Christopher I. Cazzonelli

Source to sink: regulation of carotenoid biosynthesis in plants.

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 phytohormones, 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 as well as their accumulation, storage and degradation will be the focus of this review.

Carotenoids in nature: insights from plants and beyond

Carotenoids are natural isoprenoid pigments that provide leaves, fruits, vegetables and flowers with distinctive yellow, orange and some reddish colours as well as several aromas in plants. Their bright colours serve as attractants for pollination and seed dispersal. Carotenoids comprise a large family of C40 polyenes and are synthesised by all photosynthetic organisms, aphids, some bacteria and fungi alike. In animals carotenoid derivatives promote health, improve sexual behaviour and are essential for reproduction. As such, carotenoids are commercially important in agriculture, food, health and the cosmetic industries. In plants, carotenoids are essential components required for photosynthesis, photoprotection and the production of carotenoid-derived phytohormones, including ABA and strigolactone. The carotenoid biosynthetic pathway has been extensively studied in a range of organisms providing an almost complete pathway for carotenogenesis. A new wave in carotenoid biology has revealed implications for epigenetic and metabolic feedback control of carotenogenesis. Developmental and environmental signals can regulate carotenoid gene expression thereby affecting carotenoid accumulation. This review highlights mechanisms controlling (1) the first committed step in phytoene biosynthesis, (2) flux through the branch to synthesis of α- and β-carotenes and (3) metabolic feedback signalling within and between the carotenoid, MEP and ABA pathways.

Regulation of Carotenoid Composition and Shoot Branching in Arabidopsis by a Chromatin Modifying Histone Methyltransferase, SDG8

Carotenoid pigments are critical for plant survival, and carotenoid composition is tuned to the developmental stage, tissue, and to environmental stimuli. We report the cloning of the CAROTENOID CHLOROPLAST REGULATORY1 (CCR1) gene. The ccr1 mutant has increased shoot branching and altered carotenoid composition, namely, reduced lutein in leaves and accumulation of cis-carotenes in dark-grown seedlings. The CCR1 gene was previously isolated as EARLY FLOWERING IN SHORT DAYS and encodes a histone methyltransferase (SET DOMAIN GROUP 8) that methylates histone H3 on Lys 4 and/or 36 (H3K4 and H3K36). ccr1 plants show reduced trimethyl-H3K4 and increased dimethyl-H3K4 surrounding the CAROTENOID ISOMERASE (CRTISO) translation start site, which correlates with low levels of CRTISO mRNA. Microarrays of ccr1 revealed the downregulation of 85 genes, including CRTISO and genes associated with signaling and development, and upregulation of just 28 genes. The reduction in CRTISO transcript abundance explains the altered carotenoid profile. The changes in shoot branching are additive with more axillary branching mutants, but the altered carotenoid profile may partially affect shoot branching, potentially by perturbed biosynthesis of the carotenoid substrates of strigolactones. These results are consistent with SDG8 regulating shoot meristem activity and carotenoid biosynthesis by modifying the chromatin surrounding key genes, including CRTISO. Thus, the level of lutein, the most abundant carotenoid in higher plants that is critical for photosynthesis and photoprotection, appears to be regulated by a chromatin modifying enzyme in Arabidopsis thaliana.

Alternative splicing, activation of cryptic exons and amino acid substitutions in carotenoid biosynthetic genes are associated with lutein accumulation in wheat endosperm

Endosperm carotenoid content in wheat is a primary determinant of flour colour and this affects both the nutritional value of the grain and its utility for different applications. Utilising wheat rice synteny two genes, ε-cyclase (ε-LCY) and phytoene synthase (Psy-A1), were identified as candidate genes for two of the QTL affecting lutein content in wheat endosperm. Analysis of the sequence changes in ε-LCY and Psy-A1 revealed possible causal mechanisms for both QTL. A point mutation in ε−LCY results in the substitution of a conserved amino acid in the high lutein allele. This substitution has been observed in high lutein-accumulating species from the Gentiales order. In Psy-A1, a sequence duplication at the end of exon 2 creates a new splice site and causes alternative splicing of the transcript and activation of a cryptic exon, resulting in four different transcripts: a wild-type transcript, two transcripts with early terminations and a transcript that would produce an in-frame, albeit longer protein. Only the wild-type splice variant produced an enzymatically active protein and its mRNA abundance was reduced by titration with the other splice variants. This reduction in wild-type mRNA is argued to result in a reduction in PSY protein and thus carotenoid content in wheat.

An Uncharacterized Apocarotenoid-Derived Signal Generated in ζ-Carotene Desaturase Mutants Regulates Leaf Development and the Expression of Chloroplast and Nuclear Genes in Arabidopsis

A signaling process derived from carotenoids in ζ-carotene desaturase mutants regulates the expression of a variety of chloroplast- and nucleus-encoded genes and dramatically affects early leaf development in Arabidopsis. The signaling molecule involved in this process is an apocarotenoid whose synthesis requires the activity of the carotenoid cleavage dioxygenase CCD4. In addition to acting as photoprotective compounds, carotenoids also serve as precursors in the biosynthesis of several phytohormones and proposed regulatory signals. Here, we report a signaling process derived from carotenoids that regulates early chloroplast and leaf development. Biosynthesis of the signal depends on ζ-carotene desaturase activity encoded by the ζ-CAROTENE DESATURASE (ZDS)/CHLOROPLAST BIOGENESIS5 (CLB5) gene in Arabidopsis thaliana. Unlike other carotenoid-deficient plants, zds/clb5 mutant alleles display profound alterations in leaf morphology and cellular differentiation as well as altered expression of many plastid- and nucleus-encoded genes. The leaf developmental phenotypes and gene expression alterations of zds/clb5/spc1/pde181 plants are rescued by inhibitors or mutations of phytoene desaturase, demonstrating that phytofluene and/or ζ-carotene are substrates for an unidentified signaling molecule. Our work further demonstrates that this signal is an apocarotenoid whose synthesis requires the activity of the carotenoid cleavage dioxygenase CCD4.

Cloning and characterisation of ripening-induced ethylene biosynthetic genes from non-climacteric pineapple (Ananas comosus) fruits

To gain a better understanding of non-climacteric fruit ripening, pineapple was used as a model system to clone and characterise two ripening-inducible cDNAs coding for two enzymes of the ethylene biosynthetic pathway, 1-aminocyclopropane-1-carboxylate (ACC) synthase (acacc-1) and 1-aminocyclo- propane-1-carboxylate oxidase (acaco-1) respectively. Due to the extreme acidity and high polyphenolic content of pineapple fruits, a method was optimised for the extraction of high quality RNA from fruit tissue. acacc-1 is a 1080 bp ACC synthase cDNA fragment encoding 360 amino acids including 10 of the 12 amino acid residues conserved in all aminotransferases. Comparison of the deduced amino acid sequence with previously reported ACC synthases shows between 52 and 67% similarity at the protein level. Southern analysis suggests the presence of only one copy of acacc-1 in the pineapple genome. Although some acacc-1 expression is detected in green fruits, there is a 16-fold increase in the level of acacc-1 in ripe fruit tissue. acaco-1 is a partial length cDNA clone of 611 bp which codes for 203 amino acids representing approximately 66% of the ACC oxidase open reading frame. Southern analysis suggests the presence of one or two copies of the gene in the pineapple genome. Northern analysis shows the expression of acaco-1 to be highly induced in wounded leaf tissue and to a lesser extent in ripening fruit tissue. The accumulation of ACC-synthase and ACC oxidase mRNAs during pineapple fruit ripening raises new questions about the putative role of ethylene during non-climacteric fruit ripening.

Periodic root branching in Arabidopsis requires synthesis of an uncharacterized carotenoid derivative

Significance A fundamental question in developmental biology is how patterns are established in space and time. In plants, key differences in root system architecture are attributed to the spatial distribution pattern of lateral roots (LRs), yet how the pattern of LRs is established is only beginning to be understood. We demonstrate that the establishment of sites competent to form LRs roots requires carotenoid biosynthesis. Furthermore, our results implicate an uncharacterized carotenoid-derived molecule that functions non–cell-autonomously, specifically in LR formation. The results of this study reveal novel aspects of carotenoid biology and expand the roles of carotenoid-derived molecules into root developmental patterning. In plants, continuous formation of lateral roots (LRs) facilitates efficient exploration of the soil environment. Roots can maximize developmental capacity in variable environmental conditions through establishment of sites competent to form LRs. This LR prepattern is established by a periodic oscillation in gene expression near the root tip. The spatial distribution of competent (prebranch) sites results from the interplay between this periodic process and primary root growth; yet, much about this oscillatory process and the formation of prebranch sites remains unknown. We find that disruption of carotenoid biosynthesis results in seedlings with very few LRs. Carotenoids are further required for the output of the LR clock because inhibition of carotenoid synthesis also results in fewer sites competent to form LRs. Genetic analyses and a carotenoid cleavage inhibitor indicate that an apocarotenoid, distinct from abscisic acid or strigolactone, is specifically required for LR formation. Expression of a key carotenoid biosynthesis gene occurs in a spatially specific pattern along the root’s axis, suggesting spatial regulation of carotenoid synthesis. These results indicate that developmental prepatterning of LRs requires an uncharacterized carotenoid-derived molecule. We propose that this molecule functions non–cell-autonomously in establishment of the LR prepattern.

Transcriptional control of SET DOMAIN GROUP 8 and CAROTENOID ISOMERASE during Arabidopsis development.

Carotenoids are pigments required for photosynthesis, photoprotection and the production of carotenoid-derived hormones such as ABA and strigolactones. The carotenoid biosynthetic pathway bifurcates after lycopene to produce epsilon- and beta-carotenoids and this branch is critical for determining carotenoid composition. Here, we show how the branch point can be regulated by the chromatin-modifying histone methyltransferase, Set Domain Group 8 (SDG8) targeting the carotenoid isomerase (CRTISO). SDG8 is required to maintain permissive expression of CRTISO during seedling development, in leaves, shoot apex, and some floral organs. The CRTISO and SDG8 promoters show overlapping tissue-specific patterns of reporter gene activity. Interestingly, CRTISO showed atypical reporter gene expression in terms of greater variability between different lines compared to the Cauliflower Mosaic Virus 35S promoter (CaMV35s) and epsilonLCY promoters, potentially due to chromosomal position effects. Regulation of the CRTISO promoter was dependent in part upon the presence or absence of SDG8. Knockouts of SDG8 (carotenoid and chloroplast regulation (ccr1)) and CRTISO (ccr2) result in altered carotenoid composition and this could be restored in ccr2 using the CaMV35s or CRTISO promoters. In contrast, varying degrees of GUS expression and carotenoid complementation by CRTISO overexpression using CaMV35S or CRTISO promoters in the ccr1 background demonstrated that both the CRTISO promoter and open reading frame are necessary for SDG8-mediated expression of CRTISO.

Characterization of a Strong, Constitutive Mung Bean (Vigna radiata L.) Promoter with a Complex Mode of Regulation in planta

We report the cloning and characterization in tobacco and Arabidopsis of a Vigna radiata L. (mung bean) promoter that controls the expression of VR-ACS1, an auxin-inducible ACC synthase gene. The VR-ACS1 promoter exhibits a very unusual behavior when studied in plants different from its original host, mung bean. GUS and luciferase in situ assays of transgenic plants containing VR-ACS1 promoter fusions show strong constitutive reporter gene expression throughout tobacco and Arabidopsis development. In vitro quantitative analyses show that transgenic plants harboring VR-ACS1 promoter–reporter constructs have on average 4–6 fold higher protein and activity levels of both reporter genes than plants transformed with comparable CaMV 35S promoter fusions. Similar transcript levels are present in VR-ACS1 and CaMV 35S promoter lines, suggesting that the high levels of gene product observed for the VR-ACS1 promoter are the combined result of transcriptional and translational activation. All tested deletion constructs retaining the core promoter region can drive strong constitutive promoter activity in transgenic plants. This is in contrast to mung bean, where expression of the native VR-ACS1 gene is almost undetectable in plants grown under normal conditions, but is rapidly and highly induced by a variety of stimuli. The constitutive behavior of the VR-ACS1 promoter in heterologous hosts is surprising, suggesting that the control mechanisms active in mung bean are impaired in tobacco and Arabidopsis. The ‘aberrant’ behavior of the VR-ACS1 promoter is further emphasized by its failure to respond to auxin and cycloheximide in heterologous hosts. VR-ACS1 promoter regulatory mechanisms seem to be different from all previously characterized auxin-inducible promoters.

An in vivo, luciferase-based, Agrobacterium-infiltration assay system: implications for post-transcriptional gene silencing.

An in vivo assay system for analyzing transient luciferase expression in tobacco leaves infused with Agrobacteriumtumefaciens is described. The system makes use of A. tumefaciens harboring T-DNA vectors containing either an intron-containing firefly (Photinus pyralis) luciferase (EC 1.13.12.7) gene or an intron-containing sea pansy (Renilla reniformis) luciferase (EC 1.13.12.5) gene. Single or mixed Agrobacterium lines were infiltrated into leaf tissues (Nicotiana tabacum or Nicotiana benthamiana) through stomatal openings and leaf disks from infused areas floated on reaction buffers specific to each enzyme. Photons emitted were then measured to determine reporter gene activity. Parameters affecting assay reliability and sensitivity were tested, including: buffer composition; bacterial density; infusion location; reaction kinetics; and environmental factors (light and temperature). The resulting in vivo assay system generates results comparable to those obtained using a commercially available in vitro dual-luciferase® reporter gene assay, and reports relative expression levels, as well as induction characteristics, analogous to those obtained using leaf tissue from stably transformed plants harboring the same promoter::gene constructs. Light and temperature were observed to markedly impact transient reporter activities. Co-expression of viral suppressors of post-transcriptional gene silencing (PTGS), HcPro, p19 and AC2, confirms the occurrence of PTGS within infused zones, and provides a convenient mechanism for PTGS analysis. The in vivo transient assay was used to examine the effect on PTGS of factors such as: promoter strength; incubation temperature and double-stranded RNA production. Results from these assays provide insight into the mechanism(s) used by plants to trigger and maintain PTGS.