Altered Gene Expression during Auxin-Induced Root ... - NCBI

Plant Physiol. (1987) 84, 1148-1153 0032-0889/87/84/1148/06/$O 1.00/0

Altered Gene Expression during Auxin-Induced Root Development from Excised Mung Bean Seedlings1 Received for publication September 29, 1986 and in revised form April 9, 1987

RAJINDER S. DHINDSA*, GUANGYUAN DONG2, AND LOUIS LALONDE Centerfor Plant Molecular Biology, Department ofBiology, McGill University, 1205 Avenue Docteur Penfield, Montreal, Quebec, H3A IBI, Canada ABSTRACT

Changes in the pattern of protein synthesis and in the translatable mRNA population have been examined during auxin-induced root development from excised mung bean seedlings. Several proteins, predominantly of low molecular weight and high pI, as shown by two-dimensional polyacrylamide gel electrophoresis, are synthesized specifically by auxintreated tissue. These auxin-induced proteins appear between 6 and 12 hours of auxin treatment, reach a maximum at 24 hours, and decline at 48 hours. Untreated seedlings (placed in Hoagland solution), known to produce small number of roots at the cut end probably due to endogeneous auxin accumulated at the cut end through basipetal transport, show low level synthesis of auxin-specific proteins. Antiauxin treatment that completely inhibits auxin-induced rooting also prevents the appearnce of auxin-induced proteins. The induction of a group of three to four proteins appears to be specific to antiauxin treatment. In vitro translation of mRNA from auxin-treated tissue, but not of mRNA from antianxintreated tissue, yields several polypeptides of low molecular weight and high pI. Since the auxin-induced proteins precede root development and are synthesized transitorily, it is likely that they play some regulatory role during the initiation of root development. The result show that auxininduced root formation involves altered gene expression.

Differential gene expression during organismal development, first proposed by Haldane (12) more than 50 years ago, is now well accepted. In principle it should also hold true in cases where, under special circumstances, an already mature organ gives rise to another type of organ. For example, in higher plants auxininduced rooting of excised seedlings and stem cuttings is a well documented phenomenon (1). Such adventitious organogenesis is, therefore, likely to involve an alteration of gene expression. However, there is little experimental evidence presently available for such altered gene expression. We have initiated a comprehensive study of the biochemistry and molecular biology of auxininduced root development from excised mung bean seedlings. This experimental material has been selected because of its relatively simple genome (14, 15) and the availability of a wealth of information on the effects of hormones and chemicals on its rooting response (1, 8). The results of our experiments, reported here, provide evidence that specific alterations in gene expression occur during auxin-

induced root development as shown by changes in the pattern of protein synthesis and in the population of translatable mRNAs.

MATERIALS AND METHODS Plant Material. Seedlings of mung bean (Vigna radiata cv Berken) were grown in vermiculite soaked in Hoagland solution for 8 d under a 16 h photoperiod and day/night temperature of 25°/20°C. Seedlings were excised 2.5 cm below the cotyledonary node and were used as experimental material. Treatments. Five excised seedlings were placed in 10 ml of half-strength Hoagland solution or treatment solution contained in plastic vial such that only the hypocotyl portion of the seedling (lower 2.5 cm) was in direct contact with the solution. Treatment solution either contained the auxin, IBA,3 or the antiauxin, PCIB (5, 6) in half-strength Hoagland solution. It was found that the seedlings in Hoagland solution produce a very small number of roots right at the cut end ofthe seedling as reported by Friedman et al. (8) probably stimulated by a small amount of endogenous auxin synthesized by leaves and accumulated at the cut end through basipetal transport (9). Thus seedlings treated with Hoagland solution show a low level auxin response and do not, therefore, constitute a good control. The use of 500 ,M PCIB completely abolished this residual rooting without any apparent adverse effect on the seedling. For this reason, the antiauxintreated seedlings serve as a better control. The auxin, IBA, was used at several concentrations and maximum rooting occurred at 50 ,uM. This latter concentration was used in all experiments reported here. Treatment of seedlings was carried out for 24 h except where stated otherwise. At the end of the treatment period seedlings were removed, rinsed with and placed in half-strength Hoagland solution which was replaced every 24 h. Roots became visible 72 h after the treatment and were extensively distributed on the lowermost 1.5 cm part of the hypocotyl region of the auxintreated seedling. Analysis of proteins was carried out at the time of excision of the seedling (D 0), 24 h (d 1), or 48 h (d 2) after the excision. In the case of auxin-treated seedlings, proteins were analyzed at 0, 6, 12, 18, 24, and 48 h in order to determine the time-course of the level of auxin-induced protein synthesis. In Vivo Labeling of Proteins. The lowermost 1 cm part of the seedling was used to study protein changes. It was previously determined that this part had a 2 to 3 times greater rate of protein and RNA synthesis as compared to the next upper 1 cm part. Furthermore, this is the part which produces roots most profusely. Six sections from d 0 control or variously treated seedlings

Supported by Natural Sciences and Engineering Research Council of 3Abbreviations: IBA, indolebutyric acid; PCIB, p-chlorophenoxyisoCanada grants A2724 and E1490 to R.S.D. ' Supported by a scholarship from the Canadian International Devel- butyric acid; PMSF, phenylmethylsulfonyl fluoride; PVPP, polyvinylpolypyrrolidone; 1-D, 2-D, one- and two-dimensional. opment Agency. 1148

GENE EXPRESSION AND ROOTING OF EXCISED SEEDLING mw 1

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