Received 15 Feb. 2005 Accepted 30 Apr. 2005Supported by the Gansu Provincial Natural Science Foundation of China (ZS021-A25-047-N) and Xi’an Urban Rural Construction Committee.*Author for correspondence. Tel: +86 (0)931 891 4155; E-mail: <wangcy@lzu.edu.cn>.

Journal of Integrative Plant BiologyFormerly Acta Botanica Sinica 2005, 47 (10): 1153−1158

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Ectopic Expression of the Pttkn1 Gene Induces Alterations in theMorphology of the Leaves and Flowers in Petunia hybrida Vilm.

Xin HU1, Qing-Feng WU1, Ya-Hong XIE2, Hong RU2, Feng XIE2, Xin-Yu WANG1 and Chong-Ying WANG1*

(1. School of Life Sciences, Lanzhou University, Lanzhou 730000, China;2. Xi’an Institute of Gardens, Xi’an 710065, China)

Abstract: A novel knotted1-like homeobox (knox) gene, Pttkn1 (Populus tremula×tremuloides knotted1),isolated from the cambial region of hybrid aspen, was introduced into Petunia hybrida Vilm. using the leaf-disc method mediated by Agrobacterium. A series of novel phenotypes was observed in transgenic petuniaplants, including the formation of ectopic spikes on the adaxial surface of corollas and small petals on theabaxial surface of corollas, fusion of floral organs, shortening of corolla midribs, the formation of tumor-likeknots along the midrib on the abaxial surface and serrated lobs of corolla margins, and alterations in petalcolor; except for changes in the leaves and plant architecture, RT-PCR showed that the Pttkn1 gene wasexpressed in the leaves of different petunia transgenic plants, whereas no signal was detected in wild-typeplants. The possible function of Pttkn1 in leaf and flower development is discussed.Key words: ectopic expression; knox gene; novel phenotypes; Petunia hybrida; Pttkn1 gene; RT-PCR.

Homeobox genes, encoding gene regulatory proteinsthat act as transcription factors by binding to specificcis-regulatory regions in their target genes, play impor-tant roles in the development process from yeast toanimals and plants (Matsuoka et al. 1993; Pautot et al.2001). The first homeobox gene isolated from a plantwas knotted1 (kn1) in maize (Vollbrecht et al. 1991).Subsequently, many knotted1-like homeobox (knox)genes have been cloned from different plants, includ-ing Arabidopsis, rice, barley, soybean, tobacco, tomato(Nishimura et al. 2000), and potato (Rosin et al. 2003).The knotted1-like homeobox (knox) genes have beensubdivided into two groups: class I knox genes andclass II knox genes. Class I knox genes are expressedin meristems and are excluded from developing organprimodia (Reiser et al. 2000). Ectopic expression ofclass I knox genes results in altered leaf and flowermorphology in spontaneous mutants and in transgenicplants (Sakamoto et al. 1999). It was thought that classI knox genes play an important role in maintaining

indeterminacy in the shoot apical meristem (SAM) andin subsequent shoot development ( Kim et al. 2003).We are interested in the morphological alterations offlowers from flowering plants caused by class I knoxgenes and how they function when they are expressedectopically in transgenic plants. A new class I knoxgene, Pttkn1 (Populus tremula×tremuloides knotted1),isolated from the cambial region of hybrid aspen wasintroduced recently into Petunia hybrida. Novelphenotypes, rarely seen in previous studies of knoxgenes, were observed when the Pttkn1 gene was ex-pressed ectopically in transgenic plants. Herein, weprovide a preliminary report on the expression of thePttkn1 gene, novel phenotypes of Pttkn1 petunia plants,and possible functions of Pttkn1 in plant development.

1 Materials and Methods

Petunia hybrida Vilm. was used as an experimentalmaterial and was transformed with the Pttkn1 gene.The pPCV702 plasmid, containing a cauliflower

.Rapid Communication.

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mosaic virus 35S promoter and Pttkn1 (pPCV35S::Pttkn1), was kindly supplied by Dr. Olof Olsson(Göteberg University, Göteberg, Sweden).

The Pttkn1 gene was introduced into leaf explantsof P. hybrida using the leaf-disc method, as describedby Wang and Fang (2002) with minor modifications. Asingle clone of the Agrobacterium tumefaciens strainGV3101 containing pPCV35S::Pttkn1 was cultured inYEB medium supplemented with 25 mg/L kanamycin+ 75 mg/L carbenicillin overnight. Agrobacterium cellswere collected by centrifugation (2 000g, 10 min) anddiluted with MS medium into a solution with an opticaldensity at OD600 of approximately 0.3. Leaf explants(approximately 5 mm × 5 mm) from sterilized seed-lings were soaked in Agrobacterium solution for 1 min,cocultured on MS medium containing 1 mg/L BA for 2d in the dark, and then transferred to MS medium con-taining 1 mg/L BA + 400 mg/L cefotaxime for 20 d,finally being transferred to MS-selective medium con-taining 1 mg/L BA+400 mg/L cefotaxime + 50 mg/Lkanamycin to differentiate into regenerated plants. Whenplantlets had more and stronger roots, they were trans-planted to soil containing vermiculite + perlite + sphag-num moss (1 : 1 : 1) under dim light for 1 week firstand then into natural light.

The phenotypes of Pttkn1 transgenic plants wereobserved carefully, analyzed and photographed using aSONY F 505V digital camera.

Total RNA was isolated from leaves of differenttransgenic plants using the LiCl precipitation methodas described by Verwoerd et al. (1989). RT-PCR wasperformed using the One-Step RNA PCR kit (TaKaRaBiotechnology, Dalian, China) according to themanufacturer’s instructions. The forward and reverseprimers were 5'-GCTGCTCGTCAAGAGTTTGG-3'and 5'-AATCTCAGGTAGTTCAGTCTCCC-3',respectively. The fragment was amplified under thefollowing conditions: one cycle of 50 °C for 30 min;one cycle of 94 °C for 2 min; 30 cycles of 94 °C for30 s, 55 °C for 30 s, and 72 °C for 1 min; and finallyelongated at 72 °C for 5 min. The RT-PCR productswere separated on a 1% agarose gel by electrophoresis

and photographed using an AlphaImagerTM2000Documentation Analysis System.

2 Results and Discussion

2.1 Transformation and expression of the Pttkn1gene

Twenty-one transgenic plants resistant to kanamicin,showing very different phenotypes from wild-typeplants, were obtained. In order to confirm that theseplants were, indeed, transformants induced by thePttkn1 gene, forward and reverse primers were de-signed according to the sequence of Pttkn1 and totalRNA was isolated from the leaves of both normal petu-nia plant (wild type) and different transgenic lines show-ing distinct phenotypes (with small leaves, disk-shapedleaves, lobed leaves, spoon-shaped leaves, normal leavessimilar to wild type, spoon-shaped leaves from plantswithout an inflorescence stem, and small leaves fromplants with a flattened stem). RT-PCR was performedand the results demonstrated that Pttkn1 was stronglyexpressed in four lines and was weakly expressed intwo lines, except for one in which the phenotype wassimilar to wild type (Fig. 1, lanes 6–12); no signal wasdetected for the wild type ( Fig. 1, lane 5).

According to the results of RT-PCR, we believedthat the Pttkn1 gene was introduced into P. hybridaplants and that the novel phenotypes of the

Fig. 1. RT-PCR analysis of Pttkn1 (Populus tremula ×tremuloides knotted1) gene expression. Total RNA wasisolated from the leaves of wild-type and transgenic lines.M, marker; lane 1, no RNA; lane 2, no Taq; lane 3, noreverse transcriptase; lane 4, no primer; lane 5, wild type;lane 6, small leaves; lane 7, disk-shaped leaves; lane 8,lobed leaves; lane 9, spoon-shaped leaves; lane 10, nor-mal leaves similar to wild type; lane 11, small leaves from aplant without an inflorescence stem; lane 12, small leavesfrom a plant with a flattened stem. The 300 bp indicatesPttkn1-specific bands.

Xin HU et al.: Ectopic Expression of the Pttkn1 Gene Induces Alterations in the Morphology of the Leaves and Flowers inPetunia hybrida 1155

transformants distinguished from wild-type plantswere, indeed, conferred by ectopic expression of thePttkn1 gene.2.2 Phenotypic analysis of transformants

Normal petunia plants generally have one main in-florescence stem that regularly generates shoots orovate-shaped leaves with short petioles and a smoothleaf margin (Fig. 2a), whereas transgenic petunia plantsectopically expressing Pttkn1 showed phenotypes eas-ily distinguished from the wild type. We initially dividedthese transgenic petunia plants into four classes basedon leaf and gross morphology. The first classtransformants exhibited dwarf and bushy phenotypeswith small leaves, shortened internodes or withoutinflorescence, most of which lost apical dominance (Fig.2b). The second class transformants had several shootswith shortened internodes, nearly disk-shaped leaves,which were curled (concave and convex) and bentdown at their tips (Fig. 2c). The third class transfor-mants had flattened inflorescence stems, some of which,like palm, generated very small leaves or shoots withvery small leaves, and also lost apical dominance (Fig.2d). The fourth class transformants had a main stemwith curled or lobed leaves (two to five lobes), butwithout regular phyllotaxy (Fig. 2e). These pheno-types showed that ectopic expression of Pttkn1 hadaltered the plant architecture in petunia transfor-mants.2.3 Development of leaves in transgenic plants

Leaves of petunia transformants were quite differ-ent from the wild type (Fig. 2k), including alterationsin leaf size, shape, length of the petioles, and leafvenation. The leaf morphology of Pttkn1 transformantswas also divided into four groups as follows: (i) smalland narrow leaves without prominent main veins andpetioles; (ii) small and spoon-shaped leaves withoutprominent midribs; (iii) lobed leaves (two to five lobes)without petioles, transformed simple leaves to com-pound leaves; and (iv) normal-sized or small, curled,nearly disk-shaped leaves with shortened midribs andwithout petioles (Fig. 2k). Leaf venations in transgenicpetunia plants were also changed compared with the

wild type (Fig. 2k). In addition, ectopic shoots couldbe seen on the adaxial surface of the group iv leaves.All these phenotypes suggested that ectopic expres-sion of Pttkn1 affected cell differentiation and devel-opment of the leaf. A similar phenotypic abnormalityhas been observed in transgenic plants overexpressingknox genes (Chuck et al. 1996; Tamaoki et al. 1997;Nishimura et al. 2000; Rosin et al. 2003). The similar-ity in the phenotypes of transgenic plants overexpressingknox genes suggests that knox genes from various plantsfunction in a similar manner and that these genes mayaffect common target genes when expressed at a highlevel in transgenic plants.2.4 Development of flowers in transgenic plants

Untransformed normal flowers (wild type) consistof five long and narrow sepals, five identical petals thatunite into an entire funnel-shaped corolla, four long andone short stamen, and a pistil, in which the style is alittle shorter than the stamens. The corolla of wild-typeflowers is white with five red stripes and its abaxialsurface is smoothy without knots along the midrib (Fig.2j, l). Both stamens and the pistil in wild-type flowersare not visible at the early stage of flower development(Fig. 2f). In contrast, Pttkn1 transformants displayeda series of alterations in flower development. All flow-ers of transformants were smaller and the elongationof the corollas was delayed compared with the wildtype. Bold stamens and pistils were visible, uncoveredby corollas at the early stage of flowering (Fig. 2g). Afused flower, consisting of eight different-sized sepalsand two pistils, was found in class iv transformants(Fig. 2h). Sepals in Pttkn1 transformants were muchsmaller than those of the wild type (Fig. 2m, n). Tu-mor-like knots along the midrib on the abaxial surfaceof the corolla are shown in Fig. 2m, n. One or severalectopic spikes were observed on the adaxial surfacesof the corolla in class ii transformants (Fig. 2o). Wealso found that flower shape was changed intransformants and petals could not completely fuse intoone corolla, as in the wild type, and the margins of thepetals were significantly serrated (Fig. 2o). The colorof the petals in transformants was quite different from

Journal of Integrative Plant Biology (Formerly Acta Botanica Sinica) Vol. 47 No. 10 20051156

Fig. 2. Phenotypes of both wild-type and transgenic petunia plants. a. Wild-type plant. b. Class 1 transformantsexhibiting dwarf and bushy phenotypes with small and spoon-shaped leaves. c. Class 2 plants with curled and nearlydisk-shaped leaves (arrowheads indicate concaves). d. Class 3 lines showing a flattenned stem with small leaves. e.Class 4 lines with lobed leaves. f. Wild-type flower bud without visible stamens and pistil. g. A transgenic flower budshowing visible stamens (arrowhead) and pistil (arrow). h. A fused flower showing eight different-sized sepals and twopistils (arrows). i. Ectopic shoots on leaves (arrow). j. Wild-type flower showing the adaxial surface with five red strips.k. Leaf phenotypes, wild-type leaf (wt), small and narrow leaf (I), spoon-shaped leaf (II), lobed leaf (III), and nearly disk-shaped leaf (IV). l. Wild-type flower showing a smooth abaxial surface. m. Long knots (arrows) along the midrib on theabaxial face of a transgenic corolla. n. Pocket-like knots (arrows) on the midrib. o. spikes (arrows) on the adaxial face ofthe transgenic petal. p. Ectopic small petals (arrows) on the abaxial face of the transgenic corolla. Bars, 3 cm (a–e); 0.5cm (f–i); 2 cm (j–p).

Xin HU et al.: Ectopic Expression of the Pttkn1 Gene Induces Alterations in the Morphology of the Leaves and Flowers inPetunia hybrida 1157

that of the wild type and the red color was much in-creased in transformant corollas (Fig. 2o). It isinteresting that two small petals formed ectopically onthe abaxial surface of a corolla (Fig. 2p), which hasnot been reported previously in studies of the knox genefamily. These phenotypes of the transformants indi-cate that ectopic meristems were generated on bothsurfaces of the corolla in petunia transformants andthat ectopic expression of the Pttkn1 gene intransformants altered the cell fate from determinanceinto indeterminance. It is well known that phytohor-mones regulate plant development and morphogenesis.A high auxin-to-cytokinin ratio leads to root formationin calli, whereas a low auxin-to-cytokinin ratio favorsshoot formation (Frank et al. 2000). GAs are wellknown to promote stem elongation in a variety of plants.Nester and Zeevarrt (1988) found that the corolla didnot develop in a gibberellin (GA)-deficient tomatomutant. At the early developmental stages of the petu-nia flower, the anther produces a signal that promotescorolla pigmentation and growth. This signal appearsto be GA. It seems that GA is required only for theinduction of corolla growth and anthocyanin synthesisand not for the maintenance of these processes (Weissand Halevy 1989).

Several lines of evidence suggest a link between theknox genes and hormone signaling pathways. Markeddecreases in GA1 and increases in cytokinin were ob-served in Nicotiana tabacum homeobox 15 (NTH15)-or Oryza sativa homeobox 1 (OSH1)-transformed to-bacco plants (Tamaoki et al. 1997; Kusaba et al. 1998).A decrease in GA1 was also found in potato plantsoverexpressing potato homeobox 1 (POTH1) (Rosinet al. 2003). Sakamoto et al. (2001) found that NTH15inhibited GA biosynthesis by suppressing the expres-sion of Ntc12, which is the GA 20-oxidase gene oftobacco. Frugis et al. (2001) reported that lettuce leavesoverexpressing KNOTTED-like from Arabidopsisthaliana 1 (KNAT1) acquired characteristics of inde-terminate growth typical of the shoot and that thischange in cell fate was associated with the accumula-tion of isopentenyl-type cytokinins. The phenotypes of

overexpressing KNAT2 transgenic plants suggested thatKNAT2 acts synergistically with cytokinins (Hamant etal. 2002).

In the present study, the Pttkn1 transformantsshowed ectopic shoots on the adaxial surface of leavesand ectopic spikes, petals, and tumor-knots on theadaxial or abaxial surface of corollas. The elongationof all corollas in transgenic petunia plants was delayed.In addition, we found that leaf explants from Pttkn1transgenic plants easily and rapidly differentiated outshoots under in vitro culture compared with the wildtype. These results indicate that Pttkn1 transformantscould contain high levels of cytokinins and low level ofGAs. The data presented here suggest the possibilitythat Pttkn1 functions as a morphological regulator genethat affects plant hormone metoblism either directly orindirectly, thereby causing changes in plantdevelopment. The mechanism(s) by which Pttkn1 af-fects hormonal activity remains to be elucidated.Acknowledgements The authors thank Dr. OlofOlsson (Göteberg University, Göteberg, Sweden) forproviding the plasmid containing 35S::Pttkn1.

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