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Keywords:

  • gibberellin deactivation;
  • EUI P450 monooxygenase;
  • expression pattern;
  • gibberellin profile;
  • rice traits

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The rice gene ELONGATED UPPERMOST INTERNODE1 (EUI1) encodes a P450 monooxygenase that epoxidizes gibberellins (GAs) in a deactivation reaction. The Arabidopsis genome contains a tandemly duplicated gene pair ELA1 (CYP714A1) and ELA2 (CYP714A2) that encode EUI homologs. In this work, we dissected the functions of the two proteins. ELA1 and ELA2 exhibited overlapping yet distinct gene expression patterns. We showed that while single mutants of ELA1 or ELA2 exhibited no obvious morphological phenotype, simultaneous elimination of ELA1 and ELA2 expression in ELA1-RNAi/ela2 resulted in increased biomass and enlarged organs. By contrast, transgenic plants constitutively expressing either ELA1 or ELA2 were dwarfed, similar to those overexpressing the rice EUI gene. We also discovered that overexpression of ELA1 resulted in a severe dwarf phenotype, while overexpression of ELA2 gave rise to a breeding-favored semi-dwarf phenotype in rice. Consistent with the phenotypes, we found that the ELA1-RNAi/ela2 plants increased amounts of biologically active GAs that were decreased in the internodes of transgenic rice with ELA1 and ELA2 overexpression. In contrast, the precursor GA12 slightly accumulated in the transgenic rice, and GA19 highly accumulated in the ELA2 overexpression rice. Taken together, our study strongly suggests that the two Arabidopsis EUI homologs subtly regulate plant growth most likely through catalyzing deactivation of bioactive GAs similar to rice EUI. The two P450s may also function in early stages of the GA biosynthetic pathway. Our results also suggest that ELA2 could be an excellent tool for molecular breeding for high yield potential in cereal crops.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Gibberellins (GAs) are plant hormones that play important roles in diverse aspects of plant growth and development, such as stem elongation, leaf expansion, flowering, seed development and germination (Yang et al., 1995; King et al., 2001; Ogawa et al., 2003; Fei et al., 2004; Eriksson et al., 2006; Schwechheimer, 2008; Ubeda-Tomás et al., 2009). The GA metabolism pathway has been well studied, and genetic and biochemical studies have discovered the majority of genes encoding enzymes in GA biosynthesis and deactivation (Hedden and Phillips, 2000; Olszewski et al., 2002; Sun and Gubler, 2004; Yamaguchi, 2008). Bioactive GAs are synthesized via a three-stage process involving three different organelles. Stage 1 converts geranylgeranyl diphosphate to ent-kaurene in the plastid, while stage 2 is responsible for oxidation of ent-kaurene to form GA12 or GA53 (carrying a hydroxyl group on C-13) in the endoplasmic reticulum (ER). Stage 3 converts GA12 and GA53 to GA4 and GA1, respectively, in the cytosol via sequential reactions catalyzed by GA 20-oxidase (GA20ox) and GA 3-oxidase (GA3ox).

It is well known that effective control of the steady-state concentrations of bioactive GAs in a given plant tissue/organ is crucial for regulation of plant growth and development. The levels of bioactive GAs can be regulated by controlling GA biosynthesis, conjugation of an active GA to a sugar molecule, or inactivation via further oxidation of various functional groups. A number of mechanisms are proposed to inactivate GAs; however, the major route to deactivate bioactive GAs is 2-hydroxylation catalyzed by the GA 2-oxidases (GA2ox), which adds a hydroxyl group to the C-2 position of GA1, GA4, and their immediate precursors GA20 and GA9 (Thomas et al., 1999; Sun and Gubler, 2004; Yamaguchi, 2008; Rieu et al., 2008). Recent studies have shown that 2-hydroxylation can also occur on earlier GA biosynthetic intermediates catalyzed by two members of a new class of GA2ox, AtGA2ox7 and AtGA2ox8. Interestingly, both enzymes fail to catalyze the 2-hydroxylation reaction on bioactive GAs lacking C-20, implying that they might function to regulate GA biosynthesis by removing C20 intermediates from the biosynthetic pathway (Schomburg et al., 2003; Lee and Zeevaart, 2005). In addition to 2-hydroxylation, bioactive GAs can also be inactivated by methylation. A recent study showed that two Arabidopsis genes, GAMT1 and GAMT2, encode GA methyltransferases that catalyze methylation of the C-6 carboxyl group of GAs to form the methyl esters of GAs using S-adenosyl-l-methionine as a methyl donor (Varbanova et al., 2007). Simultaneous elimination of GAMT1 and GAMT2 genes resulted in a significant increase of bioactive GAs in developing seeds whereas their ectopic expression in Arabidopsis, tobacco (Nicotiana tabacum), or petunia (Petunia hybrida) gave rise to characteristic GA-deficiency phenotypes. However, methylated GAs have not yet been detected in any plant species (Varbanova et al., 2007).

Our previous study of the recessive tall rice (Oryza sativa L.) mutant elongated uppermost internode (eui) revealed that the EUI (also known as EUI1) gene encodes a cytochrome P450 monooxygenase CYP714D1 capable of catalyzing the 16α,17-epoxidation on non-13-hydroxylated GAs, including GA4, GA9, and GA12, to reduce the biological activity of GAs in rice (Zhu et al., 2006; Yamaguchi, 2008). EUI is probably the principal GA deactivation enzyme during the heading stage because the eui mutants accumulated very high levels of bioactive GAs in the elongating uppermost internode. By contrast, ectopic expression of EUI in transgenic rice resulted in severe dwarfed phenotypes (Zhu et al., 2006; Zhang et al., 2008).

Our discovery of EUI as a GA 16,17-oxidase (GA16,17ox) provided a reasonable explanation for the presence of 16,17-[OH]2-GAs (GA 16,17-dihydrodiols) in many other plant species, including Cibotium glaucum (Yamane et al., 1988), Lupinus albus (Gaskin et al., 1992), Malus domestica (Hedden et al., 1993), Pisum sativum (Santes et al., 1995), Prunus avium (Blake et al., 2000), and Populus trichocarpa (Pearce et al., 2002). These findings suggested that 16α,17-epoxidation of GAs might be a general mechanism to inactivate GAs. The Arabidopsis genome has two close EUI-like P450s, CYP714A1 and CYP714A2 (Nelson et al., 2004; Zhu et al., 2006). Functional characterization of EUI homologs in other plant species is important to determine if 16α,17-epoxidation is a common mechanism for inactivating bioactive GAs. In this work, we genetically dissected the functions of CYP714A1 and CYP714A2 in GA-mediated growth regulation. Using a combination of genetics and profiling of endogenous GAs in transgenic plants, we were able to conclude that CYP714A1 and CYP714A2 function redundantly in regulating plant growth and development, most likely through mediating similar GA catabolism, and may also act in early steps of the GA biosynthetic pathway, in provision of precursors for GA12, resulting in GA12 accumulation. In addition, we showed that transgenic rice plants overexpressing the Arabidopsis CYP714A2 gene displayed desirable agronomic phenotypes with high yield potential.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Expression patterns and protein localization of CYP714A1 and CYP714A2

A Blast search revealed that the Arabidopsis genome encodes two EUI-like (EL) P450s, CYP714A1 and CYP714A2 (Zhu et al., 2006), referred to hereafter as ELA1 and ELA2, respectively. The two Arabidopsis proteins share 72.5% identity with each other, and 38% identity with EUI, and contain all conserved amino acids for heme binding, oxygen binding, and activation, and the ERR (Glu–Arg–Arg) triad motif known to be present in all cytochrome P450 proteins (Figure S1). Interestingly, the two ELA genes are located next to each other with only a 769-bp interval with a retrotransposon next to the ELA1 promoter, suggesting that they might be generated from a simple duplication (Figure S1). To determine the expression patterns of ELA1 and ELA2, we examined β-glucuronidase (GUS) activity histochemically in transgenic plants expressing an ELA1 or ELA2 promoter–GUS reporter fusion transgene (ELA1–GUS or ELA2–GUS). As shown in Figure 1, ELA1–GUS expression was observed in the shoot apical meristem (SAM), petioles of young leaves and emerging leaves, in sepals, stigma, anther and filaments of the developing flowers, and in the receptacle and stigma of the developing siliques (Figure 1a,c and e). Similarly, ELA2–GUS was expressed in the SAM and petioles of young leaves. In contrast, ELA2 was expressed in the leaf margin and petiole vein of cotyledons, with low levels of GUS activity detected in the filaments of developing flowers and no GUS staining detected in siliques (Figure 1b,d and f). Reverse-transcription PCR (RT-PCR) analysis of ELA1 or ELA2 transcripts confirmed that ELA1 and ELA2 exhibit partially overlapping expression patterns (Figure 1g). These results suggested that ELA1 and ELA2 might be subjected to tissue-specific functionalization through altering expression patterns as is the case with many other GA genes (Olszewski et al., 2002; Yamaguchi, 2008).

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Figure 1.  Developmental and tissue-specific expression of the ELA1 and ELA2 genes. (a) GUS activity in the shoot apical meristem (SAM) and young leaves of the ELA1-GUS seedling. (b) GUS activity in the cotyledons, SAM, veins and petiole of the ELA2-GUS seedling. (c) GUS activity in the sepal, stigma androecium of the ELA1-GUS flower. (d) GUS activity in the filament of the ELA2-GUS flower. (e) GUS activity in the receptacle and stigma of the ELA1-GUS silique. (f) Non-GUS activity was detected in the ELA2-GUS silique. (g) Detection by RT-PCR of the ELA1 and ELA2 transcripts in different tissue of the wild-type (Col-0). Actin, loading control for RT-PCR.

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Plant P450s are usually anchored on the cytoplasmic surface of the ER and are occasionally associated with the plastids (Schuler and Werck-Reichhart, 2003; Lengler et al., 2005). Our previous study showed that the rice EUI is localized on the cytoplasmic surface of the ER (Zhu et al., 2006). To determine the subcellular localizations of the two Arabidopsis ELA proteins, we transiently expressed ELA–green fluorescent protein fusions (ELA1–GFP and ELA2–GFP) in onion epidermal cells. As shown in Figures 2(a–c), microscopic analysis of the transformed onion cells revealed a similar fluorescence pattern between the two ELA–GFP proteins and the m-gfp5-ER reporter known to be localized in the ER (Haseloff et al., 1997). We also generated a fusion protein between ELA2 and the red fluorescent protein of Discosoma sp. reef coral (DsRED), co-transformed ELA2–DsRED or DsRED with the m-gfp5-ER protein into the onion epidermal cells, and examined their localization patterns. As shown in Figures (2d–i), while there was little overlap between DsRED and gfp5-ER, ELA2–DsRED was found to be co-localized with the m-gfp5-ER. These results indicated that ELA1 and ELA2 are most likely targeted to the ER similar to the rice EUI protein (Zhu et al., 2006).

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Figure 2.  Subcellular localization of the ELA1 and ELA2 proteins. (a) Cytoplasm localization of the ELA1–GFP fusion protein. (b) Cytoplasm localization of the ELA2–GFP fusion protein. (c) Endoplasmic reticulum (ER) localization of the ER-localized m-gfp5-ER control protein. (d) Localization of the ELA2–DsRED fusion protein in the onion cell co-expressing the m-gfp5-ER protein. (e) The ER localization of the mGFP5-ER control protein in the same onion cell. (f) Co-localization of ELA2–RFP and m-gfp5-ER. (g) Cytoplasm and nuclear localization of the DsRED control protein. (h) The ER localization of the m-gfp5-ER protein in the same cell as in (g). (i) DsRED was not co-localized with m-gfp5-ER. Bar: 50 μm (a–i).

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Identification of single T-DNA insertion mutants of ELA1 and ELA2

To investigate potential roles of ELA1 and ELA2 in GA metabolism, the SALK T-DNA Express database (http://signal.salk.edu/cgi-bin/tdnaexpress/) was searched and one T-DNA insertional mutant for each ELA gene [SALK_016089 (ela1) and SALK_137272 (ela2)] was identified (Figure S2a). The RT-PCR analysis indicated that the two mutants are probably true knockout mutants (Figure S2b). Neither of the single mutants exhibited any observable morphological/developmental phenotype compared with wild-type Col-0 during the entire life cycle (Figure S2c–g).

Double mutant of ELA1-RNAi/ela2 increases biomass and flowers earlier

To further investigate the role of ELA1 and ELA2 in GA metabolism and plant development, simultaneous elimination of both ELA genes was desired. However, due to their close chromosome locations, it is impossible to create a double knockout mutant from two identified T-DNA insertional mutants. Instead, we generated an RNA interference (RNAi) transgene for ELA1, transformed the resulting transgene into the ela2 mutant, and generated many independent transgenic lines. Many of the ELA1-RNAi/ela2 transgenic plants had bigger cotyledons, enlarged rosettes, and increased height in comparison with the corresponding wild-type plants (Figure 3a–c), and also flowered 3–5 days earlier than Col-0. A 30–50% increase was observed for the sizes of cotyledons and rosette leaves (Figures 4a and S3). The RT-PCR analysis showed that the expression of ELA1 was efficiently silenced in the RNAi plants exhibiting clear morphological phenotypes (Figure 3d). We observed that these ELA1-RNAi/ela2 transgenic mutants also produced larger flowers, longer siliques and bigger seeds than the wild-type during the reproductive stage (Figures 3e–f and S3). Similar phenotypes were also observed in the transgenic ela1 mutants expressing an ELA2-RNAi transgene (data not shown). Scanning electron microscopy suggested that increased plant sizes of ELA1-RNAi/ela2 probably resulted from enlarged/elongated cell size (Figure 4b–d). Taken together, these results indicated that ELA1 and ELA2 function together to inhibit plant growth.

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Figure 3.  Phenotypes of the double mutant ELA1-RNAi/ela2. (a) Ten-day-old seedling of Col-0 (wild-type) and ELA1-RNAi/ela2 plant. Three representative independent transgenic plants are shown. Bar: 0.1 cm. (b) Four-week-old plants of Col-0 and ELA1-RNAi/ela2 lines. Bar: 1 cm. (c) Adult plants of the Col-0 and ELA1-RNAi/ela2 lines during flowering. Bar: 2 cm. (Note that ELA1-RNAi/ela2 plants flowered earlier than the Col-0 plant.) (d) Detection by RT-PCR of the ELA1 and ELA2 transcripts in Col-0 and ELA1-RNAi/ela2 lines, showing that ELA1 was silenced in the ELA1-RNAi/ela2 lines. Actin, loading control for RT-PCR. (e) Comparison of the flower organs of the Col-0 and ELA1-RNAi/ela2 plants. (f) Comparison of the siliques of Col-0 and ELA1-RNAi/ela2 lines. Bar: 0.1 cm. (g) Comparison of the seeds of Col-0 and ELA1-RNAi/ela2 lines. Bar: 100 μm.

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Figure 4.  Leaf and hypocotyl phenotypes of the double mutant ELA1-RNAi/ela2. (a) Rosette leaves of Col-0 (wild-type) and ELA1-RNAi/ela2. Bar: 1 cm. (b) Sections of the sixth rosette leaves of Col-0 and ELA1-RNAi/ela2. Bar: 100 μm. (c) Scanning electron microscopy views of the epidermal cells of the sixth rosette leaves of Col-0 and ELA1-RNAi/ela2. Bar: 50 μm. (d) Scanning electron microscopy views of the epidermal cells of Col-0 and ELA1-RNAi/ela2 hypocotyls, showing the elongated cells of the ELA1-RNAi/ela2 mutant. Bar: 100 μm. (e) The sections of Col-0 and ELA1-RNAi/ela2 hypocotyls. Bar: 50 μm.

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Overexpression of ELA1 and ELA2 causes severe dwarfism with defective leaf development

Additional support for the roles of ELA1 and ELA2 in GA-mediated plant growth and development came from our analysis of transgenic Arabidopsis plants that overexpress ELA1 (ELA1-OE), ELA2 (ELA2-OE), or the rice EUI gene (EUI-OE) driven by the constitutively active cauliflower mosaic virus 35S promoter (Figure 5d,e). All transgenic lines displayed severe dwarfism at all growth stages with deep green leaves (Figure 5a–c). These results supported our prediction that ELA1 and ELA2 probably function in GA deactivation, despite our previous failure to detect the GA 16α,17-epoxidase activity for ELA1 using the yeast expression system, likely due to unfavorable assay conditions (Zhu et al., 2006). It is interesting to note that ELA1-OE plants were smallest during the seedling and rosette stages (Figure 5a,b), while at the flowering stage, EUI-OE plants were the severest dwarfs that failed to produce seeds (Figure 5c). This finding suggested that enzymatic kinetics of the three proteins might be regulated by unrecognized co-effector(s) that are developmentally regulated. Consistent with our interpretation, exogenous application with 10 nm GA1, GA3, GA4, and GA9 promoted the growth of the dwarfed ELA1-OE, ELA2-OE, and EUI-OE plants (Figure S4). As expected, GA1 and GA3 could rescue the ELA1-OE, ELA2-OE, and EUI-OE plants, similar to transgenic rice (Zhu et al., 2006). Interestingly, GA4 and GA9 also exhibited obvious rescue activity on these transgenic plants including EUI-OE, probably due to the subtle difference of GA metabolism and signaling between rice and Arabidopsis (Griffiths et al., 2006; Yamaguchi, 2008).

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Figure 5.  Overexpression of ELA1, ELA2 and EUI in Arabidopsis. (a) Ten-day-old seedlings of Col-0 (wild-type), ELA1-OE, ELA2-OE, and EUI-OE lines. Bar: 0.1 cm. (b) Four-week-old plants of Col-0, ELA1-OE, ELA2-OE, and EUI-OE plants. Bar: 1 cm. (c) Adult plants of Col-0, ELA1-OE, ELA2-OE, and EUI-OE lines. Bar: 5 cm. [Note that ELA1-OE, ELA2-OE, and EUI-OE plants were dwarfed at the entire life cycle (a–c).] (d) Detection by RT-PCR of the ELA1, ELA2, and EUI transcripts in ELA1-OE, ELA2-OE, and EUI-OE leaves. Actin, loading control. (e) Western blot detection by an anti-ELA antibody. Rubisco staining was used as loading control for immunoblot analysis. (f) Transcription levels of the GA3ox1, GA2ox2, GID1a, GID1c, and RGA were detected by quantitative RT-PCR. Similar results were obtained using a second set of samples.

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We also carefully examined rosette leaves of the transgenic plants overexpressing ELA or EUI. Interestingly, leaf vascular patterns were significantly altered in ELA1-OE and EUI-OE transgenic plants (Figure 6a). The development of leaf vasculature was disrupted in the transgenic plants, particularly in the ELA1-OE leaves without secondary vein development (Figure 6a, insert). Consistent with the growth phenotype, the leaf vascular abnormality is less obvious in ELA2-OE lines. These results not only strengthened the argument of functional similarity between ELAs and EUI but also revealed the critical role of GAs in vascular patterning. Electron microscopy analysis of the leaf transverse sections showed that cell sizes of the palisade and spongy tissues were greatly decreased in these transgenic plants compared with the wild-type control (Figure 6b). As expected, epidermis cells were also smaller on the leaves of ELA-OE or EUI-OE transgenic lines than those of the wild type (Figure 6c). Consistent with their dwarf phenotypes, the hypocotyl epidermal cells of the transgenic plants were significantly shorter than those of the wild-type control with the ELA1-OE epidermal cells being the shortest (Figure 6d). These observations indicated that overexpression of the rice EUI gene or its Arabidopsis homologs could cause multiple defects in cell and organ development.

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Figure 6.  Leaf and cellular phenotypes of ELA, ELA2, and EUI overexpression plants. (a) The sixth rosette leaves of Col-0 (wild-type), ELA1-OE, ELA2-OE, and EUI-OE plants. Insert: the magnified ELA1-OE leaf, indicating abnormal leaf vein development. Bar: 0.1 cm. (b) The sections of the sixth rosette leaves of Col-0, ELA1-OE, ELA2-OE, and EUI-OE plants. (c) Scanning electron microscopy views of the epidermal cells of the sixth rosette leaves of Col-0, ELA1-OE, ELA2-OE, and EUI-OE plants. (d) Scanning electron microscopy views of the epidermal cells of Col-0, ELA1-OE, ELA2-OE, and EUI-OE hypocotyls. Bar: 50 μm. (Note that the cell sizes of ELA1-OE, ELA2-OE, and EUI-OE were greatly decreased (b to d).)

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The rice EUI gene feedback or feedforward regulates the genes involved in GA metabolism and signaling in the eui mutant and the Eui overexpressor (Zhu et al., 2006; Zhang et al., 2008). We also found that some GA genes were indirectly regulated by ELA1 and ELA2 expression due to altered GA levels; for instance, GA3ox1 was strongly upregulated in ELA1-OE and ELA2-OE with different levels, and GID1a and GIDc were also slightly upregulated in ELA1-OE (Figure 5f). We also observed that GA2ox2 was upregulated in ELA2-OE plants as well, probably due to accumulated GA12, GA20, and GA53 in the ELA2 overexpression plants (see Figure 8a), which are GA2ox substrates. These results are consistent with the above hypothesis that there is a subtle difference between the ELA1 and ELA2 enzymatic activity, also with the observation that the GID1 genes were slightly different in GA response (Griffiths et al., 2006).

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Figure 8.  Endogenous gibberellin (GA) levels in rice and Arabidopsis plants. (a) The GA levels in the elongating second internodes of transgenic rice constitutively expressing ELA1, ELA2, EUI, and wild-type plants. The GA measurements were repeated with similar results. The level of GA19 is shown as an inset. Asterisks, undetectable. (b) The GA levels in the ELA1-RNAi/ela2 and wild-type (Col-0) plants. The GA measurements were repeated with similar results. Levels of GA12 and GA24 are shown as an inset because of their high levels. Asterisk, undetectable. (c) Partial rescue of the eui phenotype by the ectopic expression of ELA1 and ELA2 in the EUI-RNAi line (S73). Asterisks, significant difference at = 0.05.

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Ectopic expression of ELA1 and ELA2 greatly alters the agronomic traits of transgenic rice

To test if the Arabidopsis ELA genes also function in rice, we generated transgenic rice plants that overexpress ELA1 (ELA1-OER) or ELA2 (ELA2-OER) driven by the 35S promoter. The second and third generations (T1 and T2) of several independent transgenic lines were grown in isolated paddy fields with the wild-type control. Figure 7(a,b) shows that ELA1-OER lines were morphologically similar to the EUI-OE rice plants (Zhu et al., 2006), with severe dwarfism and decreased fertility (Figures 7a,b and S5). This severity of the dwarf phenotype of ELA1-OER plants correlated well with the expression level of the ELA1 transgene, indicating that ELA1 also has a strong GA deactivation activity in transgenic rice (Figure 7d). Similar to what was observed in transgenic Arabidopsis plants, ELA2-OER transgenic rice lines displayed the dose-dependent semi-dwarf phenotype (Figures 7a,e and S5). Interestingly, the ELA2-OER plants showed a good morphological pattern for high yield potential, with more fertile tillers than the wild-type (Figure 7f). As a result, grain productivity was significantly higher in the ELA2-OER lines than in the wild-type control (Figure 7g). Our results suggested that ELA2 could be a good tool for molecular breeding for higher-yield cereal crops.

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Figure 7.  Overexpression of ELA1 and ELA2 in rice. (a) Seedlings of the wild-type (TP309), ELA1-OER, and ELA2-OER lines. Four representative independent transgenic plants of ELA1-OER and ELA2-OER are shown, respectively. Bar: 2 cm. (b) Adult plants of TP309, ELA1-OER lines and ELA2-OER. Bar: 10 cm. (Note that the ELA1-OER plants were severe dwarfed, while the ELA2-OER plants were slightly reduced in plant height.) (c) Detection by RT-PCR of the expression levels of ELA1 and ELA2 in the transgenic plants. Ubi-1 was used as a RT-PCR control. (d) Protein accumulation of ELA1 and ELA2 was detected in the transgenic line by western blot using an anti-ELA antibody. Rubisco staining was used as loading control for immunoblot analysis. (e) Comparison of the plant heights of TP309 and ELA2-OER lines. (f) Tiller numbers of TP309 and ELA2 adult lines. (g) Grain weight per plant of TP309 and ELA2 adult lines. The results (e to g) indicated that the ELA2-OER rice exhibited a higher yield potential with the better agronomic traits.

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Endogenous GA levels in transgenic rice and ELA1-RNAi/ela2

To further determine the biochemical function of ELA1 and ELA2 in GA deactivation, endogenous levels of precursor and bioactive GAs in the internodes of transgenic rice constitutively expressing ELA1 and ELA2 and in Arabidopsis ELA1-RNAi/ela2 plants were measured. Our independent assays indicated that the levels of bioactive GAs, including GA4 and GA1, were decreased or undetectable in the rice ELA1-OER and ELA2-OER plants (Figure 8a), similar to those in EUI-OX (Zhu et al., 2006). Intriguingly, we detected slight higher levels of GA12 (approximately 2-fold) in the rice ELA1-OER and ELA2-OER plants, and a higher level of GA19 (3.5-fold) in the rice ELA2-OER plants, which was greatly decreased in ELA1-OER and EUI-OX (Figure 8a). Consistently, we also detected GA20 accumulation in ELA2-OER in comparison with ELA1-OER and EUI-OX (Figure 8a). Furthermore, we detected slightly higher levels of GA4 (2.4-fold) and its immediate precursor GA9 (approximately 2-fold) in the seedlings of ELA1-RNAi/ela2 than in wild-type plants (Figure 8b). Consistent with the finding in rice, the level of GA19 (approximately 0.6-fold) was slightly decreased in ELA1-RNAi/ela2 plants compared with wild-type plants (Figure 8b).

In further support of the function of ELA1 and ELA2 in GA catabolism, our additional genetic experiments showed that ectopic expression of ELA1 and ELA2 in the rice EUI-RNAi line (Zhang et al., 2008) greatly compromised the eui phenotype (Figure 8c). These results suggest that ELA1 and ELA2 probably function in the inactivating reaction of bioactive GAs similar to that mediated by the rice EUI P450, and also that the two P450s might function in early stages of the GA biosynthetic pathway, resulting in GA12 accumulation, and ELA2 might display additional activity on GA19 biosynthesis. However, those novel functions for ELA1 and ELA2 remain to be confirmed.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

ELA1 and ELA2 are new GA-deactivating P450s

Many cytochrome P450 monooxygenases have been identified to catalyze diverse reactions of many bioactive compounds including phytohormones (Mizutani et al., 1997; Hull et al., 2000; Duan and Schuler, 2005; Kim et al., 2005). EUI is the first P450 identified as catalyzing a GA inactivation reaction that converts non-13-hydroxylated bioactive GAs into bio-inactive 16,17-[OH]2-GAs. In this study, we provided evidence to argue for similar functions of the two Arabidopsis EUI P450 homologs, ELA1 (CYP714A1) and ELA2 (CYP714A2), in GA metabolism with subtle divergence in enzymatic capacity. The subtle phenotypic difference might result from amino acid sequence divergence of the CYP714 subgroup, which probably regulates enzymatic kinetics of the proteins (Zhu et al., 2006; Figure S1). The 16α,17-[OH]2-GAs were found in many other plant species (Yamane et al., 1988; Gaskin et al., 1992; Hedden et al., 1993; Santes et al., 1995; Blake et al., 2000; Pearce et al., 2002), and our study supports the prediction that EUI-like GA16,17ox catalyzes GA deactivation in a variety of plant species, which probably plays an important role in GA homeostasis and development in plants.

ELA1 and ELA2 regulate plant growth and development through fine-tuning GA homeostasis

ELA1 and ELA2 constitute a pair of duplicated genes. They have probably been subjected to sub-functionalization with overlapping but distinct expression patterns and the proposed enzymatic activities and/or kinetics. This sequence divergence might provide substrates on which natural selection could act to retain their functions, as observed in many other duplicated genes which are often sub-functionalized from their ancestral functions held by the parental genes (Prince and Pickett, 2002; Hittinger and Carroll, 2007; Des Marais and Rausher, 2008; Wang et al., 2010). With this scenario, we propose that the difference between ELA1 and ELA2 expression patterns may play an important role in fine-tuning GA homeostasis during growth and development in Arabidopsis.

Fine-tuning GA homeostasis might also result from subtle differences in enzymatic activity and/or kinetics of these EUI family members. Constitutive expression of ELA1, ELA2 and EUI generated transgenic Arabidopsis and rice plants with variable defects in growth and development strongly suggesting that the P450s should exhibit different enzymatic activity or kinetic parameters towards diverse GA substrates. Instead, we observed greatly decreased levels of the bioactive GAs, GA4 and GA1, in ELA1-OER and ELA2-OER rice plants (Figure 8a). In the ELA2-OER rice, GA1 content is about one-half of the wild-type level, higher (approximately 2-fold) than in the ELA1-OER and EUI-OE rice, whereas GA4 contents were almost the same in ELA1-OER and ELA2-OER. The changes in the active GA levels were appropriately reflected in the difference in plant heights.

It is notable that GA12, a precursor of bioactive GAs of both the early 13-hydroxylation and non-13-hydroxylation GA pathways and with no biological function recognized per se, was accumulated in ELA1-OER and ELA2-OER but not in EUI-OX rice plants. We proposed that the enzymes might have obtained capacity to function in early stages of the GA biosynthetic pathway, resulting in provision of precursors for GA12. This function was not found for EUI. Consequentially the levels of GA53 were higher in ELA1-OER and ELA2-OER than EUI-OX (Figure 8a). An additional role in GA19 biosynthesis could be also deduced for ELA2. These predicted biochemical functions of ELA1 and ELA2 fit well with and appropriately explain the phenotypes of transgenic rice and Arabidopsis plants (Figures 5–7). Based on these data, we propose that ELA1 is functionally more similar to EUI than is ELA2. However, further experiments are needed to biochemically characterize these functions of the P450s in GA metabolism with advanced techniques.

Within this scenario, and given that the Arabidopsis genome contains different components of GA signaling and metabolism in comparison with the rice genome (Griffiths et al., 2006; Yamaguchi, 2008), there certainly exists a subtle difference of GA metabolism and signaling between rice and Arabidopsis. As a consequence, exogenous application of GAs could restore the ELA1-OE, ELA2-OE, and EUI-OE plants to varying degrees (Figure S4), and GA4 and GA9 also exhibited obvious rescue activity on these transgenic plants including EUI-OE, which was not found on transgenic rice EUI-OX plants (Zhu et al., 2006). Nevertheless, the EUI-like protein-mediated fine-tuning of GA homeostasis probably plays important roles in plant growth and development in different developmental stages and organs, and in response to environmental changes, in association with other groups of GA catabolic enzymes including GA2ox (Zhu et al., 2006; Yamaguchi, 2008; Achard et al., 2009).

Gibberellins play important roles in a variety of plant growth and development processes. It is quite interesting to see that leaf vein development was greatly suppressed in the transgenic plants (Figure 6a). It is well known that leaf vein patterning is dependent on auxin signaling (Dengler and Kang, 2001; Avsian-Kretchmer et al., 2002; Megan et al., 2008). Our current study suggests that GAs might also play an important role in leaf vascular differentiation and development. However, how GAs are involved in leaf vasculature development remains elusive. One possible mechanism could be that GA homeostasis or signaling modulates auxin distribution, considering that GAs and auxin are frequently thought of as nodes in a growth network but with non-interchangeability (Fu and Harberd, 2003; Kuppusamy et al., 2009).

ELA2 provides a good tool for crop improvement

We have previously explored the potential of the EUI gene in rice molecular breeding by expressing EUI under the control of the GA biosynthesis genes GA20ox2 and GA3ox2 (Zhang et al., 2008). Like the EUI gene, ectopic expression of both ELA1 and ELA2 greatly decreased plant height of the transgenic rice (Figure 7a–d). While ELA1-OER lines were severely dwarfed, ELA2-OER lines exhibited good ‘Green Revolution’ morphological phenotypes (Monna et al., 2002; Sasaki et al., 2002; Spielmeyer et al., 2002; Sakamoto et al., 2003; Sakamoto, 2006), including semi-dwarfism with moderately decreased plant height, and more yielding tillers that resulted in a higher grain productivity than the wild-type control (Figure 7). Of particular concern, many hybrid rice varieties, including the so-called ‘Super rice’, often have an over-height problem, largely limiting their commercialization. ELA2 therefore provides a good approach for molecular design for the higher yield potential in rice. Taken together, our current study further documents that the manipulation of the GA metabolism pathway can increase the agronomic value of crops.

Experimental Procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plasmid construction and plant transformation

A 500-bp coding region of ELA1 or ELA2 was amplified by PCR and inserted into the RNAi vector pCAMBIA1300RS (provided by Dr Yinong Yang, Penn State University, PA, USA). The construct was transformed into the T-DNA mutant ela2 or ela1 to generate more than 30 independent transgenic lines of ELA1-RNAi/ela2 or ELA2-RNAi/ela1. The stable homozygous lines (T3–T6) of the double mutant were identified and used for phenotypic analysis.

Plasmids for overexpression were generated by inserting the full-length coding region of ELA1 and ELA2 into the rice expression vector 35S-C1301 (Zhu et al., 2006) or pCAMBIA2300S (for Arabidopsis transformation with the 35S promoter, provided by Dr Yinong Yang), then transformed into the wild-type Arabidopsis Col-0 and the wild-type rice cultivar TP309 to generate more than 20 independent ELA1 and ELA2 overexpression lines in Arabidopsis and rice (ELA1-OER or ELA2-OER), respectively. Six independent single-insertion transgenic Arabidopsis lines were identified in the T3 generation and used subsequently for phenotypic analyses. Rice lines were assayed with the second and third (T1 and T2) generations grown in the experimental field. Plant height and other agronomic traits were measured in the mature period with more than 30 plants for each line. ELA1 and ELA2 were ectopically expressed in an eui mutant by crossing ELA1-OER or ELA2-OER with a stable EUI RNAi line also in TP309 background (Zhang et al., 2008).

Promoter activity

The1.6-kb and 1.4-kb promoter regions of ELA1 and ELA2 were isolated and fused to PBI101.1 (ELA1-GUS and ELA2-GUS), respectively. The ELA1-GUS and ELA2-GUS reporter plasmids were introduced into Col-0 to generate independent transgenic lines. GUS activity was histochemically assayed in homozygous lines (T3).

Identification of T-DNA insertional mutants

According to the genomic locations and sequences of the CYP714A1 (ELA1, At5g24910) and CYP714A2 (ELA2, At5g24900) genes, we got the SALK T-DNA insertion lines SALK_016089 and SALK_137272 from the Arabidopsis Biological Resource Center (http://abrc.osu.edu/). The mutant SALK_016089 (ela1) contains a T-DNA insert in axon 4 of ELA1, and SALK_137272 (ela2) a T-DNA insert in axon 5 of ELA2.

Subcellular localization and protein analysis

The ELA1–eGFP, ELA2–eGFP and ELA2–DsRED fusions were made by in-frame fusion of the full-length ELA1 and ELA2 cDNA with eGFP or DsRED in the vector 35S-C1301 (Zhu et al., 2006). Transient expression of the fusion proteins and the controls eGFP, DsRED, and the ER-localized m-gfp5-ER (Haseloff et al., 1997) in onion epidermal cells was performed using a helium biolistic device (Bio-Rad PDS-1000; http://www.bio-rad.com/) as previously described (Zhu et al., 2006). For western blot analysis, antibodies (antiserum) against the conserved fragment of ELA1 and ELA2 were raised with standard procedure. Protein extracts were prepared from seedlings of Arabidopsis (Col-0, ELA1-OE and ELA2-OE) and rice (the wild-type TP309, ELA1-OER and ELA2-OER). Protein gel blotting was performed using the SuperSignal West chemiluminescence kit according to the manufacturer’s protocol (Pierce Chemical, http://www.piercenet.com/).

RNA preparation and transcript analysis

Total RNAs were isolated from 3-week-old plants using TRIzol reagent (Invitrogen, http://www.invitrogen.com/) and treated with DNaseI using the DNA-free kit (Ambion, http://www.ambion.com/). First-strand cDNA was synthesized by Superscript III reverse transcriptase (Invitrogen) with poly(T)20 primer. ELA1 and ELA2 were measured by RT-PCR that was performed for 30 cycles with 1 μl of the first-strand cDNA and gene-specific primers in a total volume of 20-μl reaction mixture. The quantitative real-time PCR experiments were performed for the Arabidopsis GA metabolism and signal genes GA3ox1, GA2ox2, GID1a, GID1c, and RGA using a Roche LightCycler with LightCycler FastStart DNA Master SYBR Green I kit (Roche, http://www.roche.com/), using the primers as reported previously (Griffiths et al., 2006; Ariizumi et al., 2008; Rieu et al., 2008). Primer sequences can be found in Table S1.

Microscope observation

Plant materials were fixed in a solution of 5% (v/v) acetic acid, 45% (v/v) ethanol, and 5% (v/v) formaldehyde at 4°C overnight and dehydrated in series with ethanol. For scanning electron microscopy, the samples were critical-point dried in liquid CO2 and coated with gold, followed by visualization with a scanning electron microscope (JSM-6360LV, JEOL, http://www.jeol.com/). For histological sections, samples were embedded in Epon812 resin (Emicron, http://www.instrument.com.cn/), and transverse sections were made at 2 μm. Sections were examined microscopically (BX51, Olympus, http://www.olympus.com/) and photographed.

Analyses of endogenous GAs

For GA measurements, elongating rice second internodes and Arabidopsis seedlings (4 weeks old) were harvested and lyophilized. The analysis methods for extraction and detection are similar to a previous report (Varbanova et al., 2007), but modified to include the following 20 targeted GAs: 8/29/23/3/1/6/5/19/13/20/44/34/53/51/7/4/24/15/9/12 [according to liquid chromatography (LC) retention time]. The LC-tandem mass spectroscopy (LC-MS/MS) system used is composed of a Shimadzu UFLC system (http://www.shimadzu.com/) coupled to an Applied Biosystems 4000 QTRAP mass spectrometer (http://www.appliedbiosystems.com/)equipped with a turbo ion spray (TIS) electrospray ion source. Due to a potentially large number of known and unknown GA and related compounds as well as tissue-specific interferences, three multiple reaction monitoring (MRM) transitions/data channels were used for detecting and quantifying each individual GA (apart from GA9-where only two usable MRM transitions are available).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We are grateful to Dr Shinjiro Yamaguchi for GA chemicals and extensive discussion and suggestion, Dr Jianming Li for critical reading of the manuscript, Professor Yuji Kamiya for useful discussion, and Professors Yinong Yang and Jim Haseloff for the plasmids. This work was supported by grants from the National Natural Science Foundation of China (90817102, 30721061 and 30670186).

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1. Amino acid alignment and genomic structure of ELA1 and ELA2. (a) Sequence alignment of the ELA1, ELA2 and EUI proteins. (b) Genomic locations of the ELA1 and ELA2 genes. A retrotransponson inserts next to ELA1.

Figure S2. Characterization of ELA1 and ELA2 loss-of-function mutants ela1 and ela2. (a) Insertion sites of T-DNA in ela1 (SALK_016089) and ela2 (SALK_137272). (b) RT-PCR detection of the ELA1 and ELA2 transcripts in wild-type (Col-0), ela1 and ela2. Actin, loading control for RT-PCR. (c) Ten-day-old seedling of the wild-type, ela1 and ela2. Bar, 0.1 cm. (d) Four-week-old plants of the wild-type, ela1 and ela2 lines. Bar, 1 cm. (e) Adult plants of the wild-type, ela1 and ela2 lines during flowering. Bar, 2 cm. (f) Comparison of flower organs of the wild-type, ela1 and ela2 lines. (g) Comparison of siliques of the wild-type, ela1 and ela2 lines. Bar, 0.1 cm.

Figure S3. Comparison of tissue and seed sizes of wild-type and ELA1-RNAi/ela2 plants. (a) Relative area of cotyledons of the wild-type (Col-0) and ELA1-RNAi/ela2 lines. (b) Relative area of the 6th rosette leaf of the wild-type and ELA-RNAi/ela2 lines. (c) Length of siliques of the wild-type and ELA1-RNAi/ela2 lines. (d) Length of seeds of the wild-type and ELA1-RNAi/ela2 lines. (e) Width of seeds of wild-type and ELA1-RNAi/ela2 lines.

Figure S4. GA sensitivity of wild-type, ELA1-OE, ELA2-OE and EUI-OE seedlings. Two-week-old seedlings of the wild-type (Col-0) and ELA1-OE, ELA2-OE and EUI-OE lines were incubated with GA1, GA3, GA4, GA9 and GA12 (10 nM) or water (mock) for 1 week. Note that the dwarfed ELA1-OE, ELA2-OE, and EUI-OE plants responded differentially to diverse GAs. Bar, 1 cm.

Figure S5. Phenotype comparison of ELA1-OER and ELA2-OER rice. (a) Plant height of the wild-type (TP309) and ELA1-OER lines. (b) Length of panicles and internodes of the wild-type and ELA1-OER lines. (c) Length of panicles and internodes of the wild-type and ELA2-OER lines.

Table S1. Primer sequences for this study.

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