The Unstable Restorer-of-fertility locus in pepper (Capsicum annuum. L) is delimited to a genomic region containing PPR genes.

KEY MESSAGE: Unstable Restorer-of-fertility (Rfu), conferring unstable fertility restoration in the pepper CGMS system, was delimited to a genomic region near Rf and is syntenic to the PPR-like gene-rich region in tomato. The use of cytoplasmic-genic male sterility (CGMS) systems greatly increases the efficiency of hybrid seed production. Although marker development and candidate gene isolation have been performed for the Restorer-of-fertility (Rf) gene in pepper (Capsicum annuum L.), the broad use of CGMS systems has been hampered by the instability of fertility restoration among pepper accessions, especially sweet peppers, due to the widespread presence of the Unstable Restorer-of-fertility (Rfu) locus. Therefore, to investigate the genetic factors controlling unstable fertility restoration in sweet peppers, we developed a segregation population (BC4F5) from crosses using a male-sterile line and an Rfu-containing line. Segregation did not significantly deviate from a 3:1 ratio for unstable fertility restoration to sterility, indicating single dominant locus control for unstable fertility restoration in this population. Genetic mapping delimited the Rfu locus to a 398 kb genomic region on chromosome 6, which is close to but different from the previously identified Rf-containing region. The Rfu-containing region harbors a pentatricopeptide repeat (PPR) gene, along with 10 other candidate genes. In addition, this region is syntenic to the genomic region containing the largest number of Rf-like PPR genes in tomato. Therefore, the dynamic evolution of PPR genes might be responsible for both the restoration and instability of fertility in pepper. During genetic mapping, we developed various molecular markers, including one that co-segregated with Rfu. These markers showed higher accuracy for genotyping than previously developed markers, pointing to their possible use in marker-assisted breeding of sweet peppers.

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.

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 ß-cyclase, ß-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.

Divergent evolution of multiple virus-resistance genes from a progenitor in Capsicum spp.

Plants have evolved hundreds of nucleotide-binding and leucine-rich domain proteins (NLRs) as potential intracellular immune receptors, but the evolutionary mechanism leading to the ability to recognize specific pathogen effectors is elusive. Here, we cloned Pvr4 (a Potyvirus resistance gene in Capsicum annuum) and Tsw (a Tomato spotted wilt virus resistance gene in Capsicum chinense) via a genome-based approach using independent segregating populations. The genes both encode typical NLRs and are located at the same locus on pepper chromosome 10. Despite the fact that these two genes recognize completely different viral effectors, the genomic structures and coding sequences of the two genes are strikingly similar. Phylogenetic studies revealed that these two immune receptors diverged from a progenitor gene of a common ancestor. Our results suggest that sequence variations caused by gene duplication and neofunctionalization may underlie the evolution of the ability to specifically recognize different effectors. These findings thereby provide insight into the divergent evolution of plant immune receptors.

Delayed-onset hearing loss in pediatric candidates for cochlear implantation.

The objective of this study was to evaluate the clinical significance of delayed-onset hearing loss in children. Seventy-three children who underwent cochlear implantation (CI) were included. They were divided into a congenital hearing loss group (n = 50) and a delayed-onset hearing loss group (n = 23). The age at diagnosis of hearing loss, age at the beginning of auditory habilitation, the age at CI, and the postimplant speech perception abilities were compared between the two groups. Children in the congenital hearing loss group were confirmed to have hearing loss at a mean age of 0.3 years, and those in the delayed-onset hearing loss group were diagnosed with hearing loss at a mean age of 2.0 years. Auditory habilitation began at a mean age of 0.4 and 2.0 years, and CI was performed at a mean age of 1.4 and 2.6 years, respectively. Children in the congenital hearing loss group had better scores on speech perception tests than those in the delayed-onset hearing loss group, but the differences were not significant. About half of the children with delayed-onset hearing loss (57 %) had risk factors associated with delayed-onset hearing loss. A high prevalence of delayed-onset hearing loss was noted in the group of children who underwent CI. Risk factors for hearing loss were not found in 43 % of children with delayed-onset hearing loss. Universal screening for delayed-onset hearing loss needs to be performed during early childhood.

Criteria for Selecting an Optimal Device for the Contralateral Ear of Children with a Unilateral Cochlear Implant.

OBJECTIVE: To identify clinical criteria for selecting the aiding device for the contralateral ear of children with a unilateral cochlear implant (CI). METHODS: Sixty-five children, including 36 bilateral CI users and 29 bimodal users, participated in the study. A speech perception test (monosyllabic word test) in noise was administered. The target speech (65 dB sound pressure level) was presented from the front loudspeaker, and noise (10 dB signal-to-noise ratio) was presented from 3 directions: from in front of the child and 90° to the child's right and left sides. The test was performed using the first CI alone and under bilateral CI or bimodal conditions. The bilateral benefits to speech perception in noise were compared between bilateral CI users and bimodal users. RESULTS: Significant benefits in speech perception in noise were evident in bilateral CI users in all 3 noise conditions. In bimodal users, the hearing threshold at low frequencies of ≤1 kHz in the nonimplanted ear affected the bilateral benefit. Bimodal users with a low-frequency hearing threshold ≤90 dB hearing level (HL) showed a significant bilateral benefit in various noise conditions. By contrast, bimodal users with a low-frequency hearing threshold >90 dB HL showed no significant bilateral benefits in all 3 noise conditions. CONCLUSIONS: Bilateral CI and bimodal listening provide better speech perception in noise than unilateral CI alone in children. The contralateral CI is better than bimodal listening for children with a low-frequency hearing threshold >90 dB HL. A hearing threshold at low frequencies of ≤1 kHz may be a good criterion for deciding on the type of device for the contralateral ear of children with a unilateral CI.

CONSTITUTIVE PHOTOMORPHOGENIC 10 (COP10) Contributes to Floral Repression under Non-Inductive Short Days in Arabidopsis.

In Arabidopsis, CONSTITUTIVE PHOTOMORPHOGENIC/DE-ETIOLATED/FUSCA (COP/DET/FUS) genes act in repression of photomorphogenesis in darkness, and recent reports revealed that some of these genes, such as COP1 and DET1, also have important roles in controlling flowering time and circadian rhythm. The COP/DET/FUS protein COP10 interacts with DET1 and DNA DAMAGE-BINDING PROTEIN 1 (DDB1) to form a CDD complex and represses photomorphogenesis in darkness. The cop10-4 mutants flower normally in inductive long days (LD) but early in non-inductive short days (SD) compared with wild type (WT); however, the role of COP10 remains unknown. Here, we investigate the role of COP10 in SD-dependent floral repression. Reverse transcription-quantitative PCR revealed that in SD, expression of the LD-dependent floral inducers GI, FKF1, and FT significantly increased in cop10-4 mutants, compared with WT. This suggests that COP10 mainly regulates FT expression in a CO-independent manner. We also show that COP10 interacts with GI in vitro and in vivo, suggesting that COP10 could also affect GI function at the posttranslational level. Moreover, FLC expression was repressed drastically in cop10-4 mutants and COP10 interacts with MULTICOPY SUPPRESSOR OF IRA1 4 (MSI4)/FVE (MSI4/FVE), which epigenetically inhibits FLC expression. These data suggest that COP10 contributes to delaying flowering in the photoperiod and autonomous pathways by downregulating FT expression under SD.

MARF and Opa1 control mitochondrial and cardiac function in Drosophila.

RATIONALE: Mitochondria interact via actions of outer and inner membrane fusion proteins. The role of mitochondrial fusion in functioning of the heart, where mitochondria comprise ≈30% of cardiomyocyte volume and their intermyofilament spatial arrangement with other mitochondria is highly ordered, is unknown. OBJECTIVE: Model and analyze mitochondrial fusion defects in Drosophila melanogaster heart tubes with tincΔ4Gal4-directed expression of RNA interference (RNAi) for mitochondrial assembly regulatory factor (MARF) and optic atrophy (Opa)1. METHODS AND RESULTS: Live imaging analysis revealed that heart tube-specific knockdown of MARF or Opa1 increases mitochondrial morphometric heterogeneity and induces heart tube dilation with profound contractile impairment. Sarcoplasmic reticular structure was unaffected. Cardiomyocyte expression of human mitofusin (mfn)1 or -2 rescued MARF RNAi cardiomyopathy, demonstrating functional homology between Drosophila MARF and human mitofusins. Suppressing mitochondrial fusion increased compensatory expression of nuclear-encoded mitochondrial genes, indicating mitochondrial biogenesis. The MARF RNAi cardiomyopathy was prevented by transgenic expression of superoxide dismutase 1. CONCLUSIONS: Mitochondrial fusion is essential to cardiomyocyte mitochondrial function and regeneration. Reactive oxygen species are key mediators of cardiomyopathy in mitochondrial fusion-defective cardiomyocytes. Postulated mitochondrial-endoplasmic reticulum interactions mediated uniquely by mfn2 appear dispensable to functioning of the fly heart.

Clinical value of persistent but downgraded vesicoureteral reflux after dextranomer/hyaluronic acid injection in children.

We aimed to investigate the clinical value of persistent but downgraded vesicoureteral reflux (VUR) after dextranomer/hyaluronic acid (Dx/HA) injection in children. The medical records of 128 children (195 ureters) who underwent Dx/HA injections for VUR were reviewed. The incidences of pre- and post-operative febrile urinary tract infections (UTIs) were analyzed in children with or without persistent VUR on voiding cystourethrography (VCUG) 3 months postoperatively. The surgical results of VUR persistent children who underwent a single additional injection were assessed. The VUR resolved completely in 100 ureters (51.3%), was persistent in 95 ureters, and newly developed in 2 ureters. The incidence of pre/post-operative febrile UTIs were 0.35 ± 0.39 per year and 0.07 ± 0.32 per year in VUR resolved children (P < 0.001), and 0.76 ± 1.18 per year and 0.20 ± 0.61 per year in VUR persistent children (P < 0.001). A single additional Dx/HA injection (44 ureters) resolved VUR in 29 ureters (65.9%), and also reduced the VUR to grade I in 7 ureters (15.9%), II in 4 (9.1%), and III in 4 (9.1%). Even in children with persistent VUR after Dx/HA injection, the incidence of febrile UTIs decreased markedly. The VUR grade significantly decreases after single additional Dx/HA injection.

Receptor-independent protein kinase C alpha (PKCalpha) signaling by calpain-generated free catalytic domains induces HDAC5 nuclear export and regulates cardiac transcription.

Receptor-mediated activation of protein kinase (PK) C is a central pathway regulating cell growth, homeostasis, and programmed death. Recently, we showed that calpain-mediated proteolytic processing of PKCα in ischemic myocardium activates PKC signaling in a receptor-independent manner by releasing a persistent and constitutively active free catalytic fragment, PKCα-CT. This unregulated kinase provokes cardiomyopathy, but the mechanisms remain unclear. Here, we demonstrate that PKCα-CT is a potent regulator of pathological cardiac gene expression. PKCα-CT constitutively localizes to nuclei and directly promotes nucleo-cytoplasmic shuttling of HDAC5, inducing expression of apoptosis and other deleterious genes. Whereas PKD activation is required for HDAC5 nuclear export induced by unprocessed PKCs activated by phorbol ester, PKCα-CT directly drives HDAC cytosolic relocalization. Activation of MEF2-dependent inflammatory pathway genes by PKCα-CT can induce a cell-autonomous transcriptional response that mimics, but anticipates, actual inflammation. Because calpain-mediated processing of PKC isoforms occurs in many tissues wherein calcium is increased by stress or injury, our observation that the catalytically active product of this interaction is a constitutively active transcriptional regulator has broad ramifications for understanding and preventing the pathological transcriptional stress response.

Detection of nickel in fish organs with a two-photon fluorescent probe.

Molecular imaging by two-photon microscopy (TPM) has become indispensable to the study of biology/medicine owing to its capability of imaging deep inside intact tissues. To make TPM a more-versatile tool, a large variety of two-photon probes are needed. Herein, we report a new two-photon fluorescent probe (ANi2) that can be excited by 750 nm femtosecond pulses and detect Ni(2+) ions in fresh fish organs at 90-175 µm depth without interference from the pH value or from other biologically relevant species through the use of TPM. TPM images of fish organs labeled with ANi2 revealed that Ni(2+) ions accumulate in fish organs in the order: kidney > heart > gill ≥ liver. Moreover, a linear relationship was found between the two-photon-excited fluorescence (TPEF) and the inductively coupled plasma mass spectrometry intensities (ICP-MS), thereby allowing the quantitative measurement of Ni(2+) ions in live tissue.

Receptor-independent cardiac protein kinase Calpha activation by calpain-mediated truncation of regulatory domains.

RATIONALE: Protein kinase (PK)Cs and calpain cysteine proteases are highly expressed in myocardium. Ischemia produces calcium overload that activates calpains and conventional PKCs. However, calpains can proteolytically process PKCs, and the potential in vivo consequences of this interaction are unknown. OBJECTIVE: To determine the biochemical and pathophysiological consequences of calpain-mediated cardiac PKCα proteolysis. METHODS AND RESULTS: Isolated mouse hearts subjected to global ischemia/reperfusion demonstrated cleavage of PKCα. Calpain 1 overexpression was not sufficient to produce PKCα cleavage in normal hearts, but ischemia-induced myocardial PKCα cleavage and myocardial injury were greatly increased by cardiac-specific expression of calpain 1. In contrast, calpain 1 gene ablation or inhibition with calpastatin prevented ischemia/reperfusion induced PKCα cleavage; infarct size was decreased and ventricular function enhanced in infarcted calpain 1 knockout hearts. To determine consequences of PKCα fragmentation on myocardial protein phosphorylation, transgenic mice were created conditionally expressing full-length PKCα or its N-terminal and C-terminal calpain 1 cleavage fragments. Two-dimensional mapping of ventricular protein extracts showed a distinct PKCα phosphorylation profile that was exaggerated and distorted in hearts expressing the PKCα C-terminal fragment. MALDI mass spectroscopy revealed hyperphosphorylation of myosin-binding protein C and phosphorylation of atypical substrates by the PKCα C-terminal fragment. Expression of parent PKCα produced a mild cardiomyopathy, whereas myocardial expression of the C-terminal PKCα fragment induced a disproportionately severe, rapidly lethal cardiomyopathy. CONCLUSIONS: Proteolytic processing of PKCα by calcium-activated calpain activates pathological cardiac signaling through generation of an unregulated and/or mistargeted kinase. Production of the PKCα C-terminal fragment in ischemic hearts occurs via a receptor-independent mechanism.

High-throughput SNP markers for authentication of Korean wheat cultivars based on seven complete plastomes and the nuclear genome.

Wheat (Triticum aestivum) has diverse uses in the food industry, and different cultivars have unique properties; therefore, it is important to select the optimal cultivar for the intended end use. Here, to establish an identification system for Korean wheat cultivars, we obtained the complete plastome sequences of seven major Korean cultivars. Additionally, the open access database CerealsDB was queried to discover single-copy genomic single-nucleotide polymorphisms (SNPs) in the hexaploid wheat genome. Ten SNPs were developed into allele-specific PCR (ASP) markers, and eight of the SNPs used for ASP markers were converted into TaqMan high-throughput genotyping markers. Phylogenetic analysis using SNP genotypes revealed the genetic diversity and relationships among 137 wheat lines from around the world, including 35 Korean cultivars. This research thus presents a high-throughput authentication system for Korean wheat cultivars that may promote food industry uses of Korean wheat. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01043-w.

Genome-wide analysis-based single nucleotide polymorphism marker sets to identify diverse genotypes in cabbage cultivars (Brassica oleracea var. capitata).

Plant variety protection is essential for breeders' rights granted by the International Union for the Protection of New Varieties of Plants. Distinctness, uniformity, and stability (DUS) are necessary for new variety registration; to this end, currently, morphological traits are examined, which is time-consuming and laborious. Molecular markers are more effective, accurate, and stable descriptors of DUS. Advancements in next-generation sequencing technology have facilitated genome-wide identification of single nucleotide polymorphisms. Here, we developed a core set of single nucleotide polymorphism markers to identify cabbage varieties and traits of test guidance through clustering using the Fluidigm assay, a high-throughput genotyping system. Core sets of 87, 24, and 10 markers are selected based on a genome-wide association-based approach. All core markers could identify 94 cabbage varieties and determine 17 DUS traits. A genotypes database was validated using the Fluidigm platform for variety identification, population structure analysis, cabbage breeding, and DUS testing for plant cultivar protection.

Genome-wide core sets of SNP markers and Fluidigm assays for rapid and effective genotypic identification of Korean cultivars of lettuce (Lactuca sativa L.).

Lettuce is one of the economically important leaf vegetables and is cultivated mainly in temperate climate areas. Cultivar identification based on the distinctness, uniformity, and stability (DUS) test is a prerequisite for new cultivar registration. However, DUS testing based on morphological features is time-consuming, labor-intensive, and costly, and can also be influenced by environmental factors. Thus, molecular markers have also been used for the identification of genetic diversity as an effective, accurate, and stable method. Currently, genome-wide single nucleotide polymorphisms (SNPs) using next-generation sequencing technology are commonly applied in genetic research on diverse plant species. This study aimed to establish an effective and high-throughput cultivar identification system for lettuce using core sets of SNP markers developed by genotyping by sequencing (GBS). GBS identified 17 877 high-quality SNPs for 90 commercial lettuce cultivars. Genetic differentiation analyses based on the selected SNPs classified the lettuce cultivars into three main groups. Core sets of 192, 96, 48, and 24 markers were further selected and validated using the Fluidigm platform. Phylogenetic analyses based on all core sets of SNPs successfully discriminated individual cultivars that have been currently recognized. These core sets of SNP markers will support the construction of a DNA database of lettuce that can be useful for cultivar identification and purity testing, as well as DUS testing in the plant variety protection system. Additionally, this work will facilitate genetic research to improve breeding in lettuce.

Reduction of Escherichia coli O157:H7 shedding in cattle by addition of chitosan microparticles to feed.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a significant human pathogen that resides in healthy cattle. It is thought that a reduction in the prevalence and numbers of EHEC in cattle will reduce the load of EHEC entering the food chain. To this end, an intervention strategy involving the addition of chitosan microparticles (CM) to feed in order to reduce the carriage of this pathogen in cattle was evaluated. Experiments with individual Holstein calves and a crossover study found that the addition of CM to feed decreased E. coli O157:H7 shedding. In the crossover study, CM resulted in statistically significant reductions in the numbers recovered from rectal swab samples (P < 0.05) and the duration of shedding (P < 0.05). The effects of feeding CM to calves differed, indicating that the optimal levels of CM may differ between animals or that other factors are involved in the interaction between CM and E. coli O157:H7. In vitro studies demonstrated that E. coli O157:H7 binds to CM, suggesting that the reduction in shedding may result at least in part from the binding of positively charged CM to negatively charged E. coli cells. Additional studies are needed to determine the impact of CM feeding on animal production, but the results from this study indicate that supplementing feed with CM reduces the shedding of E. coli O157:H7 in cattle.

Proteomic analysis reveals an elevated expression of heat shock protein 27 in preeclamptic placentas.

AIM: The purpose of this study was to identify the placental proteins that are associated with preeclampsia by performing proteomic analysis. METHODS: To identify the proteins associated with preeclampsia, we performed two-dimensional electrophoresis (2-DE), followed by silver staining. The overexpressed proteins were identified by performing matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), followed by peptide mass fingerprinting, a protein database search and Western blot analysis. Immunohistochemical staining was performed to determine the localization of the overexpressed Hsp27. RESULTS: By use of 2-DE and MALDI-TOF-MS analysis, twelve differentially expressed proteins were identified, of which four proteins were upregulated and eight proteins were downregulated. One of the upregulated spots was identified as Hsp27. Immunohistochemical analysis showed that Hsp27 was mainly located in the trophoblasts. The Western blot analysis showed that the expression of Hsp27 in the tissues of the preeclampsia placenta was significantly increased. CONCLUSIONS: Our study confirmed that four proteins are upregulated and eight proteins are downregulated in preeclampsia. These differentially expressed proteins include signal transduction protein and molecular chaperon protein, in which Hsp27 is upregulated. We suggest that the increased expression level of Hsp27 might be correlated with the pathophysiology of preeclampsia.

Genetic Diversity Assessment and Cultivar Identification of Cucumber (Cucumis sativus L.) Using the Fluidigm Single Nucleotide Polymorphism Assay.

Genetic diversity analysis and cultivar identification were performed using a core set of single nucleotide polymorphisms (SNPs) in cucumber (Cucumis sativus L.). For the genetic diversity study, 280 cucumber accessions collected from four continents (Asia, Europe, America, and Africa) by the National Agrobiodiversity Center of the Rural Development Administration in South Korea and 20 Korean commercial F1 hybrids were genotyped using 151 Fluidigm SNP assay sets. The heterozygosity of the SNP loci per accession ranged from 4.76 to 82.76%, with an average of 32.1%. Population genetics analysis was performed using population structure analysis and hierarchical clustering (HC), which indicated that these accessions were classified mainly into four subpopulations or clusters according to their geographical origins. The subpopulations for Asian and European accessions were clearly distinguished from each other (FST value = 0.47), while the subpopulations for Korean F1 hybrids and Asian accessions were closely related (FST = 0.34). The highest differentiation was observed between American and European accessions (FST = 0.41). Nei's genetic distance among the 280 accessions was 0.414 on average. In addition, 95 commercial F1 hybrids of three cultivar groups (Baekdadagi-, Gasi-, and Nakhap-types) were genotyped using 82 Fluidigm SNP assay sets for cultivar identification. These 82 SNPs differentiated all cultivars, except seven. The heterozygosity of the SNP loci per cultivar ranged from 12.20 to 69.14%, with an average of 34.2%. Principal component analysis and HC demonstrated that most cultivars were clustered based on their cultivar groups. The Baekdadagi- and Gasi-types were clearly distinguished, while the Nakhap-type was closely related to the Baekdadagi-type. Our results obtained using core Fluidigm SNP assay sets provide useful information for germplasm assessment and cultivar identification, which are essential for breeding and intellectual right protection in cucumber.

F-Box Family Genes, LTSF1 and LTSF2, Regulate Low-Temperature Stress Tolerance in Pepper (Capsicum chinense).

The F-box proteins belong to a family of regulatory proteins that play key roles in the proteasomal degradation of other proteins. Plant F-box proteins are functionally diverse, and the precise roles of many such proteins in growth and development are not known. Previously, two low-temperature-sensitive F-box protein family genes (LTSF1 and LTSF2) were identified as candidates responsible for the sensitivity to low temperatures in the pepper (Capsicum chinense) cultivar 'sy-2'. In the present study, we showed that the virus-induced gene silencing of these genes stunted plant growth and caused abnormal leaf development under low-temperature conditions, similar to what was observed in the low-temperature-sensitive 'sy-2' line. Protein-protein interaction analyses revealed that the LTSF1 and LTSF2 proteins interacted with S-phase kinase-associated protein 1 (SKP1), part of the Skp, Cullin, F-box-containing (SCF) complex that catalyzes the ubiquitination of proteins for degradation, suggesting a role for LTSF1 and LTSF2 in protein degradation. Furthermore, transgenic Nicotiana benthamiana plants overexpressing the pepper LTSF1 gene showed an increased tolerance to low-temperature stress and a higher expression of the genes encoding antioxidant enzymes. Taken together, these results suggest that the LTSF1 and LTSF2 F-box proteins are a functional component of the SCF complex and may positively regulate low-temperature stress tolerance by activating antioxidant-enzyme activities.

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.