Characterizations of the uro Mutant Suggest that the URO Gene Is Involved in the Auxin Action in Arabidopsis

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1 Acta Botanica Sinica 2004, 46 (7): Characterizations of the uro Mutant Suggest that the URO Gene Is Involved in the Auxin Action in Arabidopsis GUO Ying-Li 1, 2*, YUAN Zheng 1*, SUN Yue 1, 3**, LIU Jing 2, HUANG Hai 1*** (1. State Key Laboratory of Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, Shanghai , China; 2. School of Life Sciences, University of Science and Technology of China, Hefei , China; 3. College of Life Sciences, East China Normal University, Shanghai , China) Abstract: The Arabidopsis gene UPRIGHT ROSETTE (URO) was previously identified as a leaf developmental regulator, as all rosette leaves of the semi-dominant upright rosette (uro) mutant grow uprightly at seedling stages. Here, we report more detailed phenotypic characterizations of the uro mutant and show that the URO gene has multiple functions in plant development. In addition to its aberrant leaf-growing pattern, the uro mutant displayed pleiotropic phenotypes. Both uro/+ and uro/uro plants showed a loss of apical dominance, while such a phenotype in the uro/uro plants appeared more severe. Some secondary branches of the uro/uro plants were replaced by leaves, for which petioles were attached to the abaxial side of leaves. Flowers often exhibited varying abnormalities, with altered numbers of petals and stamens and abnormally fused organs. Stems of the uro mutant were soft, which was caused by lacking interfascicular fiber. In addition, vascular differentiation in mutant stem was delayed. The loss of apical dominance and the defects in vascular development and interfascicular fiber formation suggest that the URO function might be associated with auxin-mediated plant development. To provide more direct evidence whether the URO is involved in auxin action, we examined the URO function in auxin polar transportation pathway by analyzing pinformed1 (pin1) uro double mutant. Phenotypes of the double mutant suggest that URO and PINFORMED1 (PIN1) have partial genetic interactions in plant development, which further supports the hypothesis that the URO gene may play an important role in the auxin regulatory pathway. Key words: Arabidopsis ; auxin; interfascicular fiber; pinformed1; upright rosette The plant hormone auxin plays a critical role in the regulation of plant growth and development, including cell division and expansion, vascular tissue differentiation, root initiation, apical dominance, gravitropic and phototropic responses, fruit ripening, leaf senescence, and abscission of leaves and fruits (Eckardt, 2001). In recent years, our understanding of auxin action in several research fields has been advanced, especially in the auxin polar transport, auxin metabolism, molecular basis of gene responses to the auxin, and auxin-directed protein degradation. In particular, many genes that function in the auxin-related regulatory pathways have been uncovered mainly by studies of auxindefective mutants, and the functional characterizations of these genes provide new insights into the plant development (Kepinski and Leyser, 2002; Muday, 2002; Vogler and Kuhlemeier, 2003). It is widely expected that the forward genetic approach will be very useful in the future in dissecting auxin regulatory pathways by analyzing more novel relevant mutants. We reported in our previous work the identification of several Arabidopsis mutants that are defective in leaf development (Sun et al., 2000). One of these mutants, named upright rosette (uro) that was isolated from the T-DNAinsertional mutagenesis population, demonstrated abnormal leaf growth pattern. Each rosette leaf at seedling stages had a smaller angle to the primary inflorescence stem than that in the wild-type leaves. Genetic analysis demonstrated that the uro phenotype was caused by a semi-dominant nuclear mutation, and the URO locus was mapped to the short arm of chromosome 3, about 37 cm from telomere (Sun et al., 2000). We report in this work the more detailed phenotypic characterizations of the uro mutant. In addition to the abnormal leaf growth, the uro mutation results in pleiotropic defects in the development of stem, Received 4 Nov Accepted 2 Apr Supported by the Hi-Tech Research and Development (863) Program of China (2001AA225031) and the National Natural Science Foundation of China ( ). * Both authors contributed equally to this work. ** Author for correspondence. Tel: +86 (0) ; <sunyue928@hotmail.com>. *** Author for correspondence. Tel: +86 (0) ; <hhuang@iris.sipp.ac.cn>.

2 GUO Ying-Li et al.: Characterizations of the uro Mutant Suggest that the URO Gene Is Involved in the Auxin Action in Arabidopsis inflorescence, floral organs and fruit. The loss of apical dominance and the abnormality in interfascicular fiber in the uro mutant strongly suggest that the URO gene may play an important role in auxin action. 1 Materials and Methods 1.1 Plant materials and growth conditions Arabidopsis semi-dominant mutant uro, which is in the Landsberg erecta (Ler) genetic background, was obtained in our previous work from a T-DNA mutagenesis experiment (Sun et al., 2000). The uro mutant has been backcrossed to the wild-type Ler three times before the phenotypic characterizations. The pinformed1 (pin1) mutant was from Klaus Palme (Max-Planck-Gesellschaft, Koln, Germany). Plants were grown in soil according to our previous conditions (Chen et al., 2000). 1.2 Double mutant construction Double mutant pin1 uro was constructed by a cross between homozygous uro and heterozygous pin1 plants, and the F1 plants that showed uro phenotypes were selfed. F2 segregation data were close to a 9:3:3:1 ratio. In total 470 F2 plants, the distribution was 260 uro, 88 wild type, 87 uro pin1 that showed a novel plant stature and flower phenotypes, and 35 pin Histology Fresh tissues and organs from wild-type and mutant plants were examined under a SZH10 dissecting microscope (Olympus, Japan), and photographed using a Nikon E995 digital camera (Nikon, Japan). Hand sectioning of inflorescence stem was performed using a razor blade to generate thin sections of live material. For anatomical observation, sections were stained with 0.02% toluidine blue for 2 min, and then rinsed with water. Sections were viewed with a Zeiss light microscope (Zeiss, Germany) using bright-field illumination. 1.4 Scanning electron microscopy Fresh flowers and inflorescences were fixed with FAA at room temperature overnight, and then dehydrated through a graded alcohol series of 70%, 85%, 95% and 100% of ethanol, followed by a change to 100% ethanol once, each for 5 min. The specimens were then critical point-dried using liquid carbon dioxide, and mounted on scanning electron microscopy (SEM) stubs. The mounted specimens were sputter-coated with gold and palladdium (4:1) and examined with a scanning electron microscope (Hitachi S- 2460, Japan). 2 Results 2.1 Mutant effects on the plant architecture In contrast to the wild-type Arabidopsis plant (Fig.1A), the heterozygous uro mutant (uro/+) plant was shorter in stature with reduced apical dominance (Fig.1B). Mature wild-type plants usually have a long primary inflorescence stem with a few shorter branches arising from different parts of the primary stem (Fig.1A). However, uro/+ plants did not show such a predominant inflorescence stem, and most branches arose from lower positions of the primary inflorescence stem, very close to the rosettes (Fig.1B). The homozygous uro plant (uro/uro) displayed similar plant architecture to that of the uro/+ plant, but the phenotypes appeared more severe (Fig.1C), with a very short plant stature. In the wild-type, each branch on inflorescence stem was associated with a cauline leaf, which grew at the proximal end of the branch. Interestingly, in uro/uro plants, branches were frequently converted to leaves, for which petioles attached to the abaxial side of the lamina (Fig.1C, arrowhead). We refer to this structure as the lotus-leaf. Since it is usually thought that plant apical dominance is regulated by auxin (Eckardt, 2001), the mutant architecture suggests that the URO gene may be involved in the auxin action. 2.2 Mutant effects on the plant vegetative development During the seedling stages, all leaves except cotyledons in both uro/+ and uro/uro plants were upright (Fig.1D, right; for comparison, see a wild-type plant on the left). Although leaves of both uro/+ and uro/uro mutants were broader (Fig.1F,G) than that of the wild-type (Fig.1E), the patterns of leaf venation kept unchanged. Stems of the uro mutant were soft, and became thicker towards the basal portion (Fig.1H). In the uro/uro plants, most secondary branches were longer than the primary inflorescence stem (Fig.1I). Phenotypic analyses in the uro vegetative growth indicate that the URO gene has multiple functions in regulation of plant development. 2.3 Mutant effects on the reproductive development The uro mutation affected a number of processes in plant reproductive development. First, the flowering time was altered in the mutant plants. In comparison to the wildtype plants, the uro/+ and uro/uro plants delayed in flowering time (Fig.2). Second, although early appearing flowers of the uro/+ and uro/uro looked normal, later flowers showed varying aberrant phenotypes. These included developmentally incomplete flowers, which frequently died before seeds set (Fig.1J, arrowhead), altered petal numbers (Fig.1K), asymmetrically arranged floral organs such as stamens (data not shown) and petals (Fig.1L), and abnormally fused carpels that were sterile (Fig.1M). Finally, mutant plants formed siliques that were usually shorter than those

3 Fig.1. Phenotypes of upright rosette mutant. A C. Plant morphology. A. A wild-type Landsberg erecta (Ler) plant. B. A uro/+ plant. C. A uro/uro plant. Arrowhead shows that the uro/uro plants sometimes generate lotus-leaves. D. A wild-type (left) and a uro/uro seedlings. All rosette leaves of the uro/uro mutant at the seedling stages grow uprightly. E G. Comparison of rosette leaves. E. A Ler rosette leaf. F. A uro/+ leaf. G. A uro/uro rosette leaf. From E to G, rosette leaves are broader in uro/+ and uro/uro mutants than those in the Ler plants, but the venation patterns are similar between wild-type (E) and mutant leaves (F, G). H. A wild-type stem (left) and a uro/+ stem (right), which becomes thicker towards the basal part. I. A primary inflorescence stem (arrowhead) and some secondary inflorescence stems (arrows) in a uro/uro plant, showing that the primary inflorescence stem in the uro/uro plant is usually very short. J. Some uro/uro flowers die before seeds set (arrowhead). K. A uro/uro flower with an increased petal. L. Later uro/uro flowers contain asymmetrically arranged petals. M. Some later uro/uro flowers have increasingly fused carpels. N P. Siliques of the wild-type and mutants. The siliques in the Ler plants (N) are usually longer than those in the uro/+ (O) and uro/uro (P) mutants, and sepals, petals and stamens in the mutants do not detach from siliques even when seeds are mature. Q. A section from the elongation part of a wild-type primary stem. R. A close-up of (Q). S. A section from the elongation part of a uro/+ primary stem. T. A close-up of (S). From (Q) to (T), the endoderm cells in the mutant are proliferated while interfascicular fiber diminishes in this region of the stem. U. A section from basal part of a wild-type primary stem. V. A close-up of (U). W. A section from basal part of a uro/+ primary stem. X. A close-up of (W). From (U) to (X), although interfascicular fibers appear in the basal part of the uro/+ mutant, the cell layers are much fewer than those in the wild type. co, cortex; e, epidermis; en, endodermis; ph, phloem; pi, pith; if, interfascicular fiber; ip, interfascicular fiber precursor; mx, metaxylem; x, xylem. A and B, bars = 1 cm; C and I, bars = 0.5 cm; from D to H and from J to M, bars = 0.1 cm; from N to P, bars = 0.2 cm; Q, S, U, and W, bars = 100 µm; R, T, V, and X, bars = 50 µm. in wild-type plants (Fig.1N), regardless of uro/+ (Fig.1O) or uro/uro (Fig.1P) mutant plants. In addition, sepals, petals and stamens often firmly attached to the silique on fruit maturation (Fig.1O, P). The morphological observations of the uro mutant reveal that the URO functions are involved in the different developmental processes. 2.4 The uro mutant altered endodermis pattern and lacked interfascicular fibers in stems uro plants have very soft inflorescence stem as described (see above). To investigate the defect at the

4 GUO Ying-Li et al.: Characterizations of the uro Mutant Suggest that the URO Gene Is Involved in the Auxin Action in Arabidopsis staining in elongation part of the uro/+ stem was only concentrated in xylem (Fig.1S, T). Although interfascicular fiber precursors existed in the elongation part of the mutant stem, further lignification of the fibers did not seem to be processed. In the basal part of the uro/+ stem, blue staining was observed in interfascicular fibers as well as in xylem; however, fiber cell layers in the mutant (Fig.1W, X) were much fewer than those in the wild-type stem (Fig.1V). Furthermore, the endodermis of the uro/+ (Fig.1S,T,W, X) and the uro/uro (see below) stems were overly proliferated, resulting in a thickened endoderm zone. 2.5 Early abnormalities in uro flower development To better understand the URO regulation in flower development, we analyzed early flowers of the uro mutants by SEM. The wild-type Arabidopsis inflorescence usually produces a continuous and indeterminate number of flower primordia on its flanks (Okada et al., 1991). Even in the late stages of plant development, emergence of new flower primordia was evident (Fig.3A, arrow). In the uro/+ mutant, inflorescences at early developmental stages normally produced flower primordia, similar to that in the wild-type (Fig. 3B, arrow). However, at later stages, some uro/+ plants gave rise to a terminal flower, which was chimeric with different floral organs (Fig.3C). uro/uro plants showed the similar flower development pattern to the uro/+ plants, whereas flower termination in the uro/uro plants appeared even earlier (data not shown). In addition, some floral organs in the uro/uro mutant displayed homeotic changes: Fig.2. Flowering time of the wild-type and upright rosette mutants. Flowering rates are given by the each-day flowering number to divide the total flowers during the first 10 d after flowering. Flowers after this period were not counted because the flowering numbers were decreased to a very low level. The first day for scoring flowers was at day 22 after seed germination, when wild-type plants began to flower. A total of 138, 259 and 90 flowers in the first 10 d were analyzed for the wild-type, uro/+ and uro/uro plants, respectively. cellular level, we analyzed cell types in uro stem by cross sectioning. Fibers can be found in various parts of plants. In stems of the wild-type plants, interfascicular fibers are needed for support of the shoots. We first examined the fiber formation in wild-type plants, and a lignin staining reagent, toluidine blue, was used to show fiber distribution in stems of 5-week-old plants. In the elongation part (Fig.1Q, R) and the basal part (Fig.1U, V) of a wild-type stem, blue staining was clearly seen in vascular bundles and interfascicular fibers that are the arch-shaped cell layers between vascular tissues. In comparison, the blue Fig.3. Scanning electron microscopy of wild-type and upright rosette inflorescences and flowers. A. A wild-type inflorescence. Arrow denotes a new flower primordium emerging from central inflorescence. B. An early-stage uro/+ inflorescence, showing a newly growing flower primordium (arrow). C. A late-stage uro/+ flower inflorescence, which forms terminal flowers with different floral organs fused together. D. A uro/uro flower showing a homeotic conversion with stigmatic papillae grown on a sepal. Inflorescence and flower specimens were from 4-week-old plants.

5 stigmatic tissues that usually topped the gynoecium appeared on the tips of sepals (Fig.3D). The SEM results indicate that the URO functions are required for flower termination and may be involved in the regulation of floral homeotic genes. 2.6 Phenotypes of the pin1 uro double mutant Phenotypes of the uro mutant such as the loss of apical dominance and the block of interfascicular differentiation are generally thought to be associated with the defective auxin regulation (Zhong et al., 1997; Zhong and Ye, 2001; Booker et al., 2003). Therefore, it is possible that the URO function is involved in the auxin-regulated development. Auxin regulation is a very complex physiological process, which relates to several aspects that have been investigated extensively: auxin synthesis and metabolism, auxin polar transport, response of gene expression to auxin and auxin-directed protein degradation. To examine whether URO gene functions in one of these aspects in auxin action, we first tested auxin polar transport by construction of uro and pin-formed1 (pin1) double mutant. PIN1 is a transmembrane protein involved in the auxin efflux in auxin polar transport (Galweiler et al., 1998). Loss of function in the PIN1 protein severely affects organ initiation, and the pin1 mutation is characterized by an inflorescence meristem that does not initiate any flower, resulting in the formation of a naked inflorescence stem (Fig. 4A). Different to the pin1 single mutant, the pin1 uro/+ usually produced a few secondary inflorescences with a cauline leaf at the proximal end of each branch (Fig.4B). Although the pin1 uro/uro had a very short plant stature (Fig.4C), similar to that of the uro/uro mutant (Fig.1C), secondary branch numbers were reduced significantly (26 in 20 pin1 uro/uro plants versus 67 in 20 uro/uro plants). A novel inflorescence phenotype appeared in the pin1 uro/ uro double mutants, which was different from those in either wild-type plant (Fig.4D) or its parents (Fig.4A, E, F). The uro/+ inflorescence (Fig.4E) was quite similar to that in the wild-type (Fig.4D) at the early flowering stage. The inflorescence pattern of the uro/uro was also similar to that of the wild-type, except that the mutant inflorescence was terminal after producing a limited numbers of flowers (Fig. 4F). However, at the later flowering stages, the pin1 uro/+ double mutant usually generates flowers containing exaggerated stigmatic tissues, with a few petals and stamens (Fig.4G). The pin1 uro/uro double mutant produced many flowers. In addition to the abundant stigmatic tissues, flowers of pin1 uro/uro double mutant (Fig.4H) also contained many chimeric floral organs with more petals and stamens than those in the pin1 uro/+ flowers (Fig.4G). This kind of flower often died early and was completely sterile. The cellular pattern in the pin1 uro/uro stem (Fig.4J) reflected the additive phenotypes of both parents (Fig.4I, K), with partially restored interfascicular fibers and thick endodermis. Phenotypic analyses of the pin1 uro double mutant suggest that URO and PIN1 have partially genetic interaction in plant development. 3 Discussion The plant hormone auxin is a crucial player in plant development throughout the life cycle by directing basic developmental processes such as cell division, cell elongation and differentiation. Although recent studies have provided new insights into the molecular bases of auxin action, many aspects of the auxin regulation remain to be addressed. Therefore, characterizations of biological mutations disrupting genes that are involved in the auxinmediated developmental processes would be of great significance. After causal characterizations of the uro mutant, we propose that the URO gene may be one of the important regulators in auxin action for reasons. First, uro mutant demonstrated pleiotropic plant phenotypes, including uprightly growing leaves and the lotus-leaf structure, the thick basal part of stems, terminal inflorescence, organ-fused flowers and homeotic conversion of floral organs. Several other auxin-related mutants showed the similar phenomena, although the varying abnormalities appear in different parts of plants. For example, the predominant phenotypes of Arabidopsis mutants pin1 and pinoid (pid) are that the apical meristems produce rosette leaves and a flowering shoot, but the shoot is frequently devoid of flowers. These auxin-defective mutants also show other phenotypes such as abnormal cotyledons, leaves, stems, and floral organs (Bennett et al., 1995). ettin (ett, Sessions and Zambryski, 1995; Sessions et al., 1997) and monopters (mp, Przemeck et al., 1996; Hardtke and Berleth, 1998) are other two Arabidopsis mutants defective in auxin signaling. Abnormalities of these two mutants appear in different types of floral organs. Since auxin regulates many developmental processes in plant, mutation of a single gene that is involved in the auxin action could cause multiple and totally developmentally unrelated phenotypes. Second, several phenotypes in the uro mutant reflect typical auxin defects in plant. In plant architecture, the apical dominance, whereby the growing apical meristem suppresses the growth of axillary meristems, is mediated by auxin, as first proposed by Thimann and Skoog (1933). The uro mutant shows a clear loss of apical dominance with markedly increased shoot branches, and the secondary

6 GUO Ying-Li et al.: Characterizations of the uro Mutant Suggest that the URO Gene Is Involved in the Auxin Action in Arabidopsis Fig.4. Phenotypes of double mutant between upright rostte and pin-formed1. A C. Morphology of single and double mutants. A. A pin1 mutant plant. B. A pin1 uro/+ plant. C. A pin1 uro/uro plant. D H. Comparison of inflorescences of single and double mutants. D. A wild-type Ler inflorescence. E. A uro/+ inflorescence. F. A uro/uro inflorescence. G. A pin1 uro/+ inflorescence. H. A pin1 uro/ uro inflorescence. In (G) and (H), inflorescences of the uro/+ and uro/uro double mutants produce terminal flowers that are composed of many different floral organs fused together. I K. Analysis of cellular patterns of the stem. I. A section from the pin1 stem, showing abundant interfascicular fibers. J. A section from the pin1 uro/uro stem, showing the reduced interfascicular fiber layers in comparison with that in the pin1 stem (I). K. A section from the uro/uro stem, which contains no interfascicular fibers. All sections were from stems of 4-week-old plants at the similar positions. en, endodermis; if, interfascicular fiber; ip, interfascicular fiber precursor; ph, phloem; x, xylem; and mx, metaxylem. From A to B, bars = 1 cm; from C to H, bars = 0.2 cm; and from I to K, bars = 50 µm. branches are often longer than the primary inflorescence stem. It is generally thought that plant vascular differentiation is regulated by auxin. Arabidopsis gene AXR1 mediates auxin signal, and mutations in the AXR1 gene result in increased branches, with the degree of branching correlating with the degree of insensitivity to auxin in the different alleles (Timpte et al., 1995). Recent studies demonstrate that the expression of AXR1 in xylem and interfascicular sclerenchyma tissues is sufficient to restore wild-type shoot branching (Booker et al., 2003). In the uro mutant, the differentiation of the interfascicular fiber is markedly delayed or even completely aborted, resulting in very soft stems. Furthermore, lotus-leaves (or cup-shaped rosette leaves) are also thought to associate with auxin signal. It was reported that the pin1 mutants frequently show the cup-shaped leaves (Reinhardt et al., 2000). In the tobacco and Brasscia rape tissue culture, the media containing auxin polar transport inhibitors HFCA and TIBA resulted in the formation of lotus-leaves from explants (Ni et al., 1999). Similar to the pin1 mutants, the uro mutant produces the lotus-leaves. Therefore, it is possible that these phenotypes are resulted from the indirect effects of the auxin regulation. Third, polar auxin transport controls different developmental processes in plant, including the formation of vascular tissue. Mutations in the PIN1 gene eliminate polar auxin transport in inflorescence stem (Galweiler et al., 1998), leading to a series of abnormal plant phenotypes: altered

7 cotyledon shapes, aberrant vascular patterns in stem, suppression of the organ outgrowth, and defect in the establishment of organ boundaries (Vernoux et al., 2000). These abnormalities were not seen in the uro mutant throughout the entire plant development. In the pin1 uro double mutant, although most phenotypes are additive, the phenotypes in the inflorescence are novel. These results strongly suggest that URO and PIN1 have partially genetic interactions in the regulation of plant development and the URO gene is involved in the auxin regulation. The uro mutant was from our T-DNA mutagenesis population, and genetic analysis has revealed that a T-DNA insertion is tightly linked to the uro phenotypes (data not shown). The molecular mechanisms of URO actions and more precise gene functions will be clarified with the gene sequences being determined. Acknowledgements: The authors would like to thank Klaus Palme and Masahiko Furutani for providing pin1 seeds, XUE Hong-Wei for bring the pin1 seeds to this laboratory for the experiment, MAO Jian for technical assistance in SEM. References: Bennett S, John A, Gerd B, David R Morphogenesis in pinoid mutants of Arabidopsis thaliana. Plant J, 8: Booker J, Chatfield S, Leyser O Auxin acts in xylemassociated or medullary cells to mediate apical dominance. Plant Cell, 15: Chen C, Wang S, Huang H LEUNIG has multiple functions in gynoecium development in Arabidopsis. Genesis, 26: Eckardt N A New insights into auxin biosynthesis. Plant Cell, 13: 1 3. Galweiler L, Guan C, Muller A, Wisman E, Mendgen K, Yephremov A, Palme K Regulation of polar auxin transport by AtPIN1 in Arabidopsis vasular tissue. Science, 282: Hardtke C, Berleth T The Arabidopsis gene MONOPTEROS encodes a transcription factor mediating embryo axis formation and vascular development. EMBO J, 17: Kepinski S, Leyser O Ubiquitination and auxin signaling: a degrading story. Plant Cell, 14: S81 S95. Muday G An emerging model of auxin transport regulation. Plant Cell, 14: Ni D, Wang L, Xu Z, Xia Z Foliar modifications induced by inhibition of polar transport of auxin. Cell Res, 9: Okada K, Ueda J, Komaki M, Bell C, Shimura Y Requirement of the auxin polar transport system in early stages of Arabidopsis floral bud formation. Plant Cell, 3: Przemeck G, Mattsson J, Hardtke C, Sung Z, Berleth T Studies on the role of the Arabidopsis gene MONOPTERS in vascular development and plant cell axialization. Planta, 200: Reinhardt D, Mandel T, Kuhlemeier C Auxin regulates the initiation and radial position of plant lateral organs. Plant Cell, 12: Sessions A, Nemhauser J, McColl A, Roe J, Feldmann K, Zambryski P ETTIN patterns the Arabidopsis floral meristem and reproductive organs. Development, 124: Sessions R, Zambryski P Arabidopsis gynoecium structure in the wild and in ettin mutants. Development, 121: Sun Y, Zhang W, Li L, Guo Y, Liu T, Huang H Identification and genetic mapping of four novel genes that regulate leaf development in Arabidopsis. Cell Res, 10: Thimann K, Skoog V F Studies on the growth hormone of plants. III. The inhibiting action of the growth substance on bud development. Proc Natl Acad Sci USA, 19: Timpte C, Lincoln C, Pickett F B, Turner J, Estelle M The AXR1 and AUX1 genes of Arabidopsis function in separate auxin-response pathways. Plant J, 8: Vernoux T, Kronenberger J, Grandjean O, Laufs P, Trass J PIN-FORMED 1 regulates cell fate at the periphery of the shoot apical meristem. Development, 127: Vogler H, Kuhlemeier C Simple hormones but comlex signalling. Curr Opin Plant Biol, 6: Zhong R, Taylor J, Ye Z Disruption of interfascicular fiber differentiation in an Arabidopsis mutant. Plant Cell, 9: Zhong R, Ye Z Alteration of auxin polar transport in the Arabidopsis ifl1 mutants. Plant Physiol, 126: (Managing editor: ZHAO Li-Hui)