The Auxin-induced Maize Gene ZmSAUR2 Encodes a Short-lived Nuclear Protein Expressed in Elongating Tissues

By differential screening of a cDNA library from auxin-induced maize coleoptiles we have isolated and characterized a SAUR gene, designated ZmSAUR2, belonging to a not yet characterized subtype of the SAUR family. ZmSAUR2 encodes a 15.3-kDa protein and is specifically induced by auxin in elongating coleoptile tissue but not in primary leaves or in roots. The transcript level rapidly increased within minutes and preceded auxin-stimulated elongation of coleoptile segments. Cycloheximide also induced ZmSAUR2 transcription, as has been shown for other early auxin-induced genes, whereas abscisic acid, brassinolide, ethylene, gibberellic acid, kinetin, and methyl jasmonate did not provoke an increase in ZmSAUR2 mRNA abundance. In pulse-chase experiments using auxin-induced coleoptiles and an anti-ZmSAUR2 antibody we were able to precipitate a protein of the expected molecular mass and to determine a half-life of about 7 min, which is among the shortest known in eukaryotes. In gel shift assays binding of calmodulin to ZmSAUR2 was demonstrated, suggesting the possibility of post-transcriptional regulation. Upon transformation of onion epidermal cells with a ZmSAUR2::GUS construct the corresponding chimeric protein was detected in the nucleus. The results suggest that ZmSAUR2 encodes a short-lived nuclear protein that might be involved in auxin-mediated cell elongation. By differential screening of a cDNA library from auxin-induced maize coleoptiles we have isolated and characterized a SAUR gene, designated ZmSAUR2, belonging to a not yet characterized subtype of the SAUR family. ZmSAUR2 encodes a 15.3-kDa protein and is specifically induced by auxin in elongating coleoptile tissue but not in primary leaves or in roots. The transcript level rapidly increased within minutes and preceded auxin-stimulated elongation of coleoptile segments. Cycloheximide also induced ZmSAUR2 transcription, as has been shown for other early auxin-induced genes, whereas abscisic acid, brassinolide, ethylene, gibberellic acid, kinetin, and methyl jasmonate did not provoke an increase in ZmSAUR2 mRNA abundance. In pulse-chase experiments using auxin-induced coleoptiles and an anti-ZmSAUR2 antibody we were able to precipitate a protein of the expected molecular mass and to determine a half-life of about 7 min, which is among the shortest known in eukaryotes. In gel shift assays binding of calmodulin to ZmSAUR2 was demonstrated, suggesting the possibility of post-transcriptional regulation. Upon transformation of onion epidermal cells with a ZmSAUR2::GUS construct the corresponding chimeric protein was detected in the nucleus. The results suggest that ZmSAUR2 encodes a short-lived nuclear protein that might be involved in auxin-mediated cell elongation. The plant hormone auxin influences a variety of processes during plant growth and development, including cell elongation, cell division, differentiation, control of tropisms, and apical dominance. How auxin mediates and controls these effects on a molecular and cellular level is still far from being understood. Besides modulating membrane function it has clearly been demonstrated that auxin is able to cause rapid changes in the expression of a selected set of genes. Several sequences have been described that are up-regulated by auxin (for review, see Refs. 1Abel S. Theologis A. Plant Physiol. 1996; 111: 9-17Google Scholar, 2Sitbon F. Perrot-Rechenmann C. Physiol. Plant. 1997; 100: 443-455Google Scholar, 3Hagen G. Guilfoyle T. Plant Mol. Biol. 2002; 49: 373-385Google Scholar). Of major interest are genes that are specifically induced within minutes after auxin application in the absence of protein synthesis and are referred to as early or primary auxin-responsive genes. Three major classes are known: Aux/IAA, SAUR, and GH3 families (for review, see Ref. 3Hagen G. Guilfoyle T. Plant Mol. Biol. 2002; 49: 373-385Google Scholar). It was shown that apart from auxin, Aux/IAA and SAUR genes can also be induced by cycloheximide, and it is assumed that a pool of repressors, rapidly turning over, is involved in their transcriptional regulation. The very low abundance of the derived proteins, their short half-lives (as far as analyzed), their nuclear localization, and the absence of a catalytic domain point to an involvement in auxin signal transduction and in control of secondary downstream genes. Whereas for Aux/IAA proteins increasing evidence emerges that they act as repressors, including repressing their own transcription (for review, see Ref. 4Leyser O. Annu. Rev. Plant Biol. 2002; 53: 377-398Google Scholar), the function of SAURs still remains obscure, and even the experimental proof at the protein level is missing. SAUR genes (for small auxin up RNA) were originally identified and characterized in soybean (5McClure B.A. Guilfoyle T.J. Plant Mol. Biol. 1987; 9: 611-623Google Scholar) and found to encode a set of unstable transcripts that are induced by auxin within minutes. SAURs are abundant in the zone of elongation in soybean hypocotyls and are expressed most strongly in epidermal and cortical cells (6Gee M.A. Hagen G. Guilfoyle T.J. Plant Cell. 1991; 3: 419-430Google Scholar). Meanwhile SAURs have been identified in a variety of organisms, in Vigna radiata (7Yamamoto K.T. Mori H. Imaseki H. Plant Cell Physiol. 1992; 33: 93-97Google Scholar), Pisum sativum (8Guilfoyle T.J. Hagen G. Li Y. Ulmasov T. Liu Z. Strabala T. Gee M. Aust. J. Plant Physiol. 1993; 20: 489-502Google Scholar), Arabidopsis thaliana (9Gil P. Liu Y. Orbovic V. Verkamp E. Poff K.L. Green P.J. Plant Physiol. 1994; 104: 777-784Google Scholar), Nicotiana tabacum (10Roux C. Bilang J. Theunissen B.H. Perrot-Rechenmann C. Plant Mol. Biol. 1998; 37: 385-389Google Scholar), Raphanus sativus (11Anai T. Kono N. Kosemura S. Yamamura S. Hasegawa K. DNA Seq. 1998; 9: 329-333Google Scholar), Malus domestica (12Watillon B. Kettmann R. Arredouani A. Hecquet J.F. Boxus P. Burny A. Plant Mol. Biol. 1998; 36: 909-915Google Scholar) and more recently also in Zea mays (13Yang T. Poovaiah B.W. J. Biol. Chem. 2000; 275: 3137-3143Google Scholar). Genome analysis in A. thaliana reveals more than 70 SAUR homologs, which mostly are found in clusters (3Hagen G. Guilfoyle T. Plant Mol. Biol. 2002; 49: 373-385Google Scholar). However, it is not known how many of them are really expressed and are auxin-inducible. With one exception (AtSAUR11), all SAUR genes appear to lack introns (3Hagen G. Guilfoyle T. Plant Mol. Biol. 2002; 49: 373-385Google Scholar). SAURs encode unstable mRNAs (14McClure B.A. Guilfoyle T. Science. 1989; 243: 91-93Google Scholar, 15Franco A.R. Gee M.A. Guilfoyle T.J. J. Biol. Chem. 1990; 265: 15845-15849Google Scholar), and their high turnover rate may be due to a conserved downstream (DST) 1The abbreviations used are: DST, downstream; CaM, calmodulin; GUS, β-glucuronidase; IAA, indole-3-acetic acid; NIa, nuclear inclusion protein a; NLS, nuclear localization signal; SAUR, small auxin up RNA; MOPS, 4-morpholinepropanesulfonic acid. element in the 3′-non-translated region of the mRNA (16McClure B.A. Hagen G. Brown C.S. Gee M.A. Guilfoyle T.J. Plant Cell. 1989; 1: 229-239Google Scholar, 17Newman T.C. Ohme-Takagi M. Taylor C.B. Green P.J. Plant Cell. 1993; 5: 701-714Google Scholar) as well as elements within the coding region (9Gil P. Liu Y. Orbovic V. Verkamp E. Poff K.L. Green P.J. Plant Physiol. 1994; 104: 777-784Google Scholar, 18Gil P. Green P.J. EMBO J. 1996; 15: 1678-1686Google Scholar). There is evidence that SAURs are not only transcriptionally regulated but are also regulated at the post-transcriptional (19Li Y. Strabala T.J. Hagen G. Guilfoyle T.J. Plant Mol. Biol. 1994; 24: 715-723Google Scholar) and post-translational levels (13Yang T. Poovaiah B.W. J. Biol. Chem. 2000; 275: 3137-3143Google Scholar). Recently it was demonstrated that SAUR proteins bind calmodulin in vitro, pointing to a possible “cross-talk” between the Ca2+/calmodulin (CaM) second messenger system and auxin signal transduction (13Yang T. Poovaiah B.W. J. Biol. Chem. 2000; 275: 3137-3143Google Scholar). Auxin-induced cell elongation of maize coleoptiles is one of the fastest phytohormonal responses known, and this organ has been widely used as a model system to study regulation of cell growth and tropisms (20Evans M.L. CRC Crit. Rev. Plant Sci. 1985; 2: 317-365Google Scholar, 21Ray P.J. Dohrmann U. Hertel R. Plant Physiol. 1977; 60: 585-591Google Scholar, 22Haga K. Iino M. Plant Physiol. 1998; 117: 1473-1486Google Scholar, 23Carpita N.C. Defernez M. Findlay K. Wells B. Shoue D.A. Catchpole G. Wilson R.H. McCann M.C. Plant Physiol. 2001; 127: 551-565Google Scholar, 24Karcz W. Burdach Z. J. Exp. Bot. 2002; 53: 1089-1098Google Scholar). To detect genes involved in the elongation growth of maize, we carried out a differential screening of a cDNA library from auxin-induced coleoptiles and report here the isolation and characterization of a cDNA clone, designated as ZmSAUR2 because of its homology to SAUR genes. ZmSAUR2 shows typical characteristics of primary auxin-responsive genes; it is induced within minutes exclusively by auxin and by the protein biosynthesis inhibitor cycloheximide. In addition, sequence motifs of SAURs, such as nuclear localization (NLS) and DST can be identified. For the first time we succeeded in detecting the corresponding SAUR protein and show that it is very short-lived, having a half-life of 7 min. Coleoptile Preparation and Hormone Induction—Maize kernels were washed, soaked in water for 24 h at room temperature, and grown on wet cellulose at 25 °C in the dark. 24 h before harvest, seedlings were exposed to light for 1 min. Four-day-old etiolated coleoptiles with a length of 2.5–3.5 cm were harvested, subapical segments (10 mm long and 4 mm below the tip) were cut out, and the primary leaf was removed. Segments were incubated in aqueous solution with and for at 25 °C on a at and in To determine the in transcript one was after segments were incubated in and on how the hormone was For the and in experiments the was with a before segments were P. Plant Physiol. 1989; Scholar). and of the cDNA Z. mays cDNA library was from of isolated from auxin-induced coleoptile segments to and U. Scholar) using a cDNA synthesis were and the cDNA was by and to DNA was using the DNA was on as a was about screening was carried out with cDNA from auxin-induced was in and DNA of and at °C for h after h of in the solution the were in with for min, in with for 1 h at and was isolated from maize coleoptiles as described by R. Scholar) with plant was in a of the was to of a of and with and for at The was to the (10 at and the was more with of The aqueous was more with of aqueous were and with of and with of was with of and of at °C and at To from the was in of with and of was The was and at was The was in of with with and at for min. the in the was with of at °C and at For of was incubated in of of and 4 of for at of were on and on at for h and to for were with using the DNA at °C for h and at °C for was at °C for h in and the cDNA were with at and were by In of with and of coleoptile segments were incubated in of and of at 25 °C and a of was and incubated for min. were with mm and in the of for the and in were in a and the was in of mm 1 mm 1 mm and of and on for min. of was and at The was in 1 and in 25 mm mm by a at 1 of mm mm mm and of at antibody was to the and incubated at 4 The was to protein h at 4 by and 4 with and with the antibody was with min, was to Scholar). was and with an For a a was expressed in E. and by to the The of the coding region of ZmSAUR2 was were in the protein and by using the protein on a to ZmSAUR2 was using the 1 of with the ZmSAUR2 cDNA as an the control of the was and incubated as described in the 1 of ZmSAUR2 was incubated with and of as for 1 h at room in mm 1 mm in a of were in mm and 1 mm was at a of 25 in of 25 mm mm and 1 mm by Plant and transformation of onion cells epidermal were and on with and Plant tissue was with with DNA by using a for was to Plant Cell. 1998; Scholar). acid, was used as of the ZmSAUR2 coding sequence the was by and to the in for and DNA were carried out as described by J. T. Scholar). of ZmSAUR2 cDNA and an to genes that may be involved in auxin-mediated cell elongation, a cDNA library in was from of subapical segments of etiolated maize coleoptiles that were of auxin for and induced by for library was with cDNA from mRNA of coleoptile segments in and Of a differential of and were of them was characterized and is designated as ZmSAUR2 because of its homology to the SAUR and its by The sequence of the cDNA is shown in The ZmSAUR2 of encodes a of with a molecular mass of and an point of The sequence the first possible is with the for M. J. Cell Biol. 1989; Scholar). of an in transcript of ZmSAUR2 in a a of the expected ZmSAUR2 has an signal sequence but has a nuclear localization signal (NLS) of the C. Sci. 1991; Scholar). a conserved at can be identified. In the 3′-non-translated region a DST element is that was shown to be for the rapid of SAUR transcripts T.C. Ohme-Takagi M. Taylor C.B. Green P.J. Plant Cell. 1993; 5: 701-714Google Scholar, Y. Hagen G. Guilfoyle T.J. Plant Cell. 1991; 3: of ZmSAUR2 in transcript of ZmSAUR2 was in the with ZmSAUR2 and by Coleoptile segments were in with in the of auxin for and with an of for the for using ZmSAUR2 and by were with a and the results are shown on the of the sequence of ZmSAUR2 with other protein sequences in reveals that ZmSAUR2 can be a from SAURs characterized In Arabidopsis of them are to but also other of this the of an can be identified and that has the homology to ZmSAUR2, of this are of about and than the SAURs characterized with The of the ZmSAUR2 subtype is due to and In the region the within the ZmSAUR2 to to domain is not in the subtype by the only other described SAUR from maize, is as as ZmSAUR2 and also a long but its homology to ZmSAUR2 within this region is it might to a The region between is a for the ZmSAUR2 of its are only found in from In the region between and is not only conserved within the ZmSAUR2 but also in the subtype by and in the and as well as that might be for secondary such as or are and of ZmSAUR2 analysis of mRNA from coleoptile segments a ZmSAUR2 signal that after of in water is due to of auxin 1 and of to the to a increase of the ZmSAUR2 transcript after of time point in this it is than the in level and within the min. R. V. P. Bot. Scholar) show that application of to coleoptile segments elongation after a of about min. ZmSAUR2 transcripts appear before the segments to In is from coleoptile segments incubated for an as in but in the absence of auxin, and a of ZmSAUR2 mRNA is with the of elongation of coleoptile segments that within h after and is by the of the M. P. 1985; Scholar). of the of ZmSAUR2 coleoptile segments were incubated with abscisic acid, kinetin, ethylene, gibberellic acid, brassinolide, or methyl jasmonate as well as with the auxin and and the acid. shown in ZmSAUR2 is only induced by IAA, acid, and acid, but not by or of the other in the of other early genes an of ZmSAUR2 transcripts be in the of the protein synthesis inhibitor of protein synthesis is assumed to cause the of a of transcription or of a for S. Theologis A. Plant Physiol. 1996; 111: 9-17Google Scholar). it is also that the the of the transcripts by a not clearly T. N. Theologis A. J. Mol. Biol. Scholar). It has not yet been as to mRNA or of transcription is for the increase in ZmSAUR2 transcript is evidence for the of among SAUR genes P. Green P.J. EMBO J. 1996; 15: 1678-1686Google Scholar, Y. Strabala T.J. Hagen G. Guilfoyle T.J. Plant Mol. Biol. 1994; 24: 715-723Google Scholar). of shown in ZmSAUR2 transcripts be detected only in the coleoptile and in the but not in the and in the primary leaf of maize the of ZmSAUR2 transcripts well with the elongation growth of the coleoptile analysis of from coleoptiles and grown for in the light or 4 in the 1 in the light a ZmSAUR2 mRNA level with seedlings that were for exclusively in the dark. is with the of maize coleoptiles and In the first 4 growth of coleoptiles is of this coleoptiles and the primary leaf the whereas in coleoptiles elongating In growth is by light from the ZmSAUR2 experiments for the first time to detect a protein of the SAUR in Auxin-induced coleoptiles were with and for with as were and the protein was by an ZmSAUR2 and by gel shown in the protein by the ZmSAUR2 has an short half-life of about 7 min, as the and proteins, of the Aux/IAA S. Theologis A. Sci. U. S. A. 1994; Scholar). in and ZmSAUR2 with the we that the short-lived protein most is ZmSAUR2 of of a nuclear localization signal the that ZmSAUR2 in the nucleus. To the was to the of the coding sequence of ZmSAUR2 the control of the The chimeric construct was onion cells and the localization of the chimeric ZmSAUR2::GUS protein was Whereas the protein ZmSAUR2 did not in the cells the chimeric construct a nuclear nuclear was for the nuclear inclusion protein a used as a control M.A. Plant Cell. 1990; 2: Scholar). The results suggest that the is the cellular in which ZmSAUR2 its ZmSAUR2 a was shown to bind to protein expressed in E. or to from SAUR sequences and it was that SAURs in are proteins (13Yang T. Poovaiah B.W. J. Biol. Chem. 2000; 275: 3137-3143Google Scholar). is a protein of a domain of the proteins H. 2001; Scholar, H. Plant Sci. 1998; 3: Scholar, T. G. H. Plant Physiol. Scholar). of the sequence of ZmSAUR2 an at the that in a not shows the typical to a plant protein S. V. Poovaiah B.W. J. Biol. Chem. 1996; Scholar), or to other SAURs (13Yang T. Poovaiah B.W. J. Biol. Chem. 2000; 275: 3137-3143Google Scholar). ZmSAUR2 was by in and incubated with was by in the of and in the or absence of The results in 7 that ZmSAUR2 is able to a with in the of that with a with ZmSAUR2 incubated ZmSAUR2 was in a and the shift was was not the was suggesting that binding is cell is one of the and fastest responses in with in the of minutes (for review, see Ref. D.A. Plant Cell 1987; Scholar). the well characterized maize coleoptile system for a differential cDNA screening for auxin-responsive genes, we were able to a a very short-lived, nuclear protein that be involved in auxin-induced cell elongation. of its homology to SAUR genes, it was In addition, typical sequence elements be identified such as a in the a DST which is known to of SAUR transcripts P. Green P.J. EMBO J. 1996; 15: 1678-1686Google Scholar). The homology of ZmSAUR2 with other characterized SAUR genes is and for as a typical it to an of only this is also for the other recently identified from maize, which is the only characterized SAUR from the other the not yet characterized SAUR genes such as a within the which has homology to in on be identified in a that are more to subtype is characterized by and which these SAURs than of the subtype by was isolated from a cDNA library in a binding screening below for an it is also than its are from In the most for is far more conserved in and the ZmSAUR2 subtype as well as in the characterized SAURs to sequence typical for SAURs is the DST element in the It is also in ZmSAUR2 and well with the SAUR as shown in DST sequences were demonstrated to to the of SAUR which are among the most short-lived mRNAs in (16McClure B.A. Hagen G. Brown C.S. Gee M.A. Guilfoyle T.J. Plant Cell. 1989; 1: 229-239Google Scholar, 18Gil P. Green P.J. EMBO J. 1996; 15: 1678-1686Google Scholar, Y. Hagen G. Guilfoyle T.J. Plant Cell. 1991; 3: Scholar). In and characterized by a for an level of mRNA M.A. M.A. Green P.J. Sci. U. S. A. 2000; Scholar). Besides the DST also the coding region to SAUR mRNA Y. Liu Hagen G. T.J. Plant Physiol. 1994; Scholar). SAUR proteins have been to be detected (3Hagen G. Guilfoyle T. Plant Mol. Biol. 2002; 49: 373-385Google Scholar). be due to an of SAURs or because of their low abundance. also to detect ZmSAUR2 using by and auxin-stimulated coleoptile segments with by and gel we were able to detect a protein with the as the in it is possible that Z. not yet identified proteins we the possibility that the antibody ZmSAUR2 may with such In pulse-chase experiments a half-life of 7 was for the which is as short as that of of the Aux/IAA S. Theologis A. Sci. U. S. A. 1994; and is among the shortest known for for ZmSAUR2, also in the of the Aux/IAA proteins, was not in detecting these short-lived a Aux/IAA protein be detected by because the a of the protein and to an increased There is evidence that auxin influences the of Aux/IAA proteins N. A. C. J. Sci. U. S. A. 2001; Scholar, Hagen G. Guilfoyle T.J. Plant Cell. 2001; Scholar), and it be to this is also for SAUR by a nuclear system is to a in auxin Aux/IAA proteins are for (for review, see Ref. 4Leyser O. Annu. Rev. Plant Biol. 2002; 53: 377-398Google Scholar). which SAURs to have in with is the nuclear (9Gil P. Liu Y. Orbovic V. Verkamp E. Poff K.L. Green P.J. Plant Physiol. 1994; 104: 777-784Google Scholar) that SAURs may in the as they identified a in are found in most SAURs, and this can also be identified in ZmSAUR2, the in which it is is only that a ZmSAUR2::GUS protein is found in the is the first evidence that these are and SAURs in the nucleus. In of the nuclear localization and the of ZmSAUR2 one may SAURs their function as transcriptional downstream auxin responses to Aux/IAA possible be the recently identified secondary auxin-induced which to be an element of the in auxin-mediated transcripts are expressed in the coleoptile during and appear only after auxin K. H. S. C.S. K. G. K. G. M. R. Sci. U. S. A. Scholar), after of The transcriptional regulation of ZmSAUR2 is in with other SAURs and shown to be the signal of ZmSAUR2 mRNA at and the increase of the transcript level one may that than min, to soybean SAUR ZmSAUR2 transcripts auxin-induced cell elongation, for which in coleoptiles a of growth of about was R. V. P. Bot. Scholar, K. H. S. C.S. K. G. K. G. M. R. Sci. U. S. A. Scholar). with the of the recently identified the ZmSAUR2 is more rapidly in addition, ZmSAUR2 is not expressed in (13Yang T. Poovaiah B.W. J. Biol. Chem. 2000; 275: 3137-3143Google Scholar). With to the hormone one that brassinolide, which is also able to cause cell elongation in assays for auxin for review, see Ref. G. 1994; Scholar), did not ZmSAUR2 which is to be a for auxin is also not induced by C. Plant Physiol. 2000; Scholar). the other in the is able to elongation suggesting signal for and processes T.C. Plant Physiol. 1994; 104: Scholar). The expression in analysis ZmSAUR2 only to the coleoptile and the but not to the primary leaf or the of maize The results are in with the expression of other SAUR genes, which also with auxin-induced cell elongation, tissue is Y. Liu Hagen G. T.J. Plant Physiol. 1994; for review, see Ref. T.J. M.A. and of Plant B. Scholar). has been isolated by screening an expression library with (13Yang T. Poovaiah B.W. J. Biol. Chem. 2000; 275: 3137-3143Google Scholar). analysis of the in E. expressed protein that the binding is at the domain that is able to a a can also be identified in In shift assays using ZmSAUR2 we were able to an with The binding was because ZmSAUR2 with ZmSAUR2, whereas was not able to may that ZmSAUR2 function may be by a rapid changes in the were detected in maize coleoptiles after auxin and after and changes were within Sci. U. S. A. 1990; Scholar, D.A. 1990; Scholar). catalytic domain be identified in SAURs, may their binding to other It is known that proteins are able to bind For transcription of the it was that binding their DNA binding B. M. Y. J. B. A. T. 1994; Scholar). for the Arabidopsis transcription and an with was shown B. Plant Cell. 1996; Scholar, N. G. H. J. Biol. Chem. 1996; Scholar). one may that SAURs an element of the between the messenger system and the auxin signal the function of SAUR genes remains at this the of the of a SAUR protein for the first time with its very short its nuclear localization, and the rapid of the the that SAURs may an in early auxin signal transduction A. for in onion cell are also to for