P l a n t a (1988) 1 7 3 : 3 6 7 - 3 7 2
9 Springer-Verlag 1988
Lectin-gene expression in pea (Pisum sativum L.) roots Dominique Buffard, Pierre-Alexandre Kaminski and A. Donny Strosberg* D ~ p a r t e m e n t des Biotechnologies, Institut Pasteur, 28, rue d u D o c t e u r R o u x , F-75724 Paris C e d e x 15, F r a n c e
Abstract. The expression of a lectin gene in pea (Pisum sativum L.) roots has been investigated us-
ing the copy D N A of a pea seed lectin as a probe. An m R N A which has the same size as the seed m R N A but which is about 4000 times less abundant has been detected in 21-d-old roots. The probe detected lectin expression as early as 4 d after sowing, with the highest level being reached at 10 d, i.e. just before nodulation. In later stages (16-d- and 21-d-old roots), expression was substantially decreased. The correlation between infection by Rhizobium leguminosarum and lectin expression in pea roots has been investigated by comparing root lectin m R N A levels in inoculated plants and in plants grown under conditions preventing nodulation. Neither growth in a nitrate concentration which inhibited nodulation nor growth in the absence of Rhizobium appreciably affected lectin expression in roots. Key words: Lectin (gene expression) - Nitrate (nodulation) Pisum (root lectin) - Rhizobium R o o t (lectin).
Lectins are proteins or glycoproteins of non-immune origin which bind sugars and agglutinate cells (Goldstein et al. 1980). Legume lectins, particularly abundant in seeds, constitute a large family of homologous proteins (Strosberg et al. 1986). They have been suggested to determine the specificity of the Rhizobium-legume symbiosis through interaction with a particular carbohydrate se* To w h o m c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d
Abbreviation: c D N A = c o p y D N A ; poly(A) + R N A = p o l y a d e n ylated R N A
quence on the bacterial surface (Hamblin and Kent 1973; Bohlool and Schmidt 1974; Dazzo and Hubbell 1975). To verify this theory, extensive studies have been carried out, mostly with lectins extracted from seeds (for reviews, see Dazzo and Truchet 1983; Etzler 1986). Detection of lectins on the root surface, where infection by Rhizobium occurs, is a prerequisite for the validity of this theory. The presence of lectin on the surface of pea root hairs has been shown by indirect immunofluorescent techniques using antiserum to the seed lectin (Kato et al. 1981). Gatehouse and Boulter (1980) and Hosselet et al. (1983) have isolated lectin from young (3- to 4-dold) pea roots by affinity chromatography. This root lectin has been reported to be antigenically identical to the seed protein and to have the same electrophoretic mobility in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Its specificity has been found to be either similar to (Hosselet et al. 1983) or different from (Gatehouse and Boulter 1980) pea seed lectin. The low level of root lectin hampers the study of its synthesis and distribution during root development, but recently, sensitive enzyme-linked immunoassays (ELISA) have allowed quantification of pea root lectin in 4- and 7-d-old seedlings (Diaz et al. 1984). However, in all these studies, contamination of root tissue with lectins originating from seeds constitutes a major risk. Another approach, based on molecular hybridization with c o p y - D N A (cDNA) probes, should give more information about the regulation of synthesis of legume lectins during root development and should contribute to a better understanding of the role of lectins in plant metabolism and Rhizobium recognition. In this report, using a pea seed lectin c D N A (Higgins et al. t983), we demonstrate that a lectin
D. Buffard et al. : Lectin-mRNA levels in roots
g e n e is e x p r e s s e d in p e a r o o t s . I n a n a t t e m p t t o c o r r e l a t e t h i s e x p r e s s i o n w i t h i n f e c t i o n b y Rhizobium leguminosarum, w e a l s o c o m p a r e r o o t l e c t i n mRNA levels in i n o c u l a t e d p l a n t s a n d p l a n t s g r o w n u n d e r c o n d i t i o n s p r e v e n t i n g n o d u l a t i o n , i.e. g r o w n u n d e r a x e n i c c o n d i t i o n s o r in n i t r a t e - r i c h m e d i u m ( D a z z o a n d Brill 1978; D i a z et al. 1984).
Material and methods Plant material. Immature cotyledons were collected from Pisum sativum cv. Frisson and were quickly frozen in liquid nitrogen. Pea seedlings of the same cultivar were grown in gravel, wetted with a mineral nutrient solution devoid of NO) (or supplied with 20 mM NO; when required) and inoculated with Rhizobium leguminosarum (except where otherwise stated). Roots were carefully washed in water before excision and immediately frozen in liquid nitrogen. Isolation of RNA. The RNA fi-om pea cotyledons was isolated as described by Hall et al. (1978), and that from roots was prepared by the guanidium/hot phenol method as described in Maniatis et al. (1982). Polyadenylated (poly(A) +) RNA was selected by oligo (dT)-cellulose chromatography and titrated by [3H]polyuridylic acid hybridization as previously described (Buffard et al. 1982). Analysis by RNA blots. For Northern blots, poly(A)+RNA was electrophoresed in a 1.5% agarose/formaldehyde gel (Rave et al. 1979) and blotted onto nitrocellulose without prewashing the gel (Thomas 1980). For dot-blots, poly(A)+RNA was dissolved in 6% formaldehyde and 10 x SSC (standard saline citrate buffer); 1 x SSC = 0.15 M NaC1 and 0.015 M sodium citrate, pH 7), heated for 15 rain at 50~ C and immediately chilled on ice. Appropriate dilutions were made in 10 x SSC and 100 lal of each sample were applied to a nitrocellulose sheet (presoaked in 10 x SSC) cushioned on a layer of Whatman (Springfield Hill, UK) No. 1 paper, mounted inside a Schleicher and Schiill (Dassel, FRG) minifold filtration apparatus. Hydridization procedures. The nitrocellulose sheets were heated in a vacuum oven for 2 h at 80 ~ C and prehybridized for 2 h as described by Thomas (1980). Probes were oligolabelled (Feinberg and Vogelstein 1983) with deoxy[32p]ATP and deoxy[3aP]cytidine 5'-triphosphate, each at a specific activity of 111TBq. mmol - 1) and hybridization carried out at 40 ~ C for 16 h according to Thomas (1980) except that the final washing was made at 58~ C. Filters were dried and autoradiographed at - 80~ C with Fuji (Tokyo, Japan) RX film and intensifying screens. Autoradiographs of the dot blots were scanned using a Bio-Rad (Paris, France) densitometer coupled with a Shimadzu chromatopac C-R3A integrator/plotter (Shimadzu, Kyoto, Japan) and the relative lectinmRNA levels deduced from the peak areas.
Results A lectin gene is expressed in p e a roots. A p e a s e e d l e c t i n c D N A , p P S 15-50 ( H i g g i n s et al. 1983) w a s hybridized with blots containing mRNAs prepared from 21-d-old roots and developing seeds to determine whether lectin-gene expression occurred in
Fig. 1. Northern blot hybridization of pea seed and root mRNA with a lectin-cDNA clone, pPS 15-50. Seed and root poly(A) § RNA (23 ng and 1.1 lag, respectively) were fractionated on a 1.5% agarose/formaldehyde gel, transferred to nitrocellulose and hybridized with labeled pPS 15-50. Blots were autoradiographed for 15 h (seed mRNA) and 4 d (root mRNA) with intensifying screens at - 8 0 ~ C
p e a r o o t cells. A s s h o w n i n F i g . 1, t h e p r o b e d e t e c t e d a l e c t i n - s p e c i f i c m R N A in b o t h s e e d a n d root poly(A)+RNAs. The root lectin mRNA had t h e s a m e size a s t h e s e e d l e c t i n s e q u e n c e i.e. a b o u t 840 n u c l e o t i d e s . N o d e t e c t a b l e s i g n a l w a s o b t a i n e d with leaf mRNA, in a similar experiment, even w i t h a l a r g e a m o u n t o f m R N A (2 gg). T h e r e f o r e , the expression of the lectin gene appeared to be restricted to particular organs of this plant. Although we applied considerably more m R N A f r o m r o o t t h a n f r o m s e e d (1.1 g g v e r s u s 23 ng), t h e b a n d s e e n f o r s e e d m R N A is still m o r e intense than that seen for root mRNA indicating t h a t t h e a m o u n t o f l e c t i n m R N A e x p r e s s e d in r o o t s is c o n s i d e r a b l y l o w e r t h a n t h a t f o u n d in seeds. Different amounts of seed and root poly(A) § RNA were hybridized with the lectin-cDNA probe t o e s t i m a t e t h e level o f l e c t i n - g e n e e x p r e s s i o n in r o o t s . A s s h o w n in F i g . 2, l e c t i n m R N A i s o l a t e d f r o m 2 1 - d - o l d r o o t s w a s a b o u t 4 . 1 0 3 t i m e s less abundant than the immature-seed lectin mRNA,
D. Buffard et al. : Lectin-mRNA levels in roots
Fig. 2. Comparison of lectin-transcript levels in pea seeds and roots. Serial dilutions of root poly(A)+ RNA (from 4 gg), seed poly(A)+RNA (from 16 ng) and root poly(A) RNA (rRNA; from 4 gg) were spotted onto nitrocellulose and hybridized with labeled pPS 15-50
16d /3 [
Fig. 3A, B. Expression of the pea lectin gene in developing roots. A Decreasing quantities of root poly(A)+RNA (from 5 gg to 0.13 gg) from different developmental stages (days after sowing) were spotted onto nitrocellulose and hybridized with the labeled lectin-cDNA probe. B Densitometer scan of the autoradiograph
assuming an extensive sequence homology between the two mRNAs. No detectable signals were observed with root poly(A)- RNA, indicating that the low abundance of lectin-specific messenger in root poly(A) + R N A populations could not be accounted for by a lack of m R N A polyadenylation.
Expression of pea root lectin and infection by Rhizobium leguminosarum In the experiments above, roots were collected from 21-d old plants. At this stage, primary roots were extensively nodulated and some young nodules were developing on secondary roots. To determine whether pea lectin expression in roots is related to Rhizobium infection, we investi-
gated lectin-gene expression at earlier stages of development preceding nodulation (4- and 10-d-old roots) when pea-Rhizobium leguminosarurn recognition may occur, or at stages following nodulation (16- and 21-d-old roots). The results (Fig. 3) indicated that the lectin gene was exwessed as early as 4 d after sowing and that the highest expres'sion level was reached at 10 d. If the value for 10-d-old roots is taken as 100%, the 4-d value would be 65%, the 16-d value 40% and the 21-d value 21%. The lectinm R N A level, as determined in Fig. 3, represents the amount of lectin-specific m R N A among the total m R N A population. During the first three weeks of development, as the roots grew, the content of extractable m R N A varied (Table 1). The amount of total m R N A yielded by 100 roots in-
D. Buffard et al. : Lectin-mRNA levels in roots
Table 1. Variation of m R N A content during root development. The amount of total m R N A was determined by titration with [H3]polyuridylic acid Age of roots FW of 100 roots A m o u n t of m R N A (d) (g) gg.(g F W ) - 1 gg.(lO0 roots)- 1 4 10 16 21
5.1 19.8 37.6 42.4
5.3 5.1 3.4 2.5
26 99.5 127 108
creased between the 4-d and the 10-d-stages and then remained approximately constant. Expressed per gram fresh weight, this m R N A content decreased from the 4-d stage. Thus, the decrease in lectin-mRNA level observed after 10 d is likely to correspond to a reduced number of lectin transcripts in the 16- and 21-d-old roots. To investigate these results further, we studied the level of root lectin m R N A under culture conditions in which nodulation would not occur. Two batches of peas were grown, one with a nutrient solution devoid of NO;, the other one with 20 m M NO~. Roots were harvested after 10 d while five seedlings were grown for 21 d to verify that nodulation was indeed totally inhibited by 20 m M NO). Different amounts of poly(A) + R N A extracted from each batch were blotted onto nitrocellulose and hybridized with the labeled lectin probe. Lectin-mRNA levels were evaluated by densitometer scanning of the autoradiograph of the blot. The lectin-mRNA level in the presence of 20 m M N O ; (93%) was found to be very similar to that observed in the absence of nitrate (105%). A similar experiment was performed to study the influence of Rhizobium leguminosarum itself.' Two batches of pea seedlings were grown for 10 d in the presence or absence of Rhizobium leguminosarum. Samples of five plants were grown for 21 d to analyze nodulation or axenic conditions. A comparison of lectin-mRNA levels in inoculated and
non-nodulated roots is shown on Fig. 4. Again no appreciable difference was observed between the two batches. Discussion
Several reports have now identified lectins or related proteins at the surface or in exudates of roots of legumes (Etzler 1986). Thus, low amounts (nanogram per plant quantities, compared to milligram levels of seed lectin) of lectin-like protein have been found in young (4- and 7-d-old) roots of peas (Diaz et al. 1984), in three- to four-weekold roots of Phaseolus vulgaris (Borrebaeck 1984) or in 4-d-old roots of Le + (seed-lectin positive) or L e - (seed-lectin negative) soybean cultivars (Vodkin and Raikhel 1986). However, in these studies, the possibility of adsorption to young seedling tissues of lectin released from the imbibing seed could not be ruled out. In the present report, using a pea seed lectin c D N A as a probe, we have shown the presence of lectin p o l y ( A ) + m R N A in young pea roots, indicating that lectin biosynthesis occurs in roots. Previously, lectin-gene expression in roots has only been reported for mature soybean plants (Okamuro et al. 1986). Furthermore, Hoffman et al. (1982) and Hoffman and Donaldson (1985), using respectively a PHA-like c D N A (pPVL134) and two lectin genes (dlecl and dlec2 coding for P H A - E and PHA-L, respectively) failed to detect any lectin m R N A in roots of Phaseolus vulgaris. As in soybean (Okamuro et al. 1986), the lectin-mRNA level in pea roots is very low, being 4000 times less abundant in 21-d-old roots than in developing seeds. This difference seems even higher in soybean where a ratio of 20000 has been reported between root lectin-mRNA level and the highest level of seed lectin expression detected at mid-embryogenesis (Okamuro et al. 1986). No lectin m R N A could be detected in leaves of 10-d-old pea plants. Likewise, using a lectin-like
Fig. 4. Comparison of lectin-gene expression in pea roots grown in presence ( + Rh) or in absence ( - Rh) of Rhizobium leguminosarum. The dot-blot of poly(A)--RNA for the two batches of pea roots and root poly(A) R N A (as hybridization control) was hybridized with the labeled lectin-cDNA probe pPS 15-50
D. Buffard et al. : Lectin-mRNA levels in roots
c D N A probe, Hoffman et al. (1982) failed to observe hybridizing transcripts in primary leaves of Phaseolus vulgaris (Hoffman et al. 1982). Yet, evidence of lectin-like R N A sequences in leaves of mature pod-bearing plants of P. vulgaris has been presented (Hoffman et al. 1982), indicating that the level oflectin-gene expression in this organ may vary extensively during the life cycle of the plant. Of the four distinct D N A fragments detected in the pea genome by the lectin-cDNA probe, only one appears to correspond to a functional lectin gene (Kaminski et al. 1987). Our results confirm that expression of the gene is highly regulated in the plant and under precise developmental controls. We examined whether or not the lectin-mRNA level in pea roots, as in P. vulgaris leaves, undergoes variation with respect to the age of the plant. During the first three weeks of development, substantial changes were observed. A maximum expression level was reached 10 d after sowing but then the lectin-transcript level decreased. The change in the level of lectin transcription between 4- and 10-d-old plants correlated with the increment of the quantity of cell-wall-associated lectin per root between the 4- and 7-d stages (Diaz et al. 1984). The time-course of lectin expression, with a maximum just before Rhizobium infection, may indicate a role for lectin in the first steps of nodulation. Rhizobium binding occurs only on root hairs and on epidermal cells located just below the young hairs. Therefore, further analysis of the site of lectin expression in the different parts of the roots (root tip, root hair region...) during the first weeks of development would be required to correlate lectin localization with Rhizobium recognition. Distribution of lectin was recently studied on 5-dold pea roots by indirect immunofluorescence (Diaz et al. 1986) and indicated that lectin presence was restricted to some epidermal cells below emerging root hairs and on the tips of newly formed hairs, grouped opposite the protoxylematic poles. Moreover, a strong correlation was observed between lectin localization and susceptibility to infection, as demonstrated by spot-inoculation tests (Diaz et al. 1986). In an attempt to correlate further lectin-gene expression and Rhizobium recognition, expression of root lectin-mRNA level under growth conditions preventing nodulation was investigated. Concentrations of 16 20 m M NOa in the nutrient medium have been shown to inhibit nodulation of pea (Diaz et al. 1984) or clover roots (Dazzo and Brill 1978). These growth conditions
also decrease the amount of lectin present at the root-hair surface in 2-d-old clover roots (Dazzo and Brill 1978; Sherwood et al. 1984) as well as lectin associated with root cell walls of 4-d-old pea roots (Diaz et al. 1984). In the present study the level of lectin expression was not affected by high nitrate concentrations (20 raM), indicating that post-transcriptional events might account for the reduced level of lectin associated with the cell wall under these conditions. As NO~ seems to increase lectin in pea root slime, Diaz et al. (1984) have suggested that the decrease of detectable root lectin may be caused by changes in its secretion rather than to actual inhibition of synthesis. In an attempt to correlate lectin-gene expression with the presence of Rhizobium in the nutrient medium, we compared the lectin-mRNA level in pea seedlings grown in the presence or absence of Rhizobium. No appreciable differences were observed, indicating that no regulation, at least at the transcriptional level, was exerted by Rhizobium on lectin expression. We are grateful to Dr. T.J.V. Higgins (CSIRO, Canberra, Australia) for the generous gift of the c D N A used as a probe in the present study. We thank Dr. R. Cousin (Laboratoire du Pois, INRA, Versailles, France) and Dr. G. Duc (Station d'Am61ioration des Plantes, INRA, Dijon, France) for kindly providing pea seedlings. This work was supported by a grant ~ Fixation de l'Azote" from Elf Aquitaine, EMC, CDF-Chimie, Rh6ne-Poulenc, France. Drs. Buffard et Kaminski were supported by grants from the Minist~re de la Recherche et de la Technologie.
References Bohlool, B.B., Schmidt, E.L. (1974) Lectins: a possible basis for specificity in the Rhizobium-legume root nodule symbiosis. Science 185, 269 271 Borrebaeck, C.A.K. (1984) Detection and characterization of a leetin from non-seed tissue of Phaseotus vutgaris. Planta 161,223 228 Buffard, D., Vaillant, V., Esnault, R. (1982) Complexity of polysomal polyadenylated RNAs in Viciafaba meristematic root cells. Eur. J. Biochem. 126, 129 134 Dazzo, F.B., Brill, W.J. (1978) Regulation by fixed nitrogen of host-symbiont recognition in the Rhizobium-clover symbiosis. Plant Physiol. 62, 18-21 Dazzo, F.B., Hubbell, D.H. (1975) Cross reactive antigens and lectins as determinants of symbiotic specificity in the Rhizobium-clover association. Appl. Microbiol. 30, 1018 1033 Dazzo, F.B., Truchet, G.L. (1983) Interaction of lectin and their saecharide receptors in the Rhizobium-legume symbiosis. J. Membr. Biol. 73, 1-16 Diaz, C.L., Lems-Van Kan, P., Van Der Schaal, I.A.M., Kijne, J.W. (1984) Determination of pea (Pisum sativum L.) root lectin using an enzyme-linked immunoassay. Planta 161, 30~307 Diaz, C.L., Van Spronsen, P.C., Bakhuizen, R., Logman, G.J.J., Lugtenberg, E.J.J., Kijne, J.W. (/986) Correlation
372 between infection by Rhizobiurn leguminosarum and lectin on the surface of Pisum sativum L. roots. Planta 168, 350-359 Etzler, M.E. (1986) Distribution and function of plant lectins. In: The lectins: properties, functions and applications in biology and medicine, pp. 371-435, Liener, I.E., Sharon, N., Goldstein, J.J., eds. Academic Press, Orlando New York Feinberg, A.P., Vogelstein, B. (1983) A technique of radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6 13 Gatehouse, J.A., Boulter, D. (1980) Isolation and properties of a lectin from the roots of Pisum sativum (garden pea). Physiol. Plant. 49, 437-442 Goldstein, J.A., Hughes, R.C., Monsigny, M., Osawa, T., Sharon, N. (1980) What should be called a lectin? Nature 285, 66 Hall, T.C., Ma, Y., Buchbinder, B.U., Pyne, J.W., Sun, S.M., Bliss, F.A. (1978) Messenger RNA for G1 protein of French bean seeds: cell-free translation and product characterization. Proc. Natl. Acad. Sci. USA 75, 3196-3200 Hamblin, J., Kent, S.P. (1973) Possible role of phytohemagglutinin in Phaseolus vulgaris L. Nature (London), New Biol. 245, 28-30 Higgins, T.J.V., Chandler, P.M., Zurawski, G., Button, S.C., Spencer, D. (1983) The biosynthesis and primary structure of pea seed lectin. J. Biol. Chem. 258, 9544-9549 Hoffman, L.M., Donaldson, D.D. (1985) Characterization of two Phaseolus vulgaris phytohemagglutinin genes closely linked on the chromosome. EMBO J. 4, 883-889 Hoffman, L.M., Ma, Y., Barker, R.F. (1982) Molecular cloning of Phaseolus vulgaris Iectin mRNA and use of cDNA as a probe to estimate lectin transcript levels in various tissues. Nucleic Acids Res. 10, 7819-7828 Hosselet, M., Van Driessche, E., Van Poucke, M., Kanarek, L. (1983) Purification and characterization of an endogenous root lectin from Pisum sativum L. In: Lectins, biology, biochemistry, clinical biochemistry, vol. 3, pp. 549-558,
D. Buffard et al. : Lectin-mRNA levels in roots Bog-Hansen, T.C., Spengler, G.A., eds. de Gruyter, Berlin New York Kaminski, P.A., Buffard, D., Strosberg, A.D. (1987) The pea lectin gene family contains only one functional gene. Plant Mol. Biol. 9, 497-507 Kato, G., Maruyama, Y., Nakamura, M. (1981) Involvement of lectin in Rhizobium-pea recognition. Plant Cell Physiol. 22, 759-772 Maniatis, T., Fritsch, E., Sambrook, J. (1982) Molecular cloning. Cold Spring Harbor Laboratory, New York Okamuro, J.K., Jofuku, K.D., Goldberg, R.B. (1986) Soybean seed lectin gene and flanking nonseed protein genes are developmentally regulated in transformed tobacco plants. Proc. Natl. Acad. Sci. USA 83, 8240-8244 Rave, N., Crkvenjakov, R., Boedtker, H. (1979) Identification of pro-collagen mRNAs transferred to diazobenzyloxymethyl paper from formaldehyde agarose gels. Nucleic Acids Res. 6, 3559 3567 Sherwood, J.E., Truchet, G.L., Dazzo, F.B. (1984) Effect of nitrate supply on the in-vivo synthesis and distribution of trifoliin A, a Rhizobium trifolii-binding lectin, in Trifolium repens seedlings. Planta 162, 54C~547 Strosberg, A.D., Buffard, D., Lauwereys, M., Foriers, A. (1986) Legume lectins: a large family of homologous proteins. In: The lectins: properties, functions and applications in biology and medicine, pp. 249-264, Liener, I.E., Sharon, N., Goldstein, I.J., eds. Academic Press, Orlando New York Thomas, P. (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77, 5201-5205 Vodkin, L.O., Raikhel, N.V. (1986) Soybean lectin and related proteins in seeds and roots of Le + and Le soybean varieties. Plant Physiol. 81, 558 565
Received 20 May; accepted 21 August 1987