Copyright © Physiologia Plantarum 2000PHYSIOLOGIA PLANTARUM 108: 35–41. 2000Printed in Ireland—all rights reser6ed ISSN 0031-9317

Ovary starch reserves and flower development in apricot(Prunus armeniaca)

Javier Rodrigo*, J. Ignacio Hormaza and Marıa Herrero

Unidad de Fruticultura, SIA-DGA, Campus de Aula Dei, Apdo 727, E-50080 Zaragoza, Spain*Corresponding author, e-mail: rodro@mizar.csic.es

Received 10 June 1999; revised 29 September 1999

inversely related to an increase in ovary size and in cell numberIn histerant species where flowering takes place prior to leafemergence, a flower lifespan occurs in the absence of new in the pericarp, suggesting an intraflower, self-supported devel-photoassimilates and at the expense of pre-stored reserves opment. This process is conserved in both pollinated and

nonpollinated flowers and therefore seems to be inherent to theeither in the plant as a whole or in the flower itself. In theflower at anthesis. The onset of fruiting is preceded by thepresent study, the role that the photoassimilates stored in theestablishment of large differences among ovaries; while someflowers might play in flower development from anthesis toexperience continuous growth, others stop growing and eventu-fertilization in Prunus armeniaca L. (apricot), a histerant

species, was explored. Starch content in individual flowers was ally drop. Interestingly, large differences in starch content aremeasured with the help of an image analysis system. Starch found among flowers at anthesis. These results are discussed in

terms of the possible implications of pre-stored starch in thecontent decreased from its highest value at anthesis anddisappeared from the ovary 9 days later. This decrease was flower supporting initial flower development.

carbohydrates are mobilized from other parts of the tree orare already present in the flower at anthesis is not clear.

Starch is the most important storage carbohydrate inplant organs, especially in woody perennials (Chapin et al.1990) and it occurs in the pistil in a number of bothherbaceous (Herrero and Dickinson 1979, Kanahama andSaito 1988, Wang et al. 1993, Heitholt and Schmidt 1994,Zinselmeier et al. 1995, Aloni et al. 1996) and woody plantspecies (Schneider 1972, Sedgley 1979, Felker et al. 1983, Vuet al. 1990, Arbeloa and Herrero 1991, Fromm et al. 1995,Martınez-Palle and Herrero 1995, Gonzalez et al. 1996).This consistent presence of starch in the pistils of diverseplant species contrasts with the scarce information availableon its role. Starch reserves do play a role in the reproductiveprocess (Herrero 1992, Herrero and Hormaza 1996), sincecarbohydrates accumulated in the style support pollen tubegrowth through the style (Herrero and Dickinson 1979), andstarch within the ovule is involved in the development ofboth the ovular structures (Arbeloa and Herrero 1991) andthe embryo (Brun and Betts 1984, Arbeloa and Herrero

Introduction

In many deciduous, woody plant species, flowering occursprior to leaf emergence (Loescher et al. 1990). This is thecase in most Prunus species such as apricot (Prunus armeni-aca L.), where anthesis and fertilization take place with verylittle or complete absence of foliar area (Keller and Loescher1989). Consequently, flower growth from budbreak to earlyfruiting cannot depend on leaf photosynthesis, since by thetime the new leaves begin producing photoassimilates, fruitdevelopment has already started.

In many plants, reproductive structures fix carbon andcontribute to flower and fruit development (Antlfinger andWendel 1997 and references therein). In temperate decidu-ous trees, that contribution is highly variable (Bazzaz et al.1979). Although it reportedly plays a role in peach (Paveland DeJong 1993) and apple (Vemmos and Goldwin 1994),it is insufficient to explain flower/fruit development (Paveland DeJong 1993). Loescher et al. (1990) reported thatpreviously stored carbohydrates were the main source ofsubstrates to support the initial phases of reproductivegrowth in Prunus species. However, to what extent those

Abbre6iations – FAA, 70% ethanol:glacial acetic acid:formalin (18:1:1, v/v/v); I2KI, potassium iodide-iodine; PAS, periodic acid Schiff’sreagent.

Physiol. Plant. 108, 2000 35

1991), as well as in the determination of ovule fate (Rodrigoand Herrero 1998). However, the role played by the starchstored in the ovary in the reproductive process is stillunknown.

This lack of information may be connected to the techni-cal difficulty in approaching this question, since two indis-tinguishable populations of flowers coexist in the sameplant. One population consists of the flowers that are goingto set fruit, the other, the ones that are going to drop. Sincethere is no way to distinguish a priori between these twopopulations, and the most commonly used biochemical mea-suring methods require large amounts of plant material, anypossible effect can be masked. An alternative lies in measur-ing starch content in individual flowers. Starch content caneasily be detected with the microscope using the potassiumiodide-iodine reaction (I2KI) (Johansen 1940), although dif-ferences are qualitative only. A recently developed imageanalysis system fitted to the microscope (Rodrigo et al.1997) can overcome those technical limitations. Further-more, with this technique additional information can beobtained on the same flower by the subsequent use ofdifferent stains and by performing morphometric measure-ments after evaluating the starch content.

To explore the implications of the nutritional status of theflower in post-anthesis flower development, starch content inthe ovary was sequentially examined and related to thereproductive process and to the growth of the flowers. Toexamine whether these changes are triggered by pollinationor alternatively, they are part of an independent develop-mental program, the study was performed separately withtwo synchronized populations of flowers, one consisting ofpollinated and the other of nonpollinated flowers.

Materials and methods

Plant material

Flowers from 3 trees of the self-incompatible apricot cv.Moniqui were used. In order to compare development, onlyflowers at the same developmental stage were left on selectedbranches; both older flowers and younger buds were re-moved. Flowers were emasculated and their petals removedat balloon stage 1 day before anthesis to make the flowersunattractive to insects and to avoid self-pollination (Free1964). One group of flowers was not pollinated and anothergroup was pollinated with pollen from the cv. Canino,which is compatible with Moniqui (Rodrigo and Herrero1996). Pollen was previously obtained from flowers at theballoon stage by removing the anthers and placing them atroom temperature on paper in the laboratory. Pollen wassieved 24 h later through a 0.26-mm mesh and frozen at−20°C until required.

Microscope preparations

A total of 115 pistils were processed and observed, 35 ofthem at anthesis and 10 each on days 3, 5, 7 and 9 afteranthesis for both pollinated and nonpollinated flowers. Pis-tils were fixed in FAA (70% ethanol:glacial acetic

acid:formalin [18:1:1, v/v/v]) (Johansen 1940), dehydrated ina tertiary butyl alcohol series (70, 85, 95 and 100%, v/v) andembedded in paraffin. Five additional pistils were fixed atanthesis and 7 days later in 2.5% (v/v) glutaraldehyde in0.03 M phosphate buffer (Sabatini et al. 1963), dehydratedin an ethanol series (35, 50, 70 and 95%, v/v) and embeddedin JB4 plastic resin (Polysciences Inc., Warrington, Philadel-phia, PA, USA). Paraffin-embedded material was sectionedat 10 mm in a Reichert-Jung 1130/Biocut rotary microtome(Reichert-Jung, Heildelberg, Germany) and, prior to stain-ing, the sections obtained were rehydrated (3 washes inHistoclear [CellPath, Hemel, UK], one each in Histo-clear:ethanol [1:1, v/v] and an ethanol series [100, 70 and40%, v/v]). Resin-embedded specimens were sectioned at 2mm in a Reichert-Jung Ultracut E ultramicrotome (Reichert-Jung) and, since the resin is miscible with water, they weredirectly stained as described below.

Observation of pollen tube growth

Pollen tube growth in the style was monitored on squashpreparations of styles previously autoclaved at 0.1 MPa (at121°C) for 10 min in 5% (w/v) sodium sulphite to soften thetissues (Jefferies and Belcher 1974) and stained with 0.1%(v/v) aniline blue in 0.3 M K3PO4 (Linskens and Esser1957). The same staining procedure was used to follow thepenetration of pollen tubes into the ovary. This was ob-served on the paraffin sections previously used to quantifystarch and to measure cell number and size. Preparationswere viewed with UV epifluorescence using a BP 355-425exciter filter and an LP 460 barrier filter.

Determination of ovary size

A total of 305 pistils, 35 of them at anthesis and 15pollinated and nonpollinated pistils each per day thereafter,were individually weighed over 9 days to follow pistilgrowth. These measurements were repeated over 2 consecu-tive years. Ovary size was also measured in the paraffinsections to study the relationship between ovary size and theinformation obtained microscopically from each pistil.Preparations were observed under a Wild Heerbrugg M8binocular microscope (Wild Heerbrugg, Heerbrugg, Switzer-land) and the images collected using a Cohu 8310 RGBColour Camera (Cohu, San Diego, CA, USA) attached tothe binocular microscope and processed using a Quantiment570 Image Analysis System (Leica Cambridge, Cambridge,UK). Ovary size was recorded by measuring the width ofthe ovary wall in its central section.

Evaluation of starch content

Preparations were stained with I2KI for starch reserves(Johansen 1940), with PAS for insoluble carbohydrates(Feder and O’Brien 1968) and with PAS counterstained withtoluidine blue (Feder and O’Brien 1968) for general histo-logical observations, and observed under a Leitz Ortholux IImicroscope (Leitz, Wetzlar, Germany). The images of I2KI-stained paraffin-embedded preparations were collected and

Physiol. Plant. 108, 200036

Fig. 1. Diagram of the ovary of an apricot flower. Boxes indicatethe regions measured with the image analysis system.

Determination of cell number and size

Cell number and size were determined in the same sectionsused to measure starch content. Thus, the preparations werewashed in distilled water, stained with calcofluor white forcellulose (Hughes and McCully 1975) and observed underan Ortholux II microscope with UV epifluorescence using aBP 355-425 exciter filter and an LP 460 barrier filter. Cellswere counted along the cross-section of the ovary and meanindividual cell size was determined by dividing ovary widthby cell number.

Results

Pollen tube kinetics were monitored along the pistil inpollinated flowers (Fig. 2). Pollen grains germinated within 1day after pollination (Fig. 2A) and pollen tubes grew alongthe style in the next 3 days (Fig. 2B); some pollen tubes hadreached the base of the style in all the pistils 4 days afterpollination. On arrival at the ovary, the pollen tubes tra-versed the obturator, a placental protuberance at the ovaryentrance, 5 days after pollination (Fig. 2C), entered into theovule through the micropyle and finally penetrated thenucellus to reach the embryo sac (Fig. 2D). The first fertil-izations were observed 6 days after pollination, and fertiliza-tion was completed by 7 days after pollination.

To relate the reproductive events to the onset of fruiting,individual pistils were weighed daily from anthesis to 9 days

processed using the same image analyser described above.Starch content values were obtained by measuring the opti-cal density of the image collected, considering the sum of theoptical density of each pixel as an estimation of the starchcontent of the frame studied (Rodrigo et al. 1997). Allmeasurements were taken in the section with an embryo sacand in the consecutive section. Since two ovules are present,measurements were made on 4 sections in each pistil. Ineach section, 4 measurements of the optical density of thestained starch in a 1337 mm2 fixed frame were taken (Fig. 1),and the average value of these 16 measurements in eachpistil was considered as an indicator of starch content.

Fig. 2. Pollen tube kinetics in P.armeniaca. (A) Germinated pollen grainsat the stigma with pollen tubes growingthrough the style 1 day afterpollination. Bar=20 mm. (B) Pollentubes growing along the style 2 daysafter pollination. Bar=50 mm. (C) Apollen tube traversing the obturator inthe upper part of the ovary andgrowing through the micropyle 5 daysafter pollination. Bar=50 mm. (D) Apollen tube penetrating into the embryosac 10 days after pollination. Bar=20mm.

Physiol. Plant. 108, 2000 37

Fig. 3. Fresh weight of pistils (A) and starch content in the ovary(B) of pollinated and nonpollinated flowers of apricot cv. Moniquiduring the 9 days following anthesis. Mean9SE of the averagevalues (n=10).

(Fig. 3A). Subsequently, 15 days after pollination, pollinatedflowers began to diverge and outgrew nonpollinated flowers,which finally dropped 5 weeks after pollination. Likewise,among pollinated flowers, some outgrew others 15 daysafter pollination. These flowers finally resulted in a finalfruit set of 37%.

The reserves needed to support flower development couldnot have come from photosynthesis, since the leaves havenot yet emerged. Thus, they must have been stored either inthe vegetative tissues or in the flower itself. In order toevaluate reserves in the flower and their mobilization, thestarch content of the ovary was examined. At anthesis, thecells of the ovary were rich in starch. Starch content thendeclined in all the flowers as ovary size increased (R=−0.59***) (Fig. 3). While some flowers were devoid ofstarch 5 days after pollination, in others starch was stillpresent. However, 7 days after pollination, it had practicallydisappeared from all flowers (Figs. 3B and 4). Likewise,while mean cell size in the ovary in both pollinated andnonpollinated flowers did not increase significantly duringthe 7 days following anthesis (Figs. 4 and 5A), radial cellnumber increased following the same pattern as ovary size(R=0.82***). Mean cell number ranged from 3391 atanthesis to 6195 in pollinated and to 6594 in nonpolli-nated flowers 7 days later (Fig. 5B).

Similar patterns were evident for ovary growth (Fig. 3A),starch content (Fig. 3B), and cell number (Fig. 5B) in bothpollinated and nonpollinated flowers. The consistency ofthis pattern contrasts with the wide variability recordedwithin both pollinated and nonpollinated individual flowers;while the development of some flowers is arrested at anearly phase, others continue growing. Whereas slight differ-ences are apparent among flowers at anthesis in pistil weightand cell number, a wide variability exists in starch contentat flower opening, although these differences are not corre-lated to those recorded in ovary size (R=0.05NS). Starchcontent of flowers at anthesis follows a normal distribution(Fig. 6), ranging from a minimum value of 840 (S opticaldensity) to a maximum value of 9154, with a coefficient ofvariation (CV) of 55%. The coefficient was much lower forweight (CV=19%), which ranged from 0.01 to 0.03 g, andfor cell number (CV=10%), which ranged from 27 to 38.

later (Fig. 3A). This experiment was repeated a second year(data not shown) and the pattern of growth was consistentin both years. Differences in fresh weight among pistils atanthesis ranged from 10 to 30 mg. Although differentflowers were sampled each day, some flowers increased inweight while others did not grow and eventually dropped.Thus, 9 days after anthesis, the ovary weight in pollinatedflowers ranged from 22 to 91 mg. Since fertilization oc-curred 7 days after pollination, these differences in growthmight have been triggered by pollination. Surprisingly,growth also occurred in nonpollinated pistils (Fig. 3A); 9days after anthesis their weights ranged from 24 to 72 mg.Furthermore, an increase in ovary weight took place in bothpollinated and nonpollinated flowers prior to fertilization

Fig. 4. Sections of P. armeniacaovaries. (A) Ovary wall at anthesis.Cells contain starch reserves in theircytoplasm. (B) Ovary wall 7 days afteranthesis. While cell size is similar,starch reserves have disappeared fromthe cells. Bars=10 mm.

Physiol. Plant. 108, 200038

Fig. 5. Cell diameter (A) and cell number along the ovary width (B)of pollinated and nonpollinated flowers of apricot cv. Moniquiduring the 7 days following anthesis. Mean9SE of the averagevalues (n=10).

reserves within the ovary decline rapidly following anthesis.Since this occurs concomitantly with ovary growth, it istempting to put forward that starch reserves in the ovaryappear to be used to support early ovary growth. Othersources of carbohydrates, such as reserves translocated fromother parts of the tree or soluble carbohydrates mobilized inthe ovary, could also play a role in ovary development, butthere is little information supporting this point (Loescher etal. 1990). Likewise, the contribution of carbon fixed byreproductive structures seems to be insufficient to supportovary development (Pavel and DeJong 1993). Further workis required to evaluate the contribution of other possiblesources of carbohydrates. However, the inverse patternrecorded here between starch content in the ovary and ovarygrowth suggests an involvement of this starch in supportingovary development. Ovary growth is primarily by cell divi-sion, since cell number along the ovary increases afteranthesis, while mean cell size does not. Data for other plantspecies indicate that variations in starch content in buds andflowers may be involved in flower development. Thus, an-ther growth and pollen development have been related tochanges in their carbohydrate content in Lilium (Clement etal. 1994, 1996) and starch content in the pollen grain hasbeen associated with pollen viability in Oryza (Sheoran andSaini 1996) and Triticum (Dorion et al. 1996). However,information concerning starch reserves in post-anthesisflower development is scarce. Prunus, like other histerantspecies, provides a good experimental model to analyse theimportance of previously stored photoassimilates versusphotoassimilates originating from de novo synthesis in theinitial steps of fruit development. While our results suggestthat, in P. armeniaca, starch stored in the ovary at anthesisis involved in ovary development, whether this is restrictedto histerant species or whether it may be a more generalphenomenon in plants remains to be seen. Indirect evidencesuggests that this behaviour may occur in other plant spe-cies. First, while the presence of starch in the ovary atanthesis has not been a matter of primary concern, it hasbeen reported in a wide range of species (Sedgley 1979,Felker et al. 1983, Kanahama and Saito 1988, Vu et al.1990, Wang et al. 1993, Heitholt and Schmidt 1994, Frommet al. 1995, Zinselmeier et al. 1995, Aloni et al. 1996).Second, at least for some functions, the flower appears to bewell supplied with previously stored reserves to supportdevelopment during the days following anthesis. This is truefor pollen tube growth (Herrero and Dickinson 1979) aswell as ovule (Arbeloa and Herrero 1991, Rodrigo andHerrero 1998) and embryo development (Brun and Betts1984, Arbeloa and Herrero 1991).

Our results show that early ovary development takesplace not only in pollinated but also in nonpollinated flow-ers. Moreover, the differences in weight recorded amongindividual flowers increase progressively from anthesis to 9days after anthesis. In pollinated flowers, differences inovary growth rate have been associated with subsequentreproductive success, since growth stops before the flowerdrops (Rapoport and Rallo 1991, Yates and Sparks 1994).Thus, two groups of flowers exist in the same plant: onegroup will remain on the tree and become fruits and theother will drop. Results presented here show that this be-

Discussion

Although carbohydrate reserves can critically influence re-productive success (Charlesworth 1989) and, in woodyplants, flower growth is initially dependent on carbohydratereserves in the tree (Oliveira and Priestley 1988), the impor-tance of those reserves in the onset of fruiting is still notclear (Loescher et al. 1990). Our results indicate that starch

Fig. 6. Distribution of flowers of apricot cv. Moniqui according totheir ovary starch content at anthesis.

Physiol. Plant. 108, 2000 39

haviour is conserved in the early development of nonpolli-nated flowers. Moreover, this similar behaviour betweenpollinated and nonpollinated flowers is also conserved in thenumber of cells and in the pattern of starch depletion in theovary. While fertilization is required for further fruit devel-opment and subsequent cell division (Gillaspy et al. 1993),our results suggest that both pollinated and nonpollinatedflowers use their reserves after anthesis and, hence, ovarygrowth immediately after anthesis seems to be independentof pollination/fertilization.

Some flowers in both pollinated and nonpollinated popu-lations of flowers continue growing while development ofothers is arrested, indicating that this behaviour is indepen-dent of pollination and, therefore, inherent to the flower atanthesis. The question remains as to what determines thisdifferent behaviour among flowers. Among the parametersanalysed in this work, ovary size and cell number and sizeappear to be similar among flowers regardless whether theyabscise or become fruits. However, starch content differsconsiderably among flowers. The fact that a high percentageof pollinated flowers do not become fruits suggests that anadditional factor superimposed to pollination/fertilizationcould be involved in fruit set. Since ovary developmentseems to depend on its starch reserves and starch contentdiffers among flowers, these differences might play a role infruit set. This is supported by the fact that all the flowers donot exhaust their ovary reserves at the same time. Theduration of these reserves to support early fruiting until newphotoassimilates become available might be a critical factordetermining fruiting. However, this hypothesis needs experi-mental support and, consequently, further research is underway evaluating starch reserves in relation to fruit set underconditions leading to high and low fruit set in order toestablish whether starch reserves stored in the flower andfruit set are related. On the other hand, the mechanisms thatdetermine starch content at anthesis should be explored.Thus, differences in starch content could be related either topositional effects, since some positions in the plant are morefavoured than others from a nutritive point of view (Lloyd1980, Stephenson 1992), or to the time when differentiationtakes place. In temperate, deciduous tree species, flowerbuds are formed during the previous growing season anddifferentiation of some floral parts occurs before leaf abscis-sion in the fall. Both vegetative and floral buds store carbo-hydrates (Kozlowski 1992), although the time of storagediffers with species. Thus, in sour cherry, changes in starchcontent in flower buds occur concomitantly with chillingaccumulation (Felker et al. 1983). However, in rhododen-dron flower buds, starch accumulates before the rest period(Schneider 1972). These differences in starch content atanthesis could determine survival of a flower until newphotoassimilates are available.

Our results show that, in P. armeniaca, the mobilizationof pre-stored starch reserves in the ovary occurs betweenpollination and fertilization. Leaves appear only after starchhas disappeared from the ovary and, consequently, theproduction of new photoassimilates begins only after fruitset has begun. Moreover, flower development appears toproceed independently of pollination since it follows thesame pattern in both pollinated and nonpollinated flowers.

However, this contrasts with the fate of the flowers, some ofwhich remain on the tree and eventually become fruits whileothers stop growing and abscise. The fact that ovary growthis inversely related to starch depletion, together with theclear differences observed among flowers in their initialstarch content at anthesis, could open the way to elucidatethis different behaviour.

Acknowledgements – The authors thank Eliseo Rivas for assistancewith the image analysis. Financial support for this research wasprovided by CICYT (Project grant AGF 95-0678), INIA (Projectgrant 98-049) and CONAI (Project grant PLA 2193). J. R. wassupported by fellowships of the Spanish Ministry of Education andINIA (Spanish Ministry of Agriculture).

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Edited by L. Dolan

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