Although remarkable progress has been made toward understanding carotenoid biosynthesis, the mechanisms that regulate the transcription of carotenogenic genes remain poorly understood. Lycopene β-cyclases (LCYb) are critical enzymes located at the branch point of the carotenoid biosynthetic pathway. Here, we used the promoter sequence of as bait in a yeast one-hybrid screen for promoter-binding proteins from sweet orange (). This screen identified a MADS transcription factor, CsMADS6, that was coordinately expressed with fruit development and coloration. Acting as a nucleus-localized transcriptional activator, CsMADS6 directly bound the promoter of and activated its expression. Overexpression of in citrus calli increased carotenoid contents and induced the expression of and other carotenogenic genes, including (), (), and (). CsMADS6 up-regulated the expression of ,, and by directly binding to their promoters, which suggested the multitargeted regulation of carotenoid metabolism by CsMADS6. In addition, the ectopic expression of in tomato () affected carotenoid contents and the expression of carotenogenic genes. The sepals of -overexpressing tomato lines exhibited dramatic changes in carotenoid profiles, accompanied by changes in plastid ultrastructure. Global transcriptome analysis of transgenic sepals revealed that CsMADS6 regulates a series of pathways that promote increases in flux through the carotenoid pathway. Overall, these findings establish that CsMADS6 directly regulates and other carotenogenic genes to coordinately and positively modulate carotenoid metabolism in plants, which may provide strategies to improve the nutritional quality of crops.

BackgroundRed-flesh fruit is absent from common sweet orange varieties, but is more preferred by consumers due to its visual attraction and nutritional properties. Our previous researches on a spontaneous red-flesh mutant revealed that the trait is caused by lycopene accumulation and is regulated by both transcriptional and post-transcriptional mechanisms. However, the knowledge on post-transcriptional regulation of lycopene accumulation in fruits is rather limited so far.ResultsWe used Illumina sequencing method to identify and quantitatively profile small RNAs on the red-flesh sweet orange mutant and its wild type. We identified 85 known miRNAs belonging to 48 families from sweet orange. Comparative profiling revealed that 51 known miRNAs exhibited significant expression differences between mutant (MT) and wild type (WT). We also identified 12 novel miRNAs by the presence of mature miRNAs and corresponding miRNA*s in the sRNA libraries. Comparative analysis showed that 9 novel miRNAs are differentially expressed between WT and MT. Target predictions of the 60 differential miRNAs resulted 418 target genes in sweet orange. GO and KEGG annotation revealed that high ranked miRNA-target genes are those implicated in transcription regulation, protein modification and photosynthesis. The expression profiles of target genes involved in carotenogenesis and photosynthesis were further confirmed to be complementary to the profiles of corresponding miRNAs in WT and MT.ConclusionThis study comparatively characterized the miRNAomes between the red-flesh mutant and the wild type, the results lay a foundation for unraveling the miRNA-mediated molecular processes that regulate lycopene accumulation in the sweet orange red-flesh mutant.

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 0 cis-regulatory change at CCD4b encoding CAROTENOID CLEAVAGE DIOXYGENASE 4b is a major genetic determinant of natural variation in C 30 apocarotenoids responsible for red coloration of citrus peel. Functional analyses demonstrated that in addition the known role in synthesizing b-citraurin, CCD4b is also responsible for the production of another important C 30 apocarotenoid pigment, b-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 0 cis-regulatory enhancer within an MITE transposon and the enhanced allelic expression of CCD4b in C 30 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.

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 β,β-pathway characterized by a striking accumulation of β-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 β-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 β-carotene. In addition, a model is proposed to interpret the common appearance of a favoured β,β-pathway and the likelihood of carotenoid degradation potentially mediated by photo-oxidation and vacuolar phagocytosis in the ECMs is discussed.

Self-incompatibility (SI) is an important mechanism that prevents self-fertilization and avoids inbreeding in flowering plants. The most widespread SI system utilizes S -ribonucleases ( S-RNase s) and S -locus F-boxes ( SLF s) as S -determinants. In citrus, SI is ancestral; Citrus maxima (pummelo) is self-incompatible, while Citrus reticulata (mandarin) and its hybrids are self-compatible (SC). Here we identified nine highly polymorphic pistil-specific, developmentally expressed S-RNase s from pummelo that segregate with S -haplotypes in a gametophytic manner and cluster with authentic S-RNase s. We provide evidence that these S-RNase s function as the female S -determinants in citrus. Moreover, we found that each S-RNase is linked to ~nine SLF s. Analysis of 117 citrus SLF/SLFL genes revealed clustering into 12 types and evidence that the S-RNase s and intra-haplotypic SLFs/SLFL s co-evolved. Our data are consistent with citrus having an S -locus comprising a S-RNase and several SLF s that fit the non-self-recognition model. We identified a predominant single nucleotide mutation, S m - RNase , in SC citrus, which provides a ‘natural’ loss of function. We present evidence that SI-SC transitions due to the S m -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.

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.

Olfactory ensheathing cell (OEC) transplantation has emerged as a very promising therapy for spinal cord repair. In this study, we tested the ability of genetically modified OECs to secrete high levels of glial cell line-derived neurotrophic factor (GDNF) to promote spinal cord repair. The GDNF gene was transduced into OECs using a retroviral-based system. The engineered OECs were first characterized by their ability to express and secrete biologically active GDNF in vitro. After implantation into the spinal cord of adult rats with complete spinal cord transection, OEC survival and GDNF production were examined. The locomotor functions of animals were assessed and axon regeneration was evaluated at the morphological level. To our knowledge, we report for the first time that the genetically modified OECs are capable of producing GDNF in vivo to significantly improve recovery after spinal cord injury (SCI). This work combined the outgrowth-promoting property of OECs with the neuroprotective effects of the additionally overexpressed neurotrophic factors and opens new avenues for the treatment of SCI.

BackgroundInterest in lycopene metabolism and regulation is growing rapidly because accumulative studies have suggested an important role for lycopene in human health promotion. However, little is known about the molecular processes regulating lycopene accumulation in fruits other than tomato so far.ResultsOn a spontaneous sweet orange bud mutant with abnormal lycopene accumulation in fruits and its wild type, comparative transcripts profiling was performed using Massively Parallel Signature Sequencing (MPSS). A total of 6,877,027 and 6,275,309 reliable signatures were obtained for the wild type (WT) and the mutant (MT), respectively. Interpretation of the MPSS signatures revealed that the total number of transcribed gene in MT is 18,106, larger than that in WT 17,670, suggesting that newly initiated transcription occurs in the MT. Further comparison of the transcripts abundance between MT and WT revealed that 3,738 genes show more than two fold expression difference, and 582 genes are up- or down-regulated at 0.05% significance level by more than three fold difference. Functional assignments of the differentially expressed genes indicated that 26 reliable metabolic pathways are altered in the mutant; the most noticeable ones are carotenoid biosynthesis, photosynthesis, and citrate cycle. These data suggest that enhanced photosynthesis and partial impairment of lycopene downstream flux are critical for the formation of lycopene accumulation trait in the mutant.ConclusionThis study provided a global picture of the gene expression changes in a sweet orange red-flesh mutant as compared to the wild type. Interpretation of the differentially expressed genes revealed new insight into the molecular processes regulating lycopene accumulation in the sweet orange red-flesh mutant.