Juan M. Losada

Glycoprotein composition along the pistil of Malus x domestica and the modulation of pollen tube growth

BackgroundThe characteristics of pollen tube growth are not constant, but display distinct patterns of growth within the different tissues of the pistil. In the stigma, the growth rate is slow and autotrophic, whereas in the style, it is rapid and heterotrophic. Very little is known about the interactions between these distinct maternal tissues and the traversing pollen tube and the role of this interaction on the observed metabolism. In this work we characterise pollen tube growth in the apple flower and look for differences in glycoprotein epitope localization between two different maternal tissues, the stigma and the style.ResultsWhile immunocytochemically-detected arabinogalactan proteins were present at high levels in the stigma, they were not detected in the transmitting tissue of the style, where extensins were abundant. Whereas extensins remained at high levels in unpollinated pistils, they were no longer present in the style following pollen tube passage. Similarily, while abundant in unpollinated styles, insoluble polysaccharides such as β-glucans, were depleted in pollinated pistils.ConclusionsThe switch from autotropic to heterotrophic pollen tube growth correlates spatially with a change of glycoprotein epitopes between the stigma and the style. The depletion of extensins and polysaccharides following pollen tube passage in the style suggest a possible contribution to the acceleration of heterotrophic pollen tube growth, which would imply an active contribution of female tissues on prezygotic male–female crosstalk.This work was supported by Ministerio de Ciencia e Innovacion (MICINN)-FEDER [AGL2006-13529-C02-01, AGL 12621-C02-01, AGL 2012–40239], and Gobierno de Aragon [group A43].

Floral biology and ovule and seed ontogeny of Nymphaea thermarum, a water lily at the brink of extinction with potential as a model system for basal angiosperms.

BACKGROUND AND AIMS Nymphaea thermarum is a member of the Nymphaeales, of one of the most ancient lineages of flowering plants. This species was only recently described and then declared extinct in the wild, so little is known about its reproductive biology. In general, the complete ontogeny of ovules and seeds is not well documented among species of Nymphaea and has never been studied in the subgenus Brachyceras, the clade to which N. thermarum belongs. METHODS Flowers and fruits were processed for brightfield, epifluorescence and confocal microscopy. Flower morphology, with emphasis on the timing of male and female functions, was correlated with key developmental stages of the ovule and the female gametophyte. Development of the seed tissues and dynamics of polysaccharide reserves in the endosperm, perisperm and embryo were examined. KEY RESULTS Pollen release in N. thermarum starts before the flower opens. Cell walls of the micropylar nucellus show layering of callose and cellulose in a manner reminiscent of transfer cell wall patterning. Endosperm development is ab initio cellular, with micropylar and chalazal domains that embark on distinct developmental trajectories. The surrounding maternal perisperm occupies the majority of seed volume and accumulates starch centrifugally. In mature seeds, a minute but fully developed embryo is surrounded by a single, persistent layer of endosperm. CONCLUSIONS Early male and female function indicate that N. thermarum is predisposed towards self-pollination, a phenomenon that is likely to have evolved multiple times within Nymphaea. While formation of distinct micropylar and chalazal developmental domains in the endosperm, along with a copious perisperm, characterize the seeds of most members of the Nymphaeales, seed ontogenies vary between and among the constituent families. Floral biology, life history traits and small genome size make N. thermarum uniquely promising as an early-diverging angiosperm model system for genetic and molecular studies.

Arabinogalactan proteins mark stigmatic receptivity in the protogynous flowers of Magnolia virginiana (Magnoliaceae)

PREMISE OF THE STUDY Factors affecting floral receptivity in angiosperms remain opaque, but recent studies suggest that the acquisition of stigmatic receptivity associated with cell-wall-related arabinogalactan proteins (AGPs) may be a widespread feature of flowering plants. Here, the time during which a stigma is receptive is evaluated and related to the secretion of AGPs in Magnolia virginiana, a protogynous member of an early-divergent angiosperm clade (magnoliids) with a clearly discernible female receptive phase. METHODS Magnolia virginiana flower phenology was documented, and histochemical changes in the stigma before and after pollination were examined. Stigmatic receptivity was evaluated in relation to the secretion of AGPs detected in whole mounts and immunolocalized in sectioned stigmas. KEY RESULTS Protogynous Magnolia flowers had a precise window of stigmatic receptivity, which is concomitant with the secretion of two AGPs labeled for different epitopes. After pollen germination and tube growth, these two AGPs could no longer be detected in the stigmas, suggesting that these AGPs interact with the growing male gametophytes and could be markers of stigmatic receptivity. CONCLUSIONS These results show that the period of stigmatic receptivity is finely coordinated with the secretion of two arabinogalactan proteins on stigmas of flowers of M. virginiana. This first report of AGP presence in stigmatic tissues in a member of the magnoliids, together with recently described similar patterns in eudicots, monocots, and members of early-divergent lineages of flowering plants, suggests an ancient and widespread role for AGPs on stigmatic receptivity in angiosperms.

Maintenance of carbohydrate transport in tall trees

Trees present a critical challenge to long-distance transport because as a tree grows in height and the transport pathway increases in length, the hydraulic resistance of the vascular tissue should increase. This has led many to question whether trees can rely on a passive transport mechanism to move carbohydrates from their leaves to their roots. Although species that actively load sugars into their phloem, such as vines and herbs, can increase the driving force for transport as they elongate, it is possible that many trees cannot generate high turgor pressures because they do not use transporters to load sugar into the phloem. Here, we examine how trees can maintain efficient carbohydrate transport as they grow taller by analysing sieve tube anatomy, including sieve plate geometry, using recently developed preparation and imaging techniques, and by measuring the turgor pressures in the leaves of a tall tree in situ. Across nine deciduous species, we find that hydraulic resistance in the phloem scales inversely with plant height because of a shift in sieve element structure along the length of individual trees. This scaling relationship seems robust across multiple species despite large differences in plate anatomy. The importance of this scaling becomes clear when phloem transport is modelled using turgor pressures measured in the leaves of a mature red oak tree. These pressures are of sufficient magnitude to drive phloem transport only in concert with structural changes in the phloem that reduce transport resistance. As a result, the key to the long-standing mystery of how trees maintain phloem transport as they increase in size lies in the structure of the phloem and its ability to change hydraulic properties with plant height.The phloem is the system of ‘blood vessels’ that translocates carbohydrates from the leaves to different plant organs. Here, using new structural imaging and pressure measuring tools, the researchers show interesting phloem structural changes that ensure a passive transport mechanism in tall trees.

Pollen tube access to the ovule is mediated by glycoprotein secretion on the obturator of apple (Malus × domestica, Borkh)

Background and Aims Within the ovary, the obturator bridges the pathway of the pollen tube from the style to the ovule. Despite its widespread presence among flowering plants, its function has only been studied in a handful of species, and the molecules involved in pollen tube-obturator cross-talk have not been explored hitherto. This work evaluates the involvement of glucans and glycoproteins on pollen tube growth in the obturator of apple flowers ( Malus × domestica) . Methods Pollen tube kinetics were sequentially examined in the pistil and related to changes occurring on the obturator using histochemistry and inmunocytochemistry. To discriminate between changes in the obturator induced by pollen tubes from those developmentally regulated, both pollinated and unpollinated pistils were examined. Key Results Pollen tube growth rates were slow in the stigma, faster in the style and slow again in the ovary. The arrival of pollen tubes at the obturator was concomitant with the secretion of proteins, saccharides and glycoprotein epitopes belonging to extensins and arabinogalactan proteins (AGPs). While some of these secretions - extensins and AGPs labelled by JIM13 - were developmentally regulated, others - AGPs labelled by JIM8 - were elicited by the presence of pollen tubes. Following pollen tube passage, all these glycoproteins were depleted. Conclusions The results show a timely secretion of glycoproteins on the obturator surface concomitant with pollen tube arrival at this structure. The fact that their secretion is depleted following pollen tube passage strongly suggests their role in regulating pollen tube access to the ovule. Remarkably, both the regulation of the secretion of the different glycoproteins, as well as their association with the performance of pollen tubes exhibit similarities with those observed in the stigma, in line with their common developmental origin.

Pollen–pistil interaction in pawpaw (Asimina triloba), the northernmost species of the mainly tropical family Annonaceae

PREMISE OF THE STUDY The pawpaw, Asimina triloba, is an underutilized fruit crop native to North America that belongs to the mainly tropical, early-divergent family Annonaceae. Asimina is the only genus within the Annonaceae with species adapted to cold climates. A thorough analysis of its reproductive biology, specifically pollen-pistil interaction during the progamic phase, is essential to understand both its adaptation to cold climates and how to optimize its fertilization and fruit set. METHODS We characterized pollen-pistil interaction in Asimina triloba, including the floral cycle and anatomy, stigmatic receptivity, and the pollen tube pathway. We used a combination of histological, cytological, and immunolocalization approaches. KEY RESULTS Asimina triloba has a gynoecium formed by plicate carpels with a short stylar canal. Unicellular papillae form a continuous tissue covered by a copious secretion from the stigma to the ovary, which is most prominent on the stigma surface where it forms an extragynoecial compitum. Compared to the stigmas of other species in the Annonaceae, the stigmas of A. triloba show a long stigmatic receptivity associated with a long flowering cycle. Stigmatic receptivity is concomitant with the secretion of cell-wall-related arabinogalactan proteins (AGPs). CONCLUSIONS A long female phase with a long period of stigmatic receptivity is unusual among protogynous flowers of the magnoliid clade, suggesting a derived condition of A. triloba within the Annonaceae. This phase further correlates with the presence of cell-wall-related arabinogalactan proteins in the secretion, which may indicate the conservation of these glycoproteins during stigmatic receptivity and pollen tube growth in angiosperms.

Phloem networks in leaves

The survival of all vascular plants depends on phloem and xylem, which comprise a hydraulically coupled tissue system that transports photosynthates, water, and a variety of other molecules and ions. Although xylem hydraulics has been extensively studied, until recently, comparatively little is known quantitatively about the phloem hydraulic network and how it is functionally coupled to the xylem network, particularly in photosynthetic leaves. Here, we summarize recent advances in quantifying phloem hydraulics in fully expanded mature leaves with different vascular architectures and show that (1) the size of phloem conducting cells across phylogenetically different taxa scales isometrically with respect to xylem conducting cell size, (2) cell transport areas and lengths increase along phloem transport pathways in a manner that can be used to model Münchs pressure-flow hypothesis, and (3) report observations that invalidate da Vincis and Murrays hydraulic models as plausible constructs for understanding photosynthate transport in the leaf lamina.

Phloem structure and development in Illicium parviflorum, a basal angiosperm shrub

Recent studies in canopy-dominant trees revealed a structure-function scaling of the phloem. However, whether axial scaling is conserved in woody plants of the understory, the environments of most basal-grade angiosperms, remains mysterious. We used seedlings and adult plants of the shrub Illicium parviflorum to explore the anatomy and physiology of the phloem in their aerial parts, and possible changes through ontogeny. Adult plants maintain a similar proportion of phloem tissue across stem diameters, but scaling of conduit dimensions and number decreases the hydraulic resistance towards the base of the plant. Yet, the small sieve plate pores resulted in an overall higher sieve tube hydraulic resistance than has been reported in other woody angiosperms. Sieve elements scaled from minor to major leaf veins, but were shorter and narrower in petioles. The low carbon assimilation rates of seedlings and mature plants contrasted with a three-fold higher phloem sap velocity in seedlings, suggesting that phloem transport velocity is modulated through ontogeny. While the overall architecture of the phloem tissue in basal-angiosperm understory shrubs scales in a manner consistent with trees, modification of conduit connections may have allowed woody angiosperms to extend beyond their understory origins.

Not all ‘pine cones’ flex: functional trade-offs and the evolution of seed release mechanisms

Seed dispersal is critical for plants, but the evolution of mechanisms that actually release seeds from their parents is not well understood. We use the reproductive cones of conifers, specifically the Pinaceae clade, to explore the factors driving the evolution of different release mechanisms in plants. We combine comparative anatomical and phylogenetic analyses to test whether fundamental trade-offs in the mechanical and hydraulic properties of vasculature underlie the evolution of two seed release mechanisms: cone scale flexion and cone scale shedding. We then test whether these mechanisms are linked with differences in seed size, dispersal syndrome and reproductive allocation. Cone scale xylem in flexing species is tough, but poorly conductive. Xylem in shedding species is less extensive, fragile and highly conductive; its thin-walled tracheids allow scales to easily fracture at maturity. Shedding is also consistently associated with large, densely packed seeds. Pinaceae cones exploit a well-known trade-off in xylem mechanical strength vs hydraulic efficiency to generate release mechanisms that allow seeds of various sizes to leave the protecting cone. The linkage among release mechanisms, vascular anatomy and seed traits illustrates how a wide variety of selective pressures may influence the function and physiology of reproductive structures.

Why are the seed cones of conifers so diverse at pollination

Background and Aims Form and function relationships in plant reproductive structures have long fascinated biologists. Although the intricate associations between specific pollinators and reproductive morphology have been widely explored among animal-pollinated plants, the evolutionary processes underlying the diverse morphologies of wind-pollinated plants remain less well understood. Here we study how this diversity may have arisen by focusing on two conifer species in the pine family that have divergent reproductive cone morphologies at pollination. Methods Standard histology methods, artificial wind pollination assays and phylogenetic analyses were used in this study. Key Results A detailed study of cone ontogeny in these species reveals that variation in the rate at which their cone scales mature means that pollination occurs at different stages in their development, and thus in association with different specific morphologies. Pollination experiments nevertheless indicate that both species effectively capture pollen. Conclusions In wind-pollinated plants, morphological diversity may result from simple variation in development among lineages rather than selective pressures for any major differences in function or performance. This work also illustrates the broader importance of developmental context in understanding plant form and function relationships; because plant reproductive structures perform many different functions over their lifetime, subtle differences in development may dramatically alter the specific morphologies that they use to meet these demands.