Fruit Development, Ripening and Quality Related Genes in the Papaya Genome

Papaya (Carica papaya L.) is the first fleshy fruit with a climacteric ripening pattern to be sequenced. As a member of the Rosids superorder in the order Brassicales, papaya apparently lacks the genome duplication that occurred twice in Arabidopsis. The predicted papaya genes that are homologous to those potentially involved in fruit growth, development, and ripening were investigated. Genes homologous to those involved in tomato fruit size and shape were found. Fewer predicted papaya expansin genes were found and no Expansin Like-B genes were predicted. Compared to Arabidopsis and tomato, fewer genes that may impact sugar accumulation in papaya, ethylene synthesis and response, respiration, chlorophyll degradation and carotenoid synthesis were predicted. Similar or fewer genes were found in papaya for the enzymes leading to volatile production than so far determined for tomato. The presence of fewer papaya genes in most fruit development and ripening categories suggests less subfunctionalization of gene action. The lack of whole genome duplication and reductions in most gene families and biosynthetic pathways make papaya a valuable and unique tool to study the evolution of fruit ripening and the complex regulatory networks active in fruit ripening.     

Single‐molecule real‐time sequencing reveals diverse allelic variations in carotenoid biosynthetic genes in pepper (Capsicum spp.)

The diverse colours of mature pepper (Capsicum spp.) fruit result from the accumulation of different carotenoids. The carotenoid biosynthetic pathway has been well elucidated in Solanaceous plants, and analysis of candidate genes involved in this process has revealed variations in carotenoid biosynthetic genes in Capsicum spp. However, the allelic variations revealed by previous studies could not fully explain the variation in fruit colour in Capsicum spp. due to technical difficulties in detecting allelic variation in multiple candidate genes in numerous samples. In this study, we uncovered allelic variations in six carotenoid biosynthetic genes, including phytoene synthase (PSY1, PSY2), lycopene β‐cyclase, β‐carotene hydroxylase, zeaxanthin epoxidase and capsanthin‐capsorubin synthase (CCS) genes, in 94 pepper accessions by single‐molecule real‐time (SMRT) sequencing. To investigate the relationship between allelic variations in the candidate genes and differences in fruit colour, we performed ultra‐performance liquid chromatography analysis using 43 accessions representing each allelic variation. Different combinations of dysfunctional mutations in PSY1 and CCS could explain variation in the compositions and levels of carotenoids in the accessions examined in this study. Our results demonstrate that SMRT sequencing technology can be used to rapidly identify allelic variation in target genes in various germplasms. The newly identified allelic variants will be useful for pepper breeding and for further analysis of carotenoid biosynthesis pathways.     

Carotenoid biosynthetic genes in Brassica rapa: comparative genomic analysis,phylogenetic analysis,and expression profiling

Background

Carotenoids are isoprenoid compounds synthesized by all photosynthetic organisms. Despite much research on carotenoid biosynthesis in the model plant Arabidopsis thaliana, there is a lack of information on the carotenoid pathway in Brassica rapa. To better understand its carotenoid biosynthetic pathway, we performed a systematic analysis of carotenoid biosynthetic genes at the genome level in B. rapa.

Results

We identified 67 carotenoid biosynthetic genes in B. rapa, which were orthologs of the 47 carotenoid genes in A. thaliana. A high level of synteny was observed for carotenoid biosynthetic genes between A. thaliana and B. rapa. Out of 47 carotenoid biosynthetic genes in A. thaliana, 46 were successfully mapped to the 10 B. rapa chromosomes, and most of the genes retained more than one copy in B. rapa. The gene expansion was caused by the whole-genome triplication (WGT) event experienced by Brassica species. An expression analysis of the carotenoid biosynthetic genes suggested that their expression levels differed in root, stem, leaf, flower, callus, and silique tissues. Additionally, the paralogs of each carotenoid biosynthetic gene, which were generated from the WGT in B. rapa, showed significantly different expression levels among tissues, suggesting differentiated functions for these multi-copy genes in the carotenoid pathway.

Conclusions

This first systematic study of carotenoid biosynthetic genes in B. rapa provides insights into the carotenoid metabolic mechanisms of Brassica crops. In addition, a better understanding of carotenoid biosynthetic genes in B. rapa will contribute to the development of conventional and transgenic B. rapa cultivars with enriched carotenoid levels in the future.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1655-5) contains supplementary material, which is available to authorized users.     

Pathway analysis of a genome-wide association study in schizophrenia

Objective

The aim of this study was to identify the candidate single nucleotide polymorphisms (SNPs) and candidate mechanisms that contribute to schizophrenia susceptibility and to generate a SNP to gene to pathway hypothesis using an analytical pathway-based approach.

Methods

We used schizophrenia GWAS data of the genotypes of 660,259 SNPs in 1378 controls and 1351 cases of European descent after quality control filtering. ICSNPathway (Identify candidate Causal SNPs and Pathways) analysis was applied to the schizophrenia GWAS dataset. The first stage involved the pre-selection of candidate SNPs by linkage disequilibrium analysis and the functional SNP annotation of the most significant SNPs found. The second stage involved the annotation of biological mechanisms for the pre-selected candidate SNPs using improved-gene set enrichment analysis.

Results

ICSNPathway analysis identified fifteen candidate SNPs, ten candidate pathways, and nine hypothetical biological mechanisms. The most strongly associated potential pathways were as follows. First, rs1644731 and rs1644730 to RDH8 to estrogen biosynthetic process (p < 0.001, FDR < 0.001). The genes involved in this pathway are RDH8 and HSD3B1 (p < 0.05). All-trans-retinol dehydrogenase (RDH8) is a visual cycle enzyme that reduces all-trans-retinal to all-trans-retinol in the presence of NADPH. The chemical reactions and pathways involved result in the formation of estrogens, which are C18 steroid hormones that can stimulate the development of female sexual characteristics. Second, rs1146031 to ACVR1 to mesoderm formation and activin binding (p < 0.001, FDR = 0.032, 0.034). Two of 15 candidate genes are known genes associated with schizophrenia: KCNQ2 and APOL2. One of the 10 candidate pathways, estrogen biosynthetic process, is known to be associated with schizophrenia (p < 0.001, FDR < 0.001). However, 13 of candidate genes (RDH8, ACVR1, PSMD9, KCNAB1, SLC17A3, ARCN1, COG7, STAB2, LRPAP1, STAB1, CXCL16, COL4A4, EXOSC3) and 9 of candidate pathways were novel.

Conclusion

By applying ICSNPathway analysis to schizophrenia GWAS data, we identified candidate SNPs, genes like KCNQ2 and APOL2 and pathways involving the estrogen biosynthetic process may contribute to schizophrenia susceptibility. Further analyses are needed to validate the results of this analysis.     

Association Study of 167 Candidate Genes for Schizophrenia Selected by a Multi-Domain Evidence-Based Prioritization Algorithm and Neurodevelopmental Hypothesis

Integrating evidence from multiple domains is useful in prioritizing disease candidate genes for subsequent testing. We ranked all known human genes (n = 3819) under linkage peaks in the Irish Study of High-Density Schizophrenia Families using three different evidence domains: 1) a meta-analysis of microarray gene expression results using the Stanley Brain collection, 2) a schizophrenia protein-protein interaction network, and 3) a systematic literature search. Each gene was assigned a domain-specific p-value and ranked after evaluating the evidence within each domain. For comparison to this ranking process, a large-scale candidate gene hypothesis was also tested by including genes with Gene Ontology terms related to neurodevelopment. Subsequently, genotypes of 3725 SNPs in 167 genes from a custom Illumina iSelect array were used to evaluate the top ranked vs. hypothesis selected genes. Seventy-three genes were both highly ranked and involved in neurodevelopment (category 1) while 42 and 52 genes were exclusive to neurodevelopment (category 2) or highly ranked (category 3), respectively. The most significant associations were observed in genes PRKG1, PRKCE, and CNTN4 but no individual SNPs were significant after correction for multiple testing. Comparison of the approaches showed an excess of significant tests using the hypothesis-driven neurodevelopment category. Random selection of similar sized genes from two independent genome-wide association studies (GWAS) of schizophrenia showed the excess was unlikely by chance. In a further meta-analysis of three GWAS datasets, four candidate SNPs reached nominal significance. Although gene ranking using integrated sources of prior information did not enrich for significant results in the current experiment, gene selection using an a priori hypothesis (neurodevelopment) was superior to random selection. As such, further development of gene ranking strategies using more carefully selected sources of information is warranted.     

Over-expression of Arabidopsis thaliana carotenoid hydroxylases individually and in combination with a β-carotene ketolase provides insight into in vivo functions

Carotenoids represent a group of widely distributed pigments derived from the general isoprenoid biosynthetic pathway that possess diverse functions in plant primary and secondary metabolism. Modification of α- and β-carotene backbones depends in part on ring hydroxylation. Two ferredoxin-dependent non-heme di-iron monooxygenases (AtB1 and AtB2) that mainly catalyze in vivo β-carotene hydroxylations of β,β-carotenoids, and two heme-containing cytochrome P450 (CYP) monooxygenases (CYP97A3 and CYP97C1) that preferentially hydroxylate the ε-ring of α-carotene or the β-ring of β,ε-carotenoids, have been characterized in Arabidopsis by analysis of loss-of-function mutant phenotypes. We further investigated functional roles of both hydroxylase classes in modification of the β- and ε-rings of α-carotene and β-carotene through over-expression of AtB1, CYP97A3, CYP97C1, and the hydroxylase candidate CYP97B3. Since carotenoid hydroxylation is required for generation of ketocarotenoids by the bkt1(CrtO) β-carotene ketolase, all hydroxylase constructs were also introduced into an Arabidopsis line expressing the Haematococcus pluvalis bkt1 β-carotene ketolase. Analysis of foliar carotenoid profiles in lines overexpressing the individual hydroxylases indicate a role for CYP97B3 in carotenoid biosynthesis, confirm and extend previous findings of hydroxylase activities based on knock-out mutants, and suggest functions of the multifunctional enzymes in carotenoid biosynthesis. Hydroxylase over-expression in combination with bkt1 did not result in ketocarotenoid accumulation, but instead unexpected patterns of α-carotene derivatives, accompanied by a reduction of α-carotene, were observed. These data suggest possible interactions between the β-carotene ketolase bkt1 and the hydroxylases that impact partitioning of carbon flux into different carotenoid branch pathways.     

Selection of reliable reference genes for quantitative real-time polymerase chain reaction studies in maize grains

Key message

The stability of candidate reference genes was evaluated in maize landrace varieties and during multiple grain developmental stages to evaluate the expression of carotenoid-related genes by RT-qPCR for application to maize biofortification.

Abstract

Vitamin A deficiency affects millions of children worldwide; therefore, increasing the content of vitamin A precursors in maize grains is of interest. The study of the expression of genes involved in the carotenoid biosynthetic pathway in maize grains has provided useful information for metabolic engineering approaches. However, reliable results using real-time quantitative polymerase chain reaction (RT-qPCR) experiments are dependent on the use of the appropriate reference genes. In this study, we utilized geNorm and NormFinder softwares to identify the most stably expressed candidate reference genes in samples from seven stages of grain development and from eight landrace varieties. The results of the analysis performed using geNorm indicated that tubulin (TUB) and actin (ACT) were the most suitable reference genes among all experimental conditions, while glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) showed the least stability. The same result was obtained with the NormFinder software. The minimum number of genes required in each experimental condition to normalize the gene expression data was also determined by geNorm. The expression of phytoene synthase gene (PSY1), the first enzyme in the carotenoid biosynthetic pathway, was overestimated when the least stable candidate gene (GAPDH) was used as the internal control instead of the most stable gene pair (ACT + TUB), thus highlighting the importance of validating reference genes before conducting a RT-qPCR experiment to obtain accurate results. This study is the first survey of the stability of genes for use as reference genes to normalize RT-qPCR data from maize landraces during multiple stages of grain development.     

Dissecting the mechanism of Solanum lycopersicum and Solanum chilense flower colour formation

Flowers are the defining feature of angiosperms, and function as indispensable organs for sexual reproduction. Flower colour typically plays an important role in attracting pollinators, and can show considerable variation, even between closely related species. For example, domesticated tomato (S. lycopersicum) has orange/yellow flowers, while the wild relative S. chilense (accession LA2405) has bright yellow flowers. In this study, the mechanism of flower colour formation in these two species was compared by evaluating the accumulation of carotenoids, assessing the expression genes related to carotenoid biosynthetic pathways and observing chromoplast ultrastructure. In S. chilense petals, genes associated with the lutein branch of the carotenoid biosynthetic pathway, phytoene desaturase (PDS), ζ‐carotene desaturase (ZDS), lycopene β‐cyclase (LCY‐B), β‐ring hydroxylase (CRTR‐B) and ε‐ring hydroxylase (CRTR‐E), were highly expressed, and this was correlated with high levels of lutein accumulation. In contrast, PDS, ZDS and CYC‐B from the neoxanthin biosynthetic branch were highly expressed in S. lycopersicum anthers, leading to increased β‐carotene accumulation and hence an orange/yellow colour. Changes in the size, amount and electron density of plastoglobules in chromoplasts provided further evidence of carotenoid accumulation and flower colour formation. Taken together, these results reveal the biochemical basis of differences in carotenoid pigment accumulation and colour between petals and anthers in tomato.     

The sub-cellular localisation of the potato (Solanum tuberosum L.) carotenoid biosynthetic enzymes, CrtRb2 and PSY2

Carotenoids are isoprenoids with important biological roles both for plants and animals. The yellow flesh colour of potato (Solanum tuberosum L.) tubers is a quality trait dependent on the types and levels of carotenoids that accumulate. The carotenoid biosynthetic pathway is well characterised, facilitating the successful engineering of carotenoid content in numerous crops including potato. However, a clear understanding concerning the factors regulating carotenoid accumulation and localisation in plant storage organs, such as tubers, is lacking. In the present study, the localisation of key carotenoid biosynthetic enzymes was investigated, as one of the unexplored factors that could influence the accumulation of carotenoids in potato tubers. Stable transgenic potato plants were generated by over-expressing β-CAROTENE HYDROXYLASE 2 (CrtRb2) and PHYTOENE SYNTHASE 2 (PSY2) genes, fused to red fluorescent protein (RFP). Gene expression and carotenoid levels were both significantly increased, confirming functionality of the fluorescently tagged proteins. Confocal microscopy studies revealed different sub-organellar localisations of CrtRb2-RFP and PSY2-RFP within amyloplasts. CrtRb2 was detected in small vesicular structures, inside amyloplasts, whereas PSY2 was localised in the stroma of amyloplasts. We conclude that it is important to consider the location of biosynthetic enzymes when engineering the carotenoid metabolic pathway in storage organs such as tubers.     

Regulation of Carotenogenesis and Secondary Metabolism by Nitrogen in Wild-Type Fusarium fujikuroi and Carotenoid-Overproducing Mutants

The fungus Fusarium fujikuroi (Gibberella fujikuroi MP-C) produces metabolites of biotechnological interest, such as gibberellins, bikaverins, and carotenoids. Gibberellin and bikaverin productions are induced upon nitrogen exhaustion, while carotenoid accumulation is stimulated by light. We evaluated the effect of nitrogen availability on carotenogenesis in comparison with bikaverin and gibberellin production in the wild type and in carotenoid-overproducing mutants (carS). Nitrogen starvation increased carotenoid accumulation in all strains tested. In carS strains, gibberellin and bikaverin biosynthesis patterns differed from those of the wild type and paralleled the expression of key genes for both pathways, coding for geranylgeranyl pyrophosphate (GGPP) and kaurene synthases for the former and a polyketide synthase for the latter. These results suggest regulatory connections between carotenoid biosynthesis and nitrogen-controlled biosynthetic pathways in this fungus. Expression of gene ggs1, which encodes a second GGPP synthase, was also derepressed in the carS mutants, suggesting the participation of Ggs1 in carotenoid biosynthesis. The carS mutations did not affect genes for earlier steps of the terpenoid pathway, such as fppS or hmgR. Light induced carotenoid biosynthesis in the wild type and carRA and carB levels in the wild-type and carS strains irrespective of nitrogen availability.     

Brassinosteroid biosynthesis

Biosynthetic pathways of brassinolide from campesterol was demonstrated by studies using cultured Catharanthus roseus cells. Brassinolide is biosynthesized through two pathways, early C6-oxidation pathway and late C6-oxidation pathway, branching off at the conversion of campestanol. Recent characterization of brassinosteroid-deficient mutants of Arabidopsis, pea and tomato confirmed that the pathways operate in wide variety of plant species. Biochemical and molecular genetic studies of the mutants are providing important knowledge on genes and enzymes involved in brassinosteroid biosynthesis. The established biosynthetic pathways of brassinosteroids and the regulation of biosynthesis including up-to-date findings are introduced in this review.