Leaky mutations in the zeaxanthin epoxidase in Capsicum annuum result in bright-red fruit containing a high amount of zeaxanthin.

Fruit color is one of the most important traits in peppers due to its esthetic value and nutritional benefits and is determined by carotenoid composition, resulting from diverse mutations of carotenoid biosynthetic genes. The EMS204 line, derived from an EMS mutant population, presents bright-red color, compared with the wild type Yuwolcho cultivar. HPLC analysis indicates that EMS204 fruit contains more zeaxanthin and less capsanthin and capsorubin than Yuwolcho. MutMap was used to reveal the color variation of EMS204 using an F3 population derived from a cross of EMS204 and Yuwolcho, and the locus was mapped to a 2.5-Mbp region on chromosome 2. Among the genes in the region, a missense mutation was found in ZEP (zeaxanthin epoxidase) that results in an amino acid sequence alteration (V291 → I). A color complementation experiment with Escherichia coli and ZEP in vitro assay using thylakoid membranes revealed decreased enzymatic activity of EMS204 ZEP. Analysis of endogenous plant hormones revealed a significant reduction in abscisic acid content in EMS204. Germination assays and salinity stress experiments corroborated the lower ABA levels in the seeds. Virus-induced gene silencing showed that ZEP silencing also results in bright-red fruit containing less capsanthin but more zeaxanthin than control. A germplasm survey of red color accessions revealed no similar carotenoid profiles to EMS204. However, a breeding line containing a ZEP mutation showed a very similar carotenoid profile to EMS204. Our results provide a novel breeding strategy to develop red pepper cultivars containing high zeaxanthin contents using ZEP mutations.

A mutation in Zeaxanthin epoxidase contributes to orange coloration and alters carotenoid contents in pepper fruit (Capsicum annuum).

Phytoene synthase (PSY1), capsanthin-capsorubin synthase (CCS), and pseudo-response regulator 2 (PRR2) are three major genes controlling fruit color in pepper (Capsicum spp.). However, the diversity of fruit color in pepper cannot be completely explained by these three genes. Here, we used an F2 population derived from Capsicum annuum 'SNU-mini Orange' (SO) and C. annuum 'SNU-mini Yellow' (SY), both harboring functional PSY1 and mutated CCS, and observed that yellow color was dominant over orange color. We performed genotyping-by-sequencing and mapped the genetic locus to a 6.8-Mb region on chromosome 2, which we named CaOr. We discovered a splicing mutation in the zeaxanthin epoxidase (ZEP) gene within this region leading to a premature stop codon. HPLC analysis showed that SO contained higher amounts of zeaxanthin and total carotenoids in mature fruits than SY. A color complementation assay using Escherichia coli harboring carotenoid biosynthetic genes showed that the mutant ZEP protein had reduced enzymatic activity. Transmission electron microscopy of plastids revealed that the ZEP mutation affected plastid development with more rod-shaped inner membrane structures in chromoplasts of mature SO fruits. To validate the role of ZEP in fruit color formation, we performed virus-induced gene silencing of ZEP in the yellow-fruit cultivar C. annuum 'Micropep Yellow' (MY). The silencing of ZEP caused significant changes in the ratios of zeaxanthin to its downstream products and increased total carotenoid contents. Thus, we conclude that the ZEP genotype can determine orange or yellow mature fruit color in pepper.

Candidate Gene Analysis Reveals That the Fruit Color Locus C1 Corresponds to PRR2 in Pepper (Capsicum frutescens).

The diverse fruit colors of peppers (Capsicum spp.) are due to variations in carotenoid composition and content. Mature fruit color in peppers is regulated by three independent loci, C1, C2, and Y. C2 and Y encode phytoene synthase (PSY1) and capsanthin-capsorubin synthase (CCS), respectively; however, the identity of the C1 gene has been unknown. With the aim of identifying C1, we analyzed two pepper accessions with different fruit colors: Capsicum frutescens AC08-045 and AC08-201, whose fruits are light yellow and white, respectively. Ultra-performance liquid chromatography showed that the total carotenoid content was six times higher in AC08-045 than in AC08-201 fruits, with similar composition of main carotenoids and slight difference in minor components. These results suggest that a genetic factor in AC08-201 may down-regulate overall carotenoid biosynthesis. Analyses of candidate genes related to carotenoid biosynthesis and plastid abundance revealed that both accessions carry non-functional alleles of CCS, golden2-like transcription factor (GLK2), and PSY1. However, a nonsense mutation (C2571T) in PRR2, a homolog of Arabidopsis pseudo response regulator2-like (APRR2), was present in only AC08-201. In a population derived from a cross between AC08-045 and AC08-201, a SNP marker based on the nonsense mutation co-segregated fully with fruit color, implying that the mutation in PRR2 may cause the white color of AC08-201 fruits. Transmission electron microscopy (TEM) of AC08-201 fruit pericarp also showed less developed granum structure in chloroplast and smaller plastoglobule in chromoplast compared to those of AC08-045. Virus-induced gene silencing (VIGS) of PRR2 significantly reduced carotenoid accumulation in Capsicum annuum 'Micropep Yellow', which carries non-functional mutations in both PSY1 and CCS. Furthermore, sequence analysis of PSY1, CCS, and PRR2 in other white pepper accessions of C. annuum and Capsicum chinense showed that they commonly have non-functional alleles in PSY1, CCS, and PRR2. Thus, our data demonstrate that the fruit color locus C1 in Capsicum spp. corresponds to the gene PRR2.

Phytoene synthase 2 can compensate for the absence of PSY1 in the control of color in Capsicum fruit.

Phytoene synthase 1 (PSY1) and capsanthin-capsorubin synthase (CCS) are two major genes responsible for fruit color variation in pepper (Capsicum spp.). However, the role of PSY2 remains unknown. We used a systemic approach to examine the genetic factors responsible for the yellow fruit color of C. annuum 'MicroPep Yellow' (MY) and to determine the role of PSY2 in fruit color. We detected complete deletion of PSY1 and a retrotransposon insertion in CCS. Despite the loss of PSY1 and CCS function, both MY and mutant F2 plants from a cross between MY and the 'MicroPep Red' (MR) accumulated basal levels of carotenoids, indicating that other PSY genes may complement the loss of PSY1. qRT-PCR analysis indicated that PSY2 was constitutively expressed in both MR and MY fruits, and a color complementation assay using Escherichia coli revealed that PSY2 was capable of biosynthesizing a carotenoid. Virus-induced gene silencing of PSY2 in MY resulted in white fruits. These findings indicate that PSY2 can compensate for the absence of PSY1 in pepper fruit, resulting in the yellow color of MY fruits.

Quantitative comparison of incisal tooth wear in patients receiving one-phase or two-phase treatment for skeletal Class III malocclusion with anterior crossbite.

OBJECTIVES: The present study aimed to compare the amount of incisal tooth wear in the maxillary central incisors of patients with skeletal Class III malocclusion and anterior crossbite receiving one-phase or two-phase treatment. The hypothesis was that tooth wear would differ according to treatment modalities. MATERIALS AND METHODS: Maxillary dental casts obtained before (T1) and after (T2) orthodontic treatment were divided into three groups. Group I consisted of casts from 21 patients (7 males, 14 females; mean age 9.8 years) who received two-phase treatment (maxillary protraction followed by fixed appliance therapy). Group II comprised casts from 37 patients who underwent orthodontic camouflage treatment for crossbite, subdivided according to age. Group IIa consisted of casts from 15 adolescents (8 males, 7 females; mean age 13.5 years), and group IIb consisted of casts from 22 adults (13 males, 9 females; mean age 24.5 years). Maxillary dental casts obtained at T1 and T2 were scanned. For each pair of digital images, T2 was superimposed on T1 using the best-fit method. Tooth wear was quantified and compared among groups. RESULTS: Significantly less tooth wear was observed in group I compared to groups IIa and IIb, but no difference was found between groups IIa and IIb. Spearman correlation analysis revealed no significant correlation between tooth wear and age, treatment duration, or craniofacial morphology. CONCLUSIONS: Despite the long duration of early treatment, it caused less wear of the maxillary central incisors than did orthodontic camouflage treatment.

Relationship between the lingual frenulum and craniofacial morphology in adults.

INTRODUCTION: The purpose of this study was to determine the relationship between the length of the lingual frenulum and craniofacial morphology and test the hypothesis that skeletal Class III malocclusion is related to tongue-tie, in which the lingual frenulum is short and restricts the mobility of the tongue. METHODS: The sample consisted of 50 skeletal Class I patients (0° < ANB angle < 4°), 50 skeletal Class II patients (ANB angle > 4°), and 50 skeletal Class III patients (ANB angle <0°). Direct and indirect measuring methods were used to quantify the length of the lingual frenulum. The median lingual frenulum length was measured directly with a lingual frenulum ruler. It was evaluated indirectly by measuring the differences between the maximum mouth opening with and without the tip of the tongue touching the incisive papilla. A lateral cephalogram was taken for each subject and a computerized cephalometric analysis was used to assess the cranial morphology. Analysis of variance (ANOVA) was used to compare the differences among the 3 groups. The Pearson correlation analysis was used to detect any relationship between the lingual frenulum length and cephalometric variables. RESULTS: The median lingual frenulum length was significantly longer in the skeletal Class III subjects compared with the skeletal Class I and Class II subjects. The maximum opening of the mouth was significantly reduced in the skeletal Class III subjects compared with Class I and Class II subjects. Significant correlations were also found among the median lingual frenulum length, maximum mouth opening reduction, and the cephalometric variables such as the SNB and ANB angles, Wits appraisal, mandibular length, and the interincisal angle. CONCLUSIONS: The present study supports the hypothesis that skeletal Class III malocclusion is related to long median lingual frenulum or a tongue-tie tendency. Patients diagnosed with tongue-tie might have a tendency toward skeletal Class III malocclusion.