Differential Cene Expression in Response to Auxin ... - Plant Physiology

Plant Physiol. (1 997) 114: 1 1 97-1 206

Differential Cene Expression in Response to Auxin Treatment in the Wild Type and rac, an Adventitious Rooting-1ncompetent Mutant of Tobacco’ Steven T. Lund2*, Alan C. Smith, and Wesley P. Hackett

Departments of Horticultural Science (S.T.L., A.G.S., W.P.H.), and Plant Biology (W.P.H.), University of Minnesota, St. Paul, Minnesota 551 08 adventitious root initiation, but the physiological mechanisms underlying their root-promoting ability have not been elucidated (Jarvis, 1986; Blakesley, 1994; Haissig and Davis, 1994). Some phloem parenchyma or inner cortical parenchyma cells in adventitious rooting-competent cuttings treated with auxin form adventitious root meristems, whereas analogous cell types in rooting-incompetent cuttings generally form callus in response to auxin (Lovell and White, 1986; Hartmann et al., 1990). Thus, phloem parenchyma or inner cortical parenchyma cells in rootingincompetent cuttings can respond mitotically to auxin, but these cells are unable to undergo the organized divisions required for adventitious root initiation. Our overall goal is to understand the basis for the competence for adventitious root initiation in cuttings. Our overall hypothesis is that adventitious rooting competence in cuttings has a molecular basis at the leve1 of auxinregulated gene expression in phloem parenchyma or inner cortical parenchyma cells during adventitious root initiation. In an attempt to identify genes that are specifically regulated during auxin-induced adventitious root initiation, we compared gene expression in cuttings from shoots of adventitious rooting-competent wild-type tobacco (Nicotiana tabacum cv Xanthii) with the rooting-incompetent rac mutant (Muller et al., 1985; Caboche et al., 1987; Pelese et al., 1989; Barbier-Brygoo et al., 1991). The rac mutation is semidominant for adventitious root initiation (Lund et al., 1896). Stem cuttings from the rac genotype do not form adventitious roots in response to exogenous IBA concentrations that are optimal or 100-fold greater than optimal for adventitious root initiation in wild-type cuttings. Some phloem parenchyma or inner cortical parenchyma cells in rac cuttings form callus in response to exogenous IBA. The auxin-induced callus formations in heterozygous rac cuttings are more organized than those in the homozygous yac cuttings, but none of the callus formations develop into adventitious root meristems (Lund et al., 1996). Thus, the Yac mutation blocks adventitious root initiation prior to the first organized cell divisions that normally lead to the formation of an adventitious root meristem.

Histological analyses of auxin-treated cuttings from the wild type and the rac mutant of tobacco (Nicotiana tabacum cv Xanthii) previously revealed that some rac phloem parenchyma or inner cortical parenchyma cells form callus i n response to exogenous auxin treatment but these cells never undergo the organized divisions associated with adventitious root initiation in the wild type. Here we report the effect of the rac mutation on the temporal and spatial expression patterns of three genes previously shown to be associated with adventitious root meristems, HRGPnt3, iaa4/5, and gh3. Using histochemical staining analyses of HRGPnt3-GUS transformant cuttings, we determined that the rac mutation blocks auxin activation of the HRGPnt3 promoter. Thus, activation of the HRGPnt3 promoter occurs specifically during adventitious root initiation in tobacco cuttings. Histochemical staining analyses of iaa4/5CUS and gh3-GUS transformant cuttings revealed that the rac mutation does not repress the auxin activation of the iaa4/5 and gh3 promoters. Based on our histochemical staining analyses, we conclude that differential gene expression occurs in response to auxin treatment during adventitious root initiation i n the wild type compared with callus formation in rac cuttings. We also determined that HRGPnt3 mRNA accumulation occurs in response to components of our root-induction protocol other than auxin, indicating that HRGPnt3 expression i s regulated both developmentally and environmentally.

Some species, clones, or developmental phases within clones are competent for adventitious root formation from cuttings and others are not. The morphological basis for adventitious rooting competence is the ability of cuttings to undergo adventitious root initiation, which is the formation of an adventitious root meristem (Hartmann et al., 1990). Adventitious root meristems consist of 5 to 20 mitotically active cells containing dense cytoplasms and enlarged nuclei; these cells are dedifferentiating phloem parenchyma or inner cortical parenchyma cells and are referred to as adventitious root initials (Blakesley et al., 1991). Auxins have been shown to be effective inducers of This research was supported by the Minnesota Agricultura1 Experiment Station. This is U.S. Department of Agriculture paper no. 971,210,005. * Present address: Horticultural Sciences Department, P.O. Box 110690, University of Florida, Gainesville, FL 32611-0690. * Corresponding author; e-mail st1undQgnv.ifas.ufl.edu;fax 1-352-846 -2063.

Abbreviations: HRGP, Hyp-rich glycoprotein; IBA, indole-3butyric acid; NAA, (Y-NAA;X-gluc, 5-bromo-4-chloro-3-indolylglucuronide. 1197

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We hypothesized that differences in gene expression occur in response to auxin treatment during adventitious root initiation in the wild type versus callus formation in rac cuttings. For example, if the rac mutation blocks auxin signal transduction for adventitious root initiation, then regulatory events downstream from rac that are specific for adventitious root initiation should be aberrant in rac cuttings treated with auxin in comparison to the wild type. Any gene expression downstream from rac that is ordinarily up-regulated by auxin during adventitious root initiation in wild-type cuttings may not be induced in auxintreated rac cuttings. Because rac blocks auxin-induced adventitious root initiation prior to the first organized divisions, we postulated that we could identify genes that are specifically regulated before and during early stages of adventitious root initiation. Even if rac does not completely block a11 auxin signal transduction pathways for adventitious root initiation, gene expression that is specific for adventitious root initiation may be temporally and / or quantitatively modified. We compared the temporal and spatial expression patterns of three genes, iaa4/5, gh3, and HRGPnt3, in our rooting-competent versus rooting-incompetent experimental system. These genes were selected from previously isolated auxin-responsive or root-associated genes because they have a11 been shown to be expressed in adventitious root initials, and transgenic tobacco lines with promoter-GUS fusions for each of these genes were available. iaa4/5 (Theologis et al., 1985) and gh3 (Hagen et al., 1984; Hagen and Guilfoyle, 1985) are thought to represent early auxin response genes because induction of iaa4/5 and gk3 transcript accumulation is rapid, specific for biologically active auxins, and does not require protein synthesis. Theologis et al. (1985) isolated iaa4/5 via a differential screen of cDNA libraries constructed from pea (Pisum sativum cv Alaska) epicotyls either treated or not treated with 20 p~ IAA. Ballas et al. (1993) transformed tobacco ( N . tabacum cv Samsun) with the iaa415 promoter translationally fused to GUS and showed that in tobacco seedlings iaa4/5-GUS activity occurred in developing xylary cells and primary and lateral root meristematic cells in response to 20 p~ IAA; GUS activity was lower in the absence of exogenous IAA. Hagen et al. (1984) isolated the gk3 cDNA via a differential screen of cDNA libraries constructed from soybean (Glycine max cv Wayne) hypocotyls either treated or not treated with 50 p~ 2,4-D. In situ hybridization analyses of anti-gh3 transcripts in soybean (Gee et al., 1991) and histochemical GUS analyses in tobacco (N. tabacum cv Xanthii) transformed with a transcriptional fusion of the gk3 promoter to GUS (Hagen et al., 1991) showed that auxin induction of gk3-GUS activity occurred in many cell types, including pericycle cells, primary root meristem cells, elongating cells in the shoot, and phloem parenchyma and developing xylary cells in roots, shoots, and flowers. Very little or no GUS activity was detected in nonauxin-treated tissues. Guilfoyle et al. (1993) reported that gh3-GUS activity also occurs in adventitious root meristematic cells. Keller and Lamb (1989) isolated HRGPnt3 by screening a tobacco genomic library with a bean (Phaseolus vulgaris)

cDNA encoding a HRGP. HRGPs are members of a gene family of cell wall proteins containing the repeated pentapeptide motif SER-(HYP), (Wilson and Fry, 1986). Using RNA-blot analyses, Keller and Lamb (1989) found that the accumulation of HRGPnt3 transcripts was developmentally regulated and occurred only in primary roots; HRGPnt3 accumulation was not responsive to mechanical wounding. Histochemical staining of tobacco transformed with a HRGPnt3-GUS gene fusion revealed that the HRGPnt3 promoter is activated during lateral root initiation in pericycle cells (Keller and Lamb, 1989) and adventitious root initiation in phloem parenchyma or inner cortical parenchyma cells (Vera et al., 1994). Histochemical staining of hydroxyurea- and colchicine-treated primary roots from HRGPnt3-GUS transgenic plants indicated that the HRGPnt3 promoter is activated after treatment with auxin but prior to completion of the first division(s) in the pericycle during lateral root morphogenesis (Vera et al., 1994). MATERIALS A N D METHODS Plant Material and Growth Conditions

Wild-type, heterozygous, and homozygous rac axillary shoots of tobacco (Nicotiana tabacum cv Xanthii) were propagated in vitro as previously described (Lund et al., 1996). Heterozygous rac plants hemizygous for the iaa4/5-GUS (Ballas et al., 1993), gh3-GUS (Hagen et al., 1991), or HRGPnt3-GUS constructs (Keller and Lamb, 1989) used for the GUS analyses were generated by hand-pollinating the emasculated flowers of each of the homozygous transformant genotypes with pollen from a homozygous rac shoot that was grafted onto a N. tabacum cv Wisconsin 38 rootstock. Wild-type transformants used as controls for the GUS analyses were hemizygous for the iaa415-GUS and gh3-GUS constructs and homozygous for the HRGPnt3GUS construct. Hemizygous iaa415-GUS and gk3-GUS plants were generated by hand-pollinating the emasculated flowers of each of the homozygous transformant genotypes with pollen from a wild-type plant. Because each of the three constructs confer kanamycin resistance, a11 progeny were screened for inheritance of the transgenes by growing them on axillary shoot proliferation media containing 50 pg/mL kanamycin. The iaa4/5-GUS line segregates as having two inserts; the gk3-GUS and HRGPnt3GUS gene constructs each segregate as single loci. Auxin Treatment

Auxin treatments were carried out as previously described (Lund et al., 1996). Two-centimeter cuttings of wild-type, heterozygous, and homozygous rac axillary shoots derived from in vitro culture were placed upright on basal media containing no IBA for 24 h in continuous light to deplete endogenous IAA (d O). Cuttings were then inverted so that the morphological apical ends were in media containing or not containing filter-sterilized IBA and were subsequently placed in continua1 darkness (d 1) to minimize IBA degradation. Tissue from the morphological apical portion (1.5 cm) of each of the cuttings was used for

Cene Expression during Adventitious Root Morphogenesis a11 analyses, since the apical 75% of wild-type cuttings do not form adventitious roots in the absence of IBA treatment (Lund et al., 1996). Histochemical GUS Analyses

Every cutting assayed from each genotype was an individual progeny from a single cross; therefore, none of the individuals were clones. Transformant cuttings were treated with or without auxin for 5 d. Five to 10 wild-type or heterozygous rac transformant cuttings were tested with each auxin concentration, unless otherwise indicated. Following treatment, cross-sections 50- to 100-pm thick from the morphological apical 1.5 cm of each cutting were made by hand. Sections were incubated with X-gluc for 20 h at 37°C. X-gluc buffer was 0.05 M sodium phosphate, pH 7.0, 0.25 mM K,[Fe(CN),], 0.25 mM K,[Fe(CN),], 5 mM Na,EDTA. Based on optimization experiments (not shown), the X-gluc concentrations used for each of the three constructs were as follows: iaa4/5-GUS, 0.05 mg/mL; gh3-GUS, 1 mg/mL; HRGPnt3-GUS, 1.3 mg/mL. Quantitative Dose Response of gh3-GUS Activity to IBA Treatment

Inverted hemizygous $13-GUS cuttings and hemizygous gh3-GUS, heterozygous rac cuttings were treated for 24 h with IBA as described above. Three 1-g replicates per IBA concentration per genotype were assayed. Each I-g sample (about six cuttings per IBA concentration per genotype) was ground in liquid nitrogen containing 3 mL of extraction buffer (0.1 M potassium phosphate, pH 7.8, 1 mM Na,EDTA, 10 mM DTT, 0.8 mM PMSF, and 5% [v/v] glycerol). Samples were then homogenized for 30 s with a polytron and centrifuged at 10,OOOg for 5 min. Supernatants were collected from each sample and stored at -80°C. Samples were assayed with 0.5 mM methylumbelliferyl-6D-glucoside in extraction buffer at 37°C. Reactions were terminated and fluorescence readings were taken in filtered 0.2 M Na,CO,. Fluorescence measurements were taken during the linear phase of the reaction on a DNA fluorometer (model TKO 100, Hoechst, Charlotte, NC). Total protein extracted from each sample was measured using the bicinchoninic acid method (BCA protein assay kit, Sigma). RNA-Blot Analyses of HRGPnf3 Expression

Total RNA was extracted from 10 g of axillary shoot cuttings per genotype per IBA concentration per day with phenol/SDS, followed by precipitation in 2 M LiCl (Rochester et al., 1986). Ten grams of axillary shoot cuttings is composed of 50 to 60 cuttings; therefore, the signals shown in each lane in Figures 3 and 4 can be considered as an average HRGPnt3 mRNA accumulation response in a sample size of 50 to 60 cuttings. Twenty micrograms of total RNA was loaded in a11 lanes except lane 1 in Figure 4, in which 18 pg was loaded. Electrophoresis was carried out in 1.2% agarose gels containing formaldehyde. Equal loading was confirmed by comparisons of intensities of ethidium

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bromide staining of rRNAs. Total RNA was blotted onto a GeneScreen Plus membrane (DuPont) in 1OX SSPE (1.5 M NaCl, 0.1 M NaH,PO, *H,O, 0.01 M Na,EDTA.2H20, pH 7.4) overnight followed by UV cross-linking for 30 s and air-drying for 2 h. Prehybridizations for the analyses shown in Figure 3 were done at 48°C for 2 to 3 h (50% formamide, 5 x Denhardt's solution, 5X SSPE, 1%SDS, and 1 mg/mL denatured salmon sperm DNA). A 1.45-kb PstI-EcoRI HRGPnt3 fragment containing the coding sequences for the SER(PRO), repeated motif (Keller and Lamb, 1989) was 32Plabeled by random priming (Megaprime kit, Amersham) and used for a11 hybridizations. Hybridizations for the analyses shown in Figure 3 were carried out for 20 h at 48°C in fresh prehybridization solution. Lower-stringency washes (0.3X SSPE and 0.1% SDS) were done for 30 min at 65°C; higher-stringency washes (0.1X SSPE and 0.1% SDS) were done for 30 min at 68OC. The prehybridization and hybridization for the analysis shown in Figure 4 were both done at 42°C with 0.1 mg/mL instead of 1 mg/mL denatured salmon-sperm DNA and the wash was done at the lower stringency (see above). Because of low levels of HRGPnt3 transcript accumulation, high background-tosignal ratios and decreased quality of autoradiographs are common. RESULTS Histochemical Analyses of iaa4/5-GUS Expression

iaa415-GUS activity was detected on d 1 through 5 in the periphery of the vascular tissue of iaa415-GUS hemizygous cuttings and iaa415-GUS hemizygous, rac heterozygous cuttings whether they were treated with 5 FM IBA (Fig. 1, A-D) or without IBA (data not shown). Figure 1A shows vascular region staining in an iaa415-GUS hemizygote treated with 5 PM IBA for 5 d. We concluded that iaa4l5GUS activity occurred in adventitious root initials because the position of the cells staining for iaa415-GUS activity was at the periphery of the vascular tissue, where root initials are known to originate (Lund et al., 1996), and the shape of the groups of cells had a radial symmetry. At more advanced stages of adventitious root development, GUS activity was evident in the apical meristematic regions of adventitious root primordia in iaa415-GUS hemizygous cuttings (Fig. 1B). Examination of 5 FM IBA-treated, iaa415GUS hemizygous, rac heterozygous cuttings revealed that iaa415-GUS expression occurred in a11 callus formations (Fig. 1, C and D), indicating that auxin activation of the iaa4/5 promoter is not blocked by the rac mutation. Histochemical Analyses of gh3-GUS Expression

Histochemical staining for gh3-GUS activity was detected in less than one-half of the adventitious root meristems in gh3-GUS hemizygous cuttings treated with any concentration of IBA (data not shown). This could be explained if NAA, which was used by Hagen et al. (1991), were more effective at inducing gh3-GUS activity than IBA or if the gh3-GUS transformant line tested exhibits weak

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Figure 1. Photomicrographs of cross-sections of cuttings showing histochemical GUS staining in the indicated genotypes after treatment for 5 d with the indicated auxin concentrations. Cross-sections of each genotype were treated with X-gluc, as indicated in "Materials and Methods." Bars denote 100 jum. A, iaa4/5-CUS hemizygote, 5 /XM IBA. iaa4/5-CUS activity (Legend continues on facing page.)

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gh3-GUS activity. We subsequently found that NAA was slightly more effective at inducing gh3-GUS activity in adventitious root meristems than IBA, but gh3-GUS activity did not occur in a11 adventitious root meristems unless gh3-GUS hemizygous cuttings were treated with 50 or 100 ~ L NAA M (data not shown). Histochemical staining for gh3-GUS activity was detected in adventitious root initials (Fig. 1E) and primordia (Fig. 1F) in gh3-GUS hemizygous cuttings that were treated with 50 and 100 p~ IBA or NAA. Staining for gh3-GUS activity in adventitious root primordia was confined to the apical meristem, as was found for iaa415-GUS activity (compare F with B in Fig. 1).Histochemical staining for gh3-GUS activity was routinely detected in callus formations in gh3GUS hemizygous, rac heterozygous cuttings that were treated with 50 or 100 p~ NAA (Fig. lG), indicating that auxin activation of the gh3 promoter is not blocked by the rac mutation. As with gh3-GUS hemizygous cuttings, treatment with at least 50 PM NAA was required to detect histochemical staining for gh3-GUS activity in gh3-GUS hemizygous, rac heterozygous cuttings (data not shown). Quantitative Analyses of gh3-CUS Activity

The lack of histochemical staining for gh3-GUS activity in nonauxin-treated stems of both wild-type and rac transformant cuttings allowed us to quantitatively analyze the dose response of gh3-GUS activity to IBA treatment. Low levels of GUS activity were detected in gh3-GUS hemizygous cuttings and in gk3-GUS hemizygous, rac heterozygous cuttings treated for 1 d with O or 1 p~ IBA (Fig. 2). Based on SES, no differences in GUS activity were detected between gh3-GUS hemizygous cuttings and gh3-GUS hemizygous, rac heterozygous cuttings treated with 5,10,or 50 p~ IBA (Fig. 2); these IBA concentrations are optimal for adventitious root induction under our experimental conditions (Lund et al., 1996). Fifty micromolar IBA was the lowest concentration tested that caused a significant increase (about 5-fold) in GUS activity over non-IBA-treated controls in both gh3-GUS hemizygous cuttings and gh3GUS hemizygous, rac heterozygous cuttings (Fig. 2). Onehundred micromolar IBA was the only treatment tested that caused a significant difference in levels of GUS activity

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pM IBA Figure 2. Quantitative analysis of GUS activity i n gh3-GUS hemizygous cuttings compared with gh3-GUS hemizygous, rac heterozygous cuttings. Three replicate, 1-g samples of cuttings, from each genotype were treated with each of the indicated concentrations of IBA for 1 d. Each column represents the mean picograms of 4-methylumbelliferone (4-MU) produced per minute per milligram of protein after incubation of protein extracts from each of the replicate samples with methylumbelliferyl-(-D-glucoside for 45 min at 37OC, as measured bv a fluorometer. Bars indicate SES.

between the two genotypes; gh3-GUS activity in gh3-GUS hemizygous, rac heterozygous cuttings was about 2-fold higher than in gk3-GUS hemizygous cuttings and about 12-fold higher than in untreated cuttings from either genotype (Fig. 2). GUS activity was very low and not significantly different from non-IBA-treated controls in gh3-GUS hemizygous cuttings or gh3-GUS hemizygous, rac heterozygous cuttings treated with 500 p~ IBA (Fig. 2); however, senescence occurred in both wild-type and rac heterozygous cuttings in response to 500 ~ L IBA M treatment (Lund et al., 1996). The results shown in Figure 2 corroborate the histological analyses (Fig. 1, E-G) and further indicate that auxin activation of the gh3 promoter is not blocked by the rac mutation.

Figure 1. (Continued from facing page.) in cells at the periphery of the vascular tissue. These cells likely are adventitious root initials. B, iaa4/5-GUS hemizygote, 5 p~ IBA. Arrow indicates iaa4/5-GUS activity in the apical meristem of an adventitious root primordium. C, iaa4/5-GUS hemizygous, rac heterozygote, 5 p~ IBA. Arrow indicates iaa4/5-GUS activity in callus derived from phloem parenchyma or inner cortical parenchyma cells at an early stage of growth. D, iaa4/5-GUS hemizygous, rac heterozygote, 5 p~ IBA. Brackets indicate iaa4/5-GUS activity in phloem parenchyma or inner cortical parenchyma-derived callus a t a more advanced stage of growth than shown in C. E, gh3-GUS hemizygote, 50 p~ IBA. Arrow indicates gh3-GUS activity in cells at the periphery of the vascular region. These cells likely are adventitious root initials. F, $3-GUS hemizygote, 50 p~ IBA. Arrow indicates gh3-GUS activity in the apical meristem of an adventitious root primordium. G, gh3-GUS hemizygous, rac heterozygote, 100 p~ NAA. Brackets enclose area of gh3-GUSactivity in phloem parenchyma- or inner cortical parenchyma-derived callus. H, HRGPnt3-GUS homozygote, 5 p~ IBA. Arrow indicates HRGPnt3-GUS activity in adventitious root initials at a very early stage of adventitious root initiation. The initials appear to be about one cell layer below the surface of the section. I, HRGPnt3-GUS homozygote, 5 p~ IBA. Arrow indicates HRGPnt3-GUS activity in an adventitious root meristem prior to the organization of cells into cell files. J, HRGPnt3-GUS homozygote, 5 p~ IBA. Arrow indicates HRGPnt3-GUS activity in the apical region of an adventitious root primordium. K and L, HRGPnt3-GUS hemizygous, rac heterozygote, 5 p~ IBA. Brackets denote early (K) and more advanced (L) stages of callus formation with no HRGPnt3-GUS activity. cp, Cortical parenchyma; pp, pith parenchyma; x, xylem.

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Histochemical Analyses of HRGPnt3-GUS Expression

HRGPnt3-GUS activity was routinely detected in, and confined to, adventitious root initials and primordia in HRGPnt3-GUS homozygous cuttings that were treated with 5 JLIM IBA for 5 d. GUS activity was not detected in any tissue types in non-IBA-treated, HRGPnt3-GUS homozygous cuttings (data not shown). Histological analyses (Lund et al., 1996) demonstrated that the earliest detectable stage of adventitious root initiation in IBA-treated tobacco cuttings is when 5 to 20 mitotically active cells in a radially symmetrical shape can be detected. Figure 1H shows that histochemical staining for GUS activity was detected at this very early stage of adventitious root initiation in HRGPnt3GUS homozygous cuttings treated with 5 /J.M IBA. Figure II shows histochemical staining for GUS activity at a later stage in an adventitious root meristem. As the meristems began to form cell files and progress to the primordial stage, GUS activity was confined to the outermost apical cells of the primordia (Fig. 1J). The detection of GUS activity early during adventitious root initiation and only in cell types associated with adventitious root meristems corroborates the findings of Vera et al. (1994) for HRGPnt3-GUS activity during adventitious root morphogenesis in tobacco stems. In hemizygous HRGPnt3-GUS, heterozygous rac cuttings, GUS activity was not detected at early (Fig. IK) or more advanced (Fig. 1L) stages of auxin-induced callus formation in a total of 47 of 48 individuals in two replicate experiments. In the one remaining individual, histochemical staining was detected in the vascular and peripheral regions of the callus (data not shown). GUS activity was not detected in any cell types in non-IBA-treated, HRGPnt3-GUS hemizygous, rac heterozygous individuals (data not shown). In a separate experiment, treatment of HRGPnt3-GUS hemizygous, rac heterozygous cuttings with 10, 50, or 100 JU.M IBA did not induce GUS expression in any cell types (five cuttings per IBA concentration, data not shown). These results show that auxin activation of the HRGPnt3 promoter is blocked by the rac mutation.

RNA-Blot Analyses of HRGPnt3-GUS Expression

Equivalent levels of HRGPntS mRNA accumulation were detected in total RNA from non-IBA-treated wild-type cuttings from d 1 to 5 (Fig. 3A). When wild-type cuttings were treated with 5 JU.M IBA, HRGPnt3 mRNA accumulation was initially lower at d 1 compared with analogous non-IBAtreated cuttings. However, HRGPntS mRNA accumulation began to increase by d 3 to levels above those in non-IBAtreated cuttings, reached a maximum at d 5, and decreased by d 7 (Fig. 3B). The gradual increase followed by a decrease in HRGPntS mRNA accumulation in cuttings is similar to that found in lAA-treated primary roots of tobacco (Vera et al., 1994). In heterozygous rac cuttings treated with 5 JAM IBA, levels of HRGPnt3 mRNA accumulation were low at d 3 and 4 and then increased at d 5 to a level lower than that in IBA-treated wild-type cuttings at d 5 (Fig. 3B). In homozygous rac cuttings treated with 5 /MM IBA for 4 or

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Figure 3. RNA-blot analysis of HRGPniJ mRNA extracted from cuttings of the indicated genotypes treated ( + IBA) or not treated (-IBA) with 5 JIM IBA for the denoted number of days.

7 d, HRGPntS mRNA accumulated to lower levels than in wild-type or heterozygous rac cuttings (Fig. 3B). Because d 7 after placement in IBA was the first point at which early callus formations were histologically detected in homozygous rac cuttings (Lund et al., 1996), homozygous rac cuttings treated with IBA for 7 d likely are developmentally equivalent to the wild-type and to heterozygous rac cuttings treated with IBA for 4 to 5 d. Since HRGPs are members of a gene family, the same blot used to generate the data shown in Figure 3B was reprobed to test whether a higher-stringency wash condition reduced or eliminated the signal. Figure 3C shows that, although the signal was lower using a higher-stringency wash, the mRNA accumulation pattern for each genotype was unaltered. The result shown in Figure 3C would be expected if the bands in Figure 3B represented hybridization specific for HRGPntS mRNA and not for several species of mRNA with sequence similarity to HRGPntS. Figure 4, lane 1, shows that HRGPnt3 mRNA accumulation was very low in unwounded axillary shoots grown in the light. Placement of intact cultures in complete darkness for 24 h increased HRGPnt3 mRNA accumulation (compare lane 2 with lane 1 in Fig. 4). Treatments involving excision of shoots plus defoliation and decapitation followed by culture for 48 h caused a small but consistent increase in HRGPnt3 mRNA accumulation above that induced by darkness alone in intact shoots (compare lanes 3, 4, and 5 with lane 2 in Fig. 4), indicating a wounding effect. Maximal accumulation of HRGPntS mRNA was obtained in defoliated and decapitated shoots that were inverted for 24 h in complete darkness during the latter half of the 48-h culture period (compare lane 6 with all other lanes in Fig. 4). This treatment (Fig. 4, lane 6) is identical to d 1 of our standard root induction protocol minus IBA (see "Materials and Methods"). The results shown in Figure 4 suggest


wild type -IBA Figure 4. RNA-blot analysis ot HKGPntJ mKNA extracted from nonIBA-treated, wild-type axillary stems grown in vitro. Lane 1, Intact, continuous light-grown axillary shoots. When excised from culture for analysis, leaves and apices were removed, and the stems were immediately frozen. Lane 2, Intact, continuous light-grown axillary shoots moved to darkness for 24 h. When excised from culture for analysis, leaves and apices were removed, and the stems were immediately frozen. Lane 3, Axillary shoots excised from culture, leaves and apices removed, grown upright in light for 48 h, and then frozen. Lane 4, Axillary shoots excised from culture, leaves and apices removed, grown upright in light for 24 h, inverted in light for 24 h, and then frozen. Lane 5, Axillary shoots excised from culture, leaves and apices removed, grown upright for 48 h, 24 h of which was in light followed by 24 h in darkness, and then frozen. Lane 6, Axillary shoots excised from culture, leaves and apices removed, grown upright in light for 24 h, inverted in darkness for 24 h, and then frozen.

that there is an additive effect of wounding, inversion, and darkness on maximal accumulation of HRGPnt3 transcripts in wild-type tobacco cuttings.

DISCUSSION iaa4/5 and gh3 Expression Is Not Limiting for Adventitious

Root Initiation in rac Mutants

We conclude that iaa4/5 and gh3 promoter-driven GL/S activity is not blocked by the rac mutation in IBA-treated rac cuttings (Fig. 1, C, D, and G, respectively); this indicates that activation of the iaa4/5 and gh3 promoters is not limiting for adventitious root initiation in rac cuttings. Because the iaa4/5 promoter was found to be specifically sensitive to the presence of the auxin signal and the transcription of iaa4/5 was induced rapidly by auxin, Ballas et al. (1993) concluded that iaa4/5 expression is likely an early responsive component of auxin signal transduction. Our finding that the iaa4/5 promoter is activated both during adventitious root initiation in iaa4/5-GUS transformant cuttings and in proliferating callus derived from phloem parenchyma or inner cortical parenchyma cells in iaa4/5-GUS hemizygous, rac heterozygous cuttings treated with auxin is consistent with a role for iaa4/5 expression in auxininduced cell division but not specifically in adventitious root initiation. In contrast to iaa4/5-GUS activity, histochemical staining and quantitative analyses of gh3-GUS activity indicate that gh3 promoter activation is not correlated with the auxininduced cell divisions that occur during adventitious root initiation in wild-type cuttings or during callus formation in rac cuttings. The elevated levels of gh3-GUS activity in 50 or 100 /J.M IBA-treated cuttings of both genotypes compared with non-IBA-treated controls (Fig. 2) indicate that higher auxin concentrations than those sufficient for adventitious root initiation (5 /J.M IBA) are necessary for in-

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creased activation of the gh3 promoter in both the wildtype and rac backgrounds. gh3 expression may not be specifically correlated with developmental auxin responses such as root initiation but may be generally induced in response to the accumulation of auxin in "sinks" such as phloem parenchyma-derived callus or adventitious root initials and apical meristems. The rac mutant line was originally isolated on the basis of tolerance of mesophyll-derived cell suspensions to concentrations of NAA that were toxic to the wild type (Muller et al., 1985). We have previously shown that 100 JUM IBA is inhibitory to adventitious root formation in wildtype cuttings, suggesting that this IBA concentration may have phytotoxic effects on the cuttings; 500 JXM IBA caused senescence in both wild-type and rac cuttings after 10 d (Lund et al., 1996). Since gh3-GUS activity was higher in the rac background than in the wild type when each was treated with 100 H.M IBA (Fig. 2), elevated gh3 expression may be part of a mechanism that confers to rac tissues an increased ability to tolerate higher concentrations of auxin in comparison to the wild type. The high concentration of auxin (50 JJLM 2,4-D) used in the differential screen of soybean hypocotyls in which the gh cDNAs were isolated (Hagen et al., 1984) may have favored the selection of cDNAs that are induced by relatively high auxin concentrations. Induction of Wild-Type Levels of gh3-GUS Activity in rac Cuttings Does Not Require Higher Auxin Doses

Previous studies concluded that the rac mutation caused a 5- to 10-fold reduction in the auxin sensitivity of cell suspensions and mesophyll-derived protoplasts in comparison to the wild type, as measured by in vitro proliferation and membrane hyperpolarization, respectively (Muller et al., 1985; Barbier-Brygoo et al., 1991). We tested at the gene expression level whether rac caused a similar 5- to 10-fold reduction in auxin sensitivity in rac cuttings, using gh3-GUS activity as a molecular indicator of auxin sensitivity. We used gh3 for this purpose instead of iaa4/5, because gh3-GUS activity was never detected in histochemical staining analyses of non-auxin-treated gh3-GUS cuttings, thus providing us with a negative control. Based on a fluorometric analysis of gh3-GUS activity in gh3-GUS hemizygous cuttings compared with gh3-GUS hemizygous, rac heterozygous cuttings treated with auxin for 1 d (Fig. 2), we conclude that the rac mutation does not cause any reduction in IBA sensitivity at the level otgh3-GUS activity. Since callus formation does not occur in response to 1 d of auxin treatment in heterozygous rac cuttings (Lund et al., 1996), it is not possible that the higher gh3-GUS activity detected in gh3-GUS hemizygous, rac heterozygous cuttings treated with 100 /J,M IBA compared with analogous gh3-GUS hemizygous cuttings is correlated with greater amounts of cell division in the rac background than that in the wild-type background. Thus, the data in Figure 2 do not support the hypothesis that the rac mutation causes a general reduction in auxin sensitivity; however, these data (Fig. 2) do not preclude the possibility that a reduction in auxin sensitivity due to rac may affect other auxin signal

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transduction pathways that lead to adventitious root initiation, not including gh3. HRCPnt3 Expression 1s Developmentally and Environmentally Regulated in Tobacco Cuttings

GUS analyses (Fig. 1, H-L) and RNA-blot analyses (Fig. 3) indicate that the rac mutation affects auxin induction of HRGPnt3 expression by inhibiting HRGPnt3 promoterdriven GUS activity and by reducing HRGPnt3 mRNA accumulation in IBA-treated rac cuttings. However, our results also suggest that the factors that regulate the accumulation of HRGPnt3 transcripts may not be identical to those that induce activation of the upstream promoter sequences within 1.3 kb of the HRGPnt3-coding region (Keller and Lamb, 1989). This possibility is based on the differences in the data generated by the HRGPnt3-GUS analyses and the RNA-blot analysis shown in Figure 4. We conclude that the rac mutation acts both to block the developmental activation of the HRGPnt3 promoter that normally occurs during adventitious root initiation and to decrease the HRGPnt3 mRNA accumulation that is due to induction by some other factor(s), possibly wounding and/or ethylene. This conclusion is based on two lines of evidence. First, HRGPnt3-GUS analyses in the wild-type and rac backgrounds indicated that activation of HRGPnt3 promoter sequences within 1.3 kb upstream of the coding region occurs specifically during auxin-induced adventitious root initiation and primordia development. HRGPnt3-GUS activity was detectable very early in adventitious root initials (Fig. lH), in adventitious root meristems (Fig. lI), and in the most apical cells of adventitious root primordia (Fig. 1J)but never in any other tissue types in wild-type cuttings. HRGPnt3-GUS activity was not detectable in any tissue types in 47 of 48 HRGPnt3-GUS hemizygous, rac heterozygous cuttings, including phloem parenchyma- or inner cortical parenchyma-derived callus (Fig. 1, K and L). The detection of histochemical staining for HRGPnt3-GUS activity routinely in adventitious root initials and very rarely (less than 5%) in rac callus formations, both of which are derived from phloem parenchyma or inner cortical parenchyma cells, supports our hypothesis that differential gene expression occurs during auxininduced adventitious root initiation in the wild-type and during callus formation in rac cuttings. Second, RNA-blot analyses (Figs. 3 and 4) revealed that HRGPnt3 mRNA accumulation is not limited to auxintreated wild-type cuttings, even though our analyses of HRGPnt3-GUS activity indicated that activation of the HRGPnt3 promoter occurs only during adventitious root initiation and development in wild-type cuttings. The differences in results from the GUS and mRNA-blot analyses of HRGPnt3 expression in tobacco cuttings could be explained if the 1.3-kb 5' upstream region of the HRGPnt3 promoter-GUS construct lacks sequences responsive to some component of our root induction protocol other than that which induces adventitious root initiation. Wycoff et al. (1995) demonstrated that the promoter of a bean (P. vdgaris) HRGP-encoding gene, HRGP4.1, was activated by

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environmental cues, such as mechanical wounding and vira1 infection, and also showed a distinct developmental pattern of expression in stems and primary roots. Our data suggest that HRGPnt3 expression in tobacco cuttings, like HRGP4.1 in bean, is perhaps also responsive to environmental stimuli (e.g. wounding, darkness, orientation, and / or ethylene), as well as being a component of a developmental process (organized cell divisions during adventitious root initiation). Wound inducibility has been shown for many HRGPencoding genes (Chen and Varner, 1985; Showalter and Varner, 1989; Ebener et al., 1993). Figure 4 shows that each of the manipulations performed in our root induction protoco1 (excision, inversion, and placement of cuttings in total darkness) positively regulates levels of HRGPnt3 mRNA accumulation above those detectable in intact axillary stems grown in constant light. Figure 3A shows that after the manipulations in our root induction protocol were performed the levels of HRGPnt3 mRNA accumulation were elevated and remained relatively constant during the following 5 d on basal media. Although Keller and Lamb (1989) did not detect an increase in HRGPnt3 mRNA accumulation in response to wounding, our experimental system involving wounding, change in orientation, and darkness was sufficient to induce HRGPnt3 mRNA accumulation. Wounding and inverted orientation of explants in vitro have been found to induce ethylene evolution (Hyodo, 1991; Abeles et al., 1992), and ethylene has been shown to induce HRGP gene expression in many species (Showalter and Varner, 1989). Exogenous auxin is also known to induce ethylene biosynthesis (Mattoo and White, 1991); therefore, an elevation of HRGPnt3 mRNA levels in auxintreated wild-type cuttings (Fig. 3, B and C) above nonauxin-treated wild-type controls is consistent with HRGPnt3 mRNA accumulation being a response to an increase in ethylene over time. Furthermore, since the rac mutation causes a reduction in some types of auxin responses (e.g. cell-suspension proliferation, membrane hyperpolarization of protoplasts), it is plausible that HRGPnt3 mRNA accumulation was greatly reduced and substantially delayed in auxin-treated, rac homozygous cuttings and somewhat decreased and slightly retarded in auxin-treated, rac heterozygous cuttings. Greater HRGPnt3 mRNA accumulation in rac heterozygotes than in homozygotes is consistent with the rac copy number effect that is correlated with the inhibition of primary root growth shortly after germination of rac seedlings and reduced organization of auxin-induced callus in auxintreated rac cuttings (Lund et al., 1996). We propose, then, that environmentally induced HRGPnt3 mRNA accumulation occurs concurrently with adventitious rooting-specific HRGPnt3 mRNA accumulation and that the environmentally induced HRGPnt3 mRNAs overlap and obviate detection via RNA-blot analysis of HRGPnt3 mRNA accumulation that is specific for adventitious root initiation and development. Future studies with HRGPnt3 in tobacco cuttings should focus both on the regulatory control(s) of the HRGPnt3 promoter during adventitious root initiation and

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on the elucidation of the environmental control of HRGPnf3 mRNA accumulation.

steady-state level of auxin sensitivity in stem phloem parenchyma 01inner cortical parenchyma cell

remains level of auxin belowsensitivity minimum

Modeling the lnteraction of the rac Mutation with iaa4/5, gh3, and HRGPnt3 Expression during Adventitious Root lnitiation

If adventitious root initiation is regarded as an organized form of cell division, and auxin-induced adventitious root initiation occurs via auxin reception followed by transduction of the auxin signal, our data place iaa415 and gh3 expression upstream from rac and HRGPnf3 expression separate from rac in a linear model (Fig. 5A) or rac upstream from HRGPnt3 but on a bifurcated pathway separate from iaa415 and gh3 (Fig. 58). In both models shown in Figure 5, temporal regulation of iaa415 and gh3 occurs prior to that of rac and HRGPnt3, and rac is assumed to be involved in a signal transduction pathway that is specific for adventitious root initiation in tobacco cuttings. Alternatively, in the model shown in Figure 6, rac is assumed to be involved in modifying levels of auxin sensitivity. If the rac mutation represses increases in auxin sensitivity that normally occur in the wild type, and a higher minimum threshold(s) of auxin sensitivity is required for adventitious root initiation than for cell division, then lowered auxin sensitivity in rac cuttings may be sufficient for iaa415 and gh3 expression and cell division, but not for an adventitious rooting-specific induction of HRGPnt3 expression and adventitious root initiation (Fig. 6). Thus, rac action precedes iaa415, gh3, and HRGPnf3 expression, but only affects the expression of HRGPn f3, which is only expressed during adventitious root initiation. If gh3 expression is specifically responsive to increased auxin concentrations but not affected by rac, as was discussed earlier, then gh3 may be part of a separate pathway

A

B

AUXIN RECEPTION

AUXIN RECEPTION

t

t t

L R4c

‘root initiation’ threshold

AUXIN RECEPTION

unorganized cell divisions (iua4/5,gh3)

level of auxin sensitivity increased above minimum ‘root initiation’threshold AUXIN RECEPTION

organized cell divisions + AR1 (iaa4/5,gh3) (root-specificHRGPnt3)

Figure 6. Model in which rac modifies levels of auxin sensitivity. The rac mutation represses an increase in auxin sensitivity that is required for auxin signal transduction specifically for the organization of cell divisions, including HRGPnt3 expression, during adventitious root initiation. iaa4/5 and gh3 are expressed in both the wild-type and rac genotypes. The arrows indicate hypothetical steps in auxin signal transduction, the actual number of which is unknown. ARI, Adventitious root initiation.

than that involved in cell division or adventitious root initiation (not shown in Figs. 5 or 6). The suggestion from a11 three models (Figs. 5 and 6) is that, although early, auxin-responsive gene expression is likely a component of adventitious root initiation, it is doubtful that the expression of genes such as iaa415 and gh3 have crucial roles in the organization of the cell divisions required for adventitious root initiation. Conversely, it is plausible that genes such as HXGPnt3 that encode structural proteins may be up-regulated prior to, or coincident with, the organization of cell divisions that occur during adventitious root initiation and, thus, may be a factor in the determination of the competence for adventitious root initiation in tobacco cuttings. Since the rac mutation has a major phenotypic effect of blocking adventitious root initiation prior to the first organized cell divisions, it is likely that ruc has an important regulatory role during the early stages of adventitious root initiation in phloem parenchyma o r inner cortical parenchyma cells in tobacco cuttings. ACKNOWLEDCMENTS

‘t

i

(root-specific HRGPnt3) organized cell divisions + AR1

i

cell divisions

(iaa4/5,gh3)

(root-specific HRGPnt3) organized cell divisions ARI +

Figure 5. Linear (A) and bifurcation (6) models of the hypothetical mode of rac action on auxin signal transduction. In both models, rac is assumed to be a factor that can specifically modify downstream auxin signal transduction for adventitious root initiation. The rac mutation specifically blocks pathways directed toward the organization of cell divisions that are required for adventitious root initiation, including HRGPnt3 but not iaa4/5 or gh3 expression. The arrows indicate hypothetical steps in auxin signal transduction, the actual number of which is unknown. ARI, Adventitious root initiation.

We wish to thank Dr. M. Caboche for his gift of wild-type and homozygous rac seeds. We thank Dr. C. Lamb for providing HRGPnt3 genomic DNA and HRGPrlt3-GUS transformant tobacco seeds. We thank Dr. A. Theologis for providing iaa415-GUS transformant tobacco seeds. We thank Dr. T. Guilfoyle for providing gh3-GUS transformant tobacco seeds. We also thank Drs. D. Biesboer, G. Gardner, G. Howe, and N. Olszewski for their helpful comments when reviewing this manuscript. We thank Dr. E. Zimmermann for assistance with the RNA-blot analyses. Received February 18, 1997; accepted May 6, 1997. Copyright Clearance Center: 0032-0889/97/114/1197/10. LITERATURE CITED

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eds, The Plant Hormone Ethylene. CRC Press, Boca Raton, FL, pp 43-64 Jarvis BC (1986) Endogenous control of adventitious rooting in non-woody cuttings. In MB Jackson, ed, New Root Formation in Plants and Cuttings. Martinus Nijhoff, Dordrecht, The Netherlands, p p 191-222 Keller B, Lamb CJ (1989) Specific expression of a nove1 cell wall hydroxyproline-rich glycoprotein gene in lateral root initiation. Genes Dev 3: 1639-1646 Lovell PH, White J (1986) Anatomical changes during adventitious root formation. In MB Jackson, ed, New Root Formation in Plants and Cuttings. Martinus Nijhoff, Dordrecht, The Netherlands, pp 111-140 Lund ST, Smith AG, Hackett WP (1996) Cuttings of a tobacco mutant, rac, undergo cell divisions but do not initiate adventitious roots in response to exogenous auxin. Physiol Plant 97: 372-380 Mattoo AK, White WB (1991)Regulation of ethylene biosynthesis. In AK Mattoo, JC Suttle, eds, The Plant Hormone Ethylene. CRC Press, Boca Raton, FL, pp 21-42 Muller JF, Goujaud J, Caboche M (1985) Isolation in uitro of naphthaleneacetic acid-tolerant mutants of Nicotiana tabacum which are impaired in root morphogenesis. Mo1 Gen Genet 199: 194-200 Pelese F, Megnegneau B, Sotta B, Sossountzov L, Caboche M, Miginiac E (1989) Hormonal characterization of a non-rooting naphthaleneacetic acid-tolerant tobacco mutant by an immunoenzymatic method. Plant Physiol 89: 86-92 Rochester DE, Winter JA, Shah D M (1986) The structure and expression of maize genes encoding the major heat shock protein, hsp70. EMBO J 5: 451-458 Showalter AM, Varner JE (1989) Plant hydroxyproline-rich glycoproteins. In PK Stumpf, EE Conn, eds, The Biochemistry of Plants, Vol 15. Academic Press, New York, pp 485-520 Theologis A, Huynh TV, Davis RW (1985) Rapid induction of specific mRNAs by auxin in pea epicotyl tissue. J Mo1 Biol 183: 53-68 Vera P, Lamb CJ, Doerner PW (1994) Cell-cycle regulation of hydroxyproline-rich glycoprotein HRGPnt3 gene expression during the initiation of lateral root meristems. Plant J 6: 717-727 Wilson LG, Fry JC (1986)Extensin-a major cell wall glycoprotein. Plant Cell Environ 9: 239-260 Wycoff KL, Powell PA, Gonzales RA, Corbin DR, Lamb CJ, Dixon RA (1995)Stress activation of a bean hydroxyproline-rich glycoprotein promoter is superimposed on a pattern of tissuespecific developmental expression. Plant Physiol 109: 41-52