The Multiple Contributions of Phytochromes to the Control of Internode Elongation in Rice1[W][OA] Masao Iwamoto*, Seiichiro Kiyota, Atsushi Hanada, Shinjiro Yamaguchi, and Makoto Takano Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305–8602, Japan (M.I., S.K., M.T.); and RIKEN Plant Science Center, Yokohama, Kanagawa 230–0045, Japan (A.H., S.Y.)
Although phyAphyBphyC phytochrome-null mutants in rice (Oryza sativa) have morphological changes and exhibit internode elongation, even as seedlings, it is unknown how phytochromes contribute to the control of internode elongation. A gene for 1-aminocyclopropane-1-carboxylate oxidase (ACO1), which is an ethylene biosynthesis gene contributing to internode elongation, was up-regulated in phyAphyBphyC seedlings. ACO1 expression was controlled mainly by phyA and phyB, and a histochemical analysis showed that ACO1 expression was localized to the basal parts of leaf sheaths of phyAphyBphyC seedlings, similar to mature wild-type plants at the heading stage, when internode elongation was greatly promoted. In addition, the transcription levels of several ethylene- or gibberellin (GA)-related genes were changed in phyAphyBphyC mutants, and measurement of the plant hormone levels indicated low ethylene production and bioactive GA levels in the phyAphyBphyC mutants. We demonstrate that ethylene induced internode elongation and ACO1 expression in phyAphyBphyC seedlings but not in the wild type and that the presence of bioactive GAs was necessary for these effects. These findings indicate that phytochromes contribute to multiple steps in the control of internode elongation, such as the expression of the GA biosynthesis gene OsGA3ox2, ACO1 expression, and the onset of internode elongation.
Plants possess the means to change their shapes depending on developmental and environmental conditions. In rice plants (Oryza sativa), internode elongation is inhibited during the vegetative stage and promoted in the ensuing reproductive stage. The regulation of internode length is essential for rice plants to avoid the enclosure of the panicle within the leaf sheaths. One of the ecotypes of cultivated rice, deepwater rice, is adapted to grow in deepwater fields. This plant has a specific ability to elongate its internodes rapidly to maintain part of its foliage above the water. Plant hormones are signal molecules that are produced within plants and control growth and developmental events, such as seed germination, the formation of stems and leaves, and flowering. GA plays a significant role in internode elongation. Internodal growth has been shown to be greatly enhanced when deepwater rice plants are treated with bioactive GA (Raskin and Kende, 1984). Two loss-of-function mutations of GA biosynthesis genes, d18 in the GA 3-oxidase 2 gene (OsGA3ox2) and sd1 in the GA 20-oxidase 2 gene (OsGA20ox2), result in dwarf and 1 This work was supported by the Ministry of Agriculture, Forestry, and Fisheries of Japan (grant no. 13211109 to M.I.). * Corresponding author; e-mail
[email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Masao Iwamoto (
[email protected]). [W] The online version of this paper contains Web-only data. [OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.111.184861
semidwarf rice plants, respectively (Itoh et al., 2001; Sasaki et al., 2002). Ethylene is a gaseous plant hormone that is also associated with internode elongation. It has been reported that the ethylene concentration in submerged internodes is 50-fold higher than in nonsubmerged internodes and that ethylene applied to nonsubmerged plants promotes internode growth in deepwater rice (Me´traux and Kende, 1983). The enzyme 1-aminocyclopropane-1-carboxylate (ACC) oxidase (EC 1.14.17.4) catalyzes the final step of ethylene biosynthesis, in which the precursor ACC is converted to ethylene. A gene that encodes ACC oxidase, ACO1, has been isolated as a predominant ACC oxidase gene from submerged internodes of deepwater rice (Mekhedov and Kende, 1996). We have recently shown that ACO1 is highly expressed in elongating internodes and that it affects internode elongation at the heading stage (Iwamoto et al., 2010). This finding suggests that ACO1 is associated with the developmental control of internode elongation that is mediated by ethylene. However, it remains unknown whether the ACO1-mediated pathway influences internode elongation independently of GA signaling pathways or whether it has an effect on GA signaling pathways to induce internode elongation. Red and far-red light-absorbing phytochromes are major photoreceptors that regulate the expression of light-responsive genes and thus influence many photomorphogenic events in higher plants (Neff et al., 2000; Quail, 2002a, 2002b; Wang and Deng, 2003). In rice, there are three phytochrome genes: PHYA, PHYB, and PHYC (Kay et al., 1989; Dehesh et al., 1991; Tahir et al., 1998; Basu et al., 2000). phyAphyBphyC mutants are phytochrome-null mutants and have morpholog-
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ical changes compared with wild-type plants. In particular, phyAphyBphyC mutants show internode elongation, even in the seedling stage (Takano et al., 2009). It has previously been shown that the transcription levels and promoter activity of ACO1 are significantly increased in phyAphyBphyC mutants (Takano et al., 2009; Iwamoto et al., 2010). In this study, we used phyAphyBphyC mutants to investigate the relationship of phytochromes, ACO1 expression, and ethylene production in the control of internode elongation. Furthermore, we examined the relevance of GAs and GA-related genes to internode elongation in the phyAphyBphyC mutants.
juvenile stage. To determine the tissue localization of ACO1 promoter activity in phytochrome-null mutant seedlings, a histochemical analysis of GUS activity was performed on phyAphyBphyC seedlings by introducing the ACO1-GUS fusion gene. The ACO1-GUS fusion gene was expressed above the nodes, and a longitudinal section showed GUS activity in the basal parts of leaf sheaths of the seedlings (Fig. 1D), as was observed previously in mature wild-type plants at the heading stage (Iwamoto et al., 2010). On the contrary, no GUS staining was detected in wild-type seedlings containing the ACO1-GUS fusion gene at the juvenile stage. Expression of GA- and Ethylene-Related Genes
RESULTS Effect of Phytochromes on ACO1 Expression
To investigate which phytochromes contribute to ACO1 expression, the transcription levels of ACO1 were examined in phyAphyC, phyBphyC, and phyAphyB double mutants, which included only functional phyB, phyA, and phyC, respectively. There was a significant accumulation of the ACO1 transcript in phyAphyB mutants and a low amount in phyBphyC mutants (Fig. 1A). The ACO1 transcript was barely detectable in wild-type plants and in phyAphyC mutants. These results show that ACO1 was mainly regulated by phyA and phyB. To determine the phyA- or phyBregulated ACO1 expression, the light responsiveness of ACO1 expression was examined in etiolated coleoptiles of phyA- or phyB-deficient mutants subjected to continuous darkness for 4 d before irradiation with continuous red light. It is known that both phyA and phyB can respond to continuous red light in rice (Takano et al., 2005). The ACO1 transcript accumulated under dark conditions in wild-type plants and in phyA and phyB mutants (Fig. 1B). In wild-type plants, the ACO1 transcript levels started to decrease from 1 to 3 h after the onset of irradiation. In contrast, the transcript levels decreased slowly up to 9 h in phyA mutants, and there was no significant down-regulation in phyB mutants up to 12 h following irradiation. One of the prominent features of phyAphyBphyC mutants is the promotion of the elongation of the lower internodes. We examined the ACO1 transcript levels in the uppermost (first) to the lower elongated internodes of phyAphyBphyC mutants at the heading stage to determine whether there were any correlations between ACO1 expression and internode elongation in the phyAphyBphyC mutants. High levels of the ACO1 transcript were detected in the first to fourth elongated internodes that we examined (Fig. 1C). In contrast, the transcription levels of ACO1 were high only in the first internodes and were reduced in the lower internodes in wild-type plants at the heading stage, as reported previously (Iwamoto et al., 2010). phyAphyBphyC mutants showed internode elongation not only at the reproductive stage but also at the 1188
It has been reported that the transcription levels of two GA biosynthesis genes (OsGA3ox2 and OsGA20ox2) and a catabolism gene, GA 2-oxidase 1 (OsGA2ox1), are down- and up-regulated, respectively, in the elongating internodes of bioactive GA-overproducing rice plants (Zhu et al., 2006). In addition, another report has shown that the transcription levels of three GA signaling genes, GA-INSENSITIVE DWARF1 (GID1) and GID2 and SLENDER RICE1 (SLR1), are down-regulated in bioactive GA-overproducing rice plants and up-regulated in rice plants with reduced bioactive GA levels (Zhang et al., 2008). We examined the transcription levels of these GA-related genes in the elongating internodes of phyAphyBphyC mutants to estimate the bioactive GA levels. A comparison of the transcription levels between wild-type plants and phyAphyBphyC mutants demonstrated that OsGA3ox2 was significantly down-regulated in the phyAphyBphyC mutants (Fig. 2A). Conversely, GID1, GID2, and SLR1 were significantly up-regulated in the phyAphyBphyC mutants. There were no statistically significant differences in the expression levels of OsGA20ox2 and OsGA2ox1 between wild-type plants and phyAphyBphyC mutants. In our recent study, we showed that the ethylene biosynthesis gene, the ACC synthase gene (ACS1), and the ethylene signaling gene ETHYLENE INSENSITIVE2 (OsEIN2) were significantly up-regulated in ACO1-deficient mutants and that the ethylene signaling gene, EIN3-like1 (OsEIL1), was significantly downregulated in ACO1-overexpressing mutants (Iwamoto et al., 2010). Therefore, we examined the expression of these ethylene-related genes in the elongating internodes of phyAphyBphyC mutants at the heading stage to estimate the ethylene production levels. We found that ACO1, ACS1, and OsEIN2 were significantly up-regulated in the phyAphyBphyC mutants (Fig. 2B). The transcription levels of OsEIL1 showed no statistically significant difference between wild-type plants and phyAphyBphyC mutants, although the average OsEIL1 transcript level in phyAphyBphyC mutants was higher than in wild-type plants. ACS2, which is also expressed preferentially in elongating internodes (Iwamoto et al., 2010), showed no statistically significant difference between the mutant and the wild type. Plant Physiol. Vol. 157, 2011
The Role of Phytochromes in Internode Elongation
Plant Hormone Levels in the phyAphyBphyC Mutants
The expression analyses of three GA signaling genes (GID1, GID2, and SLR1) and two ethylene-related genes (ACS1 and OsEIN2), shown in Figure 2, indicated that the production of bioactive GAs and ethylene might be reduced in the phyAphyBphyC mutants. Thus, we examined the endogenous levels of bioactive GAs (GA1 and GA4) and ethylene production in the internodes of wild-type and phyAphyBphyC seedlings. We also measured the accumulation of abscisic acid (ABA), which is an antagonist of GA and has been shown to inhibit the growth of submerged internodes in deepwater rice (Hoffmann-Benning and Kende, 1992). The analysis of plant hormone levels demonstrated that GA1 and ethylene levels were actually lower in the phyAphyBphyC mutants than in wild-type plants (Table I). In contrast, the ABA levels were much higher in the phyAphyBphyC mutants than in wild-type plants. The amount of GA4 was too low to accurately measure in both the wild-type plants and the phyAphyBphyC mutants (data not shown). Ethylene Responsiveness of Ethylene-Related Gene Expression
Figure 1. Expression of ACO1 in phytochrome-deficient mutants. A, Transcripts of ACO1 in phytochrome double mutants, phyAphyC (AC), phyBphyC (BC), and phyAphyB (AB), identified by northern-blot hybridization. rRNA was stained by methylene blue on the same blot as a quantity control. B, ACO1 expression in phyA and phyB mutants in response to continuous red (Rc) light. The relative levels of transcripts in the coleoptiles of seedlings grown in the dark, which were sampled after being transferred to continuous red light for 12 h, are shown. The highest transcription level was defined as 1. RT-PCR amplification of RUBQ2 was performed as an internal control to normalize transcription levels. Error bars indicate SD (n = 3). C, Expression of ACO1 in the lower parts of first to fourth internodes (lanes 1–4) in phyAphyBphyC (ABC) and wild-type (WT) plants. The total number of amplification cycles needed to detect the RT-PCR products is indicated. RT-PCR amplification of RUBQ2 was performed as a loading control. D, Histochemical analysis of GUS activity in phyAphyBphyC (ABC) and wild-type seedlings. The lower portions of phyAphyBphyC seedlings (left panel) and their longitudinal section (right panel) as well as the lower portions of wild-type seedlings are shown. Arrowheads in the left panel of phyAphyBphyC seedlings denote the places where GUS Plant Physiol. Vol. 157, 2011
We examined the ethylene-responsive expression of ACO1, ACS1, and OsEIN2, whose transcription levels were significantly altered in the phyAphyBphyC mutants (Fig. 2B), to ascertain whether there were any differences in the ethylene responsiveness of gene expression between wild-type plants and the phyAphyBphyC mutants. The transcription levels of ACO1 and ACS1 were significantly increased and decreased, respectively, in the internodes of phyAphyBphyC seedlings treated with the ethylene generator ethephon (ET; Fig. 3A). The average OsEIN2 transcript level in ET-treated phyAphyBphyC mutants was lower than in control mutants (untreated phyAphyBphyC mutants), but there was no statistically significant difference in the OsEIN2 expression between the ET-treated and the control mutants. We also examined the ethylene responsiveness of ACO1, ACS1, and OsEIN2 expression in internodes that contain the shoot apical meristems of wild-type seedlings; unlike the phyAphyBphyC mutants, no internode elongation was observed. There was no significant difference in ACO1 expression between control and ET-treated wild-type seedlings (Fig. 3B). The ACO1 transcript levels in wild-type seedlings were much lower than in phyAphyBphyC seedlings, and the average transcript level in wild-type control seedlings was 12.9 when the average transcript level in phyAphyBphyC control seedlings in Figure 3A was defined as 100. The transcription levels of ACS1, in contrast, were significantly decreased in ET-treated
activity was observed. The blue bar in the left panel of phyAphyBphyC seedlings indicates the position of the longitudinal section in the right panel. IN, Internode; LS, leaf sheath; N, node. 1189
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4A). In contrast, subsequent to the application of AVG, the ACS1 transcript levels in wild-type seedlings were increased to the same levels as those in the untreated phyAphyBphyC seedlings for up to 12 h (Fig. 4B). The 12-h application of AVG also increased the ACS1 transcript levels in the phyAphyBphyC seedlings. Effects of Bioactive GAs on Ethylene-Induced Internode Elongation and Gene Expression
Figure 2. Expression of GA- and ethylene-related genes in phyAphyBphyC mutants. Transcript levels are shown for GA-related genes (A) and ethylene-related genes (B) in elongating internodes of wild-type plants (WT) and phyAphyBphyC mutants at the heading stage. Error bars indicate SE (n = 6). Asterisks indicate significant differences from wildtype plants as determined using Student’s t test (* P , 0.05, ** P , 0.01).
wild-type seedlings, similar to phyAphyBphyC seedlings. The average OsEIN2 transcript level in ETtreated wild-type seedlings was lower than in control plants, yet OsEIN2 showed no statistically significant difference in transcriptional level between them. Figure 3 shows that ACS1 was significantly downregulated by ethylene irrespective of wild-type or phyAphyBphyC seedlings. Therefore, we compared the ACS1 expression between wild-type and phyAphyBphyC seedlings by applying the precursor ACC or the ACC biosynthesis inhibitor aminoethoxyvinylglycine (AVG). When ACC was applied at a saturating concentration (10 mM), which has been reported to enhance ethylene production in the internodes of deepwater rice plants (Me´traux and Kende, 1983), the expression of ACS1 was down-regulated in the phyAphyBphyC seedlings to the same levels as in wild-type seedlings (Fig.
To understand the effect of the relationship between ethylene and GA on the regulation of internode elongation and gene expression, phyAphyBphyC seedlings that were grown in the presence or absence of the GA biosynthesis inhibitor paclobutrazol (PAC) were treated with ET. Whereas phyAphyBphyC seedlings showed enhanced internode elongation after treatment with ET or bioactive GA, PAC-pretreated phyAphyBphyC seedlings showed no enhancement of internode elongation following treatment with ET (Fig. 5A). As a control, internode elongation was enhanced in phyAphyBphyC seedlings that were grown in the presence of both PAC and bioactive GA. The ethylene- or bioactive GA-induced internode elongation was not detected in wild-type seedlings, although the same concentration of bioactive GA enhanced shoot elongation (Supplemental Fig. S1). We further examined the effects of higher concentrations of ET or bioactive GA on internode elongation in wild-type seedlings and found that the wild-type seedlings showed no enhancement of internode elongation (Supplemental Fig. S2). The ethylene-induced upregulation of ACO1 was not detected in the internodes of phyAphyBphyC seedlings that were pretreated with PAC (Fig. 5B). The transcription levels of ACS1 and OsEIN2 showed no statistically significant differences between ET-treated phyAphyBphyC seedlings and those pretreated with PAC. The analysis of the bioactive GAresponsive expression of the ethylene-related genes indicated that the bioactive GA treatment significantly increased ACS1 transcript levels in the phyAphyBphyC seedlings (Fig. 5C). The ACO1 transcript levels in control (untreated) and bioactive GA-treated phyAphyBphyC seedlings were higher than those in PACtreated phyAphyBphyC seedlings. We also examined the bioactive GA responsiveness of ACO1 and ACS1 expression in the internodes of wild-type seedlings, and there was no significant difference in expression levels among the three treatments.
Table I. Levels of internodal bioactive GA and ABA and ethylene production in wild-type and phyAphyBphyC seedlings Data represent means 6 SD of two independent measurements. Plant
GA1
ABA ng g
Wild type phyAphyBphyC 1190
0.25 6 0.06 0.08 6 0.01
21
fresh wt
3.25 6 0.23 48.47 6 1.15
Ethylene nL g
21
fresh wt h21
30.95 6 9.73 5.61 6 1.13 Plant Physiol. Vol. 157, 2011
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Expression of Plant Hormone-Related Genes in phyAphyBphyC Mutants
Figure 3. Expression of ethylene-related genes after treatment with an ethylene generator. Transcript levels are shown for ethylene-related genes in internodes of phyAphyBphyC (A) and wild-type (B) seedlings in the presence or absence of ET for 3 d. MES (10 mM) buffer alone was applied to the control. Error bars indicate SE (n = 4). Asterisks indicate significant differences from the control plants as determined using Student’s t test (P , 0.05).
The expression analyses of plant hormone-related genes indicated that three GA signaling genes (GID1, GID2, and SLR1) and two ethylene-related genes (ACS1 and OsEIN2) have altered expression in phyAphyBphyC mutants, suggesting that bioactive GA and ethylene production in phyAphyBphyC mutants may occur at lower levels than in wild-type plants. We examined GA concentrations and found that the bioactive GA1 levels were actually decreased in phyAphyBphyC mutants (Table I). In addition, OsGA3ox2 was down-regulated in phyAphyBphyC mutants (Fig. 2A). It has been reported that OsGA3ox2 is upregulated by the application of GA biosynthesis inhibitors (Sakamoto et al., 2003). These results show that the loss of phytochromes has a more dramatic effect on OsGA3ox2 transcript levels than decreased levels of bioactive GAs in the internodes of rice plants. However, these results do not exclude the possibility that the bioactive GA levels also regulate OsGA3ox2 expression at the protein level. In this study, ACS1 expression was increased in phyAphyBphyC mutants and decreased in wild-type or phyAphyBphyC seedlings treated with ethylene (Figs. 2B and 3). We have recently found that the transcription levels of ACS1 increase significantly in
DISCUSSION
In this study, we used phyAphyBphyC mutants to understand the regulatory mechanism of phytochromemediated internode elongation. It is known that ethylene production is highly induced by wounding or excision. Because phyAphyBphyC mutants show internode elongation even in the seedling stage, they are more useful for the examination of internode elongation in intact plants than mature wild-type plants at the heading stage, which may be too large to use for experiments without excision. This study reveals that phytochromes contribute to the control of internode elongation at several steps in rice. phyA and phyB Are the Main Phytochromes That Regulate ACO1 Expression
Although it has been reported that ACO1 is highly expressed in phyAphyBphyC mutants (Takano et al., 2009), it was uncertain which phytochromes contributed to ACO1 expression. Our expression analyses using phytochrome-deficient mutants indicate that phyA and phyB are the main phytochromes that play a role in the control of ACO1 expression (Fig. 1, A and B). Because the localization of ACO1 promoter activity in phyAphyBphyC seedlings is the same as in mature wild-type plants at the heading stage (Fig. 1D; Iwamoto et al., 2010), it seems that ACO1 plays the same role in the regulation of internode elongation in phyAphyBphyC seedlings and in mature wild-type plants. Plant Physiol. Vol. 157, 2011
Figure 4. Effects of an ethylene precursor or an ethylene biosynthesis inhibitor on ACS1 expression in internodes. Transcript levels are shown for ACS1 in internodes of wild-type (WT) and phyAphyBphyC (phyABC) seedlings treated with 10 mM ACC for 3 d (A) or 100 mM AVG for up to 12 h (B). Error bars indicate SE (n = 4). Asterisks indicate significant differences from untreated plants as determined using Student’s t test (* P , 0.05, ** P , 0.01). 1191
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respectively. In addition, an increase in OsEIN2 transcript levels was detected in phyAphyBphyC mutants (Fig. 2B), and it has been shown that the transcription levels of OsEIN2 increased significantly in the elongating internodes of ACO1-deficient mutants (Iwamoto et al., 2010). However, ethylene treatment did not result in a significant change in OsEIN2 expression, although its average transcript level was low in seedlings treated with ET compared with the control seedlings (Fig. 3). These results indicate that the expression of OsEIN2 could be altered, depending on the ethylene levels, but that OsEIN2 is less sensitive to ethylene than ACS1. Phytochromes Contribute to Plant Hormone Biosynthesis and the Onset of Internode Elongation
Figure 5. Effects of a GA biosynthesis inhibitor or bioactive GA on ethylene-induced internode elongation and ethylene-related gene expression. A, Total internode lengths of phyAphyBphyC (phyABC) and wild-type (WT) seedlings treated with ET, GA, PAC, PAC + ET, or PAC + GA. ET treatment was performed for 5 d, and 10 mM MES buffer alone was applied to the control. B, Transcription levels of ethylenerelated genes in internodes of phyAphyBphyC seedlings treated with ET in the presence or absence of PAC. ET treatment was performed for 5 d. C, Transcription levels of ethylene-related genes in internodes of phyAphyBphyC and wild-type seedlings treated with bioactive GA in the presence of PAC. The total number of amplification cycles needed to detect the RT-PCR products is indicated. Error bars indicate SE (n $ 3). Asterisks indicate significant differences from control (A and C), ETtreated (B), or PAC-treated (C) plants as determined using Student’s t test (* P , 0.05, ** P , 0.01).
the elongating internodes of ACO1-deficient mutants (Iwamoto et al., 2010). Taken together, these results indicate that the expression of ACS1 changes depending on the ethylene levels: up- and down-regulation of ACS1 correspond to low and high ethylene levels, 1192
The measurement of GA production revealed that phytochromes contribute to the regulation of bioactive GA1 levels. It has been reported that OsGA3ox2 is the dominant GA3ox gene in stems (Sakamoto et al., 2004) and that a loss-of-function mutation in OsGA3ox2 results in dwarfism (Itoh et al., 2001). Because OsGA3ox2 was down-regulated in phyAphyBphyC mutants (Fig. 2A) and because the difference in the GA1 precursor levels was less than the difference in the GA1 levels between wild-type plants and phyAphyBphyC mutants (Table I; Supplemental Table S1), the primary contribution of phytochromes to GA1 production was probably mediated through OsGA3ox2. It has been suggested that phytochromes induce the upregulation of bioactive GA and that an increase in bioactive GA levels causes a decrease in ABA levels in lettuce (Lactuca sativa) seeds (Toyomasu et al., 1993, 1994). Further experiments are needed to understand whether bioactive GA decreases ABA levels in the internodes of rice. It has been reported that ethylene production is enhanced in phyAphyB double mutants of pea (Pisum sativum) and phyB mutants of sorghum (Sorghum bicolor; Foo et al., 2006; Finlayson et al., 2007). Because the expression of ACO1 and internode elongation were both significantly increased in phyAphyBphyC seedlings (Takano et al., 2009), we initially supposed that phyAphyBphyC seedlings produced large amounts of ethylene, which then induced the internode elongation. To our surprise, we found that ACS1 and OsEIN2 expression was increased in the phyAphyBphyC mutants, as was also observed with ACO1-deficient mutants (Fig. 2B; Iwamoto et al., 2010), and that ethylene production was lower in phyAphyBphyC seedlings than in wild-type seedlings (Table I). Rapid internode elongation is detected in deepwater rice plants during submergence, and bioactive GA and ethylene levels are increased in submerged internodes (Me´traux and Kende, 1983; Hoffmann-Benning and Kende, 1992). Treatments with bioactive GA or ethylene indicated that the growth of internodes, from which the leaf sheath had been peeled, was greater in darkness than in light (Raskin and Kende, 1984). These reports sugPlant Physiol. Vol. 157, 2011
The Role of Phytochromes in Internode Elongation
gest that internode growth is accelerated in darkness because of the lack of activated phytochromes and that the mechanism in the internodes of phyAphyBphyC mutants is similar to that in the internodes grown in darkness. Ethylene induced the elongation of the internodes of phyAphyBphyC seedlings but not those of wild-type seedlings (Fig. 5A; Supplemental Fig. S2), which indicates that phytochromes inhibit the onset of internode elongation during the juvenile (i.e. seedling) stage and that ethylene is involved in the elongation of internodes after the onset of internode elongation. It has been reported that quintuple phytochrome mutants without all five phytochromes enhance internode elongation and that they have a low germination rate in Arabidopsis (Arabidopsis thaliana; Strasser et al., 2010). The germination rate of the quintuple phytochrome mutants was induced to normal germination levels by the application of bioactive GA. These results indicate that endogenous bioactive GA levels are low in the quintuple phytochrome mutants and that the acceleration of internode elongation is not correlated with an increase of endogenous bioactive GA levels in phytochrome-null mutants. Although rice phyAphyBphyC mutants extend their internodes, even during the vegetative growth stage, the total length of the internodes at the reproductive stage is significantly shorter in phyAphyBphyC mutants than in wild-type plants because of the short upper internodes in phyAphyBphyC mutants (Takano et al., 2009). These results suggest that both ethylene production and bioactive GA levels were not necessarily higher in the phyAphyBphyC mutants. In this study, we indicated that both ethylene production and bioactive GA1 levels were actually decreased in the phyAphyBphyC mutants (Table I). A model for phytochrome-mediated regulation of internode elongation in rice is shown in Figure 6. Phytochromes down- and up-regulate ACO1 and OsGA3ox2, respectively, and inhibit the onset of the internode elongation mediated by GA signaling pathways. The results shown in Figure 1 indicate that both phyA and phyB are the main phytochromes that control ACO1 expression. Because internode elongation during the vegetative growth stage was also observed in the phyAphyB seedlings (Supplemental Fig. S3A), phyA and phyB mainly confer inhibition of internode elongation during the vegetative growth stage. The contribution of phytochromes to OsGA3ox2 expression showed that phyB, in concert with phyA or phyC, had a considerable effect on OsGA3ox2 expression in the internodes, because OsGA3ox2 was down-regulated in phyBphyC and phyAphyB mutants but not in phyB mutants (Supplemental Fig. S3B). We observed that the OsGA3ox2 transcript levels were down- and upregulated after treatment with bioactive GA and PAC, respectively, in the internodes of both wild-type and phyAphyBphyC seedlings (Supplemental Fig. S4A). These results indicate that the OsGA3ox2 transcript levels are controlled by negative feedback regulation from bioactive GA and that phytochromes regulate the OsGA3ox2 expression independently of the negative Plant Physiol. Vol. 157, 2011
Figure 6. A model for phytochrome-mediated internode elongation. Phytochromes (phys) contribute to the down-regulation of ACO1 expression, up-regulation of OsGA3ox2, and inhibition of the onset of internode (IN) elongation. Bioactive GA is necessary for internode elongation, and ethylene contributes to internode elongation via bioactive GA-mediated signaling. A, B, and C, phyA, phyB, and phyC, respectively; SAM, S-adenosyl-Met. Dashed lines represent relationships that are biologically plausible but need further investigation.
feedback regulation. Since ethylene treatment produced no significant change in the OsGA3ox2 transcript levels in both wild-type and phyAphyBphyC seedlings (Supplemental Fig. S4B), the ethylene signal affects the GA signaling pathways downstream of the negative feedback regulation of OsGA3ox2 by bioactive GA. The results shown in Figures 3 and 5, B and C, indicate that ethylene up-regulates ACO1 via bioactive GA and that bioactive GA up-regulates ACS1 after the onset of the internode elongation. Therefore, the relationship between ethylene-related gene expression and bioactive GA is required for the concerted regulation of internode elongation by the two plant hormones. A cooperative effect of ethylene and bioactive GA on internode elongation has been reported previously in deepwater rice plants (Suge, 1985). In contrast, the ethylene-induced down-regulation of ACS1 is independent of bioactive GA or GA signaling pathways. Considering that we observed both upregulation of ACO1 and decreased ethylene production in phyAphyBphyC mutants, we hypothesized that the concentration of ACC was low in phyAphyBphyC mutants. Indeed, the results in Figure 4 show lower ACC levels in phyAphyBphyC seedlings than in wildtype seedlings. In wild-type seedlings, the decreased rate of ACS1 transcript levels after treatment with ACC was less than that after treatment with ethephon (Figs. 3B and 4A) because of the decreased ACO1 expression in the internodes of wild-type seedlings (Figs. 3 and 5C). Thus, phytochromes might play an important role in the inhibition of ACO1 expression to avoid the excessive consumption of ACC. This study indicates that ACO1 may be an appropriate indicator to demonstrate the ability of internodes to elongate, as up-regulation of ACO1 was detected in elongating internodes. In wild-type plants, the ethylene-induced up-regulation of ACO1 was detected in the elongating internodes of mature plants 1193
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but not in the internodes of seedlings (Fig. 3B; Supplemental Fig. S5). We have very recently indicated that the rice ethylene-responsive element binding protein 1-like gene, EBL1, contributes to the up-regulation of ACO1 and internode elongation of mature wildtype plants (Iwamoto and Takano, 2011). EBL1 might be one of the factors that play roles in this pathway from the onset of internode elongation to the upregulation of ACO1. Further investigation will be needed to identify additional factors.
cycles for each gene was determined prior to the RT-PCR experiments. The PCR products were fractionated on a 1.4% (w/v) agarose gel and visualized by ethidium bromide staining. The relative levels of transcripts were calculated from the intensity of the RT-PCR products stained by ethidium bromide using ImageJ version 1.33u.
Northern-Blot Analysis Total RNAs were separated by a 0.8% agarose gel and transferred to a Hybond N+ membrane (GE Healthcare). A 258-bp region in the ACO1 cDNA (nucleotides 1,070–1,327 of AK058296) was used for the generation of the DNA probes.
Histochemical Staining MATERIALS AND METHODS Plant Materials, Growth Conditions, and Chemicals Rice (Oryza sativa ‘Nipponbare’) and its phytochrome-deficient mutants were used for the experiments. Seedlings were maintained in a growth chamber at 28°C. White light (160 mmol m22 s21) was provided by cool fluorescent lights. To examine the light responsiveness of ACO1 expression, seedlings grown under continuous darkness for 4 d were transferred to continuous red (70 mmol m22 s21) light conditions, and the coleoptiles were sampled. Monochromatic light sources were provided by a red light-emitting diode panel (model LED-R; Eyela). Rice plants were grown to mature plants in a greenhouse at 27°C. Transgenic rice plants harboring the ACO1 promoter (nucleotides 21,625 to +130, with the cDNA start site of ACO1 being designated as +1)-GUS reporter gene fusions have been reported previously (Iwamoto et al., 2010). ET (Honeywell) was prepared in 10 mM MES buffer (pH 5.5) as a 1 mM solution, and the ET solution was placed in a clear plastic container (Agripot; Iwaki) containing 0.6% agar with nutrient solution (0.2 mM KNO3, 0.1 mM NH4H2PO4, 0.1 mM MgSO4, 0.1 mM CaCl2, 2.5 mM Fe-EDTA, and micronutrients). There was no direct physical contact between the ET solution and the rice plants. For the PAC treatment, PAC (Wako) was added to 0.6% agar with a nutrient solution at a concentration of 2 mM. GA3 at 0.5 mM (Sigma-Aldrich) was used for the bioactive GA treatment. ACC (Wako) or AVG (Sigma-Aldrich) was added to 0.6% agar with a nutrient solution at 10 mM or 100 mM concentration, respectively.
RNA Extraction Total RNA was extracted from the indicated tissues using an RNeasy Mini Kit (Qiagen) according to the instructions provided by the manufacturer.
Reverse Transcription and PCR Reverse transcription (RT)-PCR analysis was performed by one-step reactions (SuperScript One-Step RT-PCR system; Invitrogen). Total RNA (50 ng) was used as a template, and RT-PCR amplification reaction mixtures contained 0.2 mM of each deoxyribonucleotide triphosphate, 1.2 mM MgSO4, and 0.4 mM of each primer. The primer sets were ACO1-F (5#-CTGCGGCGATGGAGCAGCTGGA-3#) and ACO1-R (5#-CACGAACTTGGGGTACGCCACGA-3#) for ACO1; OsGA3ox2-F (5#-GAAGCCCGAGTCCGTGTGCGCGATG-3#) and OsGA3ox2-R (5#-GACGACTACCTCCTCTTCTGTGACGTG-3#) for OsGA3ox2; OsGA20ox2-F (5#-AGACCCTCTCCTTCGGCTT-3#) and OsGA20ox2-R (5#-GTCCTCAAGGTACTAGCAGTC-3#) for OsGA20ox2; OsGA2ox1-F (5#-CGAGCAAACGATGTGGAAGGGCTACA-3#) and OsGA2ox1-R (5#-TGGCTCAGGCGGAGTGAGTACATTGT-3#) for OsGA2ox1; SLR1-F (5#-CTGGAGCAGCTGGAGATGG-3#) and SLR1-R (5#-GATGCCGAAGTCGACGACG-3#) for SLR1; GID1-F (5#-CAGCGACGAGGTCAACCGCAACGAG-3#) and GID1-R (5#-CTGGCGCGTCCGTCAGGAACTCAAG-3#) for GID1; GID2-F (5#-GATCTCGGGGAGGACCTGCTGTTC-3#) and GID2-R (5#-CTTGTTGGAGTGAAGCTTATTCCAC-3#) for GID2; OsEIL1-F (5#-CAACGGAGGTGTGTTCGAAGATGTC-3#) and OsEIL1-R (5#-TCAGTAGTACCAATTCGAGCCGTC-3#) for OsEIL1; OsEIN2-F (5#-GTCTTCTTGCTCGGTGACTAAG-3#) and OsEIN2-R (5#-CCTGCTGTATTCTTGGTGGCAG-3#) for OsEIN2; and nucleotides 4,158 to 4,177 and 4,374 to 4,355 of AF184280 for the polyubiquitin gene (RUBQ2). The primer sets for ACS1 and ACS2 were reported previously by Iwai et al. (2006). PCR amplification was performed in a DNA thermal cycler (GeneAmp PCR system 9700; Applied Biosystems). The appropriate number of PCR 1194
A histochemical assay of GUS activity was performed essentially as described by Jefferson (1987). Transgenic plants were immersed in a solution of 100 mM phosphate buffer (pH 7.0) containing 1 mM 5-bromo-4-chloro-3indolyl-b-D-glucuronide as a histochemical substrate and incubated overnight at 28°C. The stained organs were treated with 70% ethanol to remove the chlorophyll and then immersed in a 50% glycerol solution.
Determination of Ethylene, GA, and ABA Measurement of ethylene emission was performed essentially as described by Iwai et al. (2006). One-week-old seedlings were grown in glass tubes, and 1 mL of gas was withdrawn from the air space of each tube after sealing with a gas-proof septum for 2 h. Samples were prepared from 1 and 1.5 g of internodal tissues from wildtype (n = 59) and phyAphyBphyC (n = 21) seedlings, respectively, for GA and ABA measurement. Quantitative analysis of GAs and ABA was performed by liquid chromatography-selected reaction monitoring, as described previously (Varbanova et al., 2007).
Supplemental Data The following materials are available in the online version of this article. Supplemental Figure S1. The shoot lengths of phyAphyBphyC and wildtype seedlings treated with bioactive GA, PAC, or PAC + GA. Supplemental Figure S2. The total internode and shoot lengths of wildtype seedlings treated with a high concentration of ET or bioactive GA. Supplemental Figure S3. Contributions of phytochromes to internode elongation and OsGA3ox2 expression. Supplemental Figure S4. Effects of bioactive GA, GA biosynthesis inhibitor, or ethylene on OsGA3ox2 expression. Supplemental Figure S5. Expression of ACO1 in elongating internodes of mature wild-type plants treated with an ethylene generator. Supplemental Table S1. Levels of GA1 precursors in internodes of wildtype and phyAphyBphyC seedlings.
ACKNOWLEDGMENTS We thank Dr. Y. Ohashi (National Institute of Agrobiological Sciences, Japan) for technical advice and support for the ethylene measurements. We are grateful to S. Ohashi (National Institute of Agrobiological Sciences, Japan) for technical assistance. Received August 5, 2011; accepted September 9, 2011; published September 12, 2011.
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