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Citrus, one of the largest fruit crops with global economic and nutritional importance, contains fruit known as hesperidium with unique morphological types. Citrus fruit ripening is accompanied by chlorophyll degradation and carotenoid biosynthesis, which are indispensably linked to color formation and the external appearance of citrus fruits. However, the transcriptional coordination of these metabolites during citrus fruit ripening remains unknown. Here, we identified the MADS-box transcription factor CsMADS3 in Citrus hesperidium that coordinates chlorophyll and carotenoid pools during fruit ripening. CsMADS3 is a nucleus-localized transcriptional activator, and its expression is induced during fruit development and coloration. Overexpression of CsMADS3 in citrus calli, tomato (Solanum lycopersicum), and citrus fruits enhanced carotenoid biosynthesis and upregulated carotenogenic genes while accelerating chlorophyll degradation and upregulating chlorophyll degradation genes. Conversely, the interference of CsMADS3 expression in citrus calli and fruits inhibited carotenoid biosynthesis and chlorophyll degradation and down-regulated the transcription of related genes. Further assays confirmed that CsMADS3 directly binds and activates the promoters of phytoene synthase 1 (CsPSY1) and chromoplast-specific lycopene β-cyclase (CsLCYb2), two key genes in the carotenoid biosynthetic pathway, and STAY-GREEN (CsSGR), a critical chlorophyll degradation gene, which explained the expression alterations of CsPSY1, CsLCYb2, and CsSGR in the above transgenic lines. These findings reveal the transcriptional coordination of chlorophyll and carotenoid pools in the unique hesperidium of Citrus and may contribute to citrus crop improvement.

As an essential horticultural crop, Citrus has carotenoid diversity, which affects its aesthetic and nutritional values. β,β-Xanthophylls are the primary carotenoids accumulated in citrus fruits, and non-heme di-iron carotene hydroxylase (BCH) enzymes are mainly responsible for β,β-xanthophyll synthesis. Previous studies have focused on the hydroxylation of BCH1, but the role of its paralogous gene in citrus, BCH2, remains largely unknown. In this study, we revealed the β-hydroxylation activity of citrus BCH2 (CsBCH2) for the first time through the functional complementation assay using Escherichia coli, although CsBCH2 exhibited a lower activity in hydroxylating β-carotene into β-cryptoxanthin than citrus BCH1 (CsBCH1). Our results showed that overexpression of CsBCH2 in citrus callus increased xanthophyll proportion and plastoglobule size with feedback regulation of carotenogenic gene expression. This study revealed the distinct expression patterns and functional characteristics of two paralogous genes, CsBCH1 and CsBCH2, and illustrated the backup compensatory role of CsBCH2 for CsBCH1 in citrus xanthophyll biosynthesis. The independent function of CsBCH2 and its cooperative function with CsBCH1 in β-cryptoxanthin biosynthesis suggested the potential of CsBCH2 to be employed for expanding the synthetic biology toolkit in carotenoid engineering.

To measure stomatal traits automatically and nondestructively, a new method for detecting stomata and extracting stomatal traits was proposed. Two portable microscopes with different resolutions (TipScope with a 40× lens attached to a smartphone and ProScope HR2 with a 400× lens) are used to acquire images of living stomata in maize leaves. FPN model was used to detect stomata in the TipScope images and measure the stomata number and stomatal density. Faster RCNN model was used to detect opening and closing stomata in the ProScope HR2 images, and the number of opening and closing stomata was measured. An improved CV model was used to segment pores of opening stomata, and a total of 6 pore traits were measured. Compared to manual measurements, the square of the correlation coefficient (R2 ) of the 6 pore traits was higher than 0.85, and the mean absolute percentage error (MAPE) of these traits was 0.02%-6.34%. The dynamic stomata changes between wild-type B73 and mutant Zmfab1a were explored under drought and re-watering condition. The results showed that Zmfab1a had a higher resilience than B73 on leaf stomata. In addition, the proposed method was tested to measure the leaf stomatal traits of other nine species. In conclusion, a portable and low-cost stomata phenotyping method that could accurately and dynamically measure the characteristic parameters of living stomata was developed. An open-access and user-friendly web portal was also developed which has the potential to be used in the stomata phenotyping of large populations in the future.

Organ allograft rejection is mainly driven by T-cell response. Studies have shown that fucosylation plays essential roles in the immune cell development and function. Terminal fucosylation inhibitor, 2-deoxy-D-galactose (2-D-gal), has been reported to suppress immunoresponse of macrophages, but its effects on T-cell-mediated immune response and transplant rejection have not been fully explored.The terminal fucosylation level in T cells was detected through ulex europaeus agglutinin-I staining. The consequences of 2-D-gal on murine T-cell proliferation, activation, cytokine secretion, and cell cycle were investigated in vitro. T-cell receptor signaling cascades were examined. Last, mouse skin transplant model was utilized to evaluate the regulatory effects of 2-D-gal on T-cell response in vivo.The expression of fucosyltransferase1 was upregulated in CD3/CD28-activated T cells along with an elevation of α(1,2)-fucosylation level as seen by ulex europaeus agglutinin-I staining. Furthermore, 2-D-gal suppressed T-cell activation and proliferation, decrease cytokines production, arrest cell cycle, and prevent the activation of T-cell receptor signaling cascades. In vivo experiments showed that 2-D-gal limited T-cell proliferation to prolong skin allograft in mice. This was accompanied by lower level of inflammatory cytokines, and were comparable to those treated with Cyclosporin A.Terminal fucosylation appears to play a role in T-cell activation and proliferation, and its inhibitor, 2-D-gal, can suppress T-cell activation and proliferation both in vitro and in vivo. In a therapeutic context, inhibiting terminal fucosylation may be a potential strategy to prevent allogeneic transplant rejection.

Senescence is a gradual physiological process involving the integration of numerous internal and environmental signals. Abscisic acid (ABA) is a well-known inducer of senescence. However, the regulatory mechanisms underlying ABA-mediated senescence remain largely unknown. Here, we report that the citrus homeodomain leucine zipper I (HD-ZIP I) transcription factor CsHB5 functions as a regulator of ABA-triggered senescence. CsHB5 acts as a nucleus-localized transcriptional activator, the expression of which appeared to be closely associated with citrus senescence. Overexpression of CsHB5 in citrus calli upregulated the expression of ABA- and reactive oxygen species (ROS)-related genes, and significantly increased the content of ABA and hydrogen peroxide (H2 O2 ), whereas silencing CsHB5 in citrus calli downregulated the expression of ABA-related genes. Additionally, heterogenous overexpression of CsHB5 in Solanum lycopersicum (tomato) and Arabidopsis thaliana (Arabidopsis) leads to early leaf yellowing under dark-induced senescence conditions. Meanwhile, the levels of ABA and H2 O2 in transgenic tomatoes increased significantly and the lycopene content decreased. Transcriptome analysis of CsHB5-overexpressing citrus calli and tomato showed that CsHB5 was involved in multiple senescence-associated processes, including chlorophyll degradation, nutrient compound biosynthesis and transport, as well as ABA and ROS signal transduction. The results of yeast one-hybrid assays, electrophoretic mobility shift assays and dual luciferase assays indicated that CsHB5 directly binds to the promoters of ABA biosynthetic genes, including β-carotene hydroxylase 1 (BCH1) and 9-cis-epoxycarotenoid dioxygenase 2 (NCED2), thereby activating their transcription. Our findings revealed that CsHB5 participates in senescence, at least partly, by directly controlling ABA accumulation. Our work provides insight into the regulatory mechanisms underlying ABA-mediated senescence.

Difficulty in preventing crops from plant viruses urges to discover novel efficient antiviral chemicals, which is sped up by precise screening methods. Fluorescence-based methods have recently been applied as innovative and rapid tools for visually monitoring the replication of viruses and screening of antivirals, whereas the quantification of fluorescence signals mainly depends on manually calculating the fluorescent spots, which is time-consuming and imprecise. In the present work, the fluorescence spots were automatically identified, and the fluorescence area was directly quantified by a program developed in our group, which avoided subjective errors from the operators. We further employed this digital and visual screening assay to identify antivirals using the tobacco mosaic virus-green fluorescence protein (TMV-GFP) construct, in which the expression of GFP intuitively reflected the efficacy of antivirals. The accuracy of this assay was validated by quantifying the activities of the commercial antiviral inhibitors ribavirin and ningnanmycin and then was applied to evaluate the subtle activity differences of a series of newly synthesized carbazole and β-carboline alkaloid derivatives. Among them, compounds 5 (76%) and 11 (63%) exhibited anti-TMV activities comparable to that of ningnanmycin (65%) at 50 μM, and they delayed the multiplication of TMV in the early stage of infection without phytotoxicity. Taken together, these findings demonstrated that the digital and visual TMV-GFP screening method was competent to test the antiviral activities of compounds with subtle modifications and facilitated the discovery of novel antivirals.

Background Drought threatens the food supply of the world population. Dissecting the dynamic responses of plants to drought will be beneficial for breeding drought-tolerant crops, as the genetic controls of these responses remain largely unknown. Results Here we develop a high-throughput multiple optical phenotyping system to noninvasively phenotype 368 maize genotypes with or without drought stress over a course of 98 days, and collected multiple optical images, including color camera scanning, hyperspectral imaging, and X-ray computed tomography images. We develop high-throughput analysis pipelines to extract image-based traits (i-traits). Of these i-traits, 10,080 were effective and heritable indicators of maize external and internal drought responses. An i-trait-based genome-wide association study reveals 4322 significant locus-trait associations, representing 1529 quantitative trait loci (QTLs) and 2318 candidate genes, many that co-localize with previously reported maize drought responsive QTLs. Expression QTL (eQTL) analysis uncovers many local and distant regulatory variants that control the expression of the candidate genes. We use genetic mutation analysis to validate two new genes, ZmcPGM2 and ZmFAB1A, which regulate i-traits and drought tolerance. Moreover, the value of the candidate genes as drought-tolerant genetic markers is revealed by genome selection analysis, and 15 i-traits are identified as potential markers for maize drought tolerance breeding. Conclusion Our study demonstrates that combining high-throughput multiple optical phenotyping and GWAS is a novel and effective approach to dissect the genetic architecture of complex traits and clone drought-tolerance associated genes.

Citrus is an important horticultural crop with global, economic and nutritional value. Carotenoids represent the main pigments in citrus fruits and contribute to the esthetic and nutritional value. The complexity of carotenoids in citrus poses a challenge for carotenoid analysis. In this chapter, we describe methods for carotenoid extraction, detection, and analysis that have been optimized for study of citrus fruits. High-performance liquid chromatography (HPLC) and Ultrahigh-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) strategies are used for carotenoid profiling of citrus fruit carotenoids and are explained in detail. We outline the applications, advantages, and disadvantages of using these methods to analyze carotenoids in the main citrus species including mandarin, orange, and pummelo.

Apomixis, or asexual seed formation, is prevalent in Citrinae via a mechanism termed nucellar or adventitious embryony. Here, multiple embryos of a maternal genotype form directly from nucellar cells in the ovule and can outcompete the developing zygotic embryo as they utilize the sexually derived endosperm for growth. Whilst nucellar embryony enables the propagation of clonal plants of maternal genetic constitution, it is also a barrier to effective breeding through hybridization. To address the genetics and evolution of apomixis in Citrinae, a chromosome-level genome of the Hongkong kumquat (Fortunella hindsii) was assembled following a genome-wide variation map including structural variants (SVs) based on 234 Citrinae accessions. This map revealed that hybrid citrus cultivars shelter genome-wide deleterious mutations and SVs into heterozygous states free from recessive selection, which may explain the capability of nucellar embryony in most cultivars during Citrinae diversification. Analyses revealed that parallel evolution may explain the repeated origin of apomixis in different genera of Citrinae. Within Fortunella, we found that apomixis of some varieties originated via introgression. In apomictic Fortunella, the locus associated with apomixis contains the FhRWP gene, encoding an RWP-RK domain-containing protein previously shown to be required for nucellar embryogenesis in Citrus. We found the heterozygous SV in the FhRWP and CitRWP promoters from apomictic Citrus and Fortunella, due to either two or three miniature inverted transposon element (MITE) insertions. A transcription factor, FhARID, encoding an AT-rich interaction domain-containing protein binds to the MITEs in the promoter of apomictic varieties, which facilitates induction of nucellar embryogenesis. This study provides evolutionary genomic and molecular insights into apomixis in Citrinae and has potential ramifications for citrus breeding.

Chromoplast-specific lycopene β-cyclase (LCYb2) is a critical carotenogenic enzyme, which controls the massive accumulation of downstream carotenoids, especially provitamin A carotenoids, in citrus. Its regulatory metabolism is largely unknown. Here, we identified a group I ethylene response factor, CsERF061, in citrus by yeast one-hybrid screen with the promoter of LCYb2. The expression of CsERF061 was induced by ethylene. Transcript and protein levels of CsERF061 were increased during fruit development and coloration. CsERF061 is a nucleus-localized transcriptional activator, which directly binds to the promoter of LCYb2 and activates its expression. Overexpression of CsERF061 in citrus calli and tomato fruits enhanced carotenoid accumulation by increasing the expression of key carotenoid pathway genes, and increased the number of chromoplasts needed to sequester the elevated concentrations of carotenoids, which was accompanied by changes in the concentrations of abscisic acid and gibberellin. Electrophoretic mobility shift and dual-luciferase assays verified that CsERF061 activates the promoters of nine other key carotenoid pathway genes, PSY1, PDS, CRTISO, LCYb1, BCH, ZEP, NCED3, CCD1, and CCD4, revealing the multitargeted regulation of CsERF061. Collectively, our findings decipher a novel regulatory network of carotenoid enhancement by CsERF061, induced by ethylene, which will be useful for manipulating carotenoid accumulation in citrus and other plants.

Carotenoids provide precursors for the biosynthesis of strigolactones, which are a new class of hormones that are essential in phosphate (Pi) signaling during plant development. Carotenoid metabolism is a finely tuned pathway, but our understanding of the regulation mechanisms is still limited. In this study, we isolated a protein designated as CsPHL3 from citrus. CsPHL3 belonged to the Pi starvation response factor (PHR)-like subclade and was upregulated by low Pi. Acting as a nucleus-localized protein with transactivation activity, CsPHL3 bound directly to activate the promoter of a key metabolic gene, lycopene β-cyclase1 (LCYb1). Transgenic analysis revealed that the CsPHL3-overexpressing tomato plants exhibited abnormal growth, like the plants grew under limited Pi conditions. The transgenic lines showed reduced carotenoid contents and elevated expression of LCYb genes but downregulation of other key carotenogenic genes, including phytoene synthase (PSY). Moreover, CsPHL3 induced anthocyanin biosynthesis and affected Pi signaling in the transgenic plants. We further demonstrated that the expression of PSY was negatively regulated by CsPHL3 and high Pi. It is concluded that CsPHL3 is a Pi starvation response factor that negatively regulates carotenoid metabolism by modulating the expression of carotenogenic genes. Establishment of the CsPHL3-CsLCYb1 network provides new valuable knowledge of the function and underlying mechanism of PHR transcription factors and expands our understanding of the complex regulation mechanisms of carotenoid biosynthesis.

Carotenoids are indispensable for both human health and plant survival. Citrus, is one of the fruit crops richest in carotenoid compounds, with approximately 115 kinds of carotenoids; tremendous diversity in carotenoids composition and concentration exists among various species, showing different colors from nearly white to crimson. The carotenoid biosynthetic pathway and the key carotenogenic genes have been identified in citrus; however, the underlying regulatory mechanisms remain unclear. In this study, among the main species of genus Citrus (primitive, wild, and cultivated), we detected carotenoids in flavedo using High-Performance Liquid Chromatography, and analyzed variations in cis-acting elements in the promoters of key carotenoid pathway genes. Intriguingly, both carotenoid composition and content were generally increased during the evolution of citrus, and the corresponding variations in the promoters were identified, including the gain or loss of critical environmental stress-responsive elements and hormone-responsive elements, which are closely associated with carotenoid enhancement. In addition, pummelo has the most heat-responsive elements, but the Mangshan mandarin does not have this element in the promoters of PSY, which is highly related to their geographical origin and indicate that temperature is a critical environmental signal influencing carotenoid accumulation. Moreover, the abscisic acid-responsive motif was rich in almost all the seven species, but the ethylene-responsive motif was deficient, which demystified the unique phytohormone regulation mechanism of carotenoid accumulation in citrus. Overall, our study provides new insights into the molecular regulatory mechanism of carotenoid enhancement in the evolution of citrus, which can facilitate breeding and cultivation efforts to improve the nutritional quality and esthetic value in citrus and hopefully other fruit crops.

Self-incompatibility (SI) is an important mechanism that prevents self-fertilization and inbreeding in flowering plants. The most widespread SI system utilizes S ribonucleases (S-RNases) and S-locus F-boxes (SLFs) as S determinants. In citrus, SI is ancestral, and Citrus maxima (pummelo) is self-incompatible, while Citrus reticulata (mandarin) and its hybrids are self-compatible (SC). Here, we identify nine highly polymorphic pistil-specific, developmentally expressed S-RNases from pummelo that segregate with S haplotypes in a gametophytic manner and cluster with authentic S-RNases. We provide evidence that these S-RNases function as the female S determinants in citrus. Moreover, we show that each S-RNase is linked to approximately nine SLFs. In an analysis of 117 citrus SLF and SFL-like (SLFL) genes, we reveal that they cluster into 12 types and that the S-RNases and intra-haplotypic SLF and SLFL genes co-evolved. Our data support the notion that citrus have a S locus comprising a S-RNase and several SLFs that fit the non-self-recognition model. We identify a predominant single nucleotide mutation, Sm-RNase, in SC citrus, which provides a 'natural' loss of function. We show that SI-SC transitions due to the Sm-RNase initially arose in mandarin, spreading to its hybrids and became fixed. Identification of an evolutionarily distant new genus utilizing the S-RNase-based SI system, >100 million years separated from the nearest S-RNase family, is a milestone for evolutionary comparative studies.

Self-incompatibility (SI) substantially restricts the yield and quality of citrus. Therefore, breeding and analyzing self-compatible germplasm is of great theoretical and practical significance for citrus. Here, we focus on the mechanism of a self-compatibility mutation in ‘Guiyou No. 1’ pummelo (Citrus maxima), which is a spontaneous mutant of ‘Shatian’ pummelo (Citrus maxima, self-incompatibility). The rate of fruit set and the growth of pollen tubes in the pistil confirmed that a spontaneous mutation in the pistil is responsible for the self-compatibility of ‘Guiyou No. 1’. Segregation ratios of the S genotype in F1 progeny, expression analysis, and western blotting validated that the reduced levels of S2-RNase mRNA contribute to the loss of SI in ‘Guiyou No. 1’. Furthermore, we report a phased assembly of the ‘Guiyou No. 1’ pummelo genome and obtained two complete and well-annotated S haplotypes. Coupled with an analysis of SV variations, methylation levels, and gene expression, we identified a candidate gene (CgHB40), that may influence the regulation of the S2-RNase promoter. Our data provide evidence that a mutation that affects the pistil led to the loss of SI in ‘Guiyou No. 1’ by influencing a poorly understood mechanism that affects transcriptional regulation. This work significantly advances our understanding of the genetic basis of the SI system in citrus and provides information on the regulation of S-RNase genes.

Lycopene is the main pigment in red-flesh citrus fruits, and its formation is a research hotspot. To explore the basis of lycopene accumulation in red-flesh mutants, we profiled the terpenoid metabolites. Compared with their respective wild types, Cara Cara (Cara) [and Red-Anliu (R-An)] oranges showed increased carotenoid and limonoid aglycone contents and decreased contents of abscisic acid (ABA) catabolites, monoterpenoid volatiles, and sesquiterpenoid volatiles. Cara contained less than half of the amount of ABA glucose ester (ABAGE), the main ABA derivative in oranges. Parallel lower transcript levels of NCED and ABA glucosyltransferase in Cara were detected at the mature green stage. These results document the changes in terpenoid profiles in Cara and show that the red flesh of citrus color mutants is related to weak ABA catabolism, especially ABAGE, and decreased transcript levels of two genes encoding uridine diphosphate (UDP)-glycosyltransferases that catalyze ABAGE biosynthesis.

In citrus, PH4–Noemi, a regulatory complex, controls proanthocyanidin accumulation by activating the transcription of proanthocyanidin biosynthetic genes, and also regulates expression of Noemi, promoting proanthocyanidin accumulation by positive feedback., Proanthocyanidins (PAs; or condensed tannins) are a major class of flavonoids that contribute to citrus fruit quality. However, the molecular mechanism responsible for PA biosynthesis and accumulation in citrus remains unclear. Here, we identify a PH4–Noemi regulatory complex that regulates proanthocyanidin biosynthesis in citrus. Overexpression of PH4 or Noemi in citrus calli activated the expression of PA biosynthetic genes and significantly increased the PA content. Interestingly, Noemi was also shown to be up-regulated in CsPH4-overexpressing lines compared with wild-type calli. Simultaneously, CsPH4 partially complemented the PA-deficient phenotype of the Arabidopsis tt2 mutant and promoted PA accumulation in the wild-type. Further analysis revealed that CsPH4 interacted with Noemi, and together these proteins synergistically activated the expression of PA biosynthetic genes by directly binding to the MYB-recognizing elements (MRE) of the promoters of these genes. Moreover, CsPH4 could directly bind to the promoter of Noemi and up-regulate the expression of this gene. These findings explain how the CsPH4–Noemi regulatory complex contributes to the activation of PA biosynthetic genes via a positive feedback loop and provide new insights into the molecular mechanisms underlying PA biosynthesis, which can be effectively employed for metabolic engineering to improve citrus fruit quality.

Summary Hongkong kumquat (Fortunella hindsii) is a wild citrus species characterized by dwarf plant height and early flowering. Here, we identified the monoembryonic F. hindsii (designated as ‘Mini‐Citrus’) for the first time and constructed its selfing lines. This germplasm constitutes an ideal model for the genetic and functional genomics studies of citrus, which have been severely hindered by the long juvenility and inherent apomixes of citrus. F. hindsii showed a very short juvenile period (~8 months) and stable monoembryonic phenotype under cultivation. We report the first de novo assembled 373.6 Mb genome sequences (Contig‐N50 2.2 Mb and Scaffold‐N50 5.2 Mb) for F. hindsii. In total, 32 257 protein‐coding genes were annotated, 96.9% of which had homologues in other eight Citrinae species. The phylogenomic analysis revealed a close relationship of F. hindsii with cultivated citrus varieties, especially with mandarin. Furthermore, the CRISPR/Cas9 system was demonstrated to be an efficient strategy to generate target mutagenesis on F. hindsii. The modifications of target genes in the CRISPR‐modified F. hindsii were predominantly 1‐bp insertions or small deletions. This genetic transformation system based on F. hindsii could shorten the whole process from explant to T1 mutant to about 15 months. Overall, due to its short juvenility, monoembryony, close genetic background to cultivated citrus and applicability of CRISPR, F. hindsii shows unprecedented potentials to be used as a model species for citrus research.

Self-incompatibility (SI) substantially restricts the yield and quality of citrus. Therefore, breeding and analyzing self-compatible germplasm is of great theoretical and practical significance for citrus. Here, we focus on the mechanism of a self-compatibility mutation in ‘Guiyou No. 1’ pummelo (Citrus maxima), which is a spontaneous mutant of ‘Shatian’ pummelo (Citrus maxima, self-incompatibility). The rate of fruit set and the growth of pollen tubes in the pistil confirmed that a spontaneous mutation in the pistil is responsible for the self-compatibility of ‘Guiyou No. 1’. Segregation ratios of the S genotype in F1 progeny, expression analysis, and western blotting validated that the reduced levels of S2-RNase mRNA contribute to the loss of SI in ‘Guiyou No. 1’. Furthermore, we report a phased assembly of the ‘Guiyou No. 1’ pummelo genome and obtained two complete and well-annotated S haplotypes. Coupled with an analysis of SV variations, methylation levels, and gene expression, we identified a candidate gene (CgHB40), that may influence the regulation of the S2-RNase promoter. Our data provide evidence that a mutation that affects the pistil led to the loss of SI in ‘Guiyou No. 1’ by influencing a poorly understood mechanism that affects transcriptional regulation. This work significantly advances our understanding of the genetic basis of the SI system in citrus and provides information on the regulation of S-RNase genes.

Curcumin is natural polyphenol from Curcuma longa rhizomes with several biological properties. Our previous studies demonstrated that curcumin inhibited functional gastric emptying disorders induced by L-arginine, the precursor of nitric oxide (NO), and atropine, an acetylcholine receptor (AChR) blocker. However, the mechanism of action of curcumin remains unclear. In the present study, mouse models of functional gastric emptying disorders induced by L-arginine and atropine were used to examine changes in interstitial cells of Cajal (ICC) and NO- and ACh-mediated regulation of gastrointestinal motility. Curcumin pre-treatment ameliorated the gastric emptying rate in mice treated with L-arginine or atropine (P

Deciphering the genetic basis of plant secondary metabolism will provide useful insights for genetic improvement and enhance our fundamental understanding of plant biological processes. Although citrus plants are among the most important fruit crops worldwide, the genetic basis of secondary metabolism in these plants is largely unknown. Here, we use a high-density linkage map to dissect large-scale flavonoid metabolic traits measured in different tissues (young leaf, old leaf, mature pericarp, and mature pulp) of an F1 pseudo-testcross citrus population. We detected 80 flavonoids in this population and identified 138 quantitative trait loci (QTLs) for 57 flavonoids in these four tissues. Based on transcriptional profiling and functional annotation, twenty-one candidate genes were identified, and one gene encoding flavanone 3-hydroxylase (F3H) was functionally verified to result in naturally occurring variation in dihydrokaempferol content through genetic variations in its promoter and coding regions. The abundant data resources collected for diverse citrus germplasms here lay the foundation for complete characterization of the citrus flavonoid biosynthetic pathway and will thereby promote efficient utilization of metabolites in citrus quality improvement.

In this study, based on automatic control and image processing, a high-throughput and low-cost maize ear traits scorer (METS) was developed for the automatic measurement of 34 maize ear traits. In total, 813 maize ears were measured using METS, and the results showed that the square of the correlation coefficient (R(2)) of the manual measurements versus the automatic measurements for ear length, ear diameter, and kernel thickness were 0.96, 0.79, and 0.85, respectively. These maize ear traits could be used to classify the type, and the results showed that the classification accuracy of the support vector machine (SVM) model for the test set was better than that of the random forest (RF) model. In addition, the general applicability of the image analysis pipeline was also demonstrated on other independent maize ear phenotyping platforms. In conclusion, equipped with image processing and automatic control technologies, we have developed a high-throughput method for maize ear scoring, which could be popularized in maize functional genetics, genomics, and breeding applications. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11032-021-01205-4.

Domesticated citrus varieties are woody perennials and interspecific hybrid crops of global economic and nutritional importance. The citrus fruit “hesperidium” is a unique morphological innovation not found in any other plant lineage. Efforts to improve the nutritional quality of the fruit are predicated on understanding the underlying regulatory mechanisms responsible for fruit development, including temporal control of chlorophyll degradation and carotenoid biosynthesis. Here, we investigated the molecular basis of the navel orange (Citrus sinensis) brown flavedo mutation, which conditions flavedo that is brown instead of orange. To overcome the limitations of using traditional genetic approaches in citrus and other woody perennials, we developed a strategy to elucidate the underlying genetic lesion. We used a multi-omics approach to collect data from several genetic sources and plant chimeras to successfully decipher this mutation. The multi-omics strategy applied here will be valuable in driving future gene discovery efforts in citrus as well as in other woody perennial plants. The comparison of transcriptomic and genomic data from multiple genotypes and plant sectors revealed an underlying lesion in the gene encoding STAY-GREEN (SGR) protein, which simultaneously regulates carotenoid biosynthesis and chlorophyll degradation. However, unlike SGR of other plant species, we found that the carotenoid and chlorophyll regulatory activities could be uncoupled in the case of certain SGR alleles in citrus and thus we propose a model for the molecular mechanism underlying the brown flavedo phenotype. The economic and nutritional value of citrus makes these findings of wide interest. The strategy implemented, and the results obtained, constitute an advance for agro-industry by driving opportunities for citrus crop improvement.

Carotenoids are a large class of structures that are important in human health and include both provitamin A and nonprovitamin A compounds. Vitamin A deficiency is a global health problem that can be alleviated by enriching provitamin A carotenoids in a range of food crops. Suitable plants for biofortification are those with high levels of the provitamin A biosynthetic precursor, lycopene, which is enzymatically converted by lycopene β-cyclase (LCYB) to β-carotene, a provitamin A carotenoid. Crops, such as citrus, naturally accumulate high levels of provitamin A and other health-promoting carotenoids. Such plants may have useful genes to expand the synthetic biology toolbox for producing a range of phenotypes, including both high provitamin A crops and crops with unique compositions of health-promoting carotenoids. To examine enzyme variants having different activity levels, we introduced two citrus LCYB alleles into tomato, a plant with fruit rich in lycopene. Overexpression in tomato of the stronger allele of the citrus chromoplast-specific lycopene β-cyclase (CsLCYb2a) produced "golden" transgenic tomato fruits with 9.3-fold increased levels of β-carotene at up to 1.5 mg/g dry weight. The use of the weaker allele, CsLCYb2b, also led to enhanced levels of β-carotene but in the context of a more heterogeneous composition of carotenoids. From a synthetic biology standpoint, these allelic differences have value for producing cultivars with unique carotenoid profiles. Overexpression of the citrus LCYB genes was accompanied by increased expression of other genes encoding carotenoid biosynthetic enzymes and increased size and number of chromoplasts needed to sequester the elevated levels of carotenoids in the transgenic tomato fruits. The overexpression of the citrus LCYB genes also led to a pleiotropic effect on profiles of phytohormones and primary metabolites. Our findings show that enzyme variants are essential synthetic biology parts needed to create a wider range of metabolic engineering products. In this case, strong and weak variants of LCYB proved useful in creating dietary sources to alleviate vitamin A deficiency or, alternatively, to create crops with a heterogeneous composition including provitamin A and healthful, nonprovitamin A carotenoids.

Cisplatin is a widely used anticancer drug that has adverse effects on gastrointestinal function. Curcumin is a natural polyphenol extracted from the rhizome of turmeric that has a wide range of biological activities. The present study investigated the effects of cisplatin on gastric emptying in mice and examined whether these can be inhibited by curcumin. We found that pretreatment with curcumin (200 mg/kg/day) for 10-30 days partly inhibited the decreases in gastric emptying rate and body weight induced by cisplatin. Furthermore, cisplatin reduced acetylcholine (ACh) concentration and the messenger RNA (mRNA) level of ACh receptor (AChR) as well as acetylcholinesterase activity in the stomach of mice; caused ultrastructural damage to interstitial cells of Cajal (ICC); and altered the expression of c-kit/stem cell factor and the gap junction protein connexin 43 in ICC. Curcumin pretreatment inhibited the effects of cisplatin on ACh indicators and ICC. These results demonstrate that curcumin can protect against cisplatin-induced gastric emptying disorder and thus has therapeutic potential for alleviating this condition in cancer patients receiving cisplatin chemotherapy.

Carotenoids in citrus contribute to the quality of the fruit, but the mechanism of its transcriptional regulation is fairly unknown. Here, we characterized a citrus FRUITFULL sub-clade MADS gene, CsMADS5, that was ripening-inducible and acted as a nucleus-localized trans-activator. Transient overexpression of CsMADS5 in citrus induced fruit coloration and enhanced carotenoid concentrations. The expression of carotenogenic genes including phytoene synthase (PSY), phytoene desaturase (PDS), and lycopene β-cyclase 1 (LCYb1) was increased in the peels of fruits overexpressing CsMADS5. Similar results were observed from stable overexpression of CsMADS5 in tomato fruits and citrus calli, even though the effect of CsMADS5 on carotenoid metabolism in transgenic citrus calli was limited. Further biochemical analyses demonstrated that CsMADS5 activated the transcription of PSY, PDS, and LCYb1 by directly binding to their promoters. We concluded that CsMADS5 positively regulates carotenoid biosynthesis in fruits by directly activating the transcription of carotenogenic genes. Moreover, CsMADS5 physically interacted with a positive regulator CsMADS6, indicating that CsMADS5 may form an enhancer complex with CsMADS6 to synergistically promote carotenoid accumulation. These findings expand our understanding of the complex transcriptional regulatory hierarchy of carotenoid biosynthesis during fruit ripening.

Combining high-throughput micro-CT-RGB phenotyping with genome-wide association study reveals the genetic architecture of tiller development in the indica subpopulation of rice., Manual phenotyping of rice tillers is time consuming and labor intensive, and lags behind the rapid development of rice functional genomics. Thus, automated, non-destructive methods of phenotyping rice tiller traits at a high spatial resolution and high throughput for large-scale assessment of rice accessions are urgently needed. In this study, we developed a high-throughput micro-CT-RGB imaging system to non-destructively extract 739 traits from 234 rice accessions at nine time points. We could explain 30% of the grain yield variance from two tiller traits assessed in the early growth stages. A total of 402 significantly associated loci were identified by genome-wide association study, and dynamic and static genetic components were found across the nine time points. A major locus associated with tiller angle was detected at time point 9, which contained a major gene, TAC1. Significant variants associated with tiller angle were enriched in the 3ʹ-untranslated region of TAC1. Three haplotypes for the gene were found, and rice accessions containing haplotype H3 displayed much smaller tiller angles. Further, we found two loci containing associations with both vigor-related traits identified by high-throughput micro-CT-RGB imaging and yield. The superior alleles would be beneficial for breeding for high yield and dense planting.

A novel biological treatment of staying with commercially mature apple fruits was proposed to promote the peel coloration of ‘Newhall’ navel orange fruit (Citrus sinensis Osbeck), which could conduce to the formation of uniform orange red peel color. At 15 days after treatment (DAT), the staying with apple (SWA) treatment led to a nearly 4-fold increase in the total carotenoid content compared with the control. Specifically, the red color apocarotenoid β-citraurin showed the highest degree of enhancement (~6 fold) under SWA treatment. Simultaneously, the expression level of β-citraurin biosynthetic gene carotenoid cleavage dioxygenase 4b (CCD4b) was dramatically induced (about 35 folds). Furthermore, SWA had the same effect of promoting coloration and obviously alleviate calyx senescence of citrus fruit compared with ethylene treatment. In conclusion, SWA treatment is a potential environment-friendly and recyclable postharvest technology to improve peel color and maintain fruit freshness for citrus.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a powerful tool for the detection and quantification of target gene expression levels. The accuracy of qRT-PCR data largely depends on the stability of the reference genes used for data normalization. Thus, the selection of suitable reference genes for qRT-PCR is necessary for accurate gene expression, especially under specific experimental conditions. In this study, stably expressed genes in peach fruit stored at various postharvest temperatures were screened based on RNA-seq data. Four newly identified stable genes and 12 traditional reference genes were selected as candidates. Their expression stability was examined in the peel and flesh of peach stored at five different temperatures (5 °C, 15 °C, 25 °C, ambient temperature, and 35 °C). Gene expression was further characterized in different plant tissues (root, stem, leaf, flower, and fruit) and fruit developmental stages. The overall performance of each candidate in all sample sets was also evaluated. As a result, the expression of PpeIF-1A was the most stable across the set of all samples, peel and flesh samples at different storage temperatures, and fruit developmental stages. PpGAPDH stability ranked highest in plant tissues. In general, PpeIF-1A and PpMUB6 were the most appropriate reference genes for all five experiments whereas PpRPS28 , PpRPT-5A, and PpAKT3 were unsuitable control genes with variable expression patterns. The relative expression of the phytoene synthase gene PpPSY1 was assessed to confirm the utility of the selected reference genes in this study. Taken together, this study identified the reference genes suitable for the accurate and reliable normalization of peach gene expression data under different experimental conditions. The novel reference gene PpMUB6 exhibited stable expression compared to most of the traditional reference genes. These results provide guidance for selecting reliable internal control genes and will benefit future gene expression analysis in a wide variety of conditions in peach.

Peach [ Prunus persica (L.)] gum exudates are produced by the trunks and fruits in peach gummosis. Clinically, these exudates have been used to treat diabetes in China, though the molecular mechanism underlying remains unclear. In the current study, a novel peach gum-derived polysaccharide was isolated, designated as PGPSD, and its anti-diabetic effect was assessed in mice. This polysaccharide was composed of arabinose, xylose and galactose in the molar ratio of 5.98:1:3.55, with the average molecular weight at 1.00 × 10 6 Da. The animal study demonstrated that the PGPSD polysaccharide significantly lowered the postprandial blood glucose in streptozotocin-induced diabetic mice. Histology and immunohistochemistry results further confirmed that the application of PGPSD polysaccharide partially restored the pancreatic islets in diabetic mice, and enhanced the expression of pancreatic duodenal homeobox-1, insulin and hexokinase1. Collectively, the data suggested that the peach gum-derived polysaccharide had a meaningful potential as a non-insulin therapeutic compound in the treatment of diabetes.

During growth cycle, the accumulation of autotoxins released from peach root into soil strongly restrains the perennial tree growth in the same soil plot. However, the toxication syndrome on plant developmental physiology and the identities of toxic substances from peach root are still elusive to date. In this study, we used pot trials to investigate the influence of peach root bark extracts (extracted either in water or ethanol) and benzoic acid (a major allelopathic agent in most plant species)on seedling performance. Subsequently, the chemical compounds from peach root extracts were characterized by Gas Chromatography-Mass. Interestingly, in our study, the major toxic cyanide was only detected in the water extracts of root bark not in the root wood part and more cyanide could be detected in the presence of β -glucosidase. After applying exogenous root extracts and benzoic acid, the seedlings physiological growth was inhibited as expected, and lipid peroxidation and the antioxidant enzymes were increased as well. The photosynthetic parameters, including net photosynthetic rate, stomatal conductance and intercellular CO 2 concentration, were all significantly reduced by the exogenous root extracts application. At cellular level, transmission electron microscopy showed that most of the organelles were lost in the root tip cells treated by ethanol-based peach root extracts, while the irregular nuclei, amorphous mitochondria in root tip cells, dissolved middle lamella and elongated plastids were observed in benzoic acid treatment. Furthermore, for the first time we identified 49 chemicals from ethanol-based peach root extracts, among which, 2, 4-bis (1, 1-dimethylethyl) -phenol (10.40%), benzoic acid (8.65%) and palmitic acid (7.12%) occurred in the highest amounts. Altogether our comprehensive physiological profile by toxication analysis and the determined major toxins from the root bark extracts could provide critical clues for peach allelopathic studies.