Tunicamycin and swainsonine stimulate Lupinus albus L. root growth in vitro

plan ience ELSEVIER

Plant Science 101 (1994) 137-142

Tunicamycin and swainsonine stimulate Lupinus albus L. root growth in vitro J61ia C o s t a a, C f i n d i d o

P. P i n t o

R i c a r d o *a'b

alnstituto de Tecnologia Quimica e Biol6giea. Universidade Nova de Lisboa. Rua da Quinta Grande. 6. Apart. 127. 2780 Oeiras, Portugal blnstituto Superior de Agronomia, Universidade Tkcnica de Lisboa. Tapada da Ajuda. 1399 Lisboa Codex, Portugal Received 2 May 1994; revision received 24 June 1994; accepted 24 June 1994

Abstract

The effects of tunicamycin, an inhibitor of lipid-linked glycosyl transfer, and swainsonine, an inhibitor of Golgi mannosidase II, on Lupinus albus L. root growth in vitro were studied. SDS-PAGE analysis of total and endomembrane fraction Con A-binding polypeptides from cultured roots indicated that: (i) tunicamycin effectively prevents Nglycosylation of root polypeptides; (ii) swainsonine inhibits Golgi mannosidase II which results in the formation of N-linked oligosaccharides with extra mannose residues. Increases in root length of 26% and 20% were observed when roots were incubated with 5 and 10 #g/ml tunicamycin, respectively, during 48 h; after this period their growth rate was inhibited. Swainsonine also had a stimulating effect on L. albus root growth during the initial 48 h, for each of the concentrations tested (5, 10 and 15/~g/ml) showing similar effects on growth (30%, 25% and 32%, respectively). During the following 48-h period (up to 96 h) a sustained stimulating effect was observed with 5 and 10 t~g/ml swainsonine (29%), while the 15-t~g/ml dose exerted a much higher stimulation (46%). Keywords: Root culture; Tunicamycin; Swainsonine; Lupinus albus

I. Introduction

The root apex is an adequate system for developmental studies since, for a particular file of cells, differentiation processes occur sequentially at increasing distances from the root apex [1,2]. Abbreviations: Con A, concanavalin A; EDTA, ethylenedinitrilo tetra acetic acid; SDS-PAGE, sodium dodecylsulphate polyacrylamide gel electrophoresis; SW, swainsonine; TM, tunicamycin. * Corresponding author, Instituto de Tecnologia Quimica e Biol6gica, Universidade Nova de Lisboa, Rua da Quinta Grande, 6, Apart. 127, 2780 Oeiras, Portugal.

Studies on the action of hormones, specially auxin, cytokinin and ethylene and environmental factors such as pressure, anaerobyosis and light on root development have been reviewed by Feldman [3]. The importance of protein glycosylation in developmental processes has been suggested in both animal [4] and plant [5-7] cells; however, its relationship with the developmental processes occurring in the root is scarcely described in the literature. The use of inhibitors of the N-glycosylation biosynthetic pathway constitutes an approach for studying the importance of correct protein glyco-

0168-9452/94/$07.00 © 1994 Elsevier Science Ireland Ltd, All rights reserved SSDI 0168-9452(94)03914-U

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sylation on development. Tunicamycin (TM) is a nucleoside antibiotic that inhibits the UDPGlcNAc-dolichyl-phosphate:GlcNAc- 1-phosphate transferase that catalyses the first step in lipid carrier-dependant protein glycosylation [8]. At the appropriate concentration, TM effectively prevents the attachment of the N-linked oligosaccharide to proteins [9]. Recently it has been shown that TM, although not affecting cellulose synthesis [10], inhibits cotton fibre elongation and secondary wall deposition [1 t]. Swainsonine (SW) is an indolizidine alkaloid that inhibits Golgi mannosidase II [12]. It has been shown to cause the formation of complex oligosaccharides with extra mannose residues in phytohemagglutinin [13]. In animal cells it blocks the synthesis of complex - but not hybrid-type- oligosaccharides [9,14]. In the present work we study the action of TM and SW on Lupinus albus root growth in vitro in order to investigate the importance of protein Nglycosylation in root development. 2. Materials and methods

Seeds of Lupinus albus L. cv. Rio Maior were surface-sterilised by immersion in a solution of 0.1% (w/v) HgC12 and 0.02% (w/v) Tween 20 for 3 min, rinsed in sterile distilled water, and germinated in 20 ml of sterile deionized water (Milli Q) in Petri dishes (9 cm diameter) (2 seeds per dish), for 2 days, at 25°C, in the dark. Apex terminal segments of 5 mm were cut from the roots and placed at 25°C, in the dark, in Petri dishes containing 15 ml of sterile root culture medium with the following composition, in mg/ml: 236.0 Ca(NOa)2.4H20 , 2.6 FeEDTA, 65.0 KCI, 12.0 KH2PO4, 81.0 KNO3, 36.0 MgSOa.7H20 and 3% (w/v) sucrose (with the pH adjusted to 5.8 before autoclaving), essentially according to Reinert and Yeoman [15]. As a control, plants were grown in sand in conditions similar to those indicated for in vitro roots. Roots were marked with waterproof ink at 5 mm from the root apex 48 h after seed imbibition and the growth of the root apex was measured after 96 h in sand. The length of the in vitro cultured roots was measured in the absence and presence of glycosylation inhibitors, every 24 h, for 96 h. TM and SW (Sigma) (2.0 mg each)

were solubilized in ethanol (1 ml) and added to the root cultures after filtration through Millipore VSWP filters (pore size, 25 nm). The addition of 112.5 ~,1 of ethanol to 15 ml of the root cultures did not cause alterations in root length. The TM preparation contained the isomers A, B, C and D in the proportions of 3%, 36%, 38% and 20%. The root endomembrane fraction was separated according to a procedure previously established [16]. Total root proteins and endomembrane root proteins were solubilised in SDS-PAGE sample buffer and were separated by SDS-PAGE according to Laemmli [17], in a Mini-Protean I1 slab gel (Bio-Rad) as previously described [16]. The stacking and running gels contained 4% and 12% acrylamide (w/v), respectively. The proteins were either detected on gel with Coomassie Blue R-250 or transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, pore size 0.45 ~m) and Con A-binding polypeptides were detected by the Con A/peroxidase affinity method according to Faye and Chrispeels [18]. Total proteins on the blots were detected with Ponceau S [19]. Protein concentration was estimated according to Bensadoun and Weinstein [20] with BSA as a standard. 3. Results and discussion

In order to study the effect of glycosylation inhibitors on root development, in vitro cultured roots were used since it allowed growth conditions to be rigorously controlled. Roots from plants grown in sand for 96 h at 25°C in the dark were 124.8 ~ 9.4 mm long, whereas cultured roots of an equivalent age were 11.5 :~ 1.9 mm long. This result suggests the absence of essential factors for normal root growth, in vitro, In order to investigate whether there was a correspondence between these growth differences and alterations in the glycosylated and non-glycosylated protein composition, SDS-PAGE patterns of total polypeptides and Con A-binding polypeptides of in vivo and in vitro roots were analysed. Very similar patterns were obtained for the two root systems when polypeptides (data not shown) and Con A-binding polypeptides of total extracts were analysed (Fig. I A). Since many glycosylated proteins are intrinsic

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J. Costa, C.P. Pinto Ricardo/Plant Sci. 101 (1994) 137-142

RC

kDa

RTM5 RTMIO RSW

R

RC RTM5RTMIO RSW

Q

66 45, 36

I

N

2924-

'

1

20-

A

B

Fig. 1. Con A-binding polypeptide patterns from roots of L. albus. (A) Total polypeptides and (B) endomembrane fraction polypeptides from (R) in vivo and (Rc) in vitro non-treated roots and in vitro roots in the presence of (RTM5 and RTMI0) 5 and 10 gg/ml tunicamycin, respectively, and of (Rsw) 15 pg/ml swainsonine. Con A-binding polypeptides differing between the in vivo and in vitro non-treated roots are indicated with arrows. On the right side of A, Con A-binding polypeptides that appear upon incubation with SW are pointed out by closed arrowheads; those that are apparently shifted to higher molecular weights are pointed out by open arrowheads. Lanes were loaded with 30 gg of total protein each.

to membranes, the SDS-PAGE patterns of Con Abinding polypeptides from endomembrane fractions of in vivo and in vitro roots were also analysed (Fig. 1B). It was found that although the patterns were quite similar, two polypeptides, one of approximately 29 kDa and the other of 24 kDa were predominantly detected in the in vivo and in the in vitro cultured roots, respectively (Fig. 1B). Although the in vitro and the in vivo root systems are not completely equivalent, the similarity of their protein and Con A-binding glycoprotein patterns supports the use of the in vitro system as a model of the in vivo system when studying the effect of N-glycosylation inhibitors on polypeptide and Con A-binding polypeptide composition. The effects of TM and SW on root development, in the concentrations of 5, 10 and 15 #g/ml have been tested. The level of detection of the root Con A-binding polypeptides, for either total extracts or endomembrane fractions decreased dramatically upon incubation with TM indicating that the inhibitor effectively prevents the attachment of the N-

linked oligosaccharides to proteins (Fig. 1A, B). The level of detection of total endomembrane root Con A-binding polypeptides was higher for the lower TM concentration (5 gg/ml) but remained constant for both 10 and 15 t~g/ml TM (Fig. 1). This result suggested that 10 gg/ml is an adequate TM concentration for N-glycosylation prevention of L. albus roots, which agrees with reported data indicating a 65-90% glycosylation inhibition at TM levels of 2-10/~g/ml [9]. The TM preparations are a mixture of several homologues some of which might eventually cause inhibition of protein synthesis as determined by the decrease of radioactive amino acid incorporation into cellular proteins [211. The effect observed may, however, also be associated to the decrease of protein stability upon glycosylation inhibition. In the present work it was observed that the total polypeptide pattern of cultured roots as detected by Coomassie Blue R remained unchanged after incubation with TM in any of the concentrations tested (data not shown). These results suggest that although differences

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Fig. 2. Effect of (A) tunicamycin and (B) swainsonine on the length of in vitro cultured L. albus roots. Concentration of the inhibitors: II, 0; T, 5; Q, 10; and A, 15 tag/ml. (C) Typical roots submitted to a 96-h incubation with 15 #g/ml of TM and SW. Magnification: × 0.8. Results are the mean of 12-20 measurements; the standard deviation is represented for the lower and the upper curves and is of similar magnitude for the other two curves.

occurred in glycosylated polypeptides the major polypeptide composition of the in vitro roots was not affected by the inhibitor. When roots were incubated in 5, 10 and 15 /~g/ml TM, increases in root length of 26%, 20% and 10%, respectively, were observed for the first 48 h of incubation, while for the remaining time the growth rate was greatly decreased (Fig. 2A). When 15/zg/ml TM was used roots lost consistency and became brownish after 48 h of incubation

{Fig. 2C}. Considering that FM completeh prevents protein glycosylation these results max suggest that after 48 h of action on the roots TM has a toxic effect on the cells. This was particularly evident for the higher concentration tested. In relation to SW, the level of detection of the root Con A-binding polypeptides, both from total extracts and from the endomembrane fraction increased upon incubation with the inhibitor• Furthermore, additional glycosylated polypeptide bands were identified and apparently some of the glycosylated polypeptides observed in the patterns of the control cultures were shifted to higher molecular masses upon inhibitor incubation (Fig. I ). These results suggest a lower level of oligosaccharide processing of the glycoproteins in accordance with the action attributable to SW, i.e.. inhibition of Golgi mannosidase 11 [12]. In fact: {i) Con A binds the structures Glc(o~l-), Man(oel-), GlcNAc(oel-) in terminal non-reducing ends and (-2)Glc(o~l-) and ( - 2 ) M a n ( ~ l - ) in the interior of the oligosaccharide chains [22,23], so the increase of the ligation of the lectin to root polypeptides indicates the presence of extra mannose residues in their oligosaccharide chains: (ill the glycosylated polypeptides have a migration rate in SDS-PAGE inferior to the corresponding non-glycosylated polypeptides, due to the covalently attached oligosaccharide chains [24], so the shift to higher molecular masses of certain glycosylated polypeptides suggests that the oligosaccharide chains synthesised in the presence of the inhibitor are larger than those normally processed. Similar results have been reported for cotyledon cells of Phaseolus vulgaris treated with SW where the oligosaccharide chains of phytohemagglutinin were abnormally processed resulting in the existence of normal oligomannose chains and of modified chains which contained fucose residues and extra mannose residues [13]. This report suggests that the Con A-binding polypeptides detected de novo and those whose apparent molecular mass was increased alter root incubation with SW are of the complex type. L. albus root growth was stimulated upon incubation with SW. During the initial 48 h, each of the concentrations tested (5, 10 and 15 >g/mll had a similar stimulating effect on growth (30%, 25"/,,

J. Costa, C.P Pinto Ricardo/Plant Sci. 101 (1994) 137-142

and 32%~, respectively) (Fig. 2B). During the following 48-h period (up to 96 h) a sustained stimulating effect was observed with 5 and 10 /zg/ml SW (29°/`0, while the 15-tzg/ml concentration exerted a much higher stimulation (46°/,,) (Fig. 2B). The roots looked healthy after the treatment with the inhibitor (Fig. 2C). Both TM and SW stimulated root growth what might be associated to the increase of cell elongation. Cell elongation probably results from alterations of the physical properties of the cell wall eventually related to cross-linking of cell wall constituents. Since most of the cell wall constitutive proteins, such as the arabinogalactoproteins and the extensins, are O-glycosylated [25], and considering that TM and SW are N-glycosylation inhibitors, it can be admitted that such proteins have not been directly affected in the experimental conditions tested in the present work. It has been reported that TM, although not affecting cellulose synthesis in cotton fibres [10], inhibits secondary wall deposition [11] that may, eventually, be associated to decreased cross-linking of cell wall constituents. If such an effect is produced by TM on root cells, then it will possibly lead to root elongation. Peroxidases, which are N-glycosylated enzymes [26], play a fundamental role in cell wall cross-linking [27-29]. In peanut the activity of these enzymes was drastically reduced after deglycosylation and their secretion inhibited by TM [30]. Thus, it could be admitted that TM and SW, being N-glycosylation inhibitors, cause alterations in peroxidase abundance or activity in L. albus root cell walls that could account for changes in cell wall extensibility and, consequently, cell elongation. The mode of action of the inhibitors in inducing root elongation can, however, be related to other abnormally glycosylated proteins of fundamental importance for cell growth. Studies concerning the cell wall of L. albus roots in the presence of the glycosylation inhibitors are required in order to further clarify the importance of N-glycosylation in root growth.

Acknowledgements This work was supported by Junta Nacional de Investigaq5.o Cientifica e Tecnol6gica, Portugal

(project PMCT/C/BIO/876/90 and grant 105/90-IF).

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