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AUSTRALIAN JOURNAL OF PLANT PHYSIOLOGY Volume 27, 2000 © CSIRO 2000

An international journal of plant function

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Aust. J. Plant Physiol., 2000, 27, 1109–1117

Histological and biochemical changes in Pinus spp. seeds during germination and post-germinative growth: triacylglycerol distribution and catalase activity Marie-Noëlle JordyAC, Susanna DantiB, Jean-Michel FavreA and Milvia Luisa RacchiB A

Laboratoire de Biologie Forestière, Université H.Poincaré, Nancy I, BP 239, F 54506 Vandoeuvre-lès-Nancy Cedex, France. B Dipartimento di Biotecnologie Agrarie, sezione di Genetica, Università degli Studi di Firenze, Piazzale delle Cascine 27, 50144 Firenze, Italia. C Corresponding author; email: [email protected] Abstract. The spatio-temporal evolution of catalase (CAT) activity and triacylglycerol distribution was investigated in seeds and seedlings from Pinus pinaster Ait., P. pinea L. and P. radiata D. Don during germination and postgermination. The high amount of triacylglycerols contained in the whole dehydrated embryo from the three species was progressively depleted, first, in the radicle and then in hypocotyl and cotyledons during post-germinative growth. In parallel, histological localisation of CAT activity and the quantitative analysis confirmed the involvement of this enzyme in cell detoxification from peroxide released during the intense lipid breakdown. Two isozymes, CAT1 and CAT-2, were identified during post-germinative growth. Both were particularly active in the hypocotyl and radicle, while CAT-2 was specifically active in the photosynthetic tissues. These results emphasise that CAT activity is also independent from lipid metabolism in certain tissues. The role of each isoenzyme is discussed in connection with the metabolic changes occurring during seed germination and seedling growth. Special attention is given to the role of the shoot apex in triacylglycerol storage and breakdown. Central mother cells have been shown as a specific lipid storage area of the shoot apical meristem, in contrast with the peripheral zone in which lipid reserves were always reduced.

Introduction Pine (Pinus spp.) seeds contain storage protein and lipid reserves in both the megagametophyte and embryo. Lipid reserves are stored in oleosomes and mainly consist of triacylglycerols including unsaturated C18–C20 fatty acids, such as linoleic acid and oleic acid (Hammer and Murphy 1994; Wolff et al. 1996). In loblolly pine (Pinus taeda), 75% of the total lipid reserves of the whole seed are stored in the megagametophyte; lipids constitute 26% of the megagametophyte and 20% of the embryo fresh weight (Groome et al. 1991). In germinating oil seeds, the acid lipase associated with the oleosome membrane hydrolyses the triacylglycerols (Hammer and Murphy 1994; Stone and Gifford 1999). Generally, the released fatty acids undergo β-oxidation and enter the glyoxylate cycle in the glyoxysome. The subsequent conversion of acetyl CoA to malate provides substrate for

sucrose synthesis during gluconeogenesis that will serve as the primary energy source for the developing young seedlings (Huang et al. 1983). In pine, both β-oxidation and the glyoxylate cycle occur in the megagametophyte but, in seedlings, the glyoxylate cycle is less active, suggesting that triacyglycerol breakdown occurs through the glyoxysomal β-oxidation pathway, releasing energy through the mitochondrial Krebs cycle (Hammer and Murphy 1994; Stone and Gifford 1999). The removal of hydrogen peroxide generated by intense oxidase activity at the glyoxysome level is catalysed by CAT. The breakdown of the lipid reserves and the key enzymes involved in their mobilisation have been extensively studied. The regulation of CAT (EC 1.11.1.6, 2H2O2→ 2H2O + O2) and glyoxylate cycle enzymes have been investigated in several Pinus species, namely Pinus edulis (Noland and Murphy 1984; Murphy et al. 1992), Pinus taeda

Abbreviations used: C, cotyledon; CAT, catalase; CAT-1, isoform 1; CAT-2, isoform 2; CMC, central mother cells of the shoot apical meristem; CP, cortical parenchyma; DTT, dithiothreitol; EDTA, ethylene diamine tetraacetic acid; H, hypocotyl; PAGE, polyacrylamide gel electrophoresis; PI, needle primordia; PM, pith meristem; PN, primary needles; PP, pith parenchyma; PR, procambium; PVPP, polyvinylpolypyrrolidone; PZ, peripheral zone of the shoot apical meristem; R, radicle; RC, root cap; RM, root meristem; SAM, shoot apical meristem; T, tannins; VS, vascular system; ε, initial rate of H2O2 decomposition at 240 nm. © CSIRO 2000

10.1071/PP00069

0310-7841/00/121109

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(Mullen 1995; Mullen and Gifford 1995a, b, 1997) and Pinus taiwanensis (Kao 1973). The loblolly pine CAT exists as multiple isoforms, four of which have been observed in the megagametophyte 7 d after the soaking of seeds. The molecular masses of the native enzyme and its subunits are 235 and 59 kDa, respectively, indicating that the pine holoenzyme is a homotetramer (Mullen and Gifford 1993). Although CAT has been well characterised in a number of gymnosperms, the study of lipid reserve mobilisation has been primarily restricted to assessing changes in enzyme activity and quantifying lipid breakdown during and following germination. Information about the histological pattern of both lipid storage and CAT activity in the different parts of embryo and seedling tissues are lacking. Therefore, in the present study, we coupled biochemical investigations of CAT activity with histological analysis of the spatio-temporal variations in lipids and CAT distribution during germination and post-germination growth. The aim of this work was to gain more insight into lipid reserve allocation during these developmental steps, identifying the tissues involved. The analysis was performed on seeds and seedlings of three different Pinus species (P. pinaster, P. radiata, P. pinea), which are representative of the south-western European pines. Materials and methods Plant material Seeds of Pinus pinaster Ait. from Landes (France), P. radiata D. Don from Alava (Spain) and P. pinea L. from Tuscany (Italy) were surfacesterilised in a 3.5% calcium hypochlorite (w/v) solution for 10 min. They were then soaked in autoclaved distilled water and placed on filter paper in Petri dishes at 25°C in the dark for 7 d. The imbibed seeds were subsequently transferred into 1-L pots containing a peat–clay mixture and grown in a climate chamber at 25°C under a 16-h photoperiod (350 µmol m–2 s–1). From this material, samples were taken at the stages mentioned in Table 1.

chromoxide (Randolph 1935). Transverse sections (40 µm) were cut with a cryomicrotome, stained for 10 min in a saturated solution of oil red O in 99% isopropanol (Lillie and Fullmer 1976), rinsed in distilled water and immediately observed under a Nikon Optiphot 2 photomicroscope. The triacylglycerols were revealed in deep red. For identification of the presence and distribution of CAT activity, fresh sections (40 µm) were incubated for 20 s in 0.01% (v/v) H2O2 and then stained in a solution of 1% (w/v) FeCl3 and 1% (w/v) K3Fe(CN)6 for 15 s. After rinsing in distilled water, the CAT activity was shown as negative staining in a blue background (Acevedo and Scandalios 1991). Preparations of crude extract Plant tissue (Table 1) was powdered in liquid nitrogen and extracted in a ratio of 1:10 (w/v) with an extraction buffer composed of 0.1 M potassium phosphate buffer, pH 7.2, 2 mM EDTA, 8 mM MgCl2, 4 mM DTT, 1% (v/v) Triton X-100 and 2% (w/v) PVPP. Concentrations of 0.1% and 0.5% (v/v) of the detergent were tested, but were inadequate to obtain a reproducible measurement of CAT activity. Homogenates were centrifuged at 4°C at 14 000 g for 10 min. The supernatant was dialysed overnight at 4°C against 1000 volumes of 0.1 M potassium phosphate buffer, pH 7.2, containing 2 mM EDTA. The dialysed samples were used for activity assays and for native PAGE. Enzyme assays CAT activity was determined by measuring the initial rate of H2O2 decomposition at 240 nm (ε: 0.0036 mM–1 cm–1) (Havir and McHale 1987). The reaction mixture contained 1 mL of 50 mM potassium phosphate, pH 7.0, and 12.5 mM H2O2. Protein content was determined by the method of Lowry et al. (1951), modified according to Peterson (1977), using bovine serum albumin as standard. Electrophoresis CAT isoenzyme patterns were identified by native PAGE on 6% acrylamide gels. Defined amounts of protein (5–10 µg) were loaded on the gel and electrophoresis was carried out in 0.03 M Tris–glycine buffer, pH 8.8, at 4°C using a Mini-Protean system (Bio-Rad, Segrate, Milano, Italy ). CAT activity staining was performed according to Chandlee and Scandalios (1983) after pretreatment of gels in 0.01% (v/v) H2O2 for 10 min. The staining mixture contained 1% (w/v) FeCl3 and 1% (w/v) K3Fe(CN)6 in distilled water.

Histology For triacylglycerol detection, samples were fixed in CRAF solution composed of 5% (v/v) formaldehyde, 3% (v/v) acetic acid and 20 mM

Table 1.

Stage

Sample stages and corresponding morphological characteristics Time after beginning of imbibition (d)

1 2 3

0 7 14

4 5 6

21 28 35

7 8

42 60

Morphological characteristics Dry seed Imbibed seed Beginning of the radicle elongation (0.5 cm long) Hypocotyl elongation Elongated hypocotyl Beginning of elongation of the first primary needles Beginning of epicotyl elongation Lateral axis of epicotyl in elongation

Results Triacylglycerol localisation Triacylglycerols were observed in dry seeds of all three species. At stages 1 and 2, triacylglycerols were distributed throughout the whole embryo (Fig. 1). During radicle emergence (stage 3), the hypocotyl, and the shoot and root tips remained intensely reactive to the oil red O (Figs 3–5). In contrast, only a few triacylglycerols were observed in the elongating parts of the radicle (Fig. 7). The newly formed primary needle primordia were totally free of lipid reserves (Fig. 5). At stage 4 (hypocotyl elongation), depletion of the triacylglycerol reserves occurred progressively in root, and then in the hypocotyl and cotyledons, faster in the cortical parenchyma and the vascular bundles than in the pith parenchyma (Fig. 8). The shoot apical meristem consists of a central zone, namely the central mother cells (CMC), surrounded by the peripheral zone (PZ) from which the leaf primordia originate; below the CMC and adjacent to the PZ

Triacylglycerol distribution and catalase activity in Pinus seedlings

is the pith meristem (PM). The PZ and the newly formed primary needles remained reduced in triacylglycerols (Figs 11–12); in contrast, a few cells located at the surface of the CMC were strongly reactive to lipid staining (Fig. 12). At the end of the hypocotyl elongation (stage 5), triacylglycerols were located only in some cells of the pith parenchyma, and in the SAM, especially at the CMC surface. At stage 6, triacylglycerols remained only in these cells (Fig. 13) and, finally, completely disappeared during elongation of the primary needles (stage 7 — not shown). At stage 8, corresponding to the elongation of the epicotyl along with its lateral buds, a new accumulation of triacylglycerols occurred in the shoot apex except in the lateral meristem (PZ) and the primary needle primordia (Fig. 17). To summarise, an ascendant mobilisation of lipid reserves in the seedlings concurrent with triacylglycerol depletion in the lateral parts of the SAM were observed during early postgerminative growth. In contrast, triacylglycerols were newly accumulated in the shoot tip during the epicotyl elongation. The SAM appeared to be a significant site for lipid reserve storage, since it was the last zone in which triacylglycerols were depleted and the first zone in which they were stored again. Histological pattern of CAT activity In dry seeds of the three species, CAT activity was distributed in the whole embryo with the exception of the shoot apical meristem, the procambial strands and the root cap (Fig. 2). During radicle elongation (stage 3), CAT activity was observed in almost all tissues, especially in the SAM, the needle primordia and the procambial strands of the hypocotyl (Fig. 6). In contrast, no activity was detected in the cortical parenchyma, in the procambial strands of the radicle and in the root cap (Fig. 4). During hypocotyl elongation (stage 4), CAT activity disappeared progressively from the root and from the hypocotyl (Fig. 9), firstly in the cortical parenchyma and then in the pith (Fig. 10). In P. pinea plantlets, CAT activity was not detected in a few randomly distributed cells of the hypocotyl pith parenchyma, while it was still observed in the other cells. It is interesting to note that, in the same tissue, tannins were also detected in randomly distributed cells. Therefore, it may be postulated that the apparent lack of CAT activity and accumulation of tannins happened in the same cells. Beyond stage 4, the parenchymatous cells surrounding the SAM no longer showed peroxidase activity (Fig. 14). Finally, at stages 6–8, corresponding to organogenesis and elongation of the primary needles, CAT activity was detected only in the SAM, the adjacent primordia of primary needles (Fig. 15) and the vascular system (Fig. 16). No activity was observed in the elongated primary needles. In summary, during post-germination growth, permanent CAT activity was observed in the procambial strands, in the vascular system, in the SAM and in the newly formed

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primary needles adjacent to the SAM. CAT activity progressively decreased in the other parts of the seedling. CAT activity and isoenzyme profile during germination Total CAT activity was assayed in the extracts from different tissues during germination and post-germination growth at stages 1, 4, 6 and 8 in the three Pinus spp. (Table 2). A strong increase in the megagametophyte and the associated embryo was observed following germination until both the radicle and hypocotyl elongated (stage 4). At that time, a gradual decrease in CAT activity was detected along the axis, the higher activity being observed in the cotyledons, the lower in the root. This decreasing trend of total CAT activity was further observed during subsequent development in all the analysed tissues. At stage 6, CAT activity was still high in the cotyledons, but finally decreased at stage 8. CAT activity did not follow exactly the same trend in all three species. The main difference noted was the lower CAT activity observed in the megagametophyte of P. pinaster in comparison with the other species at stage 4. At this time, the cotyledons were the tissues in which CAT activity was particularly high. CAT assays performed directly on non-denaturing gels revealed different bands of CAT activity in protein samples obtained from dry seeds and different tissues of germinating seeds and seedlings. In the three species, four CAT isoforms were detected in the dry seeds but only two were active, although not to the same extent, in the tissues of the germinating seeds and seedlings. In Fig. 18, the electrophoretic pattern of CAT of P. pinaster is reported. A slowly migrating activity band, which we designated CAT-2, was obvious in all tissues, particularly in the megagametophyte (stage 3/4), in the cotyledons (stages 3–4, 5–6 and 8) and in the primary needles (stage 8), in which it was the only active isoform. In contrast, a high activity level was observed associated with the faster migrating band, which we designated CAT-1, during germination and post-germinative growth in the root and hypocotyl; furthermore, the activity of this isoform was detectable also in the megagametophyte (stages 3–4) and cotyledons (stages 5–6). The isoenzyme profiles were the same in the tissues and stages considered for the three species. Discussion In the dry seeds of the three species, the lipid reserves were initially distributed in the whole embryo, including the SAM and the megagametophyte. They were progressively depleted during elongation of the pre-formed organs of the embryo, at first in the radicle, confirming previous observations (Simola 1974; Stone and Gifford 1999), and then in the hypocotyl and cotyledons (Table 3). The breakdown of the lipid reserves stored in the SAM occurred in a sequence of two steps: firstly in the PZ, concurrent with the formation of the primary needles, as soon as the radicle emerged (stage 3),

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and secondly in the PM and in the CMC, concomitantly with hypocotyl elongation (stages 4–6). The last cells that retained lipid reserves were located at the surface of the CMC area at the end of the hypocotyl elongation stage (stage 6). Riding and Gifford (1973) and Cecich (1977) also reported the disappearance of triglycerides in the CMC but, until now, the time-course variations of the lipid reserves distribution within the SAM have not yet been investigated. This parallel spatio-temporal evolution of expansion growth and organogenesis with the depletion of lipid reserves shows that, in the growing embryos which have not yet acquired completely functional vascular systems, the source (= exporting) and sink (= nutrients/energy consuming) zones overlap. This suggests that the energy presumably supplied through the glyoxysomal β-oxidation pathway (Hammer and Murphy 1994; Stone and Gifford 1999) is released at the growth sites, and thus made available directly to the mitotically and metabolically active cells. Within the SAM, the early lipid depletion observed in the PZ cells is probably involved in the initiation of the primary needle primordia, while the hydrolysis of the lipid reserves stored in the CMC might be related to the expansion growth of the hypocotyl and primary needle production. The late depletion of lipid reserves in the CMC is consistent with the low activity of these vacuolated cells. In contrast to the embryo, soluble carbohydrates issued from lipid mobilisation in the megagametophyte need to be transported to the embryo during the post-germinative growth (Kao 1973; Murphy et al. 1992; Stone and Gifford 1999). The cellular pathways of this transfer are unknown. Nevertheless, in the case of the protein bodies stored in the megagametophyte, reserve depletion occurs first in the nearest cells from the embryo (Stone and Gifford 1997). The

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Table 2.

CAT-specific activity in seed tissues of Pinus spp. during germination Values expressed as U mg–1 protein are the means ± SE of at least three independent experiments. , no data

Tissue Seed: P. pinaster P. pinea P. radiata

Stage 1: dry seed

Stage 6: Stage 8: Stage 4: rosette of the epicotyl, elongation of first primary axis with the hypocotyl needles auxiblasts

  

  

  

Megagametophyte: P. pinaster  P. pinea 38.33 ± 4.7 P. radiata 

50.38 ± 8.2 285.18 ± 36.8 696.46 ± 49.1

  

  

Cotyledons: P. pinaster P. pinea P. radiata

  

136.30 ± 15.1 40.61 ± 4.4 109.54 ± 15.9 43.07 ± 2.3 143.13 ± 49.1 44.99 ± 3.5

7.49 ± 0.7 13.70 ± 1.5 6.18 ± 5.3

Hypocotyl: P. pinaster P. pinea P. radiata

  

73.12 ± 14.1 3.12 ± 2.8 68.56 ± 15.7 32.59 ± 3.6 99.58 ± 19.7 28.79 ± 2.7

12.96 ± 0.8 31.49 ± 3.1 38.81 ± 5.1

Root: P. pinaster P. pinea P. radiata

  

31.01 ± 1.6 33.35 ± 2.1 39.11 ± 1.1

9.18 ± 1.6 26.15 ± 2.1 14.51 ± 1.1

12.24 ± 0.6 31.49 ± 1.8 37.55 ± 2.9

  

1.66 ± 0.1 3.66 ± 0.3 3.11 ± 0.7

11.76 ± 2.6 14.81 ± 1.6 8.94 ± 2.7

31.43 ± 5.1 73.18 ± 2.5 17.04 ± 3.1

Primary needles: P. pinaster  P. pinea  P. radiata 

Figs 1–17. c, cotyledon; h, hypocotyl; r, radicle; rm, root meristem; rc, root cap; sam, shoot apical meristem; cmc, central mother cells; pz, peripheral zone; pm, pith meristem; pi, needle primordia; pn, primary needles; cp, cortical parenchyma; pr, procambium; pp, pith parenchyma; vs, vascular system; t, tannins. Figs 1, 3, 5, 7, 8, 11, 12, 13 and 17: triacylglycerol staining. Figs 2, 6, 9, 10, 14, 15 and 16: catalase activity localisation. Figs 1–2. Dry seeds (stage 1). Fig. 1. Triglycerides were detected in the whole embryo (scale bar 320 µm). Fig. 2. Catalase activity was detected at high level in the whole embryo except in the sam, the root meristem, the procambium, and the root cap (scale bar 280 µm). Figs 3–7. Radicle emergence (stage 3). Fig. 3. The growing radicle contained only a few triacylglycerols. The root tip and the adjacent pith parenchyma still stored a high amount of lipids reserves (scale bar 340 µm). Fig. 4. The root tip exhibited strong catalase activity in the root meristem and in the adjacent pith parenchyma (scale bar 320 µm). Fig. 5. Shoot tip with triacylglycerols in the whole meristem and cotyledons. The needle primordia newly produced by the sam did not contain lipid reserves (scale bar 100 µm). Fig. 6. Catalase activity was detected in the whole shoot tip (scale bar 175 µm). Fig. 7. The seedling had lost its lipid reserves in the growing radicle (scale bar 900 µm). Figs 8–12. Hypocotyl elongation (stage 4). Fig. 8. The shoot tip contained less lipids than previously. The triacylglycerol content was higher in the pith than in the cortex (scale bar 300 µm). Fig. 9. Catalase activity was shown in the whole shoot tip except in cells of the sub-apical pith parenchyma (arrow) (scale bar 330 µm). Fig. 10. Hypocotyl section showing higher catalase activity in the pith than in the cortical parenchyma (scale bar 440 µm). Fig. 11. Before the end of hypocotyl elongation, primordia needles and newly formed needles were obvious and did not contain lipids in contrast with the sam (scale bar 110 µm). Fig. 12. A closer view of the sam clearly distinguishing the peripheral zone and the primordia which were reduced in lipid reserves, and cells at the periphery of the cmc area exhibiting a high content of triacylglycerols (scale bar 40 µm). Figs 13–14. End of the hypocotyl elongation (stage 5). Fig. 13. The shoot tip exhibited triacylglycerols only in cells located at the periphery of the cmc (arrow). Tannins were detected in cells of the sub-apical pith (scale bar 120 µm). Fig. 14. Higher catalase activity was detected in the sam and in the procambium (scale bar 270 µm). Fig. 15–17. During epicotyl growth (stage 8). Fig. 15. In the shoot tip, catalase activity remained high in the sam and in the needle primordia (scale bar 180 µm). Fig. 16. The epicotyl exhibited high activity in the vascular system (arrows) (scale bar 330 µm). Fig. 17. Triacylglycerols were newly accumulated in the cmc and pith meristem at the shoot tip level (scale bar 140 µm).

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exchange occurring between the growing seedling and the megagametophyte ceased when the megagametophyte was cast off at the end of hypocotyl elongation (stage 6). At this stage, the seedlings were totally devoid of lipid reserves. After the complete consumption of the lipids stored in the matured embryo, a new accumulation of triacylglycerols occurred in the seedlings at stage 8 which corresponded to the photosynthetic self-supporting period. In particular, newly synthesised reserves were accumulated in the SAM as previously mentioned by Curtis and Popham (1972) and Cecich (1977). This confirmed the CMC as a specific lipid storage area involved in both the germination and postgerminative growth. CAT activity assayed in the dry seed, and up to 2 months after imbibition, occurred in two steps. After 7 d of imbibition, the CAT-specific activity present in the mature seed increased rapidly in the megagametophyte, as expected on

M.-N. Jordy et al.

the basis of its specific role as a storage tissue. Three weeks after imbibition (stage 4), CAT still showed a high level of specific activity in the megagametophyte, but it was intense also in tissues of the developing seedling, less activity being observed in the root in which the lipid reserves were depleted. The high level of CAT activity can be associated with the need to detoxify the cells involved in the intense lipid hydrolysis at that time. Similarly, the complete disappearance of triglycerides in the root is consistent with the reduction of CAT activity in this organ. The further stages of post-germination growth are characterised by a lower enzyme activity. Between stages 4 and 6, whilst the megagametophyte was depleted and cast off, the specific activity of CAT in the cotyledons and other tissues showed a 3-fold decrease. This temporal pattern of CAT activity during germination is consistent with that expected on the basis of literature for a pine seed (Kao 1973; Hammer and Murphy

Fig. 18. Activity staining of CAT isoenzymes following native PAGE. Samples (10 µg of crude soluble proteins) were loaded onto 7.5% acryl-amide gel. Stage 3–4: lane A, megagametophyte; B, cotyledon; C, root. Stage 5–6: lane A, cotyledon; B, hypocotyl; C, root. Stage 6–7: lane A, cotyledon; B, hypocotyl; C, root; D needle. Stage 8: lane A, cotyledon; B, hypocotyl; C, root; D needle.


1994; Stone and Gifford 1999). The intense activity up to stage 4 must be linked to the activation of enzymatic defences against oxygen radicals generated by β-oxidation of fatty acids (Huang et al. 1983). Later on, with the acquisition of phototrophic independence, CAT-specific activity could be connected to the protection of photosynthesising cells against oxidative stress (Willekens et al. 1995). The presence of multiple isoforms of CAT has been reported for a number of plant sources (Willekens et al. 1995). In the Pinus species studied in this paper, four different isoforms of CAT were detected in dry seeds and two isoforms were found in embryo tissues during germination and subsequent growth. These two latter isoforms did not exhibit the same activity in the different analysed tissues (Table 3). CAT-2 was predominant in cotyledons and other seed tissues during germination and later, at stage 8, strongly active in the primary needles and cotyledons. In contrast, CAT-1 was shown to be particularly active in the root and hypocotyl at all stages. This spatio-temporal pattern of distribution of this isoform activity suggests that CAT-1 has an essential role, possibly protecting cells from reactive oxygen species generated during normal metabolic processes. In contrast, according to studies made on maize, CAT-2 would be associated with specific metabolic processes known to produce H2O2 (Tsaftaris et al. 1983; Tsaftaris and Scandalios 1986). These include the glyoxylate cycle in the postgerminative seedlings, and the removal of photorespiratory H2O2 in the bundle-sheath cells of green leaves. Therefore, it

Table 3.

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can be assumed that in Pinus as in maize leaves, both CAT-1 and CAT-2 are involved in the glyoxysomal processes from the early steps of post-germination (stages 3 and 4) until the acquisition of phototrophic independence of the seedlings. After lipid depletion, CAT-2 would be specifically involved in photorespiration. The intense activity showed by CAT-2 in the cotyledons at stage 8 could be related to the senescence occurring in this tissue and to the breakdown of the constitutive lipids. Indeed, Acevedo and Scandalios (1996) suggested that the increase of CAT activity in pollinated silks of maize might be a reflection of increased respiration during accelerated senescence. On the contrary, Del Rio et al. (1998) reported the almost complete disappearance of CAT activity in senescent pea leaves. Our results are in agreement with the hypothesis of an accelerated senescence, but it might be postulated that pine cotyledons at stage 8 are not yet completely senescent and are still active as photosynthesising tissues. The presence of CAT activity was also revealed in fresh sections by histological tests based on H2O2 conversion by endogenous enzymes. Although the negative staining used is mainly related to the CAT activity, simultaneous detection of a peroxidase activity cannot be excluded. Moreover, in certain cases, some CAT isoenzymes exhibit higher peroxidatic than catalytic activity, making it difficult to distinguish between both enzymatic effects (Havir and McHale 1987). Using this histological test, CAT activity was detected in the whole dry embryo, and then progressively disappeared from

Morphological and physiological events during seedling growth: pattern of CAT-1 and CAT-2 distribution in the different seedling tissues intensity of activity: +++++ extremely high ; ++++ very high ; +++ high; ++ moderate; + low; – absent CAT-2 activity in native PAGE

CAT-1 activity in native PAGE

Stage Morphological events

Lipid localisation

Comments

4

Hypocotyl elongation

Lipid depletion in the Cotyledons (+++++) hypocotyl and, to some Radicle (+++) extent, in the cotyledons Total depletion of lipids in the radicle

Cotyledons (+) Radicle (+++)

CAT-2 active where the lipid depletion occurs in elongating hypocotyl CAT-1 particularly active in the root zone where the lipid depletion is completed

5

Cotyledon expansion

Lipid depletion in cotyledons

Cotyledons (++++) Hypocotyl (+++) Radicle (+)

Cotyledons (++) Hypocotyl (+++) Radicle (+++)

CAT-2 active in cotyledons during lipid depletion CAT-1 active in radicle, hypocotyl and cotyledons zones where lipid depletion is completed

6

Elongation of the first primary needles

Absence of lipids

Elongating needles (–) Cotyledons (–) Hypocotyl (+) Radicle (++)

Elongating needles (–) No CAT-2 activity in cotyledons and needles Cotyledons (–) CAT-1 active in hypocotyl and in the root Hypocotyl (++) Radicle (+++)

8

Epicotyl elongation and New accumulation of senescing cotyledons lipids in the SAM

Mature needles (+++) Cotyledons (++++) Hypocotyl (+) Radicle (+)

Mature needles (–) Cotyledons (–) Hypocotyl (+++) Radicle (+++)

CAT-2 active in cotyledons and photosynthetic tissues CAT-1 active in the hypocotyl and in the root. New CAT-2 activity in primary needles probably related to their mature state CAT-2 newly active in senescing cotyledons

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the radicle to the cotyledons during germination and postgermination growth. At stage 4, negative staining revealed a higher CAT activity in the hypocotyl compared with the radicle. The enzyme assays performed at this stage confirmed this observation, and were in agreement with the histological distribution of lipids, suggesting that CAT is localised where the lipid reserves are stored. On the other hand, a permanent signal of CAT activity was detected in the vascular system. In maize, a CAT-3 isoform with enhanced peroxidase activity has been shown to be partly associated with several tissues including the stem, the coleoptile and the root. The cellular function of CAT-3 is largely unknown, but it was suggested that it might play a role in lignification (Acevedo and Scandalios 1991). We have not observed a specifically active isoform in pine vascular tissues that could be recognised as a Class II CAT like the maize CAT-3. Therefore, we can only hypothesise that in Pinus seedlings, such a function is exerted by the CAT-1 and CAT-2 isoforms. Discrepancies between lipid depletion and the negative staining of CAT activity were observed in the SAM, in the newly formed primary needles, in the procambium and in the vascular system. The detection of CAT activity was permanent in these areas, while lipids were completely hydrolysed as soon as the seedlings reached stage 3. This suggests that, in these tissues, oxidising activity is especially high, probably because they are the sites of active cellular division and differentiation (Earnshow and Johnson 1987). However, a possible peroxidase activity involved in the breakdown of auxins that stimulates primary growth of the stem and cell divisions in the vascular cambium cannot be excluded (Uggla et al. 1998). In contrast, the negative staining of CAT activity was not detected in the pith parenchyma cells below the SAM, in which fatty acid mobilisation was still not complete. A possible explanation is that the staining reaction failed in a few cells containing tannins, due to the chelation of metal ions contained in the staining solution (Asfari et al. 1993). Moreover, phenolic compounds are also involved in scavenging activity against H2O2 (Takahama and Oniki 1997). Consequently, negative results of the in situ detection test for CAT activity in these lipid-containing cells may be an artefact. The spatio-temporal pattern of CAT activity and lipid reserves in three Pinus species studied confirms the connection between lipid breakdown and CAT, which is partially involved in this process. However, further investigations by means of immunolocalisation and in situ hybridisation are needed to gain a better insight into the expression and the distribution of the different CAT isoforms involved and their specific contribution to tryglycerides metabolism. Acknowledgments This work was carried out with financial support from the Commission of the European Communities, Agriculture and Fisheries (FAIR) specific RTD programme, 3-CT-96-1445,

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Manuscript received 3 May 2000, accepted 19 July 2000

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