Nudix hydrolase 23 post-translationally regulates carotenoid biosynthesis in plants.

Carotenoids are essential for photosynthesis and photoprotection. Plants must evolve multifaceted regulatory mechanisms to control carotenoid biosynthesis. However, the regulatory mechanisms and the regulators conserved among plant species remain elusive. Phytoene synthase (PSY) catalyzes the highly regulated step of carotenogenesis and geranylgeranyl diphosphate synthase (GGPPS) acts as a hub to interact with GGPP-utilizing enzymes for the synthesis of specific downstream isoprenoids. Here, we report a function of Nudix hydrolase 23 (NUDX23), a Nudix domain-containing protein, in post-translational regulation of PSY and GGPPS for carotenoid biosynthesis. NUDX23 expresses highly in Arabidopsis (Arabidopsis thaliana) leaves. Overexpression of NUDX23 significantly increases PSY and GGPPS protein levels and carotenoid production, whereas knockout of NUDX23 dramatically reduces their abundances and carotenoid accumulation in Arabidopsis. NUDX23 regulates carotenoid biosynthesis via direct interactions with PSY and GGPPS in chloroplasts, which enhances PSY and GGPPS protein stability in a large PSY-GGPPS enzyme complex. NUDX23 was found to co-migrate with PSY and GGPPS proteins and to be required for the enzyme complex assembly. Our findings uncover a regulatory mechanism underlying carotenoid biosynthesis in plants and offer promising genetic tools for developing carotenoid-enriched food crops.

Metabolomic and transcriptomic analyses of peach leaves and fruits in response to pruning.

BACKGROUND: Pruning is an important cultivation management option that has important effects on peach yield and quality. However, the effects of pruning on the overall genetic and metabolic changes in peach leaves and fruits are poorly understood. RESULTS: The transcriptomic and metabolomic profiles of leaves and fruits from trees subjected to pruning and unpruning treatments were measured. A total of 20,633 genes and 622 metabolites were detected. Compared with those in the control, 1,127 differentially expressed genes (DEGs) and 77 differentially expressed metabolites (DEMs) were identified in leaves from pruned and unpruned trees (pdLvsupdL), whereas 423 DEGs and 29 DEMs were identified in fruits from the pairwise comparison pdFvsupdF. The content of three auxin analogues was upregulated in the leaves of pruned trees, the content of all flavonoids detected in the leaves decreased, and the expression of almost all genes involved in the flavonoid biosynthesis pathway decreased. The phenolic acid and amino acid metabolites detected in fruits from pruned trees were downregulated, and all terpenoids were upregulated. The correlation analysis revealed that DEGs and DEMs in leaves were enriched in tryptophan metabolism, auxin signal transduction, and flavonoid biosynthesis. DEGs and DEMs in fruits were enriched in flavonoid and phenylpropanoid biosynthesis, as well as L-glutamic acid biosynthesis. CONCLUSIONS: Pruning has different effects on the leaves and fruits of peach trees, affecting mainly the secondary metabolism and hormone signalling pathways in leaves and amino acid biosynthesis in fruits.

A Neighboring Aromatic-Aromatic Amino Acid Combination Governs Activity Divergence between Tomato Phytoene Synthases.

Carotenoids exert multifaceted roles to plants and are critically important to humans. Phytoene synthase (PSY) is a major rate-limiting enzyme in the carotenoid biosynthetic pathway. PSY in plants is normally found as a small enzyme family with up to three members. However, knowledge of PSY isoforms in relation to their respective enzyme activities and amino acid residues that are important for PSY activity is limited. In this study, we focused on two tomato (Solanum lycopersicum) PSY isoforms, PSY1 and PSY2, and investigated their abilities to catalyze carotenogenesis via heterologous expression in transgenic Arabidopsis (Arabidopsis thaliana) and bacterial systems. We found that the fruit-specific PSY1 was less effective in promoting carotenoid biosynthesis than the green tissue-specific PSY2. Examination of the PSY proteins by site-directed mutagenesis analysis and three-dimensional structure modeling revealed two key amino acid residues responsible for this activity difference and identified a neighboring aromatic-aromatic combination in one of the PSY core structures as being crucial for high PSY activity. Remarkably, this neighboring aromatic-aromatic combination is evolutionarily conserved among land plant PSYs except PSY1 of tomato and potato (Solanum tuberosum). Strong transcription of tomato PSY1 likely evolved as compensation for its weak enzyme activity to allow for the massive carotenoid biosynthesis in ripe fruit. This study provides insights into the functional divergence of PSY isoforms and highlights the potential to rationally design PSY for the effective development of carotenoid-enriched crops.

Carotenoid accumulation affects redox status, starch metabolism, and flavonoid/anthocyanin accumulation in citrus.

BACKGROUND: Carotenoids are indispensable plant secondary metabolites that are involved in photosynthesis, antioxidation, and phytohormone biosynthesis. Carotenoids are likely involved in other biological functions that have yet to be discovered. In this study, we integrated genomic, biochemical, and cellular studies to gain deep insight into carotenoid-related biological processes in citrus calli overexpressing CrtB (phytoene synthase from Pantoea agglomerans). Fortunella hindsii Swingle (a citrus relative) and Malus hupehensis (a wild apple) calli were also utilized as supporting systems to investigate the effect of altered carotenoid accumulation on carotenoid-related biological processes. RESULTS: Transcriptomic analysis provided deep insight into the carotenoid-related biological processes of redox status, starch metabolism, and flavonoid/anthocyanin accumulation. By applying biochemical and cytological analyses, we determined that the altered redox status was associated with variations in O2 (-) and H2O2 levels. We also ascertained a decline in starch accumulation in carotenoid-rich calli. Furthermore, via an extensive cellular investigation of the newly constructed CrtB overexpressing Fortunella hindsii Swingle, we demonstrated that starch level reducation occurred in parallel with significant carotenoid accumulation. Moreover, studying anthocyanin-rich Malus hupehensis calli showed a negative effect of carotenoids on anthocyanin accumulation. CONCLUSIONS: In citrus, altered carotenoid accumulation resulted in dramatic effects on metabolic processes involved in redox modification, starch degradation, and flavonoid/anthocyanin biosynthesis. These findings provided new perspectives to understand the biological importance of carotenogenesis and of the developmental processes associated with the nutritional and sensory qualities of agricultural products that accumulate carotenoids.

Corrigendum: The computational implementation of a platform of relative identity-by-descent scores algorithm for introgressive mapping.

[This corrects the article DOI: 10.3389/fgene.2022.1028662.].

The role of exosomes and exosomal microRNA in diabetic cardiomyopathy.

Diabetic cardiomyopathy, a formidable cardiovascular complication linked to diabetes, is witnessing a relentless surge in its incidence. Despite extensive research efforts, the primary pathogenic mechanisms underlying this condition remain elusive. Consequently, a critical research imperative lies in identifying a sensitive and dependable marker for early diagnosis and treatment, thereby mitigating the onset and progression of diabetic cardiomyopathy (DCM). Exosomes (EXOs), minute vesicles enclosed within bilayer lipid membranes, have emerged as a fascinating frontier in this quest, capable of transporting a diverse cargo that mirrors the physiological and pathological states of their parent cells. These exosomes play an active role in the intercellular communication network of the cardiovascular system. Within the realm of exosomes, MicroRNA (miRNA) stands as a pivotal molecular player, revealing its profound influence on the progression of DCM. This comprehensive review aims to offer an introductory exploration of exosome structure and function, followed by a detailed examination of the intricate role played by exosome-associated miRNA in diabetic cardiomyopathy. Our ultimate objective is to bolster our comprehension of DCM diagnosis and treatment strategies, thereby facilitating timely intervention and improved outcomes.

BCH1 expression pattern contributes to the fruit carotenoid diversity between peach and apricot.

Peach (Prunus persica L. Batsch) and apricot (Prunus armeniaca L.) are two species of economic importance for fruit production in the genus Prunus. Peach and apricot fruits exhibit significant differences in carotenoid levels and profiles. HPLC-PAD analysis showed that a greater content of ß-carotene in mature apricot fruits is primarily responsible for orange color, while peach fruits showed a prominent accumulation of xanthophylls (violaxanthin and cryptoxanthin) with yellow color. There are two ß-carotene hydroxylase genes in both peach and apricot genomes. Transcriptional analysis revealed that BCH1 expresses highly in peach but lowly in apricot fruit, showing a correlation with peach and apricot fruit carotenoid profiles. By using a carotenoid engineered bacterial system, it was demonstrated that there was no difference in the BCH1 enzymatic activity between peach and apricot. Comparative analysis about the putative cis-acting regulatory elements between peach and apricot BCH1 promoters provided important information for our understanding of the differences in promoter activity of the BCH1 genes in peach and apricot. Therefore, we investigated the promoter activity of BCH1 gene through a GUS detection system, and confirmed that the difference in the transcription level of the BCH1 gene resulted from the difference of the promoter function. This study provides important perspective to understanding the diversity of carotenoid accumulation in Prunus fruits such as peach and apricot. In particular, BCH1 gene is proposed as a main predictor for ß-carotene content in peach and apricot fruits during the ripening process.

Comprehending crystalline ß-carotene accumulation by comparing engineered cell models and the natural carotenoid-rich system of citrus.

Genetic manipulation of carotenoid biosynthesis has become a recent focus for the alleviation of vitamin A deficiency. However, the genetically modified phenotypes often challenge the expectation, suggesting the incomplete comprehension of carotenogenesis. Here, embryogenic calli were engineered from four citrus genotypes as engineered cell models (ECMs) by over-expressing a bacterial phytoene synthase gene (CrtB). Ripe flavedos (the coloured outer layer of citrus fruits), which exhibit diverse natural carotenoid patterns, were offered as a comparative system to the ECMs. In the ECMs, carotenoid patterns showed diversity depending on the genotypes and produced additional carotenoids, such as lycopene, that were absent from the wild-type lines. Especially in the ECMs from dark-grown culture, there emerged a favoured ß,ß-pathway characterized by a striking accumulation of ß-carotene, which was dramatically different from those in the wild-type calli and ripe flavedos. Unlike flavedos that contained a typical chromoplast development, the ECMs sequestered most carotenoids in the amyloplasts in crystal form, which led the amyloplast morphology to show a chromoplast-like profile. Transcriptional analysis revealed a markedly flavedo-specific expression of the ß-carotene hydroxylase gene (HYD), which was suppressed in the calli. Co-expression of CrtB and HYD in the ECMs confirmed that HYD predominantly mediated the preferred carotenoid patterns between the ECMs and flavedos, and also revealed that the carotenoid crystals in the ECMs were mainly composed of ß-carotene. In addition, a model is proposed to interpret the common appearance of a favoured ß,ß-pathway and the likelihood of carotenoid degradation potentially mediated by photo-oxidation and vacuolar phagocytosis in the ECMs is discussed.

[Connotations and extensions of comparative effectiveness research and feasibility analysis for clinical evaluation in traditional Chinese medicine].

Comparative effectiveness research (CER) is well developed in international medical research. This article analyzes the basic connotations of CER from five aspects, including selection of objective, positioning of target population, comparison of intervention scheme, identification of contrast condition and analysis of therapeutic effects. The inclusiveness and advanced nature of CER were revealed by comparing with modern medical research methods, such as evidence-based medicine, patient-important outcome and randomized controlled trials. Based on the superiority of CER, the feasibility of applying CER in clinical evaluation of traditional Chinese medicine is explored. CER should be combined with the holistic concept, syndrome differentiation and individuality-concerned treatment so as to facilitate its development in research of traditional Chinese medicine in China.

The computational implementation of a platform of relative identity-by-descent scores algorithm for introgressive mapping.

With the development of genotyping and sequencing technology, researchers working in the area of conservation genetics are able to obtain the genotypes or even the sequences of a representative sample of individuals from the population. It is of great importance to examine the genomic variants and genes that are highly preferred or pruned during the process of adaptive introgression or long-term hybridization. To the best of our knowledge, we are the first to develop a platform with computational integration of a relative identity-by-descent (rIBD) scores algorithm for introgressive mapping. The rIBD algorithm is designed for mapping the fine-scaled genomic regions under adaptive introgression between the source breeds and the admixed breed. Our rIBD calculation platform provides compact functions including reading input information and uploading of files, rIBD calculation, and presentation of the rIBD scores. We analyzed the simulated data using the rIBD calculation platform and calculated the average IBD score of 0.061 with a standard deviation of 0.124. The rIBD scores generally follow a normal distribution, and a cut-off of 0.432 and -0.310 for both positive and negative rIBD scores is derived to enable the identification of genomic regions showing significant introgression signals from the source breed to the admixed breed. A list of genomic regions with detailed calculated rIBD scores is reported, and all the rIBD scores for each of the considered windows are presented in plots on the rIBD calculation platform. Our rIBD calculation platform provides a user-friendly tool for the calculation of fine-scaled rIBD scores for each of the genomic regions to map possible functional genomic variants due to adaptive introgression or long-term hybridization.

A proteomic analysis of the chromoplasts isolated from sweet orange fruits [Citrus sinensis (L.) Osbeck].

Here, a comprehensive proteomic analysis of the chromoplasts purified from sweet orange using Nycodenz density gradient centrifugation is reported. A GeLC-MS/MS shotgun approach was used to identify the proteins of pooled chromoplast samples. A total of 493 proteins were identified from purified chromoplasts, of which 418 are putative plastid proteins based on in silico sequence homology and functional analyses. Based on the predicted functions of these identified plastid proteins, a large proportion (∼60%) of the chromoplast proteome of sweet orange is constituted by proteins involved in carbohydrate metabolism, amino acid/protein synthesis, and secondary metabolism. Of note, HDS (hydroxymethylbutenyl 4-diphosphate synthase), PAP (plastid-lipid-associated protein), and psHSPs (plastid small heat shock proteins) involved in the synthesis or storage of carotenoid and stress response are among the most abundant proteins identified. A comparison of chromoplast proteomes between sweet orange and tomato suggested a high level of conservation in a broad range of metabolic pathways. However, the citrus chromoplast was characterized by more extensive carotenoid synthesis, extensive amino acid synthesis without nitrogen assimilation, and evidence for lipid metabolism concerning jasmonic acid synthesis. In conclusion, this study provides an insight into the major metabolic pathways as well as some unique characteristics of the sweet orange chromoplasts at the whole proteome level.

Double NCED isozymes control ABA biosynthesis for ripening and senescent regulation in peach fruits.

During ripening, peach fruits (Prunus persica L. Batsch) rapidly progress to the senescent stage, resulting in a brief shelf life. Abscisic acid (ABA) plays an important role in regulating the ripening process, both in climacteric and non-climacteric fruits. A key enzyme for ABA biosynthesis in higher plants is 9-cis-epoxycarotenoid dioxygenase (NCED). In this study, two NCED isozymes, PpNCED1 and PpNCED5, were identified in peach fruits. While both NCED genes had similar transcriptional patterns (up-regulation) at the beginning of peach ripening, PpNCED5 showed a consistently lower expression level than PpNCED1. During the post-harvest stage, gene expression of PpNCED1 declined, while PpNCED5 expression increased relative to PpNCED1 expression. Considering the dynamic process of ABA accumulation during fruit ripening and senescence in peach, this study indicates that both NCED genes cooperatively control ABA biosynthesis in peach fruits. Moreover, spatio-temporal expression and transcriptional response to hormone and abiotic stress suggested that there is functional divergence between PpNCED1 and PpNCED5 genes in peach. A carotenoid-rich callus system was used to verify the function of PpNCED1 and PpNCED5. In the transgenic callus system, both PpNCED1 and PpNCED5 isozymes promoted ABA biosynthesis, which likely accelerated cell senescence through activating ROS signals. The results from this study provide evidence supporting an ABA biosynthetic regulation process via the two NCED genes in peach fruit, and suggest a mechanism of ABA-induced fruit ripening and senescence.

Functional characterization of Citrus PSY gene in Hongkong kumquat (Fortunella hindsii Swingle).

Citrus, rich in carotenoids, is the most important fruit crop based on the total annual production. In the carotenoid biosynthesis pathway, phytoene synthase (PSY, EC 2.5.1.32) catalyzes the dimerization of two molecules of geranylgeranyl pyrophosphate (GGPP) to phytoene and has been shown to be a rate-limiting enzyme for the synthesis of carotenoids. In this study, we investigated catalytic activity of CsPSY from Cara Cara navel orange (Citrus sinensis Osbeck) by heterologous expression in Escherichia coli containing a GGPP-producing plasmid. Moreover, the effects of CsPSY overexpression on carotenoid accumulation were also functionally analyzed in transgenic Hongkong kumquat (Fortunella hindsii Swingle). The resulting transgenic plants produced orange fruits, and extracts from the fruits of four overexpressing plants had a 2.5-fold average increase of phytoene with the content approximately 71.38 microg/g fresh weight. Lycopene, beta-carotene, and beta-cryptoxanthin in transgenic fruits were also markedly increased, whereas the levels of lutein and violaxanthin kept nearly unchanged with 1.1-1.3 folds variation. Transcript levels of carotenoid biosynthetic genes in the CsPSY overexpressed plants remained unaltered except that PDS and ZDS showed a minor increase. This study suggests that CsPSY plays a crucial role in citrus carotenoid biosynthesis and could be used as a means of engineering fruit crop for the production of carotenoids.

Yield-Maturity Relationships of Summer Maize from 2003 to 2017 in the Huanghuaihai Plain of China.

Information on yield-maturity relationships is important for maize breeding and cultivation, but it is seldom available in geographic zones where there are limited heat resources for summer maize. Two novel systematic crop yield models were put forward in terms of production efficiency. These models as well as three other conventional models were used to analyze the crop yield and maturity dataset of 23,691 records that were collected from the annual reports for the national summer maize zonal trials conducted in the Huanghuaihai Plain of China during 2003 to 2017. (1) Crop yield increases were usually below 14.5 kg/666.7 m2 due to longer maturity days, varying from 1 d to 15 d increments. Maize hybrids with later maturity fell into five categories: statistically significantly less, not significantly less, the same, not significantly more, or statistically significantly more output than their earlier counterparts. (2) Three yield components acted on crop yield gaps in the order of descending effects as kernel number per ear ≈ 1000-kernel weight > ear number per unit land area. (3) Space production efficiency was more important than canopy volume to crop yield. (4) Time production efficiency was dominant and maturity was negligible in crop yield formation. The findings provide insights into yield-maturity relationships in maize and useful information for summer maize breeding and cultivation strategies.

Natural Variation in CCD4 Promoter Underpins Species-Specific Evolution of Red Coloration in Citrus Peel.

Carotenoids and apocarotenoids act as phytohormones and volatile precursors that influence plant development and confer aesthetic and nutritional value critical to consumer preference. Citrus fruits display considerable natural variation in carotenoid and apocarotenoid pigments. In this study, using an integrated genetic approach we revealed that a 5' cis-regulatory change at CCD4b encoding CAROTENOID CLEAVAGE DIOXYGENASE 4b is a major genetic determinant of natural variation in C30 apocarotenoids responsible for red coloration of citrus peel. Functional analyses demonstrated that in addition the known role in synthesizing ß-citraurin, CCD4b is also responsible for the production of another important C30 apocarotenoid pigment, ß-citraurinene. Furthermore, analyses of the CCD4b promoter and transcripts from various citrus germplasm accessions established a tight correlation between the presence of a putative 5' cis-regulatory enhancer within an MITE transposon and the enhanced allelic expression of CCD4b in C30 apocarotenoid-rich red-peeled accessions. Phylogenetic analysis provided further evidence that functional diversification of CCD4b and naturally occurring variation of the CCD4b promoter resulted in the stepwise evolution of red peels in mandarins and their hybrids. Taken together, our findings provide new insights into the genetic and evolutionary basis of apocarotenoid diversity in plants, and would facilitate breeding efforts that aim to improve the nutritional and aesthetic value of citrus and perhaps other fruit crops.

Carotenoid Metabolism in Plants: The Role of Plastids.

Carotenoids are indispensable to plants and critical in human diets. Plastids are the organelles for carotenoid biosynthesis and storage in plant cells. They exist in various types, which include proplastids, etioplasts, chloroplasts, amyloplasts, and chromoplasts. These plastids have dramatic differences in their capacity to synthesize and sequester carotenoids. Clearly, plastids play a central role in governing carotenogenic activity, carotenoid stability, and pigment diversity. Understanding of carotenoid metabolism and accumulation in various plastids expands our view on the multifaceted regulation of carotenogenesis and facilitates our efforts toward developing nutrient-enriched food crops. In this review, we provide a comprehensive overview of the impact of various types of plastids on carotenoid biosynthesis and accumulation, and discuss recent advances in our understanding of the regulatory control of carotenogenesis and metabolic engineering of carotenoids in light of plastid types in plants.

[Case recruitment in large-scale clinical trials of traditional Chinese medicine].

Case recruitment of large-scale clinical trials should be strictly checked in quality and quantity for it is the key to clinical trial. This study discusses the main difficulties and countermeasures in the case recruitment of large sample, multi-center clinical trials according to the national research project "Myocardial Infarction Secondary Prevention Study in Traditional Chinese Medicine".

[Administration and quality control of large-scale clinical trials of traditional Chinese medicine].

Abstract: Large-scale clinical trial is an important measure of clinical evaluation on drugs. This paper introduces the concept and features of large-scale clinical trial, the possibility and necessity of large-scale clinical trial of traditional Chinese medicine, as well as its administration and quality control, with Myocardial Infarction Secondary Prevention Study in Traditional Chinese Medicine (MISPS-TCM), a National Program Subject, as an example.

Dynamic changes in methylome and transcriptome patterns in response to methyltransferase inhibitor 5-azacytidine treatment in citrus.

DNA methylation is known to play an important role in various developmental processes in plants. However, there is a general lack of understanding about the possible functions of DNA methylation in fruit trees. Using callus as a model, methylome, transcriptome and metabolite changes were assessed after treatment with the DNA methyltransferase inhibitor 5-azacytidine (5azaC). Genome-wide methylome analysis revealed the demethylation of a diverse of genes, including many genes encoding transcription factors (TFs), genes involved in biological processes, and the up-regulation of a wide range of transposable elements (TEs). Combined with the RNA-seq data, we observed no obvious genome-wide correlation between the changes in methylation status and expression levels. Furthermore, 5azaC treatment induced carotenoid degradation along with strong activation of carotenoid cleavage dioxygenases 1 (CpCCD1). Functional complementation analysis in bacterial system showed that CpCCD1 exhibited strong catalytic activities toward zeaxanthin, ß-carotene and lycopene. In summary, 5azaC treatments induced carotenoid degradation by CpCCD1 activation and led to a genome-wide demethylation effect.

Practice of Comparative Effectiveness Research to Identify Treatment Characteristics of Similar Chinese Patent Medicine for Angina Pectoris.

OBJECTIVE: Individualized application of TCM is not easy and may lead to undesirable results, such as poor effect or even adverse reactions. This trial aims to compare two common Chinese patent medicines with similar effects. BACKGROUND OF THE RESEARCH: Four hospitals carried out the test at the same time in Tianjin city of China. PARTICIPANTS: 144 patients were involved in this study; all patients must meet the diagnostic criteria. INTERVENTIONS: Qishen Yiqi pills, compound danshen pills, and their placebos; an efficacy analysis was conducted after the first medication and after crossover medication. PRIMARY OUTCOME MEASURES: The primary index of end point includes Seattle Angina Questionnaire score-7 and score of 7-point Likert Scale; the curative effect was compared with minimal clinically important differences value. RESULT: Two drugs have their respective advantages in treating SAP. In practical application, the two drugs shall be discriminated in use based on patients' specific symptoms. TRIAL REGISTRATION: Chinese clinical trials register is ChiCTR-TTRCC-14004406 (registered 23 March 2014).