mRNAs and a cloned histone gene are differentially expressed during

anther and pollen development in rice {Oryza sativa L.)

V. RAGHAVAN

Department of Botany, The Ohio State University, Columbus, OH 43210, USA

Summary

Spatial and temporal changes in the distribution ofmRNA sequences during anther and pollen devel-opment in rice (Oryza sativa) were investigated byin situ hybridization with [3H]polyuridylic acid([3H]poly(U)) and a cloned rice histone gene probe.Annealing of sections with [3H]poly(U) showed thatpoly (A)-containing RNA (poly (A)+RNA) was uni-formly distributed in the cells of the anther primor-dium. During the formation of the archesporialinitial, the primary parietal cell, the primary sporo-genous cell and tapetum, there was no differentialaccumulation of poly(A)+RNA in their progenitorcells. Preparatory to meiosis, there was a sharpdecrease in poly(A)+RNA concentration in the epi-dermis and middle layer of the anther wall,although the label persisted in the endothecium,

tapetum and microsporocytes. Poly(A)+RNA con-centration decreased in these cell types duringmeiosis and attained very low levels in the disinte-grating tapetum and the persistent endothecium ofthe post-meiotic anther. Pollen development wascharacterized by the absence of [3H]poly(U) bind-ing sites in the uninucleate microspores and bytheir presence in the vegetative and generative cellsof the bicellular pollen grain. In anther sectionshybridized with [3H]histone probe, gene expression'was only detected in the endothecium of the pre-meiotic anther and in the bicellular pollen grains.

Key words: anther, histone gene, in situ hybridization, Oiysasativa, poly(A)-containing RNA.

Introduction

On the basis of its size and internal structure, the antheris reckoned to be a highly reduced organ of the rice(Oryza sativa) floret. The mature anther is less than3 mm long and 0-5 mm wide, and consists of a wall ofthree concentric layers of diploid cells constituting (fromthe outside to inside) the epidermis, endothecium andmiddle layer and a layer of tapetum. The outer layers ofthe anther enclose a locule, within which are formed1200-1500 pollen grains. Since pollen grains are born outof a reduction division of the pollen mother cell (micro-sporocyte), they have the haploid or gametic number ofchromosomes. During its short life span, the pollen grainundergoes only two divisions. The first division (the firsthaploid mitosis) is characteristically asymmetric andresults in the formation of a large vegetative cell and asmall generative cell; next, the generative cell divides toform two sperm, which are involved in the act of doublefertilization in the embryo sac (Raghavan, 1988).

Besides its traditional role in the chain of events leadingto spermiogenesis, the anther of rice has attained greatimportance in recent years in the generation of pollen-derived haploids by tissue culture techniques (Raghavan,1986, for review). Thus, the anther provides an interest-Journal of Cell Science 92, 217-229 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

ing system for examining the control of gene expressionduring cell specialization in the wall layers and tapetumand during developmental transformations of the pollengrains. However, biochemical analysis of the anther walland pollen grains is limited by the heterogeneous natureof their constituent cells and by the difficulty of isolatingthem in enriched fractions. For some purposes, theuse of cytological methods for the detection of macro-molecules, such as polyadenylic acid-containing RNA(poly(A)+RNA) and mRNAs for specific proteins insquashed or sectioned preparations, offers a way toovercome these difficulties. Since many nuclear-coded mRNAs are tailed by a covalently linked poly-adenylate sequence, in situ hybridization mapping ofpoly(A)+RNA in cells and tissues by [3H]pblyuridylicacid ([ H]poly(U)) might conceivably identify the sitesof mRNA accumulation or utilization. In a similar way,spatial distribution of specific mRNAs monitored byannealing tissue sections with labelled DNA or RNAprobes of cloned genes might provide insight into the siteof synthesis of the corresponding proteins.

The spatial accumulation of mRNAs has been followedin various animal systems by in situ hybridizationmethods using [3H]poly(U) as a probe (Angerer &Angerer, 1981, for review; Phillips, 1985). Using sea-

217

urchin eggs and cleaving embryos as a model, Angerer &Angerer (1981) have defined the optimum conditions forannealing of [3H]poly(U) to poly(A)+RNA of sectionswith a minimum non-specific binding. This study hasalso shown that silver grain counts made from autoradio-graphs of sections annealed with the radioactive probe areproportional to the known poly(A) content of the system.Among plants, the spatial distribution of poly(A)+RNAin developing anthers and pollen grains of Hyoscyamusniger (Raghavan, 1981a,b) and Lilium (Porter et al.1983), and germinating spores of the fern, Onocleasensibilis (Raghavan, 1987), has been followed by anneal-ing sections with [3H]poly(U). In recent years, consider-able attention has been paid to in situ detection of specificgenes in a number of animal systems (Jamrich et al. 1984,for review; Jeffery, 1984; Angerer et al. 1984, 1985;Gizang-Ginsberg & Wolgemuth, 1985; Guelin et al.1985; Lawrence & Singer, 1986; Lawrence et al. 1988)and diverse plant organs (Anderson et al. 1986; Harris &Croy, 1986; Martineau & Taylor, 1986; Meyerowitz,1987; Aoyagi & Chua, 1988; Barker^al. 1988; Langdaleet al. 1988; McFadden et al. 1988), but no one hasapplied this tool to follow gene expression in the anther.

In the present study, I have monitored the subcellulardistribution of mRNA and a histone gene during antherand pollen development in rice by hybridization of[3H]poly(U) and cloned [3H] histone asymmetric RNAprobes, respectively, to sections. Northern blot analysishas been employed to verify the presence of histone genesequences in the anther and to corroborate informationobtained by in situ hybridization. The histone probe wasselected because histones are relatively conservedthrough evolution; moreover, on the basis of microspec-trophotometric, autoradiographic and cytochemicalanalyses, there is evidence for differential histone ac-cumulation in the vegetative and generative cells ofangiosperm pollen grains (Sauter & Marquardt, 1967a,b;Reznikova et al. 1978; Sangwan-Norreel, 1978; Bed-narska, 1981). Interest in the spatial localization ofhistone transcripts was also spurred by their possibleinvolvement in complexing with DNA of the rapidlydividing cells of the anther. The state of our knowledge ofthe structure and organization of histone genes in higherplants has recently been reviewed (Chaubet et al. 1987).

Materials and methods

Plant material

Plants raised from caryopses of Oryza sativa (IR-30) obtainedfrom The International Rice Research Institute, Manila, Phi-lippines, were induced to flower under a set of standardconditions described elsewhere (Raghavan, 1988).

Histological methodsEntire florets and anthers dissected from florets were fixed in70% ethanol/acetic acid (3:1) mixture for 24h. They weredehydrated through 90% and 100% ethanol, «-propanol and n-butanol (24 h each) and embedded in glycol methacrylate(Feder & O'Brien, 1968). Longitudinal and transverse sectionscut at 7 ,um thickness were attached to slides with drops of waterand dried overnight at 55 °C on a slide warmer before using in

the annealing reaction with [3H]poly(U). For /;/ situ hybridiz-ation with the histone gene probe, specimens fixed in aceticalcohol were dehydrated in 90 and 100% ethanol for 24 h eachand embedded in non-polar methacrylate (9 parts butyl-methacrylate and 1 part methyl-methacrylate, 1 % benzoylperoxide) (Jamrich et al. 1984). Sections cut at 0-5-1-Ofimthickness were transferred to drops of water on a slide, spreadwith chloroform vapour and dried overnight on the slide

Annealing with [3H]poly(U)In situ hybridization of sections with [3H]poly(U) essentiallyfollowed the method of Capco & Jeffery (1978), as modified inthis laboratory for use with plant materials (Raghavan, 1981a,6,1987). In the protocol followed here, 80-jt*l samples of2-OjuCiml"1 [3H]poly(U) (sp. act. 2-3-6-98 Ci mmol~{ UMP;New England Nuclear Co., Boston, MA) dissolved in thehybridization buffer (lOmM-Tris-HCl (pH7-6), 200 mM-NaCl, 5 mM-MgCl2) were applied to sections on slides that hadbeen rinsed in the same buffer. The slides were then sealed witha Corning brand coverslip and incubated for 4h at 47 °C in amoist chamber equilibrated with the hybridization buffer. Afterannealing, the slides were successively rinsed in cold tap water,hybridization buffer and RNase digestion buffer (high saltbuffer: 50mM-Tris-HCl (pH7-6), 100niM-KCl, lraM-M g C y . Unhybridized [3H]poly(U) was removed by treatmentof slides with pancreatic RNase A (Sigma Chemical Co., StLouis, MO; S0//gml~' RNase digestion buffer) for 1 h at 37°C.[3H]poly(U) binding sites were detected autoradiographicallyby dipping the dry slides in Kodak NTB-3 (Eastman KodakCo., Rochester, NY) liquid emulsion diluted with an equalvolume of water. After exposure in the dark for 4 weeks, slideswere developed using standard dark-room techniques. Theywere stained through the processed emulsion with 0-1 % eosinY, dried and mounted in Euparal.

Data analysisSlides were examined in a Zeiss Photomicroscope III with 63Xoil immersion objective. Autoradiographic quantification wasdone by counting silver grains from the surface of cells or ofpollen grains selected randomly from sections of anthers ofmore or less the same morphological stage of development. Ineach case 5-15 cells or pollen grains were counted and the datawere analysed statistically using a computer program in anApple He computer. Silver grains were also counted from areasadjacent to the sections to determine emulsion backgrounds,which have been subtracted from the values given here.

Histone gene probe preparationThe histone gene used in this study is a 1-3 kb (103 bases) insertisolated from a genomic library of 10-day-old seedlings of IR26variety of rice (Peng & Wu, 1986). The insert, which is ligatedinto the BainHl site of pBR322 includes a 405 bp (base-pairs)coding sequence that starts with ATG and terminates with astop codon TGA. According to Wu et al. (1986), the sequenceshows 80% homology with H3 histone gene from sea-urchinand 92% homology with wheat H3 histone gene.

Asymmetric RNA probes were prepared by ligating the1-3 kb insert of pRH3-2 (kindly provided by Dr Ray Wu,Cornell University) into the transcription vector pBS M13 +

(formerly known as Bluescribe; Stratagene Cloning Systems,San Diego, CA). This vector is synthesized by inserting phageT3 and T7 transcriptional promoters into the multiple cloningsite adjacent to the lacZ gene of pUC19. Following transform-ation of Escherichia coli JM101, recombinant plasmid DNAwas isolated by banding in a caesium chloride density gradient

218 V. Raghavan

and was purified according to the protocol of Cox et al. (1984).Linear templates complementary to the histone mRNA ('anti-sense' or non-coding strand) and the opposite insert orientation('sense' or coding strand) were obtained by truncation ofpurified plasmid DNA at the Hi)idlll (about 30bp down-stream) and t'coRl (about 26bp downstream) sites, respect-ively. The digested samples were extracted sequentially withphenol: chloroform, chloroform and ether and passed over G-50Sephadex column. Single-stranded RNA sense and antisenseprobes were prepared using Promega Biotec (Madison, WI)transcription system and T3 or T7 polymerase (StratageneCloning Systems) as appropriate and were labelled with[3H]ATP (sp. act. 32Cimmor ' ) , [3H]CTP (sp. act. 31 Ciminor1) and [3H]UTP (sp. act. 35 Cimmor 1 ) (all from 1CNRadiochemicals, Irvine, CA) to a specific activity of 4X106 toSxlO6disintsmin~' f.ig~ . The probes were dissolved in Den-hardt's solution (0-02% each of bovine serum albumin (SigmaChemical), Ficoll (Pharmacia, Piscataway, NJ) and polyvinyl-pyrollidone and 10% dextran sulphate (both from SigmaChemical)) containing 0-3 M-NaCl, 20mM-Tris-HCl (pH8-0),5 mM-EDTA and 50% recrystallized formamide, to a concen-tration of about 1X105 to 2xl05disintsmin~'jil"1. 32P-labelled RNA antisense probe was prepared to a specific activityof 6xlO5 to 7xl05disintsmin~',ug by including [32P]UTP(sp. act. 3000 Ci mmol"1; ICN Radiochemicals) in the reactionmixture.

Annealing with f H]histone probesDry slides carrying anther sections were dipped for 5 min eachin two changes of xylene to remove the plastic and passedthrough a decreasing ethanol series to water. The slides werenext incubated in a solution containing lOOmM-Tris-HCl(pH8-0), 50 mM-EDTA and l^gml" 1 proteinase K for 10 minat 37°C and washed briefly in distilled water and air-dried. Forannealing, 5-7 (A of the probe (7xlO5 to 8x 105 disintsmin"1,total) was placed on a 18 mm square Corning brand coverslip,which was gently inverted over sections on the slide. The edgesof the coverslip were sealed with rubber cement and the slidesincubated in a moist chamber at 45-47°C for 24 h. For eachstage of anther and pollen development, the distribution ofhistone gene was monitored by annealing sections with T7promoter-generated histone antisense probe; controls consistedof duplicate sections annealed with T3 promoter-generatedhistone sense probe of the same specific activity.

After hybridization, the slides were washed in severalchanges of 2 X SSC (1 x SSC is 0-15M-NaCl, 0-15M-sodiumcitrate) for 4h, once in RNase A ( 2 0 ^ g m r ' in 2 X SSC) for20 min, several changes in 0-1 X SSC for 2h, and 5 min each inan ascending ethanol series to 100% ethanol (all at roomtemperature). Air-dried slides were dipped in the photographicemulsion for autoradiography. Autoradiographs were stained inToluidine Blue, dried and mounted in Euparal.

RNA isolation and Northern blot hybridizationAnthers of different stages of development were collected over aperiod of 6 months and quick-frozen in liquid N2 and stored at-70°C. Total RNA was extracted from about 500 mg of anthersby a modification of Dudley & Northcote's (1978) method. Inthe protocol followed here, the material was ground in thecold in a chilled mortar with 5 ml of a medium containinglOmM-Tris-HCl, pH8-3, 5mM-ethylene glycol-bis(/3-amino-ethyl ether)-/V,yV,iV',Af'-tetraacetic acid (EGTA), 100mM-2-mercaptoethanol, 0-1 % sodium dodecyl sulphate (SDS) and50,ul diethylpyrocarbonate (DEP). After centrifugation of thehomogenate at 5000 £ (20min at 4°C), the supernatant wasshaken vigorously with an equal volume of phenol: chloroformand the aqueous phase was separated by centrifugation at

12000g (20min at 25°C). The phenol: chloroform extractionwas repeated five times. RNA was recovered from the aqueousphase by ethanol precipitation in the presence of 0'04vol. of5M-NaCl. The precipitate was dissolved in TE buffer, p H 8 0(lOmM-Tris-HCl, 1 mM-EDTA) and, following the addition of0-25 vol. each of 8M-urea and 10M-lithium chloride, the mix-ture was incubated overnight at 4°C. RNA was collected bycentrifugation at 12 000£ (20 min, 4°C), resuspended in TEbuffer and ethanol-precipitated in the presence of 0-1 vol. of3 M-potassium acetate.

For Northern blot analysis, RNA was dissolved in1 X 3-(Ar-morpholino)-propanesulphonic acid (Mops) buffer(20mM-Mops, SmM-sodium acetate, 1 mM-EDTA, p H 7 0 )containing 5% formaldehyde and 50% formamide, heated to65°C for 5 min, quick-cooled on ice and electrophoresedthrough 1 % agarose gels containing 6 % formaldehyde and1 X Mops buffer. After staining the gel in ethidium bromide,RNA fragments from the gel were transferred to a nitrocellulosefilter (Bio-Rad Laboratories, Richmond, CA) using 2 X SSC asthe solvent during blotting for 12-16h. The filter was dried,baked in a vacuum oven at 75 °C for 3 h and then incubated for3 h at 55°C in 5-10ml hybridization buffer (50% formamide,0-75M-NaCl, 0-lSM-Tris-HCl, pH8-O, 10mM-EDTA, 0-2M-phosphate buffer, pH6-8, 1 X Denhardt's solution, 10% dex-tran sulphate, 0-1% SDS). The buffer was drained off andreplaced with fresh buffer containing 2X106 to 3xl0°disintsmin~ 32P-labelled antisense RNA probe. After incubation at55 °C for an additional 16 h, the filter was washed three timeswith 2 X SSC and then incubated in 2 X SSC containingl^gml" 1 RNase A for 15 min at room temperature. After afinal wash for 30 min in O'l X SSC containing 0 1 % SDS at50°C, the filter was blotted and exposed to Kodak X-Omat filmat —70°C for 24 h using an intensifier screen. The film wasdeveloped using standard dark-room protocols.

Results

Localization of poly(A)+ RNA in the premeiotic antherSince anther and pollen development in rice IR-30 hasrecently been described (Raghavan, 1988), this infor-mation is not presented here except insofar as it isnecessary to clarify certain division sequences. Changesin the spatial distribution of [3H]poly(U) binding sites inthe cell lineages of the developing anther are presenteddiagrammatically in Fig. 1. Representative autoradio-graphs are given in Figs 2-10. There are three majorfeatures in the pattern of local poly(A)+RNA changes inthe premeiotic anthers of rice. First, in the antherprimordium containing a homogeneous mass of cells,autoradiographic silver grains were present at the samedensity over most cells following in situ hybridizationwith [ H]poly(U) (Fig. 2). Although silver grain concen-tration varied between cells of anthers of different florets,no variations in grain density were observed between thedifferent cells of the same anther. Second, prior to orfollowing the delimitation of a hypodermal cell as thearehesporial initial, there was no appreciable increase inthe number of silver grains present in this cell ascompared to other cells of the anther. As the arehesporialinitial divided to form the primary parietal cell and theprimary sporogenous cell, the number of [3H]poly(U)binding sites in the newly formed cells remained more orless the same (Fig. 3). The case in which the primary

mRNAs in rice pollen development 219

I. Stage of anther primordium

1. Epidermis 91 ±3-92. Hypodermis 11-8 ±4-43. Third layer of cells from outside 11-9 ± 4-74. Midregion 101 ±4-3

II. Stage of archesporial -initials1. Epidermis 12-9 ±4-22. Archesporial initial 12-3 ± 4-63. Hypodermis (other than the archesporial initial) 11-5 ± 3 04. Third layer from outside 13-1 ± 6-25. Midregion 12-314-5

III. Stage of primary sporogenous cells IV. Three-layered wall stage1. Epidermis 10-5 ± 1-8 1. Epidermis 10-7 ± 3-42. Primary parietal cell 12-2 ± 4-7 2. Endothecium 9-9 ±2-93. Primary sporogenous cell 12-3 ± 5-2 3. Innermost layer 13-4 ± 4-74. Layer of cells around the primary sporogenous cell 10-7 ± 1-7 4. Primary sporogenous cells 13-6 ± 5-3

V. Stage of differentiation of the tapetum1. Epidermis 4-5 ± 1-72. Endothecium 8-6 ± 3-93. Middle layer 4-7 ±2-04. Tapetum 11-5 ±5-15. Microsporocytes 121 ± 3-6

VI. Microsporocytes ready to undergo meiotic division1. Epidermis 2-9 ± 1-42. Endothecium 9-6 ± 4-23. Middle layer 1-5 ±1-14. Tapetum 12-8 ± 4-45. Microsporocytes 11-5 ± 2-9

Fig. 1. Diagrammatic representations of the early stages of anther development in rice with mean numbers of autoradiographicsilver grains (±standard error) in the cell types indicated in each diagram. Stages of anther development are traced fromphotographs (see Raghavan, 1988).

220 V. Raghavan

I. Jt•*

Fig. 2. Autoradiograph of a section of an anther primordium following in situ hybridization with [3H]poly(U). In Figs 2-4:e, epidermis; en, endothecium; i, inner layer; pc, primary parietal cell; sc, primary sporogenous cell. Bars, 10;mi.Fig. 3. A. Autoradiograph of a section of an anther lobe following in situ hybridization with [3H]poly(U). B. Same section,with focus on cells. The archesporial cell has divided to form the primary parietal cell and the primary sporogenous cell.Fig. 4. A. Autoradiograph showing annealing of [3H]poly(U) to section of an anther lobe at the stage of division of the primaryparietal cell to form the endothecium and an inner layer. B. Same section, with focus on cells.

parietal cell and other cells surrounding the primarysporogenous cell divided anticlinally to form the endo-thecium and the inner layer was similar, and also whenthe latter divided to give rise to the middle layer andtapetum (Figs 4, 5). Silver grain counts made on alimited number of archesporial initials, primary parietalcells and cells of the inner layer did not reveal anychanges in poly(A)+RNA concentrations before theirdivisions. These observations indicate that the initialevents of cell differentiation during microsporogenesis inthe rice anther are not associated with any overt changesin steady-state levels of poly(A)+RNA in the cells. Itshould, however, be noted that since the number of cellsof the anther increases markedly during premeiotic

development, there is an actual increase in the number of[3H]poly(U) binding sites per anther.

The third feature of [3H]poly(U) binding pattern isthat in anthers in which the microsporocytes were poisedto undergo meiosis, there was a sharp decline inpoly(A)+RNA concentration in the cells of the epidermisand middle layer, although the endothecium and tapetumas well as the microsporocytes had high concentrations ofsilver grains (Figs 5, 6). This finding suggests that theconcentration of poly(A)+RNA present in the two walllayers of the anther at an earlier stage was diluted duringtheir differentiation. During and after meiosis in themicrosporocytes, the endothecium and tapetum con-tinued to bind the label at a reduced rate (Fig. 7). With

mRNAs in rice pollen development 221

•••-• - e x '".» »

e ——en

—ml

*

A77• *

. 5A .• 5BFig. 5. A. Autoradiographic localization of [ H]poly(U) binding sites in the longitudinal section of an anther, at the stage offormation of the tapetum and microsporocytes. B. Same section, with focus on cells. In Figs 5, 6: e, epidermis;en, endothecium; in, microsporocytes; ml, middle layer; t, tapetum. Bar, 10/xm.

the onset of radial thickening in the walls of the endo-thecial cells, [3H]poly(U) binding sites completely disap-peared from these cells. Silver grains were, however,found even over remnants of the tapetal cells undergoingdegeneration in situ (Fig. 8).

Poly(A)+RNA accumulation during pollen developmentThe presence of high concentrations of poly(A)+RNA inthe microsporocytes made it of interest to follow the fateof mRNA during meiosis and pollen development. Asseen in Figs 5, 6, [3H]poly(U) binding occurred mostlyin the cytoplasmic region of the microsporocytes.Although it was not possible to identify specific stages ofmeiosis in autoradiographs of sections of anthers sub-jected to in situ hybridization with [3H]poly(U), therewas a reduction in silver grain density in the microsporo-cytes during meiosis and in the microspores formed(Fig. 7). Presumably, poly(A)+RNA molecules presentin the microsporocytes prior to meiosis were utilizedduring meiosis. A similar decrease in the level ofpoly(A)+RNA has been observed by biochemical analy-sis and by in situ hybridization during meiosis in themicrosporocytes of Lilium (Porter et al. 1983).

Uninucleate microspores were not targets ofpoly(A)+RNA accumulation and labelling over micro-spores following their release from the tetrad up to thevacuolate bicellular stage was barely above background(Table 1). Significant accumulation of poly(A)+RNAwas first detected with the appearance of starch grains inthe bicellular pollen grains; [3H]poly(U) binding siteswere present over both generative and vegetative cells ofsuch pollen grains (Fig. 9). Silver grain density above thepollen cytoplasm remained relatively stable during starchaccumulation, but declined as the pollen became com-pletely starch-filled (Fig. 10). At this stage, pollen grainsJacking cytoplasmic label but with binding sites localizedeither over the vegetative cell nucleus or the generativecell nucleus or over both nuclei were also found; thissuggests the possibility that with progressive maturationof pollen grains, poly(A)+RNA is not processed andexported as mRNA into the cytoplasm. The pattern of[3H]poly(U) binding in pollen grains of rice is somewhatsimilar to that reported in Hyoscyamus niger (Raghavan,19816).

Specificity of [3H]poly(U) binding to poly(A)+RNAThe specificity of [3H]poly(U) as a probe for poly (A)

222 V. Raghavan

*

f

. *x

I

I

/77

• . .

6A. . , '<*.6B

Fig. 6. A. Autoradiograph showing annealing of [3H]poly(U) to the longitudinal section of an anther at an early stage ofmeiosis. B. Same section, with focus on cells.

Table 1. Number of autoradiographic silvergrains ± standard deviation on pollen grains of rice

following in situ hybridization of sections with[JH]poly(U)

Pollen developmental stage

Soon after release from the tetradUninucleate, vacuolateBicellular, vacuolateBicellular, non-vacuolateBicellular, partly starch-filledBicellular, completely starch-filledBicellular, partially hydrolysed (from

anthers ready to dehisce)

Number of silver grains± standard deviation

4-2 ±1-93-5 ± 1-44-5 ± 1-8

30-8 ±7-477-6 ±17-234-9 ±8-421-1 ±4-8

sequences of cellular RNA was previously established forparaffin sections of various types of animal tissues (Capco& Jeffery, 1978; Angerer & Angerer, 1981; Phillips,1985) and for plastic sections of anthers and pollen grainsof Hyoscyamus niger (Raghavan, 1981a,b) and Lilium(Porter et al. 1983). Similar controls were carried outwith sections of premeiotic anthers of rice. As shown inTable 2, the interaction of [3H]poly(U) with sections ofrice anthers was sensitive to pretreatment with 0-1 M-

KOH, which presumably hydrolyses RNA into itsnucleotides. Pretreatment of sections with RNase dis-solved in a buffer containing 10 mM-KCl (low salt buffer)reduced silver grain density to 25-30 % of that observedover untreated sections, while sections pretreated withRNase A dissolved in the high-salt buffer retained nearly90 % of silver grains observed over the untreated control.Since poly(A) sequences tailed to RNA are RNase-sensitive at low salt concentrations (Beers, 1960; Darnellet al. 1971), these data are consistent with the view thatsilver grain accumulation seen in autoradiographs ofsections of rice anthers annealed with [3H]poly(U) is dueto the formation of complexes with poly(A)+RNA. Othercontrols have also tended to support this conclusion. Forexample, pretreatment of sections with RNase T2,known to hydrolyse the phosphodiester linkages withadjacent adenylic acid residues of poly(A)+RNA (Uchida& Egami, 1971), led to a significant decrease in thenumber of silver grains compared to the control, whilethe density of silver grains was barely above backgroundover sections probed with [3H]polycytidylic acid([3H]poly(C)).

In in situ hybridization experiments using [3H]-poly(U) as a probe, it is also necessary to eliminate any

inRNAs in rice pollen development 223

"7t

\• P

8A

•9

* *t

\ 9

. •

9A 19 B

Fig. 7. Autoradiographic localization of [3H]poly(U) binding sites in thelongitudinal section of an anther at the uninucleate pollen grain stage. In F7-10: e, epidermis; en, endothecium; g, generative cell or its nucleus;p, pollen grain; t, tapetum; v, vegetative cell or its nucleus. Bars, 10,um.Fig. 8. A. Autoradiograph of a longitudinal section of an anther at thebieellular pollen grain stage, following in situ hybridization with [ H]poly(B. Same section, with focus on cells.Fig. 9. A. Autoradiographic localization of [3H]poly(U) binding sites in abieellular pollen grain at an early stage of starch accumulation. B. Same pograin with focus on cell contents.Fig. 10. Autoradiograph showing annealing of [3H]poly(U) to a bieellularpollen grain at a late stage of starch accumulation.

224 V. Raghavan

Table 2. Distribution of autoradiographic silver grains on the tapetal cells and microsporocytes of rice antherfollowing in situ hybridization of sections with [3 H]poly(C) and [3H]polv(U) and of variously pretreated sections

with [3H]poly(U)

hi situ hybridization conditionsNumber of silver grains ± standard deviation

Tapetal cells Microsporocytes

(1) [3H]poly(U)»[3H]poly(C)b

(2) [3H]poly(U)pH]poly(U), after pretreatment with 0-1 M-KOHC

(3) [3H]poIy(U)[ H]poly(U), after pretreatment in RNase A dissolved in low ionic strength buffer

(4) [3H]poly(U)[3H]poly(U), after pretreatment in RNase A dissolved in high ionic strength buffer'

(5) [3H]poly(U)[ H]poly(U), after pretreatment with RNase T2

(6) [3H]poly(U)[ H]poly(U), after pretreatment with DNaseB

(7) [3H]poly(U)[3H]poly(U), after pretreatment with proteinase Kh

" [3H]poly(U) (sp. act. 2-3 Cimmol"') was used in these experiments.b [3H]poly(C) (sp. act. 1-8Cimmol" ) was used in this experiment. It was prepared according to a modification of the method of Jones el at.

(1973) involving polynucleotide phosphorylase (Boehringer-Mannheim, Indianopolis, IN) catalysed polymerization of [3H]cytidine diphosphate(sp. act. 171 Cimmol" ; Amersham Corpn, Arlington Heights, IL). For experimental purposes, the mass average length of the compound wasreduced to 50-150 bases by hydrolysis in 0-1 M-NaOH for 2h at 2S°C followed by ethanol precipitation. The precipitate was dissolved in thehybridization buffer before use.

"Incubated for 6h at 37 °C.l"1 in buffer containing lOmM-Tris-HCl (pH7-6), 10mM-KCl, 1 mM-MgCl2 for 4h at 37°C.

, ^g in RNase digestion buffer for 4h at 37°C.' RNase T2 (Sigma Chemical Co.), 100 units ml"1 in RNase digestion buffer, 24 h at 37°C.B DNase (Sigma Chemical Co.), 100 fig ml"1 in buffer containing lOOmM-Tris-HCl (pH7-6), 3 mM-MgCl2 for 1 h at 37°C.h Proteinase K, 1 fig ml"1 in buffer containing lOOmM-Tris-HCl, 50mM-EDTA (pH 8-0) for 30inin at 37°C.

15-7 ± 3-42-8 ± 1-0

10-7 ± 3-12-7 ±1-6

15-1 ±2-74-712-6

13-9 ±4-212-2 + 3-518-4 ±5-46-5 ±2-8

11-512-619-114-816-1 14-618-3 ±5-7

11-513-22-211-2

18-414-96-6 + 2-3

15-5 + 5-33-812-1

11-3 + 3-211-0 + 3-029-2 + 5-1

4-912-5

22-516-523-714-7

20-0 + 4-824-214-1

d RNase A, SO/igml"0 RNase A, SO^igml"

probable artifactual binding of the label to other cellularconstituents like DNA and proteins. Data in Table 2show that pretreatment of sections with DNase I prior toannealing did not result in a decreased binding of theisotope in the microsporocyte and tapetal cells of theanther. In fact, as observed in other studies (Capco &Jeffery, 1978; Raghavan, 1981a,6; 1987; Angerer &Angerer, 1981), hybridization signals were as high orslightly higher in sections pretreated with DNase I thanin untreated controls. These results rule out a possibleinteraction between [3H]poly(U) and A+T-rich regionsof DNA of anther cells. It is also seen that pretreatmentof sections with proteinase K did not cause a decrease inthe density of silver grains, indicating that any residualproteins present in the cells do not interfere with anneal-ing between poly(A)+RNA and the applied probe.

His tone gene expression during anther and pollendevelopmentTo monitor the expression of recognizable classes ofmRNA in anthers and pollen grains of rice, sections werehybridized with a specific histone gene probe. Autoradio-graphs of sections annealed with antisense histone RNAprobe showed no binding in the cells of the antherprimordium or in the archesporial cell, the primaryparietal cell, the primary sporogenous cell and tapetumthat subsequently differentiated in the anther; the auto-radiograph shown in Fig. 11 is a representative of theannealing pattern of the cloned gene with early stageanther sections. Duplex RNA molecules formed between

the antisense histone probe and complementary mRNAsequences of the anther were first detected in the endo-thecium at the premeiotic stage (Fig. 12), but as observedin in situ hybridization with [ H]poly(U), binding ac-tivity decreased in the endothecium of the post-meioticanther (Fig. 13) and completely disappeared as its cellsbecame radially thickened. (Silver grains seen around themicrosporocytes in Fig. 12 are probably artifactual; seealso Fig. 15, control section annealed with sense RNAprobe.) During pollen development, expression of thehistone gene was noted rather abruptly in the starch-filledbicellular pollen grains and it continued at a reduced levelin the mature pollen grains.In the former, silver grainswere distributed uniformly in the cytoplasm of thegenerative and vegetative cells (Fig. 14).

The specificity of the histone gene probe used wasdemonstrated in three ways. First, hybridization ofhistone antisense probe to sections was effectively elimi-nated by pretreatment with RNase A (lOO^gml""1 in2 X SSC at 37°C for 30min). Only background levels ofsilver grains were seen in the endothecium and in thebicellular pollen grains of pretreated sections, indicatingthat most of the annealing was to mRNA of cells. Second,in situ hybridization of duplicate sections with senseRNA probe also revealed only background levels ofhybridization signals (Fig. 15). Third, no change in thepattern or intensity of hybridization with antisense RNAprobe was observed when the slides were washed understringent conditions (0-1 X SSC at 50°C). We have alsodetected sequences complementary to rice histone

mRNAs in rice pollen development 225

/ * :

• • . • • * V. ft \en

t

11 u^ : ^ . •' H* • vi*-.. :\

Fig. 11. Autoradiograph of a section of the antherprimordium following/;; situ hybridization with [3H]histoneantisense probe. The label is barely above background. InFigs 11-15: e, epidermis; en, endothecium;m, microsporocytes; ml, middle layer; p, uninucleate pollengrain; t, tapetum; v, vegetative cell nucleus. Bars, 10 /.an.Fig. 12. Autoradiograph showing annealing of [3H]histoneantisense probe to a longitudinal section of the premeioticanther.Fig. 13. Autoradiograph showing annealing of [3H]histoneantisense probe to a longitudinal section of the post-meioticanther.Fig. 14. Autoradiograph of a section of bicellular pollengrains following in situ hybridization with [3H]histoneantisense probe.Fig. 15. Autoradiograph of a longitudinal section of thepremeiotic anther following in situ hybridization with sense[3H]histone probe.

mRNAs by Northern blot hybridization of 32P-labelledhistone antisense probe to total anther RNA (Fig. 16),suggesting that in situ localization is within the presentlevel of detection. Although it was not feasible to isolateendothecial cells or enough bicellular pollen grains fromrice anthers for RNA extraction, a single band ofapproximately 1-3 kb was evident in the blots of totalRNA isolated from anthers of different ages containingendothecium and pollen grains mixed with other celltypes. Non-specific cross hybridization, probably due tocontaminating vector sequences, to rRNA was also noted

28 S-

18 S

Fig. 16. Northern blot hybridization of rice anther RNAwith 32P-labelled antisense histone mRNA. Lane 1: calf liver28 S and 18 S rRNA markers. Lane 2: total RNA of riceanthers. Arrows point to 24-25 S and 18 S RNA; gels in bothlanes were stained with ethidium bromide. Lane 3:fluorograph of total RNA shown in lane 2, after hybridizationwith P-labelled antisense probe. Arrow indicates a bandcorresponding to 1"3 kb histone gene.

in the Northern blot of total RNA. Overall, these resultsindicate that nucleotide sequences complementary to a1-3 kb histone gene are present in the anther and thatmost, if not all, signals observed in the autoradiographs ofsections following in situ hybridization are due to theinteraction of the applied probe with these sequences.

Discussion

In the reproductive biology of angiosperms, origin of thepollen grain from a single progenitor cell in the antherprimordium and organization of a typical seven-celled,eight-nucleate embryo sac from a nucellar cell of theovule are classic examples of differentiation of specificcells along predetermined pathways. Previous studiesthat examined the distribution of mRNA in anthers ofHyoscyamus niger (Raghavan, 1981a,6) and 1.ilium(Porter et al. 1983) by cytological hybridization with[3H]poly(U) showed spatial and temporal differences inthe titre of mRNA in the cells of the developing tapetumand pollen grain. The present work involving a widerange of anther developmental stages of rice has provideda useful framework in which to compare the levels ofaccumulation of a histone mRNA with changes in thesteady-state levels of the total message.

There are interesting changes in the distribution oftotal mRNA during anther and pollen development inrice. These include a uniform distribution of the messagein the anther primordium, a progressive restriction ofmRNA molecules to the endothecium, tapetum andmicrosporocytes and their subsequent disappearancefrom these cell types, appearance of mRNA in thebicellular pollen grains and depletion of the message frommature pollen grains. If localized mRNAs specify thedevelopmental fate of cells, these observations indicatethat intense transcriptional activity is associated with theformation of wall layers and tapetum in the antherprimordium and with the differentiation of vegetativeand generative cells of the pollen grain. This could beascribed to some rare or unique mRNAs whose distri-bution may or may not bear a relationship to the rise andfall of bulk mRNA shown here. In contrast to thepresence of negligible level of mRNA in the epidermisand middle layer after they are cut off, [3H]poly(U)hybridization signals persist in the endothecium andtapetum. This is consistent with the fact that these celltypes continue to differentiate even after they are formed.In the case of the endothecium, this involves the forma-tion of radial thickening bands while the multinucleatecondition combined with endoduplication is character-istic of tapetal cells of many plants. Physiological changesleading to the formation of Ubisch bodies also occur inthe tapetum (Bhandari, 1984, for review).

There is a preferential accumulation of poly(A)+RNAmolecules in the bicellular pollen grain, but they are lostat pollen maturity. The periods of appearance anddisappearance of mRNA in the pollen grains coincideroughly with the phases of initiation and termination ofbiosynthetic activities such as starch synthesis and ac-cumulation (engorgement). A previous study of in situ

mRNAs in nee pollen development 227

hybridization binding of [3H]poly(U) to sections ofanthers of Hyoscyamus niger also showed that highconcentrations of poly(A)+RNA molecules present in thebicellular pollen are lost during their maturation(Raghavan, 19816). Recently, Stinson et al. (1987)described a striking series of changes in the levels ofseveral pollen-specific mRNAs and actin during pollenontogeny in Tradescantiapaludosa. The general pictureemerging from their work is that pollen-specific mRNAsthat are expressed soon after the first haploid mitosiscontinue to accumulate through pollen maturity, whileactin expression slows down during pollen maturation.

Results on the subcellular distribution of a cloned ricehistone gene presented here provide the first example ofdifferential accumulation of a specific mRNA duringanther development in an angiosperm. It is clear from thedata that the histone gene exhibits a pattern of cell-specific localization in the developing anther. One inter-esting finding is that the tapetum excludes histonemRNA despite the fact that its cells accumulate signifi-cant amounts of poly(A)+RNA. From this it appears thatautoradiographic silver grains seen in the tapetum follow-ing in situ hybridization with [3H]poly(U) are due to theexpression of genes other than histone H3 gene. How-ever, histone message is localized in the endothecium andpollen grains according to the same temporal pattern as[3H]poly(U), indicating the presence of histone tran-scripts in the mRNA population of these cells. Althoughit is not known whether rice histone mRNA is polyaden-ylated or not, it is noteworthy that histone mRNAaccumulates only in those cells in which poly(A)+RNAmolecules abound.

Results of this study show that the histone message isnot localized in the same pattern as poly(A)+RNA. Thissuggests that the pattern of cellular localization is specificfor this particular gene and is distinct from that of thebulk message. Absence of annealing of the histone probein the anther primordium and tapetum indicates thathistone mRNA is not necessarily found in rapidly div-iding cells or in cells that synthesize DNA, as one wouldexpect if the function of histones is to complex withDNA. Perhaps, a case can be made to show that histonesare synthesized in another organ of the floret or other cellsof the anther and transported to the dividing cells.Alternatively, as demonstrated in certain animal systems(Maxson et al. 1983), it is possible that histone synthesisis temporally uncoupled from DNA synthesis. Theobserved lack of differential binding of the probe to thegenerative cell of the bicellular pollen grain casts somedoubt about any possible role of histones (Sauter, 1969)in the regulation of transcription in this cell.

The persistent binding of the histone probe to the cellsof the endothecium was an unexpected observation. Inthis context it is a matter of interest that in two reportedinvestigations on the cloning of histone genes from rice48-h-old germinating grains (Thomas & Padayatty, 1983)and 10-day-old seedlings were used (Peng & Wu, 1986).Since dividing cells in both cases were restricted to theshoot and root apices, and parts of the leaf constitute onlya negligible fraction of the tissue mass, it can be arguedthat histone mRNA may also be found in the non-

dividing cells of the plant. In situ localization of histonetranscripts in the endothecium is consistent with thisargument. Although no evidence for the translatability ofthe message is presented, one has to bear in mind thepossibility that localization of the histone transcript in theendothecium suggests the synthesis of proteins encodedby the gene and their deposition within the cell. Whetherthese proteins are used by the endothecium or trans-ported to some other cells of the anther, such as thetapetum, needs further investigation.

This work was supported by grants GA AS 8510 and 8604from the Rockefeller Foundation, New York and grant DCB-8709092 from the National Science Foundation. Appreciation isexpressed to Dr Ray Wu, Cornell University for providing aclone of the rice histone gene, which made this work possible,and to Drs Lee F. Johnson and Philip S. Perlman (Departmentof Molecular Genetics) and their students and Mr Karl H.Joplin (MCD Program), who gave me helpful introduction tomolecular cloning methods used in this work.

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(Received 9 September 19S8 - Accepted 17 October 19SS)

mRNAs in nee pollen development 229