Correlation between changes in cell ascorbate and growth of Lupinus albus seedlings

J Plant PhysioL WJL 150. pp. 302-308 (1997)

Correlation between Changes in Cell Ascorbate and Growth of Lupinus a/bus Seedlings ORESTE AIuuGONIl, GIUSEPPE CALABRESEl, LAURA DE GARAl, ROSALIA

LIso 1

MAruA B.

I

Institute of Botany, University of Bari, Via E. Orabona 4, 70126 Bari, Italy

2

Department of Ecology, University of Calabria, 37036 Arcavacata di Rende (Cs), Italy

BITONTI2,

and

Received February 15, 1996 . Accepted June 23, 1996

Summary

The growth of Lupinus albus seedlings is stimulated when ascorbate (Asc) content is experimentally raised; on the contrary, when Asc content is lowered by lycorine - an alkaloid inhibiting Asc biosynthesis - the growth is inhibited. The stimulation of growth by Asc is due to the promotion of cell division and cell expansion. Redox enzymes of the Asc system respond in different manners to the Asc changes. Asc peroxidase is the enzyme that is most affected by Asc changes in the cell: its activity increases or decreases in parallel with the Asc content. Dehydroascorbic acid (DHA) reduction capability is affected in the opposite way: when Asc content rises, the DHA reduction ability of the cells drops, while it rises when Asc decreases. Ascorbate free radical (AFR) reductase activity does not vary with changing Asc content. A possible regulatory function of Asc redox enzymes is discussed.

Key words: Lupinus albus, growth, ascorbate, ascorbate peroxidase, ascorbate.free radical reductase, dehydroascorbic acid reductase. Abbreviations: Asc = ascorbate; AFR = ascorbate free radical; DHA = dehydroascorbic acid; GL = L-galactono-y-lactone. Introduction

Lycorine, an inhibitor of Asc biosynthesis (Arrigoni et al., 1975; Evidente et al., 1983), has been proved to be a useful tool for studying Asc-dependent metabolic reactions in Ascsynthesising organisms. Despite numerous experimental data on the role of Asc in cell metabolism (Arrigoni et al., 1976; Arrigoni et al., 1977; Arrigoni et al., 1979; Navas et al., 1994; Arrigoni, 1994; C6rdoba and Gonzales-Reyes, 1994) and on its involvement in cell division (Liso et al., 1984; Liso et al., 1988; Navas and G6mez-Diaz, 1995), the effect on plant growth of variations in Asc content has never been demonstrated. This paper aims to document some morphological and anatomical characteristics of Lupinus a/bus seedlings grown under Asc deficiency conditions. In addition, since the Asc system includes a complex net of Asc, AFR and DHA © 1997 by Gustav Fischer Verlag, lena

producing reactions (Arrigoni et al., 1992; Arrigoni, 1994), we have considered how redox enzymes of the Asc system are affected by Asc changes in the cell.

Material and Methods

Plant material Seeds of Lupinus albus L., imbibed for 20 h, were sown in moist vermiculite in a dark room at 22°C. After n h of germination, the seedlings were transferred into 250 mL glass beakers containing water or solutions of lycorine, D-glucose, L-galactono-y-Iactone or ascorbic acid at the concentrations and times specified in each case; when it was necessary, the pH of the solutions was corrected to 6.2. Fifteen seedlings were used for each beaker and were so placed that only the roots remained submerged. The medium was renewed at

Ascorbate and plant growth 24 h intervals and aerated by continuous bubbling at a rate of B-lO cm3 min-I.

Light microscopy Formalin-acetic acid - 50 % ethanol was used as a fIxative. After dehydration in an ethanol series, tissues were embedded in paraffin, sectioned at 7-101lm and stained with safranin-fast green. For mitotic index evaluation, root apices were fIXed in absolute ethanol: glacial acetic acid (3: I, v: v). After washing, only the proximal meristem zone (250 Ilm from the cap junction) was dissected and prepared as Feulgen squashes after 7 min of hydrolysis in 1 N HCI at 60·C. For each sample fIve roots were analysed with 1,000 cells being scored for each root.

Ascorbic acid determination One g of root apices (10 mm from the tip) and 3 g of hypocotyl segments (5 mm from the cotyledons) were homogenized in Band 24 mL, respectively, of a solution of 5 % metaphosphoric acid and centrifuged at 25,000 gn for 15 min. Samples of the supernatant (0.1-0.3 mL) were added to 0.1 mol/L citrate, 0.2 mol/L phosphate buffer, pH 6.2 (fInal volume 3 mL) in a quartz cuvette and the absorbance at 265 nm was measured. The Asc was oxidized by adding 1 unit of purifIed Asc oxidase (EC 1.10.3.3) (Boehringer) and the relative decrease in absorbance was measured. Asc contents were expressed as nmol/g fresh weight and calculated from a standard curve.

Enzyme determination Root apices (1.5 g) and hypocotyls (3 g) were homogenized at 4 'C in a mortar with a medium containing 50 mmol/L Tris/HCI, pH 7.B, 0.3 mol/L mannitol, 1 mmol/L EDTA and 0.05 % cysteine, in a w: v ratio of 1 : 2 for root apices and 1: 1 for hypocotyls. The homogenates were centrifuged at 40,000 gn for 20 min. Aliquots of the supernatant (2.5 mL) were passed through Sephadex G-25 columns (PD-lO columns, Pharmacia, Germany) using the grinding medium as elution buffer. The de-salted protein fractions were used to determine the enzyme activities.

303

0.1 mol/L phosphate buffer, pH 6.4, containing 4 mmol/L. Asc and 4 mmol/L H Z0 2 • The gels were then washed with distilled water and stained with a solution of 0.125 N HCI containing 0.1 % ferricyanide and 0.1 % ferrichloride (w: v). In presence of Asc, ferricyanide was reduced to ferrocyanide, which reacted with ferrichloride, dyeing the gel with Prussian blue. Under these conditions H 20 2 did not interfere with the dyes. Asc peroxidase was located as an achromatic band, due to the H 20 2-dependent oxidation of Asc, on the Prussian blue background.

Results

The growth of Lupinus albus is strongly affected by changing Asc content in the cell. Asc content in the seedlings was experimentally lowered by treatment with lycorine, an inhibitor of Asc biosynthesis that does not affect Asc utilization (Arrigoni et al., 1975; Liso et al., 1984). As can be seen from Table 1, treatment with 5 ~mollL lycorine causes decreases of 48 % and 30 % in Asc in the root and hypocotyl, respectively, conditions under which plant growth was inhibited (Fig. 1).

Table 1: Ascorbate content in seedlings of Lupinus albus treated with

lycorine and L-galactono-y-Iactone. Three-day-old seedlings were treated for IB h with lycorine or GL in the dark. The ASC content remained constant even after several days treatment in both controls and those treated with GL, whereas it decreased progressively in those treated with lycorine. The values are the means of three experiments ± SD.

Treatment

Control Lycorine 5 Ilmol/L GL 0.25 mmol/L

Asc content (nmol/g fresh wt) Root apex

Hypocotyl

1470± 102 765± 65 3470±230

BlO±50 565±39 995±77

Spectrophotometric assay APR reductase (EC 1.6.5.4) activity was tested by measuring the oxidation rate ofNADH at 340nm in a reaction mixture composed of 0.2 mmol/L NADH, 1 mmol/L ASC, and 50 mmol/L Tris-HCI, pH B. The reaction was started by adding 0.5 unit of Asc oxidase to generate saturating concentrations of APR (Arrigoni et al., 19B1). Asc peroxidase (EC 1.11.1.11) was determined using a reaction mixture composed of 50 Ilmol/L Asc, 90 Ilmol/L HzO z and 100 mmol/L phosphate buffer, pH 6.4. The HzO z dependent oxidation of Asc was followed by means of the decrease in absorbance at 265 nm. Non-enzymatic oxidation of Asc by denatured extracts was subtracted. Since no Asc was added to the grinding medium, only the cytosolic Asc peroxidase was detected (Arnako et al., 1994) DHA reductase (EC 1.B.5.1) was tested following the increase in absorbance at 265 nm due to the glutathione - dependent production of Asc. The assay mixture consisted of 1 mmol/L DHA, 2 mmol/L reduced glutathione and 100 mmol/L phosphate buffer, pH 6.2. Nonenzymatic reduction ofDHA by glutathione was subtracted. Proteins were determined by the Lowry method (1951).

Electrophoretic assay Native PAGE was performed using 7.3 % acrylamide (Davis, 1964). The gels were incubated for 15 min at room temperature in

Fig. 1: Effect of lycorine on growth of Lupinus albus seedlings. After 72 h of germination, the seedlings were transferred into water or lycorine and grown for 10 days in natural light. The treated plants appear normal, but show a reduced growth and a lower number of lateral roots than control plants.

304

ORESTE ARRIGONI, GIUSEPPE CALABRESE,

LAURA

DE GARA, MARIA B. BITONTI, and ROSALIA LISO

The Asc content was raised by adding exogenous Asc or GL (see Table 1), a compound that is promptly converted to Asc inside the cell by an active mitochondrial GL dehydrogenase (Oba et al., 1995). GL was preferentially used for the following reasons: 1) to exclude the interaction between the added Asc and the cell wall that it must cross to enter the cells; 2) to confirm that the effects obtained can be ascribed to the Asc actually present inside the cell; and 3) because the uptake of external Asc undergoes rhythmic regulation (Innocenti et al., 1994). Figure 2 shows that GL stimulates both root and hypocotyl growth of Lupinus albus and that the effect is clearly visible after a few days' treatment. The time course of root growth in Fig. 3 shows that after 72 h of incubation in GL the roots grew 60 % more than those in water, with an even greater effect for those in ascor-

Table 2: Mitotic index in root tips of Lupinus albus following treatment with L-galactono-y-lactone and ascorbic acid. Each determination was based on 5,000 cells; 1,000 cells were scored for each of five roots. Three-day-old seedlings were grown in water, GL or Asc for 48 h in the dark. The mean value of the mitotic index remained substantially unchanged in roots kept in water throughout the treatment period. Treatment

Mitotic index

Control GL 0.25 mmol/L Asc 0.1 mmol/L

3.2±0.1 4.8±0.1 5.0±0.2

Table 3: Cell length along the metaxylem line in Lupinus albus roots following treatments with L-galactono-y-lactone and ascorbic acid. The seedlings were grown in water, GL of Asc for 48 h in the dark. For each sample the result is the mean (± SD) of the measurements carried out on five roots. Treatment

Cell length (~m) distance from the root tip 4,000 ~m 1,500 ~m

Control GL 0.25 mmol/L Asc 0.1 mmol/L

«

2

Fig. 2: Effect of L-galactono-y-lactone on growth of Lupinus albus seedlings. Three-day-old seedlings were kept in water and 0.25 mmollL L-galactono-y-lactone for 4 days in the dark.

o Control

mGIucoee Ill1I

GL

•

ASC

30 I

..

···f"I' I t~:~ i f~

10

o

"

',\, ,

20

o

.a... ••'.. 24

_ 48

72

Time (hours)

Fig. 3: Time course of root growth. Lupinus albus seedlings with roots 1 em in length were selected (Time 0) and kept in water, 0.25 mmol/L glucose, 0.25 mmol/L L-galactono-y-lactone or 0.1 mmol/L ascorbic acid in the dark.

26±0.1 28±0.1 30±0.1

68±1.7 82±2.0 89±3.1

bate (80 %). To verify whether the Asc effect on root growth was due to use of Asc as a carbon source, the roots were also incubated in 0.25 mmollL glucose; no effect was observed in the presence of glucose. The Asc content in the roots in water and glucose were substantially the same and remained constant at approximately 1,500 nmollg fresh wt. throughout the treatment period. In the roots treated with GL and Asc the increase in Asc was observed in the first 24 h and remained at the same level during the following days. The increase in the Asc content was approximately 2.5 fold with GL and slightly higher with Asc. Both cell division and cell expansion are stimulated in treated roots. As shown in Table 2, the mitotic index in root meristem increases following treatment with GL or Asc, the mean value being 3.2 in water-grown roots and 5.0 in root supplied with ascorbic acid. Under the same experimental conditions, the cell length along the metaxylem line is greater in the treated roots than in control roots. The data in Table 3, related to the cell length measured on two histological areas of the root at the distances of 1,500 Jlm and 4,000 Jlm from the tip, show that both GL and Asc stimulate cell expansion. The Asc increase caused by GL or Asc treatments also induces a more rapid and abundant production of lateral roots. Examination of two batches of 30 seedlings, one kept in water and the other in 0.1 mmollL ascorbic acid for 4 days in the dark, revealed 16 ± 4.43 lateral roots in different stages of development and distributed in two lines along each root in the controls, while in the treated batch there were 23 ± 5.62 lateral roots frequently distributed in more than two lines along the root. It can be seen from the transverse root sec-

305

Ascorbate and plant growth

.. Fig. 4: Ascorbate effect on lateral root formation in Lupinus albus. Transverse sections of roots grown in water and in 0.1 mmollL ascorbic acid for 4 days in the dark. x20.

Fig. 5: Effect of L-galacrono-y-lactone on the activity of secondary meristem in Lupinus albus. Transverse sections of the roots made at the beginning of secondaty growth. The seedlings were grown in water and 0.25 mmollL L-galactono-y-lactone for 4 days in the dark. The arrows indicate the cambial zone. x 80.

-Control

. .'~}:,:~

Ascorbate

'

., J .

..:. ~:·J';'ttd 'm

tions in Fig. 4 that there are two primordia of lateral roots in nounced in the early days of treatment and then gradually the control as compared with four in the ASC treated root; decreases (Fig. 6). similar data can be obtained by adding 0.25 mmollL GL. The activities of cytosolic Asc peroxidase, DHA reductase Asc and GL also stimulate the activity of secondary meris- and AFR reductase related to experimentally induced variaterns; in the presence of Asc or GL a higher number of deri- tions of Asc content are reponed in Fig. 7. vatives are produced by the vascular cambium (Fig. 5). It can AFR reductase activity is not modified when the Asc conalso be seen from the figure that pith parenchymatic cells of tent in the cells is either decreased or increased. the roots grown in GL are larger than those in control plants. Asc peroxidase activity increases or decreases in parallel with Funher evidence for the involvement of Asc in the expan- the Asc content and this occurs both in the root apex and the sion growth was obtained by considering the lengthening of hypocotyl. It has been ascenained by native PAGE that a L. albus hypocotyls beyond 1.5-2 cm, a stage at which cell single band with Asc peroxidase activity is present both in the division has almost completely ceased and after which hypo- roots and in the hypocotyls; neither GL nor Asc treatment incotyl growth occurs through cell expansion. Hypocotyllength- duces the appearance of new bands but only an intensification ening is strongly inhibited by lycorine and stimulated by Asc of that already present in the control (data not shown). or GL. The time course of the effect of Asc and GL on hypoDHA reducing capability is affected inversely to that of cotyl growth shows that the stimulation is much more pro- Asc peroxidase. When Asc content is lowered, DHA reduc-

306

ORESTE ARRIGONI, GIUSEPPE CALABRESE, LAURA DE GARA, MARIA B. BITONTI, and ROSALIA LISO

Asc content is raised, the growth is stimulated. The growth responses to Asc variations are due to the fact that Asc stimulates both cell division and cell expansion. We have previously reported that large amounts of Asc are utilized during primary root meristem cell division and that when the Asc content of actively proliferating cells is experimentally lowered, the cell cycle is arrested in the G 1 phase (Liso et al., 1984); the addition of Asc restores DNA synthesis and cells enter the S phase (Liso et al., 1988). Present findings demonstrate that Asc also stimulates division of vascular cambium and pericycle cells: this confirms our previous results showing that the quiescence of pericycle cells is reversed or delayed by Asc treatment (Arrigoni et al., 1989; Innocenti et al., 1993) and is consistent with the data reported by Navarro et al. 2 10 4 6 8 (1992) and Citterio et al. (1994) showing that Asc is necesDays of treatment sary in both plants and animals to enable already competent Fig. 6: Time course of hypocotyl elongation. Lupinus a/bus seedlings cells to progress through the cell cycle phases, yet it is unable with hypocotyl 1.5 cm in length were selected and kept in water, to induce proliferation of non-cycling cells. 10 ~mol/L lycorine, 0.25 mmollL GL or 0.1 mmollL ASe. Data about the growth of the Lupinus albus hypocotyl and the lengthening of the cells along the metaxylem line clearly indicate that cellular expansion is also stimulated by Asc. HYPOeOTYL ROOT APEX At the present time, we do not have a clear and definitive molecular explanation for the relationship between Asc and DHA reductase· the promotion of cell division and cell expansion. However, D AFR reductase" since Asc is the physiological electron donor for peptidyl-prolyl-hydroxylase (De Gara et al., 1991), it is possible to sup1m Ase peroxidasec pose that Asc could affect these processes by regulating the hydroxylation of some proteins. In fact, it has been reported that a specific hydroxyproline-rich-protein plays a relevant role during the synthesis of new primary cell walls (Hirsinger et al., 1994) and that the inhibition of peptidyl-prolyl-hydroxylase activity modifies the normal cell differentiation pattern as well as inhibiting cell division (Cooper et al., 1994). The response of the Asc system's redox enzymes to variation in the Asc content of the cell is different for the three analysed enzymes. Cytosolic AFR reductase remains at the same level during 18-h treatment with lycorine or GL, and this result is in accordance with previous data showing that the activity of the enzyme present in the cell is regulated by Asc utilization, i.e. AFR production (Schoner and Krause, 1990; Arrigoni et al., 1992; Arrigoni, 1994; Grantz et al., Lye 1995) rather than by Asc content. Flg.7: Activities of redox enzymes of the ascorbate system in LupiIn contrast to AFR reductase, DHA reduction capability is nus a/bus seedlings kept for 18 h in different experimental condiclearly modified following treatment with lycorine. When tions. C = control; GL = 0.25 mmollL L-galactono-y-Iactone; LYC = 10 ~mol/L lycorine. a 1 unit = 1 nmol DHA reduced; b 1 unit = Asc biosynthesis is inhibited by lycorine and Asc content de1 nmol NADH oxidized; C 1 unit = 1 nmol Asc oxidised. The values creases in the cells, there is a concomitant increase in DHA recycling capability. This metabolic response to lycorine treatare the mean of three experiments ± SD. ment is common to other plants so far analysed (Cappuccio, 1993; De Gara et al., 1993), and it seems to be a homeostatic tase activity per mg protein rises from 50 ± 6 to 91 ± mechanism to protect against Asc deficiency. 8 nmol· min- DHA reduced in root apex, and from 31 ± 5 The behaviour of Asc peroxidase is also interesting as its to 54 ± 7nmol.min-1 DHA reduced in hypocotyl; conver- activity increases when the Asc content is raised, in both disely, it decreases when Asc is raised. viding (root apex) and expanding cells (hypocotyl), while its activity decreases when Asc content is lowered. This response is in some way predictable because ascorbate Discussion and Conclusions is the physiological substrate of the enzyme, but to explain the relationship between Asc peroxidase activity and growth Experimentally induced changes in Asc in the cells are ac- will require further investigation. In attempting to give an excompanied by changes of growth in Lupinus albus. When Asc planation, it is necessary to consider the following: 1) Asc content is lowered, a decrease in growth occurs, and when peroxidase is the key enzyme for scavenging HzO z in plant

o

t I

Ascorbate and plant growth

cells (Asada et aI., 1993); It is clear that its enhancement increases the efficiency of toxic H 20 2 removal and this represents a favourable condition for the cell metabolism, and 2) peroxidases represent a heterogeneous and versatile population of enzymes involved in many biochemical reactions, and it is well known that Asc peroxidase has an affinity for hydrogen peroxide higher than that of the other peroxidases. The Asc peroxidase Km for H 20 2 is reported to be in the range of 10-30 JlmollL (Tommasi et aI., 1987; Elia et aI., 1992; Koshiba et aI., 1993), while that of the other peroxidases is 70700 JlmollL (Clarkson et aI., 1992). On the basis of these data, it could be reasonably argued that when Asc peroxidase activity rises, the amount of H 2 0 2 available for the other peroxidases decreases and with it their activities. In this way the activity of the peroxidases involved in cross-linking cell wall matrix polymers could be limited. Since these peroxidases reduce cell growth by affecting the wall extensibility (Gladys et aI., 1988), it is possible to suggest that the variations of Asc peroxidase by modulating the availability of H 2 0 2 in the cell, and thus the activity of cross-linking peroxidases, perform a regulatory role in cell expansion growth. Acknowledgements

The authors wish to thank Prof. E. Evidente, University Federico II, Portici, for kindly supplying Iycorine. Part of this work was supported by funds from the CNR.

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