Biochemical markers of female receptivity in maize (Zea mays L.) assessed using in vitro fertilization

Plant Science, 70 (1990) 1 1 - 1 9 Elsevier Scientific Publishers Ireland Ltd.

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B I O C H E M I C A L M A R K E R S OF F E M A L E R E C E P T I V I T Y IN M A I Z E (ZEA M A Y S ASSESSED U S I N G IN VITRO F E R T I L I Z A T I O N

L.)

I S A B E L L E DUPUIS and CHRISTIAN D U M A S Laboratoire de Reconnaissance Cellulaire et Am~Koration des Plantes, L A I N R A ~3 879, Universit~ CL Bernard-Lyon I, Bdt. 7~1, 5~me dtage, ~3 bd du 11 novembre 1918, F-696~2 Villeurbanne Cedex {France)

(Received December 13th, 1989} (Revision received February 7th, 1990) (Accepted March 12th, 1990)

In order to better understand female receptivity in Zea mays L., the various stages of spikelet development were estimated by the total silk length, and assessed for their fertilization ability using an in vitro pollination system. Developmental variations in protein and enzyme patterns of silks and ovaries were analyzed from the young to the senescent spikelets in relationship with the receptivity. While few differences were observed in the SDS-PAGE patterns, we have characterized a set of developmentally regulated silk proteins after IEF and total protein staining, two of them likely corresponding to glycoproteins. Some of these tissue specific and stage specific proteins have been correlated with the receptivity state and may be used as markers. Whereas the esterase activity detection remains very similar throughout spikelet development, the level of general activity of peroxidase isozymes is very high in the silks and increases during the development. The peroxidase activity detection may be useful in monitoring the changes in stigma receptivity. K e y words: maize; female receptivity; in vitro pollination fertilization; protein markers; peroxydase; esterase

Introduction

Female receptivity is a major factor involved in seed set determination. The ability to assess and manipulate stigma receptivity is important for plant breeders, as it could optimize hybrid seed production. It is thus important to know the effective pollination period with respect to the stigma receptivity and ovule viability. Several classical methods are currently available to assess stigma receptivity including determination of seed set after pollination at different times of female development, analysis of stigma secretion, cytochemical tests for non-specific esterase, peroxidase and lectin binding [1]. Previous research in maize was conducted using classical in vivo pollination experiments in the field [2,3] and very little information is available concerning female receptivity. This in vivo pollination method has the disadvantage of involving several environmental factors such as time of the day, heat or cold stresses; and plant

factors such as pollen viability, pattern of silk emergence, gradient of maturity along one ear. Thus, we have used the in vitro pollination method which is a more controlled model for studying fertilization. This technique allows for the definition of the nutritional and environmental conditions, and a precise determination of the developmental stages of spikelets [4]. In the current work, we initiate the investigation of female receptivity in maize at the spikelet level using two complementary approaches. Spikelets of different silk length were tested for their receptivity using an in vitro fertilization model, and the degree of receptivity was related to protein markers using various electrophoretic and staining techniques. Materials and Methods Plant material Zea mays L. genotypes A632, LG1, F7 × F2

0168-9452/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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Fig. 1. Schematic representation of spikelet sampling procedure for in vitro pollinationand protein analysis.The total silk length and external silk length were measured as indicated from the base of ovary (o), after dissectionof the spikelets fromthe ear. (as female) and Wl17 (as male) were grown from May to September 1988, in an experimental field in Lyon. Seeds were obtained from Association G~n~rale des Producteurs de Mais (Pau, France}.

Collection and evaluation of pollen quality Pollen was collected by shaking the mature tassels over an aluminium foil. Viability was estimated with the fluorochromatic reaction [5,6]. Only pollen showing a viability score of over 90% was used immediately after collection.

Spikele t sampling procedure Immature ears were bagged before silk emergence. When the ears were collected, the point where the silks protuded from the husks was labeled with a pen. The female physiological stage was estimated after dissection, both by the total length of the silk and the external length of the silk of each spikelet as explained in Fig. 1.

In vitro pollination The in vitro pollination experiments were performed according to Dupuis and Dumas [4]. During the course of this experiment over 2400 spikelets were examined for their receptivity. Four segments containing six intact pistillate spikelets of various stages {from 1 to 30 cm total silk length) were dissected and plated on a Murashige and Skoog minerals and vitamins medium [7], with 5% sucrose, 500 rag/1 casein

hydrolysate and 0.7% agar at p H 5.6. The silks were cut to a length of 4.5 cm and pollinated on their distal end. After 24 h at 28 °C in the dark, the silks were removed and the cultures returned to the incubator for 15 days. The in vitro fertilization score was expressed as the percentage of normal and abnormal kernel development of the total ovules pollinated as previously defined [4].

Protein extraction and assay For isoelectric focusing (IEF), soluble native proteins were extracted at 4 °C. Silks (200 rag) and ovaries (55 mg) were homogenized in 200 ~l of extraction buffer [50 mM Tris--HCl (pH 8}, 15 mM dithiothreitol (DTT), 2% polyvinylpolypyrrolidone (PVPP)]. The extract was then centrifuged for 15 rain at 15 000 × g. Supernatants were recentrifuged in the same conditions and stored at - 80 °C until use. The protein content was determined using the Bradford assay [8] with bovine serum albumin (BSA) as a standard. For SDS-PAGE analysis, denatured proteins were extracted in a buffer consisting of 62.5 mM T r i s - H C 1 (pH 6.8), 2.3% SDS, 2% DTT and 10% glycerol. The extracts were heated to 1OO°C for 5 rain and centrifuged for 15 rain at 15 000 × g. The supernatants were recentrifuged in the same conditions and stored at - 8 0 ° C . The protein content was determined by the Lowry et al. assay [9] with BSA as a standard. Before electrophoresis, the proteins were precipitated with acetone, redissovled in extraction buffer, and boiled for 5 rain.

Electrophoresis IEF was performed at 4°C on 0.3 mm thick polyacrylamide gels (T = 7.50/0, C = 3%), containing 30/0 carrier ampholytes (pH 3.5-9.5, LKB) in a LKB Multiphor II unit, according to the manufacturer's prescriptions, pH gradients were estimated using pI marker proteins (Pharmacia). SDS-PAGE was performed on a discontinuous system according to Laemmli [10]. Polyacrylamide gels (0.75 mm thick, stacking gel = 4°/O, running gel -- 12%) were prepared and run in a Biorad Protean II slab cell.

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For IEF and SDS-PAGE equivalent amounts of proteins per well were loaded on the gels (SDS-PAGE: 50 ~g; IEF total proteins: 20 ~g; glycoproteins: 50 ~g; esterase and peroxidase: 75 ~g). Three to six replications were run for each experiment and for each genotype to ensure accurate observations.

Staining Total proteins were stained with silver nitrate according to Heukesoven and Dernick [11]. Concanavalin A-binding proteins were detected after IEF by the concanavalin A-peroxidase method [12] adapted to IEF by Gaude [13]. Controls were stained in presence of an inhibitor of concanavalin A (a-methyl mannose, 0.2 M). Staining for enzymes was modified from Kinzkofer and Radola [14] according to Delvall~e and Dumas [15].

Results All the results illustrated are related to the A632 genotype.

In vitro female receptivity period The female receptivity pattern assayed by in vitro fertilization is shown in Fig. 2A. The spikelets are not receptive when their total silk length is shorter than 4 cm. Then, the receptivity increases with silk length and reaches a maximum for spikelets with a 10 cm total silk length. The most receptive period lasts from 10 to 25 cm total silk length. After 25 cm, the receptivity decreases and the spikelets are no longer receptive when their total silk length is more than 30 cm. The two physiological parameters used to estimate the female stage (total silk length and external silk length) can be linearly related (Fig. 2B).

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IEF analysis Total proteins. Eight stages of the receptiv-

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Fig. 2. Female receptivity pattern of A632. (A) 2430 spikelets of different stages of development (from 4 to 33 cm total silk length) were tested for their in vitro fertilization score. Each point represents the average fertilization score of the spikelets of the same age. (Bt Relationship between the total silk length and the external silk length which allows for a prediction of the receptivity from the field plants (r2 = 0.92t.

ity were studied: from 1 cm to 33 cm total silk length. Most of the proteins detected in the silk and ovary extracts focus in the pH range 3.5-5.5, and are clustered and too numerous to allow precise analysis. The ovary protein pattern (Fig. 3A) does not change much during the development except for the later non-receptive stages, which are characterized by the increase of intensity of two protein bands (ol,2). On the other hand, the silk protein pattern (Fig. 3B) shows important modifications during the development in the pH range 6.5-7.5. Major changes are concomitent with the beginning of the receptivity period ( 8 - 1 0 cm total silk length stage): new proteins are first detected at this stage (sl,6,8) and increase in relative intensity as silk length continues (s6). Other protein bands are observed to decrease (s2) or increase (s5) in their relative intensity at the critical 10 cm total silk length stage. Band s3 increase and band s7 decreases in relative intensity throughout the maturation of the spikelet, while band s4 is no longer detectable in the receptive or senescent silks. Glycoproteins. The concanavalin A-binding

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Fig. 3. Isoelectric focusing of proteins from silk (s) and ovary (o) at different developmental stages estimated with the total silk length. The appearent pH is indicated. Changes in the patterns are noted with numbers. (A) total protein staining of ovary extract; (B) total protein staining of silk extract; (CI concanavalin A binding protein staining of ovary, oC = control (stage o15) with inhibitor of eoncanavalin A; (D) concanavalin A binding protein staining of silk, sC = control (stage s15) with inhibitor of concanavalin A, the s30 total protein staining is presented for comparison.

p a t t e r n a f t e r I E F s e p a r a t i o n is v e r y d i f f e r e n t for o v a r i e s a n d silks. T h e o v a r y p a t t e r n d i s p l a y s a v e r y faint b a n d in t h e p H r a n g e 5 . 5 - 9.5 w h e n c o m p a r e d w i t h its c o n t r o l w i t h o u t c o n c a n a v a l i n A (Fig. 3C). In c o n t r a s t , t w o s t r o n g l y labeled b a n d s a r e d e t e c t e d in t h e silk

p a t t e r n s . T h e s e silk specific g l y c o p r o t e i n s follow t h e s a m e v a r i a t i o n s d u r i n g t h e silk d e v e l o p m e n t a n d h a v e t h e s a m e a p p a r e n t p I as t h e p r o t e i n s s3 and s5 identified w i t h t h e t o t a l prot e i n s t a i n i n g (Fig. 3D). Peroxidase. T h e level of g e n e r a l a c t i v i t y of

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tion, w h e r e basic i s o z y m e s a r e only d e t e c t a b l e in the y o u n g s t a g e s . In the o v a r y p a t t e r n , no reliable c h a n g e s occur d u r i n g the d e v e l o p m e n t . T h e a p p a r e n t q u a n t i t a t i v e c h a n g e s visualized on the gel w e r e not a l w a y s r e p e a t a b l e in all the t h r e e e x p e r i m e n t a l replications and shall therefore not be discussed.

SDS-PA GE analysis T h r e e c h a r a c t e r i s t i c s t a g e s of the r e c e p t i v ity period w e r e studied by this m e t h o d : young, m a t u r e (fully r e c e p t i v e ) and s e n e s c e n t (nonr e c e p t i v e ) spikelets. Most of the d e t e c t e d poly-

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Fig. 4. Enzyme activity staining after isoelectric focusing of proteins from silk is) and ovary (o) at different developmental stages estimated with the total silk length. (A) peroxidase activity staining; (B) esterase activity staining.

p e r o x i d a s e i s o z y m e s d e t e c t e d with our s t a i n i n g technique is m u c h higher in the silk t h a n in the o v a r y (Fig. 4A). In the y o u n g silks, the s t a i n i n g is v e r y low and i n c r e a s e s d u r i n g m a t u r a t i o n while the s e n e s c e n t silks a r e c h a r a c t e r i z e d b y a v e r y intense staining. In c o m p a r i s o n , t h e general p e r o x i d a s e a c t i v i t y in t h e o v a r y s t a y s v e r y low t h r o u g h o u t the d e v e l o p m e n t , h o w e v e r , t h e s e n e s c e n t s t a g e displays a slight i n c r e a s i n g in the staining (Fig. 4A). Esterase. T h e e s t e r a s e a c t i v i t y d e t e c t i o n r e m a i n s similar t h r o u g h o u t silk and o v a r y d e v e l o p m e n t (Fig. 4B). H o w e v e r , a slight modification can be o b s e r v e d in silk m a t u r a -

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Fig. 5. SDS-PAGE of proteins from silk (s) and ovary (o) from 8, 22 and 30 cm total silk length spikelets. The molecular masses in kDa are indicated. Changes in the pattern are noted with letters.

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Fig. 6. Diagram of changes in SDS-PAGE, IEF total protein and glycoprotein patterns during silk (s) and ovary (o) development.The three sequences of receptivity correspondingto young(I), fullyreceptive (II) and senescent spikelets (III), were determinedusingin vitro pollinationfertilization.

peptides remained similar throughout ovary and silk development (Fig. 5). However, during silk development, band B is specific for the earlier stages while band A was detected first in the receptive silk. Quantitative differences were also observed for several other polypeptides: band D decreased, band C and E increased in relative intensity during the silk development. Discussion For the fertilization to occur in flowering plants, the first requirements are female receptivity and male viability. Although the pollen quality has been extensively described [16,17] there is almost no information available concerning the female receptivity mechanisms at the molecular level. In order to better understand female receptivity in Z e a mays, we correlate in the present study, in vitro fertilization success in maize to the maturation stage of the spikelets as measured from silk length, and to

tissue-specific biochemical modifications as obtained from protein analysis. Three different sequences can be distinguished in the female development in relationship to the receptivity: (1) immature partially receptive spikelets, with a O - 1 0 cm total silk length; (II) mature fully receptive spikelets, with a 10--25 cm total silk length; and (III) senescent non-receptive spikelets with a total silk length over 25 cm. This pattern of receptivity has been strongly established by in vitro pollination of nearly 2500 spikelets originating from different ears at various stages of development. These different developmental stages revealed specific and consistent changes in protein patterns as summarized in Fig. 6. Therefore, we have identified a set of protein markers that correlate to the degree of receptivity in the different maturation stages of the spikelets. Silk receptivity and ovule viability are the two components of the female receptivity, but the seed-set experiments do not allow for the

17 individual analysis of each of these components, therefore, we have separately analysed the silk and ovary proteins. The location of the production of certain protein may give a further elucidation to their function. During the ovary development, only slight differences were detected with the SDS-PAGE and IEF techniques and no major glycoproteins were identified in the pH range analyzed. However, in the senescent state two new proteins were detected (ol,2) and may be related with the disorganization of the embryo sac. In contrast, many qualitative modifications during the silk development are detected using IEF techniques. The patterns of the very young silks and the corresponding ovaries are very similar, possibly because of the differentiation of the silk from the carpellar tissue of the ovary. Later on, the silk pattern becomes more differentiated as its length increases. The 8--10 cm total silk length stage appears to be a critical developmental stage where maximum receptivity is correlated with important changes at the protein level. Furthermore, we have tested two additional genotypes (LG1, F7XF2) with the same technique, and several proteins (sl,2,3,5,6,7,8) show the similar pattern of modifications during the silk development {data not shown). Some of these gene products may have an important function in the receptivity mechanism. The tissue specificity, the stage specificity, and the fact that these proteins are detected in the three genotypes tested are consistent with this hypothesis. Furthermore two of these potential protein markers (s3,5) likely correspond to glycoproteins with a-D-mannopyranosyl and a-D-glycopyrannosyl residues. Glycoproteins have been previously detected at the cytochemical level on the stigma surface of several species [18,19] and are thought to play an important role in the pollen stigma interactions [20]. However, the fully receptive state is not correlated with one unique specific protein, and further investigations are needed to better understand the function of silk specific proteins.

We also investigated the isozyme patterns of peroxidase and esterase during female development and related the occurrence of peroxidase with the silk maturation. These enzymes were selected on their reported involvement in female receptivity. Previous cytochemical studies allowed for a localization of peroxidase and esterase activities on the stigma surface of different species. At the cytochemical level, the stigma peroxidase labeling increased throughout the duration of anthesis and a low pollen adhesion rate has been previously correlated with the absence of stigma peroxidase in Pedicularis canadensis and Clintonia borealis [21]. Earlier studies also revealed that the receptive surface in stigmas of a wide range of plants had cytochemically detectable esterase activity and this enzyme was considered as a marker of receptivity [22,23]. However, stigma non-specific esterase activity was later detected at all the developmental stages of the flower of Nicotiana sylvestris, N. tabacum, Petunia hybrida, Crinum defixum and Amaryllis vittata, although the stigma of the young stages were non-receptive according to pollen adhesion and germination [24,25]. The present approach examines peroxidases and esterases at the whole silk or ovary level. Whereas silk and ovary esterase isozymes were detected at all development stages, peroxidase isozymes were not detected in the immature silks stages and the intensity of labeling progressively increased during silk maturation. Although we can not relate this peroxidase synthesis or activation with a cellular function at this time, peroxidase activity may be useful in monitoring the changes in the stigma receptivity. To predict the female receptivity of the plant from its external characteristics in the field, our in vitro fertilization data can be applied because of the direct relationship between total silk length and external silk length (Fig. 2). For the A632 genotype, the receptivity appears before the emergence of the silks from the husks and lasts until the silk reaches a length of 30 cm. Therefore, at the effective time of exposure to pollen grains in natural conditions,

18 t h e silks a r e a l r e a d y r e c e p t i v e . H o w e v e r , t h e i m p o r t a n c e of t h e g e n o t y p e in t h e f e m a l e r e c e p t i v i t y d e t e r m i n i s m has b e e n r e p o r t e d p r e v i o u s l y b y s e v e r a l a u t h o r s [26,27] a n d it is t h u s i m p o r t a n t to d e f i n e t h e r e c e p t i v i t y for each g e n o t y p e of i n t e r e s t . I n c o n c l u s i o n , t h e d a t a p r e s e n t e d in t h i s s t u d y d e m o n s t r a t e t h a t r e c e p t i v i t y is a complex p h e n o m e n o n . H o w e v e r , t h e d e g r e e of r e c e p t i v i t y can be m o n i t o r e d using either the field c h a r a c t e r of t h e p l a n t or t h e p r o t e i n patt e r n of t h e silk. T h e a c q u i s i t i o n a n d loss of r e c e p t i v i t y w a s r e l a t e d to a s e t of t i s s u e a n d stage-specific gene products. These proteins t h a t we h a v e i d e n t i f i e d to u n d e r g o e n h a n c e d e x p r e s s i o n in silks will be u s e f u l tools in f u t u r e s t u d i e s on g e n e e x p r e s s i o n d u r i n g t h e p r o c e s s of a c q u i s i t i o n of f e m a l e r e c e p t i v i t y , e s p e c i a l l y in r e l a t i o n s h i p w i t h t h e i n i t i a l p o l l e n / s t i g m a recognition step.

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Acknowledgements 14 W e t h a n k s D r s . P. V e r g n e a n d V.T. W a g n e r for t h e i r c r i t i c a l r e a d i n g of t h e m a n u s c r i p t , a n d M r s . C. M o u l i n for h e r v a l u a b l e t e c h n i c a l assistance. This work was supported by a grant f r o m A s s o c i a t i o n G ~ n ~ r a l e des P r o d u c t e u r s de Mais.

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K.R. Shivanna and D.C. Sastri, Stigma-surface esterase activity and stigma receptivity in some taxa characterized by wet stigmas. Ann. Bot. 47 (1981) 5 3 - 6 4 . M.K. Kandasamy and U. Kristen, Developmental aspects of ultrastructure, histochemistry and receptivity of the stigma of Nicotiana sylvestris. Ann. Bot., 60 (1987} 427 -- 437.

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B.G. Gegenbach, Genotypic influences on in vitro fertilization and kernel development of maize. Crop Sci., 17 (1977)489-492. R.K. Higgins and J.F. Petolino, In vitro pollination fertilization of maize: influence of explant factors on kernel development. Plant Cell Tissue Organ. Cult., 12 (1988) 21 - 30.