Os04g0463500

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Annotated Information

Function

• 　In this study, the researchers demonstrated that TDD1 encodes the bsubunit of anthranilate synthase, OASB1, which functions as a rate-limiting enzyme for Trp biosynthesis.

• The abnormal phenotypes observed in tdd1 flowers are quite similar to those in known Arabidopsis auxin mutants, which suggest a possibility that TDD1 is involved in IAA synthesis during the floral developmental process.

Mutation

• The mature tdd1 embryo grows to more than 1.5 mm in length, which is slightly smaller than the wild-type (WT) embryo (Figure 2a,b). The growth rate of the mutant embryo is much lower than that of WT and the size of the mutant embryo at 5 days after pollination (DAP) is similar to that of the WT embryo at 3 DAP. At the early stages of embryogenesis, before organogenesis, the tdd1 embryo is morphologically indistinguishable from WT. Around 4 DAP or later, the tdd1 embryo can be distinguished from the WT embryo by the absence of both shoot and radicle in the mutant. Although the matured tdd1 embryo exhibits a simple internal structure, a cell-dense region usually forms around its center (arrowhead in Figure 2b) and a palisade-like cell layer, which may correspond to the epithelium layer, forms at its surface (Figure 2b, arrow and superimposed panel). The researchers examined the expression of two molecular markers, OSH1 and OsSCR, that are specifically expressed in the presumptive region of future shoot formation and the L2 layer of embryo, respectively. These two marker genes were expressed in the mutant embryos at the same places as in the WT embryos (Figure 2d,f). These results suggest that the tdd1 embryo succeeds in localization of the shoot region and in establishment of L2 layer identity and consequently succeeds in establishment of the basic pattern of the embryo. In contrast to severe defects in organ formation in the tdd1 embryo, the tdd1 endosperm develops normally, indicating that TDD1 is not involved in endosperm formation and development (Figure 2h compared with 2g). The growth of the mutant calli was slower than that of WT calli, which corresponds to the lower growth rate of the tdd1 embryo. When tdd1 calli were placed on regeneration medium, some of them produced adventitious leaves and shoots (Figure 2j).

Figure 2. Phenotypes of tdd1 embryos. (a, b) Median longitudinal sections of mature embryos of WT (a) and tdd1 (b). (a) Arrowhead and arrow indicate root apical meristem and shoot apical meristem, respectively. s, c and e indicate scutellum, coleoptile and epiblast, respectively. (b) Organless phenotype of tdd1 embryo. Inset shows a close-up view of an epithelium-like layer indicated by the arrow. A cell-dense region is indicated by the arrowhead. Bars = 200 lm. (c–f) in situ hybridization with the antisense RNA probe against OsSCR (c, d) and OSH1 (e, f). (c, e) WT embryos 3 DAP. (d, f) tdd1 embryos 5 DAP. Bars = 50 lm. (g, h) Mature seeds of WT (g) and tdd1 (h). Arrowheads indicate the embryos. No apparent difference was observed between the endosperms of the two genotypes. Bars = 2 mm. (i, j) Regenerated plants from calli of WT (i) and tdd1 (j). The regenerated plants of tdd1 can develop shoots and root. .

• The regenerated tdd1 plants showed a dwarf phenotype (Figure 3a) with abnormal leaf morphology (Figure 3c). Dwarfism was mainly caused by a defect in leaf elongation, especially in the leaf blade; growth reduction was not as severe in the leaf sheath (Table 1). Consequently, the ratio of blade to sheath in tdd1 was dramatically reduced (1.14) in comparison with that of WT (1.99). The angle between blade and stem (lamina joint) in the mutant leaf was increased relative to that of WT (Figure 3b, Table 1). Furthermore, the width of the mutant blade was about one-half that of WT (Figure 3c, Table 1). The growth of roots was also severely inhibited in the mutant (Figure 3d).

• The regenerated tdd1 plants were frail and almost died before flower formation. However, some plants (fewer than 1%) produced flowers with abnormal morphologies (Figure 3f–i). A normal rice flower has two glumes, one lemma and one palea (Figure 3e). Inside the flower, there are two white lodicules at the adaxial (lemma) side, which may correspond to the petals of the Arabidopsis flower; six stamens; and one pistil with two stigmas (Figure 3j). In regenerated mutant plants, about 78.8% (41/52) of the flowers contained an irregular number of one or more organs (Figures 3f–i, k–r and 4). There was considerable variation in the number of stamen, which varied from zero to seven (Figures 3k,l and 4). The abnormal variation in organ number was observed not only for stamen but also for palea, lemma, lodicule, pistil and stigma (arrowhead in Figures 3f,g, arrows in k,l and 4). In some flowers, an abnormal gap was formed between malformed palea and lemma (asterisk in Figure 3h) and a lemma-like tissue occasionally developed in the usual position of the lodicule (arrowheads in Figure 3i,l). Furthermore, several types of fused organs were sometimes observed. For example, two lodicules were fused (Figure 3m), a stamen-like organ was fused with a lodicule (Figure 3n,o) and a stigma-like organ was fused with a malformed stamen (Figure 3p,r). A few mutant plants, out of more than 1000, were able to develop mature seeds with organless embryos identical to the mutant embryos segregating in the progeny of heterozygous plants. This finding indicates that the organless phenotype in the embryo is transmitted through the regenerated plants and expressed again at the embryogenesis stage.

Figure 3. Morphological characteristics of the tdd1 mutant. (a–d) Morphology of WT (left) and tdd1 (right) plants. (a) Gross morphology of the reproductive stage. Bar = 20 cm. (b) Lamina joint of the leaves. Bar = 5 cm. (c) Leaf blades. Bar = 1 cm. (d) Roots of 3-week-old regenerated plants. Bar = 1 cm. (e–r) flower morphology of WT (e, j) and tdd1 typical mutant (f–i, k–r). Bar = 1 mm. (e–i) Gross morphology of flowers. (f–i) Four different phenotypes of tdd1 flowers are shown. Pl, palea; Le, lemma; Gl, glume. (f) Arrowheads indicate two malformed paleas. (g) Flower with defective lemma. (h) Asterisk indicates an abnormal gap between palea and lemma. (i) Arrowhead indicates an abnormal additional organ. (j–r) Morphology of the flower interior is shown. Lo, lodicule; Sg, stigma; Pi, pistil; St, stamen; St*, malformed stamen. (j–l) Numbered arrows indicate stigmas in tdd1 mutant flowers (k, l) differing in stigma number from WT (j). Arrowheads indicate abnormal additional organs. (m–r) Two lodicules (m), lodicule and stigma (n, o) and stamens and stigma (p–r) are occasionally fused. (q) Shows a magnified picture of the fused organs in (p). .

Table 1 Leaf phenotype of tdd1 regenerated plants .

• The researchers also examined the internal structure of mutant leaves by light microscopy. Figure 5 shows cross-sections of the middle part of flag leaf blades of tdd1 and WT plants. In WT leaf, five small vascular bundles were usually arranged between two large vascular bundles (Figure 5a). In contrast to WT, there were usually three small vascular bundles between two large vascular bundles in mutant leaf (Figure 5b). The reduced number of small veins in the tdd1 mutant might be the results of reduced or prematurely arrested lateral growth in the leaf blade. Furthermore, the structure of both large and small vascular bundles was abnormal in the mutant (Figure 5d,f).

Figure 5. Internal structure of leaves in regenerated tdd1 plants. (a, b) Cross sections of the leaf blades of WT (a) and tdd1 (b). Arrowheads indicate large vascular tissues. (c, d) Close-up view of the large vascular tissues of WT (c) and tdd1 (d). PH, VS and BSC indicate phloem, vessel and bundle sheath cell, respectively. Arrow in (d) shows abnormal arrangement of bundle sheath cells. (e, f) Cross sections through the small vascular tissue of WT (e) and tdd1 (f). BC indicates the bulliform cells. Arrows in (f) show the incomplete small vascular tissues and arrowheads show the abnormal bulliform cells. Bar = 100 lm. .

Expression

• The researchers next examined the expression pattern of the GUS reporter gene uidA under the control of the TDD1 promoter (TDD1-uidA) compared with uidA expression under the control of the synthetic auxin-responsive promoter DR5 (DR5-uidA) (Ulmasov et al., 1997), because we expected that TDD1 would be important for IAA synthesis. In young leaves, TDD1-uidA expression was observed at the tip region (Figure 7a). This expression pattern was similar to DR5-uidA (Figure 7b). TDD1-uidA expression was also observed in vascular bundles of young leaf (Figure 7a). In root, TDD1- uidA activity was detected in the root cap and vascular bundle (Figure 7c), whereas strong expression of DR5-uidA was also localized at the root cap (Figure 7d). uidA activity under the control of both promoters was also detected in dividing cells of the root pericycle, where lateral root initiation occurs (Figure 7e,f).

Figure 7. Localization of GUS activity in transgenic rice carrying TDD1-uidA or DR5-uidA. .

Evolution

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Labs working on this gene

• Bioscience and Biotechnology Center, Nagoya University, Nagoya, Aichi 464-8601, Japan,
• Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan,
• Genome Resource Center, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan, and
• Graduate School of Agriculture and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan