Characterization of lycopene ß-cyclase from Dunaliella bardawil for enhanced ß-carotene production and salt tolerance.

Dunaliella can accumulate more ß-carotene (10 % or even more of the dry weight of cells) than any other species. Lycopene ß-cyclase (LcyB) is the key enzyme in the catalysis of lycopene to ß-carotene. In the present research, we used Escherichia coli BL21 (DE3) as host to construct two different types of engineering bacteria, one expressing the D. bardawil LcyB and the other expressing the orthologue Erwinia uredovora crtY. The catalytic ability of LcyB and CrtY were evaluated by comparing the ß-carotene yields of the two E. coli BL21(DE3) strains, whose salt tolerance was simultaneously compared by cultivated them under different NaCl concentrations (1 %, 2 %, and 4 %). We also interfered with the LcyB gene to investigate the effect of LcyB in D. bardawil. Results displayed that the ß-carotene yield of the LcyB-transformant significantly increased by about 48 % compared with the crtY-transformant. Additionally, LcyB was verified to be able to enhance the salt tolerance of E. coli BL21 (DE3). It is concluded that D. bardawil LcyB not only has better catalytic ability but also is able to confer salt tolerance to cells. Interfering D. bardawil LcyB induced the low expression of LcyB and the changes of growth and carotenoids metabolism in D. bardawil.

The role of SlCHRC in carotenoid biosynthesis and plastid development in tomato fruit.

Chromoplasts are specialized plastids in plants involved in carotenoid synthesis, accumulation, and stress resistance. In tomatoes (Solanum lycopersicum), the Chromoplast-associated carotenoid binding protein (CHRC) regulates chromoplast development and carotenoid accumulation, although its precise mechanisms are not yet fully understood. To investigate the role of SlCHRC in carotenoid biosynthesis, we generated transgenic tomatoes using overexpression (oe-SlCHRC) and CRISPR/Cas9-mediated gene editing (cr-SlCHRC) techniques. The results demonstrated inhibited fruit ripening and delayed onset of color break in both transgenic lines. The oe-SlCHRC lines exhibited increased carotenoid accumulation, particularly (E/Z)-phytoene, lycopene, and γ-carotene, with abundant plastoglobules and carotenoid crystals observed via TEM. In contrast, cr-SlCHRC mutants showed a greener phenotype, reduced carotenoid content, and fewer plastoglobules at the BK + 10 stage. Transcriptome analysis indicated that SlCHRC influences key genes in carotenoid biosynthesis, such as SlNCED2, as well as genes related to chloroplast development, photosynthesis, and plastoglobule formation. Additionally, SlCHRC enhances heat stress tolerance in tomato fruits by upregulating heat shock proteins (HSPs), antioxidants, and proline accumulation. These findings indicate that SlCHRC plays a crucial role in improving tomato fruit quality under heat stress conditions.

Optimizing tomato waste hydrolysate for enhanced fucoxanthin biosynthesis in mixotrophic cultivation of Isochrysis galbana.

Vegetable waste, rich in bioactive compounds, offers a promising resource for producing value-added products. This study explored the use of tomato waste, containing glucose (40 mg/g), lycopene (95.12 µg/g), and ß-carotene (24.31 µg/g), for cultivating fucoxanthin-rich Isochrysis galbana. Water-soluble lycopene (2.0 µg/mL) and ß-carotene (0.4 µg/mL) effectively upregulated key carotenoid synthesis genes and boosted cell growth and fucoxanthin production (3.64 and 3.60 pg/cell, respectively) within 10 days in a mixotrophic culture. Optimized tomato waste hydrolysate achieved a high cell density of 1.21 × 107 cells/mL, 2.13 g/L biomass, and 21.02 mg/g fucoxanthin. This study highlights the potential of combining tomato waste with microalgae for a novel and innovative approach towards waste management and resource utilization.

Unraveling the Genetic Control of Pigment Accumulation in Physalis Fruits.

Physalis pubescens and Physalis alkekengi, members of the Physalis genus, are valued for their delicious and medicinal fruits as well as their different ripened fruit colors-golden for P. pubescens and scarlet for P. alkekengi. This study aimed to elucidate the pigment composition and genetic mechanisms during fruit maturation in these species. Fruit samples were collected at four development stages, analyzed using spectrophotometry and high-performance liquid chromatography (HPLC), and complemented with transcriptome sequencing to assess gene expression related to pigment biosynthesis. ß-carotene was identified as the dominant pigment in P. pubescens, contrasting with P. alkekengi, which contained both lycopene and ß-carotene. The carotenoid biosynthesis pathway was central to fruit pigmentation in both species. Key genes pf02G043370 and pf06G178980 in P. pubescens, and TRINITY_DN20150_c1_g3, TRINITY_DN10183_c0_g1, and TRINITY_DN23805_c0_g3 in P. alkekengi were associated with carotenoid production. Notably, the MYB-related and bHLH transcription factors (TFs) regulated zeta-carotene isomerase and ß-hydroxylase activities in P. pubescens with the MYB-related TF showing dual regulatory roles. In P. alkekengi, six TF families-bHLH, HSF, WRKY, M-type MADS, AP2, and NAC-were implicated in controlling carotenoid synthesis enzymes. Our findings highlight the intricate regulatory network governing pigmentation and provide insights into Physalis germplasm's genetic improvement and conservation.

Unveiling the regulatory role of DzAGL6-1 in carotenoid biosynthesis during durian (Durio zibethinus) fruit development.

KEY MESSAGE: Approximately 119 MADS-box genes have been identified in durian. Moreover, DzAGL6-1 primarily expressed during fruit development, activates the DzPSY promoter. Transient expression of DzAGL6-1 in tomatoes influences carotenoid production. MADS-box transcription factors play a crucial role in regulating plant biological processes, including fruit ripening and associated events. This study aimed to comprehend the mechanisms involved in durian fruit development and ripening and carotenoid production by conducting a genome-wide analysis of MADS-box proteins in durian (Durio zibethinus L.), an economically important fruit in Southeast Asia. A total of 119 durian MADS-box proteins were identified from the genome of the 'Musang King' cultivar. Based on the phylogenetic analysis, the proteins were classified into types I and II, which exhibited similar conserved motif compositions. Notably, only 16 durian MADS-box genes exhibited fruit-specific expression patterns. Among these genes, DzAGL6-1 was predominantly expressed during fruit development, a stage at which carotenoid biosynthesis is activated. Transient expression of DzAGL6-1 in tomato fruit increased the transcript level of the carotenoid biosynthetic gene phytoene synthase (PSY) and the ß-carotene content. Furthermore, DzAGL6-1 activated the promoter activity of DzPSY, as demonstrated by a dual-luciferase assay. These findings provide insights into the role of MADS-box transcription factors in regulating carotenoid biosynthesis during durian fruit development.

Enhancing the Catalytic Activity of Geranylgeranyl Diphosphate Synthase through Ancestral Sequence Reconstruction and Semirational Design.

Geranylgeranyl diphosphate synthase (GGPPS) is the crucial bottleneck in carotenoid biosynthesis. However, low activity limits the broad application of GGPPS. In this study, OsGGPPS1 in rice was engineered based on ancestral sequence reconstruction (ASR) and semirational design to improve the catalytic performances of existing GGPPS. The better mutant of A22R/A26P with improved enzyme activity was generated based on ASR. Additionally, the improved enzyme activity of mutants as V162A/M218S/F227Y was designed using a semirational design. The combinatorial assembly of the d-OsGGPPS1 mutant (A22R/A26P/V162A/M218S/F227Y) exhibited higher conversion of IPP and each cosubstrate of DMAPP for 9.8-fold in GPP production, GPP for 6.4-fold in FPP production, and FPP for 1.4-fold in GGPP production relative to wild-type OsGGPPS1 at 25 °C, which showed higher conversion than wild-type OsGGPPS1 at temperatures as high as 50 °C. The successful design of OsGGPPS1 was representative of protein engineering, which will shed new light on GGPPS engineering and active plant pigment resource utilization.

Carotenoid biosynthesis profiling unveils the variance of flower coloration in Tagetes erecta and enhances fruit pigmentation in tomato.

Carotenoids play a pivotal role in plant. Tagetes erecta, commonly called marigold, has increasing nutritional and economic value due to its high level of carotenoids in flower. However, the functional genes in the carotenoid biosynthesis of T. erecta have not been studied. In this work, three T. erecta varieties with flowers of yellow, yellow-orange and orange color, respectively, were examined for carotenoids composition and corresponding expression profiling of biosynthetic genes at four developmental stages. The results indicated that the varieties with higher lutein content, orange-flower 'Juwang' and yellow-orange 'Taishan', exhibited significant upregulation of genes in the upstream biosynthesis pathway, especially PDS (phytoene desaturase), PSY (phytoene synthase) and ZDS (zeta-carotene desaturase), whereas downstream carotenoid cleavage genes CCD (carotenoid cleavage dioxygenase) were markedly downregulated throughout flower development in the highest lutein containing variety 'Juwang'. Furthermore, marigold TePDS, TePSYS3 and TeZDS were isolated and transformed into tomato. Overexpression of TePDS or TeZDS resulted in the promotion of fruit ripening and accumulation of carotenoids in the transgenic lines. On the other hand, marigold TePSYS3 showed multiple effects, not only on fruit carotenogenesis but also on pigmentation patterns in vegetative tissues and plant growth. Taken together, the variations in expression profiles of the biosynthetic genes contribute to dynamic change in carotenoid levels and diversity of flower coloration in T. erecta. These functional genes of T. erecta were verified in tomato and provide targets for genetic improvement of fruit carotenoids accumulation.

A comprehensive physiological and -Omic analysis of trypsin-mediated protection of green pepper fruits from chilling injury.

Chilling injury (CI) in green pepper fruits during low-temperature storage causes a significant decline in quality. The present study utilized physiological, transcriptomic, and metabolomic analyses to idneitfy the mechanisms by which trypsin mitigates CI in green peppers stored at 4 °C for 8 days, followed by 3 days of shelf life. Results indicated that the trypsin treatment significantly reduced electrolyte leakage and the CI index in peppers, effectively extending their shelf life and preserving postharvest quality. After 4 days of storage, comparative -omic analyses identified 2514 differentially expressed genes (DEGs) and 397 differentially abundant metabolites (DAMs) between trypsin-treated and control peppers. The trypsin treatment induced changes in sugar metabolism, modulating the expression of HK, SUS, INV, and GLGC, which affected the abundance of metabolites such as CDP-glucose and α-D-p-glucose. Trypsin also enhanced carotenoid metabolism, altering the abundance of rhodopinal glucoside, 1'-hydroxyl-γ-carotene glucoside, and farnesyl 1-PP, and influencing the expression of PDS, CRTH, CRTB, and LUT5. Notably, the trypsin treatment activated the mitogen-activated protein kinase (MAPK) pathway that plays an integral role in the signal transduction of abiotic stress. Differential expression of FLS2, ELF18, PTO, PR1, PTI5, WPKY, MEKK1, and MPK6 genes in the MAPK pathway was observed, which was correlated with CI mitigation in green peppers during cold storage. In conclusion, trypsin is an effective treatment for reducing CI in green peppers during cold storage. The present study provides valuable insights into its physiological and molecular impact on green pepper fruit.

Functional Characterization of the First Bona Fide Phytoene Synthase in Red Algae from Pyropia yezoensis.

The formation of phytoene by condensing two geranylgeranyl diphosphate molecules catalyzed by phytoene synthase (PSY) is the first committed and rate-limiting step in carotenoid biosynthesis, which has been extensively investigated in bacteria, land plants and microalgae. However, this step in macroalgae remains unknown. In the present study, a gene encoding putative phytoene synthase was cloned from the economic red alga Pyropia yezoensis-a species that has long been used in food and pharmaceuticals. The conservative motifs/domains and the tertiary structure predicted using bioinformatic tools suggested that the cloned PyPSY should encode a phytoene synthase; this was empirically confirmed by pigment complementation in E. coli. This phytoene synthase was encoded by a single copy gene, whose expression was presumably regulated by many factors. The phylogenetic relationship of PSYs from different organisms suggested that red algae are probably the progeny of primary endosymbiosis and plastid donors of secondary endosymbiosis.

Two Distinct Enzymes Have Both Phytoene Desaturase and 3,4-Desaturase Activities Involved in Carotenoid Biosynthesis by the Extremely Halophilic Archaeon Haloarcula japonica.

The extremely halophilic archaeon Haloarcula japonica accumulates the C50 carotenoid, bacterioruberin (BR). To reveal the BR biosynthetic pathway, unidentified phytoene desaturase candidates were functionally characterized in the present study. Two genes encoding the potential phytoene desaturases, c0507 and d1086, were found from the Ha. japonica genome sequence by a homology search using the Basic Local Align Search Tool. Disruption mutants of c0507 and d1086 and their complemented strains transformed with expression plasmids for c0507 and d1086 were subsequently constructed. High-performance liquid chromatography (HPLC) ana-lyses of carotenoids produced by these strains revealed that C0507 and D1086 were both bifunctional enzymes with the same activities as both phytoene desaturase (CrtI) and 3,4-desaturase (CrtD). C0507 and D1086 complemented each other during BR biosynthesis in Ha. japonica. This is the first study to identify two distinct enzymes with both CrtI and CrtD activities in an extremely halophilic archaeon.

Haematococcus lacustris Carotenogensis: A Historical Event of Primary to Secondary Adaptations to Earth's Oxygenation.

(1) Background: Oxygen has exerted a great effect in shaping the environment and driving biological diversity in Earth's history. Green lineage has evolved primary and secondary carotenoid biosynthetic systems to adapt to Earth's oxygenation, e.g., Haematococcus lacustris, which accumulates the highest amount of secondary astaxanthin under stresses. The two systems are controlled by lycopene ε-cyclase (LCYE) and ß-cyclase (LCYB), which leave an important trace in Earth's oxygenation. (2) Objectives: This work intends to disclose the underlying molecular evolutionary mechanism of Earth's oxygenation in shaping green algal carotenogensis with a special focus on lycopene cyclases. (3) Methods: The two kinds of cyclases were analyzed by site-directed mutagenesis, phylogeny, divergence time and functional divergence. (4) Results: Green lineage LCYEs appeared at ~1.5 Ga after the first significant appearance and accumulation of atmospheric oxygen, the so-called Great Oxygenation Event (GOE), from which LCYBs diverged by gene duplication. Bacterial ß-bicyclases evolved from ß-monocyclase. Enhanced catalytic activity accompanied evolutionary transformation from ε-/ß-monocyclase to ß-bicyclase. Strong positive selection occurred in green lineage LCYEs after the GOE and in algal LCYBs during the second oxidation, the Neoproterozoic Oxygenation Event (NOE). Positively selected sites in the catalytic cavities of the enzymes controlled the mono-/bicyclase activity, respectively. Carotenoid profiling revealed that oxidative adaptation has been wildly preserved in evolution. (5) Conclusions: the functionalization of the two enzymes is a result of primary to secondary adaptations to Earth's oxygenation.

Molecular Mechanisms of Chlorophyll Deficiency in Ilex × attenuata 'Sunny Foster' Mutant.

Ilex × attenuata 'Sunny Foster' represents a yellow leaf mutant originating from I. × attenuata 'Foster#2', a popular ornamental woody cultivar. However, the molecular mechanisms underlying this leaf color mutation remain unclear. Using a comprehensive approach encompassing cytological, physiological, and transcriptomic methodologies, notable distinctions were discerned between the mutant specimen and its wild type. The mutant phenotype displayed aberrant chloroplast morphology, diminished chlorophyll content, heightened carotenoid/chlorophyll ratios, and a decelerated rate of plant development. Transcriptome analysis identified differentially expressed genes (DEGs) related to chlorophyll metabolism, carotenoid biosynthesis and photosynthesis. The up-regulation of CHLD and CHLI subunits leads to decreased magnesium chelatase activity, while the up-regulation of COX10 increases heme biosynthesis-both impair chlorophyll synthesis. Conversely, the down-regulation of HEMD hindered chlorophyll synthesis, and the up-regulation of SGR enhanced chlorophyll degradation, resulting in reduced chlorophyll content. Additionally, genes linked to carotenoid biosynthesis, flavonoid metabolism, and photosynthesis were significantly down-regulated. We also identified 311 putative differentially expressed transcription factors, including bHLHs and GLKs. These findings shed light on the molecular mechanisms underlying leaf color mutation in I. × attenuata 'Sunny Foster' and provide a substantial gene reservoir for enhancing leaf color through breeding techniques.

A chromosome-scale genome provides new insights into the typical carotenoid biosynthesis in the important red yeast Rhodotorula glutinis QYH-2023 with anti-inflammatory effects.

Rhodotorula spp. has been studied as one powerful source for a novel cell factory with fast growth and its high added-value biomolecules. However, its inadequate genome and genomic annotation have hindered its widespread use in cosmetics and food industries. Rhodotorula glutinis QYH-2023, was isolated from rice rhizosphere soil, and the highest quality of the genome of the strain was obtained at chromosome level (18 chromosomes) than ever before in red yeast in this study. Comparative genomics analysis revealed that there are more key gene copies of carotenoids biosynthesis in R. glutinis QYH-2023 than other species of Rhodotorula spp. Integrated transcriptome and metabolome analysis revealed that lipids and carotenoids biosynthesis was significantly enriched during fermentation. Subsequent investigation revealed that the over-expression of the strain three genes related to carotenoids biosynthesis in Komagataella phaffii significantly promoted the carotenoid production. Furthermore, in vitro tests initially confirmed that the longer the fermentation period, the synthesized metabolites controlled by R. glutinis QYH-2023 genome had the stronger anti-inflammatory properties. All of the findings revealed a high-quality reference genome which highlight the potential of R. glutinis strains to be employed as chassis cells for biosynthesizing carotenoids and other active chemicals.

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.

A transcriptional cascade mediated by two APETALA2 family members orchestrates carotenoid biosynthesis in tomato.

Carotenoids are important nutrients for human health that must be obtained from plants since they cannot be biosynthesized by the human body. Dissecting the regulatory mechanism of carotenoid metabolism in plants represents the first step toward manipulating carotenoid contents in plants by molecular design breeding. In this study, we determined that SlAP2c, an APETALA2 (AP2) family member, acts as a transcriptional repressor to regulate carotenoid biosynthesis in tomato (Solanum lycopersicum). Knockout of SlAP2c in both the "MicroTom" and "Ailsa Craig" backgrounds resulted in greater lycopene accumulation, whereas overexpression of this gene led to orange-ripe fruit with significantly lower lycopene contents than the wild type. We established that SlAP2c represses the expression of genes involved in lycopene biosynthesis by directly binding to the cis-elements in their promoters. Moreover, SlAP2c relies on its EAR motif to recruit the co-repressors TOPLESS (TPL)2/4 and forms a complex with histone deacetylase (had)1/3, thereby reducing the histone acetylation levels of lycopene biosynthesis genes. Furthermore, SlAP2a, a homolog of SlAP2c, acts upstream of SlAP2c and alleviates the SlAP2c-induced repression of lycopene biosynthesis genes by inhibiting SlAP2c transcription during fruit ripening. Therefore, we identified a transcriptional cascade mediated by AP2 family members that regulates lycopene biosynthesis during fruit ripening in tomato, laying the foundation for the manipulation of carotenoid metabolism in plants.

Potato Solanum tuberosum L. Phytoene Synthase Genes (StPSY1, StPSY2, and StPSY3) Are Involved in the Plant Response to Cold Stress.

The structure and phylogeny of the Solanum tuberosum L. phytoene synthase genes StPSY1, StPSY2, and StPSY3 were characterized. Their expression was studied in potato seedlings exposed to cold stress in the dark phase of the diurnal cycle to simulate night cooling. All of the three genes were activated as the temperature decreased, and the greatest response was observed for StPSY1. StPSY3 was for the first time shown to respond to cold stress and photoperiod. A search for cis-regulatory elements was carried out in the promoter regions and 5'-UTRs of the StPSY genes, and the regulation of all three genes proved associated with the response to light. A high level of cold-induced activation of StPSY1 was tentatively attributed to the presence of cis elements associated with sensitivity to cold and ABA.

Comparative Transcriptome Combined with Morphophysiological Analyses Revealed Carotenoid Biosynthesis for Differential Chilling Tolerance in Two Contrasting Rice (Oryza sativa L.) Genotypes.

Early spring cold spells can lead to leaf chlorosis during the rice seedling greening process. However, the physiological and molecular mechanisms underlying the rice greening process under low-temperature conditions remain unknown. In this study, comparative transcriptome and morphophysiological analyses were performed to investigate the mechanisms mediating the responses of the Koshihikari (Kos) and Kasalath (Kas) rice cultivars to chilling stress. According to their growth-related traits, electrolyte leakage, and chlorophyll fluorescence parameters, Kos was more tolerant to low-temperature stress than Kas. Moreover, chloroplast morphology was more normal (e.g., oval) in Kos than in Kas at 17 °C. The comparative transcriptome analysis revealed 610 up-regulated differentially expressed genes that were common to all four comparisons. Furthermore, carotenoid biosynthesis was identified as a critical pathway for the Kos response to chilling stress. The genes in the carotenoid biosynthesis pathway were expressed at higher levels in Kos than in Kas at 17 °C, which was in accordance with the higher leaf carotenoid content in Kos than in Kas. The lycopene ß-cyclase and lycopene ε-cyclase activities increased more in Kos than in Kas. Additionally, the increases in the violaxanthin de-epoxidase and carotenoid hydroxylase activities in Kos seedlings resulted in the accumulation of zeaxanthin and lutein and mitigated the effects of chilling stress on chloroplasts. These findings have clarified the molecular mechanisms underlying the chilling tolerance of rice seedlings during the greening process.

Transcriptome Insights into Candidate Genes of the SWEET Family and Carotenoid Biosynthesis during Fruit Growth and Development in Prunus salicina 'Huangguan'.

The Chinese plum (Prunus salicina L.) is a fruit tree belonging to the Rosaceae family, native to south-eastern China and widely cultivated throughout the world. Fruit sugar metabolism and color change is an important physiological behavior that directly determines flavor and aroma. Our study analyzed six stages of fruit growth and development using RNA-seq, yielding a total of 14,973 DEGs, and further evaluation of key DEGs revealed a focus on sugar metabolism, flavonoid biosynthesis, carotenoid biosynthesis, and photosynthesis. Using GO and KEGG to enrich differential genes in the pathway, we selected 107 differential genes and obtained 49 significant differential genes related to glucose metabolism. The results of the correlation analyses indicated that two genes of the SWEET family, evm.TU.Chr1.3663 (PsSWEET9) and evm.TU.Chr4.676 (PsSWEET2), could be closely related to the composition of soluble sugars, which was also confirmed in the ethylene treatment experiments. In addition, analysis of the TOP 20 pathways between different growth stages and the green stage, as well as transient overexpression in chili, suggested that capsanthin/capsorubin synthase (PsCCS) of the carotenoid biosynthetic pathway contributed to the color change of plum fruit. These findings provide an insight into the molecular mechanisms involved in the ripening and color change of plum fruit.

Integrative Analysis of Metabolome and Transcriptome Revealed Lutein Metabolism Contributed to Yellow Flower Formation in Prunus mume.

Prunus mume is a famous ornamental woody tree with colorful flowers. P. mume with yellow flowers is one of the most precious varieties. Regretfully, metabolites and regulatory mechanisms of yellow flowers in P. mume are still unclear. This hinders innovation of flower color breeding in P. mume. To elucidate the metabolic components and molecular mechanisms of yellow flowers, we analyzed transcriptome and metabolome between 'HJH' with yellow flowers and 'ZLE' with white flowers. Comparing the metabolome of the two varieties, we determined that carotenoids made contributions to the yellow flowers rather than flavonoids. Lutein was the key differential metabolite to cause yellow coloration of 'HJH'. Transcriptome analysis revealed significant differences in the expression of carotenoid cleavage dioxygenase (CCD) between the two varieties. Specifically, the expression level of PmCCD4 was higher in 'ZLE' than that in 'HJH'. Moreover, we identified six major transcription factors that probably regulated PmCCD4 to affect lutein accumulation. We speculated that carotenoid cleavage genes might be closely related to the yellow flower phenotype in P. mume. Further, the coding sequence of PmCCD4 has been cloned from the 'HJH' petals, and bioinformatics analysis revealed that PmCCD4 possessed conserved histidine residues, ensuring its enzymatic activity. PmCCD4 was closely related to PpCCD4, with a homology of 98.16%. Instantaneous transformation analysis in petal protoplasts of P. mume revealed PmCCD4 localization in the plastid. The overexpression of PmCCD4 significantly reduced the carotenoid content in tobacco plants, especially the lutein content, indicating that lutein might be the primary substrate for PmCCD4. We speculated that PmCCD4 might be involved in the cleavage of lutein in plastids, thereby affecting the formation of yellow flowers in P. mume. This work could establish a material and molecular basis of molecular breeding in P. mume for improving the flower color.

Functional identification of ZDS gene in apple (Malus halliana) and demonstration of it's role in improving saline-alkali stress tolerance.

Carotenoids are powerful antioxidants that mediate transfer of electrons, directly affect abiotic stress responses in plants through regulating activity of antioxidant enzymes. ζ-Carotene desaturase (ZDS) is a key enzyme in carotenoid biosynthesis pathway, which can catalyze ζ-carotene to form lycopene to regulate carotenoid biosynthesis and accumulation. However, the mechanism of its regulation of saline-alkali stress remains unclear. In this research, based on transcriptomic analysis of Malus halliana with a apple rootstock, we screened out ZDS gene (LOC103451012), with significantly high expression by saline-alkali stress, whose expression in the leaves was 10.8-fold than that of the control (0 h) under 48 h of stress. Subsequently, the MhZDS gene was isolated from M. halliana, and transgenic Arabidopsis thaliana, tobacco, and apple calli were successfully obtained through agrobacterium-mediated genetic transformation. We found that overexpression of MhZDS enhanced the tolerance of A. thaliana, tobacco and apple calli under saline-alkali stress and caused a variety of physiological and biochemical changes: compared with wild-type, transgenic plants grew better under saline stress and MhZDS-OE lines showed higher chlorophyll content, POD, SOD, CAT activities and proline content, lower electrical conductivity and MDA content. These results indicate that MhZDS plays an important role in plant resistance to saline-alkali stress, providing excellent resistance genes for the regulatory network of salinity stress response in apples and provide a theoretical basis for the breeding of apple varieties with strong saline-alkali resistance. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01333-5.