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Sucrose-induced hypocotyl elongation of Arabidopsis seedlings in darkness depends on the presence of gibberellins
Journal of Plant Physiology 167 (2010) 1130–1136
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Sucrose-induced hypocotyl elongation of Arabidopsis seedlings in darkness depends on the presence of gibberellins Yongqiang Zhang, Zhongjuan Liu, Liguang Wang, Sheng Zheng, Jiping Xie, Yurong Bi ∗ Key Laboratory of Arid and Grassland Agroecology (Ministry of Education), School of Life Sciences, Lanzhou University, Lanzhou Gansu 730000, People’s Republic of China
a r t i c l e
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Article history: Received 6 November 2009 Received in revised form 14 March 2010 Accepted 15 March 2010 Keywords: Arabidopsis Dark Gibberellin Hypocotyl elongation Sucrose
a b s t r a c t In this study, the effects of sucrose on hypocotyl elongation of Arabidopsis seedlings in light and in dark were investigated. Sucrose suppressed the hypocotyl elongation of Arabidopsis seedlings in light, but stimulated elongation in dark. Application of paclobutrazol (PAC, a gibberellin biosynthesis inhibitor) impaired the effects of sucrose on hypocotyl elongation, suggesting that endogenous GAs is required for sucrose-induced hypocotyl elongation in the dark. Exogenous GA3 application reversed the repression caused by PAC application, indicating that exogenous GA3 could substitute, at least partially, for endogenous GAs in sucrose-induced hypocotyl elongation. In addition, we found that GA 3-oxidase 1 (GA3ox1), encoding a key enzyme involved in endogenous bioactive GA biosynthesis, was up-regulated by sucrose in the dark, whereas GIBBERELLIN INSENSITIVE DWARF 1a (AtGID1a), encoding a GA receptor and playing an important role during GAs degradation to DELLA proteins (DELLAs, repressors of GA-induced plant growth), was down-regulated. These results imply that endogenous bioactive GA levels are expected to be enhanced, but the degradation of DELLAs was inhibited by sucrose in dark. Thus, our data suggest that the sucrose-induced hypocotyl elongation in the dark does not result from GA-induced degradation of DELLAs. We conclude that sucrose can stimulate hypocotyl elongation of Arabidopsis seedlings in the dark in a GA-dependent manner. Crown Copyright © 2010 Published by Elsevier GmbH. All rights reserved.
Introduction Sugars, the end products of photosynthesis, have a vital signaling function and modulate a range of important processes during plant growth and development, including seed germination, floral transition, fruit ripening, embryogenesis, and senescence (Rolland et al., 2002; León and Sheen, 2003). Responses to sugars in plants are closely integrated with many response pathways of environmental factors, among which, light is one of the important components (Gibson, 2005; Rook et al., 2006). Generally, in the presence of light, high sugar concentration inhibits seedling development, represses expression of photosynthetic genes and induces expression of storage metabolism genes (Rook et al., 2006). Light is probably one of the most influential environmental cues. It not only provides the source of energy for plant life, but as an informational signal, also affects plant growth and development
Abbreviations: AtGID1, Arabidopsis GIBBERELLIN INSENSITIVE DWARF 1; COP1, CONSTITUTIVELY PHOTOMORPHOGENIC 1; GA, gibberellin; GA2ox, GA 2-oxidases; GA3ox, GA 3-oxidases; GA20ox, GA 20-oxidases; GAI, GA INSENSITIVE; HY5, LONG HYPOCOTYL 5; HYH, HY5 HOMOLOG; PAC, paclobutrazol; PIF, PHYTOCHROMEINTERACTING FACTOR; RGA, REPRESSOR OF GA1-3; SLY1, SLEEPY1. ∗ Corresponding author. Tel.: +86 931 8912561; fax: +86 931 8912561. E-mail address: [email protected] (Y. Bi).
throughout the entire life cycle from germination to flowering (Lee et al., 2007). The development of plants in the light is referred to as photomorphogenesis, whereas development in the absence of light is referred to as skotomorphogenesis. The latter is characterized by an etiolated appearance of seedlings with a fast-growing hypocotyl or epicotyl, but the growth of the hypocotyl or epicotyl is slowed when light triggers photomorphogenetic development (Alabadí et al., 2004; Chen et al., 2004). The transition between the two development pathways is tightly regulated. Thus far, the CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1)-based complex is considered to play a critical role in the light-dependent repression of photomorphogenesis in the dark (Lee et al., 2007). COP1, as an E3 ubiquitin ligase, can constantly degrade a number of transcription factors that are required for development in light, such as the bZIP transcription factor LONG HYPOCOTYL 5 (HY5), but allows accumulation of others that promote etiolated growth, such as PHYTOCHROME INTERACTING FACTOR 1 (PIF1), PIF3, and PIF4 (Alabadí et al., 2008). Hypocotyl elongation is one of the most prominent morphological features accompanying dark-triggered etiolation, and in addition to light, endogenous gibberellins (GAs) can also control the hypocotyl elongation (Peng and Harberd, 1997; Vandenbussche et al., 2005; Achard et al., 2007). The production of bioactive GAs in plants involves GA 20-oxidases (GA20ox), GA 2-oxidases (GA2ox)
0176-1617/$ – see front matter. Crown Copyright © 2010 Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2010.03.007
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and GA 3-oxidases (GA3ox), among which GA2ox can inactivate most active GAs (Yamaguchi, 2008). Previous studies have shown that correct GA homeostasis in etiolated seedlings is essential to properly control the transition between skotomorphogenesis and photomorphogenesis (Vandenbussche et al., 2005; Alabadí et al., 2008). Plants defective in either GA biosynthesis or GA signaling are not able to fully repress photomorphogenesis after germination in the dark, and the seedlings appear to be partially de-etiolated (Achard et al., 2003, 2007; Alabadí et al., 2004, 2008; Vriezen et al., 2004). Thus, GA plays an important role during skotomorphogenetic development in the dark, including dark-induced hypocotyl elongation (Alabadí et al., 2004). GA signal transduction involves DELLA proteins, which are nuclear growth repressors, a subset of the GRAS family of candidate transcriptional factors (Yamaguchi, 2008). There are five types of DELLAs in Arabidopsis. Among these, RGA (encoded by Repressor of GA1) and GAI (encoded by GA Insensitive) are the main repressors controlling hypocotyl and stem elongation (Achard et al., 2007; Vandenbussche et al., 2007; Feng et al., 2008; Hartweck, 2008; Yamaguchi, 2008). DELLAs restrain plant growth, whereas GAs promote growth by overcoming DELLA-mediated growth restraint (Achard et al., 2007; Hartweck, 2008). GAs relieve DELLA restraint by promoting the degradation of nuclear DELLAs (Silverstone et al., 2001; Fleck and Harberd, 2002). In Arabidopsis, GAs are perceived by the GA receptor GIBBERELLIN INSENSITIVE DWARF 1 (AtGID1) (Nakajima et al., 2006). Binding of GA to AtGID1 promotes interaction of AtGID1 with the DELLAs, which promotes interaction with the F-box protein SLEEPY1 (SLY1), polyubiquitination of these proteins by the SCFSLY1 ligase complex, and eventual degradation of DELLAs in the 26S proteasome (Nakajima et al., 2006; Hartweck, 2008). It has recently been found that, while light inhibits hypocotyl growth via GA decrease and in turn DELLA accumulation, the induction of hypocotyl elongation in dark is associated with GA accumulation (Achard et al., 2007). Hence, GA is a key component of the light signaling pathway regulating hypocotyl growth (Alabadí et al., 2004; Achard et al., 2007). In comparison to the repression effect on plant growth in the presence of light (Gibson, 2005), the role of sugar in the dark remains less clear. In this study, we found that sugars stimulated Arabidopsis hypocotyl elongation in the dark and that GAs were essential for this process. Materials and methods Plant materials and growth conditions The seeds of Arabidopsis wild type Col-0 were surface-sterilized with 20% (v/v) bleach solution for about 13 min. Seeds were extensively rinsed with sterile water and sown on agar medium in Petri dishes with half strength Murashige and Skoog (MS) salts containing 0.8% (w/v) agar and without any sugars. Agar plates were kept at 4 ◦ C in the dark for 3 days, and then transferred to a growth chamber maintained at 23 ◦ C under continuous white light (about 60–70 mol m−2 s−1 ) for 4 days before treatment. For study of hypocotyl elongation, the 4-day-old seedlings were transferred to dark or still kept in light with or without sugars or other chemicals (GA3 , PAC) in the media for the indicated time shown in each figure, and then hypocotyl lengths were measured. Hypocotyl length measurement After the indicated time of growth and treatment, at least 25 seedlings were laid horizontally on an agar plate, digital pictures were taken, and hypocotyl length was measured using a
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standard 4 mm scaled ruler with ImageJ software (Alabadí et al., 2004). Reverse transcription (RT)-PCR assays Seedlings were harvested in liquid nitrogen, ground, and RNA extracted using TRIzol reagent (Invitrogen). The amount of mRNA was analyzed using semi-quantitative RT-PCR. The complementary DNA (cDNA) was then synthesized using a random hexamer primer and Moloney murine leukemia virus (M-MuLV) reverse transcriptase (Fermentas) at 42 ◦ C for 60 min. An equal amount of cDNA, estimated using reactions with ACTIN2 primers, was used as template in the following PCR reaction: 25 L reaction mixture containing 2 mM MgCl2 , 0.2 mM each dNTP, 0.5 M each gene-specific primer, and 1 units Taq DNA polymerase together with the manufacturer’s buffer using the following protocol: 5 min denaturation at 94 ◦ C followed by indicated cycles with each cycle composed of 94 C for 30 s, 57.8, 59.5, 61, 55.5, 55, 53, 53.5 and 60 ◦ C, for genes ACTIN2, GA20ox1, GA3ox1, AtGID1a, AtGID1c, SLY1, RGA, and GAI, respectively for 30 s, 72 ◦ C for 1 min, and 10 min at 72 ◦ C. PCR products were visualized by electrophoresis on agarose gels containing ethidium bromide. The sequences of the PCR primers used in this study were the following: ACTIN2F (5 -GTT GGG ATG AAC CAG AAG GA-3 ), ACTIN2R (5 -CTT ACA ATT TCC CGC TCT GC-3 ); GA20ox1F (5 -CAG CCA TTT GGG AAG GTG TATC-3 ), GA20ox1R (5 -CAA GCA GCT CTT GTA TCT ATC GT-3 ); GA3ox1F (5 -CCG AAG GTT TCA CCA TCA CTG-3 ), GA3ox1R (5 -GAG GCG ATT CAA CGG GAC TAA C-3 ); AtGID1aF (5 -GTT TGG TGG GAA TGA GAG AAC G-3 ), AtGID1aR (5 CTA AAC GCC TCA CTG TTC TTC C-3 ); AtGID1cF (5 -ACC GTC ATC TCG CAG AGT TT-3 ), AtGID1cR (5 -TCC TTG ACT CAA CCG CTC TT3 ); SLY1F (5 -GCG CAG TAC TAC CGA CTCTG-3 ), SLY1R (5 -CTT AGT GAA ACT CAT CTT CTC G-3 ); RGAF (5 -CGG AAA CGC GAT TTA TCA GT-3 ), RGAR (5 -GTC GTC ACC GTC GTT CCT AT-3 ); GAIF (5 -AGC GTC ATG AAA CGT TGA GTC AGT G-3 ), GAIR (5 -TGC CAA CCC AAC ATG AGA CAG C-3 ). Results Induction of hypocotyl elongation by sucrose in the dark To test the effect of sugars on hypocotyl growth, Arabidopsis Col-0 seedlings grown in continuous white light for 4 days (4day-old seedlings) were treated with or without 90 mM sucrose in the dark or in light. As shown in Fig. 1A and B, the hypocotyls of control seedlings in the dark significantly elongated as treatment time increased, and application of sucrose to the media further stimulated the hypocotyl elongation. For example, the hypocotyl length in the presence of sucrose was 50%, 90%, and 152% longer than that of control at days 1, 2, and 3, respectively (Fig. 1B). These results clearly suggest that sucrose plays a positive role in stimulating hypocotyl elongation in the dark. However, sucrose exhibited a slight repression effect on hypocotyl growth in the light. For example, the hypocotyl length of seedlings in the presence of sucrose was only 85% of that of the control (without sucrose) at day 3 (Fig. 1A and B). In the presence of different concentrations of sucrose, the hypocotyl length of Col-0 seedlings increased when sucrose concentration increased from 0 to 60 mM after growing in the dark for 2 days, while higher concentrations of sucrose (60–150 mM) had almost no effect in terms of increasing hypocotyl length (Fig. 1C). Thus, 90 mM sucrose was sufficient to induce significant hypocotyl elongation in the dark. In addition to sucrose, glucose and fructose also stimulated hypocotyl elongation in the dark, whereas sorbitol had no statistically significant effect on hypocotyl elongation and mannitol repressed hypocotyl elongation (Fig. 2). In addition, all the carbohydrates tested here
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Fig. 2. Effects of different sugars on hypocotyl elongation in dark. Four-day-old Col0 seedlings were treated with 90 mM sucrose (Suc), glucose (Glu), fructose (Fru), sorbitol (Sor), mannitol (Man) or 90 mM Glu together with 90 mM Fru for 2 days in light and in dark. The seedlings without any sugar treatment were used as control. Mean values and S.E. were calculated from at least 25 seedlings. Different letters denote significant differences (P < 0.05) among the means according to Student’s t-test.
transferred from light to dark conditions (Achard et al., 2007), and their important role in stimulating hypocotyl elongation (Alabadí et al., 2004, 2008). We first tested whether the endogenous GAs are involved in the sucrose-induced hypocotyl elongation in the dark. PAC (a GA biosynthesis inhibitor), widely used in many other studies (Alabadí et al., 2004; de Lucas et al., 2008; Feng et al., 2008), was used in this experiment to attenuate endogenous GA levels. Our results showed that the induction of hypocotyl elongation by sucrose was greatly impaired by 1 M PAC treatment for 2 days in the dark (Fig. 3A). This indicates that endogenous GAs are indeed required for sucrose-induced hypocotyl elongation in the dark. To further confirm this notion, 4-day-old Col-0 seedlings were treated with different concentrations of PAC (0–100 M) with or without 90 mM sucrose for 3 days in the dark. The results clearly showed that both dark-induced and sucrose-induced hypocotyl elongations decreased with increased PAC concentrations (Fig. 3B). Most notably, the hypocotyl length of seedlings grown in the presence of sucrose and 100 M PAC was reduced to a very low level, similar to that of seedlings without sucrose (Fig. 3B), suggesting that 100 M PAC could absolutely abrogate the stimulation of sucrose on hypocotyl elongation.
Fig. 1. Stimulation of sugars on hypocotyl elongation in dark. Four-day-old Col-0 seedlings were transferred to dark or kept in light with or without 90 mM sucrose treatment for 0, 1, 2, and 3 days, and the hypocotyl lengths were measured (B); representative pictures were taken at day 3 of treatment (A). (C) Four-day-old Col0 seedlings were treated with sucrose at concentrations of 0, 15, 30, 60, 90, and 150 mM in dark for 2 days, and the hypocotyl lengths were determined. Mean values and S.E. were calculated from at least 25 seedlings. Different letters denote significant differences (P < 0.05) among the means according to Student’s t-test.
showed a weak repression effect on hypocotyl elongation in the light (Fig. 2). Effect of endogenous GAs on sucrose-induced hypocotyl elongation in the dark As a next step, the possible mechanism by which sucrose promotes hypocotyl elongation in dark was studied. The well-known phytohormones GAs were selected to test their participation in the process, because of their significant accumulation in seedlings
Effect of exogenous GA3 on sucrose-induced hypocotyl elongation in darkness Based on the above results, effects of exogenous bioactive GA (GA3 ) on sucrose-induced hypocotyl elongation in the dark were investigated. Unexpectedly, we found that GA3 had little effect on promoting hypocotyl elongation in the dark regardless of the presence of sucrose. However, GA3 showed a drastic effect on stimulating hypocotyl elongation in the light (Fig. 4A and B). A possible explanation for this observation is that the endogenous GAs in the dark, but not in the light, may be already high enough for hypocotyl elongation. To test this assumption, 4-day-old Col-0 seedlings were treated with 10 M PAC plus different concentrations of exogenous GA3 (10 M to 1 mM) in the dark. Here, the percentage of the recovered hypocotyl length by exogenous GA3 to the inhibited hypocotyl length by PAC was termed the “recovery rate”, which reflects the ability of GA3 in restoring the inhibited hypocotyl length by the indicated concentration of PAC. For example, as shown in Fig. 4C, after transfer of 4-day-old Col-0 seedlings
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Fig. 3. Endogenous GAs are required for sucrose-induced hypocotyl elongation in dark. (A) Four-day-old Col-0 seedlings were treated with 1 M PAC, 90 mM sucrose (Suc), or with 1 M PAC and 90 mM Suc for 2 days in light or in dark, and then pictures were taken and hypocotyl lengths were measured. The seedlings without any chemical treatment were used as control. (B) Four-day-old Col-0 seedlings were treated with PAC at concentrations ranging from 0 to 100 M together with or without 90 mM Suc for 3 days in dark, and then pictures were taken and hypocotyl lengths were measured. Mean values and S.E. were calculated from at least 25 seedlings. Different letters denote significant differences (P < 0.05) among the means according to Student’s t-test.
to the dark for 3 days without any treatment, the hypocotyl length was 0.208 cm; with 10 M PAC treatment, the hypocotyl length was 0.175 cm; with 10 M PAC and 100 M GA3 treatment, the hypocotyl length was 0.193 cm; and the “recovery rate” was (0.193 − 0.175)/(0.208 − 0.175) = 0.52 (52%). With GA3 concentrations from 1 M to 1 mM, the recovery rates increased from 13% to 102% and from 6% to 18%, respectively, in the absence and presence of 90 mM sucrose (Fig. 4C and D). The results showed that the application of GA3 could reverse the repression caused by PAC application, suggesting that exogenous GA3 could substitute for the endogenous GAs in both dark- and sucrose-induced hypocotyl elongations. To further confirm this notion, we transferred 4-dayold Col-0 seedlings to the dark with or without 90 mM sucrose treatment in the presence of 100 M GA3 plus different concentrations of PAC (0–1 M). After 3 days, hypocotyl lengths were determined. The results showed that, in the absence of sucrose, with PAC concentrations decreased from 1 to 0.1 M, the recovery rates increased from 55% to 59%, and more notably, in the presence of sucrose, the recovery rates increased from 38% to 61% (Fig. 4E). All these data indicate that exogenous GA3 could indeed function as endogenous GAs for sucrose-induced hypocotyl elongation in dark.
Regulation of sucrose on transcript levels of several key genes involved in GA metabolism and signaling in darkness It has been reported that sugars regulate plant growth and development generally at the transcript level (León and Sheen, 2003; Gibson, 2005; Rook et al., 2006). We analyzed the effects of sucrose on the expression of several genes encoding GA3ox1, GA20ox1, GA2ox1, GA2ox2, AtGID1a, AtGID1c, SLY1, RGA, and GAI
in dark. As shown in Fig. 5, after 4-day-old Col-0 seedlings were transferred to the dark for 6 to 24 h, the expression of GA3ox1 was significantly up-regulated by sucrose, whereas the expression of AtGID1a was down-regulated by sucrose. For other genes analyzed, sucrose exhibited little effects (Fig. 5). Discussion Sugars, including sucrose, glucose and fructose, slightly repressed Arabidopsis hypocotyl elongation in the light. However, they significantly stimulated hypocotyl elongation in the dark (Fig. 1). Among these sugars, sucrose had the most significant stimulation effect in this process (Fig. 2). Sugars display absolutely opposite effects on hypocotyl elongation in dark and in light, implying that there might be different signaling pathways in regulating hypocotyl elongation in the two processes. From previous research in different fields of plant biology and in global gene expression, it is becoming clear that sugar signaling in light affects numerous metabolic and developmental processes during the entire plant life cycle (Rolland et al., 2002; Gibson, 2005; Rolland and Sheen, 2005). In our study, we found that the sucrose-induced hypocotyl elongation in dark is fully dependent on the presence of endogenous GAs (Fig. 3), implying that GA metabolism or signaling may play an important role during this process. It has long been known that GAs regulate Arabidopsis hypocotyl elongation in both light and dark by influencing the rate and final extent of cellular elongation (Cowling and Harberd, 1999). The hypocotyls of mutants deficient in GA biosynthesis (ga1) or responses (gai) were significantly shorter than that of wild types (Peng and Harberd, 1997). Dark-grown seedlings have longer hypocotyls compared to light-grown seedlings, consistent with
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Fig. 4. Effects of exogenous GA3 on both dark- and sucrose-induced hypocotyl elongation. (A) Four-day-old Col-0 seedlings were treated with 10 M GA3 , 90 mM sucrose, or with 10 M GA3 plus 90 mM sucrose (Suc) for 2 days in light or in dark, and then hypocotyl lengths were determined. Different letters denote significant differences (P < 0.05) among the means according to Student’s t-test. (B) Four-day-old Col-0 seedlings were treated with or without 10 M GA3 in light or in dark for 0, 0.5, 1, 2, and 3 days, and then the hypocotyl elongation were determined. (C) Four-day-old Col-0 seedlings were treated with 10 M PAC together with 0, 1, 10, 100, or 1000 M GA3 in dark for 3 days, and then hypocotyl lengths were determined. The seedlings without any chemical treatment were used as control. (D) Four-day-old Col-0 seedlings were treated with 90 mM sucrose (Suc), or with 90 mM Suc plus 10 M PAC together with 0, 1, 10, 100, or 1000 M GA3 in dark for 3 days, and then hypocotyl lengths were determined. (E) At different concentrations of PAC (0, 0.1, 0.25, 0.5, 0.75, 1 M), 4-day-old seedlings were treated for 3 days with or without 100 M GA3 in dark, in the presence (+Sucrose) or absence (−Sucrose) of 90 mM sucrose, hypocotyl lengths were determined. Mean values and S.E. were calculated from at least 25 seedlings. Numbers above the bars in figures (C)–(E) denote the hypocotyl “recovery rate” by the indicted concentration of GA3 as a percentage.
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Fig. 5. Effects of sucrose on the expression of genes related to GA metabolism and GA signaling. Four-day-old Col-0 seedlings were transferred to dark with (+) or without (−) 90 mM sucrose treatment for 6, 12, and 24 h, and samples were collected at each time point for RNA analyses by RT-PCR. Levels of mRNA expression are shown as the gel images.
the notion that the endogenous GA level is higher in dark-grown seedlings than that in light-grown seedlings (Alabadí et al., 2004; Feng et al., 2008). In this report, we found that when endogenous GAs were inhibited by PAC, the dark-induced hypocotyl elongation was impaired (Fig. 3A). These data clearly indicate that GAs have potential roles in dark-induced hypocotyl elongation. Nonetheless, exogenous GA3 did not exert any effect on dark- or sucrose-induced hypocotyl elongations (Fig. 4A). A possible explanation for this result is that, in the dark, the Arabidopsis seedlings have accumulated high levels of GAs. This explanation appears to be correct, because when the biosynthesis of endogenous GAs was inhibited by PAC, exogenous GA3 exhibited significant effects on enhancing both dark- and sucrose-induced hypocotyl elongations (Fig. 4C–E). Taken together, the results suggest that the presence of GAs is necessary not only for dark-induced, but also for sucrose-induced hypocotyl elongation. It has been reported that GA regulation of hypocotyl elongation in the dark occurs through at least two molecular pathways: (1) negative regulation of positive factors of light signaling such as HY5 by COP1, which determines the degree of repression of photomorphogenesis; and (2) positive regulation of the protein levels of PIF transcription factors, such as PIF3 and PIF4, through COP1 or DELLA proteins, which determine the degree of activation of skotomorphogenesis (Alabadí et al., 2008; de Lucas et al., 2008; Feng et al., 2008). However, in the null mutants cop1-4, cop1-6, pif1-1, pif3-1, and pif4-2, sucrose was found to significantly induce the hypocotyl elongations in the dark, similar to that observed in their corresponding wild types (data not shown). These data suggest that COP1 and PIFs may not be required for sugar-induced hypocotyl elongation, which appears to represent the difference between dark-induced and sugar-induced hypocotyl elongation. Achard et al. (2007) reported that, during the transition from light to dark, there were no detectable changes on transcript levels of genes encoding GA-signaling components, such as RGA, SLY1, and AtGID1a, but that the transcript level of GA2ox1, encoding an enzyme which deactivates bioactive Gas, was reduced. Thus, for the pathway of dark-induced hypocotyl elongation through degradation of DELLAs by GAs, it appears to result from increase of endogenous GA levels, but not from enhancement of GA signaling. It is therefore reasonable to think that GAs contribute to sucrose-induced hypocotyl elongation through the same mechanism. Nevertheless, in this study, we found that, on one hand,
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sucrose stimulated up-regulation of GA3ox1 in the dark (Fig. 5), and on the other hand, sucrose had almost no significant effect on GA2ox1 and GA2ox2 expression. As a result, sucrose could increase the levels of endogenous bioactive GAs. Nevertheless, sucrose down-regulated the expression of AtGID1a (encoding one of the GA receptors) (Fig. 5), which might suggest that sucrose could inhibit DELLA degradation by GAs in the dark. This means that sucrose-induced hypocotyl elongation is unlikely to result from GAmediated DELLA degradation. Therefore, dark-induced hypocotyl elongation and sucrose-induced hypocotyl elongation might be mediated through two different GA-dependent pathways. In fact, the degradation of DELLAs is just one of the mechanisms in GA regulation of plant growth and development (Cao et al., 2006). In addition, GAs can also function in plants directly via positive regulators (Sun and Gubler, 2004). Possibly, sucrose-induced hypocotyl elongation occurs through one or more of these pathways. To clearly elucidate how GAs regulate sucrose-induced hypocotyl elongation in darkness, there is more work to be done. Additionally, GAs are unlikely to function in plants alone. For example, there is evidence to suggest that GA regulation of DELLAs occurs through auxin and ethylene (Achard et al., 2003; Saibo et al., 2003), which suggests that GAs are likely to coordinate with other hormones during plant growth and development. The cross-talk may also play a role during modulation of hypocotyl elongation. In fact, interaction between GAs and brassinosteroids (BRs) in the control of Arabidopsis hypocotyl elongation has been reported (Bouquin et al., 2001; Alabadí et al., 2004), although this interaction was not found in pea (Symons and Reid, 2003; Alabadí et al., 2004). Hence, we cannot exclude the possibility that crosstalks between GAs and other phytohormones exist in Arabidopsis and are required for sugar-induced hypocotyl elongation. In summary, a new function of the sugars (including sucrose, glucose, and fructose) in prompting Arabidopsis hypocotyl elongation in the dark was found in the present study. Further investigation demonstrated that the phytohormone GAs were involved in this process. Acknowledgments We would like to thank Professor Xing-Wang Deng (Yale University, USA) for providing seeds of cop1-4, hy5-215, hyh, and hy5hyh, and Professor Peter H. Quail (University of California, USA) for providing seeds of pif1-1, pif3-1, and pif4-2. This work was supported by Specialized Research Fund for the Doctoral Program of Higher Education of China (ratification number: 20050730017), Foundation of Science and Technology of Gansu Province (3ZS051-A25-018) and the Direction Allocation Grant from the Hong Kong Research Grant Council (DAG05/06.SC09 and DAG04/05.SC08). References Achard P, Liao L, Jiang C, Desnos T, Bartlett J, Fu X, et al. DELLAs contribute to plant photomorphogenesis. Plant Physiol 2007;143:1163–72. Achard P, Vriezen WH, Van Der Straeten D, Harberd NP. Ethylene regulates Arabidopsis development via the modulation of DELLA protein growth repressor function. Plant Cell 2003;15:2816–25. Alabadí D, Gallego-Bartolomé J, Orlando L, García-Cárcel L, Rubio V, Martínez C, et al. Gibberellins modulate light signaling pathways to prevent Arabidopsis seedling de-etiolation in darkness. Plant J 2008;53:324–35. Alabadí D, Gil J, Blázquez MA, García-Martínez JL. Gibberellins repress photomorphogenesis in darkness. Plant Physiol 2004;134:1050–7. Bouquin T, Meier C, Foster R, Nielsen ME, Mundy J. Control of specific gene expression by gibberellin and brassinosteroid. Plant Physiol 2001;127:450–8. Cao D, Cheng H, Wu W, Soo HM, Peng J. Gibberellin mobilizes distinct DELLAdependent transcriptomes to regulate seed germination and floral development in Arabidopsis. Plant Physiol 2006;142:509–25. Chen M, Chory J, Fankhauser C. Light signal transduction in higher plants. Annu Rev Genet 2004;38:87–117. Cowling R, Harberd N. Gibberellins control Arabidopsis hypocotyl growth via regulation of cellular elongation. J Exp Bot 1999;50:1351–7.
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Sucrose-induced hypocotyl elongation of Arabidopsis seedlings in darkness depends on the presence of gibberellins Yongqiang Zhang, Zhongjuan Liu, Liguang Wang, Sheng Zheng, Jiping Xie, Yurong Bi ∗ Key Laboratory of Arid and Grassland Agroecology (Ministry of Education), School of Life Sciences, Lanzhou University, Lanzhou Gansu 730000, People’s Republic of China
a r t i c l e
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Article history: Received 6 November 2009 Received in revised form 14 March 2010 Accepted 15 March 2010 Keywords: Arabidopsis Dark Gibberellin Hypocotyl elongation Sucrose
a b s t r a c t In this study, the effects of sucrose on hypocotyl elongation of Arabidopsis seedlings in light and in dark were investigated. Sucrose suppressed the hypocotyl elongation of Arabidopsis seedlings in light, but stimulated elongation in dark. Application of paclobutrazol (PAC, a gibberellin biosynthesis inhibitor) impaired the effects of sucrose on hypocotyl elongation, suggesting that endogenous GAs is required for sucrose-induced hypocotyl elongation in the dark. Exogenous GA3 application reversed the repression caused by PAC application, indicating that exogenous GA3 could substitute, at least partially, for endogenous GAs in sucrose-induced hypocotyl elongation. In addition, we found that GA 3-oxidase 1 (GA3ox1), encoding a key enzyme involved in endogenous bioactive GA biosynthesis, was up-regulated by sucrose in the dark, whereas GIBBERELLIN INSENSITIVE DWARF 1a (AtGID1a), encoding a GA receptor and playing an important role during GAs degradation to DELLA proteins (DELLAs, repressors of GA-induced plant growth), was down-regulated. These results imply that endogenous bioactive GA levels are expected to be enhanced, but the degradation of DELLAs was inhibited by sucrose in dark. Thus, our data suggest that the sucrose-induced hypocotyl elongation in the dark does not result from GA-induced degradation of DELLAs. We conclude that sucrose can stimulate hypocotyl elongation of Arabidopsis seedlings in the dark in a GA-dependent manner. Crown Copyright © 2010 Published by Elsevier GmbH. All rights reserved.
Introduction Sugars, the end products of photosynthesis, have a vital signaling function and modulate a range of important processes during plant growth and development, including seed germination, floral transition, fruit ripening, embryogenesis, and senescence (Rolland et al., 2002; León and Sheen, 2003). Responses to sugars in plants are closely integrated with many response pathways of environmental factors, among which, light is one of the important components (Gibson, 2005; Rook et al., 2006). Generally, in the presence of light, high sugar concentration inhibits seedling development, represses expression of photosynthetic genes and induces expression of storage metabolism genes (Rook et al., 2006). Light is probably one of the most influential environmental cues. It not only provides the source of energy for plant life, but as an informational signal, also affects plant growth and development
Abbreviations: AtGID1, Arabidopsis GIBBERELLIN INSENSITIVE DWARF 1; COP1, CONSTITUTIVELY PHOTOMORPHOGENIC 1; GA, gibberellin; GA2ox, GA 2-oxidases; GA3ox, GA 3-oxidases; GA20ox, GA 20-oxidases; GAI, GA INSENSITIVE; HY5, LONG HYPOCOTYL 5; HYH, HY5 HOMOLOG; PAC, paclobutrazol; PIF, PHYTOCHROMEINTERACTING FACTOR; RGA, REPRESSOR OF GA1-3; SLY1, SLEEPY1. ∗ Corresponding author. Tel.: +86 931 8912561; fax: +86 931 8912561. E-mail address: [email protected] (Y. Bi).
throughout the entire life cycle from germination to flowering (Lee et al., 2007). The development of plants in the light is referred to as photomorphogenesis, whereas development in the absence of light is referred to as skotomorphogenesis. The latter is characterized by an etiolated appearance of seedlings with a fast-growing hypocotyl or epicotyl, but the growth of the hypocotyl or epicotyl is slowed when light triggers photomorphogenetic development (Alabadí et al., 2004; Chen et al., 2004). The transition between the two development pathways is tightly regulated. Thus far, the CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1)-based complex is considered to play a critical role in the light-dependent repression of photomorphogenesis in the dark (Lee et al., 2007). COP1, as an E3 ubiquitin ligase, can constantly degrade a number of transcription factors that are required for development in light, such as the bZIP transcription factor LONG HYPOCOTYL 5 (HY5), but allows accumulation of others that promote etiolated growth, such as PHYTOCHROME INTERACTING FACTOR 1 (PIF1), PIF3, and PIF4 (Alabadí et al., 2008). Hypocotyl elongation is one of the most prominent morphological features accompanying dark-triggered etiolation, and in addition to light, endogenous gibberellins (GAs) can also control the hypocotyl elongation (Peng and Harberd, 1997; Vandenbussche et al., 2005; Achard et al., 2007). The production of bioactive GAs in plants involves GA 20-oxidases (GA20ox), GA 2-oxidases (GA2ox)
0176-1617/$ – see front matter. Crown Copyright © 2010 Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2010.03.007
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and GA 3-oxidases (GA3ox), among which GA2ox can inactivate most active GAs (Yamaguchi, 2008). Previous studies have shown that correct GA homeostasis in etiolated seedlings is essential to properly control the transition between skotomorphogenesis and photomorphogenesis (Vandenbussche et al., 2005; Alabadí et al., 2008). Plants defective in either GA biosynthesis or GA signaling are not able to fully repress photomorphogenesis after germination in the dark, and the seedlings appear to be partially de-etiolated (Achard et al., 2003, 2007; Alabadí et al., 2004, 2008; Vriezen et al., 2004). Thus, GA plays an important role during skotomorphogenetic development in the dark, including dark-induced hypocotyl elongation (Alabadí et al., 2004). GA signal transduction involves DELLA proteins, which are nuclear growth repressors, a subset of the GRAS family of candidate transcriptional factors (Yamaguchi, 2008). There are five types of DELLAs in Arabidopsis. Among these, RGA (encoded by Repressor of GA1) and GAI (encoded by GA Insensitive) are the main repressors controlling hypocotyl and stem elongation (Achard et al., 2007; Vandenbussche et al., 2007; Feng et al., 2008; Hartweck, 2008; Yamaguchi, 2008). DELLAs restrain plant growth, whereas GAs promote growth by overcoming DELLA-mediated growth restraint (Achard et al., 2007; Hartweck, 2008). GAs relieve DELLA restraint by promoting the degradation of nuclear DELLAs (Silverstone et al., 2001; Fleck and Harberd, 2002). In Arabidopsis, GAs are perceived by the GA receptor GIBBERELLIN INSENSITIVE DWARF 1 (AtGID1) (Nakajima et al., 2006). Binding of GA to AtGID1 promotes interaction of AtGID1 with the DELLAs, which promotes interaction with the F-box protein SLEEPY1 (SLY1), polyubiquitination of these proteins by the SCFSLY1 ligase complex, and eventual degradation of DELLAs in the 26S proteasome (Nakajima et al., 2006; Hartweck, 2008). It has recently been found that, while light inhibits hypocotyl growth via GA decrease and in turn DELLA accumulation, the induction of hypocotyl elongation in dark is associated with GA accumulation (Achard et al., 2007). Hence, GA is a key component of the light signaling pathway regulating hypocotyl growth (Alabadí et al., 2004; Achard et al., 2007). In comparison to the repression effect on plant growth in the presence of light (Gibson, 2005), the role of sugar in the dark remains less clear. In this study, we found that sugars stimulated Arabidopsis hypocotyl elongation in the dark and that GAs were essential for this process. Materials and methods Plant materials and growth conditions The seeds of Arabidopsis wild type Col-0 were surface-sterilized with 20% (v/v) bleach solution for about 13 min. Seeds were extensively rinsed with sterile water and sown on agar medium in Petri dishes with half strength Murashige and Skoog (MS) salts containing 0.8% (w/v) agar and without any sugars. Agar plates were kept at 4 ◦ C in the dark for 3 days, and then transferred to a growth chamber maintained at 23 ◦ C under continuous white light (about 60–70 mol m−2 s−1 ) for 4 days before treatment. For study of hypocotyl elongation, the 4-day-old seedlings were transferred to dark or still kept in light with or without sugars or other chemicals (GA3 , PAC) in the media for the indicated time shown in each figure, and then hypocotyl lengths were measured. Hypocotyl length measurement After the indicated time of growth and treatment, at least 25 seedlings were laid horizontally on an agar plate, digital pictures were taken, and hypocotyl length was measured using a
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standard 4 mm scaled ruler with ImageJ software (Alabadí et al., 2004). Reverse transcription (RT)-PCR assays Seedlings were harvested in liquid nitrogen, ground, and RNA extracted using TRIzol reagent (Invitrogen). The amount of mRNA was analyzed using semi-quantitative RT-PCR. The complementary DNA (cDNA) was then synthesized using a random hexamer primer and Moloney murine leukemia virus (M-MuLV) reverse transcriptase (Fermentas) at 42 ◦ C for 60 min. An equal amount of cDNA, estimated using reactions with ACTIN2 primers, was used as template in the following PCR reaction: 25 L reaction mixture containing 2 mM MgCl2 , 0.2 mM each dNTP, 0.5 M each gene-specific primer, and 1 units Taq DNA polymerase together with the manufacturer’s buffer using the following protocol: 5 min denaturation at 94 ◦ C followed by indicated cycles with each cycle composed of 94 C for 30 s, 57.8, 59.5, 61, 55.5, 55, 53, 53.5 and 60 ◦ C, for genes ACTIN2, GA20ox1, GA3ox1, AtGID1a, AtGID1c, SLY1, RGA, and GAI, respectively for 30 s, 72 ◦ C for 1 min, and 10 min at 72 ◦ C. PCR products were visualized by electrophoresis on agarose gels containing ethidium bromide. The sequences of the PCR primers used in this study were the following: ACTIN2F (5 -GTT GGG ATG AAC CAG AAG GA-3 ), ACTIN2R (5 -CTT ACA ATT TCC CGC TCT GC-3 ); GA20ox1F (5 -CAG CCA TTT GGG AAG GTG TATC-3 ), GA20ox1R (5 -CAA GCA GCT CTT GTA TCT ATC GT-3 ); GA3ox1F (5 -CCG AAG GTT TCA CCA TCA CTG-3 ), GA3ox1R (5 -GAG GCG ATT CAA CGG GAC TAA C-3 ); AtGID1aF (5 -GTT TGG TGG GAA TGA GAG AAC G-3 ), AtGID1aR (5 CTA AAC GCC TCA CTG TTC TTC C-3 ); AtGID1cF (5 -ACC GTC ATC TCG CAG AGT TT-3 ), AtGID1cR (5 -TCC TTG ACT CAA CCG CTC TT3 ); SLY1F (5 -GCG CAG TAC TAC CGA CTCTG-3 ), SLY1R (5 -CTT AGT GAA ACT CAT CTT CTC G-3 ); RGAF (5 -CGG AAA CGC GAT TTA TCA GT-3 ), RGAR (5 -GTC GTC ACC GTC GTT CCT AT-3 ); GAIF (5 -AGC GTC ATG AAA CGT TGA GTC AGT G-3 ), GAIR (5 -TGC CAA CCC AAC ATG AGA CAG C-3 ). Results Induction of hypocotyl elongation by sucrose in the dark To test the effect of sugars on hypocotyl growth, Arabidopsis Col-0 seedlings grown in continuous white light for 4 days (4day-old seedlings) were treated with or without 90 mM sucrose in the dark or in light. As shown in Fig. 1A and B, the hypocotyls of control seedlings in the dark significantly elongated as treatment time increased, and application of sucrose to the media further stimulated the hypocotyl elongation. For example, the hypocotyl length in the presence of sucrose was 50%, 90%, and 152% longer than that of control at days 1, 2, and 3, respectively (Fig. 1B). These results clearly suggest that sucrose plays a positive role in stimulating hypocotyl elongation in the dark. However, sucrose exhibited a slight repression effect on hypocotyl growth in the light. For example, the hypocotyl length of seedlings in the presence of sucrose was only 85% of that of the control (without sucrose) at day 3 (Fig. 1A and B). In the presence of different concentrations of sucrose, the hypocotyl length of Col-0 seedlings increased when sucrose concentration increased from 0 to 60 mM after growing in the dark for 2 days, while higher concentrations of sucrose (60–150 mM) had almost no effect in terms of increasing hypocotyl length (Fig. 1C). Thus, 90 mM sucrose was sufficient to induce significant hypocotyl elongation in the dark. In addition to sucrose, glucose and fructose also stimulated hypocotyl elongation in the dark, whereas sorbitol had no statistically significant effect on hypocotyl elongation and mannitol repressed hypocotyl elongation (Fig. 2). In addition, all the carbohydrates tested here
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Fig. 2. Effects of different sugars on hypocotyl elongation in dark. Four-day-old Col0 seedlings were treated with 90 mM sucrose (Suc), glucose (Glu), fructose (Fru), sorbitol (Sor), mannitol (Man) or 90 mM Glu together with 90 mM Fru for 2 days in light and in dark. The seedlings without any sugar treatment were used as control. Mean values and S.E. were calculated from at least 25 seedlings. Different letters denote significant differences (P < 0.05) among the means according to Student’s t-test.
transferred from light to dark conditions (Achard et al., 2007), and their important role in stimulating hypocotyl elongation (Alabadí et al., 2004, 2008). We first tested whether the endogenous GAs are involved in the sucrose-induced hypocotyl elongation in the dark. PAC (a GA biosynthesis inhibitor), widely used in many other studies (Alabadí et al., 2004; de Lucas et al., 2008; Feng et al., 2008), was used in this experiment to attenuate endogenous GA levels. Our results showed that the induction of hypocotyl elongation by sucrose was greatly impaired by 1 M PAC treatment for 2 days in the dark (Fig. 3A). This indicates that endogenous GAs are indeed required for sucrose-induced hypocotyl elongation in the dark. To further confirm this notion, 4-day-old Col-0 seedlings were treated with different concentrations of PAC (0–100 M) with or without 90 mM sucrose for 3 days in the dark. The results clearly showed that both dark-induced and sucrose-induced hypocotyl elongations decreased with increased PAC concentrations (Fig. 3B). Most notably, the hypocotyl length of seedlings grown in the presence of sucrose and 100 M PAC was reduced to a very low level, similar to that of seedlings without sucrose (Fig. 3B), suggesting that 100 M PAC could absolutely abrogate the stimulation of sucrose on hypocotyl elongation.
Fig. 1. Stimulation of sugars on hypocotyl elongation in dark. Four-day-old Col-0 seedlings were transferred to dark or kept in light with or without 90 mM sucrose treatment for 0, 1, 2, and 3 days, and the hypocotyl lengths were measured (B); representative pictures were taken at day 3 of treatment (A). (C) Four-day-old Col0 seedlings were treated with sucrose at concentrations of 0, 15, 30, 60, 90, and 150 mM in dark for 2 days, and the hypocotyl lengths were determined. Mean values and S.E. were calculated from at least 25 seedlings. Different letters denote significant differences (P < 0.05) among the means according to Student’s t-test.
showed a weak repression effect on hypocotyl elongation in the light (Fig. 2). Effect of endogenous GAs on sucrose-induced hypocotyl elongation in the dark As a next step, the possible mechanism by which sucrose promotes hypocotyl elongation in dark was studied. The well-known phytohormones GAs were selected to test their participation in the process, because of their significant accumulation in seedlings
Effect of exogenous GA3 on sucrose-induced hypocotyl elongation in darkness Based on the above results, effects of exogenous bioactive GA (GA3 ) on sucrose-induced hypocotyl elongation in the dark were investigated. Unexpectedly, we found that GA3 had little effect on promoting hypocotyl elongation in the dark regardless of the presence of sucrose. However, GA3 showed a drastic effect on stimulating hypocotyl elongation in the light (Fig. 4A and B). A possible explanation for this observation is that the endogenous GAs in the dark, but not in the light, may be already high enough for hypocotyl elongation. To test this assumption, 4-day-old Col-0 seedlings were treated with 10 M PAC plus different concentrations of exogenous GA3 (10 M to 1 mM) in the dark. Here, the percentage of the recovered hypocotyl length by exogenous GA3 to the inhibited hypocotyl length by PAC was termed the “recovery rate”, which reflects the ability of GA3 in restoring the inhibited hypocotyl length by the indicated concentration of PAC. For example, as shown in Fig. 4C, after transfer of 4-day-old Col-0 seedlings
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Fig. 3. Endogenous GAs are required for sucrose-induced hypocotyl elongation in dark. (A) Four-day-old Col-0 seedlings were treated with 1 M PAC, 90 mM sucrose (Suc), or with 1 M PAC and 90 mM Suc for 2 days in light or in dark, and then pictures were taken and hypocotyl lengths were measured. The seedlings without any chemical treatment were used as control. (B) Four-day-old Col-0 seedlings were treated with PAC at concentrations ranging from 0 to 100 M together with or without 90 mM Suc for 3 days in dark, and then pictures were taken and hypocotyl lengths were measured. Mean values and S.E. were calculated from at least 25 seedlings. Different letters denote significant differences (P < 0.05) among the means according to Student’s t-test.
to the dark for 3 days without any treatment, the hypocotyl length was 0.208 cm; with 10 M PAC treatment, the hypocotyl length was 0.175 cm; with 10 M PAC and 100 M GA3 treatment, the hypocotyl length was 0.193 cm; and the “recovery rate” was (0.193 − 0.175)/(0.208 − 0.175) = 0.52 (52%). With GA3 concentrations from 1 M to 1 mM, the recovery rates increased from 13% to 102% and from 6% to 18%, respectively, in the absence and presence of 90 mM sucrose (Fig. 4C and D). The results showed that the application of GA3 could reverse the repression caused by PAC application, suggesting that exogenous GA3 could substitute for the endogenous GAs in both dark- and sucrose-induced hypocotyl elongations. To further confirm this notion, we transferred 4-dayold Col-0 seedlings to the dark with or without 90 mM sucrose treatment in the presence of 100 M GA3 plus different concentrations of PAC (0–1 M). After 3 days, hypocotyl lengths were determined. The results showed that, in the absence of sucrose, with PAC concentrations decreased from 1 to 0.1 M, the recovery rates increased from 55% to 59%, and more notably, in the presence of sucrose, the recovery rates increased from 38% to 61% (Fig. 4E). All these data indicate that exogenous GA3 could indeed function as endogenous GAs for sucrose-induced hypocotyl elongation in dark.
Regulation of sucrose on transcript levels of several key genes involved in GA metabolism and signaling in darkness It has been reported that sugars regulate plant growth and development generally at the transcript level (León and Sheen, 2003; Gibson, 2005; Rook et al., 2006). We analyzed the effects of sucrose on the expression of several genes encoding GA3ox1, GA20ox1, GA2ox1, GA2ox2, AtGID1a, AtGID1c, SLY1, RGA, and GAI
in dark. As shown in Fig. 5, after 4-day-old Col-0 seedlings were transferred to the dark for 6 to 24 h, the expression of GA3ox1 was significantly up-regulated by sucrose, whereas the expression of AtGID1a was down-regulated by sucrose. For other genes analyzed, sucrose exhibited little effects (Fig. 5). Discussion Sugars, including sucrose, glucose and fructose, slightly repressed Arabidopsis hypocotyl elongation in the light. However, they significantly stimulated hypocotyl elongation in the dark (Fig. 1). Among these sugars, sucrose had the most significant stimulation effect in this process (Fig. 2). Sugars display absolutely opposite effects on hypocotyl elongation in dark and in light, implying that there might be different signaling pathways in regulating hypocotyl elongation in the two processes. From previous research in different fields of plant biology and in global gene expression, it is becoming clear that sugar signaling in light affects numerous metabolic and developmental processes during the entire plant life cycle (Rolland et al., 2002; Gibson, 2005; Rolland and Sheen, 2005). In our study, we found that the sucrose-induced hypocotyl elongation in dark is fully dependent on the presence of endogenous GAs (Fig. 3), implying that GA metabolism or signaling may play an important role during this process. It has long been known that GAs regulate Arabidopsis hypocotyl elongation in both light and dark by influencing the rate and final extent of cellular elongation (Cowling and Harberd, 1999). The hypocotyls of mutants deficient in GA biosynthesis (ga1) or responses (gai) were significantly shorter than that of wild types (Peng and Harberd, 1997). Dark-grown seedlings have longer hypocotyls compared to light-grown seedlings, consistent with
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Fig. 4. Effects of exogenous GA3 on both dark- and sucrose-induced hypocotyl elongation. (A) Four-day-old Col-0 seedlings were treated with 10 M GA3 , 90 mM sucrose, or with 10 M GA3 plus 90 mM sucrose (Suc) for 2 days in light or in dark, and then hypocotyl lengths were determined. Different letters denote significant differences (P < 0.05) among the means according to Student’s t-test. (B) Four-day-old Col-0 seedlings were treated with or without 10 M GA3 in light or in dark for 0, 0.5, 1, 2, and 3 days, and then the hypocotyl elongation were determined. (C) Four-day-old Col-0 seedlings were treated with 10 M PAC together with 0, 1, 10, 100, or 1000 M GA3 in dark for 3 days, and then hypocotyl lengths were determined. The seedlings without any chemical treatment were used as control. (D) Four-day-old Col-0 seedlings were treated with 90 mM sucrose (Suc), or with 90 mM Suc plus 10 M PAC together with 0, 1, 10, 100, or 1000 M GA3 in dark for 3 days, and then hypocotyl lengths were determined. (E) At different concentrations of PAC (0, 0.1, 0.25, 0.5, 0.75, 1 M), 4-day-old seedlings were treated for 3 days with or without 100 M GA3 in dark, in the presence (+Sucrose) or absence (−Sucrose) of 90 mM sucrose, hypocotyl lengths were determined. Mean values and S.E. were calculated from at least 25 seedlings. Numbers above the bars in figures (C)–(E) denote the hypocotyl “recovery rate” by the indicted concentration of GA3 as a percentage.
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Fig. 5. Effects of sucrose on the expression of genes related to GA metabolism and GA signaling. Four-day-old Col-0 seedlings were transferred to dark with (+) or without (−) 90 mM sucrose treatment for 6, 12, and 24 h, and samples were collected at each time point for RNA analyses by RT-PCR. Levels of mRNA expression are shown as the gel images.
the notion that the endogenous GA level is higher in dark-grown seedlings than that in light-grown seedlings (Alabadí et al., 2004; Feng et al., 2008). In this report, we found that when endogenous GAs were inhibited by PAC, the dark-induced hypocotyl elongation was impaired (Fig. 3A). These data clearly indicate that GAs have potential roles in dark-induced hypocotyl elongation. Nonetheless, exogenous GA3 did not exert any effect on dark- or sucrose-induced hypocotyl elongations (Fig. 4A). A possible explanation for this result is that, in the dark, the Arabidopsis seedlings have accumulated high levels of GAs. This explanation appears to be correct, because when the biosynthesis of endogenous GAs was inhibited by PAC, exogenous GA3 exhibited significant effects on enhancing both dark- and sucrose-induced hypocotyl elongations (Fig. 4C–E). Taken together, the results suggest that the presence of GAs is necessary not only for dark-induced, but also for sucrose-induced hypocotyl elongation. It has been reported that GA regulation of hypocotyl elongation in the dark occurs through at least two molecular pathways: (1) negative regulation of positive factors of light signaling such as HY5 by COP1, which determines the degree of repression of photomorphogenesis; and (2) positive regulation of the protein levels of PIF transcription factors, such as PIF3 and PIF4, through COP1 or DELLA proteins, which determine the degree of activation of skotomorphogenesis (Alabadí et al., 2008; de Lucas et al., 2008; Feng et al., 2008). However, in the null mutants cop1-4, cop1-6, pif1-1, pif3-1, and pif4-2, sucrose was found to significantly induce the hypocotyl elongations in the dark, similar to that observed in their corresponding wild types (data not shown). These data suggest that COP1 and PIFs may not be required for sugar-induced hypocotyl elongation, which appears to represent the difference between dark-induced and sugar-induced hypocotyl elongation. Achard et al. (2007) reported that, during the transition from light to dark, there were no detectable changes on transcript levels of genes encoding GA-signaling components, such as RGA, SLY1, and AtGID1a, but that the transcript level of GA2ox1, encoding an enzyme which deactivates bioactive Gas, was reduced. Thus, for the pathway of dark-induced hypocotyl elongation through degradation of DELLAs by GAs, it appears to result from increase of endogenous GA levels, but not from enhancement of GA signaling. It is therefore reasonable to think that GAs contribute to sucrose-induced hypocotyl elongation through the same mechanism. Nevertheless, in this study, we found that, on one hand,
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sucrose stimulated up-regulation of GA3ox1 in the dark (Fig. 5), and on the other hand, sucrose had almost no significant effect on GA2ox1 and GA2ox2 expression. As a result, sucrose could increase the levels of endogenous bioactive GAs. Nevertheless, sucrose down-regulated the expression of AtGID1a (encoding one of the GA receptors) (Fig. 5), which might suggest that sucrose could inhibit DELLA degradation by GAs in the dark. This means that sucrose-induced hypocotyl elongation is unlikely to result from GAmediated DELLA degradation. Therefore, dark-induced hypocotyl elongation and sucrose-induced hypocotyl elongation might be mediated through two different GA-dependent pathways. In fact, the degradation of DELLAs is just one of the mechanisms in GA regulation of plant growth and development (Cao et al., 2006). In addition, GAs can also function in plants directly via positive regulators (Sun and Gubler, 2004). Possibly, sucrose-induced hypocotyl elongation occurs through one or more of these pathways. To clearly elucidate how GAs regulate sucrose-induced hypocotyl elongation in darkness, there is more work to be done. Additionally, GAs are unlikely to function in plants alone. For example, there is evidence to suggest that GA regulation of DELLAs occurs through auxin and ethylene (Achard et al., 2003; Saibo et al., 2003), which suggests that GAs are likely to coordinate with other hormones during plant growth and development. The cross-talk may also play a role during modulation of hypocotyl elongation. In fact, interaction between GAs and brassinosteroids (BRs) in the control of Arabidopsis hypocotyl elongation has been reported (Bouquin et al., 2001; Alabadí et al., 2004), although this interaction was not found in pea (Symons and Reid, 2003; Alabadí et al., 2004). Hence, we cannot exclude the possibility that crosstalks between GAs and other phytohormones exist in Arabidopsis and are required for sugar-induced hypocotyl elongation. In summary, a new function of the sugars (including sucrose, glucose, and fructose) in prompting Arabidopsis hypocotyl elongation in the dark was found in the present study. Further investigation demonstrated that the phytohormone GAs were involved in this process. Acknowledgments We would like to thank Professor Xing-Wang Deng (Yale University, USA) for providing seeds of cop1-4, hy5-215, hyh, and hy5hyh, and Professor Peter H. Quail (University of California, USA) for providing seeds of pif1-1, pif3-1, and pif4-2. This work was supported by Specialized Research Fund for the Doctoral Program of Higher Education of China (ratification number: 20050730017), Foundation of Science and Technology of Gansu Province (3ZS051-A25-018) and the Direction Allocation Grant from the Hong Kong Research Grant Council (DAG05/06.SC09 and DAG04/05.SC08). References Achard P, Liao L, Jiang C, Desnos T, Bartlett J, Fu X, et al. DELLAs contribute to plant photomorphogenesis. Plant Physiol 2007;143:1163–72. Achard P, Vriezen WH, Van Der Straeten D, Harberd NP. Ethylene regulates Arabidopsis development via the modulation of DELLA protein growth repressor function. Plant Cell 2003;15:2816–25. Alabadí D, Gallego-Bartolomé J, Orlando L, García-Cárcel L, Rubio V, Martínez C, et al. Gibberellins modulate light signaling pathways to prevent Arabidopsis seedling de-etiolation in darkness. Plant J 2008;53:324–35. Alabadí D, Gil J, Blázquez MA, García-Martínez JL. Gibberellins repress photomorphogenesis in darkness. Plant Physiol 2004;134:1050–7. Bouquin T, Meier C, Foster R, Nielsen ME, Mundy J. Control of specific gene expression by gibberellin and brassinosteroid. Plant Physiol 2001;127:450–8. Cao D, Cheng H, Wu W, Soo HM, Peng J. Gibberellin mobilizes distinct DELLAdependent transcriptomes to regulate seed germination and floral development in Arabidopsis. Plant Physiol 2006;142:509–25. Chen M, Chory J, Fankhauser C. Light signal transduction in higher plants. Annu Rev Genet 2004;38:87–117. Cowling R, Harberd N. Gibberellins control Arabidopsis hypocotyl growth via regulation of cellular elongation. J Exp Bot 1999;50:1351–7.
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