Directory UMM :Data Elmu:jurnal:S:Soil Biology And Chemistry:Vol32.Issue3.2000:

(1)

Short communication

A low molecular weight humic fraction on nitrate uptake and

protein synthesis in maize seedlings

S. Nardi

a,

*, D. Pizzeghello

a

, C. Gessa

b

, L. Ferrarese

c

, L. Trainotti

c

, G. Casadoro

c

a

Dipartimento di Biotecnologie Agrarie, UniversitaÁ di Padova, Strada Romea 16, 35020 Legnaro (Pd), Italy

b

Istituto di Chimica Agraria, UniversitaÁ di Bologna, Via B. Pichat 10, 40127 Bologna, Italy

c

Dipartimento di Biologia, UniversitaÁ di Padova, Via Ugo Bassi 58/b, 35121 Padova, Italy

Received 25 March 1999; received in revised form 26 July 1999; accepted 16 August 1999

Humic acids (HA) were found to be able to induce plant growth and dierentiation, similar to those induced by hormonal substances (Muscolo et al., 1993). The eects on ion uptake in barley seedlings appear to be selective and their magnitude is related to the concentration of humic substances and to the pH of the medium (Nardi et al., 1996). HA stimulate uptake of sodium, barium and phosphate, inhibit chloride uptake and have no eect on calcium uptake, when compared to water-aged controls (Vaughan and Malcolm, 1979). However, HA are not able to over-come the inhibitory eects on protein synthesis and sodium uptake caused by cycloheximide and D -threo-chloramphenicol (Vaughan and Malcom, 1979). HA enhanced the synthesis of invertase, but had no eect on the uptake of phosphate or on the incorporation or distribution of 14C-labelled amino acids into proteins (Vaughan and MacDonald, 1971, 1976). By utilising both transcription (6-methylpurine) and translation (cycloheximide) inhibitors, it was shown that humic matter stimulates carrier-protein synthesis at a post-transcriptional level (Nardi et al., 1996). The eects of HAs, endowed with either auxin and gibberellin-like activity or with auxin-like activity alone, on plant metabolism have been considered in previous studies (Nardi et al., 1991; Muscolo et al., 1999).

In this work low molecular weight humic fraction (LMW-HA), only characterised by gibberellin-like ac-tivity, was evaluated for its eects on growth, nitrate uptake, invertase activity and mRNA level of maize

roots. The LMW-HA was extracted from an Ah hor-izon of an Eutric Cambisol (FAO, 1990) (Treviso, Italy). The extraction, puri®cation and characterisation (elemental analysis and quantitative 13C NMR) were performed as described in Nardi et al. (1994). Auxin-and gibberellin-like activities were determined as described in Nardi et al. (1996). Three-day-old maize (Zea mays L. var. Polaris) seedlings were kept for 48 h in sterilised Hoagland's solution with either LMW-HA (0.1 mg C lÿ1 incubation period, maize roots were utilised for the de-termination of invertase activity (Arnold, 1965). For nitrate uptake, ATPase assay and measurement of pro-ton eux three-day-old seedlings were treated as described in Nardi et al. (1991).

RNA was extracted from root tissue of maize trea-ted with LMW-HA or with Hogland's solution (con-trol), as described in Logemann et al. (1987). Quanti®cation was determined spectrophotometrically and checked by ethydium bromide staining of an agar-ose gel. For each sample (treated and control) ®ve mg of total RNA were used to synthesise L-[35 S]methion-ine-labelled polypeptides using a rabbit reticulocyte lysate following the manufactures instructions (Ambion Inc., Austin, TX, USA). The trichloroacetic acid-precipitable counts were determined in 2.5 ml ali-quots of each reaction mixture. Typically, 20,000 counts per minute (cpm) per ml were obtained for each labelling reaction. Separation of 35S-labelled polypep-tides was performed as described by O'Farrell (1975). For the ®rst dimension, the isoelectric-focusing was carried out in a tube electrophoresis apparatus

www.elsevier.com/locate/soilbio

* Corresponding author. Fax: +39-49-827-2929.

(2)

fer-Pharmacia, Uppsala, Sweden) using 2 mm diameter gels composed of 4.1% (w/v) acrylamide (from a 29T-2C stock solution), 9.5 M urea, 2% (w/v) carrier ampholytes 3±10 (Pharmacia, Uppsala, Sweden), 2% (v/v) Triton X-100, 5 mM CHAPS (3-[(3-Cholamido-propyl)dimethylammonio]-1-propane-sulfonate). After electrofocusing, the gels were equilibrated for 20 min in 0.375 M Tris±HCl (pH 6.8), 3% (w/v) SDS, 0.05 M DTT, 0.01% (w/v) bromophenol-blue and loaded onto a 1520 cm slab gel apparatus. The second dimension

was performed by SDS-PAGE with the buer system of Laemmli (1970). After electrophoresis, gels were stained with Coomassie R-250 (Serva, Heidelberg, Germany), impregnated with Amplify (Amersham, Bucks, UK) according to the manufacturer's instruc-tions and dried at 808C under vacuum. The dried gels were exposed to pre¯ashed Hyper®lm MP (Amersham, Bucks, UK) atÿ808C for equivalent times.

13

C NMR spectra of the LMW-HA (Table 1) revealed a high presence of peptidic and carbohydratic (36.3%), carboxyl (29.98%) and aliphatic C (20.45%). In comparison with HAs endowed only with IAA-like activity (Muscolo et al., 1999), the LMW-HA con-tained more phenolic C, (7.32% in respect to 4.4%) and more carboxyl C (29.98% in respect to 22.5%). As shown by Vaughan and Malcom (1985), the gibber-ellin-like activity is related to the presence of phenolic compounds.

Nitrate uptake is a well known substrate-inducible transport (Clarkson and Luttge, 1991). Besides a pas-sive nitrate movement across the plasmalemma,

prob-ably via ion channels (`constitutive' system), there is a secondary active transport, requiring a proton electro-chemical gradient (Miller and Smith, 1996) generated by the activity of an H+-ATPase (primary transport) (Sze, 1985). The transport of nitrate is thought to be a symport with protons (2:1 H+:NO3ÿ stoichiometry) (Miller and Smith, 1996) and demonstrated to occur in isolated membrane vesicles (Lu and Briskin, 1993). Our LMW-HA enhanced nitrate transport (+89%) and the magnitude of the increase was higher than that induced by GA (+73%) (Table 2).

An attempt was made to verify whether the stimu-latory eect of LMW-HA on nitrate uptake depended on plasmalemma H+-ATPase. The later activity can be indirectly evaluated by both measuring the activity of K+-stimulated ATPase of microsomes and the H+ extrusion by roots. K+-stimulated ATPase activity of maize microsomes was strongly inhibited by LMW-HA and GA (Table 2) and these eects correlate well with those on H+ extrusion, although GA had only a low eect (Table 3).

These results are similar to those obtained by Mag-gioni et al. (1987), but dier from our previous data (Nardi et al., 1991). The discrepancy may be explained by considering that LMW-HA used in the previous work exhibited an IAA-like activity that was not pre-sent in the LMW-HA from the Ah soil horizon. It is also clear that H+-ATPase and proton extrusion are the major mechanisms in¯uenced by IAA (Frankenber-ger and Muhammad Arshad, 1995). In the light of these results, the stimulation of nitrate uptake by LMW-HA can not be explained by an eect on the primary transport of solutes. It is known that an increase in pH at the surface of roots does not favour nitrate uptake, while ammonium uptake increases (Barber, 1984). It is possible that LMW-HA may act by decreasing the pH at the surface of roots, thus facil-itating the H+/NO3ÿ symport. LMW-HA stimulated growth (+158%) after 48 h treatment (Table 4) and this fraction gives results similar to those observed for gibberellic acid (+128%).

The gibberellin-like activity of LMW-HA was ana-lysed by studying the eect on the invertase activity, which is a GA-marker enzyme in plant metabolism (Vaughan and Malcom, 1979). Both LMW-HA and GA stimulate invertase activity, when compared to the control (Table 4). This GA-like action was con®rmed Table 2

NO3ÿuptake and ATPase activity (mmol Pi mgÿ1prot. hÿ1) in

micro-somal fractions of three-day-old maize root seedlings preincubated for 16 h with LMW-HA and GA. The results are the means of 15 roots from 15 random plants for each experiment, which was repeated ®ve times

Treatment Concentration nmol NO3ÿ

gÿ1_{fwt min}ÿ1 _% _D_K+ _%

Values within column followed by the same lower case are not statistically dierent at p0:05 by Student±Newman Keuls test

(Sokal and Rohlf, 1969). Table 1

Elemental characteristics and percent of13_{C distribution (ppm intervals) in the NMR spectra of LMW-HA}

Composition (%) Relative intensity (total area %)

C H N Ash 0±48 aliphatic C 48±105 peptidic and

carbohydratic C

105±145 aromatic C 145±165 phenolic C 165±190 carboxyl C

(3)

by thea-amylase activity assay carried out with barley seeds (unpublished data), in which, the amounts of reducing sugars, released following treatments with either LMW-HA or GA, was comparable in the pre-sence and abpre-sence of the embryo, respectively.

Plant hormones have been shown to cause changes in the pattern of gene expression (Taiz and Zeiger, 1991) and these changes can be either quantitative or qualitative (Abeles et al., 1992). To investigate the physiological changes associated with the LMW-HA treatment of maize roots at the molecular level, total RNA from these organs was translated in a reticulo-cyte lysate system. Analysis of the synthesized polypep-tides by two-dimensional gel electrophoresis demonstrate a post-transcriptional eect of LMW-HA on protein synthesis. In fact, apart from the disappear-ance of two spots, no new polypeptides are observed in the LMW-HA treated roots, which suggests that no transcription of new mRNA occurred (Fig. 1). The main polypeptide pattern observed in controls (Fig. 1, panel A) is also visible in LMW-HA treated roots. In the latter case, however, the intensity of most of the spots appears to be highly increased after 16 h of treat-ment (Fig. 1, panel B), and this is consistent with the already suggested increase in the number of carrier proteins induced by humic matter (Nardi et al., 1991). These generalized increase in amount of the newly syn-thesized polypeptides may re¯ect a bene®cial eect of LMW-HA on the stability of the root mRNAs, similar to that described for cytokinins on an mRNA

encod-ing a chlorophyll a/b bindencod-ing polypeptide in Lemna gibba(Flores and Tobin, 1988).

The regulatory properties of the LMW-HA appeared to depend on the combination of low mol-ecular size, gibberellic-like activity and to a relevant content of phenolic and carboxyl carbon. The LMW-HA in¯uencing the protein synthesis, induced an incre-ment on the nitrate transport and on the plant growth, which could re¯ect upon a cropping increment.

1. Summary

A low molecular weight humic fraction (LMW-HA) was tested for its biological activity in maize seedlings. Results showed that LMW-HA increased nitrate uptake and strongly inhibited K+ stimulated ATPase of maize microsomes and H+ extrusion in a manner similar to gibberellic acid (GA). The stimulation of nitrate uptake by LMW-HA can not be explained by an eect on the primary transport of solutes but it is possible to suggest that it may act by decreasing the pH at the surface of roots, this facilitating the H+/ NO3ÿsymport.

Changes in the messenger RNA after the humic treatment were performed and the analysis of the syn-thesised polypeptides demonstrated a post-transcrip-tional eect of humic matter on the protein synthesis and consequently on the overall plant nutrition.

The regulatory properties of the LMW-HA Table 3

Proton extrusion from three-day-old maize root seedlings previously treated with LMW-HA and GA for 16 h. The results are the means of 15 roots from 15 random plants for each experiment, which was repeated ®ve times

Treatment Concentration mmol H+gÿ1

fwt h-1

a_{Values within column followed by the same lower case are not statistically dierent at}_p

0:05 by Student±Newman Keuls test (Sokal and

Rohlf, 1969).

Table 4

Eect of LMW-HA and GA on fresh weight and on invertase activity in three day old maize seedlings roots after an incubation period of 48 h. The results are the means of 15 roots from 15 random plants for each experiment, which was repeated ®ve times

Treatment Concentration Fresh weight (g) Invertase activity

(t0 h) (t48 h) increase % mg sucrose gÿ1prot. hÿ1 %

Values within column followed by the same lower case are not statistically dierent atp0:05 by Student±Newman Keuls test (Sokal and

(4)

appeared to depend on the combination of low mol-ecular size, gibberellin-like activity and to a relevant content of phenolic and carboxyl carbon.

Acknowledgements

This work was supported by MURST ex-40% funds. Thanks are due to Professor A. Vianello for critical reading of this manuscript and to Ms. A. Tuz for help with the English.

References

Abeles, F.B., Morgan, P.W., Saltveit Jr, M.E., 1992. Ethylene in plant biology, 2nd ed. Academic Press Inc, San Diego.

Arnold, W.N., 1965. b-Fructofuranosidase from grape berries. Biochimica et Biophysica Acta 110, 134±147.

Barber, S.A., 1984. Soil Nutrient Bioavailability: A Mechanistic Approach. Wiley & Sons, New York, pp. 184±194.

Clarkson, D.T., Luttge, U., 1991. Mineral nutrition: inducible and repressible nutrient transport systems. In: Behnke, H.D., Esser, K., Kubitzki, K., Runge, M., Ziegler, H. (Eds.), Progress in Botany, 52. Springer Verlag, Berlin, pp. 61±83.

FAO, 1990. Soil map of the world. Rome.

Flores, S., Tobin, E.M., 1988. Cytokinin modulation of LHCP mRNA levels: the involvement of posttranscriptional regulation. Plant Molecular Biology 11, 409±415.

Frankenberger, W.T., Muhammad Arshad, J.R., 1995. Phytohormones in Soils. Marcel Dekker, Inc, New York. Laemmli, U.K., 1970. Cleavage of structural proteins during the

assembly of the head of the bacteriophage T4. Nature 227, 680± 685.

Logemann, J., Schell, J., Willmitzer, L., 1987. Improved method for the isolation of RNA from plant tissue. Analytical Biochemistry 163, 16±20.

Lu, Q., Briskin, D., 1993. Modulation of the maize plasma mem-brane carrier by NO3ÿ. Phytochemistry 33, 1±8.

Maggioni, A., Varanini, Z., Nardi, S., Pinton, R., 1987. Action of soil humic matter on plant roots: stimulation of ion uptake and eects on (Mg2+ _K+_{) ATPase activity. Science of the Total}

Environment 62, 355±363.

Miller, A.J., Smith, S.J., 1996. Nitrate transport and compartmenta-tion in cereal root cells. Journal of Experimental Botany 47, 843± 854.

Muscolo, A., Bovalo, F., Gionfriddo, F., Nardi, S., 1999. Earthworm humic matter produces auxin-like eects on Daucus carota cell growth and nitrate metabolism. Soil Biology & Biochemistry 31, 1303±1311.

Muscolo, A., Felici, M., Concheri, G., Nardi, S., 1993. Eect of earthworm humic substances on esterase and peroxidase activity during growth of leaf explants of Nicotiana plumbaginifolia. Biology and Fertility of Soils 15, 127±131.

Nardi, S., Concheri, G., Dell'Agnola, G., Scrimin, P., 1991. Nitrate uptake and ATPase activity in oat seedlings in the presence of two humic fractions. Soil Biology & Biochemistry 23, 833±836. Nardi, S., Concheri, G., Dell'Agnola, G., 1996. Biological activity of

humus. In: Piccolo, A. (Ed.), Humic Substances in Terrestrial Ecosystems. Elsevier, New York, pp. 361±406.

(5)

Auxin-like eect of humic substances extracted from faeces of

Allolobophora caliginosa and A. rosea. Soil Biology & Biochemistry 26, 1341±1346.

O'Farrell, P.H., 1975. High resolution two-dimensional electophor-esis of proteins. Journal of Biological Chemistry 250, 4007±4020. Sokal, R.R., Rohlf, F.J., 1969. Biometry. W.H. Freeman and Co,

San Francisco, USA.

Sze, H., 1985. H+- translocating ATPase: advances using membrane vesicles. Annual Review of Plant Physiology 36, 175±208. Taiz, L., Zeiger, E., 1991. Plant Physiology. The Benjamin/

Cummings Publishing Company, Inc, Redwood City, CA. Vaughan, D., MacDonald, I.R., 1971. Eects of humic acid on

pro-tein synthesis and ion uptake in beet discs. Journal of Experimental Botany 22, 400±410.

Vaughan, D., MacDonald, I.R., 1976. Some eects of humic acid on cation uptake by parenchyma tissue. Soil Biology & Biochemistry 8, 415±421.

Vaughan, D., Malcom, R.E., 1979. Eect of humic acid on invertase synthesis in roots of higher plants. Soil Biology & Biochemistry 11, 247±252.