春兰AGL6-3基因的克隆及实时定量表达分析

该研究以春兰(Cymbidium goeringii)正常花及其2枚侧瓣突变成唇瓣样的花瓣(简称:蝶花)为实验材料,采用RT-PCR结合RACE技术从春兰中分离出AGL6-3基因。序列分析表明,AGL6-3基因在春兰正常花和蝶花中序列相同,该基因含有1个720bp长的开放阅读框(ORF),共编码239个氨基酸。系统进化树进行分析表明,该基因属于MADS-box基因中AP1/AGL9组的AGL6同源基因,命名为CgAGL6-3(基因登录号为KU058679)。实时荧光定量表达分析表明,CgAGL6-3在春兰正常花和蝶花各花器官中表达存在差异。在正常春兰中CgAGL6-3基因在唇瓣中强烈表达,在主萼、侧萼及蕊柱中表达量较低,在侧瓣中则微乎其微;而在蝶花中CgAGL6-3基因在唇瓣中强表达,侧瓣中的表达量次之,在主萼、侧萼和蕊柱中的表达量相近且均较低。研究说明,CgAGL6-3基因可能在春兰蝶花侧瓣唇瓣化的过程中扮演重要角色。     

The role of ABC genes in shaping perianth phenotype in the basal angiosperm Magnolia

It is generally accepted that the genus Magnolia is characterised by an undifferentiated perianth, typically organised into three whorls of nearly identical tepals. In some species, however, we encountered interesting and significant perianth modifications. In Magnolia acuminata, M. liliiflora and M. stellata the perianth elements of the first whorl are visually different from the others. In M. stellata the additional, spirally arranged perianth elements are present above the first three whorls, which suggests that they have been formed within the domain of stamen primordia. In these three species, we analysed expression patterns of the key flower genes (AP1, AGL6, AP3, PI, AG) responsible for the identity of flower elements and correlated them with results of morphological and anatomical investigations. In all studied species the elements of the first whorl lacked the identity of petals (lack of AP3 and PI expression) but also that of leaves (presence of AGL6 expression), and this seems to prove their sepal character. The analysis of additional perianth elements of M. stellata, spirally arranged on the elongated floral axis, revealed overlapping and reduced activity of genes involved in specification of the identity of the perianth (AGL6) but also of generative parts (AG), even though no clear gradient of morphological changes could be observed. In conclusion, Magnolia genus is capable of forming, in some species, a perianth differentiated into a calyx (sepals) and corolla (petals). Spirally arranged, additional perianth elements of M. stellata, despite activity of AG falling basipetally, resemble petals.     

Structure and development of ‘witches' broom’ galls in reproductive organs of Byrsonima sericea (Malpighiaceae) and their effects on host plants

Galls are anomalies in plant development of parasitic origin that affect the cellular differentiation or growth and represent a remarkable plant–parasite interaction. Byrsonima sericea DC. (Malpighiaceae) is a super host of several different types of gall in both vegetative and reproductive organs. The existence of galls in reproductive organs and their effects on the host plant are seldom described in the literature. In this paper, we present a novel study of galls in plants of the Neotropical region: the ‘witches' broom’ galls developed in floral structures of B. sericea. The unaffected inflorescences are characterised by a single indeterminate main axis with spirally arranged flower buds. The flower buds developed five unaffected brownish hairy sepals and five pairs of elliptical yellow elaiophores, five yellow fringed petals, 10 stamens and a pistil with superior tricarpellar and trilocular ovary. The affected inflorescences showed changes in architecture, with branches arising from the main axis and flower buds. The flower buds exhibited several morphological and anatomical changes. The sepals, petals and carpels converted into leaf‐like structures after differentiation. Stamens exhibited degeneration of the sporogenous tissue and structures containing hyphae and spores. The gynoecium did not develop, forming a central meristematic region, from which emerges the new inflorescence. In this work, we discuss the several changes in development of reproductive structures caused by witches' broom galls and their effects on reproductive success of the host plants.     

Bulbophyllum huangshanense sp. nov. (Orchidaceae) from Anhui,China

Bulbophyllum huangshanense, a new species of orchid from Anhui, China, is described and illustrated. This new species is close to B. taeniophyllum with pseudo‐bulbs closely spaced along the rhizome, leathery leaf, entire dorsal sepal, toothed petals, the lateral edges of lateral sepals connate and fleshy lip, but differs from the latter in having roots arising from the node with the pseudo‐bulb, inflorescence much shorter than leaf, flowers pure yellow, lip mobile, and stelidia broadly triangular.     

Organ boundary NAC‐domain transcription factors are implicated in the evolution of petal fusion

• Research rationale: Evolution of fused petals (sympetaly) is considered to be an important innovation that has repeatedly led to increased pollination efficiency, resulting in accelerated rates of plant diversification. Although little is known about the underlying regulation of sympetaly, genetic pathways ancestrally involved in organ boundary establishment (e.g. CUP SHAPED COTYLEDON [CUC] 1–3 genes) are strong candidates. In sympetalous petunia, mutations in the CUC1/2‐like orthologue NO APICAL MERISTEM (NAM) inhibit shoot apical meristem formation. Despite this, occasional ‘escape shoots’ develop flowers with extra petals and fused inter‐floral whorl organs.
• Central methods: To To determine if petunia CUC‐like genes regulate additional floral patterning, we used virus‐induced silencing (VIGS) following establishment of healthy shoot apices to re‐examine the role of NAM in petunia petal development, and uniquely characterise the CUC3 orthologue NH16.
• Key results: Confirming previous results, we found that reduced floral NAM/NH16 expression caused increased petal–stamen and stamen–carpel fusion, and often produced extra petals. However, further to previous results, all VIGS plants infected with NAM or NH16 constructs exhibited reduced fusion in the petal whorl compared to control plants.
• Main conclusions: Together with previous data, our results demonstrate conservation of petunia CUC‐like genes in establishing inter‐floral whorl organ boundaries, as well as functional evolution to affect the fusion of petunia petals.

     

Notes on the genus Quekettia (Orchidaceae) with descriptions of two new species from Colombia and Guyana

Two new species of the orchid genus Quekettia are described. Colombian Quekettia aureliae is distinguished from similar Q. microscopica by the pandurate lip with a pair of small, flap‐like calli in the isthmus between hypo‐ and epichile, and deeply emarginated epichile apex. Quekettia senghasiana found in Guyana resembles Q. papillosa but it is characterized by the free lateral sepals, oblanceolate, obtuse petals and much larger lip which is oblong–obovate in outline and with lip disc being papillate only near the base.     

A COMPARISON OF FLORAL DEVELOPMENT IN WILD TYPE AND A HOMEOTIC SEPALOID PETAL MUTANT OF CLARKIA TEMBLORIENSIS (ONAGRACEAE)

The mature wild type petals of Clarkia tembloriensis consist of a long slender claw and an expanded deltoid-shaped limb. They are pink, with a maroon spot at the base of the limb. Their surface texture is smooth. A variant of petal form, crinkled petal, occurs commonly in several natural populations of C. tembloriensis. The mature crinkled petals are elongated, greenish pink, and possess trichomes. They resemble the mature sepals of C. tembloriensis in general shape, color, and surface texture. Organ initiation and subsequent patterns of development of wild type petals, wild type sepals, and crinkled petals were examined and compared using scanning electron microscopy and allometric growth analysis. Crinkled petals are similar to wild type petals in time and position of primordia initiation, and in size and shape at inception. Crinkled petals are similar to wild type sepals in pattern of allometric growth. The crinkled petal mutant fits the broad definition of a homeotic mutant in that the petal has assumed characteristics of the sepal.     

AGAMOUS‐LIKE13, a putative ancestor for the E functional genes,specifies male and female gametophyte morphogenesis

Arabidopsis AGL13 is a member of the AGL6 clade of the MADS box gene family. GUS activity was specifically detected from the initiation to maturation of both pollen and ovules in AGL13:GUS Arabidopsis. The sterility of the flower with defective pollen and ovules was found in AGL13 RNAi knockdown and AGL13 + SRDX dominant‐negative mutants. These results indicate that AGL13 acts as an activator in regulation of early initiation and further development of pollen and ovules. The production of similar floral organ defects in the severe AGL13 + SRDX and SEP2 + SRDX plants and the similar enhancement of AG nuclear localization efficiency by AGL13 and SEP3 proteins suggest a similar function for AGL13 and E functional SEP proteins. Additional fluorescence resonance energy transfer (FRET) analysis indicated that, similar to SEP proteins, AGL13 is able to interact with AG to form quartet‐like complexes (AGL13–AG)₂ and interact with AG–AP3–PI to form a higher‐order heterotetrameric complex (AGL13–AG–AP3–PI). Through these complexes, AGL13 and AG could regulate the expression of similar downstream genes involved in pollen morphogenesis, anther cell layer formation and the ovule development. AGL13 also regulates AG/AP3/PI expression by positive regulatory feedback loops and suppresses its own expression through negative regulatory feedback loops by activating AGL6, which acts as a repressor of AGL13. Our data suggest that AGL13 is likely a putative ancestor for the E functional genes which specifies male and female gametophyte morphogenesis in plants during evolution.     

Wheat's wild relatives vary in their response to nitrogen and ozone

The wild relatives of bread wheat ( Triticum aestivum ) are valued by plant breeders for their genetic diversity. However, increasing levels of nitrogen (N) deposition and ground‐level ozone (O₃) threaten plant biodiversity in the Mediterranean and Near‐East, a hotspot for many crop wild relatives. Knowledge of the effect of these air pollutants in combination is still limited, but early indications are that effects vary depending on the level of pollutants, and on the sensitivity of the species to N and O₃. This study examined the responses of four important wheat wild relatives ( Aegilops tauschii , Aegilops speltoides , Triticum dicoccoides and Triticum monococcum ) and one modern wheat cultivar ( T. aestivum ‘Cadenza’) to treatments of N (equivalent to 50 kg ha^?1 year^?1 ammonium nitrate) and O₃ (100 ppb for 21 days), alone and in combination. Measurements included root, shoot and seed biomass, and electrolyte ratios. The O₃ sensitivity of A. tauschii and T. aestivum ‘Cadenza’ were exacerbated by the addition of N, while A. speltoides was found to be nitrophilous, with N ameliorating the negative effect of O_3. Both T. aestivum ‘Cadenza’ and T. dicoccoides produced immature seed heads, with the cultivar's seed head biomass reduced in response to O₃ and N + O₃ while that of T. dicoccoides was largely unaffected. These data suggest that all four wild relatives are likely to be affected when N and O₃ air pollutants co‐occur, and there in situ populations may therefore be at risk. Equally, the results of this study can inform use of their beneficial traits by wheat breeders, and alert them to the inadvertent inclusion of N and O₃ sensitivity.     

A flavonoid 3‐O‐glucoside:2″‐O‐glucosyltransferase responsible for terminal modification of pollen‐specific flavonols in Arabidopsis thaliana

Flavonol 3‐O‐diglucosides with a 1→2 inter‐glycosidic linkage are representative pollen‐specific flavonols that are widely distributed in plants, but their biosynthetic genes and physiological roles are not well understood. Flavonoid analysis of four Arabidopsis floral organs (pistils, stamens, petals and calyxes) and flowers of wild‐type and male sterility 1 (ms1) mutants, which are defective in normal development of pollen and tapetum, showed that kaempferol/quercetin 3‐O‐β‐d ‐glucopyranosyl‐(1→2)‐β‐d ‐glucopyranosides accumulated in Arabidopsis pollen. Microarray data using wild‐type and ms1 mutants, gene expression patterns in various organs, and phylogenetic analysis of UDP‐glycosyltransferases (UGTs) suggest that UGT79B6 (At5g54010) is a key modification enzyme for determining pollen‐specific flavonol structure. Kaempferol and quercetin 3‐O‐glucosyl‐(1→2)‐glucosides were absent from two independent ugt79b6 knockout mutants. Transgenic ugt79b6 mutant lines transformed with the genomic UGT79B6 gene had the same flavonoid profile as wild‐type plants. Recombinant UGT79B6 protein converted kaempferol 3‐O‐glucoside to kaempferol 3‐O‐glucosyl‐(1→2)‐glucoside. UGT79B6 recognized 3‐O‐glucosylated/galactosylated anthocyanins/flavonols but not 3,5‐ or 3,7‐diglycosylated flavonoids, and prefers UDP‐glucose, indicating that UGT79B6 encodes flavonoid 3‐O‐glucoside:2″‐O‐glucosyltransferase. A UGT79B6‐GUS fusion showed that UGT79B6 was localized in tapetum cells and microspores of developing anthers.     

Molecular characterization of cisgenic lines of apple ‘Gala’ carrying the Rvi6 scab resistance gene

Using resistance genes from a crossable donor to obtain cultivars resistant to diseases and the use of such cultivars in production appears an economically and environmentally advantageous approach. In apple, introgression of resistance genes by classical breeding results in new cultivars, while introducing cisgenes by biotechnological methods maintains the original cultivar characteristics. Recently, plants of the popular apple ‘Gala’ were genetically modified by inserting the apple scab resistance gene Rvi6 (formerly HcrVf2) under control of its own regulatory sequences. This gene is derived from the scab‐resistant apple ‘Florina’ (originally from the wild apple accession Malus floribunda 821). The vector used for genetic modification allowed a postselection marker gene elimination to achieve cisgenesis. In this work, three cisgenic lines were analysed to assess copy number, integration site, expression level and resistance to apple scab. For two of these lines, a single insertion was observed and, despite a very low expression of 0.07‐ and 0.002‐fold compared with the natural expression of ‘Florina’, this was sufficient to induce plant reaction and reduce fungal growth by 80% compared with the scab‐susceptible ‘Gala’. Similar results for resistance and expression analysis were obtained also for the third line, although it was impossible to determine the copy number and TDNA integration site–such molecular characterization is requested by the (EC) Regulation No. 1829/2003, but may become unnecessary if cisgenic crops become exempt from GMO regulation.     

春兰CgSEP3基因的克隆和表达分析

该研究采用RT-PCR和RACE技术从春兰(Cymbidium goeringii)中分离到1个SEPALLATA3(SEP3)基因。序列分析表明,该基因含有1个732bp的开放阅读框(ORF),共编码243个氨基酸。系统进化树分析显示,该基因是MADS-box基因家族AP1/AGL9组SEP的同源基因,其编码蛋白与其它植物SEP3类蛋白具有较高的一致性,命名为CgSEP3(登录号为KF924272)。实时荧光定量分析表明,CgSEP3在春兰花器官中均有表达,其中在唇瓣、侧瓣和萼片中的表达量较高,在子房和蕊柱中的表达量较低;而且CgSEP3在花发育各个时期都有表达,在1~2cm的花芽中表达量最高,在盛开的花中的表达量最低。研究认为,CgSEP3基因可能在春兰花瓣和萼片的形成过程中具有重要作用。     

Optimal pollinator attraction strategies in Trollius ranunculoides Hemsl. (Ranunculaceae) at different altitudes: increased floral display or promotion of nectar output?

For alpine plant species, patterns of resource allocation to functional floral traits for pollinator attraction can be highly significant in adaptation to low pollinator abundance and consequent pollen limitation. Increased pollination can be achieved either through a larger floral display or production of more pollen rewards. In this study, variation in resource allocation to different components for pollinator attraction was studied along an altitudinal gradient in Trollius ranunculoides, an obligate self‐incompatible out‐crosser of the Qinghai–Tibet Plateau. We compared resource allocation to conspicuous yellow sepals (which mainly provide visual attraction) and degenerate petals (which provide the major nectar reward) between populations at four altitudes. Furthermore, we investigated the contribution of sepals and petals to pollinator attraction and female reproductive success in an experiment with sepal or petal removal at sites at different altitudes. At the level of single flowers, resource allocation increased to sepals but decreased to petals with increasing altitude. Consistent with these results, sepals contributed much more to visitation rate and seed set than petals, as confirmed in the sepal or petal removal experiment. Sepals and petals contributed to female reproductive success by ensuring visitation rate rather than visitation duration. To alleviate increasing pollen limitation with increasing altitude, resource allocation patterns of T. ranunculoides altered to favour development of sepals rather than petals. This strategy may improve pollination and reproductive success through visual attraction (sepal) rather than nectar reward (petal) over a gradient of decreasing pollinator abundance.     

A comparison of venation pattern development in wild type and a homeotic sepaloid petal mutant of Clarkia tembloriensis (Onagraceae)

Vascular system development in sepals, petals, and sepaloid petals was compared in wild-type and crinkled petal mutant plants of Clarkia tembloriensis. Patterns of vascularization in cleared whole mounts were visualized and traced under both brightfield and polarizing illumination. Wild-type sepals exhibited a basipetal pattern of maturation, with tracheary elements maturing relatively rapidly. Mature sepals had three primary veins with numerous secondary veins. In contrast, wild-type petals exhibited an acropetal pattern of maturation, with tracheary elements maturing relatively slowly. The mature petals had only one primary vein with numerous secondary veins. Sepaloid (crinkled) petals combined characteristics of both wild-type sepals and wild-type petals. They exhibited a basipetal pattern of development and a relatively rapid maturation of the tracheary elements characteristic of wild-type sepals. Venation architecture in crinkled petal mutants showed a single primary vein with numerous secondary veins, similar to wild-type petals. The crinkled petal mutant fits the definition of a homeotic mutant in that the petal has assumed characteristics of the sepal. However, homeotic transformation from petal to sepal is incomplete since the crinkled petal still retains many of the characteristics of wild-type petals.     

Novel evidence suggests that a ‘Rickettsia felis‐like’ organism is an endosymbiont of the desert flea,Xenopsylla ramesis

Fleas are acknowledged vectors and reservoirs of various bacteria that present a wide range of pathogenicity. In this study, fleas collected from wild rodents from the Negev desert in southern Israel were tested for RickettsiaDNA by targeting the 16S rRNA (rrs) gene. Thirty‐eight Xenopsylla ramesis, 91 Synosternus cleopatrae and 15 Leptopsylla flea pools (a total of 568 fleas) were screened. RickettsiaDNA was detected in 100% of the X. ramesis and in one S. cleopatrae flea pools. None of L. algira flea pools was found positive. All positive flea pools were further characterized by sequencing of five additional genetic loci (gltA, ompB, ompA, htrA and fusA). The molecular identification of the positive samples showed all sequences to be closely related to the ‘Rickettsia felis‐like’ organisms (99–100% similarities in the six loci). To further investigate the association between ‘R. felis‐like’ and X. ramesis fleas, ten additional single X. ramesis adult fleas collected from the wild and five laboratory‐maintained X. ramesis imago, five larva pools (2–18 larvae per pool) and two egg pools (18 eggs per pool) were tested for the presence of ‘R. felis‐like’ DNA. All samples were found positive by a specific ompAPCR assay, confirming the close association of this Rickettsia species with X. ramesis in all its life stages. These results suggest a symbiotic association between ‘Rickettsia felis‐like’ and X. ramesis fleas.     

An APETALA3 homolog controls both petal identity and floral meristem patterning in Nigella damascena L. (Ranunculaceae)

Flower architecture mutants provide a unique opportunity to address the genetic origin of flower diversity. Here we study a naturally occurring floral dimorphism in Nigella damascena (Ranunculaceae), involving replacement of the petals by numerous sepal‐like and chimeric sepal/stamen organs. We performed a comparative study of floral morphology and floral development, and characterized the expression of APETALA3 and PISTILLATA homologs in both morphs. Segregation analyses and gene silencing were used to determine the involvement of an APETALA3 paralog (NdAP3–3) in the floral dimorphism. We demonstrate that the complex floral dimorphism is controlled by a single locus, which perfectly co‐segregates with the NdAP3–3 gene. This gene is not expressed in the apetalous morph and exhibits a particular expression dynamic during early floral development in the petalous morph. NdAP3–3 silencing in petalous plants perfectly phenocopies the apetalous morph. Our results show that NdAP3–3 is fully responsible for the complex N. damascena floral dimorphism, suggesting that it plays a role not only in petal identity but also in meristem patterning, possibly through regulation of perianth organ number and the perianth/stamen boundary.     

Grapevine VpPR10.1 functions in resistance to Plasmopara viticola through triggering a cell death‐like defence response by interacting with VpVDAC3

As one of the most serious diseases in grape, downy mildew caused by Plasmopara viticola is a worldwide grape disease. Much effort has been focused on improving susceptible grapevine resistance, and wild resistant grapevine species are important for germplasm improvement of commercial cultivars. Using yeast two‐hybrid screen followed by a series of immunoprecipitation experiments, we identified voltage‐dependent anion channel 3 (VDAC3) protein from Vitis piasezkii ‘Liuba‐8’ as an interacting partner of VpPR10.1 cloned from Vitis pseudoreticulata ‘Baihe‐35‐1’, which is an important germplasm for its resistance to a range of pathogens. Co‐expression of VpPR10.1/VpVDAC3 induced cell death in Nicotiana benthamiana, which accompanied by ROS accumulation. VpPR10.1 transgenic grapevine line showed resistance to P. viticola. We conclude that the VpPR10.1/VpVDAC3 complex is responsible for cell death‐mediated defence response to P. viticola in grapevine.