Plant and Cell Physiology Advance Access originally published online on April 5, 2007
Plant and Cell Physiology 2007 48(5):678-688; doi:10.1093/pcp/pcm042
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Rapid Paper |
Identification of the Gravitropism-Related Rice Gene LAZY1 and Elucidation of LAZY1-Dependent and -Independent Gravity Signaling Pathways
Botanical Gardens, Graduate School of Science, Osaka City University, Kisaichi, Katano-shi, Osaka, 576-0004 Japan
*Corresponding author: E-mail, iino{at}sci.osaka-cu.ac.jp; Fax, +81-72-891-7199.
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionMaterials and MethodsAcknowledgmentsReferences |
|---|
We identified the gene responsible for three allelic lazy1 mutations of Japonica rice (Oryza sativa L.) by map-based cloning, complementation and RNA interference. Sequence analysis and database searches indicated that the wild-type gene (LAZY1) encodes a novel and unique protein (LAZY1) and that rice has no homologous gene. Two lazy1 mutants were LAZY1 null. Confirming and advancing the previously reported results on lazy1 mutants, we found the following. (i) Gravitropism is impaired, but only partially, in lazy1 coleoptiles. (ii) Circumnutation, observed in dark-grown coleoptiles, is totally absent from lazy1 coleoptiles. (iii) Primary roots of lazy1 mutants show normal gravitropism and circumnutation. (iv) LAZY1 is expressed in a tissue-specific manner in gravity-sensitive shoot tissues (i.e. coleoptiles, leaf sheath pulvini and lamina joints) and is little expressed in roots. (v) The gravitropic response of lazy1 coleoptiles is kinetically separable from that absent from lazy1 coleoptiles. (vi) Gravity-induced lateral translocation of auxin, found in wild-type coleoptiles, does not occur in lazy1 coleoptiles. Based on the genetic and physiological evidence obtained, it is concluded that LAZY1 is specifically involved in shoot gravitropism and that LAZY1-dependent and -independent signaling pathways occur in coleoptiles. It is further concluded that, in coleoptiles, only the LAZY1-dependent gravity signaling involves asymmetric distribution of auxin between the two lateral halves and is required for circumnutation.
Keywords: Auxin - Circumnutation - Gravitropism - lazy mutants - LAZY1 - Oryza sativa
Abbreviations: CAPS, cleaved amplified polymorphic sequence; RNAi, RNA interference; RT–PCR, reverse transcription–PCR.
|
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgmentsReferences |
|---|
Gravitropism plays a central role, in association with other tropisms, in regulating the spatial orientation of plant organs (Iino 2006
). In studies of higher plant gravitropism, much attention has been focused on two major hypotheses: the starch–statolith hypothesis and the Cholodny–Went theory of tropisms. In the former hypothesis, sedimentable starch-filled amyloplasts are thought to play a key role in sensing the direction of gravity (N
mec 1901, Haberlandt 1902). The latter hypothesis proposes that tropisms, including gravitropism, are mediated by lateral translocation of the plant hormone auxin (Went and Thimann 1937). Recent studies with mutants of Arabidopsis thaliana have provided strong genetic evidence for the applicability of these hypotheses in gravitropism of both shoot organs and roots, and have uncovered some cellular components that are thought to be associated with amyloplast-based gravity sensing or lateral auxin translocation (for reviews, see Masson et al. 2002, Morita and Tasaka 2004).
The following basic questions should be answered, however, before we can successfully model gravity sensing and signaling in plants. First, it is not clear whether the amyloplast-based gravity sensing is responsible for the entire gravitropic response. In fact, hypocotyls and roots of starchless Arabidopsis mutants that do not have any sedimentable amyloplasts show some gravitropic response (e.g. Fitzelle and Kiss 2001). Secondly, it has not been resolved whether asymmetric distribution of auxin accounts for the entire gravitropic response (Haga and Iino 2006). There is a possibility that a part of the response is mediated by other plant hormones or growth-regulating substances, as in fact suggested by recent work (Aloni et al. 2004, Gutjahr et al. 2005).
A number of rice mutants showing a ‘lazy’ phenotype or prostrate growth habit have been isolated since one such mutant was reported by Jones and Adair (1938). The best characterized lazy mutants are the tester strain H79 maintained in Hokkaido University and near-isogenic lines produced by crossing this strain with the japonica cultivar Kamenoo (Goto 1978) or Taichung 69 (Yu et al. 1995). Suge and co-workers (Abe and Suge 1993, Abe et al. 1994a) used the lazy mutant in the Kamenoo background to resolve that leaf sheath pulvini and coleoptiles of the mutant have reduced gravitropic responsiveness. Extended work with the same mutant line indicated that coleoptiles and leaf sheath pulvini of the mutant have normal amyloplasts (Abe et al. 1994b) but are impaired in asymmetric distribution of auxin (Godbolé et al. 1999). Furthermore, lazy coleoptiles were found to lack circumnutation, which is observed in dark-grown wild-type coleoptiles (Yoshihara and Iino 2005, Yoshihara and Iino 2006).
The gene responsible for the above lazy mutation, symbolized as la (Kinoshita 1998), was mapped to a 1.4 cM region of chromosome 11 (Miura et al. 2003). Peijin et al. (2003) used an allelic mutant in an indica background to map the gene to a 0.4 cM region. Because it is unlikely that only one gene is responsible for all the reported lazy mutants, the lazy lines originated from the tester strain H79 and the lazy mutants shown to be allelic to these lines will be called lazy1. According to commonly used nomenclature, the mutant gene is then called lazy1 and the corresponding wild-type gene and its product, LAZY1 and LAZY1, respectively.
We identified LAZY1 as a novel and unique gene and analyzed physiological functions of LAZY1. It has become apparent that the gravitropism of coleoptiles involves LAZY1-dependent and -independent signaling pathways and that only the LAZY1-dependent pathway involves asymmetric distribution of auxin across the organ and is required for coleoptile circumnutation. Here we report these and related findings.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgmentsReferences |
|---|
Identification and analysis of LAZY1
We used the lazy1 mutant in the Kamenoo background, hereafter named lazy1-1, to identify the LAZY1 locus by map-based cloning. A mapping population of F2 caryopses was generated by crossing the lazy1-1 mutant to the indica cultivar Kasalath. F2 seedlings, grown under red light, were assayed for shoot gravitropism at the second-leaf stage and those showing clearly reduced shoot gravitropism were selected. By using the genomic DNA extracted from 884 selected individuals, the LAZY1 locus was mapped between markers RM287 and RM7275 in chromosome 11, which are located inside the previously identified LAZY1-containing region (Miura et al. 2003
, Peijin et al. 2003). With the cleaved amplified polymorphic sequence (CAPS) markers developed in this study, the LAZY1 locus was further delimited to a 39.8 kb region, which was predicted to contain 14 genes (Fig. 1A). By sequencing PCR-amplified genomic fragments, the lazy1-1 mutant was found to have a deletion of 8.0 kb in this region and an insertion of 0.76 kb at the deletion site (Fig. 1B and Supplementary Fig. S1).
|
Some retrotransposon Tos17 insertion lines of Nipponbare were recorded to show a lazy phenotype (http://tos.nias.affrc.go.jp/~miyao/pub/tos17/phenotype/63.html; Miyao et al. 2003
). We found that three of them (NC0873, NF1106 and NF1128), confirmed to show a lazy phenotype similarly to lazy1-1, are allelic to lazy1-1. These lines had no Tos17 insertion but a deletion in the LAZY1-linked region (Fig. 1B and Supplementary Fig. S1). The line NC0873 had a deletion of 20.1 kb and, in addition, an insertion of 10.9 kb. The line NF1106 had a small deletion (52 bp). The deletion in NF1128 matched exactly with that in NF1106, suggesting that the two lines originated from the same one. The lines NC0873 and NF1106 are hereafter named lazy1-2 and lazy1-3, respectively.
The genomic deletions found in the three lazy1 mutants strongly suggested that the LAZY1 locus corresponds to the gene labeled ‘LAZY1’ in Fig. 1B. The full-length rice cDNA collection (Kikuchi et al. 2003) contained a cDNA of this putative LAZY1 gene (GenBank accession No. AK067664
[GenBank]
; Supplementary Fig. S2A). We transformed the lazy1-1 mutant with this cDNA fused with the cauliflower mosaic virus 35S promoter. Some of the transformants showed no apparent lazy phenotype at the T1 generation (Fig. 2A) and those were selected for further analysis. Expression of the introduced cDNA could be confirmed in T2 coleoptiles; the transcript level was substantially higher in all the investigated lines than in the wild type (Fig. 2B). Gravitropic responses of complemented lines were compared with those of the wild type and lazy1-1 mutant. This comparison was made using red light-grown seedlings and inducing gravitropism by 90° displacement. As shown in Fig. 2C, the complemented T2 coleoptiles recovered gravitropism partially or close to the wild-type level. The circumnutation observed in dark-grown wild-type coleoptiles was also recovered in the complemented lines, although the period and amplitude were somewhat different from those of wild-type coleoptiles (Fig. 2D).
|
We further performed RNA interference (RNAi) experiments against the putative LAZY1 gene. The japonica cultivar Nipponbare was transformed with an RNAi construct (see Materials and Methods). Plants showing a lazy phenotype segregated at the T1 generation (Fig. 2A). The level of putative LAZY1 transcripts was <12% of the wild-type level in T2 coleoptiles (Fig. 2B). These coleoptiles reduced their gravitropic response to the lazy1 mutant level (Fig. 2C) and showed no coleoptile circumnutation (Fig. 2D). Taken together, the results from complementation and RNAi experiments demonstrated that the investigated gene indeed corresponds to LAZY1.
The results shown in Fig. 2 deserve more detailed attention. First, a full recovery of gravitropism was achieved in the complemented line 2 which had an eight times higher level of LAZY1 transcripts than in the wild type, whereas the recovery was partial in the complemented line 1 that had a 17 times higher level of LAZY1 transcripts (Fig. 2B). Another complemented line (line 3) showed results that were similar to those from line 1 with respect to the level of LAZY1 transcripts and the extent of gravitropism recovery (data not shown). Therefore, the partial recovery of gravitropism in complemented lines was correlated with a considerably high expression of LAZY1, suggesting that LAZY1 is inhibitory on gravitropism when it is expressed well above the wild-type level. Secondly, the lazy1-2 mutant is apparently LAZY1 null (Fig. 1B) whereas the lazy1-3 mutant is expected to produce a mutated LAZY1 that lacks a part of the C-terminus (Supplementary Fig. S1). The gravitropic response remaining in lazy1-3 coleoptiles was not greater than that remaining in lazy1-2 coleoptiles, suggesting that the mutated LAZY1 is not functional. Thirdly, the gravitropic response remaining in LAZY1-RNAi lines was not more than that remaining in the LAZY1-null lazy1-2 mutant (Fig. 2C), although some LAZY1 transcripts could be detected in these lines. At present, we have no clear explanation for this result. In the experiments shown in Fig. 2C, the gravitropism of primary roots was also investigated. The results of these experiments will be described below along with other responses in roots.
The genomic structure of LAZY1 was resolved by comparing the genomic sequence, which was identical between Kamenoo and Nipponbare, with the sequence of Nipponbare LAZY1 cDNA (Fig. 1C). The deduced amino acid sequence of LAZY1 revealed no apparent motif (Supplementary Fig. S2B). Our BLAST searches indicated that rice has no homologous gene and that Arabidopsis has one LAZY1-like gene (GenBank accession No. AY735682 [GenBank] ). The amino acid sequence predicted from this Arabidopsis gene showed 20.5% identity to LAZY1.
LAZY1 transcript distribution and effects of gravity and red light
Quantitative real-time PCR was used to investigate the distribution of LAZY1 transcripts. In the coleoptile of dark-grown seedlings, the transcript level was highest in the most apical zone and decreased basipetally (Fig. 3A). The level in leaves was much lower than that in the coleoptile base. In primary roots, the transcript level was extremely low in the most apical 1 mm zone (0.07% of the level in the coleoptile top zone) and increased acropetally. Even the highest level found in the most basal zone was only 0.6% of the level in the coleoptile base.
|
In mature green plants, the leaf-sheath pulvinus (P) had the highest level of LAZY1 transcripts (Fig. 3B). About a half of this level could be found in the lamina joint (LJ). Other leaf tissues adjacent to the leaf-sheath pulvinus or lamina joint (LS1–3, LB1 and LB2) as well as internode tissues adjacent to the node and leaf-sheath pulvinus (IN1–4) showed a much lower level. Separate experiments indicated that the transcript level in the central region of internodes or leaf blades was 3–5 or <0.1%, respectively, of the level in leaf-sheath pulvini. The transcript level in immature panicles was also low (2.6 ± 0.3%, n = 4).
In the above experiments, LAZY1 transcript levels were determined relative to the level in either the coleoptile top zone or leaf-sheath pulvinus. A direct comparison between the two tissues indicated that the coleoptile top zone had about three times more transcripts than the leaf-sheath pulvinus (relative transcript levels for the coleoptile top zone and pulvinus were 100 ± 3.2 and 28.4 ± 1.9, respectively). Therefore, the coleoptile had the highest level of LAZY1 transcripts among all the tissues investigated. Tissue-specific expression of LAZY1 in coleoptiles, pulvini and lamina joints agrees with the knowledge that these parts of rice shoots are gravity sensitive (see below).
We investigated whether the expression of LAZY1 is affected by gravitropic stimulation. The transcript levels in the upper and lower halves of horizontally placed red light-grown coleoptiles were determined at 30 and 60 min of displacement. No significant change in transcript level was found (Supplementary Fig. S3A). We further investigated, in relation to our finding that red light eliminates circumnutation (Yoshihara and Iino 2005), whether the transcript level in dark-grown coleoptiles is affected by red light. No significant effect of red light was observed within the period in which circumnutation disappears (Supplementary Fig. S3B).
Gravitropism, circumnutation and lateral root formation in primary roots of lazy1 mutants and LAZY1-complemented lines
As seen from the data in Fig. 2C, the primary roots of lazy1 mutants showed time courses of gravitropism that are almost identical to those of wild-type roots including the phase of oscillation (for this oscillation, see Haga et al. 2005). Furthermore, lazy1-1 roots showed circumnutation with a period and amplitude that were almost identical to those of wild-type circumnutation (Fig. 4B). These results indicated that LAZY1 is not required for the gravitropism and circumnutation of primary roots.
|
Although LAZY1 is little expressed in wild-type primary roots (Fig. 3A), LAZY1-complemented lines had a high level of LAZY1 transcripts in primary roots (Fig. 4A). Interestingly, these lines showed altered root gravitropism (Fig. 2C). The major difference was noted in the phase of oscillation. Furthermore, the complemented lines showed root circumnutation with a period and amplitude that were greater than those in wild-type roots (Fig. 4B). Therefore, although LAZY1 is not required for root gravitropism and circumnutation, it affects these processes if expressed in roots.
Elongation growth of primary roots and lateral root formation in these roots were not significantly affected by lazy1 mutation (Fig. 4C), indicating that LAZY1 is also not required for these processes. However, lateral root formation was strongly suppressed in the complemented plants, although primary root growth was almost normal (Fig. 4C). Therefore, LAZY1 affects lateral root formation when expressed in primary roots as it does root gravitropism and circumnutation.
Resolving LAZY1-dependent and -independent gravitropic responses
Investigation was extended to address the question of why LAZY1 null mutants, which do not have any LAZY1 homolog, have only partially reduced gravitropism. We first examined gravitropic curvature induced by a brief stimulation. The time course results shown in Fig. 5A were obtained by displacing red light-grown wild-type and lazy1-1 coleoptiles by 30 or 90° for 25 min and returning them to the vertical position. At either displacement angle, wild-type and lazy1 coleoptiles initially bent similarly, but the latter coleoptiles reached the peak earlier and returned to the vertical position earlier (Fig. 5A). A possible explanation for the observed kinetic difference is that LAZY1 simply delays the response peak while enhancing the extent of response. However, as shown in Fig. 5B, the complemented lazy1-1 lines that recovered the gravitropic response to different extents showed a response peak at a similar time. Furthermore, the time courses for wild-type and complemented lines tended to curve outward at around 45–50 min before reaching the peak (Fig. 5A, B), suggesting that the LAZY1-independent response, which peaked at 45–50 min, accompanied the LAZY1-dependent response, which peaked at 65–70 min. Together, these results indicated that the LAZY1-dependent and -independent responses are kinetically separable, the latter being induced more transiently than the former after brief stimulation.
|
In dark-grown coleoptiles of the lazy1-1 mutant, gravitropic curvature was more confined to the upper part as compared with wild-type coleoptiles (Yoshihara and Iino 2006
). This feature was not apparent in red light-grown short coleoptiles. The photographs of seedlings obtained in the above experiments as well as those obtained for Fig. 2C indicated that the lazy1 coleoptile bent along its length. Therefore, in red light-grown coleoptiles, the kinetic difference between LAZY1-dependent and -independent responses was not associated with a clear spatial difference in the location of curvature.
The 25 min displacement protocol used above and the [3H]IAA tracer method described in Haga et al. (2005) were next used to examine auxin distribution in gravitropically stimulated coleoptiles. In brief, lanolin containing [3H]IAA was applied to the coleoptile tip. After 25 min displacement, the radioactivity diffusing out of the cut surface of the basal halves was collected for 30 min and determined. Most of the collected radioactivity corresponded to [3H]IAA (Haga et al. 2005). As shown in Fig. 5C, [3H]IAA was asymmetrically distributed in wild-type coleoptiles. This asymmetry was greater at 90° than at 30°, as was the case for curvature response. In sharp contrast, however, no significant asymmetry could be detected in lazy1 coleoptiles at either displacement angle. In separate experiments, diffusible [3H]IAA was collected for a shorter period (20 min) after 25 min displacement. In this case too, no significant asymmetry could be detected in lazy1-1 coleoptiles (data not shown). Two important conclusions are reached from these results. First, auxin asymmetry occurs downstream of LAZY1, accounting most probably for LAZY1-dependent curvature. Secondly, LAZY1-independent curvature does not involve auxin asymmetry between the two halves. Discussion on LAZY1-dependent and -independent gravity signaling will be elaborated below.
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgmentsReferences |
|---|
The lazy1 mutant is characterized by partial impairment of gravitropisms in coleoptiles (Abe et al. 1994a
, Yoshihara and Iino 2006, Fig. 2C) and leaf-sheath pulvini (Abe and Suge 1993) as well as by total absence of the circumnutation in coleoptiles (Yoshihara and Iino 2006, Fig. 2D). In this study, we have additionally found that primary roots of lazy1 mutants show normal gravitropism (Fig. 2C) and circumnutation (Fig. 4B). These results indicate that LAZY1 is specifically involved in shoot gravitropism and coleoptile circumnutation.
Any possible function of LAZY1 could not be predicted from the deduced amino acid sequence. Because transcriptional expression of LAZY1 was not affected in the upper and lower halves of displaced coleoptiles and also in red light-treated coleoptiles (see Results), it is likely that LAZY1 is constitutively involved in gravity signaling. Godbolé et al. (1999) used a unique tracer method (i.e. collecting [3H]IAA from the intact coleoptile surface into agar blocks) to show that auxin asymmetry is severely impaired in lazy1-1 coleoptiles. By using a more classical method (collecting basipetally transported [3H]IAA from the basal cut end into agar blocks), we were able to demonstrate with a high resolution that auxin asymmetry does not occur in lazy1-1 coleoptiles (Fig. 5C). Clearly lateral auxin translocation occurs downstream of LAZY1 in the LAZY1-dependent gravity signaling. Coleoptiles of a maize mutant having a defect in sedimentable amyloplasts were shown to be impaired in gravity-induced auxin asymmetry (Hertel et al. 1969). Although plants may have gravity-sensing mechanisms that do not depend on amyloplasts (see below), it is most likely that the amyloplast-based gravity sensing is the one linked to the induction of auxin asymmetry. The lazy1 mutant at least has normal sedimentable amyloplasts in coleoptiles and leaf sheath pulvini (Abe et al. 1994b). Taking these results into considerations along with the above-mentioned results, it is concluded that LAZY1 functions between amyloplast sedimentation and lateral auxin translocation in gravity signal transduction. It is possible that LAZY1 interacts directly or indirectly with the plasma membrane-associated PIN proteins to regulate lateral translocation of auxin (Friml et al. 2002, Noh et al 2003). If so, however, LAZY1 must be a gravitropism-specific PIN regulator, because phototropism of coleoptiles is not impaired in lazy1 mutants (Yoshihara and Iino 2006).
This study provides genetic as well as kinetic evidence for the occurrence of LAZY1-independent gravity signaling. Although gravity-induced auxin asymmetry does not occur in lazy1 coleoptiles, gravitropism is only partially impaired in lazy1 coleoptiles. This partial impairment cannot be attributed to gene redundancy because rice has no homologous gene. Also, two of the three lazy1 mutants are apparently LAZY1 null (Fig. 1B). The most straightforward conclusion is that rice has a LAZY1-independent signaling pathway that does not involve auxin asymmetry. Furthermore, circumnutation is totally absent from lazy1 coleoptiles, strongly suggesting that circumnutation is controlled only by the LAZY1-dependent pathway.
The above conclusion on the presence of a LAZY1-independent signaling pathway is related to the following long-discussed issues. First, it was once reported that the lack of sedimentable amyloplasts only partially reduces gravitropic response in Arabidopsis hypocotyls (Caspar and Pickard 1989). The gravitropic response remaining in such mutants can become smaller under certain growth conditions, but is hard to eliminate (Fitzelle and Kiss 2001). Thus, there is no strict evidence that amyloplast-based gravity sensing accounts for the entire gravitropic response. This argument does not indicate that LAZY1-independent gravity signaling is based on a different sensing mechanism, but nevertheless would warrant investigation of alternative mechanisms such as the one proposed by Wayne and co-workers (Stevens et al. 1997 and references cited therein). Secondly, it has not yet been clearly demonstrated if the entire gravitropic response of any given organ is mediated by lateral auxin translocation (Iino 2001, Haga and Iino 2006). A recent investigation suggested that jasmonic acid mediates, at least in part, the gravitropism of rice coleoptiles (Gutjahr et al. 2005). Our results do not necessarily demonstrate that the control of auxin distribution is not involved in LAZY1-independent gravitropism, because it is possible that auxin translocation within peripheral cell layers instead of across the organ (Macdonald and Hart 1987, Haga and Iino 2006) is responsible for this gravitropism. Nevertheless, our results would warrant investigation of gravity signaling that does not involve the control of auxin distribution.
This study provided a few other results that are worth mentioning. The leaf-sheath pulvinus is the common and major gravity-responding part of mature Gramineae plants (Kaufman et al. 1987). In rice, it has additionally been shown that the lamina joint contains sedimentable amyloplasts and undertakes a gravity-induced nastic movement (Maeda 1965, Nakano and Maeda 1978). Indeed, LAZY1 is highly expressed in the lamina joint (Fig. 3B). It is probable that the LAZY1-mediated gravity signaling also takes place in the lamina joint, contributing to the maintenance and control of the upright orientation of leaf blades. Another interesting set of results came from the analysis of LAZY1-complemented lines. LAZY1 is little expressed in wild-type primary roots (Fig. 3A), and mutant analysis indicated that LAZY1 is not required for gravitropism and circumnutation of primary roots (Figs. 2C, 4B). However, LAZY1 affected these processes when expressed in primary roots (Figs. 2C, 4). It has also become apparent that overexpressed LAZY1 inhibits lateral root formation although it is not required in this process of the wild type. Auxin is known to be involved in root gravitropism and lateral root formation (for a review, see Woodward and Bartel 2005). In light of the conclusion that LAZY1 functions upstream of lateral auxin translocation in gravity signaling, it may be suggested that LAZY1 affects the auxin-mediated responses in roots by affecting the distribution of auxin.
The model shown in Fig. 6 summarizes the two gravity signaling pathways concluded to operate in rice shoots, or at least in coleoptiles. In this model, LAZY1 is considered to regulate coleoptile circumnutation, in principle, independently of gravitropism. This follows our previous conclusion that, although gravity sensing is required for circumnutation, circumnutation is not based on gravitropism itself (Yoshihara and Iino 2006). The model only represents genetic evidence and does not explain how oscillatory movement of circumnutation is created. It is anticipated that investigation of the molecular function of LAZY1 provides a clue to our understanding of the mechanism of circumnutation, a long studied subject (Darwin 1880, Brown 1991, Kitazawa et al. 2005, Yoshihara and Iino 2006), as well as of shoot gravitropism.
|
|
Materials and Methods |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgmentsReferences |
|---|
Plant growth conditions
The dark-grown and red light-grown seedlings of rice were prepared as described in Yoshihara and Iino (2005
). In brief, seedlings were raised, unless otherwise specified, in acrylic cuvettes filled with 0.7% agar. The fluence rate of the red light used to prepare red light-grown seedlings was 2.5–3.0 µmol m–2 s–1. An infrared viewer was used to handle dark-grown seedlings for experiments. Mature plants were grown in a soil mixture in a greenhouse or growth cabinets as described in Haga et al. (2005).
Map-based cloning
Map-based cloning of the LAZY1 locus was conducted as described in Haga et al. (2005) using the molecular markers obtained from the DNA bank at the National Institute of Agrobiological Sciences (http://www.dna.affrc.go.jp/). Public databases were used to design the CAPS markers for fine mapping (http://shenghuan.shtu.edu.cn/genefunction/index.htm) and to analyze the genes contained in the LAZY1-linked region (http://ricegaas.dna.affrc.go.jp/).
Complementation
The full-length LAZY1 cDNA, obtained from the Rice Genome Resource Center, was cloned into the binary vector pIG121-Hm (Ohta et al. 1990) using XbaI and SacI restriction sites. The resulting vector was introduced into Escherichia coli (strain DH5
), transferred to Agrobacterium tumefaciens (strain EHA101) and transformed into the lazy1-1 mutant by Agrobacterium-mediated transformation (Hiei et al. 1994). Control transformants were produced similarly using the empty vector.
RNAi analysis
The RNAi construct was prepares as described in Miki and Shimamoto (2004). In brief, the DNA fragment for RNAi analysis (Supplementary Fig. S2A) was amplified by PCR from LAZY1 cDNA. This fragment was cloned into the Gateway vector pENTR/D-TOPO (Invitrogen, Carlsbad, CA, USA) and transferred into the two recombination sites of the pANDA vector using LR Clonase (Invitrogen). The resulting vector was introduced into A. tumefaciens and transformed into Nipponbare by Agrobacterium-mediated transformation.
Physiological analyses
Gravitropism and circumnutation of coleoptiles were analyzed as described in Yoshihara and Iino (2005). The tracer experiments with [3H]IAA (3-[5(n)-3H]IAA, 925 GBq mmol–1; Amersham Pharmacia Biotech, Buckinghamshire, UK) were conducted as described in Haga et al. (2005) using gravitropic stimulation in place of phototropic stimulation.
To investigate primary root growth and lateral root formation, each of the surface-sterilized caryopses was placed in a hole made in a thin round sheet of silicon sponge (diameter, 18 mm; autoclaved before use), floated on water in an autoclaved and covered 25 mm test tube (20 cm long, 40 ml of deionized water), and incubated at 25 ± 0.5°C under white light (5 µmol m–2 s–1). A surface swelling of about 0.2 mm was counted as a lateral root.
Transcript analysis
Tissues were harvested, stored, and extracted for total RNA as described in Haga and Iino (2004). LAZY1 transcript abundance was analyzed by reverse transcription–PCR (RT–PCR) or real-time RT–PCR. RT–PCR was conducted as described in He et al. (2005). Real-time RT–PCR was performed on an ABI 7300 real-time PCR (Applied Biosystems, Foster City, CA, USA) using the SuperScript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen). Reverse transcription conditions were 30 min at 55°C and subsequent PCR conditions were 10 min at 95°C and 45 cycles of 15 s at 95°C, 30 s at 60°C and 30 s at 72°C, followed by 1 min at 40°C and a melting cycle (60–95°C). Samples to be compared were run simultaneously in triplicate. Each run included a concentration series of one of the analyzed samples for calibration purposes. In either analysis, OsUBQ (GenBank accession No. L31941
[GenBank]
) was amplified as an internal control. The gene-specific primers used for RT–PCR were 5'-ACGGTGACGAAGAGCAAGTT-3' (forward) and 5'-ACAGCACATTCAAGCCCTTC-3' (reverse) for LAZY1, and 5'-ATCACGCTGGAGGTGGAGT-3' (forward) and 5'-AGGCCTTCTGGTTGTAGACG-3' (reverse) for OsUBQ. These primers were designed using Primer3 (http//www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). The gene-specific primers used for real-time RT–PCR were 5'-ACGGTGACGAAGAGCAAGTT-3' (forward) and 5'-AGGAGTGTGTTCTCGGGGTA-3' (reverse) for LAZY1, and 5'-CAAGCCAAGAAGATCAAGC-3' (forward) and 5'-GTAGTGGCGATCGAAGTGGT-3' (reverse) for OsUBQ. These primers were designed using OligoPerfect Designer (Invitrogen).
Supplementary material
Supplementary material mentioned in the article is available to online subscribers at the journal website www.pcp.oxfordjournals.org.
|
Acknowledgments |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgmentsReferences |
|---|
We thank Dr. Hideyuki Takahashi (Tohoku University) for providing lazy1-1 seeds, Dr. Kenzo Nakamura (Nagoya University) for providing the binary vector pIG121-Hm, Dr. Ko Shimamoto (Nara Institute of Science and Technology) for providing the pANDA vector, and Drs. Takefumi Hattori and Keiichi Baba (Kyoto University) for allowing us to use radioisotope research facilities. We also thank Dr. Rainer Hertel for stimulating discussion, Dr. Ken Haga for technical advice, and Ms. Yuki Mitsubayashi and Mr. Makoto Nakazono for technical assistance. Tos17 insertion lines were provided by the Rice Genome Resource Center, National Institute of Agrobiological Science, Japan. This work was supported by a Grant-in-Aid for Special Research (No. 17084007) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Green Technology Project IP1006).
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgmentsReferences |
|---|
Abe K, Suge H. Role of gravitropic response in the dry matter production of rice (Oryza sativa L.): an experiment with a line having lazy gene. J. Plant Res (1993) 106:337–343.[CrossRef][ISI]
Abe K, Takahashi H, Suge H. Graviresponding sites in shoots of normal and ‘lazy’ rice seedlings. Physiol. Plant (1994a) 92:371–374.[CrossRef]
Abe K, Takahashi H, Suge H. Localization of cells containing sedimented amyloplasts in the shoots of normal and lazy rice seedlings. Biol. Sci. Space (1994b) 8:221–225.[CrossRef][Medline]
Alloni R, Langhans M, Aloni E, Ullrich CI. Role of cytokinin in the regulation of root gravitropism. Planta (2004) 220:177–182.[CrossRef][ISI][Medline]
Brown AH. Gravity perception and circumnutation in plants. Adv. Space. Biol. Med (1991) 1:129–153.[Medline]
Caspar T, Pickard BG. Gravitropism in a starchless mutant of Arabidopsis. Planta (1989) 177:185–197.[CrossRef][ISI][Medline]
Darwin C. The Power of Movements in Plants (1880) London: John Murray.
Fitzelle KJ, Kiss Z. Restoration of gravitropic sensitivity in starch-deficient mutants of Arabidopsis by hypergravity. J. Exp. Bot (2001) 52:265–275.
Friml J, Wi
niewska J, Benková E, Mendgen K, Palme K. Lateral relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis. Nature (2002) 415:806–809.[Medline]
Godbolé R, Takahashi H, Hertel R. The lazy mutation in rice affects a step between statoliths and gravity-induced auxin transport. Plant Biol (1999) 1:379–381.
Goto I. Genetic studies on resistance of rice plant to blast fungus III. Decline in the blast resistance of Ginga, a descendant variety of sensho. Ann. Phytopathol. Soc. Jpn (1978) 44:447–455.
Gutjahr C, Riemann M, Müller A, Düchting P, Weiler EW, Nick P. Cholodny–Went revisited: a role for jasmonate in gravitropism of rice coleoptiles. Planta (2005) 222:575–585.[CrossRef][ISI][Medline]
Haberlandt G. Über die Statolithefunktion der Stärkeköner. Ber. Dtsch. Bot. Ges (1902) 20:189–195.
Haga K, Iino M. Phytochrome-mediated transcriptional up-regulation of ALLENE OXIDE SYNTHASE in rice seedlings. Plant Cell Physiol (2004) 45:119–128.
Haga K, Iino M. Asymmetric distribution of auxin correlates with gravitropism and phototropism but not with autostraightening (autotropism) in pea epicotyls. J. Exp. Bot (2006) 57:837–847.
Haga K, Takano T, Neumann R, Iino M. The rice COLEOPTILE PHOTOTROPISM1 gene encoding an ortholog of Arabidopsis NPH3 is required for phototropism of coleoptiles and lateral translocation of auxin. Plant Cell (2005) 17:103–115.
He G, Tarui Y, Iino M. A novel receptor kinase involved in jasmonate-mediated wound and phytochrome signaling in maize coleoptiles. Plant Cell Physiol (2005) 46:870–883.
Hertel R, de la Fuente RK, Leopold AC. Geotropism and the lateral transport of auxin in the corn mutant amylomaize. Planta (1969) 88:204–214.[CrossRef][ISI]
Hiei Y, Ohta S, Komari T, Kumashiro T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J (1994) 6:271–282.[CrossRef][ISI][Medline]
Iino M. Phototropism in higher plants. In: Photomovement, ESP Comprehensive Series in Photosciences—Häder D, Lebert M, eds. (2001) Vol. 1. Amsterdam: Elsevier Science. 659–811.
Iino M. Toward understanding the ecological functions of tropisms: interactions among and effects of light on tropisms. Curr. Opin. Plant Biol (2006) 9:89–93.[CrossRef][ISI][Medline]
Jones JW, Adair CR. A ‘lazy’ mutation in rice. J. Hered (1938) 28:315–318.
Kaufman PB, Brock TG, Song I, Rho YB, Ghosheh NS. How cereal grass shoots perceive and respond to gravity. Amer. J. Bot (1987) 74:1446–1457.[CrossRef][ISI][Medline]
Kikuchi S, Satoh K, Nagata T, Kawagashira N, Doi K, et al. Collection, mapping, and annotation of over 28,000 cDNA clones from japonica rice. Science (2003) 301:376–379.
Kinoshita T. Linkage mapping using mutant genes in rice. Rice Genet. Newslett (1998) 15:13–74.[Medline]
Kitazawa D, Hatakeda Y, Kamada M, Fujii N, Miyazawa Y, et al. Shoot circumnutation and winding movements require gravisensing cells. Proc. Natl Acad. Sci. USA (2005) 102:18742–18747.
Macdonald IR, Hart JW. New light on the Cholodny–Went theory. Plant Physiol (1987) 84:568–570.
Maeda E. Rate of lamina inclination in excised rice leaves. Physiol. Plant (1965) 18:813–827.[CrossRef]
Masson PH, Tasaka M, Morita MT, Guan C, Chen R, Boonsirichai K. A model for the study of root and shoot gravitropism. In: The Arabidopsis Book—Somerville CR, Meyerowitz EM, eds. (2002) Rockville, MD: American Society of Plant Biologists. doi/10.1199/tab.0043.
Miki D, Shimamoto K. Simple RNAi vectors for stable and transient suppression of gene function in rice. Plant Cell Physiol (2004) 45:490–495.
Miura K, Ashikari M, Matsuoka M, Hsing YIC. Mapping of the lazy gene, la. Rice Genet. Newslett (2003) 20:29–30.
Miyao A, Tanaka K, Murata K, Sawaki H, Takeda S, Abe K, Shinozuka Y, Onosato K, Hirochika H. Target site specificity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome. Plant Cell (2003) 15:1771–1780.
Morita MT, Tasaka M. Gravity sensing and signaling. Curr. Opin. Plant Biol (2004) 7:712–718.[CrossRef][ISI][Medline]
Nakano H, Maeda E. On starch statolith in the lamina joint of rice plants. Jpn. J. Crop Sci (1978) 47:262–266.
Nmec B. Über die Wahrnehmung des Schwerkraftreizes bei den Pflanzen. Jahrb. Wiss. Bot (1901) 36:80–178.
Noh B, Bandyopadhyay A, Peer WA, Spalding EP, Murphy AS. Enhanced gravi- and phototropism in plant mdr mutants mislocalizing the auxin efflux protein PIN1. Nature (2003) 423:999–1002.[CrossRef][Medline]
Ohta S, Mita S, Hattori T, Nakamura K. Construction and expression in tobacco of a ß-glucuronidase (GUS) reporter gene containing an intron within the coding sequence. Plant Cell Physiol (1990) 31:805–813.
Peijin L, Dali Z, Xinfang L, Dan X, Dai G, Jiayang L, Qian Q. Mapping and characterization of a tiller-spreading mutant lazy-2 in rice. Chin. Sci. Bull (2003) 48:2715–2717.[CrossRef]
Stevens MP, Wayne RW, Leopold AC. The effect of the external medium on the gravitropic curvature of rice (Oryza sativa, Poaceae) roots. Amer. J. Bot (1997) 84:1522–1529.[Abstract]
Yoshihara T, Iino M. Circumnutation of rice coleoptiles: its occurrence, regulation by phytochrome, and relationship with gravitropism. Plant Cell Environ (2005) 28:134–146.[CrossRef][Medline]
Yoshihara T, Iino M. Circumnutation of rice coleoptiles: its relationships with gravitropism and absence in lazy mutants. Plant Cell Environ (2006) 29:778–792.[CrossRef][Medline]
Yu ZH, McCouch SR, Kinoshita T, Sato S, Tanksley SD. Association of morphological and RFLP markers in rice (Oryza sativa L.). Genome (1995) 38:566–574.[Medline]
Went FW, Thimann KV. Phytohormones (1937) New York: Macmillan.
Woodward AW, Bartel B. Auxin: regulation, action, and interaction. Ann. Bot (2005) 95:707–735.
(Received March 6, 2007; Accepted April 4, 2007)
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||