ABA inhibits shoot growth in rice
Seeds of rice cv. IR64 were germinated on MS agar for 48 h and then transferred to MS agar containing 0, 0.3, 1, 3 or 10 µm ABA for another 48 h. During the second 48 h period and in the absence of ABA, leaves 1, 2 and 3 grew 4, 35 and 32 mm, respectively, whereas 10 µm ABA inhibited the growth of these leaves by 75, 74 and 87%, respectively (Table 1). The growth observed in the presence of 10 µm ABA occurred mainly during the first 12 h. Lower ABA concentrations were less inhibitory, with 1 µm ABA inhibiting growth by approx. 50%.
Table 1. Growth arrest by abscisic acid (ABA) in young leaves of rice (Oryza sativa cv. IR64)
|Treatment||Leaf length (mm)|
|Leaf 1||Leaf 2||Leaf 3|
|2 d –ABA||8 ± 2b||9 ± 2g||2 ± 1k|
|4 d –ABA||12 ± 1a||44 ± 2d||34 ± 3h|
|2 d –ABA, 2 d + 1 µm ABA||10 ± 1b||30 ± 2e||17 ± 2i|
|2 d –ABA, 2 d + 10 µm ABA||9 ± 1b||18 ± 2f||6 ± 1j|
|Growth without ABA, days 3 + 4||4 ± 3q||35 ± 4p||32 ± 4p|
|Growth arrest by 10 µm ABA (%)||75||74||87|
IR64 seedlings are an order of magnitude less sensitive to ABA than Arabidopsis seedlings but the reason for this is unclear. It may reflect a lower sensitivity of the rice signal transduction pathway to ABA or a higher abundance of antagonistic gibberellins, which are released from imbibing rice embryos to promote endosperm breakdown. We compared the impacts of ABA and the synthetic gibberellin GA3 on the growth of leaf 2 (Fig. 1). In contrast to ABA, 5 µm GA3 stimulated leaf growth significantly and partly reversed the inhibition seen with 2, 5 or 10 µm ABA. Higher concentrations of GA3 did not reverse the inhibition further (data not shown). As leaf growth may be driven, in part, by sugars and amino acids released from the endosperm, the higher ABA requirement for growth arrest in rice may reflect the need to inhibit not only leaf processes but also mobilization of materials stored in the endosperm. In subsequent studies, we used 10 µm ABA in MS agar to arrest the growth of rice shoots.
Figure 1. Impact of abscisic acid (ABA) and the synthetic gibberellin, GA3, on shoot growth in rice (Oryza sativa) cv. IR64, measured in terms of the length of leaf 2 after 4 d of cultivation. Seedlings were cultured on MS medium for 2 d and then transferred to MS medium containing ABA (0, 2, 5 or 10 µm) and/or GA3 (5 µm), for a further 2 d. Arrow: growth achieved after the first 2 d of growth on MS medium. The letters below the histogram indicate groupings according to the Multiple Range Test of Duncan (1955). Error bars show SD (n = 20).
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ABA increases transcript levels of rice structural orthologues of ABI3 and ABI5 genes
Although ABA affects many aspects of leaf metabolism and growth, the ABA-regulated genes most specifically associated with growth arrest in Arabidopsis seedlings are the ABI genes. We focused on two of these genes –ABI3 and ABI5– which encode a B3 domain and a bZIP domain transcription factor, respectively. The availability of the complete genome sequences of Arabidopsis and rice facilitates the identification of structural orthologues (see the Supplementary material). blast and phylogentic analysis confirmed the long-held view (Hattori et al., 1995) that OsVP1 is the structural orthologue of ABI3 in rice (Supplementary material, Fig. S1) and established that a bZIP protein, which we denoted as OsABF1, is the structural orthologue of ABI5 in rice (Supplementary material, Fig. S2).
The question of the orthology of OsABF1 and ABI5 is complicated by a gene duplication that generated both ABI5 and AtDPBF2 in Arabidopsis. The function of the latter is poorly understood (Kim et al., 2002). However, a detailed comparison of bZIP domains (the basic DNA-binding domain and the adjacent leucine zipper domain) established that the sequence similarity between OsABF1 and ABI3 in these domains is greater than that between OsABF1 and AtDPBF2 (Supplementary material, Fig. S3).
Further support for the orthology of ABI5 and OsABF1 comes from the regulation of transcription. ABI5 transcript levels, like those of ABI3, are increased by the treatment of seedlings with ABA (Söderman et al., 1996; Kim et al., 2002; Arroyo et al., 2003), while AtDPBF2 transcript levels are not increased (Kim et al., 2002). The RT-PCR analysis showed that transcript levels for OsVP1 and OsABF1 are enhanced by ABA (Fig. 2). The gibberellin GA3 antagonized these responses to a small extent and had no inductive effect when used alone.
Figure 2. Effect of abscisic acid (ABA) and gibberellin (GA3) on gene expression in rice (Oryza sativa) cv. IR64. Transcript levels were estimated by the reverse transcription–polymerase chain reaction (RT-PCR). The genes are indicated on the left and the numbers of RT-PCR cycles are indicated on the right. rRNA, loading control of DNAse-treated rRNA.
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Abscisic acid responsiveness is not a general feature of the OsABF subfamily of bZIP genes. When we conducted RT-PCR on transcripts of the seven bZIP genes most closely related to OsABF1 (i.e. OsABF2–OsABF8), only OsABF3 also responded to ABA (Fig. 2). The nonresponsive members included OsABF5, which is also known as TRAB1, the only OsABF gene to have been previously studied (Hobo et al., 1999a). We were unable to detect transcripts of OsABF6 when we used primers based on the annotated sequence (Supplementary material, Table S1). When primers consistent with an alternative splicing pattern were used (Supplementary material, Table S1), OsABF6 transcripts were detected but proved unresponsive to ABA (data not shown). We conclude that, although the eight OsABF genes encode proteins with identical or almost identical DNA-binding domains (Supplementary material, Fig. S3), only OsABF1 and OsABF3 show ABA responsiveness at the level of transcript abundance in seedlings.
We studied the ABA and GA3 responsiveness of three additional rice genes (OsEm, OsLEA3 and WSI18). OsEm is known to be a target of TRAB1 (Hobo et al., 1999a), while OsLEA3 and WSI18 are structural orthologues of HVA1, a target of HvABI5 (Casaretto & Ho, 2003). The transcript levels of OsEm and OsLEA3 increased in response to ABA, and GA3 antagonized these responses to a small extent without having any detectable effect when used alone (Fig. 2). The transcript levels of WSI18, however, increased in response to ABA and GA3, indicating that its transcriptional control differed from that of its close paralogue, OsLEA3.
Several genes shown in Fig. 2 produced dual bands (OsABF3, OsEm and WSI18). To avoid confusing RT-PCR products of the transcripts with PCR products of contaminating genomic DNA, we designed primers to flank introns whenever possible. We also digested RNA with DNAse. In each case of dual bands in Fig. 2, the lower band was the expected band and the upper band corresponded to incompletely spliced transcripts. It is possible that incomplete splicing arises from immature tissue where some transcripts are retained in the nucleus (Fig. 3).
Figure 3. Sites of transcript accumulation for OsABF1, OsVP1 and TRAB1 in shoots of rice (Oryza sativa) cv. IR64. Left column: shoots from seedlings grown in the absence of abscisic acid (−ABA). Right column: shoots from seedlings grown in the presence of abscisic acid (+ABA). (a–h) RNA in situ hybridization conducted with antisense probes on basal sections of rice shoots; mRNA–cRNA hybrids were detected using alkaline phosphatase-linked anti-DIG IgG immunoglobulin. Sections: (a) and (b), transverse; (c)–(j), longitudinal. Genes: (a)–(d), OsABF1; (e) and (f), OsVP1; (g) and (h), TRAB1. Sections shown in (i) and (j) were stained with 4′-6-diamidino-2-phenylindole (DAPI). C, coleoptile; 1,2,3, leaf number; long arrows, abaxial sclerenchyma; short arrows, epidermal cells accumulating transcripts. Bars, 100 µm.
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Sites of transcript accumulation in the shoot base
We used RNA in situ hybridization to determine where the transcripts of OsVP1, OsABF1, TRAB1, OsEm, OsLEA3 and WSI18 accumulate in shoots. Both sense and antisense probes were used, but hybridization signals were obtained only with antisense probes. Transverse and longitudinal sections of the shoots were examined near the apex and also near the base, where the leaves are surrounded by the coleoptile.
Cellular differentiation was most active and most ABA-sensitive near the base (Fig. 3). In the transverse section for OsABF1 transcripts, all three leaves were visible in the shoot not treated with ABA (–ABA) (Fig. 3a), whereas in the shoot treated with ABA (+ABA) (Fig. 3b), growth of leaf 3 was sufficiently inhibited to be absent from the section. The antisense probe for OsABF1 transcripts showed weak hybridization to all three leaves of the transverse section of the –ABA shoot, except for a significant level of hybridization in the abaxial sclerenchyma (long, thin arrow) of leaf 1. In the +ABA shoot, hybridization of OsABF1 was very strong in the abaxial sclerenchyma of leaf 1. Longitudinal sections (Fig. 3c,d) confirmed these hybridization patterns for OsABF1 transcripts and established three additional points. First, in the sclerenchyma, hybridization was clearly present throughout the cytosol of the cortical fiber cells. Second, in the epidermis, there was a file of cells in which cells containing transcripts (small arrows) alternated with cells lacking transcripts. In the following paragraphs we show that these two cell types are silica cells and pavement cells, respectively. Third, in immature leaves of –ABA and +ABA shoots, hybridization was localized in spots that resembled DAPI-stained nuclei (Fig. 3i,j) in their size and general distribution throughout the tissue.
Transcripts of OsVP1 (Fig. 3e,f) and TRAB1 (Fig. 3g,h) showed broadly similar results to those for OsABF1 transcripts, but with two differences. First, OsVP1 transcripts were not clearly visible in immature leaves of –ABA or +ABA shoots, presumably reflecting low abundance. Second, TRAB1 transcripts were much more abundant than OsABF1 or OsVP1 transcripts in leaf 1 of –ABA shoots, a result consistent with the RT-PCR data (Fig. 2). Like transcripts of OsABF1, TRAB1 transcripts appeared to be localized in nuclei in the immature leaves of –ABA and +ABA shoots. Nuclear localization was also observed for OsEm and WSI18 transcripts (results not shown); like OsVP1 transcripts, OsLEA3 transcripts were not sufficiently abundant in immature leaves to allow their location to be seen clearly.
Figure 4 shows RNA in situ hybridization in leaf 1 of –ABA and +ABA shoots for transcripts of all six genes. For both treatments, longitudinal sections are on the left and show cytosolic hybridization within the epidermal silica cells and the cortical fiber cells of the sclerenchyma. Transverse sections are on the right and demonstrate that the files of silica cells (arrowed for OsLEA3) are separated from one another by two to four nonhybridizing epidermal cell files and are separated by the abaxial sclerenchyma from the subtending vascular bundles. In the –ABA shoots, strong hybridization was seen only for TRAB1 transcripts. By contrast, in the +ABA shoots, all six genes showed strong hybridization to cortical fiber cells and silica cells. Both of these cell types are known to be major support cells of the leaf (Savant et al., 1997; Tanaka et al., 2003; Ma et al., 2004). OsABF1, OsVP1, OsEm and OsLEA3 transcripts accumulated mainly in these support cells, whereas TRAB1 and WSI18 transcripts were abundant also in other cell types in leaf 1.
Figure 4. Sites of transcript accumulation for six genes in leaf 1 of IR64 rice (Oryza sativa) seedlings grown in the absence (left columns) and presence (right columns) of 10 µm abscisic acid (ABA). Narrow images, longitudinal sections; wide images, transverse sections. RNA in situ hybridization was conducted on basal sections using antisense probes for the indicated genes; mRNA–cRNA hybrids were detected with alkaline phosphatase-linked anti-DIG IgG immunoglobulin. Genes: (a) and (b), OsABF1; (c) and (d), OsVP1; (e) and (f), TRAB1; (g) and (h), OsEm; (i) and (j), WSI18; (k) and (l), OsLEA3. The same magnification was used for all images (bar, 100 µm). Leaf thickness decreased with distance from the mid-vein.
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We examined in more detail the epidermal cell files in which alternate cells possessed or lacked the six transcripts. In Fig. 5, acridine orange staining was compared with RNA in situ hybridization for the antisense probe of OsVP1. The hybridizing cells are easily recognized as silica cells by their dumbbell structure (Kaufman et al., 1985), and we confirmed the presence of silica in these cells by staining with methyl red (data not shown). Acridine orange staining elicited a brilliant red fluorescence from the nuclei of both the silica cells and the epidermal pavement cells that alternated with them along the file. The presence of nuclei in the pavement cells suggested that they were active but unable to accumulate transcripts of the six genes studied here. The files themselves occurred in pairs that were not immediately adjacent but separated by several files of other epidermal cells (compare with arrows in Fig. 4d).
Figure 5. Anatomical features of IR64 rice (Oryza sativa) shoots during abscisic acid (ABA)-induced growth arrest. (a) and (b) Identification of silica cells as sites of accumulation of OsVP1 transcripts in a basal longitudinal section of leaf 1 in shoots grown in the presence of ABA (+ABA). (a) Leaves stained with acridine orange. (b) RNA in situ hybridization using an antisense probe for OsVP1; mRNA–cRNA hybrids were detected using alkaline phosphatase-linked anti-DIG IgG immunoglobulin. cfc, cortical fiber cells; pc, epidermal pavement cells; sc, silica cells. (c) and (d) Acridine orange staining of leaf 1 in basal transverse sections of shoots grown in the absence (–ABA) (c) and presence (+ABA) (d) of ABA. ab, abaxial sclerenchyma; ph, phloem sclerenchyma; xy, xylem sclerenchyma. Bars, 50 µm.
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Comparison of the bases of leaf 1 in –ABA shoots (Fig. 5c) and +ABA shoots (Fig. 5d) revealed that ABA inhibited sclerenchyma development. In –ABA shoots, the abaxial sclerenchyma and the sclerenchyma of the xylem and phloem were well developed. In +ABA shoots, all three regions of sclerenchyma were poorly developed and the cortical fiber cells lacked their characteristic thick walls.
The cortical fiber cells of the sclerenchyma are thick-walled because of the formation of the secondary walls, a major component of which is cellulose. Rice contains at least 10 genes encoding cellulose synthase subunit A. Mutant analysis indicated that three of these genes (OsCesA4, OsCesA7 and OsCesA9) are essential for secondary cell-wall formation and for the strength of the stem (Tanaka et al., 2003). CesA4, CesA7 and CesA9 subunits do not have entirely redundant functions, because knocking out the gene for any one of these enzymes reduced the thickness of the sclerenchyma, weakened the stem and caused dwarfing. We compared the transcript levels of these three genes with those of OsVP1 and OsABF1 (Fig. 6). Seedlings were grown on MS agar without ABA; after 2 or 4 d, some seedlings were transferred to agar containing 10 µm ABA for 1 or 2 d, and then some were returned to MS agar without ABA for 1 d. Transcript levels for OsVP1 and OsABF1 were low on –ABA medium, increased in response to ABA and then decreased on return to –ABA medium. High transcript levels were seen for OsCesA4 and OsCesA7 throughout these treatments. By contrast, transcript levels for OsCesA9, which decreased slowly when seedlings were grown on –ABA medium, decreased rapidly when seedlings were grown on +ABA medium and then increased again when seedlings were returned to –ABA medium. The loss of OsCesA9 transcripts in response to ABA treatment may contribute to growth arrest in general and to failure of sclerenchyma development in particular.
Figure 6. Effect of 10 µm abscisic acid (ABA) on the transcript levels of OsVP1, OsABF1 and three OsCesA genes required for secondary cell-wall formation. RNA was extracted from IR64 rice (Oryza sativa) seedlings grown under the indicated conditions. Transcript levels were visualized by reverse transcription–polymerase chain reaction (RT-PCR). The genes are indicated on the left and the numbers of RT-PCR cycles are indicated on the right. rRNA, loading control of DNAse-treated rRNA. The shoot length is the length of the longest leaf at each time point.
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Sites of accumulation of transcripts in the shoot apex
We conducted RNA in situ hybridization for OsABF1, OsVP1, TRAB1, OsEm, WSI18 and OsLEA3 on transverse sections of the more mature apical region of –ABA and +ABA shoots (Fig. 7). In general, the patterns for the apex were similar to those recorded in Fig. 4 for the base, but a clear difference was the presence in the apex of leaf 1 of both abaxial and adaxial sclerenchyma, with significant hybridization in both, especially OsABF1 and OsLEA3.
Figure 7. Sites of transcript accumulation for six genes in the shoot apex of IR64 rice (Oryza sativa) seedlings grown in the absence (left column) and presence (right column) of 10 µm abscisic acid (ABA). RNA in situ hybridization was conducted on transverse sections using antisense probes (a–l), no probe (m) and sense probe (n); mRNA–cRNA hybrids were detected using alkaline phosphatase-linked anti-DIG IgG immunoglobulin. Genes: (a) and (b), OsABF1; (c) and (d), OsVP1; (e) and (f), TRAB1; (g) and (h), OsEm; (i) and (j), WSI18; (k), (l) and (n), OsLEA3; (m), without probe. Arrows, adaxial sclerenchyma. The same magnification was used for all images (bar, 100 µm). Leaf thickness decreased with distance from the mid-vein.
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