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The Plant Journal (2011) 38, 433–442 doi: 10.1111/j.1365-313X.2011.04698.x OsIAA23-mediated auxin signaling defines postembryonic maintenance of QC in rice Ni Jun, Wang Gaohang, Zhu Zhenxing, Zhang Huanhuan, Wu Yunrong and Wu Ping* State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, China Received 3 April 2011; revised 21 June 2011; accepted 2 July 2011; published online 18 August 2011. * For correspondence (fax +86 571 88206613; e-mail [email protected]). SUMMARY Although the quiescent center (QC) is crucial to root development, the molecular mechanisms that regulate its postembryonic maintenance remain obscure. In this study, a semi-dominant mutant that exhibits pleiotropic defects in root tissues, which includes the root cap, lateral and crown roots, was isolated. The mutant is characterized by a loss of QC identity during postembryonic development, and the displayed defects result from a stabilizing mutation in domain II of OsIAA23 (Os06g39590). Expression of OsIAA23 is specific to the QC of the root tip during the development of primary, lateral and crown roots. Consistent with OsIAA23 expression in the QC, the auxin signaling marked by DR5p::GUS (ß-glucuronidase) was absent in the QC region of Osiaa23. Transgenic rice plants harboring Osiaa23 under the control of the QHB promoter mimic partially the defects of Osiaa23. These results indicate that the maintenance of the QC is dependent on OsIAA23-mediated auxin signaling in the QC. These findings provide insight into Aux/IAA-based auxin signaling during postembryonic maintenance of the QC in plants. Keywords: Oryza sativa L., root development, quiescent center, OsIAA23, auxin signaling. INTRODUCTION The importance of the quiescent center (QC) to root development has been well documented in Arabidopsis. In Arabidopsis, nearly all of the cells in the root are derived from four types of stem cells. These stem cells are controlled by the QC, which is composed of a small number of mitotically inactive central cells (Benfey and Scheres, 2000). The QC acts to maintain the surrounding stem cells (SC) in an undifferentiated state, and laser ablation of the QC has been shown to cause differentiation of stem cells (van den Berg et al., 1997). Differences in the root structure of rice and Arabidopsis have been noted. In contrast with the single layer epidermis– endodermis structure in Arabidopsis, rice roots undergo eight successive asymmetrical periclinal cell divisions following the first anticlinal division. This generates the epidermis–endodermis, sclerenchyma layer, exodermis and five layers of cortex (Rebouillat et al., 2009). In Arabidopsis, the epidermis and lateral root cap cell files are derived from the same stem cell (Dolan et al., 1993). In rice, the epidermis and lateral root cap cell files derive from independent initials (Coudert et al., 2010). In spite of these differences, the basic developmental model of cell types in rice roots is similar to Arabidopsis (Kamiya et al., 2003a,b; ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd Cui et al., 2007). The molecular mechanism of QC/SC maintenance and the development of different cell files in Arabidopsis have been well studied (reviewed by IyerPascuzzi and Benfey, 2009), while knowledge of these processes in rice, the monocot model plant, remains limited. Auxin is crucial to many aspects of plant development, including lateral and crown root formation, embryonic development, and stem cell maintenance (Inukai et al., 2005; Liu et al., 2005; Woodward and Bartel, 2005; Galinha et al., 2007). Inhibition of auxin transport by N-1-naphthylphthalamic acid (NPA) causes abnormal auxin accumulation, resulting in changes in root tip cell identities (Sabatini et al., 1999). Accordingly, the disruption of auxin transport around the QC in one of the Arabidopsis PIN (PIN-FORMED) mutants, Atpin4, causes a defect in the maintenance of endogenous auxin gradients and leads to abnormal cell division (Friml et al., 2002). PLETHORA (PLT) protein expression patterns are thought to be determined by auxin gradients. The dosage can be translated into distinct cellular responses, and high levels of PLT activity promote stem cell identity and maintenance (Galinha et al., 2007). High-resolution measurements of endogenous indole-3-acetic acid (IAA) concentrations revealed a distinct maximum in the QC 433 434 Ni Jun et al. of the root apex (Petersson et al., 2009). These results indicate that auxin in the QC may play an important role, although direct evidence is not available. Auxin signaling controlled by Aux/IAA in plants has been well reviewed. Briefly, intracellular auxin is perceived by the TIR1/AFB1-3 receptors. This triggers the degradation of AUX/IAA proteins by the ubiquitin–proteasome pathway, prompted by higher intracellular auxin concentrations. Under low intracellular auxin concentrations, Aux/IAA proteins interact and inhibit the activity of AUXIN RESPONSE FACTOR (ARF), thereby blocking the activity of ARFs (Lau et al., 2008). The Aux/IAA comprises four highly conserved domains, designated as domain I, II, III and IV (Abel et al., 1994). A highly conserved core sequence, GWPPV in domain II, is responsible for the rapid degradation of Aux/IAA proteins (Ramos et al., 2001). Mutations in the core sequence block the degradation of AUX/IAAs, which results in constitutive suppression of ARF activities and ultimately causes stabilized mutants due to the interruption of the auxin signaling pathway (Ouellet et al., 2001). Many stabilized mutants of iaa have been isolated. These mutants illustrate the importance of auxin signaling via IAAs in many aspects of root development (reviewed by Overvoorde et al., 2010). This report describes the isolation and characterization of an Aux/IAA gene in rice, OsIAA23. A stabilizing mutation of OsIAA23 exhibited defects in postembryonic maintenance of the QC that caused the disintegration of the root cap and termination of root growth. The initiation of lateral root and crown root primordia was also blocked. Here, the specific expression pattern of OsIAA23 in the root tip QC during postembryogenesis is described, and the impact of OsIAA23 on root tissue development is discussed. These results provide direct evidence of the importance of Aux/IAA-mediated auxin signaling in the maintenance of the QC. RESULTS Isolation and characterization of the mutant A mutant with lateral root (LR) defects was isolated from an ethyl methane sulfonate-mutagenized population of rice ‘Nipponbare’. Mutant plants were self-pollinated and three root phenotypes were distinguished in the offspring. These phenotypes, wild type (WT), reduced number of lateral and crown roots, and defects in both lateral and crown roots, occurred in a 1:2:1 ratio (24:53:23, P = 0.857) (Figure 1a and Table S1). Based on these data, the pleiotropic defects in the root tissues of the mutant are controlled by a semi-dominant single gene. This was confirmed by the trait segregation in a backcross population between WT and the mutant (24:26, P = 0.843). Although the homozygous mutants were able to complete their life cycle, plant growth was inhibited and fertility was greatly affected. The heterozygous mutants were inter- mediate in phenotype and exhibited normal fertility (Figure 1e,f). In addition, neither auxin nor ethylene treatment was able to rescue the defects (Figure S1). To examine the lateral and crown root primordia in the mutant, the GUS reporter gene driven by the OsCYCB1;1 promoter was transformed into the heterozygous mutants and the segregating offspring were analyzed. The GUS staining and cross-sections of WT, the heterozygous mutant and the homozygous mutant plants revealed that the lateral and crown root primordia were absent in the homozygous mutant and the LR primordia were decreased in the heterozygous mutant (Figure 1b–d,g–i). The root gravitropic response in the mutant was also reduced. Wild type, heterozygous mutants and homozygous mutants were grown for 5 days in the g1 direction, followed by growth for 2 days in the g2 direction. The homozygous mutants showed a loss of gravitropic response when compared to the WT, and the heterozygous mutants showed a partial loss of gravitropic response (Figure 1j). Root QC identity is lost in the mutant during postembryonic development To determine whether the root cap, the sensor of gravity, was structurally disrupted in the mutant, the root tips of WT, heterozygous mutants and homozygous mutants were observed at 1, 5 and 10 days after germination (DAG) using a stereomicroscope. The results revealed the presence of normal root caps in both the heterozygous and homozygous mutants at 1 DAG (Figure 2a1–a3). However, at 5 DAG, the root cap was completely absent in the homozygous mutants and the size of the root cap was reduced in the heterozygous mutants (Figure 2b1–b3). At 10 DAG, root hairs in the root tip were observed, indicating the degradation of the root meristem and the termination of root growth in the homozygous mutant (Figure 2c1–c3). To further investigate the root tip cell organization in the mutant, longitudinal sections of root tips were examined. At 5 DAG, the root cap of the homozygous mutant was lost. Additionally, the cell pattern in the meristem was abnormal when compared with WT. An abnormal cell arrangement in the QC region was also observed (Figure 2h1–i2). To determine the QC identity of the mutant, the GUS reporter gene controlled by the QHB promoter (Kamiya et al., 2003b) was transformed into the heterozygous mutants, and the GUS staining was examined in WT, heterozygous mutants and homozygous mutants. Consistent with the previously reported QHB expression in the QC (Kamiya et al., 2003b), GUS staining of WT revealed GUS activity in this region (Figure 2l). In the homozygous mutant at 5 DAG, no staining was detected in the same region (Figure 2m). At 1 DAG, GUS staining as detected in both WT and mutants (Figure 2j,k). However, a detailed section analysis revealed that abnormal transverse cell divisions had occurred in the mutant when compared to the WT (Figure 2f1–g2). Considering that ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 38, 433–442 Postembryonic maintenance of QC in rice 435 Figure 1. Characterization of the mutant. (a) Root growth of 5-day-old seedlings of the wild type (WT) (left) and the heterozygous (middle) and homozygous mutant (right). Bar = 2 cm. (b–d) Cross-sections at the stem base of the WT (b), heterozygous mutant (c), and homozygous mutant (d). Bar = 0.5 mm. (e) Plants at the grain filling stage. From left to right: WT, the heterozygous mutant, and the homozygous mutant. (f) The panicle of three genotypes. From left to right: WT, the heterozygous mutant, and the homozygous mutant. Bar = 2 cm. (g–i) Stereomicroscopic images of OsCYCB1;1p::GUS in the roots of the three genotypes. From left to right: WT, the heterozygous mutant, and the homozygous mutant. Bar = 0.5 mm. (j) Gravitropic response of three genotypes. Seedlings were grown for 5 days under g1 gravitropic conditions and then moved to g2 where they were grown for 2 additional days. From top to bottom: WT, the heterozygous mutant, and the homozygous mutant. Bar = 2 cm. Figure 2. Time-lapse characterization of the root tip in the mutant. (a1–c3), Stereomicroscope images of the root apex of three genotypes at 1 DAG (a1–a3), 5 DAG (b1–b3), and 10 DAG (c1–c3). From left to right: the wild type (WT), the heterozygous mutant, and the homozygous mutant. Bars = 500 lm. (d1–i2), Vertical sections of the root tip of the WT (left) and the homozygous mutant (right) at 0 DAG (d1–e2), 1 DAG (f1–g2), and 5 DAG (h1–i2). The images shown on the right are magnifications of the images on the left. Bars = 200 lm. (j-m), QHBp::GUS expression in the root tips of the WT at 1 DAG (j) and 5 DAG (l), and of the homozygous mutant at 1 DAG (k) and 5 DAG (m). Bars = 200 lm. (a) (b) (c) (e) (g) (a1) (b1) (c1) (j) (h) (j) (a3) (b2) (d1) (d2) (e1) (e2) (f1) (f2) (g1) (g2) (h1) (h2) (i1) (i2) (b3) (c2) (k) (f) (i) (a2) (d) (c3) (l) (m) ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 38, 433–442 436 Ni Jun et al. normal converged cell files were observed in the mutant at 1 DAG, it is suspected that the abnormal transverse cell divisions occurred in the QC during the 24 h after germination. To verify this hypothesis, germinated mutant seeds without apparent roots were used (designated 0 DAG) for further investigation. The root tips that had not emerged from the seeds were separated for section analysis. No difference in the QC structure was observed between WT and mutant roots (Figure 2d1–e2). Taken together, the results suggest that the maintenance of QC structure in the mutant is impaired during postembryonic root development. (a) Cloning of the OsIAA23 gene A map-based positional approach was used to identify the gene from an F2 population derived from a cross between the heterozygous mutants and indica rice (Kasalath). The mutated gene responsible for the root tissue defects was mapped between two simple sequence repeat (SSR) markers, RM1340 and RM20328, on chromosome 6. This region includes an Aux/IAA family gene, OsIAA23 (Os06g39590). The OsIAA23 gene, sequenced from homozygous mutant genomic DNA, was found to contain a G to A base pair substitution causing a glycine (G) to glutamate (E) change (Gly64Glu) in domain II of OsIAA23 (Figure 3a). To confirm this result, a dCAPS marker was developed. Using this marker, all of the homozygous mutants produced higher mobility bands than WT, and all of the heterozygous mutants produced double bands (Figure 3b). To investigate whether the root tissue defects in the mutant were caused by the mutation of OsIAA23, WT plants were transformed with the mutated gene under the control of its native promoter. The majority of the transgenic plants exhibited phenotypes characteristic of heterozygous Osiaa23, including fewer LRs and smaller root caps. A few transgenic plants also exhibited phenotypes characteristic of the homozygous Osiaa23 (Figure 3c). Sequencing analysis found an allelic mutant of Osiaa23-2. The second allele uncovered a point mutation in the domain II of OsIAA23, resulting in a change of proline (P) to leucine (L) (Pro67Leu). The phenotypic traits of Osiaa23-2 roots were similar to those of Osiaa23 (Figure S2). These results confirm that the root tissue defects in the mutant are caused by the point mutation in domain II of OsIAA23. Response to auxin is reduced in Osiaa23 To investigate the auxin response of the Osiaa23 mutant, WT and Osiaa23 mutants were treated with a-naphthalene acetic acid (NAA). The reduction of root length in WT was more significant than that of the Osiaa23 mutant (Figures 4(a) and S1b). Moreover, the root hairs of WT were notably induced by NAA, while the development of root hairs in Osiaa23 mutant was not affected by NAA treatment (Figure 4b). These results indicated a reduced sensitivity to auxin in the (b) (c) Figure 3. Map-based cloning and complementation test of OsIAA23. (a) A point mutation was detected in the mutant, which resulted in a G to A base pair change of an Aux/IAA gene (OsIAA23), causing a change of glycine (G) to glutamate (E) in the conserved core sequence of domain II. The exons of OsIAA23 are indicated by thick rectangles, and introns are indicated by thin lines. The four domain positions are indicated by black boxes. (b) Confirmation of the point mutation in Osiaa23 using the dCAPS marker. From left to right: the WT, the heterozygous Osiaa23, and the homozygous Osiaa23, respectively. (c) Two transgenic plants containing the Osiaa23 gene exhibited phenotypes characteristic of the heterozygous and homozygous Osiaa23. Osiaa23 mutant. When the Osiaa23 mutant was treated with a higher concentration of NAA (1 lM), a few LRs were induced in the Osiaa23, although other defects were not rescued (Figure 4a). To examine the auxin response of the Osiaa23 mutant at the molecular level, the expression of OsIAA23 and two other early auxin-responsive genes, OsIAA20 (Jain et al., 2006) and ARL1/CRL1 (Inukai et al., 2005; Liu et al., 2005), were investigated in the roots of the Osiaa23 mutant using real-time reverse transcription polymerase chain reaction (RT-PCR) analysis. The results showed that the induced expression of these genes was suppressed significantly in Osiaa23. Additionally, the results showed that OsIAA23 is induced by exogenous auxin (Figure S2). To further investigate the influence of the stabilizing mutation of OsIAA23 on auxin signaling, the expression ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 38, 433–442 Postembryonic maintenance of QC in rice 437 Figure 4. Alteration of auxin response in the Osiaa23 mutant. (a) Root growth performance of the wild type (WT; left) and Osiaa23 (right) plants grown in a solution culture with 1 lM NAA for 7 days. Bar = 1 cm. (b) Proliferation of root hairs at the root apex of WT (left) and Osiaa23 (right) induced by 0.1 lM NAA treatment. Bar = 0.5 mm. (c, d) Cross-sections indicate DR5p::GUS expression in the root zone with the initiation of lateral root (LR) primordia of WT (c) and Osiaa23 (d). Bars = 20 lm. (e, f) GUS staining of cross-sections at the shoot base with the initiation of crown root primordia in WT (e) and Osiaa23 (f). Bar = 0.5 mm. (g, h) GUS staining of the root apex of 1 d-old seedlings of WT and Osiaa23. Bars = 100 lm. (a) (b) pattern of DR5p::GUS was determined. Although Osiaa23 failed to form lateral or crown roots, a significant difference in the location of lateral and crown root primordia formation was not detected between WT and Osiaa23, respectively (Figure 4c–f). The DR5p::GUS pattern in the Osiaa23 root tip was examined at 1 DAG to exclude the interference of the irregular cell pattern in the mutant. At 1 DAG, GUS staining was observed in the columella, QC and metaxylem in WT (Figure 4g). In contrast, no GUS staining was observed in the QC region of the mutant (Figure 4h). The DR5p::GUS expression pattern indicates that auxin signaling is blocked in the QC region of the Osiaa23 mutant. Expression of OsIAA23 is specific to the QC in the root tip Using RT-PCR, OsIAA23 expression in the various organs of WT plants was analyzed. OsIAA23 was found to be constitutively expressed in all of the examined tissues, although a lower abundance was observed in the embryo (Figure S4d). WT plants were transformed with a GUS (b-glucuronidase) gene driven by the OsIAA23 promoter to characterize further the expression pattern of OsIAA23. GUS staining was observed in the vascular tissues of stems and leaves (Figure S3e,f) and in the anthers (Figure S3b,cIn the maturation zone of the root, dense staining was noted in the stele and epidermis (Figure 5d,e). In contrast, the GUS staining in the root tip was exclusively located in the QC (Figure 5a,c). (c) (d) (e) (f) (g) (h) The OsIAA23 expression pattern was similar in the LRs and crown roots (Figure 5b). Because the QC structure in the root was altered during postembryonic development (Figure 2d1–i2), the temporal and spatial expression pattern of OsIAA23 was further investigated. Although OsIAA23 was expressed in the QC during postembryonic development, a uniform expression pattern was observed in the root apical meristem of the embryo (Figure S4d). During the process of LR development, OsIAA23 was expressed initially uniformly in the primordium during early stages, then the expression gradually converged until it was ultimately restricted to the QC of the lateral roots (Figure 5f,g). A similar pattern was found during the development of crown root primordia (Figure 5h–j). The maintenance of the QC is dependent on OsIAA23mediated auxin signaling The QC-specific expression of OsIAA23 in the root tip (Figure 5a,c) and the loss of auxin signaling in the QC region (Figure 4h) indicated that OsIAA23-mediated auxin signaling in the QC may be crucial to the maintenance of the QC. Five transgenic lines harboring Osiaa23 driven by the QHB promoter (Kamiya et al., 2003b) were generated to investigate this hypothesis (Figure 6a). As expected, the transgenic plants exhibited smaller root caps. Vertical sections of the root tips showed abnormal divisions of QC cells in the ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 38, 433–442 438 Ni Jun et al. (a) (c) (b) (d) (e) (g) (f) (h) Figure 5. Expression pattern of OsIAA23 revealed by a GUS reporter line. (a, b) Stereomicroscope images showing GUS staining in the primary root tip (a) and the lateral root (LR) (b). Bar = 200 lm. (c) Vertical section of the root tip of the transgenic plants. (d, e) Cross section at the matured root zone with the initiation of LR primordia of the transgenic plants and the magnified view of the stele (e). Bar = 20 lm. (f, g) OsIAA23 (f) and QHB (g) expression during the process of LR primordia development. Bar = 20 lm. (h–j) Cross-sections at the shoot base. (h), (i) and (j) are different stages of crown root primordia. Bar = 100 lm. (i) (j) transgenic plants (Figure 6e–j). Additionally, the numbers of lateral and crown roots were reduced in the transgenic plants (Figure 6b–d). These results provided evidence that OsIAA23-mediated auxin signaling regulated postembryonic maintenance of the QC in rice. DISCUSSION This study reports the isolation of a stabilized rice mutant of OsIAA23. This mutant, caused by a point mutation in the conserved core sequence GWPPV of domain II of OsIAA23, results in pleiotropic defects in the root tissues. These defects include formation of the root cap, lateral and crown roots, and the maintenance of QC identity during postembryonic development. Our report describes a stabilized Aux/IAA mutation with pleiotropic effects on root development in rice. As a distal organizer in the root apex, the disappearance of QC identity results in the disintegration of the root cap and the impairment of the root apical meristem. Previous reports demonstrated that loss-of-function of IAAs did not result in visible defects (Overvoorde et al., 2005). Screening for revertants of Osiaa23 mutant plants identified one plant that contained a mutation in domain IV of the protein encoded by Osiaa23 that displayed a normal phenotype (Figure S6). Domain IV in IAA proteins is crucial for the interaction between IAAs and ARFs (Ulmasov et al., 1997). The rescue of the iaa23-dependent phenotype suggests that the point mutation that leads to a change in amino acid in domain IV also leads to loss-of-function of the stabilized OsIAA23. This result also indicates that loss-offunction of OsIAA23 does not affect root growth, due to functional redundancy of IAA proteins. OsIAA23-mediated auxin signaling is crucial to the identity of the QC during postembryonic root development The QC controls the undifferentiated states of the nearby SC, and loss of the QC results in the differentiation of these stem cells (van den Berg et al., 1997). Two types of SC are responsible for the production of the root cap, the first type ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 38, 433–442 Postembryonic maintenance of QC in rice 439 (a) (c) (b) (d) (e) (f) (g) et al., 2003). It is notable that no clear difference in the GUS staining in the root cap and stele, except for the QC region, was found between the WT and Osiaa23 mutant (Figure 4c–f). This finding suggests that the QC may be the only tissue in which there is no functional redundancy of Aux/IAA-mediated auxin signaling. It has been proposed that auxin signaling is crucial for root stem cell niche patterning during both embryonic and postembryonic development (Aida et al., 2004). The specification of the QC in embryonic development requires auxin signaling (Berleth and Jurgens, 1993; Hardtke and Berleth, 1998; Hamann et al., 1999, 2002). The data presented here suggest that another OsIAA (or other OsIAAs) may mediate the auxin signaling of the QC during embryonic development. This hypothesis is supported by the spatio-temporal expression of OsIAA23 in both the primary root (Figure 5a) and the lateral or crown root primordial development (Figure 5f–j). QC identity during postembryonic development determines lateral root development in rice (h) (i) (j) Figure 6. Phenotype and histological characterization of the QHBp::Osiaa23 transgenic rice. (a) The construct of QHBp::Osiaa23. (b) Root phenotype of the wild type (WT) and two lines of transgenic rice. Bar = 2 cm. (c, d) Number of lateral roots (LR) (c) and crown roots (d) of 7 d-old WT and transgenic rice. The data represents a mean of five plants and the bar indicates the SD. (e–j) Vertical sections of the root tip of the WT (e) and two transgenic lines (f, g). (h), (i) and (j) are magnifications of (e), (f) and (g). Bar = 200 lm. is the columella initial cell and the second is the LR cap initial cell. These cells generate the columella cells and LR cap cells, respectively (Coudert et al., 2010). With the loss of QC identity, the root cap SC differentiate and stop supply of new root cap cells. Up to 1 DAG, no change in the root cap structure of Osiaa23 was observed when compared with the root cap of WT. However, at 5 DAG, the root cap had disappeared in Osiaa23 (Figure 2). This situation indicates that OsIAA23-mediated auxin signaling is crucial to the identity of the QC during postembryonic root development. This conclusion is supported by other evidence: the QC identity of Osiaa23 was lost gradually during postembryonic development (Figure 2), and the auxin signaling indicated by the DR5p::GUS reporter gene is blocked in the QC region of the root tip (Figure 4h). DR5, a synthetic promoter, has been widely used to monitor auxin distribution in vivo and auxin responses at the cellular level (Sabatini et al., 1999; Benkova The development of lateral and crown roots is a postembryonic event (Rebouillat et al., 2009). We investigated the earlier stage of root on the LR primordium at 1 day after germination and did not found lateral root primordium. In roots, auxin is transported acropetally through the central cylinder toward the root tip. There, it is redistributed into a basipetal stream through the LR cap and the epidermis, and then refluxes upward along the meristem back into the central downward flow to complete the auxin loop (reviewed by Kepinski and Leyser, 2005). Pericycle cells in the basal meristem require increased auxin levels to attain founder cell identity for the development of LR primordia (Dubrovsky et al., 2008). The Osiaa23 mutant exhibits defects in both the stele and root cap, which may disturb the auxin loop in the root tip and ultimately result in LR defects. Acropetal to basipetal auxin signaling loop from LR cap is important for lateral root development. Thus impairment of root cap development during postembryonic growth in Osiaa23 mutant may disturb auxin signaling for LR primordium initiation. Up to 1 DAG, the changed central cylinder structure of Osiaa23 was observed compared with WT, although the root cap structure was still maintained (Figure 2f2,g2). In this case, the mechanism of pericycle differentiation is unclear. In Arabidopsis, it was previously reported that the roots of the ahp6 (histidine phosphotransfer protein 6) mutant (Mahonen et al., 2006) and transgenic plants harboring Pro35S-VND7:SRDX (vascular-related NAC-Domain7 fused to the strong repression domain SRDX) (Kubo et al., 2005) showed perturbations in protoxylem differentiation. However, neither pericycle differentiation nor LR initiation was disturbed in these plants, which suggested that protoxylem is dispensable for these morphogenetic events (Parizot ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 38, 433–442 440 Ni Jun et al. et al., 2008). Based on the data presented here, it is hypothesized that the impairment of the acropetal and basipetal auxin signaling loop through the central cylinder and the LR cap in Osiaa23 results mainly in the impairment of lateral root primordia initiation at an earlier stage, while at a later stage, the abnormal central cylinder structure may affect the development of lateral root primordia initiation. Recent studies present evidence that a process involving oscillations in gene expression is the first step in positioning new LRs (Moreno-Risueno et al., 2010). It is possible that some oscillating transcriptional regulators may be disturbed in Osiaa23. It is proposed that OsIAA23 does not act only in the QC; it is expressed in various tissues and the reduced sensitivity to exogenous auxin(reduced root hairs numbers in response to exogenous NAA (Figure 4, 5). Transgenic rice harboring a constitutive expressed mutant Osiaa23 (QHBp::Osiaa23) resulted in a phenocopy of Osiaa23 under its native promoter, especially in the root tip (Figure 6), while did not show the reduced sensitivity to exogenous auxin (Figure S5). These results suggest that OsIAA23 does not act only in the QC, but auxin signaling mediated by OsIAA23 in the QC plays a unique role in QC maintenance. METHODS Plant material, growth conditions and mutant screening Reduction of auxin signaling mediated by OsIAA23 is important for crown root primordia development The initiation of crown root primordia, initiated from the parenchyma cells adjacent to the peripheral vascular cylinder of the shoot base, is also controlled by auxin signaling (Inukai et al., 2005; Liu et al., 2005). The data presented here reveal that the expression of CRL1 (ARL1), a key regulator for crown root primordia initiation, is not changed in Osiaa23 under normal growth conditions. However, the response of the gene to exogenous auxin is reduced markedly (Figure S3c). This situation suggests that the reduction of local auxin signaling caused by the stabilized mutation of OsIAA23 may be important for crown root primordia development. Furthermore, the OsIAA23-regulated factor(s) for crown root primordia development may be downstream of CRL1(ARL1). The emergence of crown roots can be enhanced by flooding and is mediated by ethylene action (Mergemann and Sauter, 2000). In this study, ethylene was not able to rescue the defect of crown roots in Osiaa23 (Figure S1c). This outcome is reasonable because histological analysis showed that the crown rootless phenotype of Osiaa23 was caused by the defect of primordia initiation, not the emergence of the primordia (Figure 1d). OsIAA23 does not act only in the QC, but auxin signaling mediated by OsIAA23 in the QC plays a unique role in QC maintenance. Several indirect pieces of evidence have implicated auxin signaling in QC maintenance. NPA treatment leads to the abnormal auxin accumulation in the root tip and results in unusual QC identities (Sabatini et al., 1999). AtPIN4 is expressed around the QC region. Atpin4 mutants are defective in the establishment and maintenance of endogenous auxin gradients, which results in abnormal cell divisions in the QC (Friml et al., 2002). High-resolution measurements of endogenous IAA concentrations revealed a distinct maximum in the QC of the root apex. However, the function of this high auxin concentration was unknown (Petersson et al., 2009). The mutant was isolated in an EMS-mutagenized population (Nipponbare). Rice were grown in culture solution (Yoshida et al., 1976) in a growth room with temperature regimes of 28C/22C (day/night) and 70% humidity under a 12-h photoperiod. The mutant with defects in LRs was isolated from 7-day-old seedlings. Histological observation The roots of 0 DAG were manually dissected under the stereomicroscope, and the procedures of staining, dehydration, clearing, infiltration, and embedding were performed according to Liu et al. (2005). The microtome sections were mounted on glass slides for imaging (Zeiss Axioskop, http://www.zeiss.de/en). Auxin treatment and quantitative RT-PCR analysis For auxin treatment, germinated seeds were plated on the culture solution containing 0.1 lM NAA (a-naphthalene acetic acid). After 7 days, the root hairs in the root tip were investigated using the stereomicroscope (Leica DC 300). For higher auxin treatment, 1 lM NAA was used. For quantitative RT-PCR analysis of auxin-responsive gene expression, 7-day-old seedlings were moved to the rice culture solution containing 10 lM IAA. After 3 h, the roots were harvested and total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, http://www.qiagen.com) according to the manufacturer’s instructions. First-strand cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen, http://www.invitro gen.com). Real-time RT-PCR was performed using the FastStart DNA Master SYBR Green I Kit (Roche, http://www/roche.com) on the LightCycler480 machine (Roche), following the manufacturer’s instructions. Cloning of OsIAA23 Heterozygous Osiaa23 (japonica) was crossed with Kasalath (indica). Approximately half of the F1 population that exhibited the heterozygous mutant phenotype was selected and self-pollinated. The derived F2 population was used to map OsIAA23. The gene was mapped between RM1340 and RM20328 on chromosome 6. A dCAPS marker was developed and the PCR products were digested using Hinf I. A CAPS marker was also developed to confirm the mutated site of Osiaa23-2. These PCR products were digested by MspI. To detect the gain of function allele of the mutant, the homozygous mutant genomic sequence was amplified by PCR using the primers OsIAA23-U and OsIAA23-L. A 5.2 kb PCR product that contained the mutated gene was cloned into pUCm-T (Sangon, China), followed by digestions with HindIII and EcoRV. The insert was cloned into the HindIII and SmaI sites of pCAMBIA-1300 and transformed into the wild-type plant (‘Nipponbare’) via Agrobacterium tumefaciens EHA105. ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 38, 433–442 Postembryonic maintenance of QC in rice 441 Histochemical analysis and GUS assay Histochemical GUS analysis was performed according to Jefferson et al. (1987). Transgenic plant root samples were incubated with X-gluc at 37C. After staining, the tissues were rinsed and fixed in formalin-acetic-alcohol (FAA) for 24 h, embedded in Spurr resin, and sectioned. Sections (5 lm) were mounted on slides and photographed. Construction of GUS fusion constructs For the DR5p::GUS vector, the DR5 element (Ulmasov et al., 1997) coupled to the CaMV 35S minimal promoter was amplified and digested with SalI and BamHI. The construct was then inserted into pBI101.3, which carries the structural gene for GUS and the terminator sequence of the nopaline synthase (NOS) gene. The primers used in PCR amplification were DR5-U and DR5-L. For the OsCYCB1;1p::GUS vector, the promoter of OsCYCB1;1 was amplified by PCR, digested with SalI and KpnI, and inserted into pBI101.3. The primers used for PCR were CYCB-U and CYCB-L. The QHBp::GUS vector is described by Kamiya et al. (2003b). These constructs were transformed into the heterozygous mutant of Osiaa23 via Agrobacterium tumefaciens EHA105, and the offspring were analyzed. For developing the OsIAA23p::GUS vector, the promoter sequence was amplified from Nipponbare genomic DNA and inserted into the HindIII and BamHI sites of pBI101.3. The primers used in PCR amplification are listed in Table S2. The construct was transformed into the WT (‘Nipponbare’) via Agrobacterium tumefaciens EHA105. Construction of the QHBp::Osiaa23 fusion construct The QHB promoter and the coding region of Osiaa23 were amplified by PCR to obtain the QHBp::Osiaa23 fusion. The primers QHBOsIAA23-1U and QHBOsIAA23-1L were used to amplify the promoter of QHB, and the primers QHBOsIAA23-2U and QHBOsIAA23-2L were used to amplify Osiaa23. Overlap extension PCR, using two partially overlapped PCR products as the template, was performed. The overlap extension PCR product, obtained using the primers listed in Table S2, was digested with KpnI and HindIII and cloned into pGOFS1-g (provided by Professor Wu Changying, Huazhong Agricultural University, China). The construct was transformed into the WT (‘Nipponbare’) via Agrobacterium tumefaciens EHA105. ACKNOWLEDGEMENTS We thank Dr Philip N. Benfey (Duke University, USA) for reading the manuscript critically. We also thank Drs Karen Champ and Nicola Edwards for editing the manuscript. This research is supported by the National Basic Research and Development Program of China (grant number 2005CB120900) and the Special Program of Rice Functional Genomics of China (grant number 2006AA10A102). SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. Exogenous auxin and ethylene response of Osiaa23. Figure S2. Phenotypes and molecular characterization of Osiaa23-2. Figure S3. The expression of auxin response genes in the Osiaa23. Figure S4. RT-PCR analysis of OsIAA23 in various tissues and expression pattern of OsIAA23::GUS in the tissues above ground. Figure S5. Phenotypic traits of roots and response to exogenous auxin treatment (NAA at 0.1 lM) of the transgenic plants harboring iaa23 driven by promoter of QHB. Figure S6. 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