Carbohydrate Analysis Research Articles

More small tools for sweet challenges: advances in microfluidic technologies for glycan analysis

Carbohydrates, also known glycans, are ubiquitous in nature and exhibit a wide array of biological functions essential to life. Glycans often exist as conjugates of proteins or lipids and reside predominantly at the surface of cells, where their structure and composition are known to vary in a disease-dependent fashion. This observation has encouraged the development of tools for monitoring glycan patterns on individual molecules, cells, and tissues, to elucidate the links between glycosylation and disease for therapeutic and diagnostic applications. Over the past 2 decades, microfluidic technology has emerged as an advantageous tool for profiling the glycan content of biological systems. Miniaturizing carbohydrate analysis can circumvent several challenges commonly encountered with conventional-scale analytical techniques such as low throughput and poor detection sensitivity. The latter is often complicated by the low abundance of glycans in biological specimens and the complexity of carbohydrate structures, which often necessitates extensive concentration and purification of glycans to discern their structural features. We previously examined the application of microfluidics in the synthesis of carbohydrates in a recent paper (Pinnock et al., Anal. Bioanal. Chem., 2022, 414 (18), 5139–63). This review builds upon that discussion by delving into the application of microfluidics in the complementary field of carbohydrate analysis. Special attention is given to applications related to glycomics and the ways that microfluidics have enhanced the sensitivity, reproducibility, and throughput of carbohydrate identification and structural characterization.

Ninjurin 2 Modulates Tumorigenesis, Inflammation, and Metabolism via Pyroptosis

The nerve injury-induced protein 2 (NINJ2) belongs to a family of homophilic adhesion molecules and was initially found to be involved in nerve regeneration. However, the role of NINJ2 in other cellular processes is not well studied. The Ninj2-deficient mice generated in the current study had a short lifespan and were prone to spontaneous tumors, systemic inflammation, and metabolic defects. Comprehensive carbohydrate and lipid metabolic analyses were performed to better understand the metabolic traits that contribute to these phenotypes. Carbohydrate metabolic analyses showed that NINJ2 deficiency led to defects in monosaccharide metabolism along with accumulation of multiple disaccharides and sugar alcohols. Lipidomic analyses showed that Ninj2 deficiency altered patterns of several lipids, including triglycerides, phospholipids, and ceramides. To identify a cellular process that associated with these metabolic defects, the role of NINJ2 in pyroptosis, a programmed cell death that links cancer, inflammation, and metabolic disorders, was examined. Loss of NINJ2 promoted pyroptosis by activating the NOD-like receptor protein 3 (NLRP3) inflammasome. Taken together, these data reveal a critical role of NINJ2 in tumorigenesis, inflammatory response, and metabolism via pyroptosis.

Increased belowground tree carbon allocation in a mature mixed forest in a dry versus a wet year.

Tree species differ in their carbon (C) allocation strategies during environmental change. Disentangling species-specific strategies and contribution to the C balance of mixed forests requires observations at the individual tree level. We measured a complete set of C pools and fluxes at the tree level in five tree species, conifers and broadleaves, co-existing in a mature evergreen mixed Mediterranean forest. Our study period included a drought year followed by an above-average wet year, offering an opportunity to test the effect of water availability on tree C allocation. We found that in comparison to the wet year, C uptake was lower in the dry year, C use was the same, and allocation to belowground sinks was higher. Among the five major C sinks, respiration was the largest (ca. 60%), while root exudation (ca. 10%) and reproduction (ca. 2%) were those that increased the most in the dry year. Most trees relied on stored starch for maintaining a stable soluble sugars balance, but no significant differences were detected in aboveground storage between dry and wet years. The detailed tree-level analysis of nonstructural carbohydrates and δ13 C dynamics suggest interspecific differences in C allocation among fluxes and tissues, specifically in response to the varying water availability. Overall, our findings shed light on mixed forest physiological responses to drought, an increasing phenomenon under the ongoing climate change.

Separation of All Classes of Carbohydrates by HPAEC-PAD

High performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) is a potential method of choice for the analysis of carbohydrates.

Combined pH ratiometry and fluorescence lectin-binding analysis (pH-FLBA) for microscopy-based analyses of biofilm pH and matrix carbohydrates.

Bacterial biofilms have a complex and heterogeneous three-dimensional architecture that is characterized by chemically and structurally distinct microenvironments. Confocal microscopy-based pH ratiometry and fluorescence lectin-binding analysis (FLBA) are well-established methods to characterize pH developments and the carbohydrate matrix architecture of biofilms at the microscale. Here, we developed a combined analysis, pH-FLBA, to concomitantly map biofilm pH and the distribution of matrix carbohydrates in bacterial biofilms while preserving the biofilm microarchitecture. As a proof of principle, the relationship between pH and the presence of galactose- and fucose-containing matrix components was investigated in dental biofilms grown with and without sucrose. The pH response to a sucrose challenge was monitored in different areas at the biofilm base using the ratiometric pH-sensitive dye C-SNARF-4. Thereafter, the fucose- and galactose-specific fluorescently labeled lectins Aleuria aurantia lectin (AAL) and Morus nigra agglutinin G (MNA-G) were used to visualize carbohydrate matrix components in the same biofilm areas and their immediate surroundings. Sucrose during growth significantly decreased biofilm pH (P < 0.05) and increased the amounts of both MNA-G- and AAL-targeted matrix carbohydrates (P < 0.05). Moreover, it modulated the biofilm composition towards a less diverse community dominated by streptococci, as determined by 16S rRNA gene sequencing. Altogether, these results suggest that the production of galactose- and fucose-containing matrix carbohydrates is related to streptococcal metabolism and, thereby, pH profiles in dental biofilms. In conclusion, pH-FLBA using lectins with different carbohydrate specificities is a useful method to investigate the association between biofilm pH and the complex carbohydrate architecture of bacterial biofilms.IMPORTANCEBiofilm pH is a key regulating factor in several biological and biochemical processes in environmental, industrial, and medical biofilms. At the microscale, microbial biofilms are characterized by steep pH gradients and an extracellular matrix rich in carbohydrate components with diffusion-modifying properties that contribute to bacterial acid-base metabolism. Here, we propose a combined analysis of pH ratiometry and fluorescence lectin-binding analysis, pH-FLBA, to concomitantly investigate the matrix architecture and pH developments in microbial biofilms, using complex saliva-derived biofilms as an example. Spatiotemporal changes in biofilm pH are monitored non-invasively over time by pH ratiometry, while FLBA with lectins of different carbohydrate specificities allows mapping the distribution of multiple relevant matrix components in the same biofilm areas. As the biofilm structure is preserved, pH-FLBA can be used to investigate the in situ relationship between the biofilm matrix architecture and biofilm pH in complex multispecies biofilms.

Investigation of biochemical, enzymatic, and metagenomic profiles of garbage enzymes and its application in dumping site leachate treatment.

The current article focuses on the preparation and characterization of garbage enzyme (GE) and explores its applications in treating leachate. GE is prepared from fruit and vegetable wastes and characterized via analysis of metabolites, carbohydrates, proteins, antioxidants, and enzymatic activities. This study extends our understanding of GE by reporting the presence of various metabolites. Moreover, a metagenomic analysis of GE is presented, shedding light on the microbial diversity. Firmicutes emerged as the dominant phylum, surpassing other phyla, including Proteobacteria and Actinobacteria. When exploring the potential for leachate treatment, the results indicate that vegetable GE shows 68% reduction in COD (chemical oxygen demand) and 39% reduction in ammoniacal nitrogen. Similarly, non-citrus GE also showed 64% reduction in COD and a 37% reduction in ammoniacal nitrogen, followed by citrus GE with a 33% reduction in COD and a 34% reduction in ammoniacal nitrogen compared to the control.

Characterising glycosaminoglycans in human breastmilk and their potential role in infant health.

Human breastmilk is composed of many well researched bioactive components crucial for infant nutrition and priming of the neonatal microbiome and immune system. Understanding these components gives us crucial insight to the health and wellbeing of infants. Research surrounding glycosaminoglycans (GAGs) previously focused on those produced endogenously; however, recent efforts have shifted to understanding GAGs in human breastmilk. The structural complexity of GAGs makes detection and analysis complicated therefore, research is time consuming and limited to highly specialised teams experienced in carbohydrate analysis. In breastmilk, GAGs are present in varying quantities in four forms; chondroitin sulphate, heparin/heparan sulphate, dermatan sulphate and hyaluronic acid, and are hypothesised to behave similar to other bioactive components with suspected roles in pathogen defense and proliferation of beneficial gut bacteria. Chondroitin sulphate and heparin, being the most abundant, are expected to have the most impact on infant health. Their decreasing concentration over lactation further indicates their role and potential importance during early life.

Analysis of Fiber Content and Antioxidant Activity of Bamboo Shoots (Dendrocalamus asper) to Support Functional Foods

The purpose of food consumption extends beyond meeting the body's nutritional requirements, increasingly emphasizing the potential of food as a form of medicine or health enhancer. Functional foods, which can be integrated into daily menus without specific dosage requirements, present a favorable nutritional option. In the archipelagic region of Southeast Sulawesi, characterized by extensive forests, bamboo shoots thrive abundantly. Consequently, the local community in this area has acknowledged and incorporated bamboo shoots as a dietary component. This study aims to explore the content of bamboo shoots and analyze protein, carbohydrate, and fiber levels. The research employs various methods, including phytochemical screening, alkaloid testing, flavonoid testing, saponin, tannin, triterpenoid, and steroid assessments, as well as content analysis, proximate analysis, carbohydrate analysis, and fiber level analysis. The study outcomes reveal the presence of alkaloids and flavonoids in bamboo shoots, with protein levels at 2.6 g/100 g, carbohydrate levels at 4.10 g/100 g, and fiber levels at 2.40 g/100 g. It can be concluded that bamboo shoots have important secondary metabolites, namely flavonoids and alkaloids, which may have health benefits such as being anti-inflammatory, antiviral, and inhibitory of allergies.

Viability and integrity of Pinus densiflora seeds stored for 20years at three different temperatures.

Storage temperature is one of the most important factors determining seed longevity in the genebank. This study aimed to investigate the effect of storage temperature on the seed viability and physiological integrity after a 20-year storage period of Pinus densiflora, a tree species of ecological and economic significance in South Korea. To this end, seeds were collected and stored dry for 20years at -18°C, 4°C and 25°C. Germination tests were conducted to assess seed viability and vigour, electrolyte leakage analysis was performed to assess cell membrane integrity, and carbohydrate analysis was conducted to assess metabolic integrity during germination. The results revealed that over 20years, seeds stored at -18°C maintained a high germination percentage (GP; 89%), comparable to initial GP (91%), whilst those stored at 4°C exhibited a decline in GP (44%) along with a decrease in vigour. Seeds stored at 25°C lost their viability entirely. Electrical conductivity of the leachate and leakage of inorganic compounds and soluble sugars were higher with elevated storage temperature, indicating increased imbibition damage. Additionally, changes in carbohydrate content during germination revealed that the loss of viability according to storage temperature is associated with reduced storage reserve utilization and altered carbohydrate metabolism during germination. These results enhance our understanding of the effect of seed storage temperature on longevity and physiological changes of aging in the genebank, serving as a reference for establishing conservation strategies for Pinus densiflora.

Advancing carbohydrate quantification in MALDI mass spectrometry by the rapidly freeze-drying droplet (RFDD) method.

Quantitative carbohydrate analysis faces challenges in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), including insufficient sensitivity and inconsistent spatial distribution of ion intensity. This study introduces an innovative sample preparation approach, the Rapidly Freeze-Drying Droplet (RFDD) method, aimed at overcoming these challenges by enhancing the homogeneity of the sample morphology and signal intensity in MALDI. Compared to conventional preparation methods, the RFDD method reduces the laser energy threshold and demonstrates a remarkable increase in signal intensity for carbohydrates, facilitating the detection of high-molecular-weight polysaccharides (>10 kDa). The RFDD-prepared samples exhibit a uniformly distributed signal intensity that overcomes the 'sweet spot' issue in MALDI. The enhanced signal intensity and reproducibility lead to reliable quantitative analysis of carbohydrates, eliminating the need for expensive isotopic standards in each sample. A straightforward and accessible approach is presented for general laboratories, revolutionizing carbohydrate analysis in MALDI-MS.

Deciphering biomolecular complexities: the indispensable role of surface-enhanced Raman spectroscopy in modern bioanalytical research.

Surface-enhanced Raman spectroscopy (SERS) has emerged as an indispensable analytical tool in biomolecular research, providing unmatched sensitivity critical for the elucidation of biomolecular structures. This review presents a thorough examination of SERS, outlining its fundamental principles, cataloging its varied applications within the biomolecular sphere, and contemplating its future developmental trajectories. We begin with a detailed analysis of SERS's mechanistic principles, emphasizing both the phenomena of surface enhancement and the complexities inherent in Raman scattering spectroscopy. Subsequently, we delve into the pivotal role of SERS in the structural analysis of diverse biomolecules, including proteins, nucleic acids, lipids, carbohydrates, and biochromes. The remarkable capabilities of SERS extend beyond mere detection, offering profound insights into biomolecular configurations and interactions, thereby enriching our comprehension of intricate biological processes. This review also sheds light on the application of SERS in real-time monitoring of various bio-relevant compounds, from enzymes and coenzymes to metal ion-chelate complexes and cellular organelles, thereby providing a holistic view and empowering researchers to unravel the complexities of biological systems. We also address the current challenges faced by SERS, such as enhancing sensitivity and resolution, developing stable and reproducible substrates, and conducting thorough analyses in complex biological matrices. Nonetheless, the continual advancements in nanotechnology and spectroscopy solidify the standing of SERS as a formidable force in biomolecular research. In conclusion, the versatility and robustness of SERS not only deepen our understanding of biomolecular intricacies but also pave the way for significant developments in medical research, therapeutic innovation, and diagnostic approaches.

Distribution, screening and biochemical characterization of indigenous microalgae for bio-mass and bio-energy production potential from three districts of Pakistan.

Trend of biofuel production from microalgal triacylglycerols is enhancing, because this substrate is a good sustainable and advantageous alternative to oil and gas fuel. In the present study, indigenous micro algal isolates were screened from water (n=30) and soil (n=30) samples collected from three districts of Punjab, Pakistan to evaluate their biofuel production potential. The samples were inoculated on BG - 11 agar medium plates by incubating at room temperature of 25°C providing 1000 lux for 16h light cycle followed by 8h of dark cycle for 15 d. Water samples were found to be rich in microalgae and 65.33% microalgae (49 isolates) were isolated from Faisalabad district. On the basis of microscopic morphology microalgal isolates (n=180) were selected and subjected to lipid detection by Nile red staining assay. Nile red positive isolates (n=23) were processed for biochemical (lipid, protein and carbohydrates) characterization. AIN63 isolate showed higher lipids (17.4%) content as detected by micro vanillin assay. Algal isolate AIN128 showed best protein contents (42.91%) detected by Bradford assay and AIN172 isolate showed higher carbohydrate contents (73.83%) as detected by anthrone assay. The selected algal isolates were also analyzed by Fourier transform infrared (FTIR) spectroscopy for confirmation of carbohydrate, protein and lipid analysis. These indigenous algae have the potential for in-vitro biofuel production from agricultural waste.

Recent advances in nanopore-based analysis for carbohydrates and glycoconjugates.

Saccharides are not only the basic constituents and nutrients of living organisms, but also participate in various life activities, and play important roles in cell recognition, immune regulation, development, cancer, etc. The analysis of carbohydrates and glycoconjugates is a necessary means to study their transformations and physiological roles in living organisms. Existing detection techniques can hardly meet the requirements for the analysis of carbohydrates and glycoconjugates in complex matrices as they are expensive, involve complex derivatization, and are time-consuming. Nanopore sensing technology, which is amplification-free and label-free, and is a high-throughput process, provides a new solution for the identification and sequencing of carbohydrates and glycoconjugates. This review highlights recent advances in novel nanopore-based single-molecule sensing technologies for the detection of carbohydrates and glycoconjugates and discusses the advantages and challenges of nanopore sensing technologies. Finally, current issues and future perspectives are discussed with the aim of improving the performance of nanopores in complex media diagnostic applications, as well as providing a new direction for the quantification of glycan chains and the study of glycan chain properties and functions.

Dormant carbohydrate reserves enhance pecan tree spring freeze tolerance: controlled environment observations.

Pecan (Carya illinoensis), an economically important deciduous tree, bears commercially valuable nutritional nuts. Spring freezes in April can severely injure pecan buds, decreasing bloom, and fruit set. This study determined how low temperatures affect pecan buds/flowers at different growth stages in several pecan scion/rootstock combinations. This study focused on three pecan scion/rootstock combinations: Pawnee/Peruque (PP), Kanza/Giles (KG), and Maramec/Colby (MC), grown at the Cimarron Valley Research Station, Perkins, Oklahoma. Branches at three different growth stages, i.e., outer bud scale shed, one week after bud break, and early bloom stages were collected from PP, MC, and KG. Branches were held in a Conviron E8 freezing unit at 4 temperatures (-2, 0, 2, and 4°C) for 4 and 8 hours; A total of 8 treatments. One sample set was kept as an untreated control. After 2-3 weeks, branch samples from all the temperature treatments were observed and categorized into two groups. Group one with number of branches had healthy buds/formation of healthy leaves/flowers and group two with number of dead branches. The carbohydrate content reserved from dormant was analyzed using an Anthrone reagent. Visual observations and carbohydrate analyses revealed differences in damage and carbohydrate content among the scion/rootstock combinations, low-temperature treatments, and growth stages. The MC combination had minimum visual damage to leaves, buds, and flowers and significantly lower soluble sugars and starch in bark phloem as well as significantly lower soluble sugars in woody tissue xylem. The KG combination had maximum visual damage and significantly higher soluble sugars and starches in the bark, and soluble sugars in the woody tissues. These results indicate the MC combination is more tolerant to spring freeze damage at all three growth stages compared to the other two pecan scion/rootstock combinations. The results also demonstrate the MC combination is using more non-structural carbohydrates, soluble sugars and starches, suggesting this is a possible mechanism in its freeze tolerance.

Species Diversity of Fungi Decomposers of Rhizophora stylosa Leaf Litter in Mangrove Ecosystem Pulau Sembilan Langkat Regency

Litter decomposition is a process of decomposing litter which is an important component of the nutrient cycle process because it can contribute greatly to soil fertility. Fungi are one of the microorganisms that play an important role in the decomposition process. The purpose of this study was to identify the number of species and diversity index of fungi, determine the rate of decomposition, and analyze the protein and carbohydrate content of decomposed R. stylosa leaf litter. This research was conducted on Pulau Sembilan, Langkat Regency and at the Forest Cultivation Laboratory, Faculty of Forestry, University of North Sumatra. Carbohydrate and protein analysis was carried out at the Medan Industrial Research and Standardization Center. This research method uses Litter-bag. There are 4 types of fungi namely Aspergillus sp, Fusarium sp, Penicillium sp, and Trichoderma sp. The index value of fungal diversity in R. stylosa leaf litter is 1.49. The decomposition rate of R. stylosa leaf litter is 7.7. The highest protein content was found in R. stylosa leaf litter that underwent decomposition for 30 days at 5.76% and the highest carbohydrate content was found in the control at 10.4%.

Influence of arbuscular mycorrhizal fungi density on growth and metabolism of Handroanthus serratifolius (Vahl) S.O. Grose seedlings

AbstractStudies on the relationship between Handroanthus serratifolius and arbuscular mycorrhizal fungi (AMF) are limited in the literature. The influence of AMF spore density on plant development is fundamental information to determining the degree of benefits in this relationship. Therefore, the objective of this study was to investigate the effects of different AMF spore densities on thirty‐day‐old H. serratifolius seedlings, focusing on growth and biochemical parameters using a completely randomized experimental design with three different spore densities and control. The spore densities were classified as low, medium, and high, with 1.54, 3.08, and 12.35 spores g−1, respectively. Plant growth analysis, mycorrhizal colonization, nitrogen compound concentration, and carbohydrate analysis were performed. The medium spore density treatment showed the greatest increases in biomass, height, leaf area, and root volume. Furthermore, greater absorption of phosphorus and better dynamics in nitrogen metabolism were observed in mycorrhizal plants compared to the control since the ammonium and nitrate compounds were rapidly incorporated into protein and chlorophyll compounds. The carbohydrate analysis revealed the influence of source‐sink dynamics on sugar concentration in different plant parts. These findings support the importance of determining the appropriate spore density for assessing the symbiotic relationship between forest species and AMF.

Minimal Effective Dose of Beans Required to Elicit a Significantly Lower Glycemic Response Than Commonly Consumed Starchy Foods: Predictions Based on In Vitro Digestion and Carbohydrate Analysis.

Beans elicit lower glycemic responses (GRs) than other starchy foods, but the minimum effective dose (MED) to reduce GR is unknown. We sought to determine the MED of beans compared to common starchy foods. Overnight-fasted healthy volunteers consumed ¼c (phase 1, n = 24) or ½c (phase 2, n = 18) of black, cranberry, great northern, kidney, navy and pinto beans and corn, rice, pasta and potato (controls), with blood glucose measured before and for 2 h after eating. GRs (incremental areas under the curves, iAUCs) after beans were consumed were compared to those of controls by ANOVA followed by Dunnett's test. To qualify for MED, beans had to elicit an effective reduction in GR, defined as a statistically significant reduction in iAUC of ≥20% (i.e., a relative glycemic response, RGR, ≤80). Outcomes from in vitro digestion were compared with in vivo RGR. Both doses of all six beans effectively reduced GR versus all four starchy controls, except for ¼c and ½c cranberry and pinto vs. corn, ¼c great northern and navy vs. corn and ¼c navy and pinto vs. potato. MED criteria were met for 18 comparisons of the ¼c servings, with four of the remaining six met by the ½c servings. The overall mean ± SEM RGR vs. controls was similar for the ¼c and ½c servings: 53 ± 4% and 56 ± 3%, respectively. By multiple regression analysis, RGR = 23.3 × RDS + 8.3 × SDS - 20.1 × RS + 39.5 × AS - 108.2 (rapidly digested starch, p < 0.001; slowly digested starch, p = 0.054; resistant starch, p = 0.18; available sugars, p = 0.005; model r = 0.98, p = 0.001). RGR correlated with in vitro glucose release (r = 0.92, p < 0.001). The MED of beans is ¼ cup. For n = 30 comparisons (n = 24 beans vs. controls, n = 6 controls vs. each other), an effective reduction in GR was predicted from in vitro carbohydrate analysis with 86% sensitivity and 100% specificity.

Hygienic Analysis Of Morbidity Indicators Of Patients: On The Example Of The Endocrine System

In this article, the quantitative and qualitative indicators of the hygienic analysis of protein, fat, oil, carbohydrates, vitamins and minerals in the daily diet of patients with endocrine system diseases in the Bukhara region of our country, the role of protein, fat, oil, carbohydrates, vitamins and minerals in the diet and its importance in disease prevention is assessed and practical recommendations are given to prevent unhealthy nutrition and hypodynamia during the development of the disease.

Polymer properties of softwood organosolv lignins produced in two different reactor systems.

Lignin, the second most abundant biopolymer on earth and with a predominantly aromatic structure, has the potential to be a raw material for valuable chemicals and other bio-based chemicals. In industry, lignin is underutilized by being used mostly as a fuel for producing thermal energy. Valorization of lignin requires knowledge of the structure and different linkages in the isolated lignin, making the study of structure of lignin important. In this article, lignin samples isolated from two types of reactors (autoclave reactor and displacement reactor) were analyzed by FT-IR, size exclusion chromatography, thermogravimetric analysis (TGA), and Py-GC-MS. The average molecular mass of the organosolv lignins isolated from the autoclave reactor decreased at higher severities, and FT-IR showed an increase in free phenolic content with increasing severity. Except for molecular mass and molecular mass dispersity, there were only minor differences between lignins isolated from the autoclave reactor and lignins isolated from the displacement reactor. Carbohydrate analysis, Py-GC-MS and TGA showed that the lignin isolated using either of the reactor systems is of high purity, suggesting that organosolv lignin is a good candidate for valorization.

Mechanisms for leaf color changes in Osmanthus fragrans ‘Ziyan Gongzhu’ using physiology, transcriptomics and metabolomics

BackgroundColor-leaved O. fragrans is a variety of Osmanthus fragrans, which has both the fragrance of Osmanthus and the color of color-leaved plants. However, the molecular mechanism of color change of color-leaved O. fragrans is not clear. In this study, we analyzed the regulatory mechanism of four different color leaves of ‘Ziyan Gongzhu’ through physiological, transcriptome and metabolome levels.ResultsFirstly, we measured the leaf pigments content and leaf chromatic parameters for correlation analysis, indicating a significant correlation between them. Overall, the content of chlorophyll a + b is low and the content of anthocyanin is high in T1 and T2 leaves, along with low expression of chlorophyll synthesis genes (HEMA, CHLG, and CAO, etc.) and high expression of anthocyanin synthesis genes (F3H, F3’H, DFR and ANS, etc.), resulting purple red and light purple in T1 and T2 leaves, respectively. It was also found that the pigment closely related to the color leaves of ‘Ziyan Gongzhu’ was cyanidin. The content anthocyanins, may be regulated by two putative MYB activators (OfMYB3 and OfMYB4) and two putative MYB repressors (OfMYB1 and OfMYB2). In contrast, the content of chlorophyll a + b is high and the content of anthocyanin is low in T3 and T4 leaves, along with high expression of chlorophyll synthesis genes and low expression of anthocyanin synthesis genes, resulting yellow green and dark green in T3 and T4 leaves, respectively. And abnormal chloroplast development affects chlorophyll content in T1, T2, and T3 leaves. Although the content of carotenoids first dropped in T2 leaves, it then rapidly accumulated in T4 leaves, in sync with the increase in the expression of genes related to carotenoid biosynthesis (ZDS, LHYB, and ZEP, for example). Analysis of photosynthetic, carbohydrate and hormone-related differentially abundant metabolites (DAMs) and DEGs found that they may participate in the regulation of leaf color change of ‘Ziyan Gongzhu’ by affecting pigment synthesis.ConclusionOur results pave the way for a comprehensive knowledge of the regulatory processes governing leaf color in ‘Ziyan Gongzhu’ and identify possible genes for application regarding molecular colored-leaf cultivar breeding.