Temporal and spatial regulation of anthocyanin biosynthesis provide diverse flower colour intensities and patterning in <Emphasis Type="Italic">Cymbidium</Emphasis> orchid

Main conclusion

This study confirmed pigment profiles in different colour groups, isolated key anthocyanin biosynthetic genes and established a basis to examine the regulation of colour patterning in flowers of Cymbidium orchid. Cymbidium orchid (Cymbidium hybrida) has a range of flower colours, often classified into four colour groups; pink, white, yellow and green. In this study, the biochemical and molecular basis for the different colour types was investigated, and genes involved in flavonoid/anthocyanin synthesis were identified and characterised. Pigment analysis across selected cultivars confirmed cyanidin 3-O-rutinoside and peonidin 3-O-rutinoside as the major anthocyanins detected; the flavonols quercetin and kaempferol rutinoside and robinoside were also present in petal tissue. β-carotene was the major carotenoid in the yellow cultivars, whilst pheophytins were the major chlorophyll pigments in the green cultivars. Anthocyanin pigments were important across all eight cultivars because anthocyanin accumulated in the flower labellum, even if not in the other petals/sepals. Genes encoding the flavonoid biosynthetic pathway enzymes chalcone synthase, flavonol synthase, flavonoid 3′ hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) were isolated from petal tissue of a Cymbidium cultivar. Expression of these flavonoid genes was monitored across flower bud development in each cultivar, confirming that DFR and ANS were only expressed in tissues where anthocyanin accumulated. Phylogenetic analysis suggested a cytochrome P450 sequence as that of the Cymbidium F3′H, consistent with the accumulation of di-hydroxylated anthocyanins and flavonols in flower tissue. A separate polyketide synthase, identified as a bibenzyl synthase, was isolated from petal tissue but was not associated with pigment accumulation. Our analyses show the diversity in flower colour of Cymbidium orchid derives not from different individual pigments but from subtle variations in concentration and pattern of pigment accumulation.

     

Identification and functional analysis of anthocyanin biosynthesis genes in <Emphasis Type="Italic">Phalaenopsis</Emphasis> hybrids

Phalaenopsis species are among the most popular potted flowers for their fascinating flowers. When their whole-genome sequencing was completed, they have become useful for studying the molecular mechanism of anthocyanin biosynthesis. Here, we identified 49 candidate anthocyanin synthetic genes in the Phalaenopsis genome. Our results showed that duplication events might contribute to the expansion of some gene families, such as the genes encoding chalcone synthase (PeCHS), flavonoid 3′-hydroxylase (PeF3′H), and myeloblastosis (PeMYB). To elucidate their functions in anthocyanin biosynthesis, we conducted a global expression analysis. We found that anthocyanin synthesis occurred during the very early flower development stage and that the flavanone 3-hydroxylase (F3H), F3′H, and dihydroflavonol 4-reductase (DFR) genes played key roles in this process. Over-expression of Phalaenopsis flavonoid 3′,5′-hydroxylase (F3′5′H) in petunia showed that it had no function in anthocyanin production. Furthermore, global analysis of sequences and expression patterns show that the regulatory genes are relatively conserved and might be important in regulating anthocyanin synthesis through different combined expression patterns. To determine the functions of MYB2, 11, and 12, we over-expressed them in petunia and performed yeast two-hybrid analysis with anthocyanin (AN)1 and AN11. The MYB2 protein had strong activity in regulating anthocyanin biosynthesis and induced significant pigment accumulation in transgenic plant petals, whereas MYB11 and MYB12 had lower activities. Our work provided important improvement in the understanding of anthocyanin biosynthesis and established a foundation for floral colour breeding in Phalaenopsis through genetic engineering.     

Cloning and Analysis of Promoter Regions of Flavonoid Biosynthesis Genes in Safflower

The flower of the safflower plant Carthamus tinctorius L. has been widely used in traditional Chinese medicine because of its ability to improve cerebral blood flow. Flavonoids are the main active ingredient of safflower plants. Analyses of the regulation of flavonoids are of great significance for the artificial regulation of their biosynthesis, and previous studies have mainly focused on their metabolic synthesis in safflower. For a better understanding of the regulation of flavonoid biosynthesis in safflower, promoters of flavonoid biosynthesis genes in safflower were cloned and analysed. First, the expression of the flavonoid genes in safflower was analysed. Twenty-four genes involved in flavonoid biosynthesis exhibited large differences. Four promoters of flavonoid genes that were mainly expressed in the flowers of safflower (termed pctCHI-1, pctCHS-2, pctF3H-2 and pctGT-1) were cloned and analysed. Elements that respond to light, i.e. phytohormones and the MYB regulator, were identified. The promoter activities were analysed using a protoplast system. All four promoters had activities in the flower protoplast, and the pctCHS-2 activity was the highest. Our results also demonstrate that pctCHS-2 is a flower-specific promoter. All of these results provide a basis for improved analyses of flavonoid regulation in safflower.     

Comparative Flower and Inflorescence Organogenesis among Genera of Betulaceae: Implications for Phylogenetic Relationships

Betulaceae have simple flowers but complex inflorescences. Recent phylogenetic analyses using molecular data have produced robust phylogenetic trees of Betulaceae. In this study, we evaluated the phylogenetic value of comparative organogenetic data of reproductive organs in the context of molecular phylogenies. Flower and inflorescence developmental processes of 21 species from all six genera in Betulaceae were documented with scanning electron microscopy. In each pistillate cyme, there are one primary bract, two secondary bracts, and two or three flowers in the six genera; only in Alnus are there two tertiary bracts on the abaxial side. The pistillate flower of all genera but Alnus has tepal primordia. Two tepals stop developing early on, resulting in the lack of tepals in mature flowers of Betula; while the tepals are initiated from a common circular primordium at the base of pistil in Corylus, Ostryopsis, Carpinus, and Ostrya, and the developed tepals with irregular shape and unstable number of lobes are adnate to the top of the pistil. In staminate organogenesis, each cyme has one primary bract and three flowers in all genera; two secondary bracts are only present in Alnus, Betula, and Corylus. Staminate flowers have no tepals except in Alnus and Betula, and exhibit high variation in number of stamens among genera. The number of secondary and tertiary bracts in each pistillate and staminate cyme, as well as the presence of tepals in pistillate and staminate flowers was clarified in all genera. Micro-morphological characters were used to infer the phylogenetic relationships of genera and sections of Betulaceae. Our analyses support the division of two subfamilies: Betuloideae (Alnus and Betula) and Coryloideae (Corylus, Carpinus, Ostrya, and Ostryopsis), and three tribes: Betuleae (Alnus and Betula), Coryleae (Corylus), and Carpineae (Carpinus, Ostrya, and Ostryopsis). The results agree with those from molecular phylogenetic studies, and suggest that micro-morphological characters are phylogenetically informative in Betulaceae, and reproductive organs of Betulaceae have evolved in the direction of reduction in bracts and tepals.     

Chalcone synthase homologous genes cloning and expression pattern in flowering <Emphasis Type="Italic">Fagopyrum tataricum</Emphasis> Gaertn

Tartary buckwheat (Fagopyrum tataricum Gaertn.) is highly nutritious and an excellent dietary source of flavonoid compounds. Chalcone synthase (CHS) is the first key enzyme involved in flavonoid biosynthesis. Here, three putative CHS genes (designated as FtCHS1 (GU172165), FtCHS2 (KT284884), and FtCHS3 (KT284885) were isolated from tartary buckwheat. Nucleotide sequence analysis indicated that FtCHS1 and FtCHS2 each contained one intron of 444 bp and 157 bp, respectively. FtCHS3 included two introns, one of 86 bp and another of 73 bp. The results of quantitative real-time PCR (qRT-PCR) showed the FtCHSs expression presented the same pattern in the stems and flowers, with FtCHS1>FtCHS3>FtCHS2. A different tendency was found in leaves, with FtCHS3>FtCHS2>FtCHS1. However, there was no direct correlation between the three CHS expression and total flavonoids. Furthermore, high-performance liquid chromatography (HPLC) performance reveals rutin is the most abundant flavonoid in all tissues, leaves should be the main location for quercetin storage in tartary buckwheat.     

<Emphasis Type="Italic">Aeromonas</Emphasis> and <Emphasis Type="Italic">Plesiomonas</Emphasis> species from scarlet ibis (<Emphasis Type="Italic">Eudocimus ruber</Emphasis>) and their environment: monitoring antimicrobial susceptibility and virulence

The present study aimed at evaluating the role of captive scarlet ibises (Eudocimus ruber) and their environment as reservoirs of Aeromonas spp. and Plesiomonas spp., and analyzing the in vitro antimicrobial susceptibility and virulence of the recovered bacterial isolates. Thus, non-lactose and weak-lactose fermenting, oxidase positive Gram-negative bacilli were recovered from cloacal samples (n = 30) of scarlet ibises kept in a conservational facility and from water samples (n = 30) from their environment. Then, the antimicrobial susceptibility, hemolytic activity and biofilm production of the recovered Aeromonas spp. and Plesiomonas shigelloides strains were assessed. In addition, the virulence-associated genes of Aeromonas spp. were detected. Ten Aeromonas veronii bv. sobria, 2 Aeromonas hydrophila complex and 10 P. shigelloides were recovered. Intermediate susceptibility to piperacillin-tazobactam and cefepime was observed in 2 Aeromonas spp. and 1 P. shigelloides, respectively, and resistance to gentamicin was observed in 4 P. shigelloides. The automated susceptibility analysis revealed resistance to piperacillin-tazobactam and meropenem among Aeromonas spp. and intermediate susceptibility to gentamicin among P. shigelloides. All Aeromonas isolates presented hemolytic activity, while 3 P. shigelloides were non-hemolytic. All Aeromonas spp. and 3/10 P. shigelloides were biofilm-producers, at 28 °C, while 10 Aeromonas spp. and 6/10 P. shigelloides produced biofilms, at 37 °C. The most prevalent virulence genes of Aeromonas spp. were asa1 and ascV. Scarlet ibises and their environment harbour potentially pathogenic bacteria, thus requiring monitoring and measures to prevent contamination of humans and other animals.     

The lignin synthesis related genes and lodging resistance of <Emphasis Type="Italic">Fagopyrum esculentum</Emphasis>

Lignin is closely related to the lodging resistance of common buckwheat (Fagopyrum esculentum Moench.). However, the characteristics of lignin synthesis related genes have not yet been reported. We investigated the lignin biosynthesis gene expression, activities of related enzymes, and accumulation of lignin monomers during branching stage, bloom stage, and milky ripe stage by real-time quantitative PCR, UVspectrophotometry, and gas chromatography-mass spectrometry in the 2^nd internode of three common buckwheat cultivars with different lodging resistance. The results showed that lignin content and the activity of phenylalanine ammonia lyase (PAL), 4-coumarate: CoA ligase (4CL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD) were closely related to the lodging resistance of common buckwheat. Further, we studied gene expression of cinnamate 4-hydroxylase (C4H), caffeoyl-CoA O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), cinnamoyl-CoA reductase (CCR), and caffeic acid O-methyltransferase (COMT). The lignin biosynthesis genes were divided into three classes according to their expression pattern: 1) expression firstly increasing and then descending (PAL, 4CL, CAD, C4H, CCoAOMT, F5H, and CCR), 2) expression remaining constant during maturation (C3H), and 3) expression decreasing with maturation (COMT). The present study provides preliminary insights into the expression of lignin biosynthesis genes in common buckwheat, laying a foundation for further understanding the lignin biosynthesis.     

Effect of foliar application of salicylic acid,hydrogen peroxide and a xyloglucan oligosaccharide on capsiate content and gene expression associated with capsinoids synthesis in <Emphasis Type="Italic">Capsicum annuum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>

Capsinoids are non-pungent analogues of capsaicinoids in pepper (Capsicum spp). The absence of pungency, in addition to their biological activities similar to that of capsaicinoids such as anti-inflammatory, antimicrobial, and antioxidant properties, makes capsinoids an excellent option for increasing use in human and animal nutrition, as well as health and pharmaceutical industries. There are only few sources of pepper producing capsinoids, and one of them (accession 509–45-1), Capsicum annuum L., is a potential source for increasing capsinoids content using strategies as controlled elicitation during plant production in the greenhouse. In this research we evaluated the effect of weekly and one-day-before-harvest foliar applications of hydrogen peroxide, salicylic acid and a xyloglucan oligosaccharide on the concentration of capsiate in fruits of this pepper accession, as well as the gene expression of phenylalanine ammonia-lyase (pal), putative aminotransferase (pamt), capsaicin synthase (at3) and β-keto acyl synthase (kas). Results showed that the two tested concentrations of H₂O₂ significantly increased capsiate content and gene expression associated with capsaicinoids (pamt, at3 and kas) and the phenylpropanoids (pal) pathways. Plant yield was not affected using this induction strategy. Our results indicated that the pre-harvest and weekly application of hydrogen peroxide and xyloglucan oligosaccharide improved production of capsiate in C. annuum L.     

Biofilm production and other virulence factors in <Emphasis Type="Italic">Streptococcus</Emphasis> spp<Emphasis Type="Italic">.</Emphasis> isolated from clinical cases of bovine mastitis in Poland

Background

Mastitis is a common disease in dairy cattle throughout the world and causes considerable economic losses each year. An important aetiological agent of this disease is bacteria of the genus Streptococcus; hence, exploring the mechanisms of virulence in these bacteria is an extremely important step for the development of effective prevention programmes. The purpose of our study was to determine the ability to produce biofilm and the occurrence of selected invasiveness factors among bacteria of the genus Streptococcus isolated from cattle with the clinical form of mastitis in northeastern Poland.

Results

Most of the isolates analysed demonstrated an ability to produce biofilm (over 70%). Virulence genes were searched for in the three most common streptococci in our experiment: S. agalactiae, S. uberis and S. dysgalactiae. For S. agalactiae, only four genes were confirmed: rib (33%), cylE (78%), bca (37%), and cfb (100%). The genes pavA, scpB, bac and lmb were not present in any of the tested strains. The dominant serotypes of the species were Ia (n?=?8) and II (n?=?8), in addition to some strains that were not classified in any of the groups (n?=?6). Out of the eight selected genes for S. uberis (sua, pauA/skc, gapC, cfu, lbp, hasA, hasB, hasC), only one was not found (lbp). Finally, two genes were chosen for S. dysgalactiae (eno and napr), and their presence was confirmed in 76% and 86% of the strains, respectively.

Conclusions

The experiment showed that strains of Streptococcus spp. isolated from dairy cattle with clinical cases of mastitis in the northeastern part of Poland possess several invasiveness factors that can substantially affect the course of the disease, and this should be considered when developing targeted prevention programmes.