Quantitative live imaging of floral organ initiation and floral meristem termination in Aquilegia.

In-depth investigation of any developmental process in plants requires knowledge of both the underpinning molecular networks and how they directly determine patterns of cell division and expansion over time. Floral meristems (FMs) produce floral organs, after which they undergo floral meristem termination (FMT); precise control of organ initiation and FMT is crucial to the reproductive success of any flowering plant. Using live confocal imaging, we characterized developmental dynamics during floral organ primordia initiation and FMT in Aquilegia coerulea (Ranunculaceae). Our results uncover distinct patterns of primordium initiation between stamens and staminodes compared with carpels, and provide insight into the process of FMT, which is discernable based on cell division dynamics that precede carpel initiation. To our knowledge, this is the first quantitative live imaging of meristem development in a system with numerous whorls of floral organs, as well as an apocarpous gynoecium. This study provides crucial information for our understanding of how the spatial-temporal regulation of floral meristem behavior is achieved in both evolutionary and developmental contexts. This article has an associated 'The people behind the papers' interview.

Transcriptome profiling and weighted gene co-expression network analysis of early floral development in Aquilegia coerulea.

The earliest phases of floral development include a number of crucial processes that lay the foundation for the subsequent morphogenesis of floral organs and success in reproduction. Currently, key transcriptional changes during this developmental window have been characterized in the model species Arabidopsis thaliana, but little is known about how transcriptional dynamics change over the course of these developmental processes in other plant systems. Here, we have conducted the first in-depth transcriptome profiling of early floral development in Aquilegia at four finely dissected developmental stages, with eight biological replicates per stage. Using differential gene expression analysis and weighted gene co-expression network analysis, we identified both crucial genes whose expression changes mark the transitions between developmental stages and hub genes in co-expression modules. Our results support the potential functional conservation of key genes in early floral development that have been identified in other systems, but also reveal a number of previously unknown or overlooked loci that are worthy of further investigation. In addition, our results highlight not only the dynamics of transcriptional regulation during early floral development, but also the potential involvement of the complex, essential networks of small RNA and post-translational regulation to these developmental stages.

Developmental and Molecular Changes Underlying the Vernalization-Induced Transition to Flowering in Aquilegia coerulea (James).

Reproductive success in plants is dependent on many factors but the precise timing of flowering is certainly among the most crucial. Perennial plants often have a vernalization or over-wintering requirement in order to successfully flower in the spring. The shoot apical meristem undergoes drastic developmental and molecular changes as it transitions into inflorescence meristem (IM) identity, which then gives rise to floral meristems (FMs). In this study, we have examined the developmental and gene expression changes underlying the transition from the vegetative to reproductive phases in the basal eudicot Aquilegia coerulea, which has evolved a vernalization response independently relative to other established model systems. Results from both our histology and scanning electron studies demonstrate that developmental changes in the meristem occur gradually during the third and fourth weeks of vernalization. Based on RNAseq data and cluster analysis, several known flowering time loci, including AqFT and AqFL1, exhibit dramatic changes in expression during the fourth week. Further consideration of candidate gene homologs as well as unexpected loci of interest creates a framework in which we can begin to explore the genetic basis of the flowering time transition in Aquilegia.

Comparative transcriptomics of early petal development across four diverse species of Aquilegia reveal few genes consistently associated with nectar spur development.

BACKGROUND: Petal nectar spurs, which facilitate pollination through animal attraction and pollen placement, represent a key innovation promoting diversification in the genus Aquilegia (Ranunculaceae). Identifying the genetic components that contribute to the development of these three-dimensional structures will inform our understanding of the number and types of genetic changes that are involved in the evolution of novel traits. In a prior study, gene expression between two regions of developing petals, the laminar blade and the spur cup, was compared at two developmental stages in the horticultural variety A. coerulea 'Origami'. Several hundred genes were differentially expressed (DE) between the blade and spur at both developmental stages. In order to narrow in on a set of genes crucial to early spur formation, the current study uses RNA sequencing (RNAseq) to conduct comparative expression analyses of petals from five developmental stages between four Aquilegia species, three with morphologically variable nectar spurs, A. sibirica, A. formosa, and A. chrysantha, and one that lacks nectar spurs, A. ecalcarata. RESULTS: Petal morphology differed increasingly between taxa across the developmental stages assessed, with petals from all four taxa being indistinguishable pre-spur formation at developmental stage 1 (DS1) and highly differentiated by developmental stage 5 (DS5). In all four taxa, genes involved in mitosis were down-regulated over the course of the assessed developmental stages, however, many genes involved in mitotic processes remained expressed at higher levels later in development in the spurred taxa. A total of 690 genes were identified that were consistently DE between the spurred taxa and A. ecalcarata at all five developmental stages. By comparing these genes with those identified as DE between spur and blade tissue in A. coerulea 'Origami', a set of only 35 genes was identified that shows consistent DE between petal samples containing spur tissue versus those without spur tissue. CONCLUSIONS: The results of this study suggest that expression differences in very few loci are associated with the presence and absence of spurs. In general, it appears that the spurless petals of A. ecalcarata cease cell divisions and enter the cell differentiation phase at an earlier developmental time point than those that produce spurs. This much more tractable list of 35 candidates genes will greatly facilitate targeted functional studies to assess the genetic control and evolution of petal spurs in Aquilegia.

Homologs of the STYLISH gene family control nectary development in Aquilegia.

Floral nectaries are an interesting example of a convergent trait in flowering plants, and are associated with the diversification of numerous angiosperm lineages, including the adaptive radiation of the New World Aquilegia species. However, we know very little as to what genes contribute to nectary development and evolution, particularly in noncore eudicot taxa. We analyzed expression patterns and used RNAi-based methods to investigate the functions of homologs from the STYLISH (STY) family in nectar spur development in Aquilegia coerulea. We found that AqSTY1 exhibits concentrated expression in the presumptive nectary of the growing spur tip, and triple gene silencing of the three STY-like genes revealed that they function in style and nectary development. Strong expression of STY homologs was also detected in the nectary-bearing petals of Delphinium and Epimedium. Our results suggest that the novel recruitment of STY homologs to control nectary development is likely to have occurred before the diversification of the Ranunculaceae and Berberidaceae. To date, the STY homologs of the Ranunculales are the only alternative loci for the control of nectary development in flowering plants, providing a critical data point in understanding the evolutionary origin and developmental basis of nectaries.

Leifsonia flava sp. nov., a novel actinobacterium isolated from the rhizosphere of Aquilegia viridiflora.

SYP-B2174T is a yellow-pigmented, Gram-positive, non-motile, and rod-shaped actinobacterium isolated from the rhizospheric soil of Aquilegia viridiflora Pall. collected from the Xinjiang uygur autonomous region of China. The strain's growth temperature ranges from 1 to 35°C, with an optimal growth being observed at 28°C. Growth occurs from 0 to 5% NaCl and at pH 6-8, with optimal growth being observed in 1% NaCl at pH 7. Comparative 16S rRNA gene sequence-based phylogenetic analysis placed the strain in a clade with the species Leifsonia kafniensis JCM 17021T and Leifsonia psychrotolerans DSM 22824T with similarities of 97.8 and 97.6%, respectively. The DNA-DNA hybridization values of the strain SYP-B2174T to its closest phylogenetic neighbors were significantly lower than 35.7%. The strain was identified as a novel species of the genus Leifsonia judging by the coryneform morphology, peptidoglycans based upon 2,4-diaminobutyric acid, principal phospholipids phosphatidylglycerol and diphosphatidylglycerol, major menaquinone MK-11, predominant fatty acids of anteiso-C15:0, anteiso-C17:0, and iso-C16:0, and a DNA G + C base composition of 68.7 mol%, for which the name Leifsonia flava sp. nov. is proposed. The type strain is SYP-B2174T (= CGMCC 1.15856T = DSM 105144T = KCTC 39963T).

Dissecting seed dormancy and germination in Aquilegia barbaricina, through thermal kinetics of embryo growth.

Threshold-based thermal time models provide insight into the physiological switch from the dormant to the non-dormant germinating seed. This approach was used to quantify the different growth responses of the embryo of seeds purported to have morphophysiological dormancy (MPD) through the complex phases of dormancy release and germination. Aquilegia barbaricina seeds were incubated at constant temperatures (10-25 °C) and 25/10 °C, without pre-treatment, after warm+cold stratification (W+C) and GA3 treatment. Embryo growth was assessed and the time of testa and endosperm rupture scored. Base temperatures (Tb ) and thermal times for 50% (θ50 ) of embryo growth and seed germination were calculated. W+C enabled slow embryo growth. W+C and GA3 promoted rapid embryo growth and subsequent radicle emergence. The embryo internal growth base temperature (Tbe ) was ca. 5 °C for W+C and GA3 -treated seeds. GA3 treatment also resulted in similar Tb estimates for radicle emergence. The thermal times for embryo growth (θe50 ) and germination (θg50 ) were four- to six-fold longer in the presence of GA3 compared to W+C. A. barbaricina is characterised by a multi-step seed germination. The slow embryo growth during W+C reflects continuation of the maternal programme of development, whilst the thermal kinetics of both embryo and radicle growth after the removal of physiological dormancy are distinctly different. The effects of W+C on the multiphasic germination response in MPD seeds are only partially mimicked by 250 mg·l-1 GA3 . The thermal time approach could be a valid tool to model thermal kinetics of embryo growth and radicle protrusion.

Molecular basis for three-dimensional elaboration of the Aquilegia petal spur.

By enforcing specific pollinator interactions, Aquilegia petal nectar spurs maintain reproductive isolation between species. Spur development is the result of three-dimensional elaboration from a comparatively two-dimensional primordium. Initiated by localized, oriented cell divisions surrounding the incipient nectary, this process creates a pouch that is extended by anisotropic cell elongation. We hypothesized that the development of this evolutionary novelty could be promoted by non-mutually exclusive factors, including (i) prolonged, KNOX-dependent cell fate indeterminacy, (ii) localized organ sculpting and/or (iii) redeployment of hormone-signalling modules. Using cell division markers to guide transcriptome analysis of microdissected spur tissue, we present candidate mechanisms underlying spur outgrowth. We see dynamic expression of factors controlling cell proliferation and hormone signalling, but no evidence of contribution from indeterminacy factors. Transcriptome dynamics point to a novel recruitment event in which auxin-related factors that normally function at the organ margin were co-opted to this central structure. Functional perturbation of the transition between cell division and expansion reveals an unexpected asymmetric component of spur development. These findings indicate that the production of this three-dimensional form is an example of organ sculpting via localized cell division with novel contributions from hormone signalling, rather than a product of prolonged indeterminacy.

Understanding the development and evolution of novel floral form in Aquilegia.

Flowers of the lower eudicot Aquilegia (columbine) possess morphological innovations, namely elaborate petal spurs and a fifth distinct organ identity, the staminodium, that are well suited to the investigation of key questions in developmental evolution. The recent evolution of these characteristics combined with a growing set of genetic and genomic resources has provided insight into how the traits arose and diversified. The petal spur appears to represent a key innovation that diversified largely via modification of specific aspects of cell expansion. In the case of the staminodium, gene duplication has played a role in allowing a novel organ identity to be carved out of the traditional ABC program.

Conserved roles for Polycomb Repressive Complex 2 in the regulation of lateral organ development in Aquilegia x coerulea 'Origami'.

BACKGROUND: Epigenetic regulation is necessary for maintaining gene expression patterns in multicellular organisms. The Polycomb Group (PcG) proteins form several complexes with important and deeply conserved epigenetic functions in both the plant and animal kingdoms. One such complex, the Polycomb Repressive Complex 2 (PRC2), is critical to many developmental processes in plants including the regulation of major developmental transitions. In addition, PRC2 restricts the expression domain of various transcription factor families in Arabidopsis, including the class I KNOX genes and several of the ABCE class MADS box genes. While the functions of these transcription factors are known to be deeply conserved, whether or not their regulation by PRC2 is similarly conserved remains an open question. RESULTS: Here we use virus-induced gene silencing (VIGS) to characterize the function of the PRC2 complex in lateral organ development of Aquilegia x coerulea 'Origami', a member of the lower eudicot order Ranunculales. Leaves with PRC2 down-regulation displayed a range of phenotypes including ruffled or curled laminae, additional lobing, and an increased frequency of higher order branching. Sepals and petals were also affected, being narrowed, distorted, or, in the case of the sepals, exhibiting partial homeotic transformation. Many of the petal limbs also had a particularly intense yellow coloration due to an accumulation of carotenoid pigments. We show that the A. x coerulea floral MADS box genes AGAMOUS1 (AqAG1), APETALA3-3 (AqAP3-3) and SEPALLATA3 (AqSEP3) are up-regulated in many tissues, while expression of the class I KNOX genes and several candidate genes involved in carotenoid production or degradation are largely unaffected. CONCLUSIONS: PRC2 targeting of several floral MADS box genes may be conserved in dicots, but other known targets do not appear to be. In the case of the type I KNOX genes, this may reflect a regulatory shift associated with the evolution of compound leaves.

Evolution of spur-length diversity in Aquilegia petals is achieved solely through cell-shape anisotropy.

The role of petal spurs and specialized pollinator interactions has been studied since Darwin. Aquilegia petal spurs exhibit striking size and shape diversity, correlated with specialized pollinators ranging from bees to hawkmoths in a textbook example of adaptive radiation. Despite the evolutionary significance of spur length, remarkably little is known about Aquilegia spur morphogenesis and its evolution. Using experimental measurements, both at tissue and cellular levels, combined with numerical modelling, we have investigated the relative roles of cell divisions and cell shape in determining the morphology of the Aquilegia petal spur. Contrary to decades-old hypotheses implicating a discrete meristematic zone as the driver of spur growth, we find that Aquilegia petal spurs develop via anisotropic cell expansion. Furthermore, changes in cell anisotropy account for 99 per cent of the spur-length variation in the genus, suggesting that the true evolutionary innovation underlying the rapid radiation of Aquilegia was the mechanism of tuning cell shape.

A conserved molecular framework for compound leaf development.

Diversity in leaf shape is produced by alterations of the margin: for example, deep dissection leads to leaflet formation and less-pronounced incision results in serrations or lobes. By combining gene silencing and mutant analyses in four distantly related eudicot species, we show that reducing the function of NAM/CUC boundary genes (NO APICAL MERISTEM and CUP-SHAPED COTYLEDON) leads to a suppression of all marginal outgrowths and to fewer and fused leaflets. We propose that NAM/CUC genes promote formation of a boundary domain that delimits leaflets. This domain has a dual role promoting leaflet separation locally and leaflet formation at distance. In this manner, boundaries of compound leaves resemble boundaries functioning during animal development.

Elaboration of B gene function to include the identity of novel floral organs in the lower eudicot Aquilegia.

The basal eudicot Aquilegia (columbine) has an unusual floral structure that includes two morphologically distinct whorls of petaloid organs and a clearly differentiated fifth organ type, the staminodium. In this study, we have sought to determine how Aquilegia homologs of the B class genes APETALA3 (AP3) and PISTILLATA (PI) contribute to these novel forms of organ identity. Detailed expression analyses of the three AP3 paralogs and one PI homolog in wild-type and floral homeotic mutant lines reveal complex patterns that suggest that canonical B class function has been elaborated in Aquilegia. Yeast two-hybrid studies demonstrate that the protein products of Aquilegia's AP3 and PI homologs can form heterodimers, much like what has been observed for their core eudicot homologs. Downregulation of AqvPI using virus-induced gene silencing indicates that in addition to petal and stamen identity, this locus is essential to staminodial identity but may not control the identity of the petaloid sepals. Our findings show that preexisting floral organ identity programs can be partitioned and modified to produce additional organ types. In addition, they indicate that some types of petaloid organs are not entirely dependent on AP3/PI homologs for their identity.

[Medicinal plant resources of Trib. Isopyreae and prospects of exploitation].

There are 17 species of medicinal plants from Trib. Isopyreae Schröd in China. This paper reports their resources, habitats and medicinal properties. On the basis of referring to the native and abroad medical studies on the plants from same Trilbe, their prospects of exploitation were advised.