Chromatin structure variation in successful and ...

Protoplasma (1993) 175:1-8 9 Springer-Vertag 1993 Printed in Austria

Chromatin structure variation in successful and unsuccessful arbuscular mycorrhizas of pea S. Sgorbati 1'*, G. Berta 2, A. Trotta 2, L. Sehellenbaum 3, S. Citterio 1, M. Dela Pierre 3, V. Gianinazzi-Pearson 3, and S. Scannerini 2 ~Dipartimento di Biologia, Sezione Botanica Generate, Universit/t di Milano, Milano, z Dipartimento di Biologia Vegetale, Universitfi di Torino, Torino, and 3Laboratoire de Phytoparasitologie INRA-CNRS, Station de G6n6tique et d'Am61ioration des Plantes, INRA, Dijon Received December 30, 1992 Accepted March 29, 1993

Summary, Lincoln and Frisson varietiesof endomycorrhiza-forming pea plants and isogenic mycorrhiza-resistant Frisson mutant ~ 2) plants were inoculated ~:ith Gtomus mosseae. Nuclei released from inoculated and non-inoculated (control) roots were analysed for chromatin structure and activity using flow cytometric techniques. Chromatin accessibilityto the specificDNA fluorochrome DAPI at saturating and non-saturating concentrations was measured. DNA fluorescence of nuclei of mycorrhizal Lincoln and wild genotype Frisson plants was significantlyincreased, compared to the controls, at saturating and, more strongly, at non-saturating DAPI concentrations. In contrast, the nuclei of inoculated P2 mutant roots showed a much lower increase in fluorescence, compared to uninoculated controls. Nuclei released from mycorrhiza-infectedLincoln roots were more sensitive to DNase I than those of uninfected ones. These results indicate a dramatic increase in that portion of the genome which can be transcribed in response to AM infection. Keywords: Chromatin structure; DAPI; DNase I; Flow cytometry; Pea arbuscular mycorrhizas. Abbreviations: AM arbuscular mycorrhizas; CRBCs chicken red blood cells; CV coefficient of variation; DAPI 4",6-diamidino-2phenylindole; DNase I deoxyribonucleaseI; EDTA ethylenediamine tetraacetic acid; FCM flow cytometry; TMN Tris MgC12 NaC1 buffer. Introduction Much recent work has investigated the chromatin structure o f various organisms, especially in relation to its active, partially active, or inactive state (van Holde 1989). Plant research is lagging behind in this area * Correspondence and reprints: Dipartimento di Biologia, Sezione Botanica Generale, Universit~t di Milano, Via Celoria 26, 1-20133 Milano, Italy.

(Slatter and G r a y 1991), although there have been m a n y studies of changes in chromatin structure during the cell cycle and differentiation using optical and electron microscopy (Nagl 1985). Flow cytometry (FCM) appears to be an ideal technique for studying chromatin structure "in situ" at the supranucleosomal level, since biochemical procedures to unravel chromatin superstructure destroy its native configuration (Darzynkiewicz 1990). M a n y D N A fluorochromes can be used as probes for chromatin structure in different cell systems (Cowden and Curtis 1981). In relation to cell differentiation, there are two clear examples of variation in D N A accessibility to various fluorochromes detected by flow cytometry in animal systems, i.e., erythroid differentiation and spermatogenesis (Darzynkiewicz et al. 1984, Evenson et al. 1986). Differential D N A staining of leukocyte populations with propidium iodide has also been detected at nonsaturating fluorochrome concentrations (Mazzini et al. 1983). D N A sensitivity to nuclease digestion, measured by flow cytometry, has also proved useful in assessing chromatin structure and activity in different cell types (Prentice etal. 1985, Roti Roti etat. 1985, PelIicciari etal. 1987). In plant material, F C M using various fluorochromes has been employed to show changes in chromatin structure during the cell cycle ofEuglena (Bonaly et al. 1987), or in root tissue after arbuscular mycorrhiza (.AM) infection at non-saturating dye concentrations (Berta etal. 1990). By measuring the D N A accessibility of

2 nuclei isolated f r o m leek roots to n o n - s a t u r a t i n g D A P I staining, Berta etal. (1990) showed that n u c l e a r hyp e r t r o p h y in A M is related to v a r i a t i o n i n c h r o m a t i n structure (and activity), as a response o f host cells to the fungal infection. I n the present work, we used n o n - s a t u r a t i n g D A P I c o n c e n t r a t i o n s a n d a D N a s e I digestion p r o t o c o l o n nuclei released from infected a n d uninfected pea roots, together with electron microscopy to c o n f i r m the hypothesis that the m o r e dispersed a n d active c h r o m a t i n p r o d u c e d in response to A M infection is similar in different, unrelated plants, a n d therefore a general feature of A M infection. We also report v a r i a t i o n in chrom a t i n structure between different pea genotypes which are resistant or susceptible to A M fungal infection.

Materials and methods Plant growth cultivation and infection Seeds of Pisum sativum L. cv. Lincoln, Frisson, and an isogenic mycorrhiza-resistant Frisson mutant (P 2; Duc et al. 1989)were sterilized in 1% NaC10 and sown in quartz sand. Some were inoculated with chopped roots from pot plants colonized by GIomus mosseae (Nicot and Gerd) Gerdmann and Trappe; non-inoculated pea plants served as controls. On alternate days, a low phosphorus solution was apNied (Berta et al. 1990). The seedlings were maintained in a growth chamber (20 ~ 16h photoperiod, 150 gE/ma/s of irradiance at pot height). Mycorrhizal and control roots were harvested 1month after sowing, at the time of maximal growth response in mycorrhizal plants. The degree of root infection was evaluated microscopically using staining techniques (Phillips and Hayman 1970) and colonization percentage estimates of the root system were made using a grid intersect method (Giovannetti and Mosse 1980).

Electron microscopy Small pieces (1-2ram) of mycorrhizal and non-mycorrhizal 7-8 cm long roots of P. sativum cv. Lincoln were cut 5 cm behind the apex. The samples were fixed in 3% glutaraldehyde (pH 7.2) at room temperature for 3h, rinsed, postfixed in 1% osmium tetroxide in the same buffer for 2h at room temperature, washed, dehydrated in an ethanol series and embedded according to Spurt (1969). Semithin (1-2 gin) and thin sectionswere cut with diamond knives on a Reichert Ultracnt ultramicrotome. Sectionswere stained with 1% toluidine blue in 1% borax for examination by light microscopy,or with uranyl acetate and lead citrate for transmission electron microscopy(Philips CM 10).

Sample preparationfor flow cytometry About 2 g of infected and control pea roots without apices were fixed in 4% (w/v) formaldehyde for 1h at 4 ~ in Tris buffer and then washed thoroughly in the same cold buffer (Berta etal. 1990). Embryo roots growing from ev. Lincoln germinating seeds t5h after wetting were also fixed as above. Root samples were crushed with a glass rod to release nuclei in a few milliliters of buffer. Nuclear suspensions were filtered through a nylon mesh of 60 and 15 gm pore size (Sgorbati et al. 1986). All the operations were carried out on ice. The concentration of the nuclear suspensions was adjusted

S. Sgorbati etal.: Chromatin in arbuscular mycorrhizas of pea with buffer in order to obtain 3 x 105 pea nuclei per ml in each sample.

Staining conditionsfor accessibility measurements 4',6-diamidino-2-phenylindole(DAPI) has previously been found to be superior to other fluorochromes as a probe for plant chromatin structure in root meristems using flow cytometry, with an arc lamp instrument (Berta et at. 1990). Coefficients of variation (CVs) of 2 C nuclei did not exceed 1I%, even at non-saturating dye concentrations. Saturating and non-saturating dye concentrations were established from saturating curves for each pea genotype. To test DNA accessibilityat saturating and non-saturating fluorochrome concentrations it is necessary to control instrument and staining variability; chicken red blood cells (CRBCs), prepared as previously described (Sgorbati et al. 1986), were added as an internal biological standard to each sampte before staining (proportion not exceeding 1/3 chicken to sample nuclei). Nuclear suspensions obtained from roots of Lincoln, P 2, and Frisson peas were, stained with DAPI at saturating (5.6, 28, and 56~tM) and non-saturating (0.14, 2.8, and 5.6gM) concentrations, respectively, for 30rain before FCM analysis.

DNase I digestions Nuclei of control and mycorrhizal roots of the Lincoln cultivar were subjected to DNase I digestion. For comparison, nuclei of embryo roots of germinating seeds (after 15h seed imbibition) were also subjected to DNase I digestion following the same protocol. Nuclei were released from control, mycorrhizal and embryo roots in a TMN digestion buffer (10mM Tris, 5raM MgC12, 10raM NaC1, pH 7.4) similar to that used by Roti Roti etat. (1985), but without phenylmethylsulfonylfluoride. 2 lal DNase I solution (23 Kunitz units/gI, Boehringer, Mannheim, Federal Republic of Germany) was added to each milliliter (3 x t05 nuclei) of sample, for various lengths of time at 25 ~ At each subsequent time, 0.I mI of stock staining solution (56 ~tM DAPI and 100raM EDTA in TMN buffer, to stop DNA digestion) was added to 0.9 ml of the digested nuclear suspensions, mixed and immediately analysed in the flow cytometer.

Flow eytometry The Partec PAS II system (Sonnenweg, Switzerland) was equipped with a HBO 100 W/2 are mercury lamp. UV excitation employed a UG 1 filter and TK420 dichroic mirror. DAPI fluorescence was detected using a K435 barrier filter. The data acquisition system from the same company was used to store and print data.

Results Saturating and non-saturating D A P I staining M y c o r r h i z a t roots o f F r i s s o n a n d L i n c o l n plants were highly infected (77% a n d 71%, respectively), while roots o f m u t a n t s showed only appressoria formation. T h e s a t u r a t i o n curves o f the D N A - D A P I complex o f nuclei released f r o m n o n - m y c o r r h i z a l roots o f Lincoln, Frisson, a n d P 2 pea genotypes are s h o w n i n Fig. t. T h e b i n d i n g characteristics o f D A P I to D N A varied between the different cultivars, so that dye c o n c e n t r a tions 10-40 times lower t h a n the c o r r e s p o n d i n g saturating values were used for the c h r o m a t i n structure analysis.

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Fig. 3. Fluorescence intensity distribution of nuclei released from uninfected (controls; top) and mycorrhiza-inoculated (bottom) roots of Lincoln, Frisson, and P 2 pea genotypes. Nuclei were stained at non-saturating (0.14, 5.6, and 2.8 gM, respectively) DAPI concentrations for 30rain before FCM analysis. Chicken red blood cells (CRBCs) were added as an internal biological standard. The percent increase of fluorescence (relative to the internal standard) of 2 C peak nuclei of mycorrhizal compared to control roots (I-C) is indicated

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Fig, 4. Digestion kinetics of nuclear DNA of Lincoln pea roots with DNase I at different times. The average fluorescence intensity of 2C nuclei of control (D) and mycorrhizal (0) roots was plotted along the ordinate, Nuclei released from pea embryo roots ( ~ ) after 15h of seed imbibition were also subjected to DNase I digestion following the same protocol. The initial digestion rate was much faster in mycorrhizal and embryo nuclei than in controls. After 30 min the digestion limit of mycorrhizal nuclear DNA (25.5% of the initial fluorescence) was about 34% lower than that of control roots (38.8%), and very close to that of germinating pea embryo nuclei. Inset Examples of DNA histograms of control, mycorrhizal, and pea embryo root nuclei at time 0 and after 30rain DNase I digestion. The mean fluorescence intensity of the 2 C peak at different times was taken as a reference to draw the digestion curves

S. Sgorbati et aL: Chromatin in arbuscular mycorrhizas of pea

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Fig. 5. a A root cortical cell of uninfected Pisum sativum cv. Lincoln. The nucleus, of the reticulate type, shows large patches of condensed chromatin (cch). x 17,250. b Magnification of the chromatin of a control nucleus, x 78,000 Fig, 6. a A root cortical cell of Pisum sativum cv. Lincoln, infected with Glomus mosseae. The nucleus, surrounded by arbuscular hyphae, shows a relatively low degree of chromatin condensation, ah Arbuscular hyphae; pin host plasma membrane, x 10,400. b Magnification of the less condensed chromatin of an infected cell nucleus, x 78,000

6 The staining capacity of nuclei released from roots of mycorrhiza-inoculated and control plants of the different genotypes at saturating DAPI concentrations is shown in Fig. 2. With respect to controls, the increase in fluorescence (measured as ratio to that of the internal standard) of nuclei released from inoculated roots of Lincoln and Frisson genotypes ranged from 8 to 12%, respectively. Figure 3 shows the staining capacity of nuclei released from mycorrhiza-inoculated and control roots of the different genotypes at non-saturating DAPI concentrations. An increase in fluorescence (measured as a ratio to the internal standard) of about 30 and 39%, compared to controls, was observed in the mycorrhizal Lincoln and Frisson genotypes, respectively. Interestingly, the increase in fluorescence over the control of 2 C nuclei of P 2 inoculated roots, which because of their genetic resistance to AM fungi block the infection at appressorium formation, is considerably lower (20%) than that observed in the Frisson wild genotype susceptible plants (39%). DNase I digestion

Sensitivity of active genes to DNase I seems to be a general phenomenon, reflecting the altered conformation of nucleosome particles (Weintraub and Groudine 1976, Gross and Garrad 1988). Figure 4 shows the digestion curves of nuclei from control and mycorrhizaI roots of the Lincoln cultivar, characterized by a marked decrease in DNA fluorescence. The initial decrease was faster in mycorrhizal than in control roots, reaching digestion limits of about 25 and 39% of the initial fluorescence, respectively, after 30 rain of digestion. In the same figure, the digestion curve of nuclei released from mycorrhizal roots was very close to that obtained by digestion of nuclei released from embryo roots of the same pea cultivar during seed germination, when the DNA of meristematic cells is thought to be actively transcribing (Grellet et al. 1977, Caers et al. 1979). The inset shows the corresponding DNA histograms at time 0 and alter 30 rain DNase I digestion. During the digestion time the histograms maintained their normal shape, with an unchanged fluorescence ratio between 4 C and 2 C peaks. Electron microscopy

Ultrastructural observations of root cortical cell nuclei of P. sativum cv. Lincoln showed a chromonematic or reticulate type structure, with large patches of condensed chromatin (Fig. 5 a, b). Nuclei of infected roots,

s. Sgorbati et al.: Chromatin in arbuscular mycorrhizas of pea however, were in a less condensed state, with smaller zones of condensed chromatin interspersed with wide zones of diffuse chromatin, a structure that could therefore be defined as chromomeric-chromonematic or semi-reticulate (Fig. 6 a, b).

Discussion

The modes of interaction of fluorochromes with DNA are various, including external binding and intercalation (Latt and Langlois 1990). For these reasons, DNA fluorochromes can be used as probes of chromatin structure using microfluorimetry and flow cytometry (Coweden and Curtis 1981, Darzynkiewicz 1990). We found non-saturating concentrations of DAPI particularly useful for probing chromatin structure variation which accompanies size and ultrastructural changes in nuclei released from leek roots after AM infection (Berta et al. 1990). In this study, the approach was extended to two cup tivars (Lincoln and Frisson) of a dicotyledon, P. sativum and a P 2 isogenic mutant of cv. Frisson. The saturation kinetics of root nuclei of these plants with DAPI staining were quite different and may reflect the developmental stage of root and cell differentiation. The increase in nuclear fluorescence of Lincoln and Frisson pea cultivars one month after AM inoculation, compared to controls, was evident at saturating DAPI concentrations. The accessibility of DNA to DAPI at staining equilibrium was previously reported to be significantly increased in exponentially growing compared to differentiated animN cells (Darzynkiewicz etal. 1984). In our endomycorrhizal system, it cannot be excludext that the different DNA staining capacity at saturating DAPI concentration was enhanced by- formaldehyde fixation. This is reported to alter DNA staining with different fluorochromes in relation to chromatin structure (Larsen etal. 1986). Increase in nuclear DNA fluorescence observed in inoculated roots can be explained by variation in chromatin structure, confirmed by analysis at non-saturating DAPI concentrations and (for the Lincoln cultivar) electron microscopy. However, even if polyploidization can be excluded (Berta etal. 1986, 1990; Blair and Peterson 1988), quantitative differences in DNA content, i.e., DNA amplification, cannot be completely ruled out. Moreover, DNA amplification is very frequent in plants at different developmental phases, especially under environmental stress (Nagl 1992) and/or during cell differentiation (Bassi 1990). Biochemical and cytogenetic data, together with molecular biology techniques,

s. Sgorbati et a1.: Chromatin in arbuscular mycorrhizasof pea could clarify whether there is variation in chromatin structure only, or if D N A amplification is also occurring in these endomycorrhizal systems. At non-saturating DAPI concentration (Fig. 3), the increase in nuclear fluorescence of mycorrhizal Lincoln and Frisson cultivars (30 and 39% compared to controls, respectively) was much higher than at saturating DAPI concentrations, indicating variation in chromatin structure of the host nuclei and confirming that reported previously from AM of leek (Berta et al. 1990), In addition, at least for the Lincoln cuttivar, there is ultrastructural evidence that variation in chromatin structure is induced by the fungal symbiont in pea mycorrhizae. All these observations suggest that this kind of response, although differing in intensity, could be a normal host plant reaction to fungal infection in endomycorrhizal systems. However, the D N A staining capacity of host nuclei seems to be related to the degree of infection and the development of arbuscules in the cortical parenchyma cells (Berta et N. 1989, Balestrini et al. i992). In fact, in AM resistant P 2 plants, where the presence of the fungus is limited to the surface of root epidermal cells (Duc et al. 1989), increase in D N A staining was considerably lower (20%) than in Frisson (39 %) whose cortical parenchyma cells were, contrastingly, highly infected. Although the increase in D N A staining capacity of nuclei in infected roots corresponds to increased accessibility of chromatin to fluorochromes, in relation to its more dispersed structure (Berta etal. 1990), it does not itself demonstrate higher transcriptional activity. On the other hand, it is known that D N A sensitivity to nuclease digestion is well correlated with the transcriptional potential of genes (Weisbrod 1982, Zared and Yamamoto 1984). The DNase I digestion of nuclei released from mycorrhizal roots of the Lincoln cultivar reveals a dramatic increase in sensitivity to the enzyme relative to the controls, both as rate o f digestion and digestion limit. The D N A sensitivity o f nuclei of mycorrhizal roots is also very close to that of nuclei released from pea embryo roots during germination, when cells are considered transcriptionally very active (Grellet et al. 1977). Work is in progress to elucidate the role o f histories and other chromosomal proteins in sustaining the exceptional transcriptional activity o f host cell chromatin. The possibility that AM-infected cells are transcriptionally much more active than control cells is indirectly supported by ultrastructural data (see, e.g., Toth and Miller 1984). In addition, some evidence exists that modifications in host gene expression occur during endomycorrhizal infection, since new

7 polypeptides or proteins (endomycorrhizins) have been reported in infected roots of tobacco, soybean (Dumas et al. 1990, Wyss et al. 1990) and pea cv. Frisson, which are not detected in the pea mutant resistant to AM infection (Schetlenbaum et al. 1992). In conclusion, we are convinced that AM mycorrhizae could provide an interesting model to study chromatin structure variation and function accompanying root differentiation due to the host-fungus interaction, and that F C M analysis could be a rapid and precise tool for assessing the functional condition of the infected roots. Acknowledgements

This work has been partially supported by Italian MURST (60%) and CNR, INRA (France) and Bridge (EEC) grants. References

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