BIOLOGIA PLANTARUM (PRAHA I

24 (6) : 440--445, 1982

The P ers i s t ence and Lignin- l ike A p p e a r a n c e o f the P r i m a r y Cell Wail o f M i c r o s p o r o c y t e s in Male -S ter i l e ( C M S ) Sweet Pepper ,

Caps icum annuum L.

JARMII,A HEI'IDI~YC~OV.~-ToM~:OV~. and N o u Y ] ~ TI-II HOA BIN~

Faculty of Science, Charles University, Praha*

Abstraet. Using differential staining of cell walls the anatomy of mierosporogenesis was investigated on cross-sections of anthers of fertile plants of the sweet pepper Capsicum annuum L. cv. Severka, as well as of its sterile analogues. The lignin-like staining as observed in the micro- sporocyte primary walls of fertile plants disappears with their getting independent in the course of meiosis. On the contrary, in sterile plants the stain increases in intensity, the thick-walled microsporocytes usually form a continuous block up to the period of tetradogenesis, and so far as microspores originate, they are not dissociated outwards. Moreover, in sterile anthers the external tapetum is usually not differentiated.

I n our p rev ious p a p e r (NovYE~ T m and HEND~YC~OvA-ToMxov~ 1982) a precoc ious d e g r a d a t i o n of the callose m i c r o s p o r o c y t e enve lope was obse rved on squash p r e p a r a t i o n s of an the r s as one of the m e c h a n i s m s resu l t ing in pol len steril i ty, wi th the sweet pepper . I n the p re sen t p a p e r we e m p l o y e d the t echn ique of s ta in ing a n a t o m i c a l sect ions for the d i f f e ren t i a t ion of cellulosic a n d lignified cell walls in the s ame p l a n t ma te r i a l , t hus i nves t i ga t i ng the condi t ions t h a t are decisive for the c o m m u n i c a t i o n of m i c r o s p o r o c y t e (or microspore) p r o t o p l a s t s wi th one a n o t h e r a n d wi th the i n t e rna l m e d i u m of t he a n t h e r sac, and af fec t the course of microsporogenes is .

MATERIAL AND METHODS

Material C a p s i c u m a n n u u m L., cv. S eve rka (cu l t ivar of Czechos lovak p r o v e n a n c e )

as fer t i le p lan ts . Steri le p lan ts , ana logues of the cv. Severka , were se lected f r o m the B4 a n d B5 progenies of the m a i n t e n a n c e b a c k crossing [(860/S • Se- ve rka ) • Severka] . The m a t e r i a l was p r o v i d e d b y the Vege tab le R e s e a r c h I n s t i t u t e in Olomouc where i t was de r ived f r o m the or iginal P e t e r s o n ' s steri le m a t e r i a l ( N o v ~ x e t a l . 1971). :For f u r t h e r de ta i l s see IqGVYEN THI and H E ~ - ])RYC~OV$,-To~Kov~ (1982).

Received August 24, 1981: accepted February 22, 1982 * Address: ViniSn~ 5, 128 44 Praha 2, Czechoslovakia.

440

PRIMARY CELL WALL OF MICROSPOROCYTES 441

The plants were cultivated in a usual way in a hotbed during the summer vegetation period. The fertility of individual plants was verified continuously according to the pollen grain content in anthers (staining of squash prep- arations with acetic-carmine), as well as their viability (tetrazolium test), in order to investigate microscopically extreme characteristics of sterility and fertility, not diminished by a casual seasonal variation (NGvYEN THI 1980). For the microscopic preparation proper buds of varying maturi ty were employed, the meiotic stage being checked cytologically, to co~er the entire course of microsporogenesis.

Methods

Permanent preparations of cross-sections of buds or individual anthers were made using the classic paraffin method. The material was fixed with the N~mec's bichromate-formaldehyde mixture for 24 h, the fixative always being renewed after 8 h. Following dehydration in ethanol-butanol series the objects were embedded in paraffin, and microtome sections of 5 ~m-thick- -ness were provided.

The staining of preparations (after removing paraffin from the sections) was aimed at differentiating cellulosic and lignified cell walls. The colouration of xylem cell walls in vascular bundles served as comparatives criterion. By means of the double staining method according to Cajal--Bro~ek (N]~Mv.C et al. 1962), mostly used for this purpose, the lignified cell walls stain red .with basic fuchsin, the cellulosic ones stain blue with indigoc~rmine. The mixture of malachite green with acid fuchsin gave a green colouration to lignified walls, and red to cellulosic ones.: From the preparations thus stained microphotographs were taken. To verify the results some further staining methods were employed : N~mec's crystal violet with Orange G (lignin purple, cellulose orange), safranin with light green (lignin red, cellulose green), Ehrlieh's haematoxylin with safranin (lignin red, cellulose blue), in all cases according to the directions given by N~MEc et al. (1962). The stained sections on microscopic slides were mounted in a xylene-dissolved synthetic resin Metaplex B 34/31.

RESULTS

In sweet pepper the sporogenous tissue passing lengthwise in each of the four anther loculaments is mostly composed of two cell rows. In fertile plants it is r immed from both sides with a layer of tapetal cells, on the inner side strongly, on the outer less magnified (Fig. la). In sterile analogues of the cv. Severka investigated the external tapetum was mostly absent; at this site the sporogenous cells directly neighbour on fiat cells of the anther wall (Fig. 2a). However, neither by its appearance, nor by a frequent occurrence of multiple nuclei does the internal tapetum differ from that of fertile plants.

In the anthers of genetically fertile plants the cell walls of the sporogenous tissue gradually thicken from the beginning of meiosis, most rapidly in cell corners. In its appearance the tissue becomes colenchymatous and the thickenings stain lignin-like (Fig. la). The thickening and specific stainability of cell walls proceeds all over the periphery of microsporocytes so far as they are joined in a continuous tissue. In the course of the second meiotic division the individual microsporocytes get loose, they round off, and the external layer of their walls staining lignin-like as an external contour gradually

442 J. HENDgYCHOVA-TOMKOVfit , NGUYEN THI H. ]3.

disappears. The microspore tetrads lie loosely in the anther sac cavity (Fig. lc) as far as individual microspores are not dispersed from one another as the result of dissolution of callose the tetrads were enveloped and perva- ded with.

In sterile plants the pr imary walls of sporogenous cells thicken from the beginning of meiosis and the thickenings stain lignin-like, as well. However, this process starts along the external walls at the site where the sporogenous cells neighbour on the anther wall cells, i.e. at the site where the external tapetum is lacking (Fig. 2a, b). Gradually the thickening walls form a heavy crusta that surrounds the individual microsporocytes, interconnecting them into a compact block. This situation is still observable at the stage cor- responding to tetradogenesis. In the anthers where the cavities of anther sacs have not been created at this stage, the compact block of deformed and expressively lignin-like coloured microsporoeytes is closely surrounded by hypertrophic tapetal cells (Fig. 2d, f). As far as the central cavities in anther sacs are formed, one may observe a delayed release of individual microsporo- cytes from their connection in the sporogenous tissue. These PMC's are not normally round, but they maintain their oblong and angular shape caused by a protracted connection. Their enveloping layers continue considerably thickened and lignin-like stainable (Fig. 2c). Inside these PMC's microspores were observable in some cases, in others not, but they have never been found to be dissociated outwards.

Since we paid no special at tention to the development of tape tum in this work, we only refer to the results of general observations. In fertile plants at the stage of tetradogenesis, the walls of tapetal cells lose continuity (Fig. lc); in sterile plants both the vital, hypertrophic tapetal cells with a rich content of cytoplasm persisted (Fig. 2f), and the cells practically empty with well- preserved wails (Fig. 2c, d), but not lignin-like stainable occurred.

D I S C U S S I O N

Our results verified the finding of HORI~]~R and ROGERS (1974), i.e. tha t in sterile sweet pepper with CMS the primary microsporocyte wall persists. The persistence of the middle lamella between sporogenous cells which, in our case, remained interconnected till the late stage of tetrads, is also of importance. The same is evident from the figures of the above-mentioned authors. In our material the complex of primary walls with the middle lamella appeared as expressively lignified, as inferred from their affinity to certain microscopic dyes. The middle lamella between the cells whose pr imary walls are lignified, is also impregnated in this way (WA~DRO]? 1975); under these circumstances the action of the enzyme is clearly prevented in sterile plants, which, in fertile plants, due to dissolution of the normal pectinous lamella, at the right moment disintegrates the sporogenous tissue. As far as we observed any loosening of individual microsporocytes in sterile anthers this occurred as late as at the stage of microspores. Their angular and oblong shape corresponding to their original connection in the tissue was fixed by a thickened, highly impregnated wall. The shape deformation of PMC's necessarily prevents a normal spatial distribution of microspores in the te t rad tetrahedron. This fact by itself may be a sufficient cause of pollen sterility, if, according to SAx (1935) and STEBBINS (1965) etc., the orientation

PRIMARY CELL WALL OF M1CROSPOROCYTES 443

of a microspore in the te t rad predetermines its internal polarity, as well as its future structural and functional differentiation. H o ~ N ~ and ROGERS (1974) also observed tha t inside the persisting pr imary PMC wall no micro- spore membrane formation took place. The impregnation altering the PMC wall permeability to solutions and gases, may represent a substantial change in normal communication of PMC protoplasts and young microspores with the medium of the anther sac. The formation of membrane around microspores is a significant condition for their development, as well (HESLOP-ttA~ISO~ 1971 etc.), and this process is dependent on the uptake of a series of substances from the environment (summarized in .HESLOP-HAI~I~ISON 1972). Fur ther the question offers itself, as to whether the precocious degradation of the callose layer of PMC's (in the same sterile material see INOUYEI~ T m and HE~DR~T(!Hov~-ToMKOV~ 1982), conditioned by an abrupt fall in pH and subsequent onset of callase activity, also follows from this abnormal isolation of the PMC protoplast.

In anthers of sterile plants an altered content of free amino acids has often been reported; in the case of sweet pepper (according to t~AKOUS]<,; ~ 1973), in addition to others, a decrease in the content of free phenylalanine and proline is concerned. One may consider a more intense participation of phenylalanine as the source of cinnamic acids and their derivatives in lignin biosynthesis. The lignification of cell walls is a significant fac to r in the ontogenetic differentiation of plants (LIt'ETZ 1962). I t is well-known from the papers by SIEGEL (1955), STA~FO~D (1960), etc. tha t in lignin biosynthesis peroxidases take part by creating free radicals of sinapylalcohol, coniferyl- alcohol or p-hydroxycinnamylalcohol; the phenylpropan units thus modified polymerize spontaneously into lignins of varying composition, which in itself may be important from the point of view of differentiation (No~D and STEVEiNS 1958). Peroxidases are represented by a series of isoenzymes the cytological localization of which differs (L~PETZ and (JAI~RO 1965). The factors promoting the linkage of peroxidases to the cell wall (IAA, Ca2+), also affect wall lignification (see Discussion in Dvof~XK and CEtCNO}IOI~SKA 1972). The relationship between the heteroauxin-induced peroxidase activity, its linkage to cell walls, peroxidase-promoted lignification and between cell differentiation was already considered by JE:NSEN (1955).

In sterile analogues of certain sweet pepper cultivars (also in ev. Severka) I~AKOUSK~; ~ (1973) found a different dynamics of peroxidase activity in anthers: lower at the beginning of meiosis, increasing during m'icrosporo- genesis, in comparison with an opposite course in fertile plants. The occurrence of extractible isoenzymes with peroxidase activity also was different, as well as the anatomical distribution of peroxidase activity in anther cross-sections, as evaluated by an orientation histochemical test. In sterile plants this act ivi ty was concentrated in anther walls at a premeiotic stage, during meiosis it was highest in their central parts. In fertile plants the opposite was true. The differences obtained are worth revising because o f possible connection with abnormal lignification of PMC walls we suppose in sterile plants; the comparison of the hard-extractible component of peroxidase activity in the cell-wall fraction would be of special significance.

In radish L~u and LAMt'OI~T (1968) found a highly active peroxidase iso- enzyme the composition of which is identical with hydroxyproline-rich oligopeptides that are, in glycosidic bond with arabinose, a normal corn-

444 J. HENDRYCHOV.~-TOM. KOV.~, NGUYEN THI t t . B.

ponent of plant cell walls (according to KING and BAILEY 1965). In con- nection with the problem of cell wall lignification this finding is discussed by DvorAK (1973). STEBBINS (1973) ascribes a fundamental morphogenetie importance to hydroxyproline-rich proteins, creating transverse connections between cell wall saccharides, and considers the mutation changes in hydroxy- proline metabolism a significant evolution factor. The quantitative partici- pation of hydroxyproline-rich oligopeptides in the cell wall, the proportion of hydroxyproline sequences in their molecules, the degree of substitution of their hydroxyl groups by glycosylation, etc. affect the cell wall ultra- structure, as well as its binding and osmotic properties. According to ASRFORD and NEVBERG (1980) hydroxyproline-rich proteins in plants are formed of nascent proline oligopeptides, insoluble extensins localized within the cell wall, only by their additional hydroxylation in situ. In confrontation with these findings the reduced level of free proline, a well-known phenomenon accompanying pollen sterility of various types (FuKASAWA 1962, TuP~ 1963, :BI~TIKOV et al. 1964), may also be explained in terms of linkage of this amino acid to the structures and enzymes mentioned. From this point of view the comparison of the level of free and bound proline, or hydroxyproline, would be of importance.

We tried to discuss possible causal connections between certain partial deviations, accompanying on a morphological, anatomical, biochemical, and cytological!levels, pollen sterility (type CMS) as final phenotype. So far, one cannot decide which of many partial phenomena is the primary disorder, determined by the recessive state of nuclear genes (MS), or extranuclear determinants. With regard to the variability in the intensity of their expres- sion, we also came across in our work, it will be necessary to consider more thoroughly the genotype characterizations of the material (cultivar-specific gene background of CMS, heterogeneity of sterile lines in MS genes).

Acknowledgement

We are indebted to Dr. F. J . Novhk, CSc. and :Dr. S. Rakousk~, CSc. of the Inst i tute of Ex- perimental Botany of the Czechoslovak Academy of Sciences, and to Ing. J . Betlach of the Vegetable Research Inst i tute in Olomouc for providing us with the plant material, and to Dr. Z, Pazourkov~, CSc. of the Faculty of Science, Charles' University, for her invaluable advice con- cerning the microscopic technique.

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BRITIKOV, E. A., MUSATOVA, iN. A., VL.~DI~IRTSEVA, S. V., PROTSEI~XO, )I. A.: Proline in the reproductive system of plants. -- In : Li~sxElus, H. F. (ed.) : Pollen Physiology and Fertiliz- ation. Pp. 77--85. North-Holland Publ., Amsterdam 1964.

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Figures at the end el the issue.

J . H E N D R Y C H O V / [ - T O M K O V / [ , N G U Y E N T H I I-L B. P R I M A R Y CELL W A L L OF M I C R O S P O R O C Y T E S

Fig. 1. - - a) The cross-sect ion o f a loculament of the ferti le p l an t (cv, Severka). - - s ~ sporogenous ceils pr ior to the onse t o f meiosis (grey); ar rows ~ wall th ickenings in cell corners s ta ined lignin-like; t ~ t ape tum. - - b ) The same object , sporogenous cells in t he course of the second anaphase - - te lophase; t he l ignin-like coloured th ickenings become spread a round the whole pe r iphery of sporogenous cells. - - e) Microspore t e t r ads in the an t he r cav i ty of a ferti le p l an t (cv. Severka). The ex te rna l l ignin-like coloured PMC contour disappears , the mierospores sur rounded b y eallose. Sta in ing Cajal-Bro~ek.

J. HENDRYCHOVfi~-TOMKOVA, NGUYEN THI II. B. PRIMARY (JELL WALL OF MICROSPOROCYTES

Fig. 2. -- a) The cross-section of an an ther loculament of the sterile analogue cv. Severka. -- s = sporegenous cells; t ~ t ape tum; w ~ an the r wall; the external t ape tum was no t developed here. The ar rows point to the lignin-like coloured deposits along outer walls of sporogenous cells neighbour ing directly on the an the r wall cells. -- h) The same object, the sporogenous cells in a more advanced stage of meiotic division (I telophase). The lignin-like coloured thiekenings r im the entire PMC periphery, being mos t expressive along outer walls. -- c) Object of the same origin; in the an the r sac a cavi ty was par t ly formed, some microsporocytes are being released f rom their intereonnection. Their shape is abnormal ly oblong, angular , wi th considerably thickened lignin-like coloured wall. -- d) I n the an the r sac of ano ther sterile p lant (analogue of cv. Severka) the sporogenous cells (s) main ta ined in a cont inuous block included by adjacent

J. HENDRYCHOV/~-TOMKOV-~, NGUYEN THI H. B. PRIMARY CELL WALL OF MICROSPOROCYTES

tissues. Their walls are surrounded by a thicl: lignin-like coloured layer; the arrows point to the connection of microsporocytes by the impregnating substance. -- e) The take shows a few tapetal cells with a highly degenerated content, but well-preserved walls, not staining lignin.like. They rim the anther sac cavity formed in this sterile plant at the stage of dissociated micro- sporoeytes completing their degenerative development (left). -- f) In the anther sac of a further sterile plant the cavity was not formed, the compact block of microsporocytes (s) is included by a hypertrophic tapetal tissue. The lignin-like coloured substance forms a continuous coat around the block (see arrows) that loses gradually its cell structure and passes into a band of a markedly stained amorphous matter. Staining Ca]al-Bro~ek.