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Isolation and characterization of an abscisic acid-insensitive mutation that affects specifically primary root elongation in rice (Oryza sativa L.)
Plant Science 164 (2003) 971 /978 www.elsevier.com/locate/plantsci
Isolation and characterization of an abscisic acid-insensitive mutation that affects specifically primary root elongation in rice (Oryza sativa L.) Shan-Guo Yao *, Shin Taketa, Masahiko Ichii Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan Received 6 November 2002; received in revised form 3 February 2003; accepted 5 February 2003
Abstract The isolation and detailed characterization of a monogenic recessive root growth mutant, srt6 , which was derived from an M2 population of rice (Oryza sativa L.) cv. Oochikara treated with NaN3, is described. Besides greatly reduced primary root length and diameter, the mutant at the seedling stage also shows inhibited lateral root elongation and altered root hair formation. Hydroponic cultivation reveals that root length of the mutant recovers to the level of the wild type at the booting stage, but both lateral root elongation and root hair formation in the mutant remain inhibited at all growth stages. These observations suggest that the expression of the srt6 gene is phase-specific, and that the effects of SRT6 on root growth are restricted specifically to the development of primary roots. Physiological characterization reveals that the mutant has pleiotropic defects in abscisic acid (ABA) responses, which are similar to those reported for Arabidopsis ABA-insensitive mutants abi1 and abi2 , i.e. reduced sensitivity of root growth and seed germination, and excessive water loss. Combined with its normal response of root growth to fluridone, the ABA biosynthesis inhibitor, suggesting that the action of the srt6 gene might be related to ABA perception or signal transduction but not ABA biosynthesis in the controlling of early primary root elongation in rice. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Abscisic acid; Mutant; Rice; Root elongation
1. Introduction Root system of plants functions in various aspects especially uptake of nutrients and water [1]. Because the supplies of nutrient and water in soil are always limited, local and variable, the spatial deployment of the root system, i.e. root architecture, largely determines the ability of a plant to exploit these resources [2]. Root architecture is defined by parameters such as length, number and dimension, and the root length is considered to be related to various important characters such as nutrient uptake [3], lodging tolerance [4], drought tolerance [5,6], and yield [7,8]. Thus, uncovering the mechanisms controlling root elongation will be of great importance in improving plants’ sustainability and productivity. * Corresponding author. Tel./fax: /81-87-891-3127. E-mail address: [email protected] (S.-G. Yao).
To uncover the mechanisms controlling root elongation, genetic analysis of mutants is proven to be a useful approach. In Arabidopsis , many mutants on root elongation have been isolated. These include root meristemless mutants [9,10], short-root mutants [11 / 13], and root expansion mutants [14,15]. In rice, several short-root mutants have been identified and genetically characterized [16 /20]. However, great many things related to root elongation remain largely unknown. The phyotohormone abscisic acid (ABA) plays an essential regulating role for a variety of physiological and developmental processes, including embryo development, seed dormancy, transpiration, adaptation to environmental stresses (for review, see [21]), and also root growth [22 /25]. Substantial progress has been made in the characterization of ABA signaling pathways (for review, see [26]). In particular, the extensive mutational analysis in Arabidopsis has identified several genes that control ABA responsiveness [27 /29]. How-
0168-9452/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00081-5
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ever, analysis of these mutants mainly focused on seed development or plant adaptation to abiotic environment stresses (for review, see [26]), although some of them also showed root growth resistance to exogenous ABA [27,30,31]. In rice, we identified and characterized a mutant, srt5 , in which the reduced root length was found to be partially rescued by exogenous ABA [32]. However, details on the involvement of ABA in the regulation of plant root elongation under normal conditions remain to be determined. To gain more direct information on the mechanisms of root elongation in rice, we continued a mutational analysis of root elongation mutants. In this paper, we describe the isolation and characterization of an ABAinsensitive mutation, srt6 , which affects specifically primary root elongation at early growth stage.
2. Materials and methods 2.1. Plant material All mutant lines were derived from the japonica pure line ‘Oochikara’ (Oryza sativa L.). Mutant screening was performed as reported before [32]. 2.2. Morphological characterization Morphological investigation was performed at various developmental stages in the same way as described before [32], except that lateral root morphology was characterized at day 6 for the wild type and day 7 for the mutant, respectively, because the emergence of lateral roots on the seminal root is 1 day later for the mutant than that for the wild type. To know the causes for the reduced root length in the mutant, longitudinal and transversal sections of seminal roots were observed essentially according to the procedure described by Yao et al. (2002) [32]. To investigate root hair morphology of the mutant in detail, 1 cm of seminal root tips of 4-day-old seedlings were fixed in 2% glutaraldehyde and dehydrated in an ethanol series. After dried in Critical Point Dryer (Hitachi, Japan), the samples were sputter-coated with platinum for about 10 min at 7 mA and observed under a scanning electron microscope (Hitachi, S-2150). 2.3. Physiological characterization Root growth inhibition by different hormones was carried out in the way as described before [32]. Root and shoot length of 20 seedlings cultured for 6 days were measured. Experiments were repeated three times. Because root growth of the mutant showed reduced sensitivity to ABA, seed germination test was preformed on various ABA concentrations according to Koornneef
and Jorna (1982) [33]. Germination (in terms of radicle emergence) was investigated 3 days after incubation at 30 8C with three replications. To measure water loss under conditions of water stress, aerial parts of 10-day-old seedlings were excised and stored for 90 min (25 8C, light) in open 200 ml beakers. Fresh weight was measured every 30 min during this period.
3. Results 3.1. Mutant screening From a screening of approximately 30.000 NaN3mutagenized M2 seedlings, we selected a total of 50 plants with altered root morphology. One plant with reduced primary root length (seminal root and crown roots) and altered ABA response, described below, was selected from these putative mutants. The characteristic phenotypes of the mutant were expressed stably at both M3 and M4 generations. F1 of the mutant with the wild type had normal root length, and selfed F1 plants gave an F2 population that segregated into normal and short roots fitted in a ratio to 3:1 (x2 /0.64), suggesting that the short-root phenotype is caused by a single recessive mutation. And, the insensitivity of root growth to ABA co-segregated well with the short-root phenotype. Because screening for mutants with reduced root length is of interest, and allelism test shows that the short-root phenotype of the mutant is controlled by a gene that is different from the known short-root genes (rt , srt1 , srt2 , srt3 , srt4 and srt5 ) [16 /20,32], we tentatively designated the mutant srt6 (short-root). 3.2. srt6 specifically affects primary root elongation at early growth stage Root elongation of the mutant was greatly inhibited immediately after germination (Fig. 1a), with root length of 20-day-old seedlings of about 60% of the wild type. Moreover, root diameter in the mutant was reduced to about 240 mm compared with 420 mm in the wild type (Fig. 1c). Although lateral root density in the mutant appeared the same as that in the wild type, lateral root length of the mutant was about 60% of that of the wild type (Fig. 1b). Another interesting feature in the mutant is that both seminal and crown roots develop fewer and shorter root hairs (Fig. 1c). The number and length of root hairs of seminal root around 2 cm from the tip were about 5 and 20% in the mutant than that in wild type, respectively. Microscopic observation indicated that the mutant normally developed hair cells, and these cells also formed small swellings (Fig. 1d). However, the transi-
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Fig. 1. Seedling morphology of wild type and srt6 . (a) Seedlings of wild type (upper) and srt6 (lower) at day 1/7 cultured in distilled water. Bar /5 cm. (b) Lateral root morphology of wild type and srt6 . Investigation was carried out at day 6 for wild type and day 7 for the mutant. Bar /5 mm. (c, d) Root and root hair morphology of wild type (left, upper for d) and srt6 (right, lower for d). Bar /400 mm. Fig. 2. Adult plant morphology of wild type and srt6 . (a) Root development in wild type and srt6 plants. Seedlings were water-cultured as described before (20). The values represent the means of 20 plants. Bars indicate standard deviation. (b) Root stocks of 75-day-old plants of wild type (left) and srt6 (right). Bar /20 cm. (c) Aerial parts of wild type (left) and srt6 (right) adult plants. Bar /20 cm.
tion from the swelling to tip growth was greatly inhibited. In hydroponic culture, roots of the mutant elongated significantly faster than that of the wild type at the tillering stage (Fig. 2a), and recovered root length
completely to the level of the wild type by the booting stage (Fig. 2b). The booting stage is the stage at which the head has formed but has not emerged from the leaf sheath. Adult plants of the mutant showed normal seed fertility similar to that of the wild type (Fig. 2c).
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However, root diameter, lateral root elongation and root hair formation in the mutant all remained inhibited over the whole plant growth stages. 3.3. srt6 inhibits root elongation by inhibiting cell elongation Cyto-histological observations revealed that meristem and vascular tissues were normally developed in the mutant (data not shown). Cellular organization of the mutant was also similar to that of the wild type, whereas cells of the mutant appeared irregular-shaped to some extent compared with the wild type (Fig. 3a). Furthermore, mature cortical cell length and cell width in the mutant were reduced to about 60 and 50% of that of the wild type, respectively (Fig. 3b). Considering that seedling root length of the mutant was about 50% of the wild type, we inferred that it was reduced cell size rather than cell number that is responsible for the shortroot phenotype. The reduced cell width also fitted well with the reduced root diameter in the mutant. 3.4. srt6 shows pleiotropic defects in ABA response Root growth inhibition by ABA was analyzed in wild type and srt6 mutant. Routinely, seeds were germinated directly on ABA, and root length was investigated after 6 days. Fig. 4a showed that root growth in srt6 had
higher resistance to ABA than that in the wild type over a wide range of ABA concentrations. For example, at 10 6 M ABA, primary root length on srt6 was nearly the same as that of the ABA-free control, whereas root length on wild-type seedlings was inhibited by /50% (Fig. 4b). In contrast, shoot growth in the mutant gave similar responsiveness to ABA from 10 9 to 10 7 M compared with that in the wild type (Fig. 5a). At higher concentration of 10 6 M ABA, shoot of the wild type could hardly develop, while the mutant retained about 40% length of the ABA-free control. In other respects, seed responses were also altered in srt6 plants. Germination of dry seeds from the mutant plants showed much higher resistance than the wild type (Fig. 5b), as ABA concentration of 50% inhibition for the mutant was about two times higher than that for the wild type. Moreover, the mutant also showed enhanced rate of water loss in excised aerial parts compared with the wild type (Fig. 5c). 3.5. Root growth of srt6 responds normally to fluridone and other hormones tested To investigate whether the altered ABA response in the mutant is due to defected ABA synthesis, germinated seeds were treated with various concentrations of fluridone, the ABA biosynthesis inhibitor. The results showed that roots of the mutant seedlings could not be stimulated by exogenous fluridone (Fig. 6), suggesting that the reduced root length in the mutant might not be due to the overproduction of ABA. And, no similarity of root morphology could be detected between the wild type and srt6 seedlings under the fluridone treatments, suggesting that the short-root phenotype was also unlikely caused by reduced ABA content. Because ample evidence suggested the existence of cross-talk between ABA and other hormones such as auxin [34], ethylene [35], gibberellins [36], Jasmonic acid [37] or brassinosteroid [38], we also characterized the growth of the mutant and wild-type seedlings at various concentrations of exogenously applied IAA, NAA, 2,4D, IBA, ACC, KIN, 6-BA, GA3 and JA. We found that the mutant showed wild-type sensitivities to all these hormones tested with respect to inhibition of root elongation (data not shown).
4. Discussion
Fig. 3. Cyto-histological observation of seminal root tips of 4-day-old seedlings in wild type and srt6 . (a) Transversal sections of wild type (left) and srt6 (right). EP, epidermis; C, cortex; En, endodermis; S, stele. Bar /100 mm. (b) Mature cortical cell morphology of wild type (upper) and srt6 (lower). EP, epidermis; C, cortex; S, stele. Bar /100 mm.
The srt6 mutant in rice showed much more reduced root length than the wild type at the seedling stage. Root growth of the mutant was far less inhibited by exogenous application of ABA compared with the wild type. Because the ABA insensitivity jointly segregated with the short-root phenotype in the F2 population of the mutant with the wild type, the two traits are judged to
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Fig. 4. Effect of exogenous ABA on root growth of wild type and srt6 mutant seedlings. Seedlings of wild type and srt6 grown in the presence of 10 6 M ABA for 6 days. Bar /2 cm. Dose-response curve showing the effect of ABA on root growth in wild type and srt6 plants. Primary root length of 6-day-old seedlings was measured. The values represent the means of 15 seedlings. Relative root length represents the percentage of root growth to untreated control. Root lengths of untreated control plants were: wild type, 9.79/0.3 cm; srt6 , 5.69/0.2 cm. Bars indicate standard deviation.
be caused by the same mutation. Although the mutant shows altered root morphology in the absence of exogenous ABA, we inferred that the mutant is indeed an ABA-insensitive mutant and not a simple root morphology mutant. This is because the mutant showed a substantial insensitivity to ABA compared with the wild type, but not to any other phytohormones tested that affect root growth such as auxin. A number of mutants with reduced sensitivity of ABA were previously isolated in plants such as Arabidopsis and maize, by selecting for germination in the presence of exogenous ABA [27,28,39]. Among these mutants, abi3 , abi4 , abi5 and vp1 were believed to affect specifically seed development, whereas the abi1 and abi2 mutations did not alter seed storage reserve, but primarily affected vegetative growth [40]. In this study, we describe that the srt6 mutant showed high resistance of root growth and seed germination to exogenous ABA, and excessive transpiration in isolated aerial parts, which were similar to those reported for abi1 and abi2 . Combined with the results of fluridone treatment, we assume that srt6 might be affected in the same ABA signaling pathway as abi1 and/or abi2 mutants but not in ABA biosynthesis. However, unlike abi1 , whose gene product is involved in the regulation of ABA inhibition of mitotic activity in the root meristem [30], the srt6 mutation inhibits root elongation by affecting cell elongation but not cell division. Since shoot growth in srt6 responded normally as that in the
wild type except at 10 6 M ABA, we assume that it might be due to the specific inhibitory effects of ABA at higher concentrations. Although the srt6 mutant shows similarities to that of abi1 and abi2 in the physiological traits described above, we believe that further analysis of the mutant would be helpful in understanding the mechanisms underlying root elongation in rice, because genes specifying similar phenotypes may have different functions in different species [41]. In rice, several short-root mutations have been isolated and characterized. Some of the genes showed pleiotropic effects on other root characters such as lateral roots [18] or root hairs [16,19], while the others affect only the development of primary roots [17]. However, to our knowledge, none of the rice mutants was reported to have ABA-insensitivity of root growth. Moreover, hydroponic cultivation reveals that only one mutant, srt5 , showed root length recovery at later growth stages [32]. Several phase-specifically-expressed genes have been identified in maize such as lrt1 [42], slr1 or slr2 [43]. However, all these mutations affect specifically the development of lateral roots but not primary roots. Although the effects of srt5 and srt6 gene on root elongation are both found to be of shortduration, the srt6 mutant described here clearly differs from that of srt5 in the following points: (i) srt6 inhibits root elongation by affecting cell elongation. In contrast, srt5 inhibits root elongation by affecting not only cell elongation but also cell division. (ii) Development of
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Fig. 6. Dose-response curve showing the effect of fluridone on root growth in wild type and srt6 plants. Root length of 6-day-old seedlings was measured. The values represent the means of 15 seedlings. Relative root length represents the percentage of root growth to untreated control. root lengths of untreated control plants were: wild type, 10.19/ 0.4 cm; srt6 , 6.29/0.5 cm. Bars indicate standard deviation.
Fig. 5. ABA-insensitivity of wild type and srt6 . (a) Dose-response curve showing the effect of ABA on shoot growth in wild type and srt6 plants. Shoot length of 6-day-old seedlings was measured. The values represent the means of 15 seedlings. Relative shoot length represents the percentage of shoot growth to untreated control. Shoot lengths of untreated control plants were: wild type, 4.89/0.2 cm; srt6 , 4.29/0.3 cm. Bars indicate standard deviation. (b) Dose response of ABA inhibition of germination. Germination was scored 3 days after incubation (30 8C, light). The values represent the means of 150 seeds in three replications. Bars indicate standard deviation. (c) Water loss in excised aerial parts. The values represent the means of 60 seedlings in three replications. Bars indicate standard deviation.
primary roots, but not lateral roots and root hairs, in srt6 recovers to the wild type at the booting stage. In srt5 , however, all root traits, i.e. primary roots, lateral roots and root hairs, recover completely to its wild type at the tillering stage. (iii) Root growth of srt6 is ABAinsensitive, and the mutant has wide-type sensitivity to
all the other phytohormones tested including 2,4-D, KIN and GA3. In srt5 , however, the short-root phenotype can be partially rescued by exogenous ABA, and the mutant also shows resistance to 2,4-D, KIN and GA3. Thus, unlike that of SRT5, which probably serves a general action in early rice root development that may related to ABA biosynthesis, the effects of SRT6 on primary root growth are restricted to specific developmental stage that might be related to ABA perception or signal transduction. We are now engaged in double mutant analysis between the two mutants, in an attempt to understand how the two genes interact, and how ABA regulates root elongation in rice. In summary, we have identified a mutant, srt6 , in rice. The ABA-insensitive, phase-specific and primary rootspecific traits make it a novel mutation differing from any other short-root mutations identified so far in rice, thus should be a valuable material for further understanding the physiological mechanisms underlying root elongation in rice.
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Isolation and characterization of an abscisic acid-insensitive mutation that affects specifically primary root elongation in rice (Oryza sativa L.) Shan-Guo Yao *, Shin Taketa, Masahiko Ichii Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan Received 6 November 2002; received in revised form 3 February 2003; accepted 5 February 2003
Abstract The isolation and detailed characterization of a monogenic recessive root growth mutant, srt6 , which was derived from an M2 population of rice (Oryza sativa L.) cv. Oochikara treated with NaN3, is described. Besides greatly reduced primary root length and diameter, the mutant at the seedling stage also shows inhibited lateral root elongation and altered root hair formation. Hydroponic cultivation reveals that root length of the mutant recovers to the level of the wild type at the booting stage, but both lateral root elongation and root hair formation in the mutant remain inhibited at all growth stages. These observations suggest that the expression of the srt6 gene is phase-specific, and that the effects of SRT6 on root growth are restricted specifically to the development of primary roots. Physiological characterization reveals that the mutant has pleiotropic defects in abscisic acid (ABA) responses, which are similar to those reported for Arabidopsis ABA-insensitive mutants abi1 and abi2 , i.e. reduced sensitivity of root growth and seed germination, and excessive water loss. Combined with its normal response of root growth to fluridone, the ABA biosynthesis inhibitor, suggesting that the action of the srt6 gene might be related to ABA perception or signal transduction but not ABA biosynthesis in the controlling of early primary root elongation in rice. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Abscisic acid; Mutant; Rice; Root elongation
1. Introduction Root system of plants functions in various aspects especially uptake of nutrients and water [1]. Because the supplies of nutrient and water in soil are always limited, local and variable, the spatial deployment of the root system, i.e. root architecture, largely determines the ability of a plant to exploit these resources [2]. Root architecture is defined by parameters such as length, number and dimension, and the root length is considered to be related to various important characters such as nutrient uptake [3], lodging tolerance [4], drought tolerance [5,6], and yield [7,8]. Thus, uncovering the mechanisms controlling root elongation will be of great importance in improving plants’ sustainability and productivity. * Corresponding author. Tel./fax: /81-87-891-3127. E-mail address: [email protected] (S.-G. Yao).
To uncover the mechanisms controlling root elongation, genetic analysis of mutants is proven to be a useful approach. In Arabidopsis , many mutants on root elongation have been isolated. These include root meristemless mutants [9,10], short-root mutants [11 / 13], and root expansion mutants [14,15]. In rice, several short-root mutants have been identified and genetically characterized [16 /20]. However, great many things related to root elongation remain largely unknown. The phyotohormone abscisic acid (ABA) plays an essential regulating role for a variety of physiological and developmental processes, including embryo development, seed dormancy, transpiration, adaptation to environmental stresses (for review, see [21]), and also root growth [22 /25]. Substantial progress has been made in the characterization of ABA signaling pathways (for review, see [26]). In particular, the extensive mutational analysis in Arabidopsis has identified several genes that control ABA responsiveness [27 /29]. How-
0168-9452/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00081-5
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ever, analysis of these mutants mainly focused on seed development or plant adaptation to abiotic environment stresses (for review, see [26]), although some of them also showed root growth resistance to exogenous ABA [27,30,31]. In rice, we identified and characterized a mutant, srt5 , in which the reduced root length was found to be partially rescued by exogenous ABA [32]. However, details on the involvement of ABA in the regulation of plant root elongation under normal conditions remain to be determined. To gain more direct information on the mechanisms of root elongation in rice, we continued a mutational analysis of root elongation mutants. In this paper, we describe the isolation and characterization of an ABAinsensitive mutation, srt6 , which affects specifically primary root elongation at early growth stage.
2. Materials and methods 2.1. Plant material All mutant lines were derived from the japonica pure line ‘Oochikara’ (Oryza sativa L.). Mutant screening was performed as reported before [32]. 2.2. Morphological characterization Morphological investigation was performed at various developmental stages in the same way as described before [32], except that lateral root morphology was characterized at day 6 for the wild type and day 7 for the mutant, respectively, because the emergence of lateral roots on the seminal root is 1 day later for the mutant than that for the wild type. To know the causes for the reduced root length in the mutant, longitudinal and transversal sections of seminal roots were observed essentially according to the procedure described by Yao et al. (2002) [32]. To investigate root hair morphology of the mutant in detail, 1 cm of seminal root tips of 4-day-old seedlings were fixed in 2% glutaraldehyde and dehydrated in an ethanol series. After dried in Critical Point Dryer (Hitachi, Japan), the samples were sputter-coated with platinum for about 10 min at 7 mA and observed under a scanning electron microscope (Hitachi, S-2150). 2.3. Physiological characterization Root growth inhibition by different hormones was carried out in the way as described before [32]. Root and shoot length of 20 seedlings cultured for 6 days were measured. Experiments were repeated three times. Because root growth of the mutant showed reduced sensitivity to ABA, seed germination test was preformed on various ABA concentrations according to Koornneef
and Jorna (1982) [33]. Germination (in terms of radicle emergence) was investigated 3 days after incubation at 30 8C with three replications. To measure water loss under conditions of water stress, aerial parts of 10-day-old seedlings were excised and stored for 90 min (25 8C, light) in open 200 ml beakers. Fresh weight was measured every 30 min during this period.
3. Results 3.1. Mutant screening From a screening of approximately 30.000 NaN3mutagenized M2 seedlings, we selected a total of 50 plants with altered root morphology. One plant with reduced primary root length (seminal root and crown roots) and altered ABA response, described below, was selected from these putative mutants. The characteristic phenotypes of the mutant were expressed stably at both M3 and M4 generations. F1 of the mutant with the wild type had normal root length, and selfed F1 plants gave an F2 population that segregated into normal and short roots fitted in a ratio to 3:1 (x2 /0.64), suggesting that the short-root phenotype is caused by a single recessive mutation. And, the insensitivity of root growth to ABA co-segregated well with the short-root phenotype. Because screening for mutants with reduced root length is of interest, and allelism test shows that the short-root phenotype of the mutant is controlled by a gene that is different from the known short-root genes (rt , srt1 , srt2 , srt3 , srt4 and srt5 ) [16 /20,32], we tentatively designated the mutant srt6 (short-root). 3.2. srt6 specifically affects primary root elongation at early growth stage Root elongation of the mutant was greatly inhibited immediately after germination (Fig. 1a), with root length of 20-day-old seedlings of about 60% of the wild type. Moreover, root diameter in the mutant was reduced to about 240 mm compared with 420 mm in the wild type (Fig. 1c). Although lateral root density in the mutant appeared the same as that in the wild type, lateral root length of the mutant was about 60% of that of the wild type (Fig. 1b). Another interesting feature in the mutant is that both seminal and crown roots develop fewer and shorter root hairs (Fig. 1c). The number and length of root hairs of seminal root around 2 cm from the tip were about 5 and 20% in the mutant than that in wild type, respectively. Microscopic observation indicated that the mutant normally developed hair cells, and these cells also formed small swellings (Fig. 1d). However, the transi-
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Fig. 1. Seedling morphology of wild type and srt6 . (a) Seedlings of wild type (upper) and srt6 (lower) at day 1/7 cultured in distilled water. Bar /5 cm. (b) Lateral root morphology of wild type and srt6 . Investigation was carried out at day 6 for wild type and day 7 for the mutant. Bar /5 mm. (c, d) Root and root hair morphology of wild type (left, upper for d) and srt6 (right, lower for d). Bar /400 mm. Fig. 2. Adult plant morphology of wild type and srt6 . (a) Root development in wild type and srt6 plants. Seedlings were water-cultured as described before (20). The values represent the means of 20 plants. Bars indicate standard deviation. (b) Root stocks of 75-day-old plants of wild type (left) and srt6 (right). Bar /20 cm. (c) Aerial parts of wild type (left) and srt6 (right) adult plants. Bar /20 cm.
tion from the swelling to tip growth was greatly inhibited. In hydroponic culture, roots of the mutant elongated significantly faster than that of the wild type at the tillering stage (Fig. 2a), and recovered root length
completely to the level of the wild type by the booting stage (Fig. 2b). The booting stage is the stage at which the head has formed but has not emerged from the leaf sheath. Adult plants of the mutant showed normal seed fertility similar to that of the wild type (Fig. 2c).
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However, root diameter, lateral root elongation and root hair formation in the mutant all remained inhibited over the whole plant growth stages. 3.3. srt6 inhibits root elongation by inhibiting cell elongation Cyto-histological observations revealed that meristem and vascular tissues were normally developed in the mutant (data not shown). Cellular organization of the mutant was also similar to that of the wild type, whereas cells of the mutant appeared irregular-shaped to some extent compared with the wild type (Fig. 3a). Furthermore, mature cortical cell length and cell width in the mutant were reduced to about 60 and 50% of that of the wild type, respectively (Fig. 3b). Considering that seedling root length of the mutant was about 50% of the wild type, we inferred that it was reduced cell size rather than cell number that is responsible for the shortroot phenotype. The reduced cell width also fitted well with the reduced root diameter in the mutant. 3.4. srt6 shows pleiotropic defects in ABA response Root growth inhibition by ABA was analyzed in wild type and srt6 mutant. Routinely, seeds were germinated directly on ABA, and root length was investigated after 6 days. Fig. 4a showed that root growth in srt6 had
higher resistance to ABA than that in the wild type over a wide range of ABA concentrations. For example, at 10 6 M ABA, primary root length on srt6 was nearly the same as that of the ABA-free control, whereas root length on wild-type seedlings was inhibited by /50% (Fig. 4b). In contrast, shoot growth in the mutant gave similar responsiveness to ABA from 10 9 to 10 7 M compared with that in the wild type (Fig. 5a). At higher concentration of 10 6 M ABA, shoot of the wild type could hardly develop, while the mutant retained about 40% length of the ABA-free control. In other respects, seed responses were also altered in srt6 plants. Germination of dry seeds from the mutant plants showed much higher resistance than the wild type (Fig. 5b), as ABA concentration of 50% inhibition for the mutant was about two times higher than that for the wild type. Moreover, the mutant also showed enhanced rate of water loss in excised aerial parts compared with the wild type (Fig. 5c). 3.5. Root growth of srt6 responds normally to fluridone and other hormones tested To investigate whether the altered ABA response in the mutant is due to defected ABA synthesis, germinated seeds were treated with various concentrations of fluridone, the ABA biosynthesis inhibitor. The results showed that roots of the mutant seedlings could not be stimulated by exogenous fluridone (Fig. 6), suggesting that the reduced root length in the mutant might not be due to the overproduction of ABA. And, no similarity of root morphology could be detected between the wild type and srt6 seedlings under the fluridone treatments, suggesting that the short-root phenotype was also unlikely caused by reduced ABA content. Because ample evidence suggested the existence of cross-talk between ABA and other hormones such as auxin [34], ethylene [35], gibberellins [36], Jasmonic acid [37] or brassinosteroid [38], we also characterized the growth of the mutant and wild-type seedlings at various concentrations of exogenously applied IAA, NAA, 2,4D, IBA, ACC, KIN, 6-BA, GA3 and JA. We found that the mutant showed wild-type sensitivities to all these hormones tested with respect to inhibition of root elongation (data not shown).
4. Discussion
Fig. 3. Cyto-histological observation of seminal root tips of 4-day-old seedlings in wild type and srt6 . (a) Transversal sections of wild type (left) and srt6 (right). EP, epidermis; C, cortex; En, endodermis; S, stele. Bar /100 mm. (b) Mature cortical cell morphology of wild type (upper) and srt6 (lower). EP, epidermis; C, cortex; S, stele. Bar /100 mm.
The srt6 mutant in rice showed much more reduced root length than the wild type at the seedling stage. Root growth of the mutant was far less inhibited by exogenous application of ABA compared with the wild type. Because the ABA insensitivity jointly segregated with the short-root phenotype in the F2 population of the mutant with the wild type, the two traits are judged to
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Fig. 4. Effect of exogenous ABA on root growth of wild type and srt6 mutant seedlings. Seedlings of wild type and srt6 grown in the presence of 10 6 M ABA for 6 days. Bar /2 cm. Dose-response curve showing the effect of ABA on root growth in wild type and srt6 plants. Primary root length of 6-day-old seedlings was measured. The values represent the means of 15 seedlings. Relative root length represents the percentage of root growth to untreated control. Root lengths of untreated control plants were: wild type, 9.79/0.3 cm; srt6 , 5.69/0.2 cm. Bars indicate standard deviation.
be caused by the same mutation. Although the mutant shows altered root morphology in the absence of exogenous ABA, we inferred that the mutant is indeed an ABA-insensitive mutant and not a simple root morphology mutant. This is because the mutant showed a substantial insensitivity to ABA compared with the wild type, but not to any other phytohormones tested that affect root growth such as auxin. A number of mutants with reduced sensitivity of ABA were previously isolated in plants such as Arabidopsis and maize, by selecting for germination in the presence of exogenous ABA [27,28,39]. Among these mutants, abi3 , abi4 , abi5 and vp1 were believed to affect specifically seed development, whereas the abi1 and abi2 mutations did not alter seed storage reserve, but primarily affected vegetative growth [40]. In this study, we describe that the srt6 mutant showed high resistance of root growth and seed germination to exogenous ABA, and excessive transpiration in isolated aerial parts, which were similar to those reported for abi1 and abi2 . Combined with the results of fluridone treatment, we assume that srt6 might be affected in the same ABA signaling pathway as abi1 and/or abi2 mutants but not in ABA biosynthesis. However, unlike abi1 , whose gene product is involved in the regulation of ABA inhibition of mitotic activity in the root meristem [30], the srt6 mutation inhibits root elongation by affecting cell elongation but not cell division. Since shoot growth in srt6 responded normally as that in the
wild type except at 10 6 M ABA, we assume that it might be due to the specific inhibitory effects of ABA at higher concentrations. Although the srt6 mutant shows similarities to that of abi1 and abi2 in the physiological traits described above, we believe that further analysis of the mutant would be helpful in understanding the mechanisms underlying root elongation in rice, because genes specifying similar phenotypes may have different functions in different species [41]. In rice, several short-root mutations have been isolated and characterized. Some of the genes showed pleiotropic effects on other root characters such as lateral roots [18] or root hairs [16,19], while the others affect only the development of primary roots [17]. However, to our knowledge, none of the rice mutants was reported to have ABA-insensitivity of root growth. Moreover, hydroponic cultivation reveals that only one mutant, srt5 , showed root length recovery at later growth stages [32]. Several phase-specifically-expressed genes have been identified in maize such as lrt1 [42], slr1 or slr2 [43]. However, all these mutations affect specifically the development of lateral roots but not primary roots. Although the effects of srt5 and srt6 gene on root elongation are both found to be of shortduration, the srt6 mutant described here clearly differs from that of srt5 in the following points: (i) srt6 inhibits root elongation by affecting cell elongation. In contrast, srt5 inhibits root elongation by affecting not only cell elongation but also cell division. (ii) Development of
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Fig. 6. Dose-response curve showing the effect of fluridone on root growth in wild type and srt6 plants. Root length of 6-day-old seedlings was measured. The values represent the means of 15 seedlings. Relative root length represents the percentage of root growth to untreated control. root lengths of untreated control plants were: wild type, 10.19/ 0.4 cm; srt6 , 6.29/0.5 cm. Bars indicate standard deviation.
Fig. 5. ABA-insensitivity of wild type and srt6 . (a) Dose-response curve showing the effect of ABA on shoot growth in wild type and srt6 plants. Shoot length of 6-day-old seedlings was measured. The values represent the means of 15 seedlings. Relative shoot length represents the percentage of shoot growth to untreated control. Shoot lengths of untreated control plants were: wild type, 4.89/0.2 cm; srt6 , 4.29/0.3 cm. Bars indicate standard deviation. (b) Dose response of ABA inhibition of germination. Germination was scored 3 days after incubation (30 8C, light). The values represent the means of 150 seeds in three replications. Bars indicate standard deviation. (c) Water loss in excised aerial parts. The values represent the means of 60 seedlings in three replications. Bars indicate standard deviation.
primary roots, but not lateral roots and root hairs, in srt6 recovers to the wild type at the booting stage. In srt5 , however, all root traits, i.e. primary roots, lateral roots and root hairs, recover completely to its wild type at the tillering stage. (iii) Root growth of srt6 is ABAinsensitive, and the mutant has wide-type sensitivity to
all the other phytohormones tested including 2,4-D, KIN and GA3. In srt5 , however, the short-root phenotype can be partially rescued by exogenous ABA, and the mutant also shows resistance to 2,4-D, KIN and GA3. Thus, unlike that of SRT5, which probably serves a general action in early rice root development that may related to ABA biosynthesis, the effects of SRT6 on primary root growth are restricted to specific developmental stage that might be related to ABA perception or signal transduction. We are now engaged in double mutant analysis between the two mutants, in an attempt to understand how the two genes interact, and how ABA regulates root elongation in rice. In summary, we have identified a mutant, srt6 , in rice. The ABA-insensitive, phase-specific and primary rootspecific traits make it a novel mutation differing from any other short-root mutations identified so far in rice, thus should be a valuable material for further understanding the physiological mechanisms underlying root elongation in rice.
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