类胡萝卜素生物合成调控综述

u so y B Review TRPLSC-766; No. of Pages 9 this has been covered in a number of excellent reviews [8,13–19]. In addition to providing colour to flowers and fruits, carotenoids also contribute to the production of scents and flavours that attract insects and animals for pollination and seed dispersal. All photosynthetic organisms accumu- late carotenoids where they play crucial roles in photo- system assembly, light-harvesting and photoprotection [20,21]. Typically, leaf tissues accumulate lutein, b-caro- tene, violaxanthin and neoxanthin (Figure 1), with changes in this profile altering photosynthesis, antenna assembly and photoprotection [21–23]. Carotenoids also serve as precursors for plant hormones, abscisic acid (ABA) and strigolactones (Figure 1) [6,24–27]. The strigolactone enhance total carotenoid content by >12% in Arabidopsis (Arabidopsis thaliana) seedlings, while antisense silencing of DXS reduced carotenoid levels by 13% [38,39]. The second key regulatory step is 1-hydroxy-2-methyl-2-(E)- butenyl 4-diphosphate reductase (HDR), which catalyzes the production of IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate) (Figure 1). A corre- lation between HDR mRNA abundance and carotenoid accumulation has been observed in ripening tomato fruits and greening Arabidopsis seedlings [40]. Furthermore, overexpression of tomato (Solanum lycopersicum) LeHDR in Arabidopsis increased b-carotene and lutein in chlor- oplasts, but not etioplasts [40]. Abiotic and biotic factors may influence the availability of isoprenoid precursors. Light and circardian oscillationsCorresponding author: Pogson, B.J. (barry.pogson@anu.edu.au). Source to sink: reg carotenoid biosynth Christopher I. Cazzonelli and Barry J. Pog Australian Research Council Centre of Excellence in Plant Energ University, Canberra, ACT 0200, Australia Carotenoids are a diverse group of colourful pigments naturally found in plants, algae, fungi and bacteria. They play essential roles in development, photosynthesis, root-mycorrhizal interactions and the production of phy- tohormones, such as abscisic acid and strigolactone. Carotenoid biosynthesis is regulated throughout the life cycle of a plant with dynamic changes in composition matched to prevailing developmental requirements and in response to external environmental stimuli. There are key regulatory nodes in the pathway that control the flux of metabolites into the pathway and alter flux through the pathway. The molecular nature of the mechanisms regulating carotenoid biosynthesis, including evidence for metabolite feedback, transcription and epigenetic control aswell as their accumulation, storage and degra- dation will be the focus of this review. Carotenoids: everywhere in nature and essential for life Carotenoids comprise many of the yellow, orange and red pigments of nature, including many fruits, vegetables, flowers, butterflies and crayfish. Animals are unable to synthesize carotenoids; however, they can accumulate carotenoidswhere they contribute to health and behaviour. For example, fish and birds accumulate dietary caroten- oids, which boost their immune system and advertise health, often leading to preferential selection by the sexual partner [1,2]. The human health benefits associated with carotenoids have been extensively reviewed [3–7]. In brief, carotenoids promote antioxidant activity, reduce age- related macular degeneration of the eye and can be pre- cursors for vitamin A. Metabolic engineering of caroteno- genesis has proven successful to enhance accumulation [8], develop novel compounds [9] and redirect flux [10–12], and 1360-1385/$ – see front matter � 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.20 lation of esis in plants n iology, Research School of Biology, The Australian National class of carotenoid metabolites inhibit shoot branching and stimulate a symbiotic relationship with fungi in the rhizo- sphere [25,26,28]. Consequently, carotenoid biosynthesis is regulated throughout the life cycle of a plant, with dynamic changes in composition matched to prevailing developmental requirements during germination, photomorphogenesis, photosynthesis, fruit development and in response to external environmental stimuli [29–32]. There are many reports describing altered carotenoid gene transcript abun- dance during fruit ripening, flower development or stress, which coincide with changes in carotenoid content. This review highlights recent insights into epigenetic, post- transcriptional and metabolite feedback regulation of carotenoid accumulation. Carotenoid biosynthesis depends upon the availability of isoprenoid substrates Carotenoids are derived from the plastid-localized 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (Figure 1) [33] for which glyceraldehyde-3-phosphate and pyruvate act as initial substrates leading to the synthesis of geranylgeranyl diphosphate (GGPP) (Figure 1) [34–36]. The condensation of two GGPPs by phytoene synthase (PSY) forms phytoene, the first carotenoid (Figure 1). The first steps in the MEP pathway are regulated by 1- deoxyxylulose-5-phosphate synthase (DXS) and 1-deoxy- D-xylulose 5-phosphate reductoisomerase (DXR) (Figure 1). Expression analysis of DXS showed organ specific expression and developmental regulation during tomato fruit ripening, which correlated with an increase in PSY mRNA transcripts and carotenoid accumulation [37]. The overexpression of DXS and DXR is sufficient to 10.02.003 Available online xxxxxx 1 Figure 1. Major reactions in the higher plant carotenoid biosynthetic pathway showing enzymes, carotenoids and their precursors (pipes), carotenoid sinks (barrels), carotenoid- derived signalling hormones (green signs) and other MEP isoprenoid-derived metabolites (blue sign). The windows displayed within the chrome pipes indicate abundant carotenoid pigments found in photosynthetic tissues and also represent key nodes for regulation in the pathway. Carotenoid biosynthesis is modulated by environmental factors (light), chromatin modification and metabolic feedback regulation. The side funnels represent examples of metabolic feedback control mechanisms acting upon biosynthetic gene expression as a result of altered PSY and CRTISO enzymatic activity, respectively. First, the bottleneck in phytoene biosynthesis is regulated by PSY and its overexpression increased DXS and DXR mRNA levels post-transcriptionally in etiolated tissues. Second, loss-of-function CRTISO mutants show reduced eLCY transcript levels in etiolated tissues. Abbreviations: bLCY, b-cyclase; bOHase, b-hydroxylase; CCD, carotenoid cleavage dioxygenase; CRTISO, carotenoid isomerase; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; DXS, 1-deoxyxylulose-5-phosphate synthase; eLCY, e-cyclase; eOHase, e-hydroxylase; GGPP, geranylgeranyl diphosphate; HDR, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase; NCED, 9-cis-epoxycarotenoid dioxygenase; NXS, neoxanthin synthase; PDS, phytoene desaturase; PSY, phytoene synthase; SDG8, histone methyltransferase; VDE, violaxanthin de-epoxidase; ZDS, z-carotene desaturase; and ZE, zeaxanthin epoxidase. Review Trends in Plant Science Vol.xxx No.x TRPLSC-766; No. of Pages 9 2 TRPLSC-766; No. of Pages 9 can alter the expression of nearly all MEP genes and several carotenoid genes. The colonization of plant root arbuscular mycorrhizal fungi activates the MEP pathway by increasing transcript levels of MEP, carotenoid biosyn- thetic and cleavage genes [41]. This stimulates production of carotenoids and carotenoid cleavage products, such as C13 cyclohexenone derivatives (e.g. blumenol), C14 apocar- otenoids (e.g. mycorradicin) and strigolactones in root plastids. These compounds trigger hyphal branching and a symbiotic association between the fungi and roots [41,42]. Post-transcriptional regulation of DXS and HDR at the level of protein accumulation has also been demon- strated and this has the potential to regulate carotenoid accumulation (see below). Phytoene biosynthesis is a rate-limiting step in carotenogenesis PSY is generally accepted as being the most important regulatory enzyme in the pathway. Transcriptionally, PSY genes respond to ABA, high light, salt, drought, tempera- ture, photoperiod, development cues and post-transcrip- tional feedback regulation. While there is only one PSY gene in Arabidopsis, there are two or more homologues in tomato, rice (Oryza sativa), poplar (Populus trichocarpa), bread wheat (Triticum aestivum) and maize (Zea mays) [29,43–46]. The activity of the multiple PSY enzymes appear redundant, but their expression is tissue-specific and shows unique responses to environmental stimuli in cereal roots [29,43,46]. Indeed, salt and drought induced PSY3 transcript abundance and this correlated with increased carotenoid flux and ABA in maize roots [46]. A rapid disappearance of PSY2 and PSY3 mRNA after re- watering suggests tightly controlled mRNA stability or transcription [46]. A similar finding for OsPSY3 in rice roots subjected to salt stress was found [43]. However, stress-induced changes in PSYmRNA do not always result in changes in carotenoid flux. For example, elevated tem- peratures decreased PSY1 mRNA abundance in maize leaves and unexpectedly there was a concurrent increase in carotenoids [29]. The different expression profiles of the rice PSYs correlate strongly with the presence of promoter regulatory cis-elements that mediate light (PSY1 and PSY2) and ABA responses (PSY3) [43]. Allelic variation in PSY is a mechanism that may change PSY enzymatic activity as alternative splicing of PSY-A1 allele appeared to be a major QTL determinant of flour colour in bread wheat [45]. The alternative splicing results in the generation of four different transcripts, of which one is functional, thereby titrating the level of functional PSY [45]. PSY transcript abundance is upregulated during photo- morphogenesis via a phytochrome-mediated (red-light) pathway [30,43]. Both red and far-red light treatments increase PSY mRNA and this is abolished in the phyA mutant [30]. Interestingly, photoinduction of ZmPSY2 transcription in maize leaf tissues can also be mediated by blue photoreceptors (phototrophins and cryptochromes) in addition to phytochrome [29]. There is strong evidence to show diurnal oscillations of PSY mRNA and MEP genes, Review which is consistent with their phytochrome-mediated activities [34,47–50]. A promoter study identified a cis-acting motif (ATCTA), which was present in the promoter region of other photo- synthesis- related genes and is believed to be important in mediating the transcriptional regulation of PSY [51]. How- ever, modulating mRNA levels of RAP2.2, an APETALA2 transcription factor that binds to the PSY promoter, resulted in only small pigment alterations in root calli revealing it is just one constituent of a significantly more complex regulatory network involved in carotenogenesis [52]. Finally, there are post-translational effects of photo- morphogenesis as it results in activation and relocation of PSY from the prolamellar body to the newly developing thylakoid membranes [30]. There is substantial evidence to support metabolite feedback regulation that modulates the supply of isopre- noid substrates and the accumulation of carotenoids and ABA. This includes feedback within the carotenoid path- way and between carotenoids, MEP and ABA [53–57]. A positive feedback regulation mediated by ABA affects PSY3 gene expression in rice and may play a specialized role in abiotic stress-induced ABA formation [43]. Elevated expression of PSY by a transgene resulted in increased carotenoid levels in etiolated Arabidopsis seedlings and this was via a concomitant post-transcriptional accumu- lation of DXSmRNA which reveals a feedback mechanism initiated by PSY that stimulated the supply of MEP sub- strates (Figure 1) [32,58]. However, the overexpression of just DXS in dark-grown seedlings does not increase carotenoid accumulation [32]. Therefore, regulation of the first committed step in carotenogenesis by PSY is tightly coordinated and controlled by source and sink metabolites (Figure 1). Regulation of lycopene biosynthesis by desaturases, isomerases and chromatin modifiers The production of all trans-lycopene from phytoene requires a complex set of four reactions requiring phy- toene desaturase (PDS), z–carotene isomerase (Z-ISO), z- carotene desaturase (ZDS) and carotenoid isomerase (CRTISO), as well as a light-mediated photoisomeriza- tion (Figure 1) [57,59–65]. PDS may play a rate-limiting role in the generation of 9,15,90-tri-cis-z-carotene as tran- script abundance is slightly upregulated during photo- morphogenesis via a phytochrome-mediated pathway [30]. The Arabidopsis variegated mutant, IMMUTANS contains lesions in a plastid-targeted alternative oxidase (PTOX) required for phytoene desaturase (PDS) activity, thereby links desaturation to chloroplast electron trans- port [66]. The accumulation of phytoene has been postu- lated to involve negative feedback regulation [56]. For example, in the pds3 mutant, genes encoding down- stream enzymes such as ZDS and lycopene cyclase (LCY), were downregulated, as were upstream genes such as IPI, GGPS and PSY [56]. Alternatively, the absence of downstream carotenoids in pds3 mutants could modulate the signal. Evidence for this is an analysis of a tomato PDS promoter–GUS fusion that demonstrated end-product regulation in photosynthetic tissues [67]. The regulatory roles for ZDS and Z-ISO in Trends in Plant Science Vol.xxx No.x the catalysis of z-carotene, the product of PDS, to tetra- cis-lycopene, the substrate for CRTISO, as well as control 3 TRPLSC-766; No. of Pages 9 by photoisomerization under day- length- limiting con- ditions, have not yet been described. CRTISO, which catalyses cis–trans reactions to isomer- ase the four cis-bonds introduced by the desaturases, has emerged as a regulatory node in the pathway [61,68]. CRTISO mutants, such as ccr2 and tangerine, result in accumulation of cis-carotenes, such as 7,70,9,90-tetra-cis- lycopene, in the etioplasts (dark-grown plastids) of seed- lings and chromoplasts of fruit [63,64]. Despite this block in etioplasts and chromoplasts, the biosynthetic pathway proceeds in chloroplasts via photoisomerization, but there is delayed greening and substantial reduction in lutein in Arabidopsis and varying degrees of chlorosis in tomato and rice [63,64,69]. Interestingly, a chromatin-modifying histone methyl- transferase enzyme (SET DOMAIN GROUP 8, SDG8) was shown to be required for CRTISO expression (Box 1) [68]. The absence of SDG8 alters the methylation of chromatin associated with the CRTISO gene, thereby reducing gene expression, impairing lutein biosynthesis and increasing shoot branching, in part by possibly limit- ing strigolactone biosynthesis [68]. This was the first report implicating epigenetic regulatory mechanisms in the control of carotenoid composition [68]. SDG8 is required to maintain expression of CRTISO in seedlings, leaves, shoot apexes, anthers and pollen [70]. The CRTISO and SDG8 promoters show overlapping tissue-specific expression patterns in many tissues essential for defining plant architecture and development, including germinat- ing seedlings, meristems, shoot apexes, floral anthers and pollen (Box 1) [70]. Regulation of lutein and other xanthophylls Carotenoid biosynthesis bifurcates after lycopene to pro- duce epsilon- and beta-carotenoids by enzymatic activity of the two lycopene cyclases, eLCY and bLCY, and this branch point has a major regulatory role in modulating the ratio of the most abundant carotenoid, lutein to the beta-carotenoids [55,68] (Figure 1). Investigations into lutein biosynthesis have yielded lut1, e-hydroxylase [71]; lut2, eLCY [72,73]; ccr2, CRTISO [63]; and lut5, an additional b-hydroxylase [74] as well as the SDG8 chro- matin regulatory mutant, ccr1 [68]. Intriguingly, lutein levels can be altered by producing lycopene via an alternate pathway that does not require the formation of cis-carotenes [75]. Furthermore, the absence of CRTISO or specific carotene isomers results in less lutein [63,64]. The question is whether this reflects altered lycopene substrate preference by the cyclases or metabolite feedback regulation. Flux through the two branches can be controlled at the level of eLCY mRNA [55,73,76] and recent experiments indicate that both CRITSO (ccr2) and SDG8 (ccr1) mutants have some effect on e-cyclase (eLCY) transcript levels (Figure 1), suggesting feedback may account for at least part of the reduction in lutein (Figure 1) [55,68]. Natural genetic variation in maize was found to be regulated by eLCY, for which four natural elcy polymorph- isms explained 58% of the variation in lutein and beta- Review carotenoids [77] while cosuppression of eLCY in Arabidop- sis also altered the ratio of lutein to beta-carotenoid [76]. In 4 Brassica napus the downregulation of lycopene eLCY by RNAi in seeds showed a higher total carotenoid content, specifically increased levels of b-carotene, zeaxanthin, vio- laxanthin and, unexpectedly, lutein [78]. This unexpected increase in lutein was inconsistent with another report that showed tuber specific silencing of eLCY increased b- carotene levels in potato (Solanum tuberosum) [79]. Clearly, the evidence supports a hypothesis that lutein composition is largely rate-determined by eLCY expres- sion, but feedback regulation can reveal complex regulat- ory mechanisms, such as that in B. napus. A molecular synergism between eLCY and bLCY activi- ties is an overall major determinant of flux through the branch leading to production of lutein, b-carotene and the xanthophyll cycle (XC) carotenoids [53,78]. The bLCY gene from the eubacterium Erwinia herbicola and daffodil (Nar- cissus pseudonarcissus) flowers were introduced into the tomato plastid genome and lycopene was channelled into the beta-branch, resulting in increased accumulation of XC carotenoids in leaves and predominantly b-carotene in fruits [12]. Unexpectedly, transplastomic tomatoes again showed a >50% increase in total carotenoid accumulation [12]. Conversely, in the absence of bLCY, eLCY produces a number of unusual carotenes, including d-carotene, e-car- otene and lactucaxanthin (e,e-carotene-3,30-diol), in endo- sperm tissue. Several genes encoding enzymes in isoprenoid (DXR and DXS) and carotenoid biosynthesis (b-OHase and ZE) appear to be the subject of negative transcriptional regulation, mediated by a carotenoid or a molecule derived from a carotenoid [53] and these epsilon carotenoids are candidates. With respect to accumulation of the b-xanthophylls, light stress results in synthesis of zeaxanthin from b- carotene [80] and it is worth noting that beta-hydroxylase (b-OHase) and violaxanthin de-epoxidase (VDE) mRNA are high-light inducible and repressible, respectively [81]. Post-translation modulation of VDE activity by the lumi- nal pH and ascorbate content are also critical for determin- ing the levels of zeaxanthin during high light [82,83]. Carotenoid degradation and turnover A long-standing question for carotenoid accumulation in photosynthetic tissues has been the rate of synthesis and presumed slow rate of turnover implied by the persistent yellow of senescing leaves. However, recent data using 14CO2 uptake demonstrates that synthesis, and by infer- ence turnover, is much greater than expected [84]. Furthermore, the incorporation of 14C in different caroten- oids was not uniform and varied in different mutants and under high light [84]. Given the continued synthesis in mature leaves is much greater than expected, then there must be active degradation. A mechanism for enzymatic turnover in addition to that due to oxidative damage has now been provided. Studies in Arabidopsis seeds, strawberry (Fragar- ia � ananassa), grape (Vitis vinifera L.) and citrus fruits (Ci