Anthocyanic vacuolar inclusions--their nature and significance in flower colouration

The petals of a number of flowers are shown to contain similar intensely coloured intravacuolar bodies referred to herein as anthocyanic vacuolar inclusions (AVIs). The AVIs in a blue-grey carnation and in purple lisianthus have been studied in detail. AVIs occur predominantly in the adaxial epidermal cells and their presence is shown to have a major influence on flower colour by enhancing both intensity and blueness. The latter effect is especially dramatic in the carnation where the normally pink pelargonidin pigments produce a blue-grey colouration. In lisianthus, the presence of large AVIs produces marked colour intensification in the inner zone of the petal by concentrating anthocyanins above levels that would be possible in vacuolar solution. Electron microscopy studies on lisianthus epidermal tissue failed to detect a membrane boundary in AVI bodies. AVIs isolated from lisianthus cells are shown to have a protein matrix. Bound to this matrix are four cyanidin and delphinidin acylated 3,5-diglycosides (three, new to lisianthus), which are relatively minor anthocyanins in whole petal extracts where acylated delphinidin triglycosides predominate. Flavonol glycosides were not bound. A high level of anthocyanin structural specificity in this association is thus implied. The specificity and effectiveness of this anthocyanin "trapping" is confirmed by the presence in the surrounding vacuolar solution of only delphinidin triglycosides, accompanied by the full range of flavonol glycosides. "Trapped" anthocyanins are shown to differ from solution anthocyanins only in that they lack a terminal rhamnose on the 3-linked galactose. The results of this study define for the first time the substantial effect AVIs have on flower colour, and provide insights into their nature and their specificity as vacuolar anthocyanin traps.     

Greenish blue flower colour of Strongylodon macrobotrys

The flower colour of Strongyledon macrobotrys is luminous blue green and attracts bats for pollination. The chemical basis for development of the flower colour was investigated. The flower contained an anthocyanin (malvin) and a flavone (saponarin), approximately 1:9 (malvin: saponarin) in molar ratio. The pH of the pigmented epidermal cell sap of the jade vine petal was exceptionally high, 7.90, while the pH value of the colourless inner tissue was 5.60. Copigmentation test using the mixtures of malvin and saponarin (1:9 M ratio) at various pH values revealed that the characteristic blue green colour of the jade vine is developed by copigmentation of malvin with saponarin in slightly alkaline cell sap, pH 7.9. In the copigmentation in slightly alkaline condition, saponarin shows a strong yellow colour, which gives a greenish tone to the flower colour.     

Inflorescence and floral development of Dahlstedtia species (Leguminosae: Papilionoideae: Millettieae)

Inflorescence and floral development of two tropical legume trees, Dahlstedtia pinnata and Dahlstedtia pentaphylla, occurring in the Atlantic Forest of south-eastern and southern Brazil, were investigated and compared with other papilionoids. Few studies have been made of floral development in tribe Millettieae, and this paper is intended to fill that gap in our knowledge. Dahlstedtia species have an unusual inflorescence type among legumes, the pseudoraceme, which comprises axillary units of three or more flowers, each with a subtending bract. Each flower exhibits a pair of opposite bracteoles. The order of flower initiation is acropetal; inception of the floral organs is as follows: sepals (5), petals (5), carpel (1) plus outer stamens (5) and finally inner stamens (5). Organ initiation in sepal, petal and inner stamen whorls is unidirectional; the carpel cleft is adaxial. The vexillum originates from a tubular-shaped primordium in mid-development and is larger than other petals at maturity, covering the keels. The filament tube develops later after initiation of inner-stamen primordia. Floral development in Dahlstedtia is almost always similar to other papilionoids, especially species of Phaseoleae and Sophoreae. But one important difference is the precocious ovule initiation (open carpel with ovules) in Dahlstedtia, the third citation of this phenomenon for papilionoids. No suppression, organ loss or anomalies occur in the order of primordia initiation or structure. Infra-generic differences in the first stages of ontogeny are rare; however, different species of Dahlstedtia are distinguished by the differing distribution pattern of secretory cavities in the flower.     

Formation of UV-honey guides in Rudbeckia hirta

The UV-honey guides of Rudbeckia hirta were investigated by UV-photography, reflectance spectroscopy, LC-MS analysis and studies of the enzymes involved in the formation of the UV-absorbing flavonols present in the petals. It was shown for the first time that the typical bull’s eye pattern is already established at the early stages of flower anthesis on the front side of the petal surface, but is hidden to pollinators until the buds are open and the petals are unfolded. The rear side of the petals remains UV-reflecting during the whole flower anthesis. Studies on the local distribution of 19 flavonols across the petals confirmed that the majority are concentrated in the basal part of the ray flower. However, in contrast to the earlier studies, eupatolitin 3-O-glucoside (6,7-dimethoxyquercetin 3-O-glucoside) was present in both the basal and apical parts of the petals, whereas eupatolin (6,7-dimethoxyquercetin 3-O-rhamnoside) was exclusively found in the apical parts. The enzymes involved in the formation of the flavonols in R. hirta were demonstrated for the first time. These include a rare flavonol 6-hydroxylase, which was identified as cytochrome P450-dependent monooxygenase and did not accept any methylated flavonol as substrate. All enzymes were present in the basal and apical parts of the petals, although some of them clearly showed higher activities in the basal part. This indicates that the local accumulation of flavonols in R. hirta is not achieved by a locally restricted presence of the enzymes involved in flavonol formation.     

Pelargonium elandsmontanum (Geraniaceae: section Hoarea), a new species from the Western Cape Province, South Africa

The new species Pelargonium elandsmontanum is a local endemic from Elandsberg Nature Reserve near Malmesbury in the Western Cape Province, South Africa. One of six species of sect. Hoarea with just the posterior two petals developed, it resembles P. ternifolium in its trifoliolate leaves and pink petals but is distinguished from that species by the short, stout petioles, rhombic, acute leaflets with the upper surface glabrous or with spreading hairs (vs cuneate, apically incised leaflets with both surfaces adpressed-hairy), and five (vs four) fertile stamens.     

Spatial patterns of flower colour variation in native and introduced ranges of Convolvulus arvensis (Convolvulaceae) revealed by citizen-science data and machine learning

• Flower colour polymorphism refers to the presence of multiple colour variants in plant populations. Investigation of this phenomenon led to multiple discoveries, including the principles of heredity and the foundations of population genetics. I examined flower colour variation across native and introduced ranges of Convolvulus arvensis, which exhibits flower colour polymorphism (individuals have white or pink petals).
• To study flower colour variation of this species throughout large geographic scale, I used observations gathered from the iNaturalist platform. To handle a large amount of data, I trained a neural network to classify the plants' morphs based on photographs. After which I performed spatial analyses to examine the patterns of the colour frequency, also in relation to environmental factors.
• The results show that flower colours are polymorphic across the whole species range, but the frequency of pink versus white flowers varies. In the Palearctic, I observed geographic clines of colour morph frequencies: a higher frequency of the pink morph in populations from Northwest Europe, whereas in South and East Europe, towards the eastern edge of the range, the white morph was dominant. In contrast, pattern of colour distribution in North America (where the species is invasive) seems random, but the model indicates a link between higher proportions of pink morphs in mild and humid climates.
• The mechanisms behind the observed patterns remain largely unknown, as changes in a morphs' frequency are not strongly linked to abiotic factors. To understand the spatial pattern, a detailed investigation, accounting for the species' phylogeography is needed. This study provides another example of how the general public may collect data relevant to ecological studies, even when the data are not collected for a specific project.

     

Why do so many petals have conical epidermal cells?

Background

The conical epidermal cells found on the petals of most Angiosperm species are so widespread that they have been used as markers of petal identity, but their function has only been analysed in recent years. This review brings together diverse data on the role of these cells in pollination biology.

Scope

The published effects of conical cells on petal colour, petal reflexing, scent production, petal wettability and pollinator grip on the flower surface are considered. Of these factors, pollinator grip has been shown to be of most significance in the well-studied Antirrhinum majus/bumble-bee system. Published data on the relationship between epidermal cell morphology and floral temperature were limited, so an analysis of the effects of cell shape on floral temperature in Antirrhinum is presented here. Statistically significant warming by conical cells was not detected, although insignificant trends towards faster warming at dawn were found, and it was also found that flat-celled flowers could be warmer on warm days. The warming observed is less significant than that achieved by varying pigment content. However, the possibility that the effect of conical cells on temperature might be biologically significant in certain specific instances such as marginal habitats or weather conditions cannot be ruled out.

Conclusions

Conical epidermal cells can influence a diverse set of petal properties. The fitness benefits they provide to plants are likely to vary with pollinator and habitat, and models are now required to understand how these different factors interact.     

Comparative study of floral development in Onobrychis melanotricha, Hedysarum varium and Alhagi persarum (Leguminosae: Papilionoideae: Hedysareae)

Floral initiation and development of Hedysarum varium, Onobrychis melanotricha and Alhagi persarum was studied using epi-illumination light-microscopy techniques. The studied species belong to the tribe Hedysareae of the inverted repeat loss clade (IRLC clade), which is characterized by missing the large inverted repeat in the chloroplast genome. The main aim of our study was to determine developmental bases for similarities and differences among the three taxa and to verify the position of Alhagi relative to other genera of the IRLC clade. According to our observations, bracteoles are missing in Onobrychis melanotricha, but are present in the other two species. All three species share unidirectional sepal initiation starting with a median abaxial sepal and bidirectional petal initiation. Stamen initiation is unidirectional in all except in the outer stamen whorl of Hedysarum varium, where it is bidirectional. An important ontogenetic feature in O. melanotricha is the existence of five common primordia, which give rise to petal and stamen primordia. Although in H. varium and O. melanotricha common primordia are observed at some stages in floral organ initiations, in Alhagi all organs are initiated separately. Moreover, overlap in time of floral organs initiation occurs in H. varium and O. melanotricha, but not in A. persarum. The carpel initiates concurrently with the petal primordia in all. It might be presumed that Alhagi is primitive in relation to the other studied Hedysareae taxa, due to the presence of bracteoles, the absence of common primordia, and the lack of overlap in time of different organ initiations.     

Over-expression of the Gerbera hybrida At-SOC1-like1 gene Gh-SOC1 leads to floral organ identity deterioration

Background and Aims

The family of MADS box genes is involved in a number of processes besides controlling floral development. In addition to supplying homeotic functions defined by the ABC model, they influence flowering time and transformation of vegetative meristem into inflorescence meristem, and have functions in roots and leaves. Three Gerbera hybrida At-SOC1-like genes (Gh-SOC1–Gh-SOC3) were identified among gerbera expressed sequence tags.

Methods

Evolutionary relationships between SOC1-like genes from gerbera and other plants were studied by phylogenetic analysis. The function of the gerbera gene Gh-SOC1 in gerbera floral development was studied using expression analysis, protein–protein interaction assays and reverse genetics. Transgenic gerbera lines over-expressing or downregulated for Gh-SOC1 were obtained using Agrobacterium transformation and investigated for their floral phenotype.

Key Results

Phylogenetic analysis revealed that the closest paralogues of At-SOC1 are Gh-SOC2 and Gh-SOC3. Gh-SOC1 is a more distantly related paralogue, grouping together with a number of other At-SOC1 paralogues from arabidopsis and other plant species. Gh-SOC1 is inflorescence abundant and no expression was seen in vegetative parts of the plant. Ectopic expression of Gh-SOC1 did not promote flowering, but disturbed the development of floral organs. The epidermal cells of ray flower petals appeared shorter and their shape was altered. The colour of ray flower petals differed from that of the wild-type petals by being darker red on the adaxial side and greenish on the abaxial surface. Several protein–protein interactions with other gerbera MADS domain proteins were identified.

Conclusions

The At-SOC1 paralogue in gerbera shows a floral abundant expression pattern. A late petal expression might indicate a role in the final stages of flower development. Over-expression of Gh-SOC1 led to partial loss of floral identity, but did not affect flowering time. Lines where Gh-SOC1 was downregulated did not show a phenotype. Several gerbera MADS domain proteins interacted with Gh-SOC1.     

梅花"粉皮宫粉"花色色素的花青苷实质和花色的动态变化

特征颜色反应和紫外-可见光谱分析初步表明梅花"粉皮宫粉"的粉红色花色色素为花青素-3-糖苷.用分光光度法检测梅花"粉皮宫粉"不同花发育时期、在树冠不同着生部位花朵花瓣的相对花青苷含量,结果表明"粉皮宫粉" 的花色主要存在着花发育时期而导致的时间变化.花色在蕾期最浓艳,花瓣展开后便逐渐变淡;在整个花发育时期,同一朵花不同层次花瓣的颜色浓淡均为外层花瓣>中层花瓣>内层花瓣,且不同层次花瓣颜色的变化趋势几乎一致.虽然树冠下部单花的花色浓于上部的、树冠内层的浓于外层的,但花朵在树冠的着生部位导致的花色差异并不显著.花青苷除了导致"粉皮宫粉"的粉红花色外,还可能增强其花的抗寒性,为花的凌寒而开创造了条件.本文可为梅花的美学鉴赏、梅花红色花色的机理探索及其色素的分子结构鉴定提供参考.     

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.

     

单瓣与重瓣垂丝海棠花器官形态发育比较观察

为探究垂丝海棠重瓣花成花原因,该研究以单瓣垂丝海棠和重瓣垂丝海棠为实验材料,应用体式显微镜和扫描电镜观察垂丝海棠单瓣、重瓣品种花器官分化过程;解剖观察重瓣垂丝海棠大蕾期的花与盛开的花,统计其花器官的形态与数目;应用R语言对重瓣垂丝海棠的花瓣数目与其余各轮花器官数目进行相关性分析。结果显示：(1)单瓣和重瓣垂丝海棠的花器官分化均分为萼片原基分化期、花瓣原基分化期、雄蕊原基分化期、雌蕊原基分化期,且各轮花器官按照向心顺序依次分化发育。(2)在花瓣原基分化期,单瓣垂丝海棠仅分化出一轮(5枚)均匀分布于两枚萼片交汇处的花瓣原基,而重瓣垂丝海棠分化出两轮分布散列的花瓣原基,第一轮为5~7枚,第二轮为7~10枚。(3)在重瓣垂丝海棠各轮花器官中存在较多萼片瓣化、雄蕊瓣化、雌雄蕊异常发育的情况。(4)重瓣垂丝海棠各轮花器官数目间相关性分析结果显示,其花瓣数目与雄蕊数目以及瓣化中的雄蕊数目间存在明显的正相关关系,该现象与常规雄蕊瓣化植物表现的雄蕊数目减少、花瓣数目增多的现象不同。研究表明,重瓣垂丝海棠花瓣数目的增多并不完全依赖于雄蕊变瓣,暗示垂丝海棠重瓣花成花原因的多元性与复杂性。     

丽格海棠花青素苷成分及分布对花色的影响

以红色、红心白边、粉红、玫红、黄色、黄心红边、浅粉和白色8种花色丽的格海棠花瓣为试验材料,采用目视测色法、RHSCC比色法和色差仪测定花瓣表型,通过组织切片法观察花瓣色素细胞的显微结构和分布特点,采用双光束紫外-可见光分光光度计和高效液相色谱-电喷雾离子化-质谱连用技术(HPLC-ESI-MS)测定分析花瓣中花青素苷的成分和含量,为探讨丽格海棠花色的呈色机理和花色育种提供参考。结果显示:(1)丽格海棠的明度L*随花瓣颜色变深而降低,红度a*则表现出相反趋势,红度(a*)和彩度(C*)值与明度(L*)呈显著负相关关系,且a*和C*是影响L*的主要因素。(2)红花品种花瓣色素主要分布于上表皮细胞和海绵组织中;红白花品种花瓣色素主要分布于上下表皮中,且下表皮积累量更多;粉色花和玫红花品种花瓣色素主要分布于上下表皮细胞;黄红花和粉白色花品种花瓣上表皮中含有少量色素,而黄花和白花品种花瓣几乎没有色素积累。各花色丽格海棠花瓣上表皮细胞均为圆锥形,且红花和红白花品种锥形化程度最高,它们花瓣下表皮细胞均呈扁平的长方形。(3)8个丽格海棠品种花瓣中共检测出15种花青素苷,其中10种为芍药素苷,3种为矢车菊素苷,1种为锦葵素苷,1种为飞燕草素苷,酰化花青素苷占多数;红花品种花瓣中总花青素苷含量最高,玫红花品种次之,黄花和白花品种中未检出;除粉红花品种外,其余含花青素苷的品种中芍药素苷含量最高,均占总花青素苷含量的50%以上,是花瓣的主要呈色物质。(4)丽格海棠花瓣中总花青素苷含量与其红度(a*)、彩度(C*)值呈正相关关系、与其L*值呈负相关关系。研究表明,花青素苷的积累有利于丽格海棠花瓣红色化,并影响其花瓣彩度(C*)及明度(L*);色素分布细胞数量和上表皮细胞锥形化明显影响花瓣呈色,且花瓣主要的呈色物质为芍药素苷,酰基化修饰可能影响其明度。     

Differential gene expression of the honey bees Apis mellifera and A. cerana induced by Varroa destructor infection

Varroa destructor mite is currently the most serious threat to the world bee industry. Differences in mite tolerance are reported between two honey bee species Apis mellifera and Apis cerana. Differential gene expression of two honey bee species induced by V. destructor infection was investigated by constructing two suppression subtractive hybridization (SSH) libraries, as first steps toward elucidating molecular mechanisms of Varroa tolerance. From the SSH libraries, we obtained 289 high quality sequences which clustered into 132 unique sequences grouped in 26 contigs and 106 singlets where 49 consisted in A. cerana subtracted library and 83 in A. mellifera. Using BLAST, we found that 85% sequences had counterpart known genes whereas 15% were undescribed. A Gene Ontology analysis classified 51 unique sequences into different functional categories. Eight of these differentially expressed genes, representative of different regulation patterns, were confirmed by qRT-PCR. Upon the mite induction, the differentially expressed genes from both bee species were different, except hex 110 gene, which was up-regulated in A. cerana but down-regulated in A. mellifera, and Npy-r gene, which was down-regulated in both species. In general, most of the differential expression genes were involved in metabolic processes and nerve signaling. The results provide information on the molecular response of these two bee species to Varroa infection.     

Flower color change accelerated by bee pollination in Tibouchina (Melastomataceae)

Floral color changes are common among Melastomataceae and have been interpreted as a warning mechanism for bees to avoid old flowers, albeit increasing long-distance flower display. Here the reproductive systems of Tibouchina pulchra and T. sellowiana were investigated by controlled pollinations. Their pollinators were identified, and experiments on floral color and fragrance changes were conduced to verify if those changes affect the floral visitation. Both Tibouchina species are self compatible. The flowers lasted three days or more, and the floral color changed from white in the 1st day to pink in the following days. Pollen deposition on stigma induced floral color change. The effectiveness of the pollination is dependent on bees’ size; only large bees were regarded as effective pollinators. In experimental tests, the bees in T. pulchra preferred the natural white flowers while the visitors of T. sellowiana were attracted by both natural and mimetic 1st-day flowers (2nd-day flowers with experimentally attached 1st-day flower petals). During the experiments on floral fragrance, the bees visited both natural and mimetic 1st-day flowers (2nd-day flowers with 1st-day flower scents). In both experiments, the bees avoided natural 2nd-day flowers, but seldom visited modified 2nd-day flowers. The attractiveness of T. pulchra and T. sellowiana flowers cannot be attributed exclusively to the color or the fragrance separately, both factors seemingly act together.     

Ineffective crypsis in a crab spider: a prey community perspective

Cryptic coloration is assumed to be beneficial to predators because of an increased encounter rate with unwary prey. This hypothesis is, however, very rarely, if ever, studied in the field. The aim of this study was to quantify the encounter rate and capture success of an ambush predator, in the field, as a function of its level of colour-matching with the background. We used the crab spider Misumena vatia, which varies its body colour and can thereby match the colour of the flower it hunts upon. We carried out a manipulative field experiment using a complete factorial design resulting in six different colour combinations of crab spiders and flowers differing in their degree of colour-matching. A rich and diverse set of naturally occurring insects visited the flowers while we continuously video-recorded the spider''s foraging activity. This enabled us to test the crypsis, the spider avoidance and the flower visitor attraction hypotheses, all three supported by previous studies. Flower visitors of different groups either avoided crab spiders independent of colour-matching, such as solitary bees and syrphid flies, or ignored them, such as bumble-bees and honeybees. Moreover, colour-matched spiders did not have a higher encounter rate and capture success compared to the visually apparent ones. Thus, our results support the spider avoidance hypothesis, reject the two other hypotheses and uncovered a fourth behaviour: indifference to predators. Because flower visitors reacted differently, a community approach is mandatory in order to understand the function of background colour-matching in generalist predators. We discuss our results in relation to the size and sociality of the prey and in relation to the functional significance of colour change in this predator.