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Cytokinin inhibits lateral root initiation but stimulates lateral root elongation in rice (Oryza sativa)
ARTICLE IN PRESS Journal of Plant Physiology 162 (2005) 507—515
www.elsevier.de/jplph
Cytokinin inhibits lateral root initiation but stimulates lateral root elongation in rice (Oryza sativa) Bakul Rani Debi, Shin Taketa, Masahiko Ichii Faculty of Agriculture, Kagawa University, Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0701, Japan Received 20 June 2004; accepted 7 August 2004
KEYWORDS Cytokinin; Inhibition; Lateral root; Rice; Stimulation
Summary Research in lateral root (LR) development mainly focuses on the role of auxin. This article reports the effect of cytokinins (kinetin and trans-zeatin) on LR formation in rice (Oryza sativa L.). Our results showed that cytokinin has an inhibitory effect on LR initiation and stimulatory effect on LR elongation. Both KIN and ZEA at a concentration of 1 mM and above completely inhibited lateral root primordium (LRP) formation. The inhibitory effect of cytokinin on LR initiation required a continuous presence of KIN or ZEA in the growth solution. Cytokinin did not show any inhibitory effect on LR emergence from the seminal root once LRPs had been formed. The LRPs that developed in cytokinin-free solution can emerge normally in the solution containing inhibitory concentration (1 mM) of KIN and ZEA. The KIN and ZEA treatment dramatically stimulated LR elongation at all the concentrations tested. Maximum LR elongation was observed at a concentration of 0.01 mM KIN and 0.001 mM ZEA. The epidermal cell length increased significantly in LRs of cytokinin treated seedlings compared to those of untreated control. This result indicates that the stimulation of LR elongation by cytokinin is due to increased cell length. Exogenously applied auxin counteracted the effect of cytokinin on LR initiation and LR elongation, suggesting that cytokinin acts on LR elongation through an auxin dependent pathway. & 2004 Elsevier GmbH. All rights reserved.
Abbreviations: IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; KIN, kinetin; LR, lateral root; LRP, lateral root primordium; TTC, 2, 3, 5-triphenyltetrazolium chloride; ZEA, zeatin Corresponding author. Tel./fax: +81 87 891 3127. E-mail addresses: [email protected] (B. Rani Debi), [email protected] (M. Ichii). 0176-1617/$ - see front matter & 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2004.08.007
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Introduction Lateral root (LR) development is a multiphase process including at least initiation and emergence of lateral root primordia (LRP) and activation of LR meristem. LRs originate in the root pericycle, in which individual quiescent cells are stimulated to dedifferentiate and proliferate to form the LRP. The LRP then grows via both cell division and cell expansion. The emergence of LRP from the parent root occurs primarily by cell expansion. Soon after the emergence, the LRP undergoes an activation step to form a fully functional LR meristem (Malamy and Benfey, 1997) that directs growth from this stage onward. Numerous studies have shown that auxin is necessary for the initiation and subsequent growth of LRs (Blakely et al., 1988; Celenza et al., 1995; Reed et al., 1998). Exogenous application of auxin or an enhancement of endogenous auxin synthesis results in a significant increase in LRs number (Klee et al., 1987; Kares et al., 1990; Boerjan et al., 1995; Laskowski et al., 1995). Cytokinins are a class of plant hormones that influences many developmental processes including leaf senescence, apical dominance, chloroplast development, anthocyanin production and regulation of cell division. Cytokinin, together with auxin plays an essential role in plant morphogenesis, having a profound influence on the formation of roots and shoots and their relative growth. Cytokinins act antagonistically to auxin and determine cell fate by promoting shoot and root differentiation in callus culture (Skoog and Miller, 1957). On initiation of LR primordia, the interaction of auxin and cytokinin is antagonistic, with auxin stimulating and cytokinin inhibiting cell division in the root pericycle. Cytokinins are formed in the root tips and interact with auxin in the regulation of LR formation (Wightman et al., 1980; Hinchee and Rost, 1986; Torrey, 1986). The auxin:cytokinin ratio has been reported to play an important role in LR development in pea seedlings. Application of exogenous auxin promoted LR initiation and application of cytokinin reduced the number of LRs (Bo ¨ttgor, 1974; Goodwin and Morris, 1979; Hinchee and Rost, 1986). There are many reports described the inhibitory effect of cytokinin on the initiation of LRs. However, there are only few reports described the effect of cytokinin on the advanced stages of LR formation. There are contradictory results obtained on the effect of cytokinin on LR emergence. For example, Wightman et al. (1980) reported that cytokinin is inhibitory for LR emergence and they described that the LR emergence was about twice
B. Rani Debi et al. as sensitive as the initiation of primordia to the presence of cytokinins. Hinchee and Rost (1986) observed inhibitory effect of cytokinin on LR emergence in pea seedlings with detached cotyledons. However, the inihibitory effect of cytokinin on LR emergence diminished when the cotyledons remained intact. Furthermore, compared to other stages of LR formation, the effect of cytokinin on LR elongation is really poorly understood. So the purpose of our present study is to elucidate the effect of cytokinin on LR formation in plant species other than dicots as the previous studies were carried out mainly in dicots. We have observed the effect of kinetin (KIN) and zeatin (ZEA) on various stages of LR formation using japonica rice (Oryza sativa L.), Nipponbare as the plant material. Our results showed that the effect of cytokinin varies depending on developmental stages of LR formation, with an inhibitory effect on LR initiation, no inhibitory effect on LR emergence and a dramatic stimulatory effect on LR elongation.
Materials and methods Plant materials and growth conditions Japonica rice, Nipponbare (Oryza sativa L. subsp. japonica) was used in the experiments. Seeds were surface sterilized with 0.2% (W/V) benomyl solution in an incubator at 30 1C for 1 day, and rinsed with tap water several times. Then the seeds were soaked in tap water for another 1 day at the same incubator for germination. The germinated seeds were used in the hormone experiments. Experiments were conducted in a plant growth cabinet (ML R350, Sanyo) at 25 1C and under continuous white fluorescent light at a light intensity of 500 mmol m2 s1.
Experiments on the effect of cytokinin on LR formation The germinated seeds were placed in plastic nets on a float and transferred to black cups containing 200 ml solution of each KIN and ZEA of indicated concentrations or distilled water as the control. The culture solutions were renewed every 2 days. After 10 days, all the emerged LRs on seminal roots were counted. Lateral root density was expressed as the number of LRs per centimeter of seminal root obtained by dividing the LRs number by the length of the respective seminal root. To investigate the effect of cytokinin on the emergence of
ARTICLE IN PRESS The effect of cytokinin on lateral root development in rice LRs, germinated seeds were placed in plastic nets and transferred to black cups containing 200 ml of distilled water and grown for 3 days to allow the roots to form LRP, and then 15 seedlings were transferred to black cups containing 200 ml of 1 mM of each KIN, ZEA or distilled water as the control. The seedlings were grown for an additional 4 days and the LRs that had emerged from the seminal roots were counted everyday. To investigate the effect of cytokinin on the elongation of LRs, seedlings were grown similarly for 4 days in distilled water and then transferred to the indicated concentrations of each KIN, ZEA or distilled water and grown for an additional 6 days. The length of 20 longest LRs from each seminal root was measured.
Observation of LRP Formation of LRP was observed according to Wang et al. (2003) with some modifications. Briefly, the seminal roots of 3-day old seedlings grown in distilled water and in 1 mM of each KIN or ZEA were stained in the mixture of 25 ml 0.4% (W/V) 2,3,5triphenyltetrazolium chloride (TTC) and 25 ml of 0.1 M H3PO4 buffer (pH 7.2) in dark for 2 h at 30 1C in an incubator. Then the roots were degassed in a vacuum drier for 30 min and then transferred to a separate solution of 10% HCl for 20 min and a 10% glycerol solution for 20 min. The roots were then placed in a slide on a drop of glycerol and a cover glass was placed and tapped gently by the blunt end of a needle and observed under a microscope and photographed.
Measurement of epidermal cell length of LRs Histological observation of LRs was performed according to Yao et al. (2003). To estimate the epidermal cell length of LRs with or without cytokinin treatment, 1 cm of LR tips of 10-day-old seedlings were fixed in 2% gluteraldehyde 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).
Combined treatment of KIN and ZEA with auxin To know whether auxin has any influence on the action of cytokinin on LR formation, we observed the effect of KIN and ZEA in the presence of indole3-acetic acid (IAA) and indole-3-butyric acid (IBA). To see the combined effect of cytokinin and auxin
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on LR initiation, germinated seeds were placed in black cups containing 200 ml of 0.1 mM of each KIN or ZEA with that of 1 mM IAA or 0.1 mM IBA. Moreover, 1 mM IAA, 0.1 mM IBA, 0.1 mM of each KIN or ZEA were used as the control. After 10 days of growth, the number of LRs per centimeter of seminal root was determined. To see the combined effect of cytokinin and auxin on LR elongation, the germinated seeds were grown in distilled water for initial 4 days and then transferred to black cups containing 200 ml of 0.01 mM KIN or 0.001 mM ZEA in combination with 0.1, 1 and 10 mM IAA or 0.1, 1 and 10 mM IBA solution and in 0.1, 1 and 10 mM IAA or 0.1, 1 and 10 mM IBA as the control and grown for an additional 6 days and the length of 20 longest LRs from each seminal roots of 10 seedlings were measured.
Results Inhibitory effect of cytokinin on LR initiation To assess the effect of cytokinin on LR formation in rice, germinated seeds of Nipponbare were grown in indicated concentrations of both KIN and ZEA or distilled water as the control. After 10 days of growth, LR density was calculated. The results revealed that both KIN and ZEA treatment reduced significantly the LR density in a dose-dependent manner (Fig. 1). The number of LRs per centimeter decreased with increasing the concentration of KIN and ZEA. At a concentration of 1 mM and above (Figs. 1, 2), complete inhibition of LR formation occurred in case of both KIN and ZEA treated seedlings. ZEA is stronger inhibitor of LR formation than KIN. The above results showed that 1 mM of both KIN and ZEA blocked LR formation completely. So to know which stage of LR formation was specifically affected by cytokinin, the germinated seeds were grown in 1 mM of KIN or ZEA or distilled water for 3 days and formation of LRP on seminal root was observed. Many LRP were formed in the roots grown in distilled water, but no LRP was found to be formed in the roots treated with 1 mM of KIN or ZEA (Fig. 2B). This result indicates that cytokinin inhibits LR initiation in the root pericycle.
Cytokinin is required continuously for inhibition of LR formation To test whether cytokinin is required continuously for inhibition of LR formation or an initial treatment is enough to trigger the inhibition of LR
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and grown for an additional 4 days. The results showed that when the seedlings were transferred from KIN or ZEA solution to distilled water, LRs formed on the lower part of the root that grew newly in distilled water after transfer (Fig. 2C). However, no LRs were formed on the upper part of the root that grew in 1 mM KIN or ZEA (Fig. 2C) before transfer. This result indicates that the inhibitory effect of cytokinin on LR formation is reversible and that a continuous presence of cytokinin is required for the inhibition of LR formation.
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Figure 1. Effect of KIN (A) and ZEA (B) on lateral root formation in rice. Seedlings were grown in different concentrations of KIN, ZEA or in distilled water as the control. After 10 days of growth, the total number of emerged lateral roots on each seminal root was counted and divided by the length of the respective seminal root length to get the number of lateral roots per centimeter of seminal root. Each data point represents the averages of 10 seedlings and the error bar represents the standard deviations of the means.
Figure 2. Comparison of LR formation in KIN treated and non-treated seminal roots. (A) Seminal root of 10-day old seedlings grown in control solution showing LR formation (left) and in 1 mM KIN solution showing inhibition of LR formation (right). (B) Formation of LRs primordia on seminal root of 3-day old seedling grown in control solution (left, arrow indicates LRP) and in 1 mM KIN solution (right). (C) The seminal root of the seedling that was grown initial 3 days in 1 mM KIN solution showing inhibition of LR formation (root segment a) and then transferred to distilled water and grown for an additional 4 days showing formation of LR (root segment b).
formation, we performed a shift experiment. The seeds were germinated and grown for 3 days in 1 mM KIN or ZEA and then transferred to distilled water
After formation of LRP in the pericycle cells, the next step is the emergence of LRP through the epidermis of the parent roots. To analyze the effect of cytokinin on the emergence of LRP, the germinated seeds were grown in distilled water for 3 days to allow them to initiate LRP and then the seedlings were transferred to inhibitory concentration (1 mM) of each KIN and ZEA solution. Total number of emerged LRs on each seminal root was counted everyday for 4 days after transfer. The results showed that the roots in KIN and ZEA solution produced similar number of LRs as the roots in control after 1 day of transfer (Fig. 3A). After 2 days of transfer, the KIN and ZEA treated roots were found to produce less number of total LRs compared to control. Thereafter, the KIN and ZEA treated roots did not increase the total number of LRs. On the contrary, the roots in control solution increased the total number of LRs until 3 and 4 days after transfer (Fig. 3A, B left). The LRs in the KIN and ZEA treated roots were found to be emerged only in the upper part of the root that grew in distilled water before transfer to the KIN and ZEA solution, indicating that cytokinin treatment did not inhibit the emergence of already formed LRP (Fig. 3B right). However, after transfer, the lower part of the root that grew newly in KIN and ZEA produced no LRs, indicating that LRP initiation is inhibited by cytokinin (Fig. 3B right). All these results indicate that cytokinin does not inhibit the emergence of LRP from the seminal root. Once LRP is formed, it can increase cell number and emerge from the parent root normally in the presence of cytokinin.
Stimulatory effect of cytokinin on LR elongation The above results showed that cytokinin inhibits LR initiation and that it has no adverse effect on
ARTICLE IN PRESS The effect of cytokinin on lateral root development in rice
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Figure 3. Effect of cytokinin on LR emergence. (A) Seedlings were grown in distilled water for initial 3 days and then transferred to 1 mM KIN and 1 mM ZEA solution and in distilled water as the control and were grown for additional 4 days. The number of all LRs that emerged from the seminal root was counted everyday after transfer. (B) The seminal root of the seedlings grown in initial 3 days in distilled water and then transferred to distilled water and grown for 4 days (left). The seminal root of the seedlings grown initial 3 days in distilled water (right, root segment a) and then transferred to 1 mM KIN and grown for additional 4 days (right, root segment b).
LRP emergence. To test the effect of cytokinin on LR elongation, seeds were grown in distilled water for 4 days and then transferred to different concentrations of KIN and ZEA or distilled water as the control. As shown in Figs. 4 and 5, a dramatic LR elongation was found at all the concentrations of KIN and ZEA treated seedlings compared to untreated control. Maximum LR elongation was observed at a concentration of 0.01 mM for KIN and 0.001 mM for ZEA. Even the inhibitory concentration (1 mM) of KIN or ZEA that completely blocked LR initiation also promoted LR elongation significantly. The epidermal cell length increased significantly in LRs of KIN or ZEA treated seedlings compared to those of untreated LRs (Fig. 6).
Effect of exogenous auxins on the cytokinin induced LR initiation and elongation As auxins are known to play a key role in LR formation, we further examined whether exogenously applied auxin influence the inhibition of LR formation by KIN and ZEA. For this experiment, we grew the seeds in various concentrations of IAA and IBA or distilled water together with 0.1 mM of each KIN and ZEA. The results showed that the exogenously applied IAA at a concentration of 1 mM and that of IBA at a concentration of 0.1 mM counteracted the inhibitory effect of KIN and ZEA on LR initiation (Fig. 7A). However, the inhibition by 1 mM or above concentration of KIN or ZEA could not
overcome by IAA or IBA at concentration as high as 10 mM (data not shown). As maximum LR elongation was observed upon treatment with 0.01 mM KIN or 0.001 mM ZEA, we examined the elongation of LRs in combined solutions of different concentrations of IAA and IBA or distilled water with 0.01 mM KIN or 0.001 mM ZEA. The results revealed that both IAA and IBA inhibited in a dose-dependent manner the promotion of LR elongation by KIN and ZEA (Fig. 7B, C).
Discussion This article reports the effect of cytokinins on initiation, emergence and elongation of LRs in rice. Our results revealed that both the KIN and ZEA at 1 mM and higher concentration inhibit LR formation by inhibiting the initiation of LRs primordia (Figs. 1 and 2). Such inhibition of LR formation by cytokinins is consistent with the previous results observed in other plant species (Torrey, 1962; Bo ¨ttgor, 1974; Goodwin and Morris, 1979; Wightman et al., 1980). Cytokinins were reported to inhibit cell division during early phase of organization of adventitious root primordia in pea stem cuttings (Bollmark et al., 1986). Eriksen (1974) reported that in pea, cytokinin at high concentrations may have an inhibitory effect on an early stage of rooting by blocking the activity of auxin. Exogenous application of cytokinins to maize (Bourquin and Pilet,
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Figure 5. Effect of KIN and ZEA on LR elongation. Seedlings were grown in distilled water for initial 4 days and then transferred to 0.01 mM KIN, 0.001 mM ZEA or distilled water as the control and grown for an additional 6 days and photographed. (A) LR elongation in control solution. (B, C) LR elongation by 0.01 mM KIN and 0.001 mM ZEA, respectively is shown. Bars ¼ 10 mm.
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Figure 4. Effect of KIN (A) and ZEA (B) on lateral root elongation. Seedlings were grown in distilled water for initial 4 days and then transferred to different concentrations of KIN (A) and ZEA (B) or distilled water as the control and grown for another 6 days. Then the length of 20 longest LRs from each seminal root was measured. Each data point represents the averages of 10 seedlings and error bars represent the standard deviation of the means.
1990) or pea (Bertell and Eliasson, 1992) roots increased free IAA. Such increase of free IAA was also observed in cytokinin overproducing lines of Nicotina glutinosa transformed with the ipt gene (Binns et al., 1987). Soon after the formation of LR primordia, the next step is the emergence of LR primordia through the epidermis of the parent root. Cytokinins were shown to be inhibitors of LR emergence (Wightman et al., 1980; Biddington and Dearman, 1982). In contrast, our results revealed that cytokinins have no adverse effect on the emergence of LRP. The present results showed that the inhibitory effect of cytokinin is restricted to the initiation of LRP in the pericycle. But once LRPs are formed, it can normally emerge even in the presence of inhibitory concentration (1 mM) of cytokinins (Fig. 3A, B). Our
results are consistent with the findings of Forsyth and Staden (1981) in pea where they showed that the LRPs begin synthesizing cytokinins immediately after primordia induction, and therefore do not react to the exogenous application of cytokinin. Hinchee and Rost (1986) reported that cytokinin is inhibitory for LR emergence in pea seedlings with detached cotyledons. However, the inhibitory effect of cytokinin on LR emergence diminished when the cotyledons remained intact. The presence of cotyledons or shoot is required for LR emergence, probably because these organs supply auxins acropetally to the root (Rowntree and Morris, 1979; Tsurumi and Wada, 1980). Our results showed that both KIN and ZEA significantly stimulated LR elongation once LRPs had been formed (Figs. 4 and 5). To our knowledge, this is the first report that cytokinins stimulate LR elongation. The LR length increased about 4–5 times by 0.01 mM KIN or 0.001 mM ZEA compared to that of untreated control (Fig. 4). Histological observation also revealed a 4–5 times increased epidermal cell length of the LRs treated by 0.01 mM KIN and 0.001 mM ZEA compared to untreated control (Fig. 6), indicating that the increased LR elongation by cytokinin is primarily due to an increased cell length of the existing cells rather than enhanced cell division. This stimulation effect of cytokinin is unique to LRs because, in rice, the inhibitory concentration (1 mM) of cytokinin significantly inhibited the seminal root elongation (Fig. 2A), while LR length was still stimulated
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Figure 6. Effect of cytokinin on epidermal cell length of LRs (A). (B) Electron microscopy of LRs showing epidermal cells. Left: seedlings were grown in distilled water, middle: seedlings were grown in 0.01 mM KIN and right: seedlings were grown in 0.001 mM ZEA. Seedlings were grown in distilled water for 4 days and then transferred to 0.01 mM KIN and 0.001 mM ZEA or distilled water and grown for another 6 days. Then 1 cm of LR segment were fixed in 2% glutaraldehyde and dehydrated in an ethanol series. After dried in critical point dryer, the samples were sputter-coated with platinum for about 10 min at 7 mA. The root samples were then observed and photographed under a scanning electron microscope (Hitachi, S-2150) at 10 kV. Data on epidermal cell length was taken from four LRs of each of the treatment and 30 cells were measured from each LR.
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at this concentration (Fig. 4). Our results are consistent with the cytokinin-responsive Arabidopsis response regulator (ARR5) gene promoter expression pattern in Lotus japonicus observed by Lohar et al. (2004). They reported that ARR5 expression was undetectable in the dividing cells of the early LR primordium, but later they observed high expression in the base of the cone-shaped LRP, in the root tip and in the LR meristem for the subsequent growth of the LRs. The expression patterns of ARR5 indicate that cytokinins have an inhibitory effect on LRP initiation and cytokinins play important role in subsequent growth of LRs. The auxin:cytokinin ratio has been shown to play a role in co-ordinating LR development in pea seedlings (Bo ¨ttgor, 1974; Hinchee and Rost, 1986). So we also examined the effect of cytokinin on LR formation in rice in the presence of auxins. Our
Figure 7. Effect of combined application of cytokinin with auxin on LR formation. Seedlings were grown in combined solution of 0.1 mM KIN with 1 mM IAA or 0.1 mM IBA and in 0.1 mM ZEA with 1 mM IAA or 0.1 mM IBA. For control treatment, seedlings were grown in 0.1 mM KIN, 0.1 mM ZEA, 1 mM IAA or 0.1 mM IBA solution. After 10 days of growth, the number of emerged LRs on each seminal root were counted and divided by the length of the respective seminal root (A). Seedlings were grown in initial 4 days in distilled water and then transferred to combined solutions of 0.01 mM KIN or 0.001 mM ZEA with indicated concentrations of IAA (B) or IBA (C) and in indicated concentrations of IAA (B) or IBA(C) as the control and grown for another 6 days. Then the length of 20 longest LRs was measured. Each data point represents the averages of 10 seedlings and error bars represent the standard deviation of the means.
ARTICLE IN PRESS 514 results showed that addition of auxins in the growth solution effectively counteracted the inhibitory effect of cytokinin on LR formation (Fig. 7A). In contrast, the stimulatory effect of cytokinin on LR elongation was inhibited gradually with increasing concentration of auxins in the growth solution (Fig. 7B, C). These results suggest that the inhibitory and stimulatory action of cytokinin depends on its concentration relative to that of auxin concentration. In summary, we analyzed the effect of cytokinin on LR development in rice and found that although cytokinin is inhibitory for LR initiation, it has no adverse effect on LR emergence and it shows dramatic stimulatory effect on LR elongation in rice. Further study with other species and plant hormone mutants are needed to better understand the mechanisms of cytokinin regulation of LR development.
Acknowledgements The financial support of the Ministry of Education, Science, Sports and Culture of Japan under scholarship program for foreign students is greatly acknowledged.
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Cytokinin inhibits lateral root initiation but stimulates lateral root elongation in rice (Oryza sativa) Bakul Rani Debi, Shin Taketa, Masahiko Ichii Faculty of Agriculture, Kagawa University, Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0701, Japan Received 20 June 2004; accepted 7 August 2004
KEYWORDS Cytokinin; Inhibition; Lateral root; Rice; Stimulation
Summary Research in lateral root (LR) development mainly focuses on the role of auxin. This article reports the effect of cytokinins (kinetin and trans-zeatin) on LR formation in rice (Oryza sativa L.). Our results showed that cytokinin has an inhibitory effect on LR initiation and stimulatory effect on LR elongation. Both KIN and ZEA at a concentration of 1 mM and above completely inhibited lateral root primordium (LRP) formation. The inhibitory effect of cytokinin on LR initiation required a continuous presence of KIN or ZEA in the growth solution. Cytokinin did not show any inhibitory effect on LR emergence from the seminal root once LRPs had been formed. The LRPs that developed in cytokinin-free solution can emerge normally in the solution containing inhibitory concentration (1 mM) of KIN and ZEA. The KIN and ZEA treatment dramatically stimulated LR elongation at all the concentrations tested. Maximum LR elongation was observed at a concentration of 0.01 mM KIN and 0.001 mM ZEA. The epidermal cell length increased significantly in LRs of cytokinin treated seedlings compared to those of untreated control. This result indicates that the stimulation of LR elongation by cytokinin is due to increased cell length. Exogenously applied auxin counteracted the effect of cytokinin on LR initiation and LR elongation, suggesting that cytokinin acts on LR elongation through an auxin dependent pathway. & 2004 Elsevier GmbH. All rights reserved.
Abbreviations: IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; KIN, kinetin; LR, lateral root; LRP, lateral root primordium; TTC, 2, 3, 5-triphenyltetrazolium chloride; ZEA, zeatin Corresponding author. Tel./fax: +81 87 891 3127. E-mail addresses: [email protected] (B. Rani Debi), [email protected] (M. Ichii). 0176-1617/$ - see front matter & 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2004.08.007
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Introduction Lateral root (LR) development is a multiphase process including at least initiation and emergence of lateral root primordia (LRP) and activation of LR meristem. LRs originate in the root pericycle, in which individual quiescent cells are stimulated to dedifferentiate and proliferate to form the LRP. The LRP then grows via both cell division and cell expansion. The emergence of LRP from the parent root occurs primarily by cell expansion. Soon after the emergence, the LRP undergoes an activation step to form a fully functional LR meristem (Malamy and Benfey, 1997) that directs growth from this stage onward. Numerous studies have shown that auxin is necessary for the initiation and subsequent growth of LRs (Blakely et al., 1988; Celenza et al., 1995; Reed et al., 1998). Exogenous application of auxin or an enhancement of endogenous auxin synthesis results in a significant increase in LRs number (Klee et al., 1987; Kares et al., 1990; Boerjan et al., 1995; Laskowski et al., 1995). Cytokinins are a class of plant hormones that influences many developmental processes including leaf senescence, apical dominance, chloroplast development, anthocyanin production and regulation of cell division. Cytokinin, together with auxin plays an essential role in plant morphogenesis, having a profound influence on the formation of roots and shoots and their relative growth. Cytokinins act antagonistically to auxin and determine cell fate by promoting shoot and root differentiation in callus culture (Skoog and Miller, 1957). On initiation of LR primordia, the interaction of auxin and cytokinin is antagonistic, with auxin stimulating and cytokinin inhibiting cell division in the root pericycle. Cytokinins are formed in the root tips and interact with auxin in the regulation of LR formation (Wightman et al., 1980; Hinchee and Rost, 1986; Torrey, 1986). The auxin:cytokinin ratio has been reported to play an important role in LR development in pea seedlings. Application of exogenous auxin promoted LR initiation and application of cytokinin reduced the number of LRs (Bo ¨ttgor, 1974; Goodwin and Morris, 1979; Hinchee and Rost, 1986). There are many reports described the inhibitory effect of cytokinin on the initiation of LRs. However, there are only few reports described the effect of cytokinin on the advanced stages of LR formation. There are contradictory results obtained on the effect of cytokinin on LR emergence. For example, Wightman et al. (1980) reported that cytokinin is inhibitory for LR emergence and they described that the LR emergence was about twice
B. Rani Debi et al. as sensitive as the initiation of primordia to the presence of cytokinins. Hinchee and Rost (1986) observed inhibitory effect of cytokinin on LR emergence in pea seedlings with detached cotyledons. However, the inihibitory effect of cytokinin on LR emergence diminished when the cotyledons remained intact. Furthermore, compared to other stages of LR formation, the effect of cytokinin on LR elongation is really poorly understood. So the purpose of our present study is to elucidate the effect of cytokinin on LR formation in plant species other than dicots as the previous studies were carried out mainly in dicots. We have observed the effect of kinetin (KIN) and zeatin (ZEA) on various stages of LR formation using japonica rice (Oryza sativa L.), Nipponbare as the plant material. Our results showed that the effect of cytokinin varies depending on developmental stages of LR formation, with an inhibitory effect on LR initiation, no inhibitory effect on LR emergence and a dramatic stimulatory effect on LR elongation.
Materials and methods Plant materials and growth conditions Japonica rice, Nipponbare (Oryza sativa L. subsp. japonica) was used in the experiments. Seeds were surface sterilized with 0.2% (W/V) benomyl solution in an incubator at 30 1C for 1 day, and rinsed with tap water several times. Then the seeds were soaked in tap water for another 1 day at the same incubator for germination. The germinated seeds were used in the hormone experiments. Experiments were conducted in a plant growth cabinet (ML R350, Sanyo) at 25 1C and under continuous white fluorescent light at a light intensity of 500 mmol m2 s1.
Experiments on the effect of cytokinin on LR formation The germinated seeds were placed in plastic nets on a float and transferred to black cups containing 200 ml solution of each KIN and ZEA of indicated concentrations or distilled water as the control. The culture solutions were renewed every 2 days. After 10 days, all the emerged LRs on seminal roots were counted. Lateral root density was expressed as the number of LRs per centimeter of seminal root obtained by dividing the LRs number by the length of the respective seminal root. To investigate the effect of cytokinin on the emergence of
ARTICLE IN PRESS The effect of cytokinin on lateral root development in rice LRs, germinated seeds were placed in plastic nets and transferred to black cups containing 200 ml of distilled water and grown for 3 days to allow the roots to form LRP, and then 15 seedlings were transferred to black cups containing 200 ml of 1 mM of each KIN, ZEA or distilled water as the control. The seedlings were grown for an additional 4 days and the LRs that had emerged from the seminal roots were counted everyday. To investigate the effect of cytokinin on the elongation of LRs, seedlings were grown similarly for 4 days in distilled water and then transferred to the indicated concentrations of each KIN, ZEA or distilled water and grown for an additional 6 days. The length of 20 longest LRs from each seminal root was measured.
Observation of LRP Formation of LRP was observed according to Wang et al. (2003) with some modifications. Briefly, the seminal roots of 3-day old seedlings grown in distilled water and in 1 mM of each KIN or ZEA were stained in the mixture of 25 ml 0.4% (W/V) 2,3,5triphenyltetrazolium chloride (TTC) and 25 ml of 0.1 M H3PO4 buffer (pH 7.2) in dark for 2 h at 30 1C in an incubator. Then the roots were degassed in a vacuum drier for 30 min and then transferred to a separate solution of 10% HCl for 20 min and a 10% glycerol solution for 20 min. The roots were then placed in a slide on a drop of glycerol and a cover glass was placed and tapped gently by the blunt end of a needle and observed under a microscope and photographed.
Measurement of epidermal cell length of LRs Histological observation of LRs was performed according to Yao et al. (2003). To estimate the epidermal cell length of LRs with or without cytokinin treatment, 1 cm of LR tips of 10-day-old seedlings were fixed in 2% gluteraldehyde 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).
Combined treatment of KIN and ZEA with auxin To know whether auxin has any influence on the action of cytokinin on LR formation, we observed the effect of KIN and ZEA in the presence of indole3-acetic acid (IAA) and indole-3-butyric acid (IBA). To see the combined effect of cytokinin and auxin
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on LR initiation, germinated seeds were placed in black cups containing 200 ml of 0.1 mM of each KIN or ZEA with that of 1 mM IAA or 0.1 mM IBA. Moreover, 1 mM IAA, 0.1 mM IBA, 0.1 mM of each KIN or ZEA were used as the control. After 10 days of growth, the number of LRs per centimeter of seminal root was determined. To see the combined effect of cytokinin and auxin on LR elongation, the germinated seeds were grown in distilled water for initial 4 days and then transferred to black cups containing 200 ml of 0.01 mM KIN or 0.001 mM ZEA in combination with 0.1, 1 and 10 mM IAA or 0.1, 1 and 10 mM IBA solution and in 0.1, 1 and 10 mM IAA or 0.1, 1 and 10 mM IBA as the control and grown for an additional 6 days and the length of 20 longest LRs from each seminal roots of 10 seedlings were measured.
Results Inhibitory effect of cytokinin on LR initiation To assess the effect of cytokinin on LR formation in rice, germinated seeds of Nipponbare were grown in indicated concentrations of both KIN and ZEA or distilled water as the control. After 10 days of growth, LR density was calculated. The results revealed that both KIN and ZEA treatment reduced significantly the LR density in a dose-dependent manner (Fig. 1). The number of LRs per centimeter decreased with increasing the concentration of KIN and ZEA. At a concentration of 1 mM and above (Figs. 1, 2), complete inhibition of LR formation occurred in case of both KIN and ZEA treated seedlings. ZEA is stronger inhibitor of LR formation than KIN. The above results showed that 1 mM of both KIN and ZEA blocked LR formation completely. So to know which stage of LR formation was specifically affected by cytokinin, the germinated seeds were grown in 1 mM of KIN or ZEA or distilled water for 3 days and formation of LRP on seminal root was observed. Many LRP were formed in the roots grown in distilled water, but no LRP was found to be formed in the roots treated with 1 mM of KIN or ZEA (Fig. 2B). This result indicates that cytokinin inhibits LR initiation in the root pericycle.
Cytokinin is required continuously for inhibition of LR formation To test whether cytokinin is required continuously for inhibition of LR formation or an initial treatment is enough to trigger the inhibition of LR
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and grown for an additional 4 days. The results showed that when the seedlings were transferred from KIN or ZEA solution to distilled water, LRs formed on the lower part of the root that grew newly in distilled water after transfer (Fig. 2C). However, no LRs were formed on the upper part of the root that grew in 1 mM KIN or ZEA (Fig. 2C) before transfer. This result indicates that the inhibitory effect of cytokinin on LR formation is reversible and that a continuous presence of cytokinin is required for the inhibition of LR formation.
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Figure 1. Effect of KIN (A) and ZEA (B) on lateral root formation in rice. Seedlings were grown in different concentrations of KIN, ZEA or in distilled water as the control. After 10 days of growth, the total number of emerged lateral roots on each seminal root was counted and divided by the length of the respective seminal root length to get the number of lateral roots per centimeter of seminal root. Each data point represents the averages of 10 seedlings and the error bar represents the standard deviations of the means.
Figure 2. Comparison of LR formation in KIN treated and non-treated seminal roots. (A) Seminal root of 10-day old seedlings grown in control solution showing LR formation (left) and in 1 mM KIN solution showing inhibition of LR formation (right). (B) Formation of LRs primordia on seminal root of 3-day old seedling grown in control solution (left, arrow indicates LRP) and in 1 mM KIN solution (right). (C) The seminal root of the seedling that was grown initial 3 days in 1 mM KIN solution showing inhibition of LR formation (root segment a) and then transferred to distilled water and grown for an additional 4 days showing formation of LR (root segment b).
formation, we performed a shift experiment. The seeds were germinated and grown for 3 days in 1 mM KIN or ZEA and then transferred to distilled water
After formation of LRP in the pericycle cells, the next step is the emergence of LRP through the epidermis of the parent roots. To analyze the effect of cytokinin on the emergence of LRP, the germinated seeds were grown in distilled water for 3 days to allow them to initiate LRP and then the seedlings were transferred to inhibitory concentration (1 mM) of each KIN and ZEA solution. Total number of emerged LRs on each seminal root was counted everyday for 4 days after transfer. The results showed that the roots in KIN and ZEA solution produced similar number of LRs as the roots in control after 1 day of transfer (Fig. 3A). After 2 days of transfer, the KIN and ZEA treated roots were found to produce less number of total LRs compared to control. Thereafter, the KIN and ZEA treated roots did not increase the total number of LRs. On the contrary, the roots in control solution increased the total number of LRs until 3 and 4 days after transfer (Fig. 3A, B left). The LRs in the KIN and ZEA treated roots were found to be emerged only in the upper part of the root that grew in distilled water before transfer to the KIN and ZEA solution, indicating that cytokinin treatment did not inhibit the emergence of already formed LRP (Fig. 3B right). However, after transfer, the lower part of the root that grew newly in KIN and ZEA produced no LRs, indicating that LRP initiation is inhibited by cytokinin (Fig. 3B right). All these results indicate that cytokinin does not inhibit the emergence of LRP from the seminal root. Once LRP is formed, it can increase cell number and emerge from the parent root normally in the presence of cytokinin.
Stimulatory effect of cytokinin on LR elongation The above results showed that cytokinin inhibits LR initiation and that it has no adverse effect on
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Figure 3. Effect of cytokinin on LR emergence. (A) Seedlings were grown in distilled water for initial 3 days and then transferred to 1 mM KIN and 1 mM ZEA solution and in distilled water as the control and were grown for additional 4 days. The number of all LRs that emerged from the seminal root was counted everyday after transfer. (B) The seminal root of the seedlings grown in initial 3 days in distilled water and then transferred to distilled water and grown for 4 days (left). The seminal root of the seedlings grown initial 3 days in distilled water (right, root segment a) and then transferred to 1 mM KIN and grown for additional 4 days (right, root segment b).
LRP emergence. To test the effect of cytokinin on LR elongation, seeds were grown in distilled water for 4 days and then transferred to different concentrations of KIN and ZEA or distilled water as the control. As shown in Figs. 4 and 5, a dramatic LR elongation was found at all the concentrations of KIN and ZEA treated seedlings compared to untreated control. Maximum LR elongation was observed at a concentration of 0.01 mM for KIN and 0.001 mM for ZEA. Even the inhibitory concentration (1 mM) of KIN or ZEA that completely blocked LR initiation also promoted LR elongation significantly. The epidermal cell length increased significantly in LRs of KIN or ZEA treated seedlings compared to those of untreated LRs (Fig. 6).
Effect of exogenous auxins on the cytokinin induced LR initiation and elongation As auxins are known to play a key role in LR formation, we further examined whether exogenously applied auxin influence the inhibition of LR formation by KIN and ZEA. For this experiment, we grew the seeds in various concentrations of IAA and IBA or distilled water together with 0.1 mM of each KIN and ZEA. The results showed that the exogenously applied IAA at a concentration of 1 mM and that of IBA at a concentration of 0.1 mM counteracted the inhibitory effect of KIN and ZEA on LR initiation (Fig. 7A). However, the inhibition by 1 mM or above concentration of KIN or ZEA could not
overcome by IAA or IBA at concentration as high as 10 mM (data not shown). As maximum LR elongation was observed upon treatment with 0.01 mM KIN or 0.001 mM ZEA, we examined the elongation of LRs in combined solutions of different concentrations of IAA and IBA or distilled water with 0.01 mM KIN or 0.001 mM ZEA. The results revealed that both IAA and IBA inhibited in a dose-dependent manner the promotion of LR elongation by KIN and ZEA (Fig. 7B, C).
Discussion This article reports the effect of cytokinins on initiation, emergence and elongation of LRs in rice. Our results revealed that both the KIN and ZEA at 1 mM and higher concentration inhibit LR formation by inhibiting the initiation of LRs primordia (Figs. 1 and 2). Such inhibition of LR formation by cytokinins is consistent with the previous results observed in other plant species (Torrey, 1962; Bo ¨ttgor, 1974; Goodwin and Morris, 1979; Wightman et al., 1980). Cytokinins were reported to inhibit cell division during early phase of organization of adventitious root primordia in pea stem cuttings (Bollmark et al., 1986). Eriksen (1974) reported that in pea, cytokinin at high concentrations may have an inhibitory effect on an early stage of rooting by blocking the activity of auxin. Exogenous application of cytokinins to maize (Bourquin and Pilet,
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Figure 5. Effect of KIN and ZEA on LR elongation. Seedlings were grown in distilled water for initial 4 days and then transferred to 0.01 mM KIN, 0.001 mM ZEA or distilled water as the control and grown for an additional 6 days and photographed. (A) LR elongation in control solution. (B, C) LR elongation by 0.01 mM KIN and 0.001 mM ZEA, respectively is shown. Bars ¼ 10 mm.
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Figure 4. Effect of KIN (A) and ZEA (B) on lateral root elongation. Seedlings were grown in distilled water for initial 4 days and then transferred to different concentrations of KIN (A) and ZEA (B) or distilled water as the control and grown for another 6 days. Then the length of 20 longest LRs from each seminal root was measured. Each data point represents the averages of 10 seedlings and error bars represent the standard deviation of the means.
1990) or pea (Bertell and Eliasson, 1992) roots increased free IAA. Such increase of free IAA was also observed in cytokinin overproducing lines of Nicotina glutinosa transformed with the ipt gene (Binns et al., 1987). Soon after the formation of LR primordia, the next step is the emergence of LR primordia through the epidermis of the parent root. Cytokinins were shown to be inhibitors of LR emergence (Wightman et al., 1980; Biddington and Dearman, 1982). In contrast, our results revealed that cytokinins have no adverse effect on the emergence of LRP. The present results showed that the inhibitory effect of cytokinin is restricted to the initiation of LRP in the pericycle. But once LRPs are formed, it can normally emerge even in the presence of inhibitory concentration (1 mM) of cytokinins (Fig. 3A, B). Our
results are consistent with the findings of Forsyth and Staden (1981) in pea where they showed that the LRPs begin synthesizing cytokinins immediately after primordia induction, and therefore do not react to the exogenous application of cytokinin. Hinchee and Rost (1986) reported that cytokinin is inhibitory for LR emergence in pea seedlings with detached cotyledons. However, the inhibitory effect of cytokinin on LR emergence diminished when the cotyledons remained intact. The presence of cotyledons or shoot is required for LR emergence, probably because these organs supply auxins acropetally to the root (Rowntree and Morris, 1979; Tsurumi and Wada, 1980). Our results showed that both KIN and ZEA significantly stimulated LR elongation once LRPs had been formed (Figs. 4 and 5). To our knowledge, this is the first report that cytokinins stimulate LR elongation. The LR length increased about 4–5 times by 0.01 mM KIN or 0.001 mM ZEA compared to that of untreated control (Fig. 4). Histological observation also revealed a 4–5 times increased epidermal cell length of the LRs treated by 0.01 mM KIN and 0.001 mM ZEA compared to untreated control (Fig. 6), indicating that the increased LR elongation by cytokinin is primarily due to an increased cell length of the existing cells rather than enhanced cell division. This stimulation effect of cytokinin is unique to LRs because, in rice, the inhibitory concentration (1 mM) of cytokinin significantly inhibited the seminal root elongation (Fig. 2A), while LR length was still stimulated
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Figure 6. Effect of cytokinin on epidermal cell length of LRs (A). (B) Electron microscopy of LRs showing epidermal cells. Left: seedlings were grown in distilled water, middle: seedlings were grown in 0.01 mM KIN and right: seedlings were grown in 0.001 mM ZEA. Seedlings were grown in distilled water for 4 days and then transferred to 0.01 mM KIN and 0.001 mM ZEA or distilled water and grown for another 6 days. Then 1 cm of LR segment were fixed in 2% glutaraldehyde and dehydrated in an ethanol series. After dried in critical point dryer, the samples were sputter-coated with platinum for about 10 min at 7 mA. The root samples were then observed and photographed under a scanning electron microscope (Hitachi, S-2150) at 10 kV. Data on epidermal cell length was taken from four LRs of each of the treatment and 30 cells were measured from each LR.
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at this concentration (Fig. 4). Our results are consistent with the cytokinin-responsive Arabidopsis response regulator (ARR5) gene promoter expression pattern in Lotus japonicus observed by Lohar et al. (2004). They reported that ARR5 expression was undetectable in the dividing cells of the early LR primordium, but later they observed high expression in the base of the cone-shaped LRP, in the root tip and in the LR meristem for the subsequent growth of the LRs. The expression patterns of ARR5 indicate that cytokinins have an inhibitory effect on LRP initiation and cytokinins play important role in subsequent growth of LRs. The auxin:cytokinin ratio has been shown to play a role in co-ordinating LR development in pea seedlings (Bo ¨ttgor, 1974; Hinchee and Rost, 1986). So we also examined the effect of cytokinin on LR formation in rice in the presence of auxins. Our
Figure 7. Effect of combined application of cytokinin with auxin on LR formation. Seedlings were grown in combined solution of 0.1 mM KIN with 1 mM IAA or 0.1 mM IBA and in 0.1 mM ZEA with 1 mM IAA or 0.1 mM IBA. For control treatment, seedlings were grown in 0.1 mM KIN, 0.1 mM ZEA, 1 mM IAA or 0.1 mM IBA solution. After 10 days of growth, the number of emerged LRs on each seminal root were counted and divided by the length of the respective seminal root (A). Seedlings were grown in initial 4 days in distilled water and then transferred to combined solutions of 0.01 mM KIN or 0.001 mM ZEA with indicated concentrations of IAA (B) or IBA (C) and in indicated concentrations of IAA (B) or IBA(C) as the control and grown for another 6 days. Then the length of 20 longest LRs was measured. Each data point represents the averages of 10 seedlings and error bars represent the standard deviation of the means.
ARTICLE IN PRESS 514 results showed that addition of auxins in the growth solution effectively counteracted the inhibitory effect of cytokinin on LR formation (Fig. 7A). In contrast, the stimulatory effect of cytokinin on LR elongation was inhibited gradually with increasing concentration of auxins in the growth solution (Fig. 7B, C). These results suggest that the inhibitory and stimulatory action of cytokinin depends on its concentration relative to that of auxin concentration. In summary, we analyzed the effect of cytokinin on LR development in rice and found that although cytokinin is inhibitory for LR initiation, it has no adverse effect on LR emergence and it shows dramatic stimulatory effect on LR elongation in rice. Further study with other species and plant hormone mutants are needed to better understand the mechanisms of cytokinin regulation of LR development.
Acknowledgements The financial support of the Ministry of Education, Science, Sports and Culture of Japan under scholarship program for foreign students is greatly acknowledged.
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