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1 1 Identification and characterization of abl mutant defected in leaf development in Arabidopsis thaliana Gan-Der Ho and Chang-Hsien Yang1 Graduate Institute of Biotechnology National Chung Hsing University Taichung, Taiwan 40227 ROC 1 To whom correspondence should be addressed 2 2 Abstract To investigate the mechanism regulating leaf formation, a recessive mutation, abnormal leaf (abl), affecting leaf development was isolated from transformants of Arabidopsis tagged with T-DNA in our laboratory. The abl mutants showed dwarf phenotype with abnormal filamentous leaves, short hypocotyl and short roots. A severe reduction (more than 3 times) in plant and leaf size was observed for abl mutants. The anatomical analysis revealed that the ABL gene represents a class of gene which is strictly required for normal leaf sharp, leaf expansion and root elongation after meristematic division in both the root and shoot. abl mutants did not display photomorphogenic development in darkness, indicating that the mutant is not likely impaired in the biosynthesis of brassinosteroids. In contrast to wild-type plants which contained dark green and round rosette leaves, abl mutants produced yellow-green leaves which were not smooth in the edge. abl mutants also showed late-flowering phenotype by producing more rosette leaves and more secondary inflorescence than that in wild-type plants. The early raising flowers in abl mutants were male sterile due to the production of short stamen and immature pollens. The flower produced during late stage were however fertile and generated normal siliques and seeds. To clone ABL gene, IPCR and TAIL-PCR approaches were applied to isolate the DNA sequence flanking the border of the T-DNA. Further cloning and characterization of ABL gene is in progress and should help to understand the cell division and differentiation during leaf development in plants. 3 3 Introduction The maturation of a plant body is accompanied by the continuous production of cells that are sequentially differentiated into the above-ground portion such as leaves, stem, flowers, and the underground portion such as roots (Meyerowitz, 1997; van den Berg et al., 1997). The region harboring the ability to generate new cells, is called the meristem, and is established in the tip of the embryonic shoot and root, named shoot apical meristem (SAM) and root apical meristem (RAM) respectively, during embryogenesis (Medford, 1992; Dolan et al., 1993). Recently, extensive studies on various plant species have shown that meristematic activity is controlled not only by environmental signals such as light but also by endogenous factors such as hormones. For example, darkness can inhibit shoot apical meristem activity (Smith, 1994; McNellis and Deng, 1995), and the balance and transport of auxin can influence meristem maintenance as well as root meristem organization (Avasarala et al., 1996; Kerk and Feldman, 1995). Many different mutants having defects in either shoot or root development have been isolated and characterized and used to identify genes involved in the formation of shoots or roots (Medford et al., 1992; Borton and Poethig, 1993; Talbert et al., 1995; Clark et al., 1995; Laufs et al., 1998; Keddie et al., 1998; Cheng et al., 1995; Benfey and Schiefelbein, 1994; Willemsen et al., 1998). For example, shoot meristemless (stm) and root meristemless (rml) mutants in Arabidopsis are disrupted in the embryonic shoot and root meristem respectively, resulting in the absence of shoot formation in stm and extremely short root in rml after embryogenesis (Borton and Poethig, 1993; Cheng et al., 1995). Other root and shoot mutations in Arabidopsis affect the normal root cell expansion such as cobra and lion's tail (Benfey and Schiefelbein, 1994; Hauser et al., 1995), and the differentiation and expansion in the developing leaf primordia such as curly (crl) (Medford et al., 1992). All of the organs formed from the shoot apical 4 4 meristem in crl mutants are abnormal, including leaf and flower development (Medford et al., 1992). To study the process of development, mutants selection is the easiest way to achieve a clear result and can directly know how a gene work in the pathway. In Arabidopsis, many mutants defected in leaf development have been identified through the EMS mutagenesis or the T-DNA tagging (Berna et al., 1999, Serrano-cartagena et al., 1999). Genes affected leaf development have been cloned such as AGO1, encoded a cytoplasmic protein, ROT3, encoded cytochrome P-450, PFL, encoded ribosomal protein S18 (Berna et al., 1999). However, the exploration of the leaf development compared with other organs and tissues such as flower and root in plants is still remains investigated. Genes that affect the leaf development such as the destruction of the photosynthesis, affection of the cell proliferation, shape and size (Donnelly et al., 1999; Ori et al., 2000), and abnormal venation pattern have been cloned (Candela et al., 1999). The immutans (im) is a mutant of Arabidopsis that has green and white leaf sectors. Leaf anatomy is radically altered in the green and white sectors of im mutants. Mesophyll cell sizes are dramatically enlarged in the green sectors and palisade cells fail to expand in the white sectors. This gene encodes the IM protein, which is a chloroplast homolog of the mitochondrial alternative oxidase (Aluru et al., 2001). Arabidopsis top1 α mutants exhibit irregular pattern of leaf and flower initiation and affects spiral phyllotaxis and organ architecture. The TOP1α gene encodes a type-I DNA topoisomerase and promote the relaxation of supercoiled DNA by introducing a transient single-strand break in the duplex and acts in a number of different DNA metabolism, such as DNA replication, transcription, and repair and chromatin compaction (Takahashi et al., 2002). The asymmetric leaves2 (as2) mutant of Arabidopsis generated leaf lobes and leafletlike structures from the petioles of leaves in a bilaterally asymmetric manner. AS2 gene has been identified that involved in the establishment of a prominent 5 5 midvein and of networks of other veins as well as in the formation of the symmetric leaf lamina, which might be related to repression of class 1 knox homeobox genes in leaves (Semiarti1 et al., 2001). Sometimes destruction of the gene related to leaf development do not show only a single phenotype change to mutants and always accompany with dwarf, sterile, pale leaves. In this study, a leaf development defected mutant caused by T-DNA tagging has been identified and named abnormal leaf (abl). Interestingly, the abl mutant shows multiple phenotypes such as dwarf, filamentous leaves, short roots, shrunk and pale leaves and sterile at the early raising flower. Our findings imply that ABL represents a novel type of regulatory gene in controlling different developmental processes in Arabidopsis. Results Identification and characterization of Arabidopsis abnormal leaf (abl) mutant In a process to transform 35S::TFL construct into Arabidopsis for functional analysis, more than 10 independent transgenic T2 families were generated. A morphological mutant showing variations in shape, color, size, and flowering were observed in a T2 family (Fig. 1A, right). This mutant segregated 1/4 (12/50) in T2 family indicated it is a recessive mutation (Table 1). After germination, the most distinct and interesting phenotypes observed in this mutant were the early disruption of root growth by producing short roots and the production of an abnormal shoot with filamentous leaves (Fig. 1A). In this mutation was identified a gene, named ABNORMAL LEAF (ABL). The expansion of the cotyledons is slower in the abl mutant than in the wild-type plant 3 to 5 days after germination, but, the subsequent development of the cotyledons seems normal (Fig. 1B, right). The hypocotyl of the ars mutants is similar to that in wild-type seedlings (Fig. 1B, C). 6 6 abl mutant produced small and light green leaves Despite the short root and abnormal leaves, the abl mutants can be transferred to soil and grow in greenhouses (Fig. 2A, left). In contrast to wild-type plants (Fig. 2A, right), a severe reduction (more than 5 times) in plant size was observed for abl mutant plants (Fig. 2A, left) during early developmental stage. The mature rosette leaves of abl mutants are much smaller than that in wild-type plants (Fig. 2B-E). Different from wild-type leaves that were dark green in color, the leaves of abl mutants showed much light color (Fig. 2B-E). Beside their small size, the shape of leaves of abl mutants is also significantly different from that in wildtype plants (Fig. 2B-E). abl leaves were not smooth in the edge compared to wild-type leaves which were round shape and showed a smooth in the edge (Fig. 2B-E). abl mutant flowered later and produced more inflorescence shoot than wildtype plants When the inflorescence and flower organs was produced in the wild-type plants. abl mutant, however, still produced small leaves without internode elongation (Fig. 2A). During late development stage, inflorescence was finally produced in abl mutants. Wild-type plants produced about 8 rosette leaves before flowering (Table 2). By contrast, more than 20 leaves in average were produced in abl mutants before flowering (Table 2). Therefore, abl showed the late-flowering phenotypes. Different from wild-type plants that produced only a main inflorescence and 2-3 secondary inflorescence (Fig. 2A, right and 3A, Table 3), the number of inflorescence produced in abl mutant was significantly increased up to three and the secondary inflorescence produced were about 5-7 (Fig. 3B, C, Table 2). 7 7 abl mutant produced abnormal flowers with short flower organs during early development Flowers were produced on the inflorescence of abl mutants. When the flowers were examined, novel flower phenotype with short floral organs was observed in abl mutants. Although all four floral organs were observed in these flowers, they apparently ceased growth early without further development (Fig. 4A, B). These flowers failed to open during whole flower development and remained unsenescent for a long period in the inflorescence (Fig. 4A, B). This phenotype was significantly different from wild-type inflorescence which contained flowers with elongated floral organs (Fig. 4C). When these abl mutant flowers were opened manually, the petals, stamens and carpels failed to elongate (Fig. 4A, B) and were significantly shorter than those in wild-type flowers (Fig. 4C and 3B), resulting in the formation of unopened flowers during flower development (Fig. 4A and B). These early arising abl mutant flowers were sterile since the immature of stamens and carpel and the anthers were unable to produce functional pollens (Fig. 4A, B) when compared to wild-type flowers (Fig. 4C). The severity of alteration in floral organs were gradually reduced in late arising flowers (Fig. 4D, E). These late arising flowers produced functional pollens since seeds were produced. abl mutant did not exhibit photomorphogenic characteristics in darkness Since the ars mutant showed some characteristics of the dwarf phenotype, for example the stunted growth, an investigation of whether cell elongation regulated by light is altered in abl mutant became interesting. When grown in the dark, wild-type seedlings displayed normal skotomorphogenic development by producing elongated hypocotyls, unopened apical hooks, undeveloped (small and 8 8 unopened) cotyledons; and no true leaf development can be observed (Fig. 1D, left). Dark-grown abl mutant seedlings (Fig. 1D, right) showed the similar characteristics of dark-grown wild-type plants (Fig. 1D, left). Dark-grown ars seedlings produced hypocotyls (Fig. 1D, right) longer than those grown in the light (Fig. 1C, right, Table 3). Dark-grown wild-type seedlings (Fig. 1D, left) also produced several times longer hypocotyls than those grown in the light (Fig. 1C, left, Table 3). In addition, the apical hooks of cotyledons were observed in dark-grown abl seedlings (Fig. 1D, right) similar to that observed in dark-grown wild-type seedlings (Fig. 1D, left). In dark-grown abl mutants, roots developed similarly to and were as short as those in light-grown mutants (Fig. 1C, D, Table 3). These results indicate that abl mutation is not due to a defect in brassinosteroids biosynthesis. Discussion A transgenic Arabidopsis mutant abnormal leaf (abl) with defects in both leaf and root development was identified. ABL gene is different from genes such as RML and HOBBIT (HBT) in which the short root phenotype is caused by a defect in meristematic activity at the tip of the root once the genes have been mutated (Cheng et al., 1995; Willemsen et al., 1998). In addition, the abl mutant root is not caused by the radial expansion of single cortical cells. In Arabidopsis mutants, lion's tail is caused by the expansion of the stele cells whereas pompom is caused by cell expansion in both the epidermis and cortex (Hauser et al., 1995). The ABL gene is therefore different from other genes characterized previously and is responsible for the normal cell elongation and cellular organization of the root. Since abl mutants are also defective in leaf development, it is interesting to know exactly what ABL gene function is and if there are any mutant characteristics in common between mutant roots and leaves. The abl mutant can 9 9 initiate leaf primordia indicating that it should contain a functional SAM. This observation differs ars mutants from mutants such as stm and mgoun (mgo) in Arabidopsis in which the function of SAM has been altered (Borton and Poethig, 1993; Laufs et al., 1998). The inability of the young leaf primordia to further develop and expand in the abl mutant leaf may be due to the impairment in cell elongation. This is quite different from the mutant phenotype of some shoot expansion mutants such as crl in which cell elongation seems normal (Medford et al., 1992). This indicated that the function of the ABL gene is required for the expansion of cells which generate from the meristem. Without normal cell expansion for those cells, further cell arrangement and differentiation are altered as observed in abl mutants. This indicates that the ABL gene is also responsible for the maturation and normal cellular organization for these divided cells. Although most light-regulation-defect mutants characterized in Arabidopsis such as de-etiolated (det), constitutive photomorphogenic (cop) and drawf (dwf) also showed a dwarf phenotype (Chory et al., 1991; Deng and Quail, 1992; Azpiroz et al., 1998), the absence of photomorphogenic characteristics appearing in the dark-grown abl mutant, i.e., the developed cotyledons and the production of true leaves, lead to the assumption that ars mutation is not related to a defect in the light regulatory process.. In addition, the overall morphology of these mutants is quite distinct from abl mutants suggesting a different function from the ABL gene. The abl mutants characterized in this study are apparently different from other root or leaf mutants described elsewhere. The ABL gene may represent a novel class of genes required for cell expansion as well as cell organization and further differentiation after meristematic division. The abl mutation has been shown to co-segregate with insertional T-DNA. Isolation of the ABL gene using genomic sequence flanking this T-DNA is in progress, and should lead to a deeper understanding of the function of ABL and the regulation of root and leaf development in plants. 10 10 Materials and Methods Plant material and mutant isolation Primary transgenic lines (T1) were generated by introducing 35S promoter::TFL into Arabidopsis ecotype Columbia. T1 plants were self-pollinated and used to generate T2 progeny. T2 seeds were used to screen for the appearance of abnormal mutations. Wild-type T2 plants from this mutant population were selfpollinated and T3 progeny were used to further test the inheritance of the mutation. Plant growth conditions Seeds were sterilized and planted on agar plates containing 1/2 MS medium (Murashige and Skoog, 1962), kept at 4oC for 2 d, and then grown in growth chambers at 25oC under long-day conditions (16 h light/8 h dark) for 14 d before being transplanted into the greenhouse. The light intensity of the growth chambers was 150 E m-2 s-1. For the dark treatment, the germinated seeds were covered with foil and germinated in growth chambers at 25 oC without any light supplement. Wild-type and homozygous abl mutant seedlings were transplanted to soil and grown in greenhouses. The greenhouses were maintained at 25oC with 16 h of light for long-day conditions. Acknowledgements This work was supported by grants to C-H Y from the National Science Council, Taiwan, Republic of China, grant number: NSC89-2311-B-005-050. 11 11 References Aluru, M.R., Bae, H., Wu, D. and Rodermel, S.R. (2001). The Arabidopsis immutans mutation affects plastid differentiation and the morphogenesis of white and green sectors in variegated plants. Plant Physiology 127: 67– 77. Avasarala, S., Yang, J. and Caruso, J.L. (1996). Production of phenocopies of the Lanceolate mutant in tomato using auxin transport inhibitors. J. Exp. Bot. 47: 709-712. Azpiroz, R., Wu, Y., LoCascio, J.C. and Feldmann, K.A. (1998). An Arabidopsis brassinosteroid-dependent mutant is blocked in cell elongation. Plant Cell 10: 219-230. Benfey, P.N. and Schiefelbein, J.W. (1994). Getting to the root of plant development: The genetic of Arabidopsis root formation. Trends Genet. 10: 84-88. Berna´, G., Robles, P. and Micol J. (1999). 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The REVOLUTA gene is necessary for apical meristem development and for limiting cell divisions in the leaves and stems of Arabidopsis thatiana. Development 121: 2723-2735. van den Berg, C., Willemsen, V., Hendriks, G., Weisbeek, P. and Scheres, B. (1997). Short-range control of cell differentiation in the Arabidopsis root meristem. Nature 390: 287-289. 14 14 Willemsen, V., Wolkenfelt, H., de Vrieze, G., Weisbeek, P. and Scheres, B. (1998). The HOBBIT gene is required for formation of the root meristem in the Arabidopsis embryo. Development 125: 521-531. 15 15 Legends to figures Fig. 1. Phenotypes of the abl mutant seedlings. (A). An abl mutant seedlings grown in the light for 10 days. (B). Wild-type (left) and abl mutant (right) seedlings grown in the light for 10 days. (C). Wild-type (left) and abl mutant (right) seedlings grown in the light for 10 days. (D). Wild-type (right) and ars mutant (left) seedlings grown in darkness for 12 days. Fig. 2. Phenotypes of the mature abl mutant plants. (A). Wild-type (right) and abl mutant (left) plants grown on soil for 5 weeks. During this time, wild-type plants already flowered and produced only a single main inflorescence containing floral bud on the shoot apex whereas abl mutant plants still produced vegetative rosette leaves. (B). The adaxial surface of a mature wild-type (left) and abl mutant (right) leaves. (C). Close-up of the adaxial surface of abl mutant leaf from (B). (D). The abaxial surface of a mature wild-type (left) and abl mutant (right) leaves. (E). Close-up of the abaxial surface of abl mutant leaf from (D). Fig. 3. Inflorescence development in abl mutant plants. (A). Close-up of the main inflorescence containing floral bud on the shoot apex of a wild-type plant. (B). Close-up of the main inflorescence containing floral bud on the shoot apex of a abl mutant plant. (C). abl mutant plants produced several main inflorescence containing floral bud on the shoot apex. 16 16 Fig. 4. Flower development in abl mutant plants. (A) Close-up of the top of inflorescence in an abl mutant plant. Unopened flowers were observed in this inflorescence. Sepals were detached from two mature flowers to explore the inner petal, stamen and carpel. (B) Close-up of an abl mutant flower from (A). (C) Close-up of the flowers on top of inflorescence in wild-type plant. (D) Close-up of an late-arising abl mutant flower. (E) Close-up of another late-arising abl mutant flower.