Role of AUX1 in the control of organ identity during in vitro organogenesis and in mediating tissue speciWc auxin and cytokinin interaction in Arabidopsis
Abstract Classic plant tissue culture experiments haveshown that exposure of cell culture to a high auxin to cyto-kinin ratio promotes root formation and a low auxin tocytokinin ratio leads to shoot regeneration. It has beenwidely accepted that auxin and cytokinin play an antagonis-tic role in the control of organ identities during organogene-sis in vitro. Since the auxin level is highly elevated in theshoot meristem tissues, it is unclear how a low auxin tocytokinin ratio promotes the regeneration of shoots. Toidentify genes mediating the cytokinin and auxin interac-tion during organogenesis in vitro, three allelic mutants thatdisplay root instead of shoot regeneration in response to alow auxin to cytokinin ratio are identiWed using a forwardgenetic approach in Arabidopsis. Molecular characteriza-tion shows that the mutations disrupt the AUX1 gene, whichhas been reported to regulate auxin inXux in plants. Mean-while, we Wnd that cytokinin substantially stimulates auxinaccumulation and redistribution in calli and some speciWctissues of Arabidopsis seedlings. In the aux1 mutants, thecytokinin regulated auxin accumulation and redistributionis substantially reduced in both calli and speciWc tissues ofyoung seedlings. Our results suggest that auxin elevationand other changes stimulated by cytokinin, instead of lowauxin or exogenous auxin directly applied, is essential forshoot regeneration.
The interaction between auxin and cytokinin plays anessential role in a wide range of plant growth and develop-mental processes. Studies on whole plants and excised tis-sues have demonstrated the existence of synergistic,antagonistic, and additive interactions of these two hor-mones, dependent on the plant species and tissues used(Coenen and Lomax 1997; Swarup et al. 2002; Hartig andBeck 2006). Classic plant tissue culture experiments initi-ated in tobacco pith tissue culture have demonstrated thatthe ratio of auxin to cytokinin is critical in cell fate determi-nation in vitro (Skoog and Miller 1957). Exposing calluscultures to a high auxin to cytokinin ratio promotes the for-mation of roots, whereas a low auxin to cytokinin ratioresults in generation of shoots. In addition, application ofthese two hormones in ratios of intermediate range pro-motes callus proliferation. The device of adjusting auxin tocytokinin ratio to induce the production of calli andregulate shoot and root regeneration is now a well-established practice for a variety of plants in both researchand industry.
Another classic example of auxin and cytokinin interac-tion is apical dominance. Studies have shown that auxinproduced at the apex represses the outgrowth of lateral
Electronic supplementary material The online version of this article (doi:10.1007/s00425-008-0846-6) contains supplementary material, which is available to authorized users.
A. Kakani · G. Li · Z. Peng (&)Department of Biochemistry and Molecular Biology, Mississippi State University, Mail box 9650, Starkville, MS 39762, USAe-mail: [email protected]; [email protected]
buds. In contrast, cytokinin applied to lateral buds promotestheir release from apical dominance. Further research hasdemonstrated that removal of the endogenous auxin sourcevia decapitation results in up to 40-fold increase of cytoki-nin in xylem exudates (Bangerth 1994; Li et al. 1995).Recent studies in Arabidopsis have shown that auxin medi-ates a very rapid negative control of the cytokinin pool bymainly suppressing its biosynthesis via the isopentenylade-nosine-5�-monophosphate independent pathway (Nord-strom et al. 2004). In addition, auxin has been found tostimulate both oxidative breakdown and glucosylation ofactive cytokinins in a tissue-dependent manner (Coenenand Lomax 1997). On the other hand, an increase in freeIAA (active form) has been observed both in cytokininoverproducing lines of Nicotiana glutinosa transformedwith the bacterial cytokinin biosynthesis gene ipt (isopente-nyl transferase) and in maize and pea treated with exoge-nously applied cytokinin (Binns et al. 1987; Bourquin andPilet 1990; Besrtell and Eliasson 1992). Although itremains to be proven, a putative mechanism is cytokinininhibition of the enzymes that conjugate free IAA (Yip andYang 1986). Meanwhile, it has been reported that cytokininoverexpression can lead to down regulation of the IAA poolin tobacco (Eklöf et al. 1997). The discrepancy suggeststhat further studies are required in this Weld to gain a com-plete picture of the auxin–cytokinin interaction.
The auxin and cytokinin control of cell division inundiVerentiated cells presents a good example of the syner-gistic interaction of these two hormones. Studies haveshown that auxin increases the expression of a CDC2 classof cyclin-dependent kinases in tobacco pith explants. Whilethe expression of the CDC2 like kinase is induced inresponse to auxin, its catalytic activity is increased onlywhen the explants are also treated with cytokinin (Johnet al. 1993). Cyclin �3 is a D cyclin whose expression ishighly dependent on cytokinin (Soni et al. 1995). It isbelieved that �3 may be the factor required to activate theCDC2 kinase; therefore, the auxin and cytokinin synergisti-cally control the expression and activity of the CDC2 likekinase, which renders the cell competent for cell division(Coenen and Lomax 1997).
In contrast to synergistic control of cell division in cal-lus, auxin and cytokinin demonstrate antagonistic eVects onthe initiation of lateral root primordia. The root primordiumis derived from pericycle cells opposite the xylem poles ofthe root vasculature, where auxin promotes while cytokinininhibits cell division. It has been shown that auxin increasesthe expression of CDC2 like protein in the extracts of pearoots, but cytokinin reduces the levels of the CDC2 likekinase (John et al. 1993; Coenen and Lomax 1997). Com-pared with the observations of cell division in callus, theresults above suggest that the eVect of auxin and cytokinininteraction is highly tissue speciWc. Recently, Müller and
Sheen reported that cytokinin and auxin interact antagonis-tically in root stem-cell speciWcation in early embryogene-sis (Muller and Sheen 2008). However, how cytokinin andauxin interact during shoot stem-cell speciWcation has notbeen reported.
The auxin eZux and inXux complexes have been shownto be essential to pattern formation during embryogenesisand development in plants (Review, De Smet and Jurgens2007; Lucas et al. 2008). AUX 1 is an amino acid perme-ase-like membrane protein initially identiWed via screeningof auxin resistant mutants (Bennett et al. 1996). AUX1 hasbeen shown to act as an auxin inXux facilitator. AUX1 reg-ulates gravitropic curvature by acting in unison with theauxin eZux carrier to co-ordinate the localized redistribu-tion of auxin within the Arabidopsis root apex (Marchantet al. 1999). Mutations in the AUX1 gene also confer resis-tance to ethylene and cytokinin (Timpte et al. 1995). Inaddition, it has been reported that subcellular traYcking ofAUX1 and PIN1 use two distinct pathways (Kleine-Vehnet al. 2006). The binding activity of AUX1 to IAA has beenextensively studied recently (Carrier et al. 2008). In addi-tion, an AUX1/LAX-type gene, PaLAX1 from a wild cherrytree (Prunus avium), has also been shown to promoteuptake of auxin into cells (Hoyerova et al. 2008). Enhancedauxin uptake may result in intense auxin Xow and thus con-nect to pattern formation.
The essential role of the interaction between auxin andcytokinin in the control of organogenesis in vitro has beendiscovered over half a century. Nevertheless, the process ormechanism mediating the interaction between auxin andcytokinin has not been identiWed. In this article, we reportthe isolation, characterization, and molecular cloning ofthree allelic mutants which display abnormal organ identi-ties in response to the ratio of auxin to cytokinin duringorganogenesis in vitro. Molecular cloning of the mutatedgenes demonstrated that the mutation occurred within theaux1locus. We further demonstrated that cytokinin substan-tially stimulated auxin accumulation in calli and speciWc tis-sues of young seedlings and AUX1 plays an essential role inregulating cytokinin controlled auxin elevation and redistri-bution in calli and the speciWc tissues of young seedlings.
Materials and methods
Arabidopsis mutant screening
Arabidopsis seeds were sterilized and placed on Gamborg’sB5 medium (pH 5.7) supplemented with 2,4-Dichlorophe-noxyacetic acid (2,4-D) and Kinetin (KIN) as indicated in thetext and 1% sucrose and 0.9% agar. After 5 days of coldtreatment at 4°C, the Petri dishes were transferred to a 22°Cgrowth chamber for callus induction under 16 h of dim light
123
Planta (2009) 229:645–657 647
(50 �mol m¡2s¡2) and 8 h of dark. One-month-old calli weretransferred to regeneration media containing 0.5 mg l¡1
(2.8 �M) IAA (indole-3-acetic acid) and 0.5 mg l¡1 (2.2 �M)6-benzyladenine (6-BA). The images of the regeneratedorgans were recorded after 1 month of culture.
The isolated putative mutants were crossed with wildtype. The phenotype in F1 and F2 generations were exam-ined and recorded to test if a mutation was recessive ordominant. Co-segregation of the phenotype with T-DNAwas examined by PCR ampliWcation of a DNA fragmentwithin the T-DNA. The primers used are: Primer 1: 5�
CAACTTTATCCGCCTCC 3�; Primer 2: 5� TGTCGC-CCTTATTCCCT 3�.
DR5:GUS and DR5:GFP transgenic lines in aux1-7 background
The Arabidopsis seeds of DR5:GUS transgenic line inwild-type background were kindly provided by Dr. ThomasJ. Guilfoyle (Ulmasov et al. 1997). The pollen from thisline was used to pollinate aux1-7 homozygous mutantordered from the Arabidopsis Biotechnology ResearchCenter. F3 progeny homozygous for both aux1-7 andDR5:GUS reporter were used for experiments. TheDR5:GFP transgenic line (Ottenschlager et al. 2003) in theaux1-7 background was generated in the same way.
IAA2:GUS, AUX1:GUS, and AUX1:AUX1-YFP transgenic lines
The IAA2:GUS transgenic lines in wild type (Ws) andaux1-100 background (Swarup et al. 2001), the AUX1pro-moter:uidA GUS transgenic line (Marchant et al. 1999),and AUX1pro::AUX1-114-YFP transgenic line (Swarupet al. 2004) were kindly provided by Dr. Malcolm Bennett.
Histochemical analyses of GUS activity
The histochemical stain of GUS was carried out as reported bySessions et al. (1999) except without sectioning. BrieXy, plantmaterials were stained in GUS staining solution [100 mMsodium phosphate at pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 1 mM potassium ferricyanide, 1 mM potassium ferrocya-nide and 1 mg ml¡1 of X-Gluc (Gold Bio Technology, Inc.)].The samples were incubated at 37°C overnight after beingplaced under a vacuum for 10 min in a desiccator. The stain-ing solution was removed and the tissues were cleaned byincubating with several rinses of 70% ethanol.
Microscopy
Fluorescent images were acquired using a Zeiss LSM 510Confocal Laser Scanning Microscope (Carl Zeiss Microi-
maging, Inc.) with an Inverted Zeiss Axiovert 200 M lightmicroscope and a Plan Apochromat 5X/0.16 NA objectivelens. For the observation of YFP, an EYFP LP 530 Wlter setwas used with Excitation and Emission wavelengths of 514and 530 nm, respectively. A 512 £ 512-pixel Scan-Formatwas used to capture the images. For GFP observation, anEGFP LP505 Wlter set was used with Excitation and Emis-sion wavelengths of 488 and 505 nm, respectively. A512 £ 512-pixel Scan-Format was used to capture images.The GUS images were acquired using a Zeiss Stemi SV11(Apo) light microscope.
Plant growth and phytohormone application for RNA extraction
Arabidopsis (Columbia) and mutant aux1-7 seeds wereplated on B5 medium for germination at 22°C under 16 h oflight and 8 h of darkness. Auxin and cytokinin treatmentswere initiated by submerging 12-day-old seedlings in B5liquid medium containing 2.2 mg l¡1 (10 �M) 2,4-D or3.0 mg l¡1 (14 �M) kinetin for 5 min. The liquid wasdrained and kept for 1 h at 22°C. Tissues were collectedand snap frozen with liquid nitrogen.
Total RNA isolation and cDNA synthesis
Total RNA was prepared using a RNeasy Plant Mini Kit(QIAGEN), and quantiWed using a Nanodrop ND-1000Spectrophotometer (NanoDrop Technologies, Inc.). TotalRNA (5 �g) was reverse transcribed using SuperScript™First-Strand Synthesis System for RT-PCR following man-ufacturer’s instructions (Invitrogen).
Real-Time quantitative PCR analyses
Arabidopsis auxin inducible genes IAA3 (At1g04240), IAA6(At1g52830), IAA14 (At4g14550), and IAA17 (At1g04250)and cytokinin inducible genes ACR4 (At1g69040) and AP2domain-containing transcription factor (At4g23750) wereused for Real-Time PCR analysis (Himanen et al. 2004;Kiba et al. 2005). The transcript levels for these genes werequantiWed using the iQ™ SYBR® Green Supermix Kitfollowing manufacturer’s instructions (BIO-RAD). Tonormalize the variations, the stably expressed housekeepinggene Ubiquitin was used as an internal control. Gene-speciWc primer pairs were designed against each gene withamplicons ranging from 72 to 128 bp (IAA3, F: 5�-GGTGCACCATACTTGAGGAA-3�, R: 5�- TCCCACAGAGAATTTGAACATC-3�; IAA6, F: 5�-AGGTCTAGCACTCGAGATCACA-3�, R: 5�-TTCTTCTTACTCGATCCGCATA-3�;IAA14, F: 5�-AAGCAGAGGAGGCAATGAGT-3�, R: 5�-TCCATGGAAACCTTCACAAA-3�; IAA17, F: 5�-CATACCGGAAGAACGTGATG-3�, R: 5�-GCTCCGTCCATTG
123
648 Planta (2009) 229:645–657
ATACCTT-3�; ACT domain-containing protein (ACR4),F: 5�-TAACGGTCACAAGAGCTGAAGT-3�, R: 5�-TTGGCCTATGGTTTGTCTTATG-3�; AP2 domain-containingtranscription factor, F: 5�-CCAGTGACGACGAAGAAGAA-3�, R: 5�-CGACTCCATTTGACCACAAC-3�; Ubiqui-tin, F: 5�-GAGTCCTCAGACACCATTGACAAC-3�, R:5�-GTGCTCTCCTTCTGGATGTTGTAG-3�). The Real-Time quantitative PCR reactions were performed in aniCycler iQ™ Real-Time PCR Detection System (BIO-RAD). The thermal proWle was 95°C, 5 min, 1 cycle; 95°C,10 s, 60°C, 30 s, 55 cycle. Cycle threshold (Ct) values forthe PCR product growth curve were determined based onthree independent replicates for each biological sample.The corresponding number of transcripts was deduced fromindividual standard curves for each gene, based on quantiW-cation of gene-speciWc PCR products with Nanodrop ND-1000 Spectrophotometer (NanoDrop Technologies, Inc.) ina series of dilutions.
Results
Optimizing hormone combinations for regeneration mutant screening in Arabidopsis
To identify the cellular component that regulates organidentity during organogenesis in vitro, we tested diVerenthormone combinations in callus induction and organ regen-eration to determine optimal conditions for mutant screen-ing in Arabidopsis. Callus is very sensitive toenvironmental stimuli in addition to hormones. Environ-mental factors often have an irreversible impact on theregeneration capability. For example, prior exposure tostresses (Ikeda-Iwai et al. 2003) will induce embryogenesisin Arabidopsis. Other environmental factors, such as lightintensity, also aVect organ regeneration in plants (Lillo1989). These reports suggest that reducing the time of cal-lus culture, thus the exposure of the cell culture to environ-mental factors, may facilitate the identiWcation of cellularcomponent(s) mediating hormone regulation in organogen-esis. To reduce the time of cell culture, we directly inducedcalli with diVerent ratios of auxin to cytokinin and exam-ined their eVect on organ regeneration. In addition, we usedintact germinating seeds (Columbia) to induce calli to avoidinterference from wounds, which is known to aVect callusformation. Since calli were produced from roots Wrst whenintact seeds were used, only the calli generated from roottissues were selected for regeneration studies in this report.All calli induced with diVerent hormones and their combi-nations were transferred to a common regeneration mediumfor further observation of development. We found thatwhen a Gamberg’s B5 organic medium supplemented with0.5 mg l¡1 (2.8 �M) IAA and 0.5 mg l¡1 (2.2 �M) 6-BA
was used as the common organ regeneration medium(ORM1), the identities of regenerated organs were solelydetermined by the hormone used in the callus induction andthe results were highly repeatable (Supplemental Fig. 1).Calli induced in a medium with 0.5 mg l¡1 (2.2 �M) 2,4-Dregenerated only roots after being transferred to the ORM1medium (Supplemental Fig. 1). In contrast, the calliinduced in media with 0.05 mg l¡1 (0.22 �M) 2,4-Dplus1.0 mg l¡1 (4.6 �M) KIN or 2.0 mg l¡1 (9.3 �M) KINregenerated only shoots after being transferred to ORM1medium (Supplemental Fig. 1). In addition, calli inducedby 0.25 mg l¡1 (1.1 �M) 2,4-D plus 2.0 mg l¡1 (9.3 �M)KIN also regenerated only shoots. Further, most of thecalli induced in media with 0.25 mg l¡1 (1.1 �M) 2,4-D + 0.2 mg l¡1 (0. 9 �M) KIN; 0.25 mg l¡1 (1.1 �M) 2,4-D + 1.0 mg l¡1 (4.6 �M) KIN; 0.5 mg l¡1 (2.2 �M) 2,4-D +0.2 mg l¡1 (0. 9 �M) KIN; 0.5 mg l¡1 (2.2 �M) 2,4-D +1.0 mg l¡1 (4.6 �M) KIN, and 0.5 mg l¡1 (2.2 �M) 2,4-D + 2.0 mg l¡1 (9.3 �M) KIN continued callus proliferationafter being transferred to the ORM1 medium for 1 month.These observations were consistent with the concept that ahigh auxin/cytokinin ratio led to root regeneration and alow auxin/cytokinin ratio led to shoot regeneration (Skoogand Miller 1957). The established correlation between thehormones used for callus induction and culture and theidentities of the regenerated organs enabled us to carry outa mutant screening in Arabidopsis. Highly consistentresults are the advantage of this system.
Screening and characterization of mutants with an altered response to auxin/cytokinin ratios
We were interested in genes in which mutations aVect theidentities of regenerated organs, including mutants thatregenerate roots in media for shoot regeneration and viceversa. We expected that mutants with such a phenotypewere caused by disruption of genes encoding products thatconfer organ identity in vitro. Since our experiments hadshown that calli produced in medium with 0.05 mg l¡1 2,4-D plus 2.0 mg l¡1 KIN (CM1 medium) only regeneratedshoots in ORM1 medium, our original plan was to inducecalli in the CM1 medium and search for mutants that onlyregenerated roots after being transferred to the ORM1medium. The Arabidopsis T-DNA activation mutant seedswere used for screening (Weigel et al. 2000). Among theover 50,000 seeds screened, we found three plants thatgrew healthy roots instead of producing calli in the CM1medium, suggesting that the hormone concentrations weused were too low for these plants. We transferred theseseedlings into soil for seed production and re-screened theproduced seeds in a medium with higher auxin (CM2medium, 0.25 mg l¡1 2,4-D and 2.0 mg l¡1 KIN). Mean-while, we also re-examined the phenotype of the regener-
123
Planta (2009) 229:645–657 649
ated seeds in the CM1 medium. The progeny of the threeplants still grew healthy roots instead of producing calli inthe CM1 medium. However, all of them produced calli inCM2, suggesting that the three plants required a higherauxin level for callus induction (Fig. 1a). Interestingly, theinduced calli regenerated only roots instead of shoots whentransferred to ORM1 medium after 1 month of culture(Fig. 1b). In addition, the mutant calli regenerated rootswhen induced in media that resulted in callus proliferationin wild type (compare Supplemental Fig. 1 and Fig. 1c).Further, some belt-shaped root structures were observed inmedia that usually led to root regeneration in wild type(Fig. 1c, see arrows). These results suggested that the mutantcalli displayed strong root regeneration capability in mediadesigned for shoot regeneration and callus proliferation.
Genetic analyses found that these three mutants wereallelic to each other and recessive although they werescreened using the T-DNA activation mutant seed stock,suggesting that the gene was disrupted instead of beingactivated in these mutants. PCR analyses of a T-DNA frag-ment in the F2 populations revealed that all three mutantsco-segregated with T-DNA in over 50 mutant plants testedfor each of the 3 mutants. In contrast, T-DNA was notdetected in about one-third of the wild-type plants, suggest-ing that these mutants harbored only one T-DNA insertion.To further characterize these mutants, we examined theirgrowth response to auxin and cytokinin, respectively. Themutant seeds were germinated in media with diVerent con-centrations of 2,4-D and KIN, respectively. The root lengthof 10-day-old seedlings was measured and compared withthe wild-type control. The results showed that mutant-1 andmutant-2 were insensitive to both auxin and cytokinin(Fig. 2). In medium with 0.04 mg l¡1 2,4-D, the root lengthof wild-type seedlings was about 8% of the untreated con-trol while the root length of mutant-1 and mutant-2 wasabout 65 and 72% of the length of the control roots, respec-
tively (Fig. 2a). When KIN concentration was higher than1.0 mg l¡1, the roots of mutant-1 and mutant-2 seedlingswere signiWcantly longer than the wild-type roots and thediVerence between wild type and mutants increased athigher KIN concentrations (Fig. 2b), suggesting that themutated gene might play a role in the cytokinin responsepathway in addition to the auxin response pathway. Thehormone response curves of mutant-3 were almost identicalto mutant-2; therefore, the results were not presented.
Fig. 1 Phenotype of isolated mutants in callus induction and regener-ation media. a Mutant-1 phenotype in callus induction medium CM1(0.05 mg l¡1 2,4-D and 2.0 mg l¡1 KIN) and CM2 (0.25 mg l¡1 2,4-Dand 2.0 mg l¡1 KIN). The mutant-1 and wild type (Columbia) are indi-cated in the Wgure, respectively. Mutant-1 did not form calli in CM1medium but formed calli in CM2 medium (see calli formed in the rootregion). b Mutant phenotype in organ regeneration. The calli were in-duced in medium containing 0.25 mg l¡1 2,4-D and 2.0 mg l¡1 KIN(CM2) and then transferred to the regeneration medium with0.5 mg l¡1 IAA and 0.5 mg l¡1 6-BA. The phenotype was recorded af-ter 1 month of regeneration. The wild type and mutants are indicated inthe Wgure, respectively. c mutant-1 regeneration response to diVerentcallus induction media. Sterilized Arabidopsis seeds were used to in-duce calli in media containing 2,4-D and KIN in concentrations as indi-cated in the Wgure. One-month-old calli were then transferred to theregeneration medium containing 0.5 mg l¡1 IAA and 0.5 mg l¡1 6-BAfor organ regeneration. The organ regeneration images were recordedafter 1 month of induction. The belt-shaped organs are marked with ar-row heads
�
123
650 Planta (2009) 229:645–657
Molecular cloning of the mutated gene
After we conWrmed that the mutant phenotype co-segre-gated with T-DNA in these mutants, we cloned the mutatedgenes via plasmid rescue. A 540-bp genomic DNA fragmentof mutant-1 was rescued and sequenced after digesting thegenomic DNA with E coR I and two identical 125-bpgenomic DNA fragments were rescued and sequenced afterdigesting the genomic DNA of mutant-2 and mutant-3 withSpe I. The results indicated that mutant-2 and mutant-3were probably the progeny of the same mutant, because theseeds obtained from ABRC were a mixture of 100 lines.DNA sequencing analysis and Blast search indicated thatthe mutated sites were located within the previously identi-Wed AUX1 gene. The T-DNAs in the mutant-1 and mutant-2/mutant-3 were inserted in the seventh exon of the AUX1
gene, about 34 amino acids apart from each other as shownin Fig. 3. The disruption of the AUX1 gene was consistentwith the recessive nature of these mutants. AUX1 is a puta-tive auxin inXux facilitator localized on the plasma mem-brane with similarities to plant amino acid permeases(Bennett et al. 1996). The function of AUX1 in auxinuptake, auxin binding, lateral root development, and gravit-ropism has been extensively studied (Timpte et al. 1995;Marchant et al. 1999, 2002;Swarup et al. 2001; Kleine-Vehn et al. 2006; Carrier et al. 2008). However, the role ofAUX1 in the regulation of organ regeneration in vitro andin the control of organ identity in response to auxin/cytoki-nin ratio has not been addressed. To conWrm our observa-tions, we used the aux1-7 mutant (Pickett et al. 1990) torepeat the callus induction and organ regeneration experi-ments in CM1, CM2, and ORM1 media as described in themutant screening experiments above. The phenotype of cal-lus induction and root regeneration of the aux1-7 mutantwere similar to mutant-1 and mutant-2/3 (data not shown).Since mutant-1, mutant-2/3, and aux1-7 have the same phe-notype and disrupted the same gene, our results suggestedthat the phenotypes observed were due to the mutation inthe AUX1 gene.
Cytokinin induced auxin accumulation and redistribution in calli and speciWc tissues of Arabidopsis seedlings
Since low ratios of auxin/cytokinin lead to shoot regenera-tion and high ratios of auxin/cytokinin lead to root regener-ation, we examined how the ratios of auxin/cytokinin aVectthe expression of DR5:GUS and DR5:GFP in calli andseedlings. The synthetic promoter DR5 is highly inducibleby auxin and has an expression pattern similar to that of thenative GH3 promoter when fused with the reporter genes(Ulmasov et al. 1997). It has been reported that the GUSreporter activity driven by the DR5 promoter correlateswith direct auxin measurements in plants (Casimiro et al.2001; Friml et al. 2002). Therefore, the DR5:GUS andDR5:GFP reporter system is a useful tool in revealing cel-lular active auxin level in plants and has been widely usedin auxin related studies. To our surprise, we found thatcytokinin can substantially induce the expression of the
Fig. 2 Dose-response of the isolated mutants to 2,4-D and KIN. Thepercentage of root length is shown and the root lengths without hor-mone treatment were taken as 100% for the wild type and mutants,respectively. The root length was measured when seedlings were10 days old. The data presented is the average of 3 biological replicaswith at least 30 seedlings each. The hormone treatments are indicatedin the Wgure. a Dose-response to 2,4-D treatment. b Dose-response toKIN treatment
Fig. 3 T-DNA insertion sites in isolated mutants. The nine exons ofthe AUX1 gene are shown by the black bars. The T-DNA insertion sitesof mutant-1 and mutant-2/mutant-3 are marked and the correspondingamino acids in the insertion sites are labeled. The DNA sequences inthe T-DNA insertion sites are also presented at the bottom
123
Planta (2009) 229:645–657 651
DR5 promoter driving reporters in calli and speciWc tissuesof Arabidopsis seedlings (Figs. 4, 5). The GUS level in calliinduced with 0.05 mg l¡1 2,4-D plus 2.0 mg l¡1 KIN wassubstantially higher than those induced by 0.05 mg l¡1 2,4-D alone in wild-type calli (Fig. 4a, a-1, a-2). Similarly, calliinduced with 0.25 mg l¡1 2,4-D plus 2 mg l¡1 KIN hadhigher GUS expression than those induced with0.25 mg l¡1 2,4-D alone (Fig. 4a, a-3, a-4). In addition, thedistribution pattern of the GUS reporter had also changed.The GUS was limited to regions close to the surface in calliinduced by a low level of 2,4-D (0.05 mg l¡1). When2.0 mg l¡1 KIN was added, the GUS was distributed acrossthe entire calli although some regions might still have amore intense expression (Fig. 4a, a-1, a-2). The GUS leveland distribution pattern change in calli in response to cyto-kinin treatments were supported by DR5:GFP reporteractivities with the same treatments, which was more clearlydisplayed under the confocal microscopy (Fig. 4b). TheDR5 driving GFP reporter was sharply enriched in manybright granule spots when 2.0 mg l¡1 cytokinin was added
into the medium (Fig. 4b, b-2, b-4). In contrast, this kind ofbright granule spots were not common in calli induced by2,4-D alone (Fig. 4b, b-1–b-3). The calli induced by 2,4-Dplus cytokinin grew much faster than those induced by 2,4-D alone, and the callus texture was also diVerent. The callusfresh weight induced with 0.05 mg l¡1 2,4-D plus2.0 mg l¡1 KIN was about 2.5 § 0.2 times higher than thatinduced by 0.05 mg l¡1 2,4-D after 1 month of culture. Thecalli induced by 2,4-D alone were soft and watery. The calliinduced by 2,4-D plus high KIN were more compact andwith granule features. It is clear that auxin elevation stimu-lated by KIN was diVerent from that caused by directlyapplying exogenous auxin in the medium. The diVerencesincluded auxin distribution pattern in calli and the auxinlevel. These observations suggested that KIN did muchmore than just stimulating auxin accumulation. Because ourobservations were recorded in the early stage of callusdevelopment, it was unknown if any of the callus struc-tures, such as the bright granule spots, were related to theshoot primordium formation later.
Fig. 4 Histochemical assay of DR5:GUS and DR5:GFP expression incalli of wild type and aux1-7 mutant. The calli were induced for amonth in B5 media with hormone combinations as indicated below. aDR5:GUS activities in response to hormone treatments in calli; a-1 toa-4 are images of wild type. a-5 to a-8 are images of aux1-7 mutant.a-1 and a-5 tissues cultured in B5 medium with 0.05 mg l¡1 2,4-D; a-2and a-6 tissues cultured in B5 medium with 0.05 mg l¡1 2,4-D plus2.0 mg l¡1 KIN; a-3 and a-7 tissues cultured in B5 medium with0.25 mg l¡1 2,4-D; a-4 and a-8 tissues cultured in B5 medium with
0.25 mg l¡1 2,4-D plus 2.0 mg l¡1 KIN. b DR5:GFP activity in re-sponse to hormone treatments in calli. The images were acquired usinga Zeiss LSM 510 Confocal Laser Scanning Microscope (Carl Zeiss Mi-croimaging, Inc.). b-1 to b-4 are images of wild type. b-5 to b-8 are im-ages of aux1-7 mutant. b-1 and b-5 tissues cultured in B5 medium with0.05 mg l¡1 2,4-D; b-2 and b-6 tissues cultured in B5 medium with0.05 mg l¡1 2,4-D plus 2.0 mg l¡1 KIN; b-3 and b-7 tissues cultured inB5 medium with 0.25 mg l¡1 2,4-D; b-4 and b-8 tissues cultured in B5medium with 0.25 mg l¡1 2,4-D plus 2.0 mg l¡1 KIN
123
652 Planta (2009) 229:645–657
The prominent role of cytokinin in the control of auxinlevel in calli was not expected. To test if such a phenome-non was unique in calli, we examined the impact of cyto-kinin on auxin distribution in Arabidopsis youngseedlings harboring the DR5:GUS and DR5:GFP reportergenes. As shown in Fig. 5a, high concentration of cytoki-nin stimulated DR5:GUS accumulation in root elongationzone and the junction of hypocotyl and cotyledons in 5-day-old young seedlings (Fig. 5a, a-1–a-4). The eVect ofcytokinin on auxin accumulation in roots was moreclearly displayed when young seedlings with DR5:GFPreporter were examined using confocal microscopy(Fig. 5b, b-1–b-4, see regions indicated by the arrows).Meanwhile, auxin level appeared to be reduced in cotyle-dons and the middle region of hypocotyls (Fig. 5a, a-1–a-4). These observations suggested that cytokinin also
caused auxin redistribution and tissue speciWc accumula-tion in young seedlings.
IAA2:GUS is another auxin inducible promoter drivingreporter construct widely used in auxin studies (Swarupet al. 2001). To verify that the cytokinin inducedDR5:reporter accumulation in speciWc tissues was not aDR5 promoter speciWc response but a general response ofthe plants to the cytokinin treatment, we examined theIAA2:GUS reporter expression in response to cytokinintreatments in wild-type seedlings, ecotype Ws instead ofColumbia. As shown in Supplemental Fig. 2, the resultswere the same as revealed by using the DR5:GUS andDR5:GFP reporters. Cytokinin had induced auxin accumu-lation in root elongation zone and the junction betweenhypocotyl and cotyledons, including the shoot meristemregion (Supplemental Fig. 2).
Fig. 5 Histochemical assay of DR5:GUS and DR5:GFP response tocytokinin treatments in seedlings. The seedlings were germinated inB5 media supplemented with KIN in concentrations as indicated be-low. The seedlings and roots were 5 days old. a DR5:GUS activities inresponse to cytokinin treatments in seedlings; a-1 to a-4 are images ofwild type. a-5 to a-8 are images of aux1-7 mutant. a-1 and a-5 seed-lings grown in B5 medium; a-2 and a-6 seedlings grown in B5 mediumwith 0.2 mg l¡1 KIN; a-3 and a-7 seedlings grown in B5 medium with1.0 mg l¡1 KIN; a-4 and a-8 seedlings grown in B5 medium with
2.0 mg l¡1 KIN. b DR5:GFP activities in response to KIN treatmentsin roots. The images were acquired using a Zeiss LSM 510 ConfocalLaser Scanning Microscope (Carl Zeiss Microimaging, Inc.). b-1 tob-4 are images of wild-type roots. b-5 to b-8 are images of aux1-7mutant roots. b-1 and b-5 roots cultured in B5 medium; b-2 and b-6roots cultured in B5 medium with 0.2 mg l¡1 KIN; b-3 and b-7 rootscultured in B5 medium with 1.0 mg l¡1 KIN; b-4 and b-8 roots culturedin B5 medium with 2.0 mg l¡1 KIN
123
Planta (2009) 229:645–657 653
Cytokinin induced auxin accumulation and redistribution is substantially reduced in the seedlings and calli of aux1 mutant
We crossed DR5:GUS and DR5:GFP reporters into aux1-7mutant background, and examined the reporters expressionresponses to cytokinin and auxin treatments. As shown inFig. 5a, while wild-type seedlings accumulated GUS in theroot elongation zone and the junction of cotyledons andhypocotyl after being treated with KIN, the GUS reporteraccumulation in the aux1-7 mutant was substantiallyreduced compared to the wild type in 5-day-old seedlings(Fig. 5a, a-5–a-8). In addition, the mutant roots were longerthan the wild type (Fig. 5a). The results were the samewhen DR5:GFP reporter was examined (Fig. 5b). Since theGFP samples were examined using exactly the same condi-tion under confocal microscopy, the intensity shown inFig. 5b could be compared quantitatively. AUX1 mutationalso aVected cytokinin stimulated auxin accumulation incalli (Fig. 4). The aux1-7 seedlings treated with 0.05 mg l¡1
2,4-D did not produce calli (Fig. 4a, a-5). Instead, somenodule like nodes could be observed along the roots and theGUS reporter was highly expressed in the tip regions of thenodes. aux1-7 seedlings grown in medium with 0.05 mg l¡1
2,4-D plus 2.0 mg l¡1 KIN did not produce calli either(Fig. 4a, a-6). It was evident that KIN did not substantiallystimulate DR5:GUS expression in aux1-7 mutant (Fig. 4a,compare a-6 with a-5). Studies with DR5:GFP reportershowed the same results (Fig. 4b, compare b-6 with b-5).When 0.25 mg l¡1 2,4-D was applied, callus clumps gener-ated from the roots of aux1-7 seedlings. Adding 2.0 mg l¡1
KIN enhanced callus formation (Fig. 4, a-7 vs. a-8; b-7 vs.b-8). However, the DR5:GFP reporter level appeared to belower in aux1-7 calli than wild-type calli grown under thesame condition as shown in Fig. 4b, which were observedusing a confocal microscopy under exactly the same condi-tion (Fig. 4b, b-4, b-8). The results described above indi-cated that KIN stimulated auxin accumulation in calli andspeciWc tissues of Arabidopsis was reduced in aux1-7mutant, suggesting a role of AUX1 in mediating cytokininregulated auxin redistribution and elevation.
AUX1 is highly expressed in calli
To understand how AUX1 was involved in the regulation oforgan regeneration in calli, we induced calli from theAUX1:AUX1-116-YFP transgenic line (Marchant et al.1999; Swarup et al. 2004) under diVerent conditions andexamined the expression of YFP reporter using a Zeiss LSM510 Confocal Laser Scanning Microscope. As shown inFig. 6, the AUX1 promoter driving AUX1-116-YFPexpressed in calli induced by 0.05 mg l¡1 2,4-D and0.25 mg l¡1 2,4-D, respectively. When 2.0 mg KIN was
added, the AUX1-YFP distribution pattern switched tostructures like small granules instead of smooth distribution,which was probably due to the structure and texture changeof calli (Fig. 6c, d). Since we were examining the calli in thecallus induction stage, it was unknown if there was a corre-lation between YFP pattern and the primordia of the regen-erating organs. AUX1 is highly expressed within the shootapical meristem and primary root tip in plants (Bennett et al.1996; Marchant et al. 1999; Swarup et al. 2004). It remainsto be tested if the high expression of the AUX1 in calli isassociated with de novo apical meristem formation.
Cytokinin treatment induces the expression of auxin inducible genes
Our results above had indicated that cytokinin treatmentresulted in auxin accumulation in speciWc tissues in plants,including calli. To investigate how the auxin and cytokininpathways interact with each other, we examined the expres-sion of selected auxin and cytokinin inducible genes usingReal-Time PCR under auxin and cytokinin treatments,respectively. As shown in Fig. 7, auxin inducible genesIAA3, IAA6, IAA14, and IAA 17 were induced by the appli-cation of 2.0 mg l¡1 KIN in both wild type and aux1-7.However, the induction levels of these genes in aux1-7
Fig. 6 Histochemical image of AUX1 promoter driving YFP in calliinduced with diVerent hormones. The YFP images are acquired usinga Zeiss LSM 510 Confocal Laser Scanning Microscope. The calli(Columbia) harboring an AUX1:AUX1-116-YFP reporter constructwere induced in B5 medium for 1 month with hormones indicated be-low. a calli induced with 0.05 mg l¡1 2,4-D; b calli induced with0.25 mg l¡1 2,4-D; c calli induced with 0.05 mg l¡1 2,4-D plus2.0 mg l¡1 KIN; d calli induced with 0.25 mg l¡1 2,4-D plus2.0 mg l¡1 KIN
123
654 Planta (2009) 229:645–657
mutant were lower than in the wild type in all treatmentsexcept for the expression of the IAA3, whose expression inaux1-7 was slightly higher than wild type. In addition, theinduction of cytokinin inducible genes ACR4 (At1g69040)and AP2 (At4g23750) (Rashotte et al. 2003; Kiba et al.2005) by cytokinin were substantially reduced in the aux1-7 mutant. These observations suggested that the cross-regu-lation between auxin and cytokinin in gene expression waspartially disrupted in the aux1-7 mutant and the aux1-7mutation disturbed the response of the mutant to cytokininas well.
Discussion
Establishing a procedure for screening organ identity mutants in response to the ratio of auxin to cytokinin in organogenesis
Classic plant tissue culture experiments have shown thatthe ratio of auxin to cytokinin determines the organ identityduring organ regeneration (Skoog and Miller 1957). Whilelow auxin to cytokinin ratio leads to shoot development,high auxin to cytokinin ratio leads to root development.The particular ratio requirements for shoot and root devel-
opments are species and ecotype dependent. Although ithas been widely accepted that the ratio of auxin to cytoki-nin plays a critical role in determining organ identity duringorgan regeneration, no cellular components that controlorgan identity in response to the ratio of auxin to cytokininduring organogenesis have been identiWed. Meanwhile, ithas also been reported that many environmental factors,including light, osmotic stress, etc., irreversibly change thecell fate of cells in culture thus regulating the frequency andpattern of regeneration, (Lillo 1989; Ikeda-Iwai et al.2003). In order to identify mutants which are truly involvedin hormone regulation of organogenesis instead of environ-mental eVect, we designed a mutant screening procedure inwhich the cell fates of the cultured cells were determined inthe very early stage of cell culture. In addition, the inducedcalli were transferred to a common regeneration mediumfor further observation of organ development and for com-parison. One advantage of this method is that the impact ofenvironmental factors on organogenesis has been mini-mized due to the short cell culture time. Therefore, theidentities of the regenerating organs are highly repeatablefor every hormone concentration used. Using this approach,we have isolated AUX1 as a cellular component regulatingorgan identity of in vitro regeneration. The calli of aux1mutant, induced with 0.25 mg l¡1 2,4-D plus 2.0 mg l¡1
Fig. 7 Gene expression in response to auxin and cytokinin treatmentsin aux1-7 mutant revealed by Real-Time PCR. The 12-day-old seed-lings were used for auxin and cytokinin treatment, respectively. Theseedlings were submerged in hormone solution for 5 min and incu-bated for another hour. The 2,4-D concentration was 2.2 mg l¡1
(»10 �M) and KIN concentration was 3.0 mg l¡1 (»15 �M). RNAsamples extracted with RNeasy Plant Mini Kit (QIAGEN) were used
for cDNA synthesis and Real-Time PCR. Ubiquitin gene was includedas an internal control and used to normalize the gene expression level.The relative expression level of ubiquitin gene was assigned as1,000 units in all the treatments. The gene identities and hormone treat-ments are indicated in each graph. The gene expression level in wildtype is represented by the white bar and in aux1-7 is represented by theblack bar
123
Planta (2009) 229:645–657 655
KIN, regenerate roots instead of shoots. In addition, somebelt like abnormal root structures have been observed whencultured in media optimized for root regeneration (Fig. 1c),suggesting that mutations in this gene lead to organ identitychange during regeneration. When calli were induced in amedium with very high concentrations of cytokinin, forexample, a medium containing 3.0 mg l¡1 KIN and0.25 mg l¡1 2,4-D, about 2–10% of the mutant calli pro-duced shoots while the rest still regenerated roots or stayedas calli (data not shown), suggesting that probably othercellular components are also involved in determining theorgan identity when the ratio of auxin to cytokinin isextremely low in Arabidopsis. We are characterizing moremutants and expect more genes to be identiWed. Theseresults indicate that our approach can be eVectively used toidentify cellular components regulating organ identity inorganogenesis.
Shoot regeneration is associated with auxin elevationin calli
We Wnd that cytokinin substantially stimulates auxin eleva-tion in calli and speciWc tissues in Arabidopsis seedlings,including the root elongation zone, the junction of hypocotyland cotyledons, and probably the meristem region in 5-day-old seedlings (Figs. 4, 5, Supplemental Fig. 2). The fact thatan increase of cytokinin concentration in medium causeselevation of cellular auxin level in calli suggests that regen-eration of shoot, which requires high cytokinin, is associatedwith a high auxin level instead of a low auxin level. Thisobservation Wts well with the fact that auxin is highly ele-vated in shoot meristem tissues. Since root regeneration isassociated with high auxin/cytokinin ratio and shoot regen-eration associates with low auxin/cytokinin ratio, it is gener-ally believed that high auxin is required for root regenerationbut not for shoot regeneration. Our results suggest that highauxin is also associated with shoot regeneration. However, itis clear that the auxin elevation caused by cytokinin is diVer-ent from that resulting from direct administration of exoge-nous auxin in both auxin distribution pattern and auxin levelas revealed by reporters driven by auxin responsive promot-ers (Figs. 4, 5, Supplemental Fig. 2). In addition, the callustexture and growth rate are also diVerent between calligrown in media with and without KIN. These observationssuggest that cytokinin does more than just stimulating auxinaccumulation in calli, the other functions may be critical forspecifying the shoot identity.
Tissue speciWc interaction between auxin and cytokininin Arabidopsis seedling
Auxin and cytokinin are two major plant hormones whosecomplex interaction has been reported in various aspects of
plant growth and development. The interaction can be syn-ergistic, antagonistic, or additive depending on the type oftissues, developmental stages, and plant species (Coenenand Lomax 1998). Coenen and Lomax (1997) have identi-Wed a tomato mutant (diageotropica, dgt) whose roots areresistant to both auxin and cytokinin but the shoot growthand hypocotyl elongation are sensitive to cytokinin. Inaddition, organ regeneration from the calli derived from dgthypocotyls show reduced sensitivity to auxin but normalsensitivity to cytokinin. These results suggest that plantroot and shoot tissues can respond to cytokinin with sepa-rate signaling pathways. Several studies have reported themutual control of auxin and cytokinin abundance and gen-erated controversial conclusions. Nordstrom et al. (2004)found that auxin mediates a very rapid negative control ofthe cytokinin pool by mainly suppressing the biosynthesisof the isopentenyladenosine-5�-monophosphate indepen-dent pathway. On the other hand, the eVect of cytokininoverproduction on the auxin pool was slower. An increaseof auxin level has been reported after administration ofexogenous cytokinin or elevating the cytokinin by overex-pression of the bacterial ipt gene (Binns et al. 1987; Bour-quin and Pilet 1990; Besrtell and Eliasson 1992).Meanwhile, it has been reported that cytokinin overexpres-sion has led to down regulation of IAA pool in tobacco(Eklöf et al. 1997).
Using the DR5:GFP, DR5:GUS and IAA2:GUS report-ers, we have demonstrated a tissue speciWc response ofauxin to applied exogenous cytokinin in Arabidopsis youngseedlings. While cytokinin stimulates auxin accumulationin the root elongation zone, the junction between hypocotyland cotyledons, and the shoot meristem (see SupplementalFig. 2), the auxin level appeared to be decreased in cotyle-don blade, middle region of the hypocotyl, and the rootmeristem (see Fig. 5b). It will be very interesting to testwhether cytokinin stimulate de novo shoot regeneration invitro via enhancing local auxin Xow and accumulation inregions that form shoot meristem.
Our Real-Time PCR analyses of gene expression dem-onstrate that auxin inducible genes IAA3, IAA14, IAA17,and IAA6 are signiWcantly induced by the application ofexogenous cytokinin and the gene expression level isreduced in the aux1 mutants compared with the expressionin wild type except the IAA3 gene. Meanwhile, cytokinininducible gene ACR4 (At1g69040) and AP2 (At4g23750)are slightly inhibited by auxin in wild type but not in theaux1-7 mutant (Fig. 7). These results suggest a multifacetedinteraction of these two hormones at a molecular level andAUX1 is critical to the multifaceted interaction. Consistentwith our observation, cytokinin induction of IAA3 andIAA17 expression has also been observed in DNA oligoarray studies in Arabidopsis (Che et al. 2002; Rashotteet al. 2003).
123
656 Planta (2009) 229:645–657
Possible role of AUX1 in organ identity control in organogenesis
AUX1 has been identiWed as an auxin inXux facilitator local-ized on the plasma membrane with similarities to plantamino acid permeases (Bennett et al. 1996). The function ofAUX1 in auxin uptake has been well documented (Marchantet al. 1999). Although the aux1-7 mutant has been shown tobe resistant to auxin, ethylene, and cytokinin (Timpte et al.1995), there is no speciWc report on the role of AUX1 inorgan identity control during organogenesis in vitro. In addi-tion, there is no report on the role of AUX1 in mediating theinterplay between cytokinin and auxin pathways.
We identiWed AUX1 gene using a forward geneticapproach, while searching for mutants with abnormalresponse to the auxin to cytokinin ratio in regeneration. Wefound that the aux1 mutants were insensitive to both auxinand cytokinin. When calli were induced, the mutant callidid not regenerate shoots in media with a low ratio of auxinto cytokinin, in which the wild-type control only regener-ates shoots. We further demonstrated that mutation inAUX1 gene reduced auxin accumulation stimulated bycytokinin in the root elongation zone, calli and also in theshoot meristem region of young seedlings (Figs. 4, 5, Sup-plemental Fig. 2). In addition, our results showed thatAUX1:AUX1-116-YFP was highly expressed in calli andthe YFP expression pattern was similar to the DR5:GFPdistribution pattern in calli (please compare Fig. 6 withFig. 4). Since cytokinin stimulates auxin accumulation incalli and the accumulation is substantially reduced in aux1mutants, it clearly suggests that AUX1 plays a role in medi-ating cytokinin regulated auxin accumulation and redistri-bution in calli either directly or indirectly. Ozawa et al.(1998) proposes that callus has a root competent stage and ashoot competent stage. The shoot competent stage is on topof the root competent stage. Cells which have lost the com-petency of shoot regeneration can still regenerate roots,suggesting that shoot regeneration requires additional com-ponents or pathways. AUX1 might be one of the compo-nents that controls shoot regeneration via regulatingcytokinin induced auxin accumulation and redistribution,which relates to auxin Xow and patterning.
We have established a mutant screening procedure toidentify cellular components that regulate organogenesis inresponse to auxin to cytokinin ratio in Arabidopsis. Theaux1 mutant identiWed using this method promotes rootregeneration in media that usually lead to shoot regenera-tion. In addition, we Wnd that high cytokinin promotes auxinaccumulation in several speciWc tissues in young seedlingsand calli via an AUX1 dependent pathway. Our results sug-gest that shoot regeneration is associated with auxin accu-mulation in calli and this observation is consistent with thefact that shoot meristem cells contain a high level of auxin.
Acknowledgments We are grateful to Dr. Thomas J. Guilfoyle forDR5:GUS transgenic line and Dr. Malcolm Bennett for theIAA2:GUS, AUX1:GUS, and AUX1:AUX1-YFP lines and for his val-ued comments on the manuscript. We thank Dr. John Boyle, Dr. Din-Pow Ma, and Dr. Kenneth Willeford for a critical reading of the man-uscript. This research was approved for publication as Journal ArticleJ-10944 of the MAFES, Mississippi State University.
References
Bangerth F (1994) Response to cytokinin concentration in the xylemexudate of bean (Phaseolus vulgaris L.) plants to decapitation andauxin treatment and relationship to apical dominance. Planta194:439–442
Bennett MJ, Marchant A, Green HG, May ST, Ward SP, Millner PA,Walker AR, Schulz B, Feldmann KA (1996) Arabidopsis AUX1gene: a permease-like regulator of root gravitropism. Science273:948–950
Besrtell G, Eliasson L (1992) Cytokinin eVects on root growth and pos-sible interactions with ethylene and indole-3-acetic acid. PhysiolPlant 84:255–261
Binns AN, Labriola J, Black RC (1987) Initiation of auxin autonomyin Nicotiana glutinosa cells by the cytokinin-biosynthesis genefrom Agrobacterium tumefaciens. Planta 171:539–548
Bourquin M, Pilet PE (1990) EVect of zeatin on the growth and indo-lyl-3-acetic acid and abscisic acid levels in maize roots. PhysiolPlant 80:342–349
Carrier DJ, Baker NTA, Swarup R, Callaghan R, Napier RM, BennettMJ, Kerr ID (2008) The binding of auxin to the Arabidopsis auxininXux transporter AUX1. Plant Physiol 148:529–535
Casimiro I, Marchant A, Bhalerao RP, Beeckman T, Dhooge S, Swa-rup R, Graham N, Inze D, Sandberg G, Casero PJ, Bennett M(2001) Auxin transport promotes Arabidopsis lateral root initia-tion. Plant Cell 13:843–852
Che P, Gingerich DJ, Lall S, Howell SH (2002) Global and hormone-induced gene expression changes during shoot development inArabidopsis. Plant Cell 14:2771–2785
Coenen C, Lomax TL (1997) Auxin–cytokinin interactions in higherplants: old problems and new tools. Trends Plant Sci 2:351–356
Coenen C, Lomax TL (1998) The diageotropica gene diVerentiallyaVects auxin and cytokinin responses throughout development intomato. Plant Physiol 117:63–72
De Smet I, Jurgens G (2007) Patterning the axis in plants—auxin incontrol. Curr Opin Genet Dev 17:337–343
Eklöf S, Åstot CJB, Moritz T, Olsson O, Sandberg G (1997) Auxin–cytokinin interactions in wild-type and transgenic tobacco. PlantCell Physiol 38:225–235
Friml J, Benkova E, Blilou I, Wisniewska J, Hamann T, Ljung K,Woody S, Sandberg G, Scheres B, Jurgens G, Palme K (2002)AtPIN4 mediates sink-driven auxin gradients and root patterningin Arabidopsis. Cell 108:661–673
Hartig K, Beck E (2006) Crosstalk between auxin, cytokinins, and sug-ars in the plant cell cycle. Plant Biol (Stuttg) 8:389–396
Himanen K, Vuylsteke M, Vanneste S, Vercruysse S, Boucheron E,Alard P, Chriqui D, Van Montagu M, Inze D, Beeckman T (2004)Transcript proWling of early lateral root initiation. Proc Natl AcadSci USA 101:5146–5151
Hoyerova K, Perry L, Hand P, Lankova M, Kocabek T, May S,Kottova J, Paces J, Napier R, Zazimalova E (2008) Functionalcharacterization of PaLAX1, a putative auxin permease, in heter-ologous plant systems. Plant Physiol 146:1128–1141
Ikeda-Iwai M, Umehara M, Satoh S, Kamada H (2003) Stress-inducedsomatic embryogenesis in vegetative tissues of Arabidopsis thali-ana. Plant J 34:107–114
123
Planta (2009) 229:645–657 657
John PCL, Zhang K, Dong C, Diederich L, Wightman F (1993)p34[cdc2] related proteins in control of cell cycle progression, theswitch between division and diVerentiation in tissue development,and stimulation of division by auxin and cytokinin. Aust J PlantPhysiol 20:503–526
Kiba T, Naitou T, Koizumi N, Yamashino T, Sakakibara H, Mizuno T(2005) Combinatorial microarray analysis revealing arabidopsisgenes implicated in cytokinin responses through the His-> AspPhosphorelay circuitry. Plant Cell Physiol 46:339–355
Kleine-Vehn J, Dhonukshe P, Swarup R, Bennett M, Friml J (2006)Subcellular traYcking of the Arabidopsis auxin inXux carrierAUX1 uses a novel pathway distinct from PIN1. Plant Cell18:3171–3181
Li C-J, Guevara E, Herrera J, Bangerth F (1995) EVect of apex excisionand replacement by 1-naphthylacetic acid on cytokinin concentra-tion and apical dominance in pea plants. Physiol Plant 95:465–469
Lillo C (1989) EVects of media components and environmental factorson shoot formation from protoplast-derived calli of Solanum tu-berosum. Plant Cell Tissue Organ Cult 19:103–111
Lucas M, Godin C, Jay-Allemand C, Laplaze L (2008) Auxin Xuxes inthe root apex co-regulate gravitropism and lateral root initiation.J Exp Bot 59:55–66
Marchant A, Kargul J, May ST, Muller P, Delbarre A, Perrot-Rechen-mann C, Bennett MJ (1999) AUX1 regulates root gravitropism inArabidopsis by facilitating auxin uptake within root apical tis-sues. EMBO J 18:2066–2073
Marchant A, Bhalerao R, Casimiro I, Eklof J, Casero PJ, Bennett M,Sandberg G (2002) AUX1 promotes lateral root formation byfacilitating indole-3-acetic acid distribution between sink andsource tissues in the Arabidopsis seedling. Plant Cell 14:589–597
Muller B, Sheen J (2008) Cytokinin and auxin interaction in root stem-cell speciWcation during early embryogenesis. Nature 453:1094–1097
Nordstrom A, Tarkowski P, Tarkowska D, Norbaek R, Astot C, Dole-zal K, Sandberg G (2004) Auxin regulation of cytokinin biosyn-thesis in Arabidopsis thaliana: a factor of potential importance forauxin–cytokinin-regulated development. Proc Natl Acad SciUSA 101:8039–8044
Ottenschlager I, WolV P, Wolverton C, Bhalerao RP, Sandberg G, Is-hikawa H, Evans M, Palme K (2003) Gravity-regulated diVeren-tial auxin transport from columella to lateral root cap cells. ProcNatl Acad Sci USA 100:2987–2991
Ozawa S, Yasutani I, Fukuda H, Komamine A, Sugiyama M (1998)Organogenic responses in tissue culture of srd mutants of Arabid-opsis thaliana. Development 125:135–142
Pickett FB, Wilson AK, Estelle M (1990) The aux1 mutation of Ara-bidopsis confers both auxin and ethylene resistance. Plant Physiol94:1462–1466
Rashotte AM, Carson SD, To JP, Kieber JJ (2003) Expression proWlingof cytokinin action in Arabidopsis. Plant Physiol 132:1998–2011
Sessions A, Weigel D, Yanofsky MF (1999) The Arabidopsis thalianaMERISTEM LAYER 1 promoter speciWes epidermal expressionin meristems and young primordia. Plant J 20:259–263
Skoog F, Miller CO (1957) Chemical regulation of growth and organformation in plant tissues cultured in vitro. Symp Soc Exp Biol54:118–130
Soni R, Carmichael JP, Shah ZH, Murray JA (1995) A family of cyclinD homologs from plants diVerentially controlled by growth regu-lators and containing the conserved retinoblastoma protein inter-action motif. Plant Cell 7:85–103
Swarup R, Friml J, Marchant A, Ljung K, Sandberg G, Palme K, Ben-nett M (2001) Localization of the auxin permease AUX1 suggeststwo functionally distinct hormone transport pathways operate inthe Arabidopsis root apex. Genes Dev 15:2648–2653
Swarup R, Parry G, Graham N, Allen T, Bennett M (2002) Auxincross-talk: integration of signalling pathways to control plantdevelopment. Plant Mol Biol 49:411–426
Swarup R, Kargul J, Marchant A, Zadik D, Rahman A, Mills R, YemmA, May S, Williams L, Millner P, Tsurumi S, Moore I, Napier R,Kerr ID, Bennett MJ (2004) Structure-function analysis of thepresumptive Arabidopsis auxin permease AUX1. Plant Cell16:3069–3083
Timpte C, Lincoln C, Pickett FB, Turner J, Estelle M (1995) TheAXR1 and AUX1 genes of Arabidopsis function in separate aux-in-response pathways. Plant J 8:561–569
Weigel D, Ahn JH, Blazquez MA, Borevitz JO, Christensen SK, Fan-khauser C, Ferrandiz C, Kardailsky I, Malancharuvil EJ, NeVMM, Nguyen JT, Sato S, Wang ZY, Xia Y, Dixon RA, HarrisonMJ, Lamb CJ, Yanofsky MF, Chory J (2000) Activation taggingin Arabidopsis. Plant Physiol 122:1003–1013
Yip WK, Yang SF (1986) EVect of thidiazuron, a cytokinin-active ureaderivative, in cytokinin-dependent ethylene production systems.Plant Physiol 80:515–519
![Relevance of somatostatin receptors and other peptide ... · homeostasis of an organ, whereas quanti- ... subtypes ss h and sst 4 [3,4]. In Vitro Detection of Somatostatin Receptors](https://static.documents.pub/doc/80x56/5f32aff17c45c5451b2e5a86/relevance-of-somatostatin-receptors-and-other-peptide-homeostasis-of-an-organ.jpg)
