3,144 .HY5 is shown to be phosphorylated by SPA kinases 145 .The regulatory roles of these PKs and PPs have been studied in respect to plant growth development 144,146 ; however, it remains to be explored whether the PKs and PPs are involved in light-regulated SM biosynthesis.
SUMOylation is the reversible conjugation of small ubiquitinrelated modifier (SUMO) peptides to the target protein and has emerged as an important posttranslational regulation mechanism in plants and animals 147 .SUMOylation has been implicated in many biological processes such as growth, development, biotic and abiotic stress, as well as phytohormone signaling and flowering.In Arabidopsis, SUMOylation regulates the stability of the bHLH TF ICE1 (Inducer of CBF Expression) by blocking its degradation which is required for activating freezing tolerance 148 .The bHLH TF MYC2 is crucial for Arabidopsis seedling development in blue light.Blue light induces the MYC2 SUMOylation, which is essential for MYC2 to function under blue light.SUMOylation increases the MYC2 stability and enhances its DNA binding ability.As MYC2 is also a key component of JA signaling and a regulator of many JA-responsive SM biosynthesis, future studies should include the investigation of crosstalk between light signaling and hormones, as well as SUMOylation with regard to SM biosynthesis 149 .In Arabidopsis, SUMOylation by the SUMO E3 ligase SIZ1 (SAP and MIZ1 domain containing ligase 1) increases PAP1 stability and is required for light-mediated anthocyanin accumulation 150 .It is reasonable to expect light-activated SUMOylation also affects other regulatory proteins; however, experimental verifications will be necessary.
Light affects the expression of approximately 20 percent of genes in the Arabidopsis and rice genomes 151 .The massive change in the transcriptomic landscape indicates that this process is modulated at chromatin level 152 .Plant epigenome is indeed dynamic, and extensive research reveals the effects of lightinduced epigenetic changes, such as histone methylation, histone acetylation/deacetylation, and incorporation of histone variants, on plant growth and development.The core components of the light signaling pathway, such as HY5 and PIFs, interact with and recruit epigenetic factors to regulate gene expression 153 .In addition to epigenome, epitranscriptome (posttranscriptional modification of RNA such as methylation) is considered as a part of the epigenetic system and affects many aspects of plant development 153,154 .However, information is limited regarding the light and epigenetic regulation of SM biosynthesis in plants.In Arabidopsis, MYBD overexpression affects the acetylation status of the MYBL2 promoter and represses MYBL2 expression to promote anthocyanin accumulation 40 .Light-induced epigenetic changes affecting the biosynthesis of other SM, and the chromatin factors involved in this process remain to be characterized.The advances in omics technology will offer promising information on epigenetic regulation of SM biosynthesis.Recently, scATACseq (single-cell Assay for Transposase-Accessible Chromatin by sequencing) has emerged as a powerful technique to study epigenomic landscape of plants at a single-cell resolution in response to biotic or abiotic factors.Single-cell RNAseq has provided valuable insights into the spatial organization and the identification of new transporters in C. roseus TIA pathway 119,155 .A well-assembled and annotated genome is fundamental to the success of these approaches.Although challenging, integrating scATACseq and scRNAseq in response to light or other factors will yield significant information on the regulation of SM pathways in plants.

Fig. 3
Fig. 3 Posttranscriptional and posttranslational regulation of specialized metabolite biosynthesis in response to light.a Light and miRNA-mediated regulatory network of anthocyanin biosynthesis.Light induces the expression of several transcription factors (including HY5, MYB1, and PIFs) and miRNAs (miR858, miR7125, and miR156).The expression of miRNAs is differentially regulated by HY5, MYB1, and PIFs.MYB1 positively regulates the MBW complex, while HY5 represses the MBW repressor MYBL2.The light-inducible miR156 positively regulates MBW by repressing the repressor SPL9.MYB16, an activator of lignin pathway gene CCR, also represses MBW, thus maintaining the homeostasis between anthocyanin and lignin in response to light.b Light and protein kinase-mediated regulatory network of anthocyanin biosynthesis.Light induces the expression of MAP kinases (MPK4 and MPK6) which phosphorylate HY5, as well as PAP and MYB1, positively regulating anthocyanin biosynthesis.The kinase SnRK1, a negative regulator of the MBW complex, is suppressed by light.Phosphorylation of the components in the MBW complex leads to its dissociation and degradation, resulting in reduced anthocyanin accumulation.CCR CINNAMOYL-CoA REDUCTASE.MPK4 Mitogen-Activated Protein Kinase 4, MPK6 Mitogen-Activated Protein Kinase 6, PAP1 PRODUCTION OF ANTHOCYANIN PIGMENT 1, PIFs PHYTOCHROME INTERACTING FACTORs, SnRK1 SNF1-related protein kinase 1, SPL9 SQUAMOSA PROMOTER BINDING PROTEIN-LIKE9, CCR Cinnamoyl-CoA reductase, WD40,TRANSPARENT TESTA GLABROUS1.The arrows and T-bars represent positive and negative regulation, respectively.The double-headed arrows indicate the physical interactions between kinases and their target proteins.The phosphate moieties (P) with a positive effect on regulation are depicted in yellow while those with a negative effect are in red.