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Achene Structure, Development and Lipid Accumulation in SunflowerCultivars Differing in Oil Content at Maturity

A N I T A I . M A N T E S E1,*, D IEG O M E D A N 1 and A N TO N IO J . H A LL 2

1Departamento de Recursos Naturales y Ambiente and2IFEVA, CONICET/FAUBA, Facultad de Agronoma,

Universidad de Buenos Aires, Av. San Martin 4453, (1417) Buenos Aires, Argentina

Received: 4 August 2005 Returned for revision: 10 October 2005 Accepted: 19 January 2006 Published electronically: 4 May 2006

Background and Aims Sunflower cultivars exhibit a wide range of oil content in the mature achene, but therelationship between this and the dynamics of oil deposition in the achene during grain filling is not known.Information on the progress, during the whole achene growth period, of the formation of oil bodies in thecomponents of the achene and its relationship with variations in final oil content is also lacking. Methods The biomass dynamics of achene components (pericarp, embryo, oil) in three cultivars of very differentfinal oil concentration (3056 % oil) were studied. In parallel, anatomical sections were used to follow the formationof oil and protein bodies in the embryo, and to observe pericarp anatomy. Key Results In all cultivars, oil bodies were first observed in the embryo 67 daa after anthesis (daa). The per-cellnumber of oil bodies increased rapidly from 1012 daa until 2530 daa. Oil bodies were absent from the outer celllayers of young fruit and from mature pericarps. In mature embryos, the proportion of cell cross-sectional areaoccupied by protein bodies increased with decreasing embryo oil concentration. The sclerenchymatic layer of themature pericarp decreased in thickness and number of cell layers from the low-oil cultivar to the high-oil cultivar.Different patterns of oil accumulation in the embryo across cultivars were also found, leading to variations in ripeembryo oil concentration. In the high-oil cultivar, the end of oil deposition coincided with cessation of embryogrowth, while in the other two cultivars oil ceased to accumulate before the embryo achieved maximum weight. Conclusions Cultivar differences in mature achene oil concentration reflect variations in pericarp proportion andthickness and mature embryo oil concentration. Cultivar differences in protein body proportion and embryo andoil mass dynamics during achene growth underlie variations in embryo oil concentration.

Key words: Sunflower, oil body, lipid accumulation, protein body, pericarp.

I NT R ODUC T I ON

Oil concentration of the sunflower fruit (achene) at maturityis known to vary among cultivars, and much effort by plantbreeders has been aimed at increasing the oil concentrationin mature achenes of modern oilseed cultivars (e.g. LopezPereira et al., 2000; Leon et al., 2003). Attempts have alsobeen made to identify the effects of factors such as radiation(Dosio et al., 2000; Aguirrezabal et al., 2003), temperature(Harris and Bojinger, 1980; Rondanini et al., 2003) and timeof sowing (de la Vega and Hall, 2002) on this trait. A limitedamount of attention has been paid to the dynamics of oildeposition in the achene in individual cultivars throughoutthe whole of the achene growth phase in single cultivars(e.g. Villalobos et al., 1996). However, studies of particular

issues affecting final oil concentration (e.g. the formation ofoil and protein bodies, embryo/pericarp proportions, etc.)have usually covered only part of the achene growth phasein single cultivars or related only to the situation found inthe mature achene. Some work has been done on thechanges in achene histology during oil and protein bodyformation (Tzen et al., 1993; Bolyakina and Raikhman,1999), but again coverage of these processes during theachene growth phase is only partial and cultivar differenceshave usually not been an object of this research. Conse-quently, although much is known about oil and proteindeposition in sunflower achenes at levels which range

from the biochemical to gross achene morphology, and

about some of the factors which control achene final oilconcentration, there is only partial understanding of theorigin of cultivar differences. The work reported hereaimed at documenting the growth of achene components(pericarp, embryo, oil) throughout the whole of the achenegrowth phase in three cultivars with contrasting final acheneoil concentration and at providing a histological basis for theobserved changes in component mass.

Gross anatomy of the sunflower fruit, particularly thepericarp, is relatively well known (Hanausek, 1902; Roth,1977; Saenz, 1981; Percie Du Sert and Durrieu, 1988;Denis et al., 1994; Lindstrom et al., 2000). Genetic factorsare important in determining the constitution of the peri-carp, although environmental and management practices

also affect it (Baldini and Vannozzi, 1996). In a study of27 different populations of sunflower, McWilliam andEnglish (1978) found that the embryo : achene ratio is agood indicator of achene oil concentration, and changes inembryo : pericarp ratios have been a feature of breedingfor modern oilseed sunflower cultivars (Sadras andVillalobos, 1994; Lopez Pereira et al., 2000). However,it is not clear how these variations in mature achenepericarp : embryo ratios between cultivars arise duringthe growth of the achene, as dynamics of these compo-nents of the achene have usually been studied in individ-ual cultivars (e.g. Villalobos et al., 1996; Ploschuk andHall, 1997).* For correspondence. E-mail [email protected]

Annals of Botany 97: 9991010, 2006

doi:10.1093/aob/mcl046, available online at www.aob.oxfordjournals.org

The Author 2006. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.For Permissions, please email: [email protected]

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In sunflower, lipid accumulation rate in the embryo is lowin the days following anthesis, and later increases to a fairlystable rate which is maintained until close to physiologicalmaturity (Villalobos et al., 1996; Connor and Hall, 1997;Rondanini et al., 2003). Available biochemical evidence isconsistent with these observations. Thus, Garces and

Mancha (1989) observed that the incorporation of radio-active precursors into polar lipids (components of the cel-lular membranes) predominated until 12 d after anthesis(daa), preferential incorporation in triacylglycerol synthesistaking place only from 12 to 25 daa. Reserve lipids in sun-flower and other oilseeds are laid down in subcellular struc-tures known as oil bodies, formed by a hydrophobic matrixof triacylglycerols surrounded by a layer of phospholipids,on which proteins called (oleosins and caleosins) aredeposited (Huang, 1992; Murphy, 2001). Oleosins andthe phospholipid layer confer stability to the oil bodiesand mantain them as separate entities. There has been con-siderable interest in the ontogeny of oil bodies (Tzenet al.,1993; Murphy, 2001). A recent study in sunflower(Beaudoin et al., 1999) revealed that oleosins are synthe-sized on endoplasmic reticulum-bound ribosomes and latertransferred to areas of the endoplasmic reticulum involvedin oil-body biosynthesis, and Mazhar et al. (1998) alsofound that oleosin synthesis began in early stages of embryogrowth. Tzen et al. (1993) found average diameters ofbetween 065 and 20 mm for rape, mustard, cotton, linseed,corn, peanuts and sesame seed oil bodies, and reported thatoil-body size in 11 more species (including sunflower) werewithin this range. What is still lacking is a comparativestudy of the dynamics of oil deposition in achene structuresacross cultivars and the provision of histological informa-tion on the connections between oil-body formation in

the various parts of the achene and overall progress ofoil deposition in the achene.Besides oil, the embryo cells of sunflower contain protein

bodies as a reserve component (Vintejoux, 1992; Shoar-Ghafari and Vintejoux, 1997; Bolyakina and Raikhman,1999). Steer et al. (1984), Goffner et al. (1988) andPloschuk and Hall (1997) found that protein accumulationin sunflower occurs at a greater rate during the first part ofthe achene filling period. Achene oil and protein concen-trations in the mature achene are often found to exhibitnegative associations (Blanchet and Merrien, 1982;Connor and Sadras, 1992; Lopez Pereira et al., 2000).Across cultivars, however, higher mature achene oil con-centrations are only partially associated with reduced pro-

tein production; the increase in oil concentration at achenematurity was somewhat more in proportion than thedecrease in protein concentration (Lopez Pereira et al.,2000). The connections between oil body and proteinbody density in embryo tissues of cultivars of differingfinal oil concentration has not been explored.

Work reported in this paper aimed at studying thedynamics of the mass of achene components (pericarp,embryo, oil) in three sunflower cultivars differing in finaloil concentration. Highest growth rates for each achenecomponent are staggered in time and the pericarp ceasesto grow well before the other components achieve maxi-mum mass (e.g. Villalobos et al., 1996). Consequently,

particular attention was paid to the timing of initiationand when the growth of each component ended, and tothe rate of growth of each component during its majorphase of growth. In parallel, the dynamics of oil-bodyformation in the embryo were followed, the area of proteinbodies and oil bodies was compared in sections of embryo

tissues, and cultivar differences in pericarp histology weredocumented.

MAT E R I AL S AND ME T HODS

Crop management and treatments

Experiments were carried out using three commercial sun-flower cultivars of contrasting mature achene oil concen-tration (Dakar, approx. 30 % oil; 11051, approx. 44 %oil; and Prosol 35, approx. 56 % oil) (Table 1). Seeds ofthe first two cultivars were provided by Advanta Argentina,and the last by Produsem SA, Pergamino Argentina. Cropswere hand sown on 17 November 1997 on a vertic argiudol

at the Facultad de Agronoma, Universidad de BuenosAires, using a fully randomized complete block designwith three replicates (35 m 35 m plots). The plotswere totally surrounded by two border rows. Crop densitywas 48 plants m2, with 03 m between plants in the rowsand 07 m between rows. Nitrogen was applied as urea, at50 kg ha1, 20 d after emergence. Insects were controlledwith carbofuran (5 L ha1 at 48 %) applied before sowing,and plots were weeded manually until the canopy closed.When rainfall was insufficient, irrigation was applied. Dur-ing the achene filling period the mean daily temperature was225 C and mean daily global radiation 198 MJ m2d1.

Sampling protocol

Approximately 2 months after sowing (16 January 1998for the low and high concentration cultivars, and 19 January1998 for the medium oil cultivar), 40 plants per plot weretagged at the start of anthesis of each plant. Twenty of theseplants were assigned to samplings for achene componentmass dynamics and achene histology, the remaining 20 tosamplings for achene oil concentration. To study growth inmass of the pericarp and embryo, plants were sampled threetimes a week over a period of 5 weeks, commencing 0 daysafter anthesis (daa) for a total of 16 samplings. At eachsampling five fruits per plant from each of three plantswere taken from the outer region of the capitulum (first

four rings). Fruit were removed using fine forceps, takingcare not to damage the head. The same head was not sam-pled more than five times and fruit was never taken fromareas close to a previous sample on the same head. Embryoswere manually separated from the surrounding ovary tissueand the dry weight of embryos and pericarps determined. Todetermine achene oil content dynamics, all fruit from theoutermost four rings of the head were collected from oneplant per replicate on the same dates as the samples usedfor embryo and pericarp mass dynamics. Each plant wassampled only once. Achenes were dried for at least 48 h at60 C, and achene oil percentage was determined on 10-gsubsamples of entire dried seeds using nuclear magnetic

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resonance (NMR) analysis. These data were used to calcu-late embryo oil content and concentration (on a dry-weightbasis) using the equations:

achene weight mg % oil achene=100

= oil content per embryo mg 1

oil content per embryo=embryo weight 100

= embryo oil concentration % 2

All values for oil concentration in the whole achene or in theembryo shown in this paper are on a dry-weight basis.

At 1, 45, 67, 89, 1012, 1415, 2530 and 4045daa,additional samples were taken for achene histology fromplants assigned to sampling for embryo and pericarp massdynamics. Particular care was taken to ensure that thesefruits were representative (in size and appearance) of theachenes that formed part of the larger sample taken forachene component mass dynamics. Groups of three periph-eral flowers/developing fruits were sampled from three dif-ferent plants at each sampling date. Embryo sections wereprepared for all samples starting with the 45 daa harvest,and pericarp sections were prepared from samples harvested1, 89 and 4045 daa.

During the summers of 199495 and 199697 experi-ments similar to the one described above were conductedat the same site using the same protocol with a single excep-tion : achene oil contents were determined on samples madeup of all achenes on the capitulum instead of being restrictedto peripheral achenes only.

Estimation of duration and rates of growth of embryo,

pericarp and oil

Duration of, and rates of, mass increase for embryo,pericarp and oil were estimated using the non-linear routineof SYSTAT (Wilkinson, 1986) to fit bilinear regressions tothe data. This routine uses an optimization technique todetermine the breakpoint between linear sections, theslope and intercept of an initial straight line defined by:

p = a1 + bx for x < x0; and of a second straight line definedby: p = a2 for x > x0; where p is the weight of pericarp,embryo or oil, a1 and a2 are the intercepts and b the slope.

x is expressed in units of daa and x0 is the point of inter-section between the two straight lines indicating the endof the growth of the achene component. The duration ofachene component growth periods was defined as theinterval between anthesis of the flowers on the peripheralportion of the head and x0 (pericarp); or the interval betweenthe intercept of the initial line on the time axis and x0(embryo). The intercepts on the time axis of the bilinearfunctions were taken as the effective initiation of embryo oroil deposition and the slopes of the initial time as the effec-tive rates of growth of these achene components. Embryodry weight increase and oil deposition begin with somedelay in respect to anthesis, so values for the first threesamples (5, 8 and 10 daa) were not used when fitting bilinearfunctions. Because durations and rates of achene componentgrowth can be affected by temperature, data were alsoexpressed on a thermal time basis with a base temperatureof 1 C (Chimenti et al., 2001) and bi-linear functions

fitted to mass/thermal time relationships. Data for air tem-peratures during the experiment were obtained from a stan-dard meteorological station situated 500 m from the plots.

Dynamics of oil-body formation

Freshly collected fruits were dissected and either wholeembryos or small cubical portions (sides 051 mm long)were cut from the different embryo parts. Theses wereplaced in 3 % glutaraldehyde in phosphate buffer(pH 72), dehydrated in an ascending series (7095 %) ofethanol concentrations and included in JB4 resin (Poly-sciences Inc., Warrington, USA) for optical microscopy,or in Spurr resin (Spurr, 1969) for electron microscopy,

following the standard methodology of OBrien andMcCully (1981). Semi-thin (1 mm) and ultra-thin (7080 A) sections were obtained with a Ultracut III Reichertultramicrotome (Reichert-Jung, Austria). Semi-thin sec-tions of early (low lipid content in cells) samplings stainedbest with 01 % Sudan black, and later (higher lipid content)sections stained better with 05 % toluidine blue. Conse-quently, staining procedures were adjusted according toachene age. Sections were photographed with a Zeiss Axio-plan (Oberkochen, Germany) optical microscope. Ultra-thinsections were stained in uranile acetate and lead citrate(Reynolds, 1963) and photographed with a Jeol JEM/1200 EXII (Jeol, Japan) transmission electron microscope.

T A B L E 1. Peripheral achene final mature dry weight in experiments conducted during the summers of 199495, 199697 and199798 for three cultivars of sunflower differing in oil content at maturity

Mature achene dry weight (mg) Mature achene oil concentration (%, d. wt basis)

Cultivar 199495 199697 199798 199495 199697 199798

Dakar 164.5 6 0.92a 134.3 6 1.35b 158.5 6 0.53c [33.1 6 0.19a] [33.3 6 1.06a] 30.4 6 0.13b

11051 84.3 6 2.03a 93.5 6 1.56b 112 6 0.23c [44.9 6 0.71a] [42.7 6 0.93a] 44 6 0.38a

Prosol 35 70.5 6 0.77a 89.5 6 0.75b 93.4 6 0.46c [44.7 6 0.44a] [50.5 6 1.43b] 55.8 6 0.22c

Valuesof themature acheneoil concentration shownin thelast three columns were determined forachenes from allpositions onthe capitulumin 199495and199697(valuesin square brackets)and fromachenesfrom the peripheralposition onlyin 199798. Values are means(n = 3)6 s.e. andare derived frombilinear functions fitted to experimental data.

Within each column, values followed by different letters are significantly ( P < 005) different.

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Intra-embryo distribution and mean size of oil bodies

at 4045daa

Cotyledons, shoot axis and radicle of mature embryoswere sectioned freehand approx. 2530mm thick. Thesections were stained in 01 % aqueous nile blue and viewedwith a Zeiss fluorescence microscope (Axioplan,Oberkochen, Germany) to record the distribution patternof oil bodies among the various parts of the embryo. Todetermine oil body final size, the isolation procedure ofTzen et al. (1993) was followed with slight modifications.The tissue (25 g of cotyledon of mature embryos per 10 mLgrinding medium) was ground with a mortar at 20 C in agrinding medium containing 06 M sucrose, 1 mM EDTA,10mM KCl, 1mM MgCl2, 2 mM DTT and 05 M Tricinebuffer adjusted to pH 75 with KOH. The homogenatewas filtered and a 75-mL portion of the filtrate was placedin a15-mL centrifuge tube, and 75 mL of a flotation med-ium (grinding medium containing 04 M instead of 06 Msucrose) was layered on top. The tube was centrifuged at

11^000 g for 30 min in a Sorvall RC5C ultracentrifuge. Thesupernatant was collected and resuspended in 75 mL of

grinding medium containing an additional 75 m L 2 MNaCl. The resuspended material was placed in a 15-mLcentrifuge tube, 75 mL of flotation medium (grinding med-ium containing 75 mL 2 M NaCl and 025 M instead of 06 Msucrose) was layered on top, and the tube was centrifuged at11000 g for 30 min. The supernatant was collected, and theoil bodies were stained with 01 % aqueous nile blue andexamined by fluorescence microscopy.

Distribution, size and relative importance of protein

bodies in the mature achene

Whole mature embryos harvested at 4045 daa wereembedded in paraffin and serially cut at 1015 mm witha Minot-type rotary microtome. Sections were stainedwith safranin-fast green (Johansen, 1940) to observe theprotein bodies. Photographs of selected embryo tissues(mesophyll of cotyledon, and parenchyma of the shootradicle axis) were taken with a Zeiss fluorescence micro-scope. The photographs were digitized with a HewlettPackard Scan Jet 4c scanner, and images of at least tencells per tissue per cultivar were analysed with ImageTool 1.28 for Windows (Wilcox et al., 1996) to determineper-cell average sectional area of protein bodies, and wholecell sectional area.

Pericarp structure

Thin slices (approx. 2 mm thick) were cut from the cen-tral region of young developing fruits (1 daa and 89 daa)and mature fruits (4045 daa) of all three cultivars. Sliceswere placed in 3 % glutaraldehyde in phosphate buffer (pH72), dehydrated in an ascending series (7095 %) of ethanolconcentrations, and included in Historesin (Leica) (youngfruits) or JB4 resin (Polysciences, Inc.) (mature fruits).Semi-thin (13 mm) sections were obtained with a UltracutIII Reichert ultramicrotome and stained with 01 % cresylviolet. In addition, portions of mature fruits of all three

cultivars were embedded in paraffin and serially cut at1015 mm with a Minot-type rotary microtome, and sectionswere stained with safranin-fast green (Johansen, 1940) toobserve pericarp thickness. Two measurements of maturepericarp thickness were made on each of five different sam-ples per cultivar.

Statistical analyses

Standard analyses of variance were performed on thedata, and the Tukey test was used to determine significanceof differences between means (Steel and Torrie, 1980). Inthe text, means are given 6 s.e.

R E SUL T S

Embryo structure and oil-body dynamics

At 56 daa embryo length was 0508 mm in 11051 andProsol 35, and 081 mm in Dakar, and the cotyledonsand the hypocotylradicle axis were already discernible.Maximum embryo length was reached at 1617 daa,ranging from 8510 mm in 11051 and Prosol 35, and1316 mm in Dakar. Mature achene weights decreased inthe order Dakar ! 11051 ! Prosol 35, while matureachene oil concentration increased in the same order(Table 1). At a very early growth stage (45 daa) cells inall tissues were generally isodiametric and scarcely differ-entiated, with a dense central nucleus and optically clearcytoplasm (Fig. 1A). During the following 4 weeks cellsexpanded and tissues attained moderate histological differ-entiation. Cell walls remained thin and intercellular spacesscarce, while large quantities of oil and reserve protein

accumulated in oil bodies and protein bodies.In all three cultivars the dynamics of oil-body formationand distribution among embryo tissues were similar. Thefirst oil bodies (up to ten per cell transection) appeared in thecotyledons at 67 daa as relatively uniform globular bodiesapprox. 23 mm in diameter, adjacent to the cell wall butdistinctly separated from each other (Fig. 1B). At 89 daaoil-body number per cell had increased (Fig. 1CD). Oilbodies appeared simultaneously in the epidermis and meso-phyll of the cotyledons (Fig. 1D). At 1012daa (Fig.1E) and1415 daa (Fig. 1F) oil bodies (1520 per cell transection)occupied much of the periphery of the cell and startedappearing in the centre (Fig. 1F) of the cell. At 2530 daa there were 3035 oil bodies per transection, and

oil bodies occupied most of the cell transection (Fig. 2A).There was no detectable difference in oil body number percell transection between samples taken at 2530 daa and at4045 daa (Fig. 2BD).

In the mature embryos (4045 daa) the nucleus, the oilbodies and the protein bodies were the most conspicuouscell components (Fig. 2BD). The oil bodies in most cellswere tightly packed and became somewhat polygonalbecause of compression, but their individuality was stillpreserved (Fig. 2E, F). Oil bodies were present in the epi-dermis and mesophyll of the cotyledons, and in the epider-mis, cortex and central cylinder of the hypocotylradicleaxis of mature embryos of all three cultivars (data not


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shown), with apparently similar abundance in all tissues. Oilbodies isolated from embryos of the three cultivars hadsimilar shapes and diameters ranging from 1 to 4mm.Mean oil-body diameter tended to increase in the sequencelow-oil to medium-oil to high-oil cultivar (1966 016 mm,213 6 018 mm and 25 6 03 mm, respectively) but these

differences were not statistically significant. The diametersof isolated oil bodies were consistent with those observedin cotyledon sections of the three cultivars.

Protein bodies

Protein bodies became visible at 89 daa as numerous,small unstained bodies which contrasted with the moder-ately stained cytoplasm and the Sudan-black dark-stainedoil bodies (Fig. 1C, D). At embryo maturity, toluidine blue-stained protein bodies were easily distinguishable as large,dark, approximately globular structures with irregular con-tours due to pressure from oil bodies (Fig. 2B2F). In ultra-thin sections protein bodies showed many electron-cleargloboids (Fig. 2B2F). Protein bodies appeared to be lessabundant in mature embryos of the high-oil than in the low-oil cultivar (cf. Fig. 2B with Fig. 2C). The average areasoccupied by protein bodies in the whole cell sections of themature embryo were 372 6 116 mm2 cell1, 281 6106 mm2 cell1 and 286 6 145 mm2 cell1, equivalent

to 358 % 6 194, 236 % 6 138 and 249 % 6 123 ofthe whole cell area, in the low-, medium- and high-oilcultivars, respectively. These differences in percentage ofcell area occupied by protein bodies were statisticallysignificant (P < 005).

Pericarp anatomy

Transections of very young developing fruits (1 daa,Fig. 3A; 89 daa, Fig. 3B) showed no traces of oil bodiesat any pericarp layer. Sections of mature achenes (Fig. 3CE) showed several layers (epidermis, hypodermis, blacklayer or pigmented cells, sclerenchymatic tissue andparenchymatic tissue, the latter adjacent to the seed), butoil bodies were not present in any of these cell types. Thesclerenchymatic layer was noticeable due to the massiveamount of thick-walled cells, distributed in discrete areasseparated by thin parenchymatic radii. The thickness ofsecondary walls decreased towards the pericarp interior(best seen in Fig. 3D and E). The average thickness ofthe sclerenchymatic layer decreased from the low-oil cul-tivar to the medium- and high- oil cultivars (6236 035mm,341 6 03 mm and 239 6 05 mm, respectively). Thesevalues reflected differences in the number of cell layers,which was higher in the low-oil cultivar (186 6 02,Fig. 3C) than in the medium-oil (116 6 03, Fig. 4D)and in the high-oil cultivars (68 6 01, Fig. 4E). The dif-

ferences among cultivars for pericarp thickness and numberof cell layers were highly significant (P < 001).

Pericarp and embryo growth and oil deposition

The growth patterns of achene components (pericarp,embryo, oil) were broadly similar across the three cultivars,with the pericarp growing first and rapid increases ofembryo and oil mass taking place later, and the pericarpceasing to grow before the embryo and oil components hadreached their maximum values (Fig. 4). The detail of thesepatterns, however, varied greatly between cultivars(Tables 2 4). Differences in initial mass and effective

A B

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F I G . 1. Transections of sunflower cotyledons at early developmentalstages: (A) Prosol 35 at 45 daacells show meristematic featuresthroughout the tissue; (B) Prosol 35at 67daaoilbodies become visible;(C) and(D) Dakar and11051,respectivelyat89 daa oilbodies occupyonly the cell periphery; (E) and (F) Prosol 35 at 1012 daa and 1415 daa,respectively. Fixation with 3 % glutaraldehyde, included in JB4 resin andstained with Sudan black 01 %. n, Nucleus; o, oil body; p, protein body.

Scale bars = 10 mm.


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rates and durations of growth of the pericarp were thebasis for very different final pericarp weights acrosscultivars (Table 2 and Fig. 4). The effective durations ofembryo growth did not differ among cultivars, but embryosof Dakar grew faster than those of the remaining two

cultivars (Table 3). Embryo weights at the first harvest atwhich this component could be distinguished (5 daa) were536 031, 346 024and356 033 mgfor low-, mid- andhigh-oil cultivars, respectively. The outcome of these dif-ferences in initial mass and rate of growth was that mature

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F I G . 2. Transections of sunflowercotyledonsat latedevelopmentalstages:(A) Prosol 25at 30 daathe numberof oilbodiesincreasesconsiderably andthelight intercellular areas correspond to the cytoplasm and protein bodies (fixation with 3 % glutaraldehyde, inclusion in JB4 resin and coloration with Sudanblack01 %);(B),(C) and(D) 4045 daa(Dakar, 11051 andProsol 35, respectively)the protein bodiesare stained dark, oilbodiesare lighter dueto thestaining used(05 % toluidineblue) (fixationwith glutaraldehyde 3 %, inclusionin JB4 resin. (E) and (F) Ultra-thinsectionsshowing ultrastructural details ofthe cotyledon at 4045 daa (Dakar andProsol35, respectively).Note theproteinbodies,the compression of theoil bodies andthe reduced areaoccupiedbyother cytoplasm fractions. Fixed with glutaraldehyde 3 %, inclusion in Spurr resin and stained with uranile acetate and lead citrate. g, Globoids; o, oil body;

p, protein body. Scale bars: AD = 10mm; E and F = 2mm.


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embryo weights decreased from the low oil cultivar to thehigh oil one, although the range between the largest andsmallest embryos was much more restricted than for thecorresponding pericarps. There was some indication thatinitiation of rapid embryo growth lagged further behindanthesis in the mid- and high-oil cultivars with respect tothe low-oil one (Table 3). However, the overall duration ofachene growth (i.e. time until effective initiation of embryogrowth + effective duration of embryo growth) was very

similar between cultivars, unlike what was found for thepericarps (Fig. 4 and Table 2). Consequently, the finalproportion of embryo in the achene differed widely acrosscultivars (Fig. 4 and Table 3).

Final oil mass per embryo was rather similar across cul-tivars, although in the high-oil cultivar it was significantlyhigher than the other two (Table 4). Cultivar ranking forrates of oil deposition during the effective growth period forthis variable was similar to that found for embryo growth

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F I G . 3. Transections of sunflower pericarp: (A and B) early developmental stages of Prosol 35. (C), (D) and (E) mature pericarp (Dakar, 11051 andProsol 35, respectively). Note differences in thickness of sclerenchymatic tissue between cultivars. Fixation with 3 % glutaraldehyde, inclusion in JB4resin and stained with 01 % cresyl violet. e, Epidermis; h, hypodermis; pa, parenchyma; s, sclerenchyma. Scale bars: A and B = 10mm; C = 100mm;

D and E = 45mm.


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rates. However, the effective duration of achene oil massincrease was significantly (P < 005) greater in Prosol 35(Table 4), in contrast to the effective duration of embryogrowth which was similar across cultivars (Table 3). Animportant consequence of this was that embryo oil concen-tration (%) actually fell during the last phase of achenefilling in the low- and medium-oil cultivars (Fig. 5). Inthe high-oil cultivar, embryo oil concentration (%)remained stable in the middle third of the oil depositionphase, but rose again in the final phase of oil deposition(Fig. 5). These interactions between the patterns of growth

of embryo and oil masses outweighed the effects of laterinitiation of the effective period of oil deposition in the mid-and high-oil cultivars and determined quite large differ-ences in embryo final oil concentration (%) (Table 4).Results for the experiments conducted in 199596 and199697 were broadly consistent, for peripheral achenes

of all three cultivars, with those presented here (Fig. 4)for patterns of growth of the pericarp, the embryo andthe whole achene, and for cultivar rankings for mature ach-ene weight, mature achene oil concentration and matureembryo oil concentration (data not shown). Achene oil con-tent in these early experiments was determined on samplesdrawn from all achene positions on the head, rather than onperipheral achenes only as in present results, hence patternsof oil mass per achene dynamics are not comparable topresent data. Anthesis (and, consequently, the start of oildeposition in the achene) progresses from the periphery tothe centre of the head over a period of 57 d. Consequently,mean oil content per achene in samples drawn from allpositions of the head continues to increase over a longerperiod than that found in peripheral achenes only. In spite ofthis effect, apparent relative peripheral embryo oil con-centration (i.e., that estimated using oil content values mea-sured on achenes from all positions) fell during the finalstages of achene filling in Dakar in both early experiments,and in one out of two years in 11051 (Mantese, 1998),exhibiting an effect similar to that seen in Fig. 5 for thesetwo cultivars. This result indicates that the fall in embryo oilconcentration shown in Fig. 5 for Dakar and 11051 is aconsistent feature of these cultivars that is stable acrossyears.

Figure 6 illustrates the contrasting patterns of relativeembryo oil concentration dynamics in the three cultivars

using data normalized to remove the effect of cultivar dif-ferences in embryo oil concentration. The dynamics of rel-ative embryo oil content may be of agronomic interest,particularly when crops are exposed to stresses whichmay curtail achene filling.

DI SC USSI ON

The present temporally and histologically referenced studyof oil accumulation in the sunflower achene across cultivarsof very different final oil concentration has produced someresults that are consistent with studies using mature achenesor those based on a single cultivar. It has also served to

highlight the pitfalls inherent in extrapolating resultsobtained in a single cultivar to others.

The lag between anthesis and the appearance of the firstoil bodies in the embryo cells at 67 daa is consistent withboth the present observations (e.g. Fig. 4) and those ofothers (e.g. Villalobos et al., 1996) on the dynamics ofoil deposition in sunflower achenes. They are also consistentwith biochemical evidence (Garces and Mancha, 1989) thatlipid synthesis early in achene filling (up to 12 daa) isassociated more with cell membranes than with the depo-sition of reserve triacylgycerols. In sunflower, all cell typesin the embryo formed oil bodies, including the epidermalcells. Oil-body diameters, measured in both isolated oil

Dryw

eight(mggrain1)

Dryweight(mggrain1)

Dryweight(mggrain1)

A

B

C

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0 10 20 30 40 50

Time (daa)

Pericarp

Embryo

Oil

F I G . 4. Dynamics of pericarp, embryo and oil weights per achene for threesunflower cultivarsDakar (A), 11051 (B) and Prosol 35 (C). Eachpointrepresents the mean of three replicates, vertical bars are 6 1 s.e. The con-tinuous lines show the adjusted bi-linear functions (r2 range 089099) andthe dashed lines show the extrapolations used to estimate the start ofthe effective filling. The arrows indicate the end of the oil growth periodsfor the three achene components. Mean values for timing of initiation andrates and durations of growth for each achene component are given in

Table 2 Table 4.


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bodies and in cell sections, were slightly larger (range 19625 mm) than the upper limit (2 mm) of the range found byTzen et al. (1993) in their survey of 18 species (includingsunflower) and the 0520 mm range suggested by Murphy(2001) for orthodox seeds. No evidence was found forchanges in oil-body size in different tissues of the embryo,unlike maize in which oil bodies from the scutellum arelarger than those from the embryonic axis (Trelease, 1969).The appearance of the first oil bodies in sunflower embryostakes place fairly early during achene filling, earlier than the2 weeks after anthesis reported for rapeseed (Murphy,1990). However, this species difference may be more appar-ent than real in ontogenic terms, as achene-filling takesplace in rapeseed over a longer period of time. There hasbeen some uncertainty about the presence, or otherwise, ofreserve lipids in the pericarp of the sunflower achene. It wasnot possible to find any indication of oil bodies in sectionstaken from pericarp samples harvested up to 89 daa or inthose from mature achenes. Harris et al. (1978) assumed

3 % lipids in pericarps, presumably associated with cellmembranes. However, even if this were a reasonableassumption when applied to the early stages of achenegrowth, it is unlikely to remain so throughout achene filling.While sections of pericarp from very young achenes showednucleated cells and cytoplasm (Fig. 3A, B), both of thesecell components were absent in sections taken from maturepericarp. An additional issue here is that the proportion ofsclerenchyma (with presumably low membrane : wallratios) in the pericarp increases rapidly after 89 daa, andin some cultivars more than others. Although this wholeissue could do with more attention, the available informa-tion suggests that any lipids extracted from the pericarp areassociated with membranes, and the contribution of these

lipids to oil extracted using solvents is likely to lose impor-tance as achene growth progresses. Results presented here(Figs 4 6) are based on the assumption that all lipids arelocated in the embryo, but it should be noted that otherassumptions (e.g. a constant 2 % of pericarp mass assignedto lipids, or 2 % until 12 daa followed by a linear decrease to0 % at physiological maturity) does not alter the temporalpatterns or the differences between cultivars shown in thesefigures.

Protein bodies also appeared in embryo cells early(89 daa) during embryo growth, which is consistent withobservations by Goffner et al. (1988) and Mazhar et al.(1998) about deposition of reserve proteins in sunflower

embryos. The present data also show that the proportionof mature embryo cell sectional area occupied by proteinbodies was significantly greater in the low-oil cultivar thanin the other two, at least partially consistent with the oftennoted trade-off between achene oil and protein concentra-tions (Blanchet and Merrien, 1982; Connor and Sadras,1992; Lopez Pereira et al., 2000). Interestingly, however,the proportions of embryo cell cross-sectional areas takenup by protein bodies was similar between the mid- and highoil embryos, in spite of the much greater embryo oil con-centration of the latter cultivar.

The order in which rapid growth of the components(pericarp, embryo and oil) of the achene initiated and ceasedwas broadly similar, across all three cultivars, to thoseobserved in previous studies using single cultivars(e.g. Villalobos et al., 1996; Rondanini et al., 2003). How-ever, it is also clear that the origins of cultivar differencesin mature achene oil concentration are complex andstrongly linked to the patterns of growth of each component

and its interactions with the remaining ones. Importantly,this study has demonstrated that variations in initial size andrate and duration of growth of the pericarp can impactstrongly on the proportion of the embryo in the acheneand, following on from that, mature achene oil concentra-tion. Equally, the data also show that although the propor-tion of embryo in the achene is an important source ofcultivar differences in final achene oil concentration, culti-var differences in embryo oil concentration also contributeto this effect. Finally the degree of synchrony betweencessation of growth of the whole embryo and of oil hasbeen shown to be a factor in altering final oil concentrationof the embryo and achenes (Figs 5 and 6), in addition todifferences in rates and durations of oil deposition and

embryo growth (Tables 3 and 4). When only the final oilconcentration of the achene is considered, interactions of thetype shown in this work are masked. Taken as a whole, thepresent information also fills the previously existing voidabout the anatomical bases of observations derived exclu-sively from mature achenes (Percie Du Sert and Durrieu,1988; Shoar-Ghafari and Vintejoux, 1997; Bolyakina andRaikhman, 1999). Some interest has been shown in thepatterns of gene expression at the start of achene growth(Mazhar et al., 1998); present results suggest that attentionalso needs to be paid to the switching off of genes close tothe end of achene filling and the factors which may controlthe latter.

T A B L E 2. Dynamics of the growth of pericarp in the three cultivars of sunflower differing in oil content at maturity

Pericarp weight (mg) Duration of growth Growth rate

Cultivar Initial (0 daa) Final daa Cd mg d1 mg (Cd)1

Dakar 4.9 6 0.90a 72.7 6 1.12a 18.6 6 0.21a [429.2 6 8.7a] 3.7 6 0.07a [0.16 6 0.002a]

11051 3.0 6 0.62b 40.7 6 0.71b 15.4 6 0.35b [352.4 6 7.9b] 2.7 6 0.11b [0.10 6 0.002b]Prosol 35 2.7 6 0.66b 26.0 6 0.08c 13.7 6 0.29c [323.4 6 8.4c] 1.8 6 0.06c [0.07 6 0.003c]

Data in square brackets are values of duration and rates on a thermal time basis (base temperature = 1 C).Values are means (n = 3) 6 s.e. and are derived from bilinear functions fitted to experimental data.Within each column, values followed by different letters are significantly (P < 005) different.


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TABLE

4.

Dynamicsofoilmassintheembryo

inthetreecultivarsofsunflowerdiffe

ringinoilcontentatmaturity

Initiationofeffectiveperiodof

oilformation

Effectivedurationofoilcontent

growth

Effectiverateofoilcontent

growth

Totaldurationofoilcontent

growth

Cultivar

Finaloilweight

(mgperembryo)

daa

Cd

d

Cd

mgd1

mg(Cd)1

daa

Cd

%oilinembryo

(d.wtbasis)

Dakar

48.46

0.78a

7.36

0.57a

[168.46

13.7

a]

18.36

0.30a

[455.36

6.9

a]

2.66

0.04a

[0.116

0.003a]

25.66

0.37a

[623.06

8.5

a]

55.96

0.26a

11051

49.46

0.20a

8.56

0.83a

[195.46

20.0

a]

19.16

0.66a

[481.66

15.1

a]

2.56

0.06a

[0.116

0.003a]

27.66

0.47b

[677.06

10.8b]

69.36

0.61b

Prosol35

52.06

0.37b

9.56

0.44a

[216.06

10.6

a]

23.06

0.68b

[512.06

15.6

a]

2.26

0.05b

[0.106

0.004a]

32.46

0.44c

[728.06

10.1c]

72.46

0.25c

Datainsquarebracketsarevaluesofdurationandratesonathermaltimebasis(base

temperature=

1C).Valuesaremeans(n=

3)6

standarderrorandarederivedfrombili

nearfunctionsfittedto

experimentaldata.

Withineachcolumn,valuesfollowedby

differentlettersaresignificantly(P

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