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37 Pag., 1 Tabl., 6 Fig. The definitive version is available at: http://aob.oxfordjournals.org/ ; Background and Aims An intense pollen–pistil interaction precedes fertilization. This interaction is of particular relevance in agronomically important species where seeds or fruits are the edible part. Over time some agronomically species have been selected for the ability to produce fruit without seeds. While this phenomenon is critical for commercial production in some species, very little is known about the events behind the production of seedless fruit. In this work, the relationship between pollen–pistil interaction and the onset of fruiting was investigated in citrus mandarin. Methods Pistils were sequentially examined in hand-pollinated flowers paying attention to pollen-tube behaviour, and to cytochemical changes along the pollen-tube pathway. To evaluate which of these changes were induced by pollination/fertilization and which were developmentally regulated, pollinated and unpollinated pistils were compared. Also the onset of fruiting was timed and changes in the ovary examined. ; Key Results Conspicuous changes occurred in the pistil along the pollen-tube pathway, which took place in a basipetal way encompassing the timing of pollen-tube growth. However, these changes appear to be developmentally regulated as they happened in the same way and at the same time in unpollinated flowers. Moreover, the onset of fruiting occurred prior to fertilization and the very same changes could be observed in unpollinated flowers. Conclusions Pollen–pistil interaction in citrus showed similarities with unrelated species and families belonging to other taxa. The uncoupling of the reproductive and fruiting processes accounts for the parthenocarpic ability of unpollinated flowers to produce fruit in citrus. However, the maintenance of a functional reproductive process reflects the potential to produce seeded fruits, providing a basis for the understanding of the production of seeded or unseeded fruits and further understanding of the process of parthenocarpy in other species. ; This work was supported by: the Italian Ministry of the University – Project PRIN "The productive process in fruit tree species: molecular, physiological and agronomical aspects of floral incompatibility and strategies for its control"; the Minister of Science and Innovation – EU Feder [Project grant CICYT AGL-2006-13529-CO2-01/AGR and AGL-2009-12621-CO2-01/AGR]; the Aragon Government A-43 support to the group. ; Peer reviewed

5 Pags. The definitive version is available at: https://www.ishs.org/ishs-book/1231 ; Mango (Mangifera indica) is a tropical fruit tree originated in southeastern Asia, which is cultivated in regions with tropical and subtropical climates worldwide. Mango cultivation has significantly increased in the last decades, often spreading to areas where environmental conditions are not the most favorable for optimal fruit set. In this work, we describe the reproductive biology of mango in the coastal areas of southern Spain, the furthest region from the equator where mango is produced commercially at a significant scale. As in other regions, mango trees produced several inflorescences per tree with hundreds to thousands of male and hermaphrodite flowers, depending on the cultivar, but only a very small percentage of the flowers are able to set fruits due to a massive drop of flowers and fruitlets. Although about half of the flowers in the inflorescence are male flowers and, consequently, contribute to this massive drop, a high proportion of the hermaphrodite flowers were not receptive to pollen germination. In receptive flowers, the characterization of the pollen tube pathway within the pistil showed chalazogamy. A detailed study of the ovules indicates that a high proportion of them showed degenerated embryo sacs, both before and after fertilization. Paternity analysis with molecular markers showed a high proportion of embryos resulting from self-pollination in orchards planted with a single cultivar. However, without pollination restrictions, the offspring shows a higher proportion of genotypes resulting from cross-pollination, suggesting preferential cross-fertilization. The variable response of this parameter, together to the variable proportion of hermaphrodite/male flower ratio, also obtained in response to environmental conditions, suggests a reproductive plasticity in this species. ; This research was supported by the Ministerio de Economía y Competitividad – European Regional Development Fund, European Union (AGL2012-40239 and AGL2013-43732-R) and Gob. Aragón A-43. V. Pérez was supported by a Cabildo de La Palma-CSIC PhD scholarship. ; Peer reviewed

7 Pags.- 1 Tabl.- 3 Figs. Article derived of a contribution of the authors to "VIII International Cherry Symposium". The definitive version is available at: https://www.actahort.org/index.htm ; In sweet cherry, as other temperate woody perennials, cycles of growth and dormancy are synchronized with the seasons. Flower primordia survive to low winter temperatures entering a dormant stage, but chilling is also required for a proper blooming. However, the biological mechanisms behind dormancy and chilling requirements are still not fully understood. In other species, pollen meiosis has been reported as one of the first biological events occurring after dormancy, but this process has not been studied in sweet cherry. In this work, pollen development has been characterized under the microscope in the cultivars Burlat and Bing in relation to winter dormancy up to anthesis during five years. The chilling requirements of both cultivars were empirically determined, and the dates of chilling fulfilment were related to meiosis and flowering time. During endodormancy, the anthers presented the sporogenous tissue formed by the pollen mother cells. First changes in pollen mother cells were detected about three weeks after chilling fulfillment as prelude of anther meiosis, which was completed within a week. Then pollen development continued during two or three weeks up to anthesis. Positive correlations were found between the dates of chilling fulfilment, pollen meiosis and full bloom for both cultivars. These results showed the influence of chilling winter temperatures on pollen meiosis time. ; This research was supported by Ministerio de Economía, Industria y Competitividad (MEIC) - European Regional Development Fund, European Union (AGL2012-40239); Instituto Nacional de Investigación Agraria (INIA) (RTA2014-00085-00, RFP2015-00015-00); Gobierno de Aragón - European Social Fund, European Union (Grupo Consolidado A-43). E. F. was supported by a grand BES-2010-037992. ; Peer reviewed

6 Pags.- 2 Figs. Article derived of a contriution of the authors to " II International Workshop on Floral Biology and S-Incompatibility in Fruit Species". The definitive version is available at: https://www.actahort.org/index.htm ; In temperate fruit trees, chilling accumulation during winter dormancy is required for proper flowering and is one of the main drawbacks of growing temperate trees in warmer latitudes. Chilling requirements are specific for each cultivar, suggesting a tight genetic control. The importance of chilling requirements, for cultivar adaptation to particular areas, has resulted in the development of different empirical models to quantify low temperatures. However, the biological processes behind these requirements remain unveiled. In Prunus species flower development requires several months from differentiation, at the end of the previous summer, until bloom the following spring. Development is halted during the winter, when the flower buds enter a dormant stage. To evaluate any possible changes behind the requirements for chilling, in this work flower development has been examined in sweet cherry (Prunus avium L.) and related to temperature, light, and chilling requirements. Anatomical characterization of flower buds, from the autumn to the spring, showed that the flower buds survive the winter at a particular stage of flower development. While no anatomical changes were detected during the theoretical chilling fulfillment, further cytochemical characterization revealed that conspicuous changes occurred in the pistil. Quantification of these changes with an image analysis system fitted to the microscope sheds light on the biological basis for chilling requirements. ; Financial support for this research was provided by Ministerio de Economía y Competitividad (MINECO) - European Regional Development Fund, European Union (AGL2012-40239 and BES- 2010-037992 to E. F.); Instituto Nacional de Investigación Agraria (RFP2015-00015-00, RTA2014-00085-00); Gobierno de Aragón - European Social Fund, European Union (Grupo Consolidado A-43). ; Peer reviewed

10 pags.- 6 Figs. ; Temperate woody perennials survive to low temperatures in winter entering a dormant stage. Dormancy is not just a survival strategy, since chilling accumulation is required for proper flowering and arbitrates species adaptation to different latitudes. In spite of the fact that chilling requirements have been known for two centuries, the biological basis behind remain elusive. Since chilling accumulation is required for the normal growth of flower buds, it is tempting to hypothesize that something might be going on at this particular stage during winter dormancy. Here, we characterized flower bud development in relation to dormancy, quantifying changes in starch in the flower primordia in two sweet cherry cultivars over a cold and a mild year. Results show that, along the winter, flower buds remain at the same phenological stage with flower primordia at the very same developmental stage. But, surprisingly, important variation in the starch content of the ovary primordia cells occurs. Starch accumulated following the same pattern than chilling accumulation and reaching a maximum at chilling fulfillment. This starch subsequently vanished during ecodormancy concomitantly with ovary development before budbreak. These results showed that, along the apparent inactivity during endodormancy, flower primordia were physiologically active accumulating starch, providing a biological basis to understand chilling requirements. ; This research was supported by Ministerio de Economía, Industria y Competitividad (MEIC) – European Regional Development Fund, European Union (AGL2012-40239 and BES-2010-037992 to EF); Instituto Nacional de Investigación Agraria (INIA) (RTA2014-00085-00, RFP2015-00015-00, and RTA2017-00003-00); Gobierno de Aragón – European Social Fund, European Union (Grupo Consolidado A-43). ; Peer reviewed

7 Pags.- 2 Figs. Original work presented by the authors at VII International Cherry Symposium (Plasencia, Spain. June 23-June 27 2013). The definitive version of the article is available at: http://www.actahort.org/index.htm ; In order to survive low temperatures, fruit trees in temperate regions stop their growth and enter dormancy. This is not just a survival strategy; chilling is also a prerequisite for adequate flowering. Chilling requirements are specific for each genotype and determine the possible geographical distribution of the different cultivars. In spite of the relevance of dormancy for fruit production, very little is known on the biological events during this time in flower buds, in which no visible changes can be observed until bud burst. In this work, flower bud development has been sequentially examined in sweet cherry (Prunus avium L. 'Bing') grafted on Santa Lucia 64 rootstock, in relation to dormancy and chilling accumulation. The work was performed over two consecutive years, paying attention to the possible anatomical and cytochemical changes accompanying dormancy. Results showed that, while no anatomical variations occurred along dormancy, conspicuous cytochemical changes could be tracked along this period that may help to understand the requirement for chilling. ; This work was supported by Ministerio de Economı́a y Competitividad (MINECO) - European Regional Development Fund, European Union (Project grants: AGL2012-40239- C02, INIA RFP2012-00017-C03 and INIA RTA2014-00085-00-00) by Grupo de Investigación de Aragón A-43 (Gobierno de Aragón - European Social Fund). E.F. was supported by the Ministerio de Economı́a y Competitividad by a doctoral fellowship (FPI BES-2010-037992). ; Peer reviewed

12 Pags.- 1 Fig. ; Plants, unlike animals, alternate multicellular diploid, and haploid generations in their life cycle. While this is widespread all along the plant kingdom, the size and autonomy of the diploid sporophyte and the haploid gametophyte generations vary along evolution. Vascular plants show an evolutionary trend toward a reduction of the gametophyte, reflected both in size and lifespan, together with an increasing dependence from the sporophyte. This has resulted in an overlooking of the importance of the gametophytic phase in the evolution of higher plants. This reliance on the sporophyte is most notorious along the pollen tube journey, where the male gametophytes have to travel a long way inside the sporophyte to reach the female gametophyte. Along evolution, there is a change in the scenery of the pollen tube pathway that favors pollen competition and selection. This trend, toward apparently making complicated what could be simple, appears to be related to an increasing control of the sporophyte over the gametophyte with implications for understanding plant evolution. ; This work was supported by Ministerio de Economía y Competitividad – European Regional Development Fund, European Union (AGL2012-40239, AGL2013-43732-R, and Recupera2020) and Gob. Aragón A-43. JL was supported by a Juan de la Cierva postdoctoral grant of the Spanish Ministry of Science and Innovation. ; Peer reviewed

25 Pags. 1 Tabl. 5 Figs. The definitive version is available at: http://www.sciencedirect.com/science/journal/03044238 ; In recent years, a growing interest to widen the cherry (Prunus avium L.) production calendar results in cultivation out of the traditional cultivation areas. Since cherry has high chilling requirements, this often causes erratic cropping related to phenological alterations. However, appropriate phenological characterisation and comparison is hampered, due to the lack of a consensus phenological scale for this species. In this work, we have characterised flower development in sweet cherry, framing it in the BBCH scale. For this purpose, the phenology of two cherry cultivars has been characterized over 2 consecutive years and adapted to the BBCH code, and flower development has been framed within the principal growth stages of this code. This provides a unified standardised approach for phenological comparative studies. ; This work was supported by Ministerio de Economía y Competitividad (MINECO) – European Regional Development Fund, European Union (Project grants: AGL2009-12621-C02-00, AGL2012-40239-C02, INIA RF2011-00029-C03 and INIA RFP2012-00017-C03) and Gobierno de Aragón (Grupo Consolidado A-43). E. Fadón was supported by a FPI fellowship of MINECO [BES-2010-037992]. ; Peer reviewed

27 pag., 3 fig., 2 suppl. fig., 1 tabl. Available online 14 December 2013. The definitive version is available at: http://www.sciencedirect.com/science/journal/00988472 ; In temperate woody plants, flowering time is adjusted to the environmental temperature, and survival to cold winter temperatures is achieved through dormancy. But also chilling temperatures during dormancy are required for proper flower bud development and flowering. The time the flower bud remains dormant is both genetically and environmentally controlled, and is a major factor determining the adaptation of species and cultivars to particular ecological conditions because chilling requirements vary greatly among genotypes. Recently this adaptation is jeopardised with climate changing conditions. However, little is known on the biological milestones underpinning these events, and when differences in flowering time are established. In this work we evaluate the hypothesis that anther meiosis time is related to winter cold temperatures, and that differences in flowering time are set up by differences in the time when pollen meiosis occurs. For this purpose, anther development has been characterised in five apricot cultivars with different chilling requirements. The work was done over two years with different weather conditions, a cold and a mild winter. The sporogenous tissues differentiated prior to dormancy and remained in this apparently quiescent stage during the winter. Once chilling requirements were fulfilled, meiosis closely followed and was highly correlated to breaking of endodormancy. Meiosis was completed within one week and was followed by a change in the colour of the anthers from green to yellowish, which could be a useful visual indicator to know that breaking of endodormancy had already occurred. The fact that this sequence of events was consistent in all the cultivars, and different climatic years analysed, supports the hypothesis that winter cold temperatures are related to the time of pollen meiosis, which in turn reflects in different flowering times. ; Financial support for this research was provided by Ministerio de Economía y Competitividad - European Regional Development Fund, European Union (AGL2009-12621-C02, AGL2012-40239) and 24 Gobierno de Aragón - European Social Fund, European Union (Grupo de Investigación de Aragón A-43). C. J. was supported by a fellowship of INIA (Instituto Nacional de Investigación y Tecnología Agroalimentaria) ; Peer reviewed

Pags.- Figs.- Tabls. The definitive version is available at: http://www.amjbot.org/ ; • Premise of the study: Permanent tetrads are the most common form of pollen aggregation in flowering plants. The production of pollen in monads is plesiomorphic in angiosperms, but the aggregation into tetrads has arisen independently different times during the evolution of flowering plants. The causes behind the recurrent evolution of pollen aggregation from monads remain elusive. Permanent tetrad pollen is quite common in the Annonaceae, the largest family in the early-divergent order Magnoliales. In some genera, such as Annona, both tetrad- and monad-producing species can be found. • Methods: In this comparative study of pollen development, we use immunolocalization, cytological characterization, and enzymatic assays of four species in the genus Annona and one species in its closely related genus Asimina that release pollen in tetrads and two species in the genus Annona that release pollen in monads. • Key results: The main difference between species with tetrad and monad pollen is a delayed digestion of callose and cellulose at the pollen aperture sites that resulted in nonlayering of the exine in these areas, followed by a rotation and binding of the young microspores at the aperture sites. • Conclusions: Small changes in development resulted in clear morphological changes on pollen dispersal time and open a window on the possible selective advantage of the production of aggregated pollen. ; This work was supported by Ministerio de Economía y Competitividad–European Regional Development Fund, European Union (AGL2010-15140, AGL2012-40239, and AGL2013-43732-R), the BBVA Foundation (BIOCON 08-184/09), INIA (RF2012-00010 and RFP2012-00016), and Gob. Aragón A-43. J.L. was supported by a Juan de la Cierva postdoctoral grant of the Spanish Ministry of Science and Innovation. ; Peer reviewed

10 Pags. The definitive version is available at: http://link.springer.com/journal/497 ; Pollen performance is an important determinant for fertilization success, but high variability in pollen behavior both between and within species occurs in different years and under varying environmental conditions. Annona cherimola, an early-divergent angiosperm, is a species that releases a variable ratio of bicellular and tricellular hydrated pollen at anther dehiscence depending on temperature. The presence of both bi- and tricellular types of pollen is an uncommon characteristic in angiosperms and makes Annona cherimola an interesting model to study the effect of varying environmental conditions on subsequent pollen performance during the final stages of pollen development. In this work, we study the influence of changes in temperature and humidity during the final stages of pollen development on subsequent pollen performance, evaluating pollen germination, presence of carbohydrates, number of nuclei, and water content. At 25 °C, which is the average field temperature during the flowering period of this species, pollen had a viability of 60–70 %, starch hydrolyzed just prior to shedding, and pollen mitosis II was taking place, resulting in a mixture of bi- and tricellular pollen. This activity may be related to the pollen retaining 70 % water content at shedding. Temperatures above 30 °C resulted in a decrease in pollen germination, whereas lower temperatures did not have a clear influence on pollen germination, although they did have a clear effect on starch hydrolysis. On the other hand, slightly higher dehydration accelerated mitosis II, whereas strong dehydration arrested starch hydrolysis and reduced pollen germination. These results show a significant influence of environmental conditions on myriad pollen characteristics during the final stages of pollen development modifying subsequent pollen behavior and contributing to our understanding of the variability observed in pollen tube performance. ; Financial support for this work was provided by the Spanish Ministry of Science and Innovation—European Regional Development Fund (Project Grants AGL2009-12621-CO-01 and AGL2010-15140), INIA (RF2009-00010), GIE-Aragón 43, Junta de Andalucía (FEDER AGR2742), and the European Union under the INCO-DEV program (Contract 015100). J.L. was supported by a grant from Junta de Andalucía. ; Peer reviewed

Trabajo presentado en el XXVIII International Horticultural Congress on Science and Horticulture for People (IHC2010): International Symposium on Plant Physiology from Cell to Fruit Production System, celebrado en Lisboa (Portugal), del 22 al 27 de agosto de 2010 ; Cherimoya (Annona cherimola Mill.) is a subtropical fruit tree with an incipient but promising commercial production niche in subtropical climates. However, the expansion of this crop from its areas of origin has taken place without its natural pollinating agent and crop production relies heavily on hand pollination. Erratic yields are often obtained without apparent causes, and they could be related to the lack of knowledge on the biological requirements of pollen in this species. Since pollen performance in the pistil is dependent on previous pollen development in the anther, here we perform a study on the influence of temperature and water content on final pollen development stages and, as a consequence, on pollen performance in A. cherimola. The results show a significant influence of temperature and relative humidity on pollen performance. These results provide biological support for adequate pollen handling to optimize production in this species. ; Financial support for this work was provided by the Spanish Ministry of Education (Project Grants AGL2009-12621 and AGL2010-15140), INIA (RF2009- 00010), GIE-Aragón 43, Junta de Andalucía (FEDER AGR2742), and the European Union under the INCO-DEV program (Contract 015100). J.L. was supported by a grant from Junta de Andalucía.

9 Pag., 8 Fig. ; • Premise of the study: A variety of mechanisms to prevent inbreeding have arisen in different angiosperm taxa during plant evolution. In early-divergent angiosperms, a widespread system is dichogamy, in which female and male structures do not mature simultaneously, thus encouraging cross pollination. While this system is common in early-divergent angiosperms, it is less widespread in more recently evolved clades. An evaluation of the consequences of this system on outbreeding may provide clues on this change, but this subject has been little explored. • Methods: In this work, we characterized the cycle and anatomy of the flower and studied the influence of temperature and humidity on stigmatic receptivity in Annona cherimola, a member of an early-divergent angiosperm clade with protogynous dichogamy. • Key results: Paternity analysis reveals a high proportion of seeds resulting from self-fertilization, indicating that self-pollination can occur in spite of the dichogamous system. Stigmatic receptivity is environmentally modulated—shortened by high temperatures and prolonged by high humidity. • Conclusions: Although spatial and temporal sexual separation in this system seems to effectively decrease selfing, the system is modulated by environmental conditions and may allow high levels of selfing that can guarantee reproductive assurance. ; Financial support for this work was provided by the Spanish Ministry of Education (Project Grants AGL2007-60130, AGL2009-12621, and AGL2010-15140), INIA (RF2009-00010), GIE-Aragón 43, Junta de Andalucía (FEDER AGR2742), and the European Union under the INCO-DEV program (Contract 015100). J.L. was supported by a grant from Junta de Andalucía. ; Peer reviewed

26 Pag. The definitive version is available at: http://aob.oxfordjournals.org/ ; Background and Aims: In temperate woody perennials, flower bud development is halted during the winter, when the buds enter dormancy. This dormant period is a prerequisite for adequate flowering, is genetically regulated, and plays a clear role in possibly adapting species and cultivars to climatic areas. However, information on the biological events underpinning dormancy is lacking. Stamen development, with clear differentiated stages, appears as a good framework to put dormancy in a developmental context. Here, stamen developmental changes are characterized in apricot (Prunus armeniaca) and are related to dormancy. Methods: Stamen development was characterized cytochemically from the end of August to March, over 4 years. Developmental changes were related to dormancy, using the existing empirical information on chilling requirements. Key Results: Stamen development continued during the autumn, and the flower buds entered dormancy with a fully developed sporogenous tissue. Although no anatomical changes were observed during dormancy, breaking of dormancy occurred following a clear sequence of events. Starch accumulated in particular places, pre-empting further development in those areas. Vascular bundles developed and pollen mother cells underwent meiosis followed by microspore development. Conclusions: Dormancy appears to mark a boundary between the development of the sporogenous tissue and the occurrence of meiosis for further microspore development. Breaking of dormancy occurs following a clear sequence of events, providing a developmental context in which to study winter dormancy and to evaluate differences in chilling requirements among genotypes. ; This work was supported by Ministerio de Ciencia e Innovación - European Regional Development Fund, European Union [Grant numbers: AGL2006-13529-CO2-00, AGL2009-12621-C02-00]; Gobierno de Aragón [Grupo de Excelencia de Aragón A- 43]; Instituto Nacional de Investigación y Tecnología Agraria y Agroalimentaria [Doctoral fellowship INIA2003-8 to C.J.]. ; Peer reviewed

33 Pag. The definitive version, with the figures, is available at: http://aob.oxfordjournals.org/ ; Background and Aims: Recent studies of reproductive biology in ancient angiosperm lineages are beginning to shed light on the early evolution of flowering plants, but comparative studies are restricted by fragmented and meagre species representation in these angiosperm clades. In the present study, the progamic phase, from pollination to fertilization, is characterized in Annona cherimola, which is a member of the Annonaceae, the largest extant family among early-divergent angiosperms. Beside interest due to its phylogenetic position, this species is also an ancient crop with a clear niche for expansion in subtropical climates. Methods: The kinetics of the reproductive process was established following controlled pollinations and sequential fixation. Gynoecium anatomy, pollen tube pathway, embryo sac and early post-fertilization events were characterized histochemically. Key Results: A plesiomorphic gynoecium with a semi-open carpel shows a continuous secretory papillar surface along the carpel margins, which run from the stigma down to the obturator in the ovary. The pollen grains germinate in the stigma and compete in the stigma-style interface to reach the narrow secretory area that lines the margins of the semi-open stylar canal and is able to host just one to three pollen tubes. The embryo sac has eight nuclei and is well provisioned with large starch grains that are used during early cellular endosperm development. Conclusions: A plesiomorphic simple gynoecium hosts a simple pollen–pistil interaction, based on a support–control system of pollen tube growth. Support is provided through basipetal secretory activity in the cells that line the pollen tube pathway. Spatial constraints, favouring pollen tube competition, are mediated by a dramatic reduction in the secretory surface available for pollen tube growth at the stigma–style interface. This extramural pollen tube competition contrasts with the intrastylar competition predominant in more recently derived lineages of angiosperms. ; This work was supported by the Spanish Ministry of Education (Project Grants AGL2004-02290/AGR, AGL2006-13529 and AGL2007-60130/AGR), GIC-Aragón 43, Junta de Andalucía (AGR2742), and the European Union under the INCO-DEV program (Contract 015100). J.L. was supported by a grant from Junta de Andalucía. ; Peer reviewed