Insights into the molecular mechanism of yellow cuticle coloration by a chitin-binding carotenoprotein in gregarious locusts.

Carotenoids are hydrophobic pigments binding to diverse carotenoproteins, many of which remain unexplored. Focusing on yellow gregarious locusts accumulating cuticular carotenoids, here we use engineered Escherichia coli cells to reconstitute a functional water-soluble ß-carotene-binding protein, BBP. HPLC and Raman spectroscopy confirmed that recombinant BBP avidly binds ß-carotene, inducing the unusual vibronic structure of its absorbance spectrum, just like native BBP extracted from the locust cuticles. Bound to recombinant BBP, ß-carotene exhibits pronounced circular dichroism and allows BBP to withstand heating (T0.5 = 68 °C), detergents and pH variations. Using bacteria producing distinct xanthophylls we demonstrate that, while ß-carotene is the preferred carotenoid, BBP can also extract from membranes ketocarotenoids and, very poorly, hydroxycarotenoids. We show that BBP-carotenoid complex reversibly binds to chitin, but not to chitosan, implying the role for chitin acetyl groups in cuticular BBP deposition. Reconstructing such locust coloration mechanism in vitro paves the way for structural studies and BBP applications.

Integrative physiology and transcriptome reveal salt-tolerance differences between two licorice species: Ion transport, Casparian strip formation and flavonoids biosynthesis.

BACKGROUND: Glycyrrhiza inflata Bat. and Glycyrrhiza uralensis Fisch. are both original plants of 'Gan Cao' in the Chinese Pharmacopoeia, and G. uralensis is currently the mainstream variety of licorice and has a long history of use in traditional Chinese medicine. Both of these species have shown some degree of tolerance to salinity, G. inflata exhibits higher salt tolerance than G. uralensis and can grow on saline meadow soils and crusty saline soils. However, the regulatory mechanism responsible for the differences in salt tolerance between different licorice species is unclear. Due to land area-related limitations, the excavation and cultivation of licorice varieties in saline-alkaline areas that both exhibit tolerance to salt and contain highly efficient active substances are needed. The systematic identification of the key genes and pathways associated with the differences in salt tolerance between these two licorice species will be beneficial for cultivating high-quality salt-tolerant licorice G. uralensis plant varieties and for the long-term development of the licorice industry. In this research, the differences in growth response indicators, ion accumulation, and transcription expression between the two licorice species were analyzed. RESULTS: This research included a comprehensive comparison of growth response indicators, including biomass, malondialdehyde (MDA) levels, and total flavonoids content, between two distinct licorice species and an analysis of their ion content and transcriptome expression. In contrast to the result found for G. uralensis, the salt treatment of G. inflata ensured the stable accumulation of biomass and total flavonoids at 0.5 d, 15 d, and 30 d and the restriction of Na+ to the roots while allowing for more K+ and Ca2+ accumulation. Notably, despite the increase in the Na+ concentration in the roots, the MDA concentration remained low. Transcriptome analysis revealed that the regulatory effects of growth and ion transport on the two licorice species were strongly correlated with the following pathways and relevant DEGs: the TCA cycle, the pentose phosphate pathway, and the photosynthetic carbon fixation pathway involved in carbon metabolism; Casparian strip formation (lignin oxidation and translocation, suberin formation) in response to Na+; K+ and Ca2+ translocation, organic solute synthesis (arginine, polyamines, GABA) in response to osmotic stresses; and the biosynthesis of the nonenzymatic antioxidants carotenoids and flavonoids in response to antioxidant stress. Furthermore, the differential expression of the DEGs related to ABA signaling in hormone transduction and the regulation of transcription factors such as the HSF and GRAS families may be associated with the remarkable salt tolerance of G. inflata. CONCLUSION: Compared with G. uralensis, G. inflata exhibits greater salt tolerance, which is primarily attributable to factors related to carbon metabolism, endodermal barrier formation and development, K+ and Ca2+ transport, biosynthesis of carotenoids and flavonoids, and regulation of signal transduction pathways and salt-responsive transcription factors. The formation of the Casparian strip, especially the transport and oxidation of lignin precursors, is likely the primary reason for the markedly higher amount of Na+ in the roots of G. inflata than in those of G. uralensis. The tendency of G. inflata to maintain low MDA levels in its roots under such conditions is closely related to the biosynthesis of flavonoids and carotenoids and the maintenance of the osmotic balance in roots by the absorption of more K+ and Ca2+ to meet growth needs. These findings may provide new insights for developing and cultivating G. uralensis plant species selected for cultivation in saline environments or soils managed through agronomic practices that involve the use of water with a high salt content.

Mutation mapping of a variegated EMS tomato reveals an FtsH-like protein precursor potentially causing patches of four phenotype classes in the leaves with distinctive internal morphology.

BACKGROUND: Leaf variegation is an intriguing phenomenon observed in many plant species. However, questions remain on its mechanisms causing patterns of different colours. In this study, we describe a tomato plant detected in an M2 population of EMS mutagenised seeds, showing variegated leaves with sectors of dark green (DG), medium green (MG), light green (LG) hues, and white (WH). Cells and tissues of these classes, along with wild-type tomato plants, were studied by light, fluorescence, and transmission electron microscopy. We also measured chlorophyll a/b and carotene and quantified the variegation patterns with a machine-learning image analysis tool. We compared the genomes of pooled plants with wild-type-like and mutant phenotypes in a segregating F2 population to reveal candidate genes responsible for the variegation. RESULTS: A genetic test demonstrated a recessive nuclear mutation caused the variegated phenotype. Cross-sections displayed distinct anatomy of four-leaf phenotypes, suggesting a stepwise mesophyll degradation. DG sectors showed large spongy layers, MG presented intercellular spaces in palisade layers, and LG displayed deformed palisade cells. Electron photomicrographs of those mesophyll cells demonstrated a gradual breakdown of the chloroplasts. Chlorophyll a/b and carotene were proportionally reduced in the sectors with reduced green pigments, whereas white sectors have hardly any of these pigments. The colour segmentation system based on machine-learning image analysis was able to convert leaf variegation patterns into binary images for quantitative measurements. The bulk segregant analysis of pooled wild-type-like and variegated progeny enabled the identification of SNP and InDels via bioinformatic analysis. The mutation mapping bioinformatic pipeline revealed a region with three candidate genes in chromosome 4, of which the FtsH-like protein precursor (LOC100037730) carries an SNP that we consider the causal variegated phenotype mutation. Phylogenetic analysis shows the candidate is evolutionary closest to the Arabidopsis VAR1. The synonymous mutation created by the SNP generated a miRNA binding site, potentially disrupting the photoprotection mechanism and thylakoid development, resulting in leaf variegation. CONCLUSION: We described the histology, anatomy, physiology, and image analysis of four classes of cell layers and chloroplast degradation in a tomato plant with a variegated phenotype. The genomics and bioinformatics pipeline revealed a VAR1-related FtsH mutant, the first of its kind in tomato variegation phenotypes. The miRNA binding site of the mutated SNP opens the way to future studies on its epigenetic mechanism underlying the variegation.

Activation of the native PHYTOENE SYNTHASE 1 promoter by modifying near-miss cis-acting elements induces carotenoid biosynthesis in embryogenic rice callus.

KEY MESSAGE: Modification of silent latent endosperm-enabled promoters (SLEEPERs) allows the ectopic activation of non-expressed metabolic genes in rice callus Metabolic engineering in plants typically involves transgene expression or the mutation of endogenous genes. An alternative is promoter modification, where small changes in the promoter sequence allow genes to be switched on or off in particular tissues. To activate silent genes in rice endosperm, we screened native promoters for near-miss cis-acting elements that can be converted to endosperm-active regulatory motifs. We chose rice PHYTOENE SYNTHASE 1 (PSY1), encoding the enzyme responsible for the first committed step in the carotenoid biosynthesis pathway, because it is not expressed in rice endosperm. We identified six motifs within a 120-bp region, upstream of the transcriptional start site, which differed from endosperm-active elements by up to four nucleotides. We mutated four motifs to match functional elements in the endosperm-active BCH2 promoter, and this promoter was able to drive GFP expression in callus and in seeds of regenerated plants. The 4 M promoter was not sufficient to drive PSY1 expression, so we mutated the remaining two elements and used the resulting 6 M promoter to drive PSY1 expression in combination with a PDS transgene. This resulted in deep orange callus tissue indicating the accumulation of carotenoids, which was subsequently confirmed by targeted metabolomics analysis. PSY1 expression driven by the uncorrected or 4 M variants of the promoter plus a PDS transgene produced callus that lacked carotenoids. These results confirm that the adjustment of promoter elements can facilitate the ectopic activation of endogenous plant promoters in rice callus and endosperm and most likely in other tissues and plant species.

Distinct growth patterns in seedling and tillering wheat plants suggests a developmentally restricted role of HYD2 in salt-stress response.

KEY MESSAGE: Mutants lacking functional HYD2 homoeologs showed improved seedling growth, but comparable or increased susceptibility to salt stress in tillering plants, suggesting a developmentally restricted role of HYD2 in salt response. Salinity stress threatens global food security by reducing the yield of staple crops such as wheat (Triticum ssp.). Understanding how wheat responds to salinity stress is crucial for developing climate resilient varieties. In this study, we examined the interplay between carotenoid metabolism and the response to salt (NaCl) stress, a specific form of salinity stress, in tetraploid wheat plants with mutations in carotenoid ß-hydroxylase 1 (HYD1) and HYD2. Our investigation encompassed both the vulnerable seedling stage and the more developed tillering stage of wheat plant growth. Mutant combinations lacking functional HYD2 homoeologs, including hyd-A2 hyd-B2, hyd-A1 hyd-A2 hyd-B2, hyd-B1 hyd-A2 hyd-B2, and hyd-A1 hyd-B1 hyd-A2 hyd-B2, had longer first true leaves and slightly enhanced root growth during germination under salt stress compared to the segregate wild-type (control) plants. Interestingly, these mutant seedlings also showed decreased levels of neoxanthin and violaxanthin (xanthophylls derived from ß-carotene) and an increase in ß-carotene in roots. However, tillering hyd mutant and segregate wild-type plants generally did not differ in their height, tiller count, and biomass production under acute or prolonged salt stress, except for decreases in these parameters observed in the hyd-A1 hyd-B1 hyd-A2 hyd-B2 mutant that indicate its heightened susceptibility to salt stress. Taken together, these findings suggest a significant, yet developmentally restricted role of HYD2 homoeologs in salt-stress response in tetraploid wheat. They also show that hyd-A2 hyd-B2 mutant plants, previously demonstrated for possessing enriched nutritional (ß-carotene) content, maintain an unimpaired ability to withstand salt stress.

Enhancing astaxanthin biosynthesis and pathway expansion towards glycosylated C40 carotenoids by Corynebacterium glutamicum.

Astaxanthin, a versatile C40 carotenoid prized for its applications in food, cosmetics, and health, is a bright red pigment with powerful antioxidant properties. To enhance astaxanthin production in Corynebacterium glutamicum, we employed rational pathway engineering strategies, focused on improving precursor availability and optimizing terminal oxy-functionalized C40 carotenoid biosynthesis. Our efforts resulted in an increased astaxanthin precursor supply with 1.5-fold higher ß-carotene production with strain BETA6 (18 mg g-1 CDW). Further advancements in astaxanthin production were made by fine-tuning the expression of the ß-carotene hydroxylase gene crtZ and ß-carotene ketolase gene crtW, yielding a nearly fivefold increase in astaxanthin (strain ASTA**), with astaxanthin constituting 72% of total carotenoids. ASTA** was successfully transferred to a 2 L fed-batch fermentation with an enhanced titer of 103 mg L-1 astaxanthin with a volumetric productivity of 1.5 mg L-1 h-1. Based on this strain a pathway expansion was achieved towards glycosylated C40 carotenoids under heterologous expression of the glycosyltransferase gene crtX. To the best of our knowledge, this is the first time astaxanthin-ß-D-diglucoside was produced with C. glutamicum achieving high titers of microbial C40 glucosides of 39 mg L-1. This study showcases the potential of pathway engineering to unlock novel C40 carotenoid variants for diverse industrial applications.

Cloning and Analysis of Expression of Genes Related to Carotenoid Metabolism in Different Fruit Color Mutants of Pepper (Capsicum annuum L.).

The formation of fruit color in pepper is closely related to the processes of carotenoid metabolism. In this study, red wild-type pepper XHB, SP01, PC01 and their corresponding mutants H0809 (orange), SP02 (yellow), and PC02 (orange) were used as research materials. The Ggps, Psy, Lcyb, Crtz, Zep, and Ccs genes involved in carotenoid biosynthesis were cloned, and bioinformatics and expression analyses were carried out. The results showed that the full lengths of the six genes were 1110 bp, 2844 bp, 1497 bp, 2025 bp, 510 bp, and 1497 bp, and they encoded 369, 419, 498, 315, 169, and 498 amino acids, respectively. Except for the full-length Ccs gene, which could not be amplified in the yellow mutant SP02 and the orange mutant PC02, the complete full-length sequences of the other genes could be amplified in different materials, indicating that the formation of fruit color in the SP02 and PC02 mutants could be closely related to the deletion or mutation of the Ccs gene. The analytical results of real-time quantitative reverse transcription PCR (qRT-PCR) showed that the Ggps, Psy, Lcyb, Crtz, and Zep genes were expressed at different developmental stages of three pairs of mature-fruit-colored materials, but their patterns of expression were not consistent. The orange mutant H0809 could be amplified to the full Ccs gene sequence, but its expression was maintained at a lower level. It showed a significant difference in expression compared with the wild-type XHB, indicating that the formation of orange mutant H0809 fruit color could be closely related to the different regulatory pattern of Ccs expression. The results provide a theoretical basis for in-depth understanding of the molecular regulatory mechanism of the formation of color in pepper fruit.

Outdoor cultivation and metabolomics exploration of Chlamydomonas engineered for bisabolene production.

Demonstrating outdoor cultivation of engineered microalgae at considerable scales is essential for their prospective large-scale deployment. Hence, this study focuses on the outdoor cultivation of an engineered Chlamydomonas reinhardtii strain, 3XAgBs-SQs, for bisabolene production under natural dynamic conditions of light and temperature. Our preliminary outdoor experiments showed improved growth, but frequent culture collapses in conventional Tris-acetate-phosphate medium. In contrast, modified high-salt medium (HSM) supported prolonged cell survival, outdoor. However, their subsequent outdoor scale-up from 250 mL to 5 L in HSM was effective with 10 g/L bicarbonate supplementation. Pulse amplitude modulation fluorometry and metabolomic analysis further validated their improved photosynthesis and uncompromised metabolic fluxes towards the biomass and the products (natural carotenoids and engineered bisabolene). These strains could produce 906 mg/L bisabolene and 54 mg/L carotenoids, demonstrating the first successful outdoor photoautotrophic cultivation of engineeredC. reinhardtii,establishing it as a one-cell two-wells biorefinery.

Metabolome and Transcriptome Reveal Chlorophyll, Carotenoid, and Anthocyanin Jointly Regulate the Color Formation of Triadica sebifera.

The Chinese tallow tree (Triadica sebifera) is an economically important plant on account of its ornamental value and oil-producing seeds. Leaf colour is a key characteristic of T. sebifera, with yellow-, red- and purple-leaved varieties providing visually impressive displays during autumn. In this study, we performed metabolomic and transcriptomic analyses to gain a better understanding of the mechanisms underlying leaf colour development in purple-leaved T. sebifera at three stages during the autumnal colour transition, namely, green, hemi-purple, and purple leaves. We accordingly detected 370 flavonoid metabolites and 10 anthocyanins, among the latter of which, cyanidin-3-xyloside and peonidin-3-O-glucoside were identified as the predominant compounds in hemi-purple and purple leaves. Transcriptomic analysis revealed that structural genes associated with the anthocyanin biosynthetic pathway, chlorophyll synthesis pathway and carotenoid synthesis pathway were significantly differential expressed at the three assessed colour stages. Additionally, transcription factors associated with the MYB-bHLH-WD40 complex, including 22 R2R3-MYBs, 79 bHLHs and 44 WD40 genes, were identified as candidate regulators of the anthocyanin biosynthetic pathway. Moreover, on the basis of the identified differentially accumulated anthocyanins and key genes, we generated genetic and metabolic regulatory networks for anthocyanin biosynthesis in T. sebifera. These findings provide comprehensive information on the leaf transcriptome and three pigments of T. sebifera, thereby shedding new light on the mechanisms underlying the autumnal colouring of the leaves of this tree.

Genetic Basis and Evolutionary Forces of Sexually Dimorphic Color Variation in a Toad-Headed Agamid Lizard.

In the animal kingdom, sexually dimorphic color variation is a widespread phenomenon that significantly influences survival and reproductive success. However, the genetic underpinnings of this variation remain inadequately understood. Our investigation into sexually dimorphic color variation in the desert-dwelling Guinan population of the toad-headed agamid lizard (Phrynocephalus putjatai) utilized a multidisciplinary approach, encompassing phenotypic, ultrastructural, biochemical, genomic analyses, and behavioral experiments. Our findings unveil the association between distinct skin colorations and varying levels of carotenoid and pteridine pigments. The red coloration in males is determined by a genomic region on chromosome 14, housing four pigmentation genes: BCO2 and three 6-pyruvoyltetrahydropterin synthases. A Guinan population-specific nonsynonymous single nucleotide polymorphism in BCO2 is predicted to alter the electrostatic potential within the binding domain of the BCO2-ß-carotene complex, influencing their interaction. Additionally, the gene MAP7 on chromosome 2 emerges as a potential contributor to the blue coloration in subadults and adult females. Sex-specific expression patterns point to steroid hormone-associated genes (SULT2B1 and SRD5A2) as potential upstream regulators influencing sexually dimorphic coloration. Visual modeling and field experiments support the potential selective advantages of vibrant coloration in desert environments. This implies that natural selection, potentially coupled with assortative mating, might have played a role in fixing color alleles, contributing to prevalence in the local desert habitat. This study provides novel insights into the genetic basis of carotenoid and pteridine-based color variation, shedding light on the evolution of sexually dimorphic coloration in animals. Moreover, it advances our understanding of the driving forces behind such intricate coloration patterns.

Investigation of the morphological, physiological, biochemical, and catabolic characteristics and gene expression under drought stress in tolerant and sensitive genotypes of wild barley [Hordeum vulgare subsp. spontaneum (K. Koch) Asch. & Graebn.].

BACKGROUND: Barley (H. vulgare L.) is an important cereal crop cultivated across various climates globally. Barley and its ancestor (H. vulgare subsp. spontaneum) are an economically valuable model for genetic research and improvement. Drought, among various abiotic stresses, is a substantial threat to agriculture due to its unpredictable nature and significant impact on crop yield. RESULTS: This study was conducted in both greenhouse and laboratory settings. Prior to the study, wild barley accessions were pre-selected based on their sensitivity or tolerance to drought as determined from fieldwork in the 2020-2021 and 2021-2022 cropping seasons. The effects of three levels of drought stress were evaluated (control, 90-95% field capacity [FC]; mild stress, 50-55% FC; and severe stress, 25-30% FC). Several parameters were assessed, including seedling and root growth, enzymatic activity (CAT, SOD, POD), soluble protein levels, chlorophyll content, carotenoids, abaxial and adaxial stomatal density and dimensions, and relative gene expression of Dhn1, SOD, POD, and CAT. Drought stress significantly increased enzyme activities, especially at 25-30% FC, and more in the tolerant genotype. On the other hand, sensitive genotypes showed a notable increase in stomatal density. Under drought stress, there was a general decline in seedling and root growth, protein content, chlorophyll and carotenoids, and stomatal dimensions. Importantly, gene expression analysis revealed that Dhn1, SOD, POD, and CAT were upregulated under drought, with the highest expression levels observed in the drought-tolerant genotype under severe stress conditions (25-30% FC). CONCLUSIONS: Our investigation highlights the distinct morphological, physiological, biochemical, and gene-expression profiles of drought-resistant and drought-sensitive wild barley genotypes under varying degrees of drought.

Comparative transcriptome and coexpression network analysis reveals key pathways and hub candidate genes associated with sunflower (Helianthus annuus L.) drought tolerance.

BACKGROUND: Drought severely limits sunflower production especially at the seedling stage. To investigate the response mechanism of sunflowers to drought stress, we utilized two genotypes of sunflower materials with different drought resistances as test materials. The physiological responses were investigated under well-watered (0 h) and drought-stressed conditions (24 h, 48 h, and 72 h). RESULTS: ANOVA revealed the greatest differences in physiological indices between 72 h of drought stress and 0 h of drought stress. Transcriptome analysis was performed after 72 h of drought stress. At 0 h, there were 7482 and 5627 differentially expressed genes (DEGs) in the leaves of K55 and K58, respectively, and 2150 and 2527 DEGs in the roots of K55 and K58, respectively. A total of 870 transcription factors (TFs) were identified among theDEGs, among which the high-abundance TF families included AP2/ERF, MYB, bHLH,and WRKY. Five modules were screened using weighted gene coexpressionnetwork analysis (WGCNA), three and two of which were positively and negatively, respectively, related to physiological traits. KEGG analysis revealedthat under drought stress, "photosynthesis", "carotenoid biosynthesis", "starch and sucrose metabolism", "ribosome", "carotenoid biosynthesis", "starch and sucrose metabolism", "protein phosphorylation" and "phytohormone signaling" are six important metabolic pathways involved in the response of sunflower to drought stress. Cytoscape software was used to visualize the three key modules, and the hub genes were screened. Finally, a total of 99 important candidate genes that may be associated with the drought response in sunflower plants were obtained, and the homology of these genes was compared with that in Arabidopsis thaliana. CONCLUSIONS: Taken together, our findings could lead to a better understanding of drought tolerance in sunflowers and facilitate the selection of drought-tolerant sunflower varieties.

Water saving irrigation mediates bioactive pigments metabolism and storage capacity in tomato fruit.

Tomato fruit consumption is influenced by flavor and nutrient quality. In the present study, we investigate the impact of water saving irrigation (WSI) as a pre-harvest management on flavor and nutrient quality of tomato fruit. Our results demonstrate that WSI-treated tomato fruit exhibited improved sensory scores as assessed by a taste panel, accompanied by elevated levels of SlGLK2 expression, sugars, acids, and carotenoid contents compared to non-treated fruit. Notably, WSI treatment significantly enhanced the development of chloroplast and plastoglobulus in chromoplast, which served as carotenoid storage sites and upregulated the expression of carotenoid biosynthetic genes. Furthermore, integrated transcriptome and metabolome analysis revealed heightened expression of sugar and flavonoid metabolism pathways in WSI-treated tomato fruit. Remarkably, the master regulator SlMYB12 displayed a substantially increased expression due to WSI. These findings suggest that WSI is an effective and sustainable approach to enhance the pigments metabolism and storage capacity as well as the organoleptic characteristics and nutritional value of tomato fruit, offering a win-win solution for both water conservation and quality improvement in agro-food production.

The CrMYB33 transcription factor positively coordinate the regulation of both carotenoid accumulation and chlorophyll degradation in the peel of citrus fruit.

Citrus, cultivated extensively across the globe, possesses considerable economic importance and nutritional value. With the degradation of chlorophyll and accumulation of carotenoids, mature citrus fruits develop an orange-yellow peel, enhancing fruit value and consumer preference. MYB transcription factors (TFs) exert a significant role in diverse plant developmental processes and investigating their involvement in fruit coloration is crucial for developing new cultivars. This work aimed to characterize a citrus TF, CrMYB33, whose expression was found to be positively correlated with carotenoid biosynthesis during fruit ripening. The interference of CrMYB33 expression in citrus fruit resulted in inhibition of carotenoid accumulation, down-regulation of carotenoid biosynthetic genes, and a slower rate of chlorophyll degradation. Conversely, overexpression of CrMYB33 in tomato (Solanum lycopersicum) enhanced chlorophyll degradation and carotenoid biosynthesis, resulting in a deeper red coloration of the fruits. Furthermore, the transcription of associated genes was upregulated in CrMYB33-overexpressing tomato fruits. Additional assays reveal that CrMYB33 exhibits direct links and activation of the promoters of lycopene ß-cyclase 2 (CrLCYb2), and ß-carotene hydroxylases 2 (CrBCH2), both crucial genes in the carotenoid biosynthetic pathway. Additionally, it was found to inhibit chlorophyllase (CrCLH), a gene essential in chlorophyll degradation. These findings provide insight into the observed changes in LCYb2, BCH2, and CLH expression in the transgenic lines under investigation. In conclusion, our study revealed that CrMYB33 modulates carotenoid accumulation and chlorophyll degradation in citrus fruits through transcriptionally activating genes involved in metabolic pathways.

Natural Albino Mutant of Daylily (Hemerocallis spp.) Reveals a Link between Drought Sensitivity and Photosynthetic Pigments Metabolism.

BACKGROUND: Mutant analysis remains one of the main genetic tools for characterising unclarified gene functions in plants, especially in non-model plants. Daylily (Hemerocallis spp.) is a popular perennial ornamental plant grown worldwide. Analysis of daylily mutants can enhance understanding of genes regulating the albino phenotype and improve the cultivar quality of daylily. METHODS: The natural albino mutant (Alb-⁣/-) was isolated by screening a self-pollinated progeny of daylily cultivar 'black-eyed stella'. Transmission electron microscopy was used in analysing the structure of plastids between mutant and wild-type seedlings. The content of chlorophyll, carotenoids and chlorophyll precursors in plants was measured by ultraviolet spectrophotometry. RNA sequencing and physiological measurements were performed to explore the association between drought tolerance and mutation. RESULTS: All the seedlings of the daylily albino mutants died spontaneously within fifteen days after germination when grown in soil. The carotenoid and chlorophyll content in the leaves of the mutant plants significantly decreased compared with those of the wild-type control. The mutant plants displayed stunted growth, and their leaves were white or light yellow in color. Abnormal plastids such as those showing endomembrane vesiculation and lacking stacking were discovered in the leaves of mutant plants. Furthermore, genetic analysis revealed that a single recessive nuclear gene mutation led to the albino trait, RNA sequencing and real-time quantitative PCR validation showed extensive differences in gene expression between the mutant plants and the wild-type control, and most of the genes related to chlorophyll metabolism were down-regulated, with foldchange ranging from 0.20-0.49. Additionally, the surviving homozygous plants (Alb+⁣/+), which do not contain this mutation, were also isolated by analysing the phenotype of their self-pollinated progeny. The net photosynthesis rate and light saturation point of Alb+⁣/+ were higher than those of heterozygous (Alb+⁣/-) plants. Additionally, the Alb+⁣/+ plants were more tolerant to drought conditions than the Alb+⁣/- plants, suggesting that a heterozygous Alb- mutation is sufficient to negatively affect photosynthetic efficiency and drought tolerance. CONCLUSIONS: The albino mutation negatively affects photosynthetic efficiency and drought tolerance, and homozygous mutation is required for the characteristic albino phenotype. This work highlights the link between albino mutation, photosynthetic pigment metabolism and drought sensitivity in daylily.

SlERF109-like and SlNAC1 Coordinately Regulated Tomato Ripening by Inhibiting ACO1 Transcription.

As a typical climacteric fruit, tomato (Solanum lycopersicum) is widely used for studying the ripening process. The negative regulation of tomato fruits by transcription factor SlNAC1 has been reported, but its regulatory network was unclear. In the present study, we screened a transcription factor, SlERF109-like, and found it had a stronger relationship with SlNAC1 at the early stage of tomato fruit development through the use of transcriptome data, RT-qPCR, and correlation analysis. We inferred that SlERF109-like could interact with SlNAC1 to become a regulatory complex that co-regulates the tomato fruit ripening process. Results of transient silencing (VIGS) and transient overexpression showed that SlERF109-like and SlNAC1 could regulate chlorophyll degradation-related genes (NYC1, PAO, PPH, SGR1), carotenoids accumulation-related genes (PSY1, PDS, ZDS), ETH-related genes (ACO1, E4, E8), and cell wall metabolism-related genes expression levels (CEL2, EXP, PG, TBG4, XTH5) to inhibit tomato fruit ripening. A dual-luciferase reporter and yeast one-hybrid (Y1H) showed that SlNAC1 could bind to the SlACO1 promoter, but SlERF109-like could not. Furthermore, SlERF109-like could interact with SlNAC1 to increase the transcription for ACO1 by a yeast two-hybrid (Y2H) assay, a luciferase complementation assay, and a dual-luciferase reporter. A correlation analysis showed that SlERF109-like and SlNAC1 were positively correlated with chlorophyll contents, and negatively correlated with carotenoid content and ripening-related genes. Thus, we provide a model in which SlERF109-like could interact with SlNAC1 to become a regulatory complex that negatively regulates the tomato ripening process by inhibiting SlACO1 expression. Our study provided a new regulatory network of tomato fruit ripening and effectively reduced the waste of resources.

Modulating spectral response of raw photosynthetic pigments via ternary cadmium chalcogenide quantum dots: simultaneous enhancement at green spectrum and inhibition at UV region.

Photosynthesis relies on the absorption of sunlight by photosynthetic pigments (PPs) such as chlorophylls and carotenoids. While these pigments are outstanding at harvesting light, their natural structure restricts their ability to harvest light at specific wavelengths. In this study, Oleic acid-capped CdSeS and CdTeS ternary quantum dots (QDs) were synthesized using a novel two-phase synthesis method. Then, these QDs were used to interact with raw PPs, a mixture of chlorophylls and carotenoids isolated from spinach. Our findings revealed the following: (1) Interacting QDs with raw PPs effectively inhibited the chlorophyll fluorescence of the pigments upon excitation in UV light region (250-400 nm) without causing any damage to their structure. (2) By forming an interaction with QDs, the chlorophyll fluorescence of raw PPs could be induced through excitation with green-light spectrum. (3) The composition of the QDs played a fundamental role in their interaction with PPs. Our study demonstrated that the photophysical properties of isolated PPs could be modified by using cadmium-based QDs by preserving the structure of the pigments themselves.

De Novo Biosynthesis of Lutein in Yarrowia lipolytica.

Lutein is a high-value tetraterpenoid carotenoid that is widely used in feed, cosmetics, food, and drugs. Microbial synthesis of lutein is an important method for green and sustainable production, serving as an alternative to plant extraction methods. However, an inadequate precursor supply and low catalytic efficiency of key pathway enzymes are the main reasons for the low efficacy of microbial synthesis of lutein. In this study, some strategies, such as enhancing the MVA pathway and localizing α-carotene synthase OluLCY within the subcellular organelles in Yarrowia lipolytica, were adopted to enhance the synthesis of precursor α-carotene, which resulted in a 10.50-fold increase in α-carotene titer, reaching 38.50 mg/L. Subsequently, by improving hydroxylase activity with truncated N-terminal transport peptide and locating hydroxylases to subcellular organelles, the final strain L9 producing 75.25 mg/L lutein was obtained. Eventually, a lutein titer of 675.40 mg/L (6.13 mg/g DCW) was achieved in a 5 L bioreactor by adding the antioxidant 2,6-ditert-butyl-4-methylphenol. This study realizes de novo synthesis of lutein in Y. lipolytica for the first time and achieves the highest lutein titer reported so far.

Quenching of bacteriochlorophyll a triplet state by carotenoids in the chlorosome baseplate of green bacterium Chloroflexus aurantiacus.

To capture weak light fluxes, green photosynthetic bacteria have unique structures - chlorosomes, consisting of 104-5 molecules of bacteriochlorophyll (BChl) c, d, e. Chlorosomes are attached to the cytoplasmic membrane through the baseplate, a paracrystalline protein structure containing BChl a and carotenoids (Car). The most important function of Car is the quenching of triplet states of BChl, which prevents the formation of singlet oxygen and thereby provides photoprotection. In our work, we studied the dynamics of the triplet states of BChl a and Car in the baseplate of Chloroflexus aurantiacus chlorosomes using picosecond differential spectroscopy. BChl a of the baseplate was excited into the Qy band at 810 nm, and the corresponding absorption changes were recorded in the range of 420-880 nm. It was found that the formation of the Car triplet state occurs in ∼1.3 ns, which is ∼3 times faster than the formation of this state in the peripheral antenna of C. aurantiacus according to literature data. The Car triplet state was recorded by the characteristic absorption band T1 → Tn at ∼550 nm. Simultaneously with the appearance of absorption T1 → Tn, there was a bleaching of the singlet absorption of Car in the region of 400-500 nm. Theoretical modeling made it possible to estimate the characteristic time of formation of the triplet state of BChl a as ∼0.5 ns. It is shown that the experimental data are well described by the sequential scheme of formation and quenching of the BChl a triplet state: BChl a* → BChl aT → CarT. Thus, carotenoids from green bacteria effectively protect the baseplate from possible damage by singlet oxygen.

Microbiome of seventh-century old Parsurameswara stone monument of India and role of desiccation-tolerant cyanobacterium Lyngbya corticicola on its biodeterioration.

The Parsurameswara stone monument, built in the seventh century, is one of the oldest stone monuments in Odisha, India. Metagenomic analysis of the biological crust samples collected from the stone monument revealed 17 phyla in the microbiome, with Proteobacteria being the most dominant phylum, followed by cyanobacteria. Eight cyanobacteria were isolated. Lyngbya corticicola was the dominant cyanobacterium in all crust samples and could tolerate six months of desiccation in vitro. With six months of desiccation, chlorophyll-a decreased; however, carotenoid and cellular carbohydrate contents of this organism increased in the desiccated state. Resistance to desiccation, high carotenoid content, and effective trehalose biosynthesis in this cyanobacterium provide a distinct advantage over other microbiomes. Comparative metabolic profiles of the biological crust and L. corticicola show strongly corrosive organic acids such as dichloroacetic acid, which might be responsible for the biocorrosion of stone monuments.