Multi-omics analyses reveal the importance of chromoplast plastoglobules in carotenoid accumulation in citrus fruit.

Chromoplasts act as a metabolic sink for carotenoids, in which plastoglobules serve as versatile lipoprotein particles. PGs in chloroplasts have been characterized. However, the features of PGs from non-photosynthetic plastids are poorly understood. We found that the development of chromoplast plastoglobules (CPGs) in globular and crystalloid chromoplasts of citrus is associated with alterations in carotenoid storage. Using Nycodenz density gradient ultracentrifugation, an efficient protocol for isolating highly purified CPGs from sweet orange (Citrus sinensis) pulp was established. Forty-four proteins were defined as likely comprise the core proteome of CPGs using comparative proteomics analysis. Lipidome analysis of different chromoplast microcompartments revealed that the nonpolar microenvironment within CPGs was modified by 35 triacylglycerides, two sitosterol esters, and one stigmasterol ester. Manipulation of the CPG-localized gene CsELT1 (esterase/lipase/thioesterase) in citrus calli resulted in increased lipids and carotenoids, which is further evidence that the nonpolar microenvironment of CPGs contributes to carotenoid accumulation and storage in the chromoplasts. This multi-feature analysis of CPGs sheds new light on the role of chromoplasts in carotenoid metabolism, paving the way for manipulating carotenoid content in citrus fruit and other crops.

Verbascum species as a new source of saffron apocarotenoids and molecular tools for the biotechnological production of crocins and picrocrocin.

Crocins are glucosylated apocarotenoids present in flowers and fruits of a few plant species, including saffron, gardenia, and Buddleja. The biosynthesis of crocins in these plants has been unraveled, and the enzymes engineered for the production of crocins in heterologous systems. Mullein (Verbascum sp.) has been identified as a new source of crocins and picrocrocin. In this work, we have identified eight enzymes involved in the cleavage of carotenoids in two Verbascum species, V. giganteum and V. sinuatum. Four of them were homologous to the previously identified BdCCD4.1 and BdCCD4.3 from Buddleja, involved in the biosynthesis of crocins. These enzymes were analyzed for apocarotenogenic activity in bacteria and Nicotiana benthamiana plants using a virus-driven system. Metabolic analyses of bacterial extracts and N. benthamiana leaves showed the efficient activity of these enzymes to produce crocins using ß-carotene and zeaxanthin as substrates. Accumulations of 0.17% of crocins in N. benthamiana dry leaves were reached in only 2 weeks using a recombinant virus expressing VgCCD4.1, similar to the amounts previously produced using the canonical saffron CsCCD2L. The identification of these enzymes, which display a particularly broad substrate spectrum, opens new avenues for apocarotenoid biotechnological production.

ZmPTOX1, a plastid terminal oxidase, contributes to redox homeostasis during seed development and germination.

Maize plastid terminal oxidase1 (ZmPTOX1) plays a pivotal role in seed development by upholding redox balance within seed plastids. This study focuses on characterizing the white kernel mutant 3735 (wk3735) mutant, which yields pale-yellow seeds characterized by heightened protein but reduced carotenoid levels, along with delayed germination compared to wild-type (WT) seeds. We successfully cloned and identified the target gene ZmPTOX1, responsible for encoding maize PTOX-a versatile plastoquinol oxidase and redox sensor located in plastid membranes. While PTOX's established role involves regulating redox states and participating in carotenoid metabolism in Arabidopsis leaves and tomato fruits, our investigation marks the first exploration of its function in storage organs lacking a photosynthetic system. Through our research, we validated the existence of plastid-localized ZmPTOX1, existing as a homomultimer, and established its interaction with ferredoxin-NADP+ oxidoreductase 1 (ZmFNR1), a crucial component of the electron transport chain (ETC). This interaction contributes to the maintenance of redox equilibrium within plastids. Our findings indicate a propensity for excessive accumulation of reactive oxygen species (ROS) in wk3735 seeds. Beyond its known role in carotenoids' antioxidant properties, ZmPTOX1 also impacts ROS homeostasis owing to its oxidizing function. Altogether, our results underscore the critical involvement of ZmPTOX1 in governing seed development and germination by preserving redox balance within the seed plastids.

Leaky mutations in the zeaxanthin epoxidase in Capsicum annuum result in bright-red fruit containing a high amount of zeaxanthin.

Fruit color is one of the most important traits in peppers due to its esthetic value and nutritional benefits and is determined by carotenoid composition, resulting from diverse mutations of carotenoid biosynthetic genes. The EMS204 line, derived from an EMS mutant population, presents bright-red color, compared with the wild type Yuwolcho cultivar. HPLC analysis indicates that EMS204 fruit contains more zeaxanthin and less capsanthin and capsorubin than Yuwolcho. MutMap was used to reveal the color variation of EMS204 using an F3 population derived from a cross of EMS204 and Yuwolcho, and the locus was mapped to a 2.5-Mbp region on chromosome 2. Among the genes in the region, a missense mutation was found in ZEP (zeaxanthin epoxidase) that results in an amino acid sequence alteration (V291 → I). A color complementation experiment with Escherichia coli and ZEP in vitro assay using thylakoid membranes revealed decreased enzymatic activity of EMS204 ZEP. Analysis of endogenous plant hormones revealed a significant reduction in abscisic acid content in EMS204. Germination assays and salinity stress experiments corroborated the lower ABA levels in the seeds. Virus-induced gene silencing showed that ZEP silencing also results in bright-red fruit containing less capsanthin but more zeaxanthin than control. A germplasm survey of red color accessions revealed no similar carotenoid profiles to EMS204. However, a breeding line containing a ZEP mutation showed a very similar carotenoid profile to EMS204. Our results provide a novel breeding strategy to develop red pepper cultivars containing high zeaxanthin contents using ZEP mutations.

The MdMYB44-MdTPR1 repressive complex inhibits MdCCD4 and MdCYP97A3 expression through histone deacetylation to regulate carotenoid biosynthesis in apple.

Carotenoids are photosynthetic pigments and antioxidants that contribute to different plant colors. However, the involvement of TOPLESS (TPL/TPR)-mediated histone deacetylation in the modulation of carotenoid biosynthesis through ethylene-responsive element-binding factor-associated amphiphilic repression (EAR)-containing transcription factors (TFs) in apple (Malus domestica Borkh.) is poorly understood. MdMYB44 is a transcriptional repressor that contains an EAR repression motif. In the present study, we used functional analyses and molecular assays to elucidate the molecular mechanisms through which MdMYB44-MdTPR1-mediated histone deacetylation influences carotenoid biosynthesis in apples. We identified two carotenoid biosynthetic genes, MdCCD4 and MdCYP97A3, that were confirmed to be involved in MdMYB44-mediated carotenoid biosynthesis. MdMYB44 enhanced ß-branch carotenoid biosynthesis by repressing MdCCD4 expression, whereas MdMYB44 suppressed lutein level by repressing MdCYP97A3 expression. Moreover, MdMYB44 partially influences carotenoid biosynthesis by interacting with the co-repressor TPR1 through the EAR motif to inhibit MdCCD4 and MdCYP97A3 expression via histone deacetylation. Our findings indicate that the MdTPR1-MdMYB44 repressive cascade regulates carotenoid biosynthesis, providing profound insights into the molecular basis of histone deacetylation-mediated carotenoid biosynthesis in plants. These results also provide evidence that the EAR-harboring TF/TPL repressive complex plays a universal role in histone deacetylation-mediated inhibition of gene expression in various plants.

Carbon usage in yellow-fleshed Manihot esculenta storage roots shifts from starch biosynthesis to cell wall and raffinose biosynthesis via the myo-inositol pathway.

Cassava is a crucial staple crop for smallholder farmers in tropical Asia and Sub-Saharan Africa. Although high yield remains the top priority for farmers, the significance of nutritional values has increased in cassava breeding programs. A notable negative correlation between provitamin A and starch accumulation poses a significant challenge for breeding efforts. The negative correlation between starch and carotenoid levels in conventional and genetically modified cassava plants implies the absence of a direct genomic connection between the two traits. The competition among various carbon pathways seems to account for this relationship. In this study, we conducted a thorough analysis of 49 African cassava genotypes with varying levels of starch and provitamin A. Our goal was to identify factors contributing to differential starch accumulation. Considering carotenoid levels as a confounding factor in starch production, we found that yellow- and white-fleshed storage roots did not differ significantly in most measured components of starch or de novo fatty acid biosynthesis. However, genes and metabolites associated with myo-inositol synthesis and cell wall polymer production were substantially enriched in high provitamin A genotypes. These results indicate that yellow-fleshed cultivars, in comparison to their white-fleshed counterparts, direct more carbon toward the synthesis of raffinose and cell wall components. This finding is underlined by a significant rise in cell wall components measured within the 20 most contrasting genotypes for carotenoid levels. Our findings enhance the comprehension of the biosynthesis of starch and carotenoids in the storage roots of cassava.

An ependymin-related blue carotenoprotein decorates marine blue sponge.

Marine animals display diverse vibrant colors, but the mechanisms underlying their specific coloration remain to be clarified. Blue coloration is known to be achieved through a bathochromic shift of the orange carotenoid astaxanthin (AXT) by the crustacean protein crustacyanin, but other examples have not yet been well investigated. Here, we identified an ependymin (EPD)-related water-soluble blue carotenoprotein responsible for the specific coloration of the marine blue sponge Haliclona sp. EPD was originally identified in the fish brain as a protein involved in memory consolidation and neuronal regeneration. The purified blue protein, designated as EPD-related blue carotenoprotein-1, was identified as a secreted glycoprotein. We show that it consists of a heterodimer that binds orange AXT and mytiloxanthin and exhibits a bathochromic shift. Our crystal structure analysis of the natively purified EPD-related blue carotenoprotein-1 revealed that these two carotenoids are specifically bound to the heterodimer interface, where the polyene chains are aligned in parallel to each other like in ß-crustacyanin, although the two proteins are evolutionary and structurally unrelated. Furthermore, using reconstitution assays, we found that incomplete bathochromic shifts occurred when the protein bound to only AXT or mytiloxanthin. Taken together, we identified an EPD in a basal metazoan as a blue protein that decorates the sponge body by binding specific structurally unrelated carotenoids.

Lycopene ε-cyclase mediated transition of α-carotene and ß-carotene metabolic flow in carrot fleshy root.

The accumulation of carotenoids, such as xanthophylls, lycopene, and carotenes, is responsible for the color of carrot (Daucus carota subsp. sativus) fleshy roots. The potential role of DcLCYE, encoding a lycopene ε-cyclase associated with carrot root color, was investigated using cultivars with orange and red roots. The expression of DcLCYE in red carrot varieties was significantly lower than that in orange carrots at the mature stage. Furthermore, red carrots accumulated larger amounts of lycopene and lower levels of α-carotene. Sequence comparison and prokaryotic expression analysis revealed that amino acid differences in red carrots did not affect the cyclization function of DcLCYE. Analysis of the catalytic activity of DcLCYE revealed that it mainly formed ε-carotene, while a side activity on α-carotene and γ-carotene was also observed. Comparative analysis of the promoter region sequences indicated that differences in the promoter region may affect the transcription of DcLCYE. DcLCYE was overexpressed in the red carrot 'Benhongjinshi' under the control of the CaMV35S promoter. Lycopene in transgenic carrot roots was cyclized, resulting in the accumulation of higher levels of α-carotene and xanthophylls, while the ß-carotene content was significantly decreased. The expression levels of other genes in the carotenoid pathway were simultaneously upregulated. Knockout of DcLCYE in the orange carrot 'Kurodagosun' by CRISPR/Cas9 technology resulted in a decrease in the α-carotene and xanthophyll contents. The relative expression levels of DcPSY1, DcPSY2, and DcCHXE were sharply increased in DcLCYE knockout mutants. The results of this study provide insights into the function of DcLCYE in carrots, which could serve as a basis for creating colorful carrot germplasms.

An R-R-type MYB transcription factor promotes non-climacteric pepper fruit carotenoid pigment biosynthesis.

Carotenoids are major accessory pigments in the chloroplast, and they also act as phytohormones and volatile compound precursors to influence plant development and confer characteristic colours, affecting both the aesthetic and nutritional value of fruits. Carotenoid pigmentation in ripening fruits is highly dependent on developmental trajectories. Transcription factors incorporate developmental and phytohormone signalling to regulate the biosynthesis process. By contrast to the well-established pathways regulating ripening-related carotenoid biosynthesis in climacteric fruit, carotenoid regulation in non-climacteric fruit is poorly understood. Capsanthin is the primary carotenoid of non-climacteric pepper (Capsicum) fruit; its biosynthesis is tightly associated with fruit ripening, and it confers red pigmentation to the ripening fruit. In the present study, using a coexpression analysis, we identified an R-R-type MYB transcription factor, DIVARICATA1, and demonstrated its role in capsanthin biosynthesis. DIVARICATA1 encodes a nucleus-localised protein that functions primarily as a transcriptional activator. Functional analyses showed that DIVARICATA1 positively regulates carotenoid biosynthetic gene (CBG) transcript levels and capsanthin levels by directly binding to and activating CBG promoter transcription. Furthermore, an association analysis revealed a significant positive association between DIVARICATA1 transcription level and capsanthin content. ABA promotes capsanthin biosynthesis in a DIVARICATA1-dependent manner. Comparative transcriptomic analysis of DIVARICATA1 in Solanaceae plants showed that its function likely differs among species. Moreover, the pepper DIVARICATA1 gene could be regulated by the ripening regulator MADS-RIN. The present study illustrates the transcriptional regulation of capsanthin biosynthesis and offers a target for breeding peppers with high red colour intensity.

A desert Chlorella sp. that thrives at extreme high-light intensities using a unique photoinhibition protection mechanism.

While light is the driving force of photosynthesis, excessive light can be harmful. Photoinhibition is one of the key processes that limit photosynthetic productivity. A well-defined mechanism that protects from photoinhibition has been described. Chlorella ohadii is a green micro-alga, isolated from biological desert soil crusts, which thrives under extreme high light (HL). Here, we show that this alga evolved unique protection mechanisms distinct from those of the green alga Chlamydomonas reinhardtii or plants. When grown under extreme HL, a drastic reduction in the size of light harvesting antennae occurs, resulting in the presence of core photosystem II, devoid of outer and inner antennas. This is accompanied by a massive accumulation of protective carotenoids and proteins that scavenge harmful radicals. At the same time, several elements central to photoinhibition protection in C. reinhardtii, such as psbS, light harvesting complex stress-related, photosystem II protein phosphorylation and state transitions are entirely absent or were barely detected. In addition, a carotenoid biosynthesis-related protein accumulates in the thylakoid membranes of HL cells and may function in sensing HL and protecting the cell from photoinhibition. Taken together, a unique photoinhibition protection mechanism evolved in C. ohadii, enabling the species to thrive under extreme-light intensities where other photosynthetic organisms fail to survive.

Genome-wide identification and in-silico expression analysis of CCO gene family in sunflower (Helianthus annnus) against abiotic stress.

Carotenoid cleavage oxygenases (CCOs) enzymes play an important role in plant growth and development by producing a wide array of apocarotenoids and their derivatives. These compounds are vital for colouring flowers and fruits and synthesizing plant hormones such as abscisic acid and strigolactones. Despite their importance, the gene family responsible for CCO enzymes in sunflowers has not been identified. In this study, we identify the CCO genes of the sunflower plant to fill this knowledge gap. Phylogenetic and synteny analysis indicated that the Helianthus annnus CCO (HaCCO) genes were conserved in different plant species and they could be divided into three subgroups based on their conserved domains. Analysis using MEME tool and multiple sequence alignment identified conserved motifs in the HaCCO gene sequence. Cis-regulatory elements (CREs) analysis of the HaCCO genes indicated the presence of various responsive elements related to plant hormones, development, and responses to both biotic and abiotic stresses. This implies that these genes may respond to plant hormones, developmental cues, and drought stress, offering potential applications in the development of more resistant crops. Genes belonging to the 9-cis-epoxy carotenoid dioxygenases (NCED) subgroups predominantly exhibited chloroplast localization, whereas the genes found in other groups are primarily localized in the cytoplasm. These 21 identified HaCCOs were regulated by 60 miRNAs, indicating the crucial role of microRNAs in gene regulation in sunflowers. Gene expression analysis under drought stress revealed significant up-regulation of HaNCED16 and HaNCED19, genes that are pivotal in ABA hormone biosynthesis. During organ-specific gene expression analysis, HaCCD12 and HaCCD20 genes exhibit higher activity in leaves, indicating a potential role in leaf pigmentation. This study provides a foundation for future research on the regulation and functions of the CCO gene family in sunflower and beyond. There is potential for developing molecular markers that could be employed in breeding programs to create new sunflower lines resistant to biotic and abiotic stresses.

The transcriptional regulatory module CsHB5-CsbZIP44 positively regulates abscisic acid-mediated carotenoid biosynthesis in citrus (Citrus spp.).

Carotenoids contribute to fruit coloration and are valuable sources of provitamin A in the human diet. Abscisic acid (ABA) plays an essential role in fruit coloration during citrus fruit ripening, but little is known about the underlying mechanisms. Here, we identified a novel bZIP transcription activator called CsbZIP44, which serves as a central regulator of ABA-mediated citrus carotenoid biosynthesis. CsbZIP44 directly binds to the promoters of four carotenoid metabolism-related genes (CsDXR, CsGGPPs, CsBCH1 and CsNCED2) and activates their expression. Furthermore, our research indicates that CsHB5, a positive regulator of ABA and carotenoid-driven processes, activates the expression of CsbZIP44 by binding to its promoter. Additionally, CsHB5 interacts with CsbZIP44 to form a transcriptional regulatory module CsHB5-CsbZIP44, which is responsive to ABA induction and promotes carotenoid accumulation in citrus. Interestingly, we also discover a positive feedback regulation loop between the ABA signal and carotenoid biosynthesis mediated by the CsHB5-CsbZIP44 transcriptional regulatory module. Our findings show that CsHB5-CsbZIP44 precisely modulates ABA signal-mediated carotenoid metabolism, providing an effective strategy for quality improvement of citrus fruit and other crops.

Differences of skin carotenoids and adherence to the Mediterranean Diet pattern in adults from Southern Italy and Dominican Republic.

BACKGROUND: The measurement of the skin carotenoids using the Veggie Meter® has emerged as a rapid objective method for assessing fruit and vegetable intake, highly recommended by the Mediterranean Diet (MD), which represents one of the healthiest dietary patterns, worldwide. This study aimed to examine differences in skin carotenoid content and degree of adherence to the MD pattern between two adult populations from Southern Italy and the Dominican Republic. METHODS: This cross-sectional study enrolled a total of 995 adults, 601 subjects from Italy and 394 from the Dominican Republic. All participants underwent anthropometric measurements and skin carotenoid assessment by Veggie Meter®. Adherence to the MD and lifestyle were evaluated using the Mediterranean Diet Adherence Screener (MEDAS) and the Mediterranean Lifestyle Index (MEDLIFE) questionnaires. Correlations between the skin carotenoid and MEDAS score were estimated using Pearson's correlation coefficient. Multiple linear regression models were created to determine variables that affect skin carotenoid score for both populations. RESULTS: Mean total skin carotenoids were higher in the Italian compared to the Dominican Republic population (342.4 ± 92.4 vs 282.9 ± 90.3; p < 0.005) regardless of sex (women: 318.5 ± 88.9 vs 277.3 ± 91.9, p < 0.005 and men: 371.7 ± 88.3 vs 289.5 ± 88.1, p < 0.005), and remaining statistically significant after age-adjustment of the Dominican Republic sample. Using the MEDAS questionnaire, we found a higher MD adherence score in the Italian than in the Dominican Republic population also after age-adjusting data (7.8 ± 2.1 vs 6.2 ± 3.7; p < 0.005) and even when categorized by sex (Italian vs age-adjusted Dominican Republic women: 7.9 ± 2.1 vs 6.3 ± 2.6; Italian vs age-adjusted Dominican Republic men: 7.7 ± 2.2 vs 6.0 ± 4.7; p < 0.005). Using the MEDLIFE test, total Italians presented a lower score with respect to the age-adjusted Dominican Republic population (3.2 ± 1.2 vs 3.4 ± 1.4; p < 0.05). In multiple regression analysis, skin carotenoids were associated with sex and negatively associated with BMI in the Italian population (sex: ß: 54.95; 95% CI: 40.11, 69.78; p < 0.0001; BMI: ß: - 1.60; 95% CI: - 2.98,0.86; p = 0.03), while they resulted associated with age and sex in the Dominican Republic population (age: ß: 2.76; 95% CI: 1.92, 3.56; p < 0.001; sex: ß: 23.29; 95% CI: 5.93, 40.64; p = 0.009). Interestingly, skin carotenoids were positively correlated with MEDAS score in both populations (Italy: r = 0.03, p < 0.0001, Dominican Republic: r = 0.16, p = 0.002). CONCLUSIONS: This study provides the assessment of the adherence to the MD and skin carotenoid content in adults living in Southern Italy and the Dominican Republic, showing a higher MD adherence score and a skin carotenoid content in inhabitants from the Mediterranean region. Our findings highlight the need to globally encourage fruit and vegetable intake, particularly in non-Mediterranean area.

Intramolecular charge transfer and the function of vibronic excitons in photosynthetic light harvesting.

A widely discussed explanation for the prevalence of pairs or clusters of closely spaced electronic chromophores in photosynthetic light-harvesting proteins is the presence of ultrafast and highly directional excitation energy transfer pathways mediated by vibronic excitons, the delocalized optical excitations derived from mixing of the electronic and vibrational states of the chromophores. We discuss herein the hypothesis that internal conversion processes between exciton states on the <100 fs timescale are possible when the excitonic potential energy surfaces are controlled by the vibrational modes that induce charge transfer character in a strongly coupled system of chromophores. We discuss two examples, the peridinin-chlorophyll protein from marine dinoflagellates and the intact phycobilisome from cyanobacteria, in which the intramolecular charge-transfer (ICT) character arising from out-of-plane distortion of the conjugation of carotenoid or bilin chromophores also results in localization of the initially delocalized optical excitation on the vibrational timescale. Tuning of the ground state conformations of the chromophores to manipulate their ICT character provides a natural photoregulatory mechanism, which would control the overall quantum yield of excitation energy transfer by turning on and off the delocalized character of the optical excitations.

Relationship between non-photochemical quenching efficiency and the energy transfer rate from phycobilisomes to photosystem II.

The chromophorylated PBLcm domain of the ApcE linker protein in the cyanobacterial phycobilisome (PBS) serves as a bottleneck for Förster resonance energy transfer (FRET) from the PBS to the antennal chlorophyll of photosystem II (PS II) and as a redirection point for energy distribution to the orange protein ketocarotenoid (OCP), which is excitonically coupled to the PBLcm chromophore in the process of non-photochemical quenching (NPQ) under high light conditions. The involvement of PBLcm in the quenching process was first directly demonstrated by measuring steady-state fluorescence spectra of cyanobacterial cells at different stages of NPQ development. The time required to transfer energy from the PBLcm to the OCP is several times shorter than the time it takes to transfer energy from the PBLcm to the PS II, ensuring quenching efficiency. The data obtained provide an explanation for the different rates of PBS quenching in vivo and in vitro according to the half ratio of OCP/PBS in the cyanobacterial cell, which is tens of times lower than that realized for an effective NPQ process in solution.

Roles of ApcD and orange carotenoid protein in photoinduction of electron transport upon dark-light transition in the Synechocystis PCC 6803 mutant deficient in flavodiiron protein Flv1.

Flavodiiron proteins Flv1/Flv3 accept electrons from photosystem (PS) I. In this work we investigated light adaptation mechanisms of Flv1-deficient mutant of Synechocystis PCC 6803, incapable to form the Flv1/Flv3 heterodimer. First seconds of dark-light transition were studied by parallel measurements of light-induced changes in chlorophyll fluorescence, P700 redox transformations, fluorescence emission at 77 K, and OCP-dependent fluorescence quenching. During the period of Calvin cycle activation upon dark-light transition, the linear electron transport (LET) in wild type is supported by the Flv1/Flv3 heterodimer, whereas in Δflv1 mutant activation of LET upon illumination is preceded by cyclic electron flow that maintains State 2. The State 2-State 1 transition and Orange Carotenoid Protein (OCP)-dependent non-photochemical quenching occur independently of each other, begin in about 10 s after the illumination of the cells and are accompanied by a short-term re-reduction of the PSI reaction center (P700+). ApcD is important for the State 2-State 1 transition in the Δflv1 mutant, but S-M rise in chlorophyll fluorescence was not completely inhibited in Δflv1/ΔapcD mutant. LET in Δflv1 mutant starts earlier than the S-M rise in chlorophyll fluorescence, and the oxidation of plastoquinol (PQH2) pool promotes the activation of PSII, transient re-reduction of P700+ and transition to State 1. An attempt to induce state transition in the wild type under high intensity light using methyl viologen, highly oxidizing P700 and PQH2, was unsuccessful, showing that oxidation of intersystem electron-transport carriers might be insufficient for the induction of State 2-State 1 transition in wild type of Synechocystis under high light.

Flash-kinetics as a complementary analytical tool in PAM fluorimetry.

A new measuring system based on the already existing Multi-Color-PAM Fluorimeter (Schreiber et al. in Photosynth Res 113:127-144, 2012) was developed that in addition to standard PAM measurements enables pump-and-probe flash measurements and allows simultaneous measurements of the changes in chlorophyll fluorescence yield (F) during application of saturating flashes (ST). A high-power Chip-on-Board LED array provides ST flashes with close to rectangular profiles at wide ranges of widths (0.5 µs to 5 ms), intensities (1.3 mmol to 1.3 mol 440 nm quanta m-2 s-1) and highly flexible repetition times. Using a dedicated rising-edge profile correction, sub-µs time resolution is obtained for assessment of initial fluorescence and rise kinetics. At maximal to moderate flash intensities the flash-kinetics (changes of F during course of ST, STK) are strongly affected by 'High Intensity Quenching' (HIQ), consisting of Car-triplet quenching, TQ, and donor-side-dependent quenching, DQ. The contribution of TQ is estimated by application of a second ST after 20 µs dark-time. Upon application of flash trains (ST sequences with defined repetition times) typical period-4 oscillations in dark fluorescence yield (F0) and ST-induced fluorescence yield, FmST, are obtained which can be measured in vivo both with suspensions and from the surface of leaves. Examples of application with dilute suspensions of Chlorella and an intact dandelion leaf are presented. It is shown that weak far-red light (730-740 nm) advances the S-state distribution of the water-splitting system by one step, resulting in substantial lowering of FmST and also of the I1-level in the polyphasic rise of fluorescence yield induced by a multiple-turnover flash (MT). Based on comparative measurements of STK and the polyphasic rise kinetics with the same Chlorella sample, it is concluded that the generally observed lower values of maximal fluorescence yields using ST-protocols compared to MT-protocols are due to a higher extent of HIQ (mainly DQ) and the contribution of variable PSI fluorescence to FmST.

Far-red light inhibits lateral bud growth mainly through enhancing apical dominance independently of strigolactone synthesis in tomato.

The ratio of red light to far-red light (R:FR) is perceived by light receptors and consequently regulates plant architecture. Regulation of shoot branching by R:FR ratio involves plant hormones. However, the roles of strigolactone (SL), the key shoot branching hormone and the interplay of different hormones in the light regulation of shoot branching in tomato (Solanum lycopersicum) are elusive. Here, we found that defects in SL synthesis genes CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7) and CCD8 in tomato resulted in more lateral bud growth but failed to reverse the FR inhibition of lateral bud growth, which was associated with increased auxin synthesis and decreased synthesis of cytokinin (CK) and brassinosteroid (BR). Treatment of auxin also inhibited shoot branching in ccd mutants. However, CK released the FR inhibition of lateral bud growth in ccd mutants, concomitant with the upregulation of BR synthesis genes. Furthermore, plants that overexpressed BR synthesis gene showed more lateral bud growth and the shoot branching was less sensitive to the low R:FR ratio. The results indicate that SL synthesis is dispensable for light regulation of shoot branching in tomato. Auxin mediates the response to R:FR ratio to regulate shoot branching by suppressing CK and BR synthesis.

Reducing PHYTOENE SYNTHASE activity fine-tunes the abundance of a cis-carotene-derived signal that regulates the PIF3/HY5 module and plastid biogenesis.

PHYTOENE SYNTHASE (PSY) is a rate-limiting enzyme catalysing the first committed step of carotenoid biosynthesis, and changes in PSY gene expression and/or protein activity alter carotenoid composition and plastid differentiation in plants. Four genetic variants of PSY (psy-4, psy-90, psy-130, and psy-145) were identified using a forward genetics approach that rescued leaf virescence phenotypes and plastid abnormalities displayed by the Arabidopsis CAROTENOID ISOMERASE (CRTISO) mutant ccr2 (carotenoid and chloroplast regulation 2) when grown under a shorter photoperiod. The four non-lethal mutations affected alternative splicing, enzyme-substrate interactions, and PSY:ORANGE multi-enzyme complex binding, constituting the dynamic post-transcriptional fine-tuning of PSY levels and activity without changing localization to the stroma and protothylakoid membranes. psy genetic variants did not alter total xanthophyll or ß-carotene accumulation in ccr2, yet they reduced specific acyclic linear cis-carotenes linked to the biosynthesis of a currently unidentified apocarotenoid signal regulating plastid biogenesis, chlorophyll biosynthesis, and photomorphogenic regulation. ccr2 psy variants modulated the PHYTOCHROME-INTERACTING FACTOR 3/ELONGATED HYPOCOTYL 5 (PIF3/HY5) ratio, and displayed a normal prolamellar body formation in etioplasts and chlorophyll accumulation during seedling photomorphogenesis. Thus, suppressing PSY activity and impairing PSY:ORANGE protein interactions revealed how cis-carotene abundance can be fine-tuned through holoenzyme-metabolon interactions to control plastid development.

Galactinol synthase 2 influences the metabolism of chlorophyll, carotenoids, and ethylene in tomato fruits.

Galactinol synthase (GolS), which catalyses the synthesis of galactinol, is the first critical enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs) and contributes to plant growth and development, and resistance mechanisms. However, its role in fruit development remains largely unknown. In this study, we used CRISPR/Cas9 gene-editing technology in tomato (Solanum lycopersicum) to create the gols2 mutant showing uniformly green fruits without dark-green shoulders, and promoting fruit ripening. Analysis indicated that galactinol was undetectable in the ovaries and fruits of the mutant, and the accumulation of chlorophyll and chloroplast development was suppressed in the fruits. RNA-sequencing analysis showed that genes related to chlorophyll accumulation and chloroplast development were down-regulated, including PROTOCHLOROPHYLLIDE OXIDOREDUCTASE, GOLDEN 2-LIKE 2, and CHLOROPHYLL A/B-BINDING PROTEINS. In addition, early color transformation and ethylene release was prompted in the gols2 lines by regulation of the expression of genes involved in carotenoid and ethylene metabolism (e.g. PHYTOENE SYNTHASE 1, CAROTENE CIS-TRANS ISOMERASE, and 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE2/4) and fruit ripening (e.g. RIPENING INHIBITOR, NON-RIPENING, and APETALA2a). Our results provide evidence for the involvement of GolS2 in pigment and ethylene metabolism of tomato fruits.