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Changes in Anthocyanin Production during Domestication of Citrus
Eugenio Butelli, Andrés Garcia-Lor, Concetta Licciardello, Giuseppina Las Casas, Lionel Hill, Giuseppe Reforgiato Recupero, Manjunath L. Keremane, Chandrika Ramadugu, Robert Krueger, Qiang Xu, Xiuxin Deng, Anne-Laure Fanciullino, Yann Froelicher, Luis Navarro, Cathie Martin
Eugenio Butelli
John Innes Centre, Norwich NR4 7UH, United Kingdom (E.B., L.H., C.M.);
ORCID record for Eugenio Butelli
For correspondence: eugenio.butelli@jic.ac.uk
Andrés Garcia-Lor
Concetta Licciardello
Giuseppina Las Casas
Lionel Hill
ORCID record for Lionel Hill
Giuseppe Reforgiato Recupero
ORCID record for Giuseppe Reforgiato Recupero
Manjunath L. Keremane
ORCID record for Manjunath L. Keremane
Chandrika Ramadugu
Robert Krueger
Qiang Xu
ORCID record for Qiang Xu
Xiuxin Deng
ORCID record for Xiuxin Deng
Anne-Laure Fanciullino
Yann Froelicher
Luis Navarro
ORCID record for Luis Navarro
Cathie Martin
ORCID record for Cathie Martin
Mandarin (Citrus reticulata), citron (Citrus medica), and pummelo (Citrus maxima) are important species of the genus Citrus and parents of the interspecific hybrids that constitute the most familiar commercial varieties of Citrus: sweet orange, sour orange, clementine, lemon, lime, and grapefruit. Citron produces anthocyanins in its young leaves and flowers, as do species in genera closely related to Citrus, but mandarins do not, and pummelo varieties that produce anthocyanins have not been reported. We investigated the activity of the Ruby gene, which encodes a MYB transcription factor controlling anthocyanin biosynthesis, in different accessions of a range of Citrus species and in domesticated cultivars. A white mutant of lemon lacks functional alleles of Ruby, demonstrating that Ruby plays an essential role in anthocyanin production in Citrus. Almost all the natural variation in pigmentation by anthocyanins in Citrus species can be explained by differences in activity of the Ruby gene, caused by point mutations and deletions and insertions of transposable elements. Comparison of the allelic constitution of Ruby in different species and cultivars also helps to clarify many of the taxonomic relationships in different species of Citrus, confirms the derivation of commercial varieties during domestication, elucidates the relationships within the subgenus Papeda, and allows a new genetic classification of mandarins.
Anthocyanins are phenolic compounds responsible for the red, blue, and purple colors in most angiosperm plants. They are accumulated in different tissues, particularly in flower petals, fruit peel, and leaves (Winkel-Shirley, 2001).
The pathway of anthocyanin biosynthesis is well characterized, and the structural genes that encode the core biosynthetic enzymes have been identified from many species (Tanaka et al., 2008). These genes are coordinately induced by a conserved regulatory complex formed by the interaction between MYB transcription factors, basic helix-loop-helix transcription factors, and WD40-repeat proteins, called the MBW complex (Quattrocchio et al., 1993; Koes et al., 2005; Ramsay and Glover, 2005). DNA-binding specificity is provided principally by the MYB factors. Mutations in the MYB regulatory genes often result in a lack of expression of the biosynthetic genes and loss of anthocyanin pigmentation, as exemplified by the occurrence of red and white varieties of grape (Vitis vinifera; Kobayashi et al., 2004), apple (Malus domestica; Espley et al., 2007), and species of Petunia (Quattrocchio et al., 1999; Hoballah et al., 2007) and Antirrhinum (Schwinn et al., 2006).
In Citrus, the ability to accumulate anthocyanins is not a universal feature (Fig. 1). In lemons and limes, purple pigmentation is clearly present in young leaves and flowers (new growth). In contrast, other cultivated Citrus varieties of economic importance are completely unable to synthesize anthocyanins (Hodgson, 1967). Sweet oranges, with the notable exception of the striking fruit-specific, red-colored, blood varieties, do not display any anthocyanin pigmentation.
(Figure continues on following page.)
The taxonomy of Citrus, a genus of plants belonging to the family Rutaceae (Supplemental Fig. S1), is complicated, as illustrated by the two most widely accepted classification systems, those proposed by Swingle and Tanaka, recognizing 16 and 162 species, respectively (Swingle, 1946; Swingle and Reece, 1967; Tanaka, 1977). In Citrus, the definition of a species is obscured by the ability of Citrus species to hybridize easily within the genus and also with closely related genera. Sexual promiscuity and thousands of years of human cultivation have generated many hybrids whose origin cannot be inferred solely on the basis of morphological and geographical data. However, a growing body of evidence supports the view that there are three basic or primary species: mandarin (Citrus reticulata), pummelo (Citrus maxima), and citron (Citrus medica; Scora, 1975; Barrett and Rhodes, 1976). All the other so-called species are hybrids derived from these three primary species (Moore, 2001). A fourth species, belonging to the subgenus Papeda, was involved in the derivation of some limes (Nicolosi et al., 2000). The term secondary species has been used widely to define Citrus hybrids, since most of them are apomictic, polyembryonic, and normally propagated vegetatively by grafting. This ensures that plants maintain their hybrid genetic constitution over generations but also makes genetic studies challenging and crop improvement by conventional breeding almost impossible. Within a given species or hybrid, phenotypic differences between cultivars usually are the result of spontaneous or induced somatic mutations that have been selected and propagated.
We have shown previously that, in different blood varieties of sweet orange, the insertion of retrotransposons in the promoter of Ruby, a regulatory gene encoding a MYB transcription factor, is responsible for the fruit-specific accumulation of anthocyanins (Butelli et al., 2012). Here, our objective was to determine whether Ruby controls the accumulation of anthocyanins in young leaves and flowers of pigmented Citrus species and hybrids and whether mutations in this gene can account for the inability of some Citrus varieties to develop pigmentation. We used sequence information to determine whether the phylogeny of Ruby is congruent with the generally accepted phylogeny of Citrus and the proposed ancestry of commercially important hybrids.
The Ruby Locus in Citrus and Related Genera
Most taxonomists divide the group recognized as true Citrus fruit trees into six related genera: Citrus, Poncirus, Microcitrus, Eremocitrus, Fortunella, and Clymenia (Swingle and Reece, 1967; Supplemental Fig. S1). The genus Citrus is further divided into two subgenera: Citrus, which includes the common cultivated plants with commercially important fruit, and Papeda (Swingle, 1946; Krueger and Navarro, 2007).
We attempted a comprehensive analysis of the sequence variation at the Ruby locus in the three primary Citrus species, in four members of the subgenus Papeda, in several interspecific hybrids, and in accessions belonging to four genera related to Citrus that are relevant to understanding the regulation of anthocyanin production. For each, we sequenced the Ruby gene and at least 1.6 kb of the region upstream of the coding sequence. The species and hybrids we have studied are listed in Table I.
Table I. Species and hybrids used in this study
Ruby is involved in the fruit-specific accumulation of anthocyanins in blood varieties of sweet orange (Butelli et al., 2012). A second gene, which we named Ruby2, also encodes a putative R2R3 MYB transcriptional activator with the potential to regulate anthocyanin biosynthesis. The Ruby2 and Ruby genes are arranged in tandem on chromosome 6 and are separated by an intergenic region ranging from 1.6 to 12 kb in different species. In Citrus, all the Ruby2 alleles are predicted to encode nonfunctional proteins because of the presence of different combinations of deletions, frame shifts, and stop mutations (Supplemental Data Set S1).
No other genes with similarity to Ruby and encoding proteins belonging to subgroup 6 of the R2R3 MYB family (known to regulate anthocyanin biosynthesis; Stracke et al., 2001) were detected in the high-quality reference haploid clementine genome or in two different genome annotation projects of sweet orange (https://phytozome.jgi.doe.gov and http://citrus.hzau.edu.cn/orange/). A previous genome-wide targeted analysis of R2R3 MYB genes in sweet orange (Liu et al., 2014) and examination of the genome sequences of other Citrus species (Q. Xu and X. Deng, unpublished data) also failed to identify other paralogs of known anthocyanin-related MYB genes.
Based on these observations, the hypothesis was developed that, in all the accessions belonging to the genus Citrus, Ruby is the only R2R3 MYB transcriptional activator involved in anthocyanin biosynthesis. Our objective was to determine whether variation within the Ruby locus is responsible for the differential ability of different accessions to produce anthocyanins.
Ruby in Citron
Citron (C. medica) is a true species with a very low level of heterozygosity. Most varieties are able to accumulate anthocyanins in young leaves and flowers (Fig. 1A), where cyanidin 3-glucoside and peonidin 3-(6″-malonyl)-glucoside accounted for approximately 80% of the total anthocyanin content (Fabroni et al., 2016).
The three varieties of citron we tested (Table I) contained the same Ruby allele, predicted to be active and named RC, in homozygous form. RC was inherited, identical in sequence, in many hybrids, including lemon, where its expression was characterized.
Lying at position −902 relative to the start of transcription in RC is a 5,435-bp retrotransposon that we named Tcl3 (Supplemental Fig. S2, A and B). Tcl3 is 92% and 91% identical in sequence to the two retroelements that are responsible for anthocyanin accumulation in Mediterranean (Tcs1) and Chinese (Tcs2) blood oranges, respectively (Butelli et al., 2012). All three elements are members of the same family of Copia-like long terminal repeat (LTR) retrotransposons, but while Tcs1 and Tcs2 are intact, active, and recently inserted elements, Tcl3 in citron is defective and is the result of an ancient retrotransposition event, as deduced by the divergence of LTR sequences that can be used to estimate the time of integration of LTR retrotransposons (SanMiguel et al., 1998). The two LTRs of Tcl3 differ by seven nucleotides and a 10-bp InDel, and the open reading frame of Tcl3 contains four stop mutations and one 5-bp deletion causing a frame-shift mutation that would inactivate the encoded polyprotein (Supplemental Fig. S2, B and C). These observations, together with its insertion at a considerable distance from the start of transcription, suggest that the presence of the Tcl3 retrotransposon likely has little or no impact on Ruby expression.
Ruby in Pummelo
Pummelo (C. maxima) is a true species of Citrus with many cultivars and high genetic diversity. Anthocyanin pigmentation has never been reported in pummelo. The four accessions we initially considered (Chandler, Reinking, Siamese acidless, and Kao Pan) contain different combinations of three Ruby alleles that we named rSTOPp, rTATA, and rCAAT (Table I).
The rSTOPp allele is characterized by the presence of a stop mutation (TGG to TGA at positions equivalent to +1,481 in the RC reference allele) in the third exon that would result in the truncation of the Ruby protein involving loss of its C-terminal 116 amino acids. This mutation results in a complete loss of function of the Ruby protein, as demonstrated by the lack of anthocyanin production when a truncated cDNA was transiently overexpressed in Nicotiana benthamiana (Fig. 2B). In contrast, the full-length Ruby cDNA induced the accumulation of visible levels of delphinidin-3-rutinoside in N. benthamiana (Fig. 2A).
Functionality of different Ruby alleles. N. benthamiana leaves were infiltrated with cDNA constructs corresponding to three different Ruby alleles. The phenotypes of leaves, the color of the leaf extracts, and the HPLC scans at 525 nm 7 d after infiltration are shown. A, Transient overexpression of Ruby cDNA encoding a full-length protein isolated from the flesh of Moro blood orange. B, Transient overexpression of a cDNA construct encoding a protein with a terminal deletion of 116 amino acids corresponding to the alleles found in some pummelo and mandarin accessions and hybrids derived from them. C, Transient overexpression of Ruby cDNA isolated from the fruit peel of M. australasica var Sanguinea encoding a protein with a 27-amino acid (aa) subterminal deletion. D, Evidence of the identity of delphinidin-3-rutinoside in sample C by positive mode electrospray MS1 and MS2. For this compound, the expected mass is 611.1607 (found mass, 611.1633; error, 4.25 ppm). Note the fragment representing delphinidin (expected mass, 303.0499) and the losses of a hexose moiety (expected loss, 162.0528) and a rhamnose moiety (expected loss, 146.0579).
Although the coding sequences of the other two alleles identified in pummelo encoded intact proteins, we detected a number of changes in the core promoter that might disrupt Ruby expression. The rTATA allele contains a T-to-C change in the center of the putative TATA box. This mutation lies at −32 bp relative to the start of transcription (as determined in lemon; see below). In the allele rCAAT, we identified another T-to-C transition, this time within a putative CAAT box (at position −73 bp relative to the start of transcription and also present in rSTOPp). TATA and CAAT boxes are cis-acting elements that determine the efficiency of the promoter in many eukaryotic genes (Cooper and Hausman, 2016), and the two mutations, specific for the pummelo accessions, lie in a region that is well conserved in Citrus and related genera (Supplemental Fig. S3A). Sweet orange carries the rTATA allele from its pummelo parent (see below; Butelli et al., 2012), and retrotransposon insertions into this allele provide either a new TATA box or activate a cryptic TATA box to activate Ruby expression in blood oranges (Butelli et al., 2012).
To confirm the prediction that both rCAAT and rTATA alleles are not expressed, we analyzed Chandler pummelo, since both alleles are present in this variety (Table I; Supplemental Fig. S3B). We were unable to isolate Ruby cDNA fragments, and we could detect no Ruby expression in leaves of Chandler pummelo (Supplemental Fig. S12C).
Although, theoretically, the lack of expression of Ruby from the rTATA and rCAAT alleles could have resulted from mutations in a closely linked regulator of Ruby, these alleles failed to complement alleles of Ruby with interruptions in the Ruby coding sequence (such as rSTOPp) in pummelo, and rTATA failed to complement the alleles from mandarin (which carry large deletions in the Ruby gene) when combined in sweet orange (Butelli et al., 2012).
All four pummelo varieties analyzed contained at least one copy of a Ruby gene with an intact coding sequence. Therefore, pummelo appears to maintain the potential to produce anthocyanins, a potential that could be fulfilled by recombination of sequences lying between the mutated CAAT box allele and the mutated TATA box allele. Such an event is possible because pummelo, unlike the majority of cultivated Citrus plants, produces true zygotic, monoembryonic seeds. Although purple pummelo varieties have not been reported, an individual wild tree bearing purple/red-skinned fruit was identified by Xiuxin Deng in a mountainous region of Hubei province, China (Supplemental Fig. S3C). Sequence analysis indicated that this pigmented variety carried a Ruby allele with both an active CAAT box and an active TATA box that might be the result of such a recombination event. This unique allele, named RP, was predicted to be fully active, since an intact coding sequence was under the control of a core promoter without mutations in the CAAT or TATA box (Fig. 3; Supplemental Fig. S3). Although there were mismatches in the sequence of the region upstream of the TATA box in the RP alleles, this sequence bore a greater similarity to the equivalent regions of rTATA than to rCAAT, including the absence of a 22-bp deletion. Downstream of the active TATA box motif in RP, the sequence differed from rTATA, and most of the single-nucleotide polymorphisms present in the area sequenced were identical between RP and rCAAT (Supplemental Fig. S4). This new active RP allele supports the possibility that it might be the result of recombination. Although the occurrence of such a recombination event is likely to be rare, this particular individual might have been identified and preserved by the local population because of its colored fruit peel. The phenotype of the RP allele might reflect the expression pattern of Ruby in a pigmented ancestor of pummelo, before loss-of-function Ruby alleles began to predominate as a result of evolution or human selection.
Schematic representation of different Ruby alleles identified in primary species of Citrus and related genera. Functional alleles (R) associated with anthocyanin pigmentation are highlighted in purple.
Ruby in Mandarin
The mandarin group is composed of numerous varieties with considerable genetic diversity but is treated as a single species under the name C. reticulata according to Swingle (Garcia-Lor et al., 2015).
We analyzed two common types of mandarin: Ponkan, an old variety thought to be a pure, prototypical C. reticulata (Hodgson, 1967), and Avana, a Mediterranean variety also known as Willowleaf. The structure and the sequence of the Ruby locus was identical in the two varieties and carried one allele (rSTOPm) with the same stop mutation identified in pummelo and another (rDEL2) with a 2-kb deletion involving the first two exons and a large portion of the upstream regulatory region of Ruby (Table I; Fig. 3; Supplemental Fig. S5). Sequence alignments suggested that the rSTOPm allele was derived from pummelo because the two alleles, rSTOPm and rSTOPp, showed 99% nucleotide identity and contained identical polymorphisms, including the distinctive stop mutation, that were not present in Ruby alleles from other Citrus or related species (Supplemental Fig. S5). Comparative sequencing of different Citrus varieties (Wu et al., 2014) has indicated a substantial admixture from pummelo in Ponkan and Willowleaf mandarins. In particular, the reported nucleotide heterozygosity in the terminal region of chromosome 6, where Ruby is located, supports the likely pummelo origin of a Ruby allele in some varieties of mandarin.
Since an ancestral mandarin may have had a distinct allelic composition at the Ruby locus, we looked among existing mandarins for different alleles of Ruby.
Tanaka (1977) recognized 36 mandarin species separated into five taxonomic groups. We sequenced the Ruby alleles from 17 mandarin varieties, representative of accessions from all five groups (Table I).
On the basis of their constitution of Ruby alleles, these mandarin accessions could be divided into three groups (Fig. 4). Group A (rDEL0.8 and rDEL0.8) was characterized by being homozygous for a deletion of ∼0.8 kb that spans the first two exons of the Ruby gene (between positions −175 and +629 relative to the start of transcription in the reference allele RC). Mandarins of group B (rDEL2 and rDEL2) were homozygous for a larger deletion of ∼2 kb (upstream of position +729 in RC). Mandarins of group C (rDEL2 and rSTOPm) were polymorphic for Ruby alleles, containing one allele introgressed from pummelo (rSTOPm) and one allele with the 2-kb deletion (rDEL2) also present in group B. This polymorphic genetic constitution has probably been maintained because group C varieties, including Ponkan and Willowleaf, are highly apomictic. None of the alleles in mandarin encode an active Ruby protein, explaining why mandarins have never been associated with anthocyanin pigmentation.
Classification of mandarins based on the analysis of the Ruby gene. Mandarins can be classified into three groups (A, B, and C) on the basis of two deletions and an introgression from pummelo. Many mandarin varieties are hybrids between members of these three groups or interspecific hybrids involving other Citrus species. C. indica, recognized by both Tanaka and Swingle as a mandarin species, is not related to mandarins. Roman numbers indicate the five taxonomic groups recognized by Tanaka (Hodgson, 1967). The gradation in color shading indicates the contribution from genomes of other species. Common names for the mandarin accessions are provided in Table I.
The short deletion allele (rDEL0.8) is highly polymorphic (Supplemental Fig. S5). Group A includes varieties of Japanese origin that show admixture from other species (Kitajima et al., 2007; Curk et al., 2015; Garcia-Lor et al., 2015). Our sequencing of genes other than Ruby in satsumas (C. unshiu) suggested introgression from pummelo that is different from that documented in Ponkan and Willowleaf (Wu et al., 2014). C. tachibana, also in group A, contained a Ruby2 allele derived from an unknown species as defined by distinct single-nucleotide polymorphisms.
Mandarins in group B include small-fruited sour mandarins (C. sunki and C. reshni) of reputed Chinese origin. Unlike rDEL0.8, the Ruby allele of this group with the larger deletion (rDEL2) did not show polymorphisms within or between accessions. The long 2-kb deletion encompassed the short one (0.8 kb), from which it may have been derived relatively recently.
Our data suggested that group C mandarins were derived from group B following hybridization with pummelo, an event that did not restore anthocyanin biosynthesis. This hybridization may have been a significant step in mandarin evolution that introduced new features, including fruit quality and general plant fitness.
Based on the allelic composition of Ruby, many mandarin varieties are likely to be hybrids between members of the three subgroups (Fig. 4). The different combinations of the three distinct alleles could help identify the origin and classify mandarin varieties that tend to cluster together in phylogenetic studies.
The allelic constitution at the Ruby locus also indicated the involvement of other Citrus species in important varieties usually classified as mandarins (Fig. 4). In particular, our analysis supports the conclusion that C. nobilis originated as a hybrid between mandarin (group A) and sweet orange (Hodgson, 1967). Within C. nobilis, the common accessions King and Kunembo inherited different Ruby alleles from their sweet orange parent, confirming that they had independent origins (Penjor et al., 2013; Table I; Fig. 4). Our analysis also confirmed a major contribution of papeda (C. micrantha) to the Nasnaran mandarin C. amblycarpa (Froelicher et al., 2011). C. indica, which was considered by both Tanaka and Swingle to be a wild mandarin species, shows no relationship to any of the other mandarins and most likely is a true species distantly related to citron (Supplemental Fig. S6).
Ruby in Papeda
Most Citrus plants are hybrids derived from different combinations of the three true species of Citrus (pummelo, mandarin, and citron). Contributions from a fourth species belonging to the subgenus Papeda have been demonstrated in some limes (Nicolosi et al., 2000). Papeda is considered to be an old and primitive type of Citrus, but its monophyletic status and its subdivision from Citrus are debated (Federici et al., 1998; Bayer et al., 2009; Carbonell-Caballero et al., 2015). We analyzed four different species of Papeda (C. micrantha, C. ichangensis, C. macroptera, and C. hystrix; Table I). All of them were able to accumulate anthocyanins in new growth but to differing extents. In C. ichangensis, pigmentation is strong (Fig. 1), whereas in C. micrantha, pigmentation is often not visible (Fig. 1) or is limited to a trace of purple on the outside of young buds (Hodgson, 1967). The Ruby alleles in C. ichangensis (RPap1 and RPap1) and C. micrantha (RPap2 and RPap2) were unrelated, while, surprisingly, C. macroptera and C. hystrix appeared to be identical hybrids of C. micrantha and C. ichangensis (Table I). All the Ruby alleles in Papeda had intact open reading frames, consistent with their pigmented phenotypes. Both alleles from C. micrantha, however, had an insertion of a defective Copia-like retrotransposon (named Tcm7), lacking both LTRs and inserted in antisense orientation 0.3 kb upstream of the predicted start of transcription (Fig. 3). This insertion also was associated with a large deletion of the promoter region that extends to include part of the upstream Ruby2 gene. The insertion of the retrotransposon and the deletion of part of the promoter are likely to affect the expression of Ruby and may explain the relatively weak pigmentation in new growth of C. micrantha compared with C. ichangensis (Fig. 1).
Ruby in Citrus Hybrids
Many commercial Citrus varieties have a hybrid origin (Scora, 1975; Barrett and Rhodes, 1976; Nicolosi et al., 2000; Moore, 2001). Hybrids derived from citron display intense purple pigmentation in young, developing leaves and flowers, where anthocyanins can provide protection for vulnerable tissues undergoing new growth. In contrast, hybrids involving mandarin and pummelo have inherited only nonfunctional Ruby alleles and do not produce anthocyanins (Fig. 5; Supplemental Fig. S7).
Segregation analysis of Ruby in some of the commercially important Citrus species and hybrids considered in this study. This simplified diagram shows a single existing variety of pummelo and mandarin as potential parents of both sweet and sour orange, which is highly unlikely. Light purple boxes indicate species and hybrids that are able to accumulate anthocyanins and that are always associated with active Ruby alleles from citron (RC) or papeda (RPap2). Pink and blue arrows indicate the maternal and paternal contributions to the generation of a hybrid based on specific mitotypes and chlorotypes as determined in previous studies. The gradation in color shading indicates the contribution from different species to the derivation of the hybrids.
Citron is one parent of lemon and of several hybrids often collectively defined as limes. Citron is usually fertilized by self-pollination and always served as the male parent in the formation of cultivated hybrids (Nicolosi et al., 2000; Curk et al., 2016).
To determine the role of Ruby in pigmented hybrids, we focused on lemon (C. limon) because of the availability of a unique white mutant, Zagara Bianca, which is completely unable to accumulate anthocyanins and probably arose as a bud sport of the variety Femminello (Cultrone et al., 2010). The anthocyanin composition in young leaves of the wild type accession was determined by ultra-high-performance liquid chromatography-electrospray ionization-mass spectrometry. Two compounds, cyanidin 3-(6″-malonyl)-β-glucoside and cyanidin 3-glucoside, accounted for most of the anthocyanin content (Fig. 6A; Supplemental Figs. S8–S10). The same compounds are the main anthocyanins identified in fruit of blood varieties of sweet orange and in new growth of many types of Citrus cultivars, including lemon (Hillebrand et al., 2004; Fabroni et al., 2016). No anthocyanins could be detected in the white mutant. Ruby was expressed in pigmented young flowers and leaves of the wild type accession but not in the corresponding tissues of the mutant (Fig. 6B).
Analysis of the wild type (WT) and the white mutant of lemon. A, HPLC scans recorded at 525 nm of methanol extracts from young leaves of Femminello wild-type (purple line) and Zagara Bianca (green line) lemons. Peaks marked with roman numbers indicate the main compounds identified in Femminello and absent in Zagara Bianca: Ia, cyanidin 3-glucoside; Ib, cyanidin 3-rutinoside; II, cyanidin 3-(6″-malonyl)-β-glucoside; and III, peonidin 3-(6′′-malonylglucoside). The identification of peaks is based on ultra-high-performance liquid chromatography-electrospray ionization-mass spectrometry analysis (Supplemental Figs. S8–S10) and comparison with literature data. B, Relative quantitative reverse transcription-PCR quantification of Ruby expression in flower buds and young leaves of Femminello and Zagara Bianca lemons. Error bars represent se. C, Schematic diagram of the two alleles of Ruby identified in lemon, RC and rSTOPp. The different symbols are explained in Figure 3 and Supplemental Figure S2A. The HindIII restriction sites and the genomic region used as a probe are shown. D, Southern-blot analysis of genomic DNA from different lemon varieties digested with HindIII and probed with a 32P-labeled probe of the Ruby gene. Zagara Bianca, the only variety completely unable to produce anthocyanins, does not display the hybridization band corresponding to the active allele RC.
The two lemon accessions were compared by Southern-blot analysis. Genomic DNA, hybridized to a probe of the full-length Ruby gene (Fig. 6C), displayed two distinct HindIII bands in wild-type lemon, one of which was clearly absent in the mutant (Fig. 6D). The common band present in both accessions corresponded to the Ruby allele rSTOPp carrying the nonsense mutation that disrupts protein function (Fig. 6C). This nonfunctional allele was inherited from pummelo via sour orange (Fig. 5). The allele RC, derived from citron, was missing in the white mutant, showing that Ruby is essential for anthocyanin biosynthesis in lemon and, by inference, in other species of Citrus.
The active allele of Ruby, RC, encodes a transcript that translates into a predicted protein of 262 amino acids with only eight differences compared with the active Ruby protein in blood varieties of sweet orange. The start of Ruby transcription (designated +1) in lemon was mapped using 5′ RACE. A TATA box and a potential CAAT box were identified at positions −34 and −74, respectively. The Ruby gene contains three exons at positions +103 to +220, +319 to +447, and +1,243 to +1,783.
Citron was involved in the independent origin of different varieties of lime. The most common are Mexican lime (C. aurantifolia) and Persian lime (C. latifolia). The composition of Ruby alleles in Mexican lime (RPap2 and RC) was in agreement with Papeda (C. micrantha) being its female parent (Nicolosi et al., 2000). Persian lime (rSTOPp and RC) displayed the same Ruby alleles as lemon, which is compatible with it having been derived from a cross between lemon and Mexican lime, as proposed by Bayer et al. (2009) based on chloroplast DNA marker analysis and Curk et al. (2016) using nuclear and cytoplasmic marker analyses (Fig. 5).
The origin of other citron-derived hybrids of minor commercial importance has received little attention. Palestine sweet lime (C. limettioides) carried the rDEL0.8 allele in all the accessions analyzed (including the acidic form Soh Synteng; Table I), which argues against the contribution of either sweet or sour orange, as suggested by Nicolosi et al. (2000), and, rather, suggests the presence of a pummelo × mandarin hybrid in its parentage, as proposed by Curk et al. (2014, 2016), based on haplotyping and nuclear and cytoplasmic DNA marker analyses. This hybrid, yet to be identified, also might have been involved in the derivation of Meyer lemon, which also contains the rDEL0.8 allele. The composition of Ruby alleles (rDEL2 and RC) in limetta (C. limetta) was consistent with the involvement of sour orange and citron as parents (Curk et al., 2014, 2016). For Rangpur lime (C. limonia), the composition of Ruby alleles (rDEL2 and RC) suggested a mandarin (group B) × citron origin.
The absence of anthocyanin pigmentation in flowers and leaves of many commercially important hybrids is associated with the absence of citron in their parentage (Fig. 5). For example, sour orange (C. aurantium), important for the production of marmalade and also used widely as a rootstock in the past, is a direct pummelo × mandarin F1 hybrid (Wu et al., 2014), confirmed by its composition of Ruby alleles (rSTOPp and rDEL2), and never produces anthocyanins.
Grapefruit (C. paradisi) was obtained in the West Indies in the 18th century, probably as a natural hybrid between pummelo and sweet orange (Morton, 1987; Moore, 2001). This origin is supported by its Ruby alleles (rSTOPp and rDEL2), and grapefruit never produces anthocyanins.
Clementine (C. clementina) is considered to have arisen as a natural hybrid of sweet orange and Mediterranean mandarin, based on morphological markers and sequence data (Hodgson, 1967; Nicolosi et al., 2000; Wu et al., 2014). Its lack of anthocyanins is consistent with its composition of Ruby alleles (rSTOPm and rDEL2) and this hybrid origin.
Sweet orange (C. sinensis) is considered a complex hybrid involving mandarin (providing the predominant genetic material) and pummelo (Garcia-Lor et al., 2013b; Xu et al., 2013; Wu et al., 2014), and the allelic composition of the Ruby locus (rTATA and rDEL2) confirmed this. We have been unable to detect any expression of Ruby in different tissues of common varieties of sweet orange (Butelli et al., 2012), consistent with the complete absence of anthocyanin pigmentation. As discussed for pummelo, from which it is derived, the allele rTATA in sweet orange is characterized by a T-to-C transition within the TATA box that prevents the gene from being expressed. In two independent blood varieties of sweet orange, however, the insertion of transposable elements has either provided a new TATA box and transcriptional start site (Tcs1) or promoted the use of a cryptic alternative start site (Tcs2) upstream of the T-to-C mutation (Supplemental Fig. S2, A and D; Butelli et al., 2012). Tcs1 and Tcs2 also provide new instructions for cold-dependent, fruit-specific expression of Ruby but are unable to restore the normal pattern of anthocyanin accumulation in new growth of leaves and flowers. These data confirm that rTATA fails to express Ruby because it has a defective TATA box.
Allelic Constitution of Ruby in Genera Related to Citrus
In addition to Citrus, we considered three distinct genera (Poncirus, Microcitrus, and Fortunella) that are classified within the true Citrus fruit trees and a more distant genus, Severinia, considered a primitive Citrus fruit tree (Swingle and Reece, 1967; Supplemental Fig. S1). These genera are relevant in the context of anthocyanin pigmentation.
Trifoliate orange (Poncirus trifoliata) is a species, unusually deciduous, that is commonly used as a rootstock for Citrus scions. Young and senescent P. trifoliata leaves are pigmented with anthocyanins (Supplemental Fig. S11A). Ruby is predicted to be functional in P. trifoliata (Fig. 3), and the two alleles we identified (RPonA and RPonB) were very similar in sequence, and both were expressed in pigmented leaves (Supplemental Fig. S11, B and C).
In Microcitrus, we initially analyzed a variety (M. australasica var Sanguinea) that is native to Australia, where it arose spontaneously (Netzel et al., 2006). This variety accumulates high levels of anthocyanins not only in new growth but also in fruit peel and pulp. We identified two Ruby alleles, RMic1 and RMic2, that could be distinguished by the presence of a 0.5-kb deletion in the promoter (between positions equivalent to −887 to −364 in the RC reference allele) of the RMic1 allele or a subterminal in-frame 81-bp deletion (between positions +1,650 and +1,730) in the coding region of the RMic2 allele (Fig. 3). In different tissues, the expression of Ruby was associated with anthocyanin pigmentation (Supplemental Fig. S12). Interestingly, while both alleles were expressed in leaves, in fruit, we could detect only monoallelic expression of RMic2 (Supplemental Fig. S12E), suggesting that the subterminal deletion, affecting the amino acid sequence of a region that is not conserved in anthocyanin-specific MYB regulatory genes, is unlikely to affect protein function. To confirm the functionality of the transcription factor encoded by RMic2, we transiently overexpressed the corresponding cDNA in leaves of N. benthamiana. As shown in Figure 2C, the construct was able to induce anthocyanin biosynthesis fully with no significant differences compared with the full-length Ruby cDNA isolated from Moro blood orange.
Further analysis of the promoter region of RMic2 revealed the presence of an insertion of a 0.4-kb repetitive element with an accompanying 7.5-kb deletion involving the entire Ruby2 gene and a translocation of 0.5 kb of DNA from chromosome 5 at a position equivalent to −856 in the RC reference allele (Fig. 3). The insertion, flanked by target duplication sites, corresponded to a repetitive element containing consensus motifs for SINE nonautonomous retrotransposons. Although it was difficult to assess the relative effects of the insertion, the deletion, and the translocation on the expression of the downstream Ruby gene, the presence of this rearrangement provided further evidence of the importance of transposable elements in reshaping gene structure and expression. This was confirmed by the analysis of a different variety of M. australasica with anthocyanin production in new growth but green-yellow fruit that do not accumulate anthocyanins. In this accession (Table I), the complex mutation in the promoter of Ruby was not present and both alleles corresponded to RMic1.
We analyzed two ornamental species belonging to the genus Fortunella: Nagami oval kumquat (F. margarita) and Malayan kumquat (F. polyandra), which do not accumulate anthocyanins, and we found only nonfunctional Ruby alleles (rFor) in both species, all carrying a single nucleotide insertion at position +444 in the second exon that resulted in a frame-shift mutation (Fig. 3). Calamondin (Citrofortunella microcarpa), another common variety that is anthocyaninless, also contained this defective allele from Fortunella (rFor) together with the rDEL0.8 allele. This allelic composition confirmed the presence of mandarin in its ancestry but argued against a contribution by the sour variety Sunki, frequently suggested as a candidate parent (Lai and Chen, 2008).
We sequenced Ruby in Chinese box orange (Severinia buxifolia) because this species is a distant relative of the genus Citrus that is often used as an outgroup in phylogenetic studies. S. buxifolia produces highly pigmented, small fruit that becomes black at maturity. Both Ruby alleles were characterized by a repetitive SINE-like element of 0.5 kb inserted 0.4 kb upstream of the predicted start of transcription (Fig. 3). As in M. australasica var Sanguinea, it is possible that the presence of SINE-like elements could influence the strong anthocyanin pigmentation in fruit.
Phylogenetic Analysis
We conducted a phylogenetic analysis of 18 Ruby alleles representing the diversity of Citrus and related genera. Alleles from primary species only were used in the analysis, because the Ruby sequences in the hybrids are identical to those identified in the primary parental species (Fig. 5).
Among all of the models tested via the Phylemon Web site (Sánchez et al., 2011), the one with the best fit was HKY+G+F. The phylogenetic relationships between Citrus species and their relatives inferred from the maximum-likelihood method using this model are shown in Figure 7.
Phylogenetic relationship between primary species of Citrus and related genera based on the sequence of Ruby. The phylogenetic trees were generated using the Phylemon software program. Branch support is given at all branches. The different Ruby alleles are color coded as in Table I. The sequences of the Ruby gene and its promoter used for phylogenetic analysis have been deposited in GenBank under accession numbers KT591672 to KT591689.
The model that best fitted the data using the MEGA software was the T92 model (Kimura, 1980; Tamura, 1992; Tamura et al., 2011). The phylogenetic relationships between Citrus species and their relatives inferred using MEGA software are shown in Supplemental Figure S13.
Phylemon and MEGA phylogenies suggested the same relationships between Ruby alleles, indicating a clear separation between Citrus and related genera (strong branch support). Poncirus and Microcitrus were quite distant from Citrus (high bootstrap values), while Fortunella was more closely related. This analysis supported the phylogenetic relationships in the Citrinae established using other markers (Nicolosi et al., 2000; Garcia-Lor et al., 2013a, 2013b; Penjor et al., 2013; Ramadugu et al., 2013) but did not reveal the distinct phylogenetic cluster of citron and Microcitrus established using the complete chloroplast DNA of 34 species (Carbonell-Caballero et al., 2015).
A division between the two subgenera, Citrus and Papeda, was not supported by our analysis and has been challenged in other studies (Penjor et al., 2010; Carbonell-Caballero et al., 2015). Instead, our data suggested a polyphyletic origin of the Papeda group. The Ruby alleles in C. micrantha are related to those of C. medica and C. indica, while the Ruby alleles of C. ichangensis clustered with the alleles from mandarin. We propose that the subgenus Papeda contains DNA from at least two distinct unrelated species (C. micrantha and C. ichangensis) and several hybrids between them (such as C. hystrix and C. macroptera). This distinction could explain much of the conflicting data on the origin and classification of Papeda. Whole-genome sequencing would confirm or refute these suggestions.
The analysis in Figure 7 also confirmed that rSTOPm, one of the alleles identified in common mandarin varieties such as Ponkan and Willowleaf, lies within the clade of pummelo alleles and has been introgressed into mandarin from pummelo, as suggested by Wu et al. (2014).
Anthocyanins are widely distributed plant pigments with a diverse range of functions and tissue distributions. Since they are not essential for plant survival, they provide a unique visual marker for the occurrence of mutations and the activation of transposable elements, which may result in changes to the patterns of plant pigmentation, as demonstrated in maize (Zea mays), Antirrhinum, Petunia, and blood varieties of sweet orange (Malinowski, 1935; Fincham, 1967; McClintock, 1984; Coen et al., 1986; Butelli et al., 2012).
Ruby is a regulatory gene encoding a transcription factor that is essential for anthocyanin accumulation in Citrus and related genera. The Ruby locus contains two potential MYB regulators of anthocyanin biosynthesis (Ruby and the inactive Ruby2). Many angiosperm plants have experienced extensive duplication of these regulatory genes. The amplification of MYB genes belonging to subgroup 6 that regulate anthocyanin biosynthesis may be a general mechanism underlying the diversity of floral pigmentation patterns, as observed in Petunia (Bombarely et al., 2016) and Antirrhinum (Schwinn et al., 2006), but also might be the result of domestication and human selection, as proposed for grape (Matus et al., 2008). In Citrus, the presence of no more than one functional MYB regulator suggests that the production of anthocyanins is not under strong selective pressure and is largely dispensable. In Cucurbitaceae, the only family of plants where anthocyanin accumulation has never been reported, two R2R3 MYB genes of subgroup 6 also are present, but, according to our analysis, neither gene encodes a functional MYB activator. Therefore, while in many plant species the expansion of MYB genes has resulted in evolutionary novelty, in other lineages the loss of anthocyanin production is associated with molecular decay of the MYB regulators.
The Ruby locus is very dynamic. In a limited number of accessions, we uncovered six large deletions, six insertions of transposable elements, one translocation, and four critical mutations within the Ruby gene and its promoter region (Fig. 3). Most of these events have had consequences on anthocyanin accumulation, ranging from a complete suppression of production to complex reconfiguration of the distribution of anthocyanins in different tissues.
Transposable elements are major forces in the evolution and rearrangement of genomes and important determinants of genome size and gene expression (McClintock, 1984). The insertion of Tcl3 in citron likely did not result in obvious changes in pigmentation, but the presence of Tcm7 in C. micrantha, accompanied by a deletion, was associated with almost complete abolition of anthocyanin pigmentation in new growth.
The strong fruit-specific pigmentation of blood varieties of sweet orange also was the result of the insertion of retrotransposons (Butelli et al., 2012). Comparative analysis of core promoter regions in many different accessions and the mapping of the start of transcription of Ruby in lemon allowed us to identify a key mutation in the TATA box of Ruby in an allele derived from the pummelo parent in the hybrid, sweet orange. As a result, sweet orange has completely lost the ability to accumulate anthocyanins. In blood varieties of sweet orange, the insertion of retrotransposons into this nonfunctional promoter prompted the use of alternative TATA boxes that reestablished anthocyanin production and also determined the cold-induced expression of Ruby, specifically in fruit (Butelli et al., 2012).
The accumulation of anthocyanins in fruit of species distantly related to Citrus (M. australasica and S. buxifolia) also was associated with the insertion of transposable elements upstream of Ruby. In these species, the retrotransposons belong to a different family, SINE, and may have been involved in changing the activity of the Ruby promoter and the control of anthocyanin production. Normally, anthocyanin production in plants is induced by light (Steyn et al., 2002), and the evolution of anthocyanin production within the fruit might have required the development of light-independent regulatory control of Ruby expression, as observed with blood oranges (Butelli et al., 2012).
The loss of anthocyanins may have occurred naturally, as a consequence of the different climates in which Citrus species originated. Citron arose in high-altitude, dry regions where anthocyanins are important in providing protection from high light and UV irradiation (Moore, 1866; Steyn et al., 2002). In contrast, mandarin and pummelo thrive in lowland tropics with high humidity and rain throughout the year (Webber, 1943). In these conditions, light stress may be less severe and anthocyanins less important for the protection of young tissues.
Citrus plants have been grown for millennia in Asia for reasons other than for food. Citron has an ancient history as a sacred plant, and its powerful symbolism is associated with the fruit. Believed to be indigenous to northeastern India, it was absorbed into Jewish tradition and is still used in ancient religious rituals (Swingle and Reece, 1967). China is the center of origin of most Citrus species. In Chinese tradition, early use of Citrus was strictly religious and mainly associated with flowers. Kumquat flowers represented luck and prosperity, while the white flowers of mandarin and orange were symbols of purity and innocence. This symbolism has persisted into modern Christianity (Heilmeyer, 2001; Moore and Garibaldi, 2003). The simultaneous occurrence of golden fruit and white blossom remains a central element in the symbolism of C. tachibana, a sacred tree in Japan. It is impossible to know the extent to which human intervention played a significant role in the selection of varieties of mandarin, pummelo, and kumquat with pure white flowers for ornamental and religious purposes. Our study, however, indicates that the ability to accumulate anthocyanins has been lost independently, several times during Citrus evolution, as a result of mutations in the Ruby gene.
Analysis of the Ruby locus has informed us of the origin of present-day varieties of Citrus and the history of their spread around the world. Although phylogenetic inference based on the analysis of a single gene may lead to conclusions that require further support, our analysis of Ruby has confirmed the widely accepted hybrid origin of the most important cultivated Citrus varieties (Fig. 5) and the phylogenetic relationships between primary species and related genera (Fig. 7). The analysis of Ruby also has shed new light on the origin of poorly studied hybrids, on the classification of mandarins, on members of the diverse group Papeda, and on the occurrence of ancient introgression events across species.
The analysis of Ruby explains why many Citrus varieties have lost the ability to accumulate anthocyanins and provides a useful tool to help in the interpretation of the mosaic structure of Citrus genomes.
Leaves were obtained from different sources as indicated in Table I. Plant materials were ground in liquid nitrogen, and DNA and RNA were extracted using the DNeasy Plant Mini Kit and the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions.
Isolation of Ruby Alleles
For most accessions, Ruby alleles were isolated by PCR using primers designed to amplify fragments spanning the region 1.6 kb upstream of the start of the coding sequence to 136 bp downstream of the Ruby stop codon. PCR fragments were used directly for sequencing or cloned into the pGEM-T Easy vector (Promega). For accessions where only direct sequencing was used, nucleotide polymorphisms were depicted using the IUPAC code. The active Ruby alleles in citron (RC) and in Microcitrus australasica var Sanguinea (RMic2) were isolated by inverse PCR after restriction enzyme digestion with PstI and ClaI, respectively, followed by self-ligation. Primer sequences are listed in Supplemental Table S1.
DNA Gel Blot
DNA from leaves of lemons (C. limon) was digested with HindIII, separated by electrophoresis, transferred to nitrocellulose membranes, and hybridized as described previously (Butelli et al., 2012).
Determination of the Transcriptional Start Site of Ruby
The transcriptional start site in active alleles of Ruby was determined using RNA extracted from young purple leaves of Femminello lemon. DNase-treated RNA was reverse transcribed using the 5′/3′ RACE Kit (Roche). The cDNA product was sequenced to determine the transcriptional start nucleotide in the sequences lying upstream of the Ruby coding sequences. Gene-specific primers listed in Supplemental Table S1 were used for 5′ end cDNA amplification according to the manufacturer's instructions.
Expression Analysis of Ruby
Quantification of Ruby expression in new growth of Femminello and Zagara Bianca lemons and Chandler pummelo and in pigmented fruit and leaves of M. australasica var Sanguinea was performed by qRT-PCR as described previously (Butelli et al., 2012). The allelic expression of Ruby in pigmented leaves of Poncirus trifoliata and in fruit skin of M. australasica var Sanguinea was determined by RT-PCR followed by sequencing.
Transient Expression in Nicotiana benthamiana Leaves
Transient expression of different Ruby cDNA constructs was carried out using the HyperTrans system (Sainsbury et al., 2009). Ruby cDNAs were isolated from the flesh of Moro blood orange or the fruit peel of M. australasica var Sanguinea, PCR amplified using primers listed in Supplemental Table S1, and transferred into pEAQ-HT-DEST1 vectors. A truncated cDNA containing the mutations identified in the alleles rSTOPm and rSTOPp was obtained using a reverse primer with a stop codon and cDNA from Moro blood orange as a template. The plasmids obtained were introduced into Agrobacterium tumefaciens GV3101, and the strains were infiltrated into the abaxial side of leaves of 4-week-old plants. Leaves were harvested 7 d after infiltration.
Analysis and Identification of Anthocyanins
Leaves of lemon or N. benthamiana were frozen in liquid nitrogen and ground to a fine powder. Approximately 200 mg of powder was extracted with 800 μL of 80% methanol containing 1% HCl. Liquid chromatography-tandem mass spectrometry of anthocyanins was carried out on a Nexera ultra-high-performance liquid chromatography system attached to an ion-trap time of flight mass spectrometer (Shimadzu). Separation was on a 100- × 2.1-mm 2.6μ Kinetex XB-C18 column (Phenomenex) using the following gradient of acetonitrile (solvent B) versus 0.1% formic acid in water (solvent A), run at 0.5 mL min−1 and 40°C: 0 min, 2% B; 0.5 min, 2% B; 2 min, 10% B; 11 min, 30% B; 15 min, 90% B; 15.8 min, 90% B; 16 min, 2% B; 20.1 min, 2% B. Anthocyanins were monitored by UV/visible absorbance (200–600 nm) and positive mode electrospray mass spectrometry (full spectra from m/z 200 to 2,000 and data-dependent MS2 of the most abundant precursor ions at an isolation width of m/z 3, 50% collision energy, and 50% collision gas). Anthocyanins were identified on the basis of their mass, and the mass of their fragments in MS2, as well as by comparison with published spectra.
The sequences of the Ruby alleles were aligned using BioEdit (Hall, 1999), SeqMan version 7.0 (http://www.dnastar.com), and SATé-II (Liu et al., 2012) software applications. Analyses were performed to determine which model best matched the data using Phylemon 2.0 (http://phylemon.bioinfo.cipf.es; Sánchez et al., 2011). The HKY+G+F model took into account the nucleotide substitution model of Hasegawa-Kishino-Yano (HKY; number of substitution types = 2), which allowed variable base frequencies, one transition rate, and one transversion rate, as well as the nucleotide frequency (F) and the gamma distribution (G). The construction of the maximum-likelihood tree was performed using MEGA5 software with bootstrap support calculated from 1,000 samples to assess the branch support.
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers KT591672 to KT591689.
Supplemental Figure S1. Classification of Citrus according to Swingle.
Supplemental Figure S2. Active Ruby alleles associated with LTR retrotransposons in blood varieties of sweet orange and citron.
Supplemental Figure S3. Analysis of promoter sequences in pummelo.
Supplemental Figure S4. Multiple sequence alignment of Ruby in pummelo.
Supplemental Figure S5. Multiple sequence alignment of Ruby in mandarin.
Supplemental Figure S6. Multiple sequence alignment of Ruby in different species of Citrus.
Supplemental Figure S7. PCR analysis of Ruby in primary species and hybrids of Citrus.
Supplemental Figure S10. Mass spectrometry data for peak identification of anthocyanins in leaves of lemon.
Supplemental Figure S11. Expression analysis of Ruby in leaves of P. trifoliata.
Supplemental Figure S12. Expression analysis of Ruby in different tissues of M. australasica var Sanguinea.
Supplemental Figure S13. Phylogenetic relationship between primary species of Citrus and related genera based on the sequence of Ruby using the MEGA software program.
Supplemental Table S1. Sequences of the primers used in this study.
Supplemental Data Set S1. Nucleotide sequence of Ruby2 in different accessions of the genus Citrus.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Eugenio Butelli (eugenio.butelli{at}jic.ac.uk).
E.B. and C.M. planned and designed the research; E.B., C.M., A.G.-L., C.L., G.L.C., and L.H. performed experiments; M.L.K., C.R., R.K., G.R.-R., A.-L.F., Y.F., L.N., Q.X., and X.D. provided plant material, DNA, information, and images; E.B. and C.M. wrote the article with input and comments from all the other authors.
↵1 This work was supported by the Institute Strategic Program Understanding and Exploiting Plant and Microbial Secondary Metabolism from the Biotechnology and Biological Sciences Research Council (grant no. BB/J004596/1 to C.M. and E.B.), the European Union FP7 ATHENA collaborative project (grant no. 245121 to C.M., E.B., C.L., G.L.C, and G.R.R.), the Ministry of Economia y Competitividad (grant no. AGL2011-26490 to A.-G.L. and L.N.), the Ministry of Economía, Industria y Competitividad-Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria Fondo Europeo de Desarrollo Regional (grant no. RTA2015-00069-00-00 to A.-G.L. and L.N.), and the networking activities within the European Union-funded COST ACTION FA1106 QualityFruit.
Received November 7, 2016.
Published February 14, 2017.
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(1975) Symposium on Biochemical Systematics, Genetics and Origin of Cultivated Plants. 9. History and Origin of Citrus. Bull Torrey Bot Club 102: 369–375
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(2002) Anthocyanins in vegetative tissues: a proposed unified function in photoprotection. New Phytol 155: 349–361
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(2001) The R2R3-MYB gene family in Arabidopsis thaliana. Curr Opin Plant Biol 4: 447–456
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(1946) The botany of Citrus and its wild relatives of the orange subfamily (family Rutaceae, subfamily Aurantioideae). In W Reuther, H Webber, L Batchelor, eds, The Citrus Industry. University of California Press, Berkeley, pp 129–474
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(1967) The botany of Citrus and its wild relatives. In W Reuther, H Webber, L Batchelor, eds, The Citrus Industry. University of California Press, Berkeley, pp 190–430
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(2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731–2739
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(1977) Fundamental discussion of citrus classification. Studia Citrologica 14: 1–6
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(2008) Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. Plant J 54: 733–749
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(1943) Plant characteristics and climatology. In W Reuther, H Webber, L Batchelor, eds, The Citrus Industry, Vol 1. University of California Press, Berkeley, pp 41–69
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(2014) Sequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during citrus domestication. Nat Biotechnol 32: 656–662
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You are going to email the following Changes in Anthocyanin Production during Domestication of Citrus
GENES, DEVELOPMENT, AND EVOLUTION
Arabidopsis Leaf Flatness Is Regulated by PPD2 and NINJA through Repression of CYCLIN D3 Genes
NMT1 and NMT3 N-Methyltransferase Activity Is Critical to Lipid Homeostasis, Morphogenesis, and Reproduction
Evidence for Exaptation of the Marchantia polymorpha M20D Peptidase MpILR1 into the Tracheophyte Auxin Regulatory Pathway
Show more GENES, DEVELOPMENT, AND EVOLUTION


text:
Betanzos - Betanzos, A Coruña, Galicia, España - Coats of Arms on Waymarking.com
Betanzos - Betanzos, A Coruña, Galicia, España
in Coats of Arms
Posted by: Ariberna
29T E 563909 N 4791595
Quick Description: Coat of Betanzos town
Location: Galicia, Spain
Date Posted: 7/10/2021 3:39:39 PM
Waymark Code: WM14HD7
Published By: Alfouine
In this medieval bridge, when the english soldiers destroy the bridge, in XIX century put this shield of stone.
"The Betanzos shield is made up, in an azure field , of a tower with two silver bodies supported by a bridge with three arches of the same metal, over natural river waters. Flanking the tower, three on each side, six roels of gold . For stamp, a royal crown.
The shield represents one of the medieval entry bridges to the city of Betanzos, characterized in the past by having a defensive tower located in its center, called "Caramona". It is not clear, however, to which of these bridges it corresponds: if the "New Bridge", which crossed the Mendo River and was demolished in 1969 to extend the route of the N-VI highway; or to the "Old Bridge", which still today crosses the Mandeo River as it passes through the town. As for the roels, they represent the six monterias that surround the city.
The oldest known coat of arms of Betanzos dates from the 15th century, and is sculpted in one of the arches of the Church of San Francisco in that city. Discovered in 1961 , it lacks the six roels that today, and since at least the 16th century, flank the tower."
(visit link)
Bearer of Coat of Arms: Town
Full name of the bearer: Betanzos
Where is Coat of Arms installed (short description) ?:
Material / Design: Stone
Blazon (heraldic description):
The Betanzos shield is made up, in an azure field , of a tower with two silver bodies supported by a bridge with three arches of the same metal, over natural river waters. Flanking the tower, three on each side, six roels of gold . For stamp, a royal crown
Old Bridge in Betanzos
Web page about the bearer of Coat of Arms (if exists): [Web Link]
Web page about the structure where is Coat of Arms installed (if exists): Not listed
Logging requirements: Please upload your own personal photo of the coat of arms. You or your GPS can be in the picture, but it's not a requirement.
Nearest Coats of Arms
Date Logged Log
7/12/2021 Ariberna visited it

redpajama_set_name: RedPajamaCommonCrawl


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INA SCE2010 Needle Roller Bearing, Steel Cage, Open End, Inch, 1-1/4" ID, 1-1/2" OD, 5/8" Width, 8800rpm Maximum Rotational Speed, 6500lbf Static Load Capacity, 3700lbf Dynamic Load Capacity: Amazon.com: Industrial & Scientific.
7. Febr. 2012 Gegen Ende 2008 haben das Bundesamt für Umwelt BAFU und das Bundesamt für. Landwirtschaft BLW gemeinsam Umweltziele für die Landwirtschaft publiziert. Darin sind Ziele formuliert, die aus Gesetzen, Verordnungen, internationalen Abkommen und. Bundesratsbeschlüssen hergeleitet worden sind.
This paper presents a brief overview of TIVET in Kenya (history and present), the laws governing TIVET, and its structure and challenges today. A robust review of various relevant concepts that are in use in TVET is presented and solutions to identified shortcomings of the Kenyan TIVET system proposed. The Develop a.
698 ,,,IKO,INA.,! :CZB ;:SCE2010 ;:31.75 mm;:38.1 mm;:SCE2010 ;:15.875 mm;:0.030 kg;:;:;:;:.
INA bearings have many optional features available in- AA040 - Shell type sealed bearings come standard with 40% Retinax LX2 (Example: SCE8799 AA040) SCE2010. BCE2010. 1.250. 31.750 1.500. 38.100. 0.625. 15.875. 0.066. 30. 3,700. 16,500. 6,500. 29,000. 8,800. SCE2012. BCE2012. 1.250. 31.750 1.500.
SCE 2010 – July 2013. Southern California Edison 2007 – 2010. Southern. San Antonio, Texas Area Refueling talent in a highperforming culture within the Oil & Gas industry. Human Resources Skills:. Microwave September 2009 – November 2012. GGB Bearing Technology div of BF Goodrich May 1995 – April 2009.


From 12 – 14 March 2019, the international corrugated and folding carton industry will get together for the 4th edition of the specialised trade fair CCE International at the Munich Trade Fair Centre in Germany. On a total of 5,000 m2 net exhibition space, 155 exhibitors from 23 countries will present raw materials, machinery, accessories and services for the production and conversion of corrugated and cartonboard, covering the entire production chain. Live demonstrations at the exhibitor stands will give insights into the range of technological developments and current innovations. The most important exhibitor countries are Italy, Germany, China, the Netherlands and the United Kingdom.
The growing online trade and trend towards recyclable and sustainable packaging have a positive impact on the corrugated and folding carton industry: No other material is better known for its sustainability than corrugated and cartonboard. Challenges include the constantly accelerating retail business as well as the increasing demand for digitalised packaging and individualised products. Digital print offers enhanced opportunities to meet these demands. 'We see enormous potential for digital print on corrugated and cartonboard. The question is how the industry sector can make optimum use of this technology', says Liljana Goszdziewski, Exhibition Director of CCE International, on behalf of the organisers, Mack Brooks Exhibitions. 'At this year's show, visitors will discover the diverse applications of the latest printing and finishing techniques. A key feature of our trade fair is the intense exchange of ideas at the stands, which facilitates the joint developm ent of specific production solutions. We currently see a boost of innovations in the industry, primarily due to digital technologies', Liljana Goszdziewski continues.
Important technology trends will also be covered in the free seminar programme at CCE International 2019. On all three days of the show, industry experts will talk about the key topic 'Digital Print' as well as 'Sustainability' and 'Innovative Converting Techniques', share their knowledge with the audience, and lead open discussions. The many presentations include seminars such as 'Digital Trends in Corrugated – The Perspective from a MIS' by the company Kiwiplan Europe, 'Maximising Folder-Gluer Performance' by the company Leary Co., W. H., and 'Start with Finishing! The Smart Way to go Digital' by the company Highcon.
Europe's only trade fair focussing solely on manufacturing and converting corrugated and cartonboard is, once again, characterised by its high internationality. The European market is booming and therefore attracting manufacturing and converting companies from around the world. On a total of 5,000 m2 net exhibition space, visitors will meet 155 exhibitors from 23 countries. The majority of exhibitors are from Europe, primarily Germany, Italy, the Netherlands, and the United Kingdom. Exhibitors from other regions are mainly from China, Taiwan, and the United States.
At CCE International, visitors will discover a comprehensive product portfolio and exhibitors covering the entire production chain. The exhibition profile includes raw materials, machinery, accessories and services for the production and conversion of corrugated and cartonboard: Papers – corrugated and carton board; consumables; corrugating line, equipment and components; corrugated and carton converting machinery; design and CAD/CAM; printing processes and equipment; cutting, creasing and die cutting equipment; ancillary equipment; materials handling and warehousing; pallet strapping and handling systems; MIS and plant scheduling systems as well as waste extraction and baling. Thus, CCE International addresses buyers and users of machinery, accessories and services for the production and conversion of corrugated and cartonboard.
Once again, CCE International will take place alongside ICE Europe, the World's Leading Exhibition for Paper, Film, and Foil Converting. ICE Europe addresses converters and packaging specialists and presents the entire spectrum of technological innovations for the production and conversion of flexible, web-based materials. A total of 463 exhibitors will showcase their product innovations. All visitors of CCE International and ICE Europe can attend both shows with their tickets.
CCE International 2019 will take place in hall B6 at the Munich Trade Fair Centre in Germany. ICE Europe 2019 will take place in halls A5 and A6. Both shows will be open on Tuesday, 12 March 2019, and Wednesday, 13 March 2019, from 9:00 AM to 5:00 PM, and on Thursday, 14 March 2019, from 9:00 AM to 4:00 PM. Tickets for both shows are available in the Online Ticket Shop on the show website. Day tickets are available for €24 online and €38 on-site, season tickets are available for €34 online and €50 on-site.


You are right. In many cases the better low light ability of FF ends up in sloppy exposure and longer raw development sessions. Not that much with professional photographers but the rest of the pack.
Replied in Anybody had problems with a Panasonic 100-400mm recently?
Richard, it is indeed telling me something I already know. The issue is that way too often comment sections and threads are hijacked to discuss the equivalency stuff ad nausea.
The sheer volume of contributions dealing with equivalency overly emphasizes one aspect of the lens discussed while others are effectively pushed into the background.
Don't know why you are speading this nonsense here. In terms of exposure it is an f4.5 lens period.
I have never noticed this on D800/D800E. AF-speed will always also depend on the camera used. What camera did you use? Faster with MF-lens must be a joke anyway, unless an AF lens requires calibration.
'only a little bit better' than the Nikon fifties? You must either be kidding or using it on a D700 or even older body.
Replied in Best quality 50mm prime for Nikon FF?
At the price of the 58 'poor copies' are hard to digest. Build quality leaves things to be desired. Thus you really need a 'love affair' to find this acceptable.
light_bulb has not added any gear yet.


We share and enjoy a great guided tour in Barcelona. Our guide was funny, interesting and dynamic he give us a lot of information about spot to visit. Recommanded of course 😀 !
We've been there to rent 2 bikes. The owner was smiling and professional, he give us a lot of advices to visit the city easily. Recommanded !
Get quick answers from Bcn Classic Rides staff and past visitors.


text:
Huskers, Sooners A Day Closer to Renewing Football Rivalry
It's not number 1 against number 2 like it was on Thanksgiving Day in 1971 when Nebraska topped Oklahoma 35-31 in the Game of the Century. But, after 50 years the rivalry and history between the former Big 8 and Big 12 rivals is still being reflected on by fans and the media.
On Saturday, unranked NU will take a 2-1 record to Norman. The Blackshirts have allowed only 10 points combined in the past two games, albeit against Fordham and Buffalo. Third-ranked OU is 2-0. The Sooners had a shaky 40-35 season-opening win over Tulane, before romping over Western Carolina 76-0 last Saturday.
Kickoff this Saturday is set for 11 a.m. The pregame starts at 7 a.m. on News Talk 900 KJSK. It will be joined at 10:30 on 93.5 The Hawk.


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Running Start is a program for high school juniors and seniors who want to challenge themselves academically.
*Important: Sophomores & juniors who want to enter in the Fall, must wait until their first semester grades are posted at the high school is using GPA to qualify.
**The Assessment Center will tell you if your ALEKS Math test score is college-level.
For more information, Clark College's website.
Call us at (360)885-5331 or send us an email for more information.


Synthetic ion channels have been known for nearly three decades, but it is only in the past decade that analysis of the currents these ionic conductors carry has become a standard technique. A broad range of structural types have been explored and these reports have produced a very diverse collection of ion channel conductance behaviours. In this critical review we describe a notational method to extract salient information from reported ion channel experiments. We use an activity grid to represent quantitative information on conductance and opening duration with a five-level colour code to represent qualitative information on the nature of the conductance–time profile. Analysis of the cumulative dataset suggests that the reported conductance data can reflect the structural features of the compounds prepared, but does also reflect the energetic landscape of the bilayer membrane in which synthetic ion channels function (143 references).


text:
Forsyth County Teacher Aide Fired
Educator Made Discriminatory Comments on Facebook
A screenshot of Jane Wood Allen's original post in which she directed racially charged insults at First Lady, Michelle Obama, is shown above. Photo credited to Georgia Newsday.
Ashton Bruce, Staff Writer
October 11, 2016|38 Views
As of Monday, Oct. 3, Jane Wood Allen, a teacher aide in Forsyth Country, was fired from Chestatee Elementary School for racist comments directed towards Michelle Obama posted on Facebook.
Allen's most controversial post called First Lady, Michelle Obama, a "gorilla", a word with historical connotations as a racially charged insult, and a "disgrace to America". The post, made on Sept. 29, had hundreds of "shares" on Facebook in only hours and a thousand by the next day. After the racist comment made by an elementary school employee transcended across different social networking sites, including Twitter, people angry by the comments scrolled through other posts on Allen's Facebook wall. Allen apparently had a history of making racist claims towards African-Americans and Hispanic people, as well as Islamophobic remarks.
Allen deleted her Facebook page three days after she made the post due to backlash.
Hundreds of people protested Allen's employment in an elementary school system through social media. Most of the claims against Allen remarked how unethical her opinions were, how inappropriate it was that they were made publicly, and how those opinions could potentially be inflicted upon her students.
The Forsyth County district made a public statement regarding the issue, saying that racism and discrimination are not tolerated in the entirety of the school district. The district provided no other statement, but Allen has been terminated from her duty in the elementary school.


text:
Update on Support for Black Lives Matter Uprisings
To honor our ongoing commitments to transparency and accountability to the larger community that makes all our work possible, we want to provide an update on how CCBF has been supporting people arrested in connection with recent Black Lives Matter uprisings. This includes the reparations taking and property destruction in downtown Chicago on Sunday night. If you would like more context on that, please read some of the articles linked at the bottom of this post!
Since Friday, August 7th, CCBF has paid $93,145 to free 36 people in three counties who were arrested while supporting the Black Lives Matter movement or otherwise rejecting police violence against Black people. This includes one person in DuPage County, 10 people in Winnebago County, and 25 people in Cook County. We expect to pay more bonds in Cook County today and will continue to do so as needed.
This is only possible because of the work of Black liberation organizers on the ground, CCBF volunteers, and our partners in the Cook County Public Defender's Office, National Lawyers Guild of Chicago, Showing Up for Racial Justice (SURJ) Chicago and everyone running jail support, and all the people who donated to ensure the resources to pay bond were available. Yesterday, two CCBF volunteers spent a combined 10 hours in the jail paying ransoms to free 23 people! This is a good reminder that it takes a lot more than just money to pay bonds and free people. While it also takes lots of money, CCBF does not need more donations at this time. Please continue to direct money to grassroots community organizing groups!
For more context on the uprisings, resistance to police violence, and how you can get involved, read this press release from and support the work of Black Lives Matter Chicago! For additional context, we recommend these two articles: In Defense of Looting and All Power to the Looters.
Update on Support for Black Lives Matter Uprisings2020-08-122020-08-12https://chicagobond.org/wp-content/uploads/2018/10/ccbf_horizontal-sixpoint-0white.pngChicago Community Bond Fundhttps://chicagobond.org/wp-content/uploads/2020/08/8-12-2020-blm-bonds-update.png200px200px
CCBF Welcomes Two New Staff Members
CCBF is Hiring Interns for Spring Semester 2022
CCBF is Hiring a Legal Advocacy Coordinator!
CCBF Welcomes Two New Directors


My name is Ian Lutz and I am the creator and host of the podcast INSIDEtrack! This is a podcast that interviews elite athletes weekly to understand better what sets these athletes apart. We are all students of the sport of running so why not learn from the very very best in the field.
Please let me know what you think and if there is anyone you'd like to hear on the program! I also would be very thankful if you would share this link with anyone who wants to learn more about the sport!
If you're interviewing elite athletes why would you talk with Chris Solinsky?
Ian, thank you for these! I look forward to diving in.
Holding an American Record makes one elite. Even if it get's broken.
Didn't say elite in 2015.


text:
import React from 'react'

export default props => {
  return (

UPDATE

      <form onSubmit={props.updateDog}>
        <input type="text" name="update_dogId" placeholder="Document ID" onChange={props.handleChange} value={props.dogId} />
        <input type="text" name="update_name" placeholder="Dog's Name" onChange={props.handleChange} value={props.name} />
        <input type="text" name="update_favoriteToy" placeholder="Dog's Favorite Toy" onChange={props.handleChange} value={props.favoriteToy} />
        <button type="submit">Update Dog</button>
      </form>

  )
}

redpajama_set_name: RedPajamaGithub


This afternoon, Instagram released a new video app called Hyperlapse. As the name might suggest, it's made for shooting and condensing video footage into time-lapse segments, and it's stupid simple. You open the app, hit a button to start recording and tap the same one to stop the video, pick a playback speed (1-12x) and save that to your camera roll or share it to Instagram or Facebook.
It's going to get so much use during fashion week.
At this point, nearly every editor peppers his or her Instagram with runway shots throughout Fashion Month, which kicks off in New York next Wednesday. It's really hard to get a good shot, though, even from a front row seat: As models whiz by, most turn out blurry, unless a particularly quick-fingered spectator is able to hit focus and then snap a shot in a matter of seconds. Video on Instagram is similarly a hot mess for runway footage, but Hyperlapse makes it possible for editors to capture the full show, crank up the speed a bit and present a shortened version to their followers.
And the cool part is, that person could be anybody. The behind-the-scenes tech includes stabilization that makes even a rookie look more like a professional. Having tested the app out this afternoon, we'd still recommend giving it some help and not make too many sudden movements.
So fashion folks, consider this a call to arms. Download this app and bring some magic to the masses this fashion week.
Today, Instagram announced the addition of its own Vine-eqsue video service--and fashion brands are already jumping on board.
Want to take your blog mobile? There's an app for that.
Invent as many fancy fashion apps as you want, tech world. Instagram will always (okay, well, maybe not always) be the fashion crowd's app of choice, and its influence has never been as prominent as it was during this past New York Fashion Week. Not only was everyone posting Instagram pics of everything, at all times, but, at times, it almost felt like designers and producers actually had Instagram in mind while planning shows and presentations.
These days, being at a runway show is as much — if not more — about capturing moments for social media as it is about the live experience. At what cost?


From this, we learned that we needed to offer more than one solar pump product to meet farmer needs. The pilot taught us the importance of building on the critical components of Juhudi's financial model: customer education and demonstrations accompanied by a strong pitch and bundled solutions. We decided to innovate and to be more ambitious in the next iteration.
1. Expand the pilot to 1 year. If there's anything the credit business has taught us, it is that the appetite for credit is cyclical. We see higher demand for credit at the end and beginning of the year. Extending the pilot period to one year ensures that we'll observe the demand for this product throughout the agricultural season.
2. Take it to multiple branches across different counties of Kenya. By expanding the pilot to at least 10 field branches, we can gauge the potential of the product in different geographic regions with distinct climatic conditions. Also, failing to fully secure staff buy-in at a branch may kill the potential of a product.
3. Innovate on the technology offering. We added a new solar water pump product with a battery, so that farmers can work the pump very early and late in the day, or even in cloudy weather. The product also has a higher lift and a lower price point.
4. Broaden the concept of solar irrigation to holistic water resource management. We learned that sometimes farmers want to invest in an irrigation kit or water tank before purchasing the water pump. To incorporate that decision-making, we will include "smart water" solutions, such as water tanks, irrigation kits, wells and water filters as well as staff training on technical water resource management topics.
5. Hire dedicated staff. We hope to hire at least six loan officers whose key performance indicators would be selling these products alongside the traditional parameters common across Juhudi loan officers. Although these newly hired staff will have the power to form new groups and disburse loans, they will also be tasked to sell to the existing portfolios of our loan officers.
6. Introduce incentives for farmers. In the last pilot, we introduced staff incentives. We now want to take it a step further by targeting farmer group officials to market this product for us. They will now be rewarded for successful sales conversions, helping spread sustainable water solutions far and wide across Kenya.


text:
Mortgage Servicing
Featured Transactions
Jordan A. Smith
jsmith@medalistcapital.com
3105 Glenwood Avenue, Suite 301
Analyst Raleigh
BA, University of North Carolina
Jordan joined Medalist Capital in the spring of 2016 as a full-time analyst after completing an internship with the firm. Before that, he worked as an investment sales intern at Cushman Wakefield and a development and leasing intern at Commercial Realty Advisors.
Jordan earned a BA in English from the University of North Carolina at Chapel Hill, where he also played on the men's lacrosse team and was a member of the Carolina Leadership Academy and Carolina Outreach Program.
< Back to Team
Charlotte Raleigh Charleston Atlanta


text:
package de.hsbremen.chat.client;

import net.miginfocom.swing.MigLayout;

import javax.swing.*;
import java.awt.*;

/**
 * Created by cschaf on 31.03.2015.
 */
public class Gui extends JFrame{
    public JPanel pnlMain;
    private JLabel lblUsername;
    public JTextField tbxUsername;
    private JLabel lblPort;
    public JTextField tbxPort;
    private JLabel lblIp;
    public JTextField tbxIp;
    public JButton btnConnect;
    public JButton btnDisconnect;
    private JScrollPane scrollPanelMessages;
    public JTextArea traMessages;
    public JTextField tbxMessage;
    public JButton btnSend;
    private JScrollPane scrollPanelUsers;
    public JList listUsers;

    public Gui(){
        super("Client Gui");
        this._initComponents();
        this._addComponents();

        setContentPane(this.pnlMain);
        setDefaultCloseOperation(JFrame.EXIT_ON_CLOSE);
        setSize(637, 500);
        setPreferredSize(new Dimension(637, 500));
        pack();
        setVisible(true);
    }

    private void _addComponents(){
// row 1
        pnlMain.add(lblUsername, "left, split 2, span 4");
        pnlMain.add(tbxUsername, "pushx, growx");
        pnlMain.add(lblIp, "split");
        pnlMain.add(tbxIp, "pushx, growx");
        pnlMain.add(lblPort, "split");
        pnlMain.add(tbxPort, "pushx, growx");
        pnlMain.add(btnConnect, "sg navBtn");
        pnlMain.add(btnDisconnect, "sg navBtn, wrap");
// row 2
        pnlMain.add(scrollPanelMessages, "push, grow, span 6, split");
        pnlMain.add(scrollPanelUsers, "wrap, pushy, growy");
// row 3
        pnlMain.add(tbxMessage, "pushx, growx, split, span 6");
        pnlMain.add(btnSend);

    }

    private void _initComponents(){
        pnlMain = new JPanel(new MigLayout());
        lblUsername = new JLabel("Username:");
        tbxUsername = new JTextField("Guest");
        lblIp = new JLabel("IP:");
        tbxIp = new JTextField("localhost");
        lblPort = new JLabel("Port:");
        tbxPort = new JTextField("1337");
        btnConnect = new JButton("Connect");
        btnDisconnect = new JButton("Disconnect");
        traMessages = new JTextArea(10,10);
        traMessages.setEditable(false);
        //traMessages.setLineWrap(true);
        scrollPanelMessages = new JScrollPane(traMessages);
        listUsers = new JList();
        scrollPanelUsers = new JScrollPane(listUsers);
        tbxMessage = new JTextField("");
        btnSend = new JButton("Send");

        btnDisconnect.setEnabled(false);
        tbxMessage.setEnabled(false);
        btnSend.setEnabled(false);
    }

    public void changeNavbarEnableState(boolean state){
        tbxUsername.setEnabled(state);
        tbxIp.setEnabled(state);
        tbxPort.setEnabled(state);
        btnConnect.setEnabled(state);
        btnDisconnect.setEnabled(!state);
        btnSend.setEnabled(!state);
        tbxMessage.setEnabled(!state);
    }
}