Transcriptomic and Metabolic Analyses Reveal the Mechanism of Ethylene Production in Stony Hard Peach Fruit during Cold Storage.

Stony hard (SH) peach (Prunus persica L. Batsch) fruit does not release ethylene and has very firm and crisp flesh at ripening, both on- and off-tree. Long-term cold storage can induce ethylene production and a serious risk of chilling injury in SH peach fruit; however, the regulatory mechanism underlying ethylene production in stony hard peach is relatively unclear. In this study, we analyzed the phytohormone levels, fruit firmness, transcriptome, and lipidome changes in SH peach 'Zhongtao 9' (CP9) during cold storage (4 °C). The expression level of the ethylene biosynthesis gene PpACS1 and the content of ethylene in SH peach fruit were found to be upregulated during cold storage. A peak in ABA release was observed before the release of ethylene and the genes involved in ABA biosynthesis and degradation, such as zeaxanthin epoxidase (ZEP) and 8'-hydroxylase (CYP707A) genes, were specifically induced in response to low temperatures. Fruit firmness decreased fairly slowly during the first 20 d of refrigeration, followed by a sharp decline. Furthermore, the expression level of genes encoding cell wall metabolic enzymes, such as polygalacturonase, pectin methylesterase, expansin, galactosidase, and ß-galactosidase, were upregulated only upon refrigeration, as correlated with the decrease in fruit firmness. Lipids belonging to 23 sub-classes underwent differential rearrangement during cold storage, especially ceramide (Cer), monoglycosylceramide (CerG1), phosphatidic acid (PA), and diacyglyceride (DG), which may eventually lead to ethylene production. Exogenous PC treatment provoked a higher rate of ethylene production. We suspected that the abnormal metabolism of ABA and cell membrane lipids promotes the production of ethylene under low temperature conditions, causing the fruit to soften. In addition, ERF transcription factors also play an important role in regulating lipid, hormone, and cell wall metabolism during long-term cold storage. Overall, the results of this study give us a deeper understanding of the molecular mechanism of ethylene biosynthesis during the postharvest storage of SH peach fruit under low-temperature conditions.

Plant LHC-like proteins show robust folding and static non-photochemical quenching.

Life on Earth depends on photosynthesis, the conversion of light energy into chemical energy. Plants collect photons by light harvesting complexes (LHC)-abundant membrane proteins containing chlorophyll and xanthophyll molecules. LHC-like proteins are similar in their amino acid sequence to true LHC antennae, however, they rather serve a photoprotective function. Whether the LHC-like proteins bind pigments has remained unclear. Here, we characterize plant LHC-like proteins (LIL3 and ELIP2) produced in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). Both proteins were associated with chlorophyll a (Chl) and zeaxanthin and LIL3 was shown to be capable of quenching Chl fluorescence via direct energy transfer from the Chl Qy state to zeaxanthin S1 state. Interestingly, the ability of the ELIP2 protein to quench can be acquired by modifying its N-terminal sequence. By employing Synechocystis carotenoid mutants and site-directed mutagenesis we demonstrate that, although LIL3 does not need pigments for folding, pigments stabilize the LIL3 dimer.

Antenna Protein Clustering In Vitro Unveiled by Fluorescence Correlation Spectroscopy.

Antenna protein aggregation is one of the principal mechanisms considered effective in protecting phototrophs against high light damage. Commonly, it is induced, in vitro, by decreasing detergent concentration and pH of a solution of purified antennas; the resulting reduction in fluorescence emission is considered to be representative of non-photochemical quenching in vivo. However, little is known about the actual size and organization of antenna particles formed by this means, and hence the physiological relevance of this experimental approach is questionable. Here, a quasi-single molecule method, fluorescence correlation spectroscopy (FCS), was applied during in vitro quenching of LHCII trimers from higher plants for a parallel estimation of particle size, fluorescence, and antenna cluster homogeneity in a single measurement. FCS revealed that, below detergent critical micelle concentration, low pH promoted the formation of large protein oligomers of sizes up to micrometers, and therefore is apparently incompatible with thylakoid membranes. In contrast, LHCII clusters formed at high pH were smaller and homogenous, and yet still capable of efficient quenching. The results altogether set the physiological validity limits of in vitro quenching experiments. Our data also support the idea that the small, moderately quenching LHCII oligomers found at high pH could be relevant with respect to non-photochemical quenching in vivo.

Xanthophyll carotenoids stabilise the association of cyanobacterial chlorophyll synthase with the LHC-like protein HliD.

Chlorophyll synthase (ChlG) catalyses a terminal reaction in the chlorophyll biosynthesis pathway, attachment of phytol or geranylgeraniol to the C17 propionate of chlorophyllide. Cyanobacterial ChlG forms a stable complex with high light-inducible protein D (HliD), a small single-helix protein homologous to the third transmembrane helix of plant light-harvesting complexes (LHCs). The ChlG-HliD assembly binds chlorophyll, ß-carotene, zeaxanthin and myxoxanthophyll and associates with the YidC insertase, most likely to facilitate incorporation of chlorophyll into translated photosystem apoproteins. HliD independently coordinates chlorophyll and ß-carotene but the role of the xanthophylls, which appear to be exclusive to the core ChlG-HliD assembly, is unclear. Here we generated mutants of Synechocystis sp. PCC 6803 lacking specific combinations of carotenoids or HliD in a background with FLAG- or His-tagged ChlG. Immunoprecipitation experiments and analysis of isolated membranes demonstrate that the absence of zeaxanthin and myxoxanthophyll significantly weakens the interaction between HliD and ChlG. ChlG alone does not bind carotenoids and accumulation of the chlorophyllide substrate in the absence of xanthophylls indicates that activity/stability of the 'naked' enzyme is perturbed. In contrast, the interaction of HliD with a second partner, the photosystem II assembly factor Ycf39, is preserved in the absence of xanthophylls. We propose that xanthophylls are required for the stable association of ChlG and HliD, acting as a 'molecular glue' at the lateral transmembrane interface between these proteins; roles for zeaxanthin and myxoxanthophyll in ChlG-HliD complexation are discussed, as well as the possible presence of similar complexes between LHC-like proteins and chlorophyll biosynthesis enzymes in plants.

Chlorophyll-carotenoid excitation energy transfer and charge transfer in Nannochloropsis oceanica for the regulation of photosynthesis.

Nonphotochemical quenching (NPQ) is a proxy for photoprotective thermal dissipation processes that regulate photosynthetic light harvesting. The identification of NPQ mechanisms and their molecular or physiological triggering factors under in vivo conditions is a matter of controversy. Here, to investigate chlorophyll (Chl)-zeaxanthin (Zea) excitation energy transfer (EET) and charge transfer (CT) as possible NPQ mechanisms, we performed transient absorption (TA) spectroscopy on live cells of the microalga Nannochloropsis oceanica We obtained evidence for the operation of both EET and CT quenching by observing spectral features associated with the Zea S1 and Zea●+ excited-state absorption (ESA) signals, respectively, after Chl excitation. Knockout mutants for genes encoding either violaxanthin de-epoxidase or LHCX1 proteins exhibited strongly inhibited NPQ capabilities and lacked detectable Zea S1 and Zea●+ ESA signals in vivo, which strongly suggests that the accumulation of Zea and active LHCX1 is essential for both EET and CT quenching in N. oceanica.

Complete genome sequence of Flavobacterium kingsejongi WV39, a type species of the genus Flavobacterium and a microbial C40 carotenoid zeaxanthin producer.

A novel species, Flavobacterium kingsejongi WV39, isolated from feces of Antarctic penguins and a type species of the genus Flavobacterium, is yellow because it synthesizes a C40 carotenoid zeaxanthin. The complete genome of F. kingsejongi WV39 is made up of a single circular chromosome (4,224,053bp, 39.8% G+C content). Annotation analysis revealed 3,955 coding sequences, 72 RNAs (18 rRNA+54 tRNA), and five genes involved in zeaxanthin biosynthesis. The key gene encoding ß-carotenoid hydroxylase (CrtZ), which is the last enzyme in the zeaxanthin biosynthetic pathway, was cloned and subjected to complementary analysis in a heterologous E. coli strain. The CrtZ of F. kingsejongi WV39 showed a higher activity than other reported CrtZs.

Photoautotrophic production of macular pigment in a Chlamydomonas reinhardtii strain generated by using DNA-free CRISPR-Cas9 RNP-mediated mutagenesis.

Lutein and zeaxanthin are dietary carotenoids reported to be protective against age-related macular degeneration. Recently, the green alga Chlamydomonas reinhardtii has received attention as a photosynthetic cell factory, but the potential of this alga for carotenoid production has not yet been evaluated. In this study, we selected the C. reinhardtii CC-4349 strain as the best candidate among seven laboratory strains tested for carotenoid production. A knock-out mutant of the zeaxanthin epoxidase gene induced by preassembled DNA-free CRISPR-Cas9 ribonucleoproteins in the CC-4349 strain had a significantly higher zeaxanthin content (56-fold) and productivity (47-fold) than the wild type without the reduction in lutein level. Furthermore, we produced eggs fortified with lutein (2-fold) and zeaxanthin (2.2-fold) by feeding hens a diet containing the mutant. Our results clearly demonstrate the possibility of cost-effective commercial use of microalgal mutants induced by DNA-free CRISPR-Cas9 ribonucleoproteins in algal biotechnology for the production of high-value products.

The high content of ß-carotene present in orange-pulp fruits of Carica papaya L. is not correlated with a high expression of the CpLCY-ß2 gene.

We investigated the transcriptional regulation of six genes involved in carotenoid biosynthesis, together with the carotenoid accumulation during postharvest ripening of three different papaya genotypes of contrasting pulp color. Red-pulp genotype (RPG) showed the lowest content of yellow pigments (YP), such as ß-cryptoxanthin, zeaxanthin, and violaxanthin, together with the lowest relative expression levels (REL) of CpLCY-ß2 and CpCHX-ß genes. On the contrary, the yellow-pulp genotype (YPG) showed the highest content of YP and the highest REL of CpLCY-ß2 and CpCHX-ß genes. Interestingly, the orange-pulp genotype (OPG) showed intermediate content of YP and intermediate REL of CpLCY-ß2 and CpCHX-ß genes. The highest content of ß-carotene shown by OPG despite having an intermediate REL of the CpLCY-ß2 genes, suggests a post-transcriptional regulation. Thus, the transcriptional level of the genes, directing the carotenoid biosynthesis pathway, can partially explain the accumulation of carotenoids during the postharvest ripening in C. papaya genotypes of contrasting pulp color.

Dissecting and modeling zeaxanthin- and lutein-dependent nonphotochemical quenching in Arabidopsis thaliana.

Photosynthetic organisms use various photoprotective mechanisms to dissipate excess photoexcitation as heat in a process called nonphotochemical quenching (NPQ). Regulation of NPQ allows for a rapid response to changes in light intensity and in vascular plants, is primarily triggered by a pH gradient across the thylakoid membrane (∆pH). The response is mediated by the PsbS protein and various xanthophylls. Time-correlated single-photon counting (TCSPC) measurements were performed on Arabidopsis thaliana to quantify the dependence of the response of NPQ to changes in light intensity on the presence and accumulation of zeaxanthin and lutein. Measurements were performed on WT and mutant plants deficient in one or both of the xanthophylls as well as a transgenic line that accumulates lutein via an engineered lutein epoxide cycle. Changes in the response of NPQ to light acclimation in WT and mutant plants were observed between two successive light acclimation cycles, suggesting that the character of the rapid and reversible response of NPQ in fully dark-acclimated plants is substantially different from in conditions plants are likely to experience caused by changes in light intensity during daylight. Mathematical models of the response of zeaxanthin- and lutein-dependent reversible NPQ were constructed that accurately describe the observed differences between the light acclimation periods. Finally, the WT response of NPQ was reconstructed from isolated components present in mutant plants with a single common scaling factor, which enabled deconvolution of the relative contributions of zeaxanthin- and lutein-dependent NPQ.

Cloning and Functional Characterization of a Lycopene ß-Cyclase from Macrophytic Red Alga Bangia fuscopurpurea.

Lycopene cyclases cyclize the open ends of acyclic lycopene (ψ,ψ-carotene) into ß- or ε-ionone rings in the crucial bifurcation step of carotenoid biosynthesis. Among all carotenoid constituents, ß-carotene (ß,ß-carotene) is found in all photosynthetic organisms, except for purple bacteria and heliobacteria, suggesting a ubiquitous distribution of lycopene ß-cyclase activity in these organisms. In this work, we isolated a gene (BfLCYB) encoding a lycopene ß-cyclase from Bangia fuscopurpurea, a red alga that is considered to be one of the primitive multicellular eukaryotic photosynthetic organisms and accumulates carotenoid constituents with both ß- and ε-rings, including ß-carotene, zeaxanthin, α-carotene (ß,ε-carotene) and lutein. Functional complementation in Escherichia coli demonstrated that BfLCYB is able to catalyze cyclization of lycopene into monocyclic γ-carotene (ß,ψ-carotene) and bicyclic ß-carotene, and cyclization of the open end of monocyclic δ-carotene (ε,ψ-carotene) to produce α-carotene. No ε-cyclization activity was identified for BfLCYB. Sequence comparison showed that BfLCYB shares conserved domains with other functionally characterized lycopene cyclases from different organisms and belongs to a group of ancient lycopene cyclases. Although B. fuscopurpurea also synthesizes α-carotene and lutein, its enzyme-catalyzing ε-cyclization is still unknown.

Dynamic control of the mevalonate pathway expression for improved zeaxanthin production in Escherichia coli and comparative proteome analysis.

Engineered heterologous multi-gene metabolic pathways often suffer from flux imbalance and toxic metabolites, as the production host typically lacks the regulatory mechanisms for the heterologous pathway. Here, we first coordinated the expression of all genes of the mevalonate (MEV) pathway from Saccharomyces cerevisiae using the tunable intergenic regions (TIGRs), and then dynamically regulated the TIGR-mediated MEV pathway to prevent the accumulation of toxic metabolites by using IPP/FPP-responsive promoter. After introduction of the dynamically controlled TIGR-mediated MEV pathway into Escherichia coli, the content and concentration of zeaxanthin in shaker flask cultures were 2.0- and 2.1-fold higher, respectively, than those of the strain harboring the statically controlled non-TIGR-mediated MEV pathway. The content and concentration of zeaxanthin in E. coli ZEAX (pZSPgadE-MevTTIGR-MevBTIGRIS-2) reached 722.46mg/L and 23.16mg/g dry cell weight (DCW), respectively, in 5.0L fed-batch fermentation. We also comparatively analyzed the proteomes between E. coli ZEAX and E. coli ZEAX (pZSPgadE-MevTTIGR-MevBTIGRIS-2) to understand the mechanism of zeaxanthin biosynthesis. The results of the comparative proteomes demonstrate that zeaxanthin overproduction may be associated with increased precursor availability, increased NADPH availability, increased ATP availability, oxidative stress response, and increased membrane storage capacity for zeaxanthin due to changes in both cellular shape and membrane composition.