Diff erengal effects of glutamine and arginine on 7s globulin accumulation during the maturation of oil palm somatic embryos

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1 Plant Cell Reports (1999) 18: O Springer-Verlag 1999 Diff erengal effects of glutamine and arginine on 7s globulin accumulation during the maturation of oil palm somatic embryos Received: 22 July 1997 I Revision received: 6 January 1998 I Accepted 1 December 1998 ' Abstract The low vigour of plantlets resulting from oil palm somatic embryos may be due to insufficient levels of deposited storage proteins. Thus, in order to improve embryonic maturation and the vigour of regenerated plantlets, we investigated the effects of modifying the culture conditions with respect to the accumulation of the major oil palm storage proteins, the 7s globulins. In this study, the effect of arginine and glutamine on globulin accumulation was studied using somatic embryos of two different geno- ' types. Arginine and glutamine were both found to enhance protein accumulation but in different ways, which were best illustrated by measurements of soluble proteins per embryo and 7s globulin content per dry weight. Arginine increased the level of soluble proteins by 46% irrespective of the clone, and glutamine by 19% and 63% depending on the clone. The clone which accumulated the least protein in the presence of glutamine was that which contained the more protein initially. Only arginine favoured the accumulation of 7s globulin content per dry weight, irrespective of the clone considered (+26%). This study will enable further investigations of specific storage proteins as potential markers for regenerated plantlets vigour. Key words Amino acid. Proteosynthesis. Somatic embryogenesis. Storage proteins Abbreviations ELISA Enzyme-linked immunoabsorbent assay Communicated by G. Pelletier E Morcillo (E$. F. Aberlenc-Bertossi. M. Noirot S. Hamon. Y. Duval ORSTOM/CIRAD-CP, Laboratoire GeneTrop, B. P. 5045, F Montpellier, Cedex 01, France Fax: _ - \ r - Introduction Individual somatic embryos with synchronised development can be mass produced in oil palm from embryogenic suspension culture (de Touchet et al. 1991; Teixeira et al. 1995; Duval et al. 1995). However, unlike zygotic embryos cultured in vitro, under the same germination conditions somatic embryos regenerate plantlets with poor vigour. This lack of vigour would seem to be due to their incomplete maturation under the in vitro development conditions practised (Roberts et al. 1990). In order to improve oil palm somatic embryo maturation, the effects of modifying the culture conditions have beeninvestigated. Merkle et al. (1995) suggested that storage proteins could be maturation markers and, consequently, markers of the quality of the regenerated plantlets. 7s globulins, the dominant storage proteins in oil palm zygotic embryos (Morcillo et al a), were chosen as biochemical markers in order to determine somatic embryo maturation. Morcillo et al. (1997b) identified 7s globulins in somatic embryos using Western blotting. The 7s globulin content, as measured using an ELISA-based method, was found to be 80 times lower than that of zygotic embryos, with around 30% glutamine/glutamic acidand asparagine/aspartic acid type nitrogenous amino acids and high levels of arginine (around 11 %). These amino acids, which are important precursors of 7s globulin synthesis, could play a role in oil palm somatic embryo maturation. In fact, in many species, the addition of organic nitrogen sources to the culture mediumleads to an increase in embryo dry weight, protein content and conversion rate into plants (Stuart et al. 1985). In the study presented here, we investigated the effects of adding glutamine and arginine to the culture medium on the accumulation of 7s globulins in somatic embryos obtained from two different clones.

2 E Materials and methods Plant material Two embiryogenic oil palm (EZaeis gziineensis Jacq.) clones of different genetic origin (LMC 121 and LMC 221) were maintained at ORSTOM-CIRAD (Montpellier). Somatic embryos were obtained from embryogenic suspension cultures using the process developed by de Touchet et al. (1991). After 4 weeks in hormone-free liquid medium, embryonic clumps were sieved (mesh size: 1 mm) and directly plated (40 mg of fresh weight per petri dish) on solidified (0.8% agar) basal medium supplemented with sucrose (30 g/l) and casein hydrolysate (0.5 g/l). Embryos were subcultured every 7 days on the same medium. During the first week, 5 pm BA (6-benzyladenine) was supplemented to the above-mentioned medium (Aberlenc-Bertossi and Duval 1993; Duval et al. 1995). The first epidermised round embryos (1-2 mm) appeared during the beginning of the third week of culture; these rapidly became ovoid (2-3 mm) and progressively elongated (4-6 mm) up to the end of the fourth week of culture. Experimental design The two factors (arginine and glutamine) were studied in a randomised 2' factorial design. Each factor was represented by two treatments, GO (O mm) and G20 (20 mm) for glutamine and AO (O mm) and A5 (5 I&) for arginine, which were defined as being not toxic for embryo regeneration. Each combination was replicated six times, i.e. six petri dishes, and each replicate constituted an experimental unit. Maturation trials Arginine (A-3784, Sigma) and glutamine (G-5763, Sigma) were added to the in vitro culture medium during embryo development from the second week of culture on a solid medium, for a total of 3 weeks. Arginine was added before sterilisation of the medium. Glutamine was sterilised by filtering (0.22 pm) and added after autoclaving. Protein analysis The storage proteins were extracted from three batches of 50 freezedried embryos in a phosphate buffer (25 mm, ph 7) with NaCl (0.2 M), leupeptine (1 r*m) and PMSF (10 r*m). The amount of soluble proteins was estimated by Bradford's method (1976). 7s globulins from this crude protein extract were then quantified by indirect ELISA. Protein extracts (25 ng) from embryos were diluted in 50 mm bicarbonate (ph 9.6) buffer, and immunoplate wells were coated with the protein solution (100 pl) for 18 hat 4 C. The plate wells were then blocked with 3% gelatin in PBS for 2 h at 25 C. Anti-7s globulin antibodies were prepared by Biocytex (Marseille, Franke) from purified 7s globulin of oil palm zygotic embryos according to Morcillo et al. (1997 a). Polyclonal antibodies were diluted to a suitable concentration in blocking solution (1 % BSA-PBS) and incubated in the plate wells for 2 h at 25 C after washing with 0.2% Tween in double-distilled water. Thereafter, the plate wells were washed again with the Tween solution. The second antibody (anti-rabbit immunoglobulin bound to alkaline phosphatase), previously diluted by a factor of U9000 in BSA-PBS, was added to each well and incubated for 1 h at 37 "C. Plate wells were washed with PBS-Tween, then 100 pj of alkaline phosphate substrate (pnpp) (Sigma 104@ phosphatase substrate ) was added to each well prior to incubation for min at 37 OC. The optical density was then assayed at 405 nm. Results were expressed in 7s globulin equivalents (Eq. GLO in pg/50 embryos). A modification of the model of Huet et al. (1988) was used for the adjustment of the calibration curve. Observations 869 The embryos were sampled after three l-week culture cycles with the supplemented amino acids, weighed and freeze-dried. The following traits were measured: the number of isolated embryos with an epidermis (NE) produced per dish, embryo dry weight (mg150 embryos) (DW) and the amount of soluble protein (SP) and 7s globulins (GLO) accumulated (mg/50 embryos). The results were used to calculate soluble protein content on dry weight basis (SPDW) and the 7s globulin content on a dry weight or soluble protein basis (GLOOW, mglg DW, GLO/SP, mg/g of SP). Statistical analyses Comparison of clones before treatment The clones were compared under standard culture conditions using a one-way analysis of variance with fixed effect. The clone effects studied were NE, DW, SP, GLO, GLODW and GLO/SP. Search for and interpretation of the main components Data were expressed relative to the control. Effects of arginine and glutamine were summarised using principal components a@ysis (PCA) (Hotelling 1933). This analysis revealed new independent components (or factors) which were interpreted according to their correlation with the observed traits. Only the components with an eigenvalue higher than l were considered. The most strongly correlatedcharacters (negatively or positively) were thosethat best explained the component in statistical terms and enabled its interpretation. Moreover, the square of this correlation (equivalent to the coefficient of determination) was used to quantify the component contribution to the variance observed for each character. Factor scores were then calculated for each experimental unit. Comparison of the effect of ghtanzine and arginine on the components The effects of the clone, arginine and glutamine were tested on the factor scores using an analysis of variance with three fixed classification criteria (clone, arginine, glutamine). In the event of a significant effect with more than two modes, which is the ;ase with significant interactions, the Newman and Keuls multiple comparison of means test (Newman 1939; Keuls 1952) was used. Lastly, to illustrate the effects of the components, we present the means observed for the most strongly correlated characters. Results Comparison of clones Under standard culture conditions, the somatic embryos produced from the two embryogenic cell lines, LMC 121 and LMC 221, had similar development and growth characteristics (Table 1). The number of regenerated embryos per dish was not different between the two clones.= 4.74; not significant (NS)], nor was the dry weight per 50 embryos = 0.93; NS). However, they differed in their protein content. There were 1.5 times more soluble proteins in the embryos of clone. 221 (3.90 mg/50 embryos) than in those of clone 121 (2.40 mg/50 embryos = 21i37; P<O.OOl). 7s globulins, which represented around 2-7% of the total soluble proteins, accu-

3 v 1' 870 Table 1 Intra-clone effects of glutamine and arginine on maturation somatic embryos. The data are given as the mean" of six independent experiments ~ Treatment (mm) - Arg Glu O 0 o Number of embryos " " " b " a*b " b Dry weight (mg)/ 50 embryos 47.42" 49.80" 65.43b 65.24b 51.21" 57.27' 78.24' 75.20d Soluble proteins (mg)/ 50 embryos 2.40" 3.90" 4.13b 6.01b 3.73b 6.05b 6.04' 8.00' Soluble proteins/ dry weight "g) 7s globulins (mg)/ 50 embryos 51.2" 82.0OaSb 0.052" 0.261" 62.73b *b*C 0.070b 0.360b 72.10' C*d 0.076b 0.390b 76.55' 111.5b*c*d 0.11' 0.520' 7s globulins/ dry weight (mg/g> 1.06" 5.34" 1.04" 5.66" 1.48b 7.00b 1.3Sb 6.8gb 7s globulins/ soluble proteins "g) 21.00a 65.25" 16.71" 60.38a 20.76" 64.79" 18.26" 65.13a " The intra-clone means followed by the same letter are not significantly different at the 0.05 probability threshold determined by the Newman and Keuls test Table 2 Correlation between new components and initial traits Variables Component 1,Component 2 PRODE GLOENR r R2% r R2S Numbers of embryos -0.13NS *** 35 per petri dish Dry weight (mg)/50 embryos 0.87 *** NS 0.6 Soluble proteins (mg)/ 0.99*"* NS embryos Soluble proteinddry weight 0.79*** NS 7.8 " ( 7s globulins (mg)/50 embryos 0.78*** *** 30 7s globulins/dry weight (mg/g) 0.20 NS *** 85 7s globulins/soluble proteins *** *** 50 (mgk) splanation of variance Total Prp NS, **, *** Not significant or significant at P10.01 or 0.001, respectively mulated more in the embryos of clone 221 (0.261 mg/50 embryos) than in clone 121 (0.052 mg150 embryos) (FI,,,, = ; P<O.OOl). Likewise, globulin contents in relation to total proteins and dry weight were three to five times greater in the LMC 221 embryos (F,,,, = ; P<O.OOl and F,,,, = ; P<O.OOl, respectively) (Table 1). Identification of two types of.effects, protein accumulation ("PRODE') and specific 7s globulin enrichment ("GLOENR') The effects linked to the clone, to arginine and to glutamine on the traits observed (Table 1) could primarily be attributed (78%) to two independent components (Table 2). The first explained 61.5% of the variance observed by the two components and accounted for 98% of the variation in soluble protein contents per organ (Table 2); it could.be interpreted as protein deposition. Thus, the first component was arbitrarily named "PRODE" for PROtein DEposition. The second component statistically explained 85% of the variation in 7s globulin content in relation to dry weight (Table 2). It was interpreted as the relative accumulation of 7s globulins leading to enrichment of the dry weight in these storage proteins. Thus, the second component was named "GLOENR' for GLObulin ENRichment. Two traits, total soluble proteins per embryo and 7s globulin content on the dry weight basis, were chosen to characterise the accumulation of storage proteins. These two traits were not statistically correlated with each other (r = 0.250, IZ = 43). Effects of glutamine and arginine on protein deposition (PRODE) and specific enrichment in 7s globulins (GLOENR). Impact on soluble protein quantities (SP) and on globulin content on the dry weight basis (GLODW) The PRODE component depended on the clone, arginine, glutamine and the clone x glutamine interaction (Table 3). Arginine and glutamine had a positive effect on the component and, consequently, on soluble proteins (Table 1). Arginine had the same effect on both clones in terms of the relative increase in soluble protein quantities (+46%). However, the relative effect of glutamine differed depending on the clone (Table l), which was reflected in a variation in levels of soluble proteins of 19-66% for clones 221 and 121, respectively. In this case, solubleprotein content was 5.09 mg/50 embryos for clone 121 and 5.89 mg/50 embryos for clone 221. The fact that there was no "arginine x glutamine" interaction dembnstrated that a combination of the two amino acids has an effect corresponding to the sum of the two separate effects (additivity of effects) (Table 3). Under these conditions, the embryos accumulated 6.04 and 8.00 mg of soluble proteind50 embryos for clones 121 and 221, respectively (Table 1). Arginine was the only amino acid to affect the GLOENR component, resulting in specific 7s globulin enrichment of somatic embryos (Table 3). As a result, 7s globulin content on the dry weight basis increased by 26%.

4 87 1 Table 3 Effects of clone, arginine (5 mm) andor glutamine (20 mm) on the two major components, PRODE and GLOENR F test: tested effects - df(1.35) Clone Component 1 MS effect PRODE MS error P-level. P<O.OOO Component 2 MS effect GLOENR MS error P-level P=0.057 Arginine (Arg) Glutamine (Glu) ClonexArg P <o. ou0 P<U.OOU P= P<O.OOO P=0.51 P=0.26 ClonexGlu ArgxGln ClonexArgxGlu i P<O.OOO P=0.59 P= * P=0.07 P=0.7 P=0.7 [ The specific cases of the proportion of 7s globulin to total soluble proteins (GLO/SP) The PRODE and GLOENR components accounted for 32% and 50% of the differences in 7s globulin content in relation to total soluble proteins (GLO/SP), respectively (Table 2). However, their effects were antagonistic. Any compound that simultaneously increases protein synthesis and globulin enrichment will have the opposite effect on the GLO/SP ratio, in which case the effects can cancel each other out, as with arginine. However, since glutamine affected only PRODE (positive effect), it resulted in areduction in the GLO/SP ratio for clone 121 = 8.236, P = In this case, and in the absence of an arginine xglutamine interaction, the relative 7s globulin amounts on a soluble protein basis was mg/g without glutamine addition and mg/g with glutamine. The specific cases of the number of embryos (NE) Of the variability in the number of embryos 35% was statistically explained by GLOENR and a negligible proportion by PRODE (Table 2). In fact, 65% of the variation was of an unknown origin: This may explain both the differences seen between the clones (10%) for the number of embryos produced after 3 weeks of treatment (152 embryos per dish on average with clone 121 compared to 193 for clone 221) and the negative effect of glutamine (-10%) on both clones. On average, 116 and 152 embryos were produced per dish supplemented with glutamine by clones 121 and 221, respectively, compared to 129 and 170 without glutamine. c Discussion Two independent principal components are used to describe the effects of arginine and/or glutamine on protein accumulation in somatic embryos. These two independent components were mainly described by two traits: the amount of soluble proteins per embryo and the ratio of 7s globulins to dry weight. These two traits were not statistically correlated with each other. This observation leads to two conclusions: (1) these two traits are sufficient to describe the effects of arginine and/or glutamine on somatic embryos, (2) their impact on embryo maturation has to be studied separately. In oil palm somatic embryos, arginine and glutamine supplied in the medium enhanced the levels of accumulated proteins. However, these two amino acids did not show the same effect on storage protein accumulation, and arginine seemed to be more propitious to 7s globulin enrichment. Glutamine, on the other hand, led to a reduction in 7s globulin content over total soluble proteins for one of the two clones studied. This decrease could be due to a preferential synthesis of proteins other than 7s globulins, such as albumins, which have also been identified in oil palm zygotic embryos (Morcillo et al a). Using glutamine at higher concentrations should favour protein synthesis. However, this amino acid is known to be toxic (Oztur and Palsson 1990), and a substantial reduction in the number of regenerated embryos has been seen in oil palm at concentrations of over 20 mm (our unpublished data). Our results on oil palm and those of Khlifi and Tremblay on black pine (1995) revealed differences in performance between clones. In the case of oil palm, the clone which accumulates the least protein in the presence of glutamine is that which contained the most protein initially. Arginine and glutamine play a different role in protein enrichment, but both enhanced total soluble protein synthesis. In fact, a combination of the two amino acids in the medium had an additive effect on the accumulation of both soluble proteins and 7s globulins, which were found to be 2.5 and 1.3 times more abundant, respectively, than in standard cultures. Further work will be carried out to investigate whether glutamine and arginine exert their effect by providing reduced amino groups for the synthesis of the other amino acids which limit the rate or extent of storage protein synthesis. Alternatively, they might play a regulatory role in the transcriptional or translational regulation of storage proteins gene expression, as has been suggested for other species (Lai et al. 1992). The addition of a nitrogen source has a positive effect on somatic embryogenesis of oil palm and other species (Stuart et al. 1985). In most cases, the nitrogen source is added in an organic nitrogen form such as glutamine and arginine, which are the major amino acids of storage pro-..

5 872 teins. In alfalfa, for instance, amino acid enrichment of the medium increases the number of embryos regenerated (Skokut et al. 1985), protein reserve accumulation in the embryos (Lai et al. 1992; Lai and McKersie 1994) or embryo conversion into in vitro plantlets (Stuart and Strickland 1984). In gymnosperms, glutamine is used to increase the number of embryos regenerated (Khlifi and Tremblay 1995) and encourage their development (Finer et al. 1989). Likewise, in carrot somatic embryos, total protein contents are three times higher when glutamine i$ added to the culture medium (Dodeman 1995). In oil palm, adding organic nitrogen favours storage protein accumulation, hence improving somatic embryo maturation. Moreover, our work revealed that 7s globulin enrichment depends on the genotype and the type of amino acid used. Further work will now have to be carried out to study the effect of total soluble protein amount and 7s globulin enrichment in the somatic embryo on the vigour of regenerated plantlets. Acknowledgements The authors are grateful to Dr. Rival and S. Dussert for their comments on the manuscript and Dr. Tregear for English corrections. This work was conducted under a joint research programme between ORSTOM (Institut Français de Recherche Scientifique pour le Développement en Coopération) and CIRAD (Centre de Coopération Internationale efl Recherche Agronomique pour le Développement). References Aberlenc-Bertossi F, Duval Y (1993) Effet des cytokinines sur la maturation des embryons de palmier?i huile. In: CIRAD- ORSTON University of Montpellier II editors. XIIème Colloq Sec Fr IAPTC. Montpellier, p 27 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem 72: Dodeman VL (1995) Comparaison des embryogenbses zygotique et somatique chez la carotte (Daucus carota L.). Identification et induction de protéines de maturation. PhD thesis, université Paris XI, Orsay Duval Y, Engelmann F, Durand-Gasselin T (1995) Somatic emdryogenesis in oil palm (Elaeis guineensis Jacq.) In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 30: somatic embryogenesis and synthetic Seed I. Springer, Berlin Heidelberg New York, pp Finer JJ, Kriebel HW, Becwar MR (2989) Initiation of embryogenic callus and suspension cultures of eastem white pine (Pinus strobus L.). Plant Cell Rep 8: Hotelling H (1933) Analysis of a complex of statistical variables into principal components. J Educ Psycho1 24: & Huet S, Laporte J, Vautherot JF (1988) Statistical methods for the comparison of antibody levels in serums assayed by enzymelinked immuno sorbent assay. Biom Praxim 28:61-80 Keuls M (1952) The use of a studentized range in conn$ction with analysis of variance. Euphytica 1: Khlifi S, Tremblay FM (1995) Maturation of black spruce somatic embryos. Part I. Effect of L-glutamine on the number and germinability of somatic embryos. Plant Cell Tissue Organ Cult 41:23-32 Lai FM, McKersie BD (199;) Regulation of storage protein synthesis by nitrogen and sulfur nutrients in alfalfa (Medicago sativa L.) somatic embryos. Plant Sci 103: Lai FM, Senaratna T, Mc Kersie BD (1992) Glutamine enhances storage protein synthesis in Medicago sativa L. somatic embryos. Plant Sci 87: Merkle SA, Parrott WA, Flinn BS (1995) Morphogenic aspects of somatic embryogenesis. In: Thorpe TA (ed) In vitro embryogenesis in plants. Kluwer Academic Publishers Dordrecht, pp MorcilIo F, Bertossi-Aberlenc F, Trouslot P, Duval Y (1997a) Characterization of 2s and 7s storage proteins in embryos of oil palm. Plant Sci 122: Morcillo F, Bertossi-Aberlenc F, Hamon S, Duval Y (1997 b) Accumulation of storage protein and 7s globulins during zygotic and somatic embryo development in Elaeis guineensis. Plant Physio1 Biochem, 1998,36 (7): Newman D (1939) The distribution of range in samples from a normal population expressed in terms of an independant estimate of standard deviation. Biometrika 31:20-30 Ozturk SS, Palsson BO (1990) Chemical decomposition of glutamine in cell culture media: effect of media type, ph, and serum concentration. Biotechnol Prog 6: Roberts DR, Flinn BS, Webb DT, Webster FB, Sutton BCS (1990) Abscisic acid and indole-3-butyric acid regulation of maturation and accumulation of storage proteins in somatic embryos of interior spruce. Physiol Plant 78: Skokut TA, Manchester J, Schaefer J (1985) Regeneration in alfalfa tissue culture: stimulation of somatic embryo production by amino acids and N-15 NMR determination of nitrogen utilization. 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