The plant hormones gibberellin (GA), ethylene and auxin can promote hypocotyl elongation of Arabidopsis seedlings grown in the light on a low nutrient medium (LNM). In this study, we used hypocotyl elongation as a system to investigate interactions between GA and ethylene or auxin and analysed their influence on the development of stomata in the hypocotyl. When applied together, GA and ethylene or auxin exerted a synergistic effect on hypocotyl elongation. Stimulated cell elongation is the main cause of hypocotyl elongation. Furthermore, hypocotyls treated with GA plus either ethylene or auxin show an increased endoreduplication. In addition, a small but significant increase in cell number was observed in the cortical cell files of hypocotyls treated with ethylene and GA together. However, studies with transgenic seedlings expressing CycB1::uidA genes revealed that cell division in the hypocotyl occurs only in the epidermis and mainly to form stomata, a process strictly regulated by hormones. Stomata formation in the hypocotyl is induced by the treatment with either GA or ethylene. The effect of GA could be strongly enhanced by the simultaneous addition of ethylene or auxin to the growth medium. Gibberellin is the main signal inducing stomata formation in the hypocotyl. In addition, this signal regulates hypocotyl elongation and is modulated by ethylene and auxin. The implication of these three hormones in relation to cell division and stomata formation is discussed.
Plant growth is regulated by environmental stimuli such as light, temperature and touch, and by endogenous regulators known as phytohormones. In many cases, these stimuli act interdependently; for instance, gibberellin (GA) biosynthesis (Ait-Ali et al., 1999; Yamaguchi et al., 1998) and responsiveness (Reed et al., 1996) are phytochrome regulated, and ethylene inhibits hypocotyl elongation in the dark, but stimulates it in the light (Neljubow, 1901; Smalle et al., 1997). In addition, nutrient availability can also influence the hormonal effect (Collett et al., 2000). Besides hormonal cross-talk with environmental conditions, various hormones also interact in the regulation of plant growth responses. Auxin is known to enhance ethylene biosynthesis (Abel et al., 1995), and ethylene has been shown to influence auxin transport in roots and hypocotyls (Lehman et al., 1996; Luschnig et al., 1998). Recently, it has been reported that auxin promotes GA biosynthesis in decapitated stems of pea and tobacco (Ross et al., 2002 and references therein).
The Arabidopsis hypocotyl is a simple organ, the growth of which is accomplished by cell elongation. Cell division is limited to stomata formation (Gendreau et al., 1997). Hypocotyl elongation is strongly influenced by different hormones; thus, it is a convenient system to study hormonal interactions controlling plant growth. Gibberellins are known to promote hypocotyl growth (Cowling and Harberd, 1999) and are strictly required for hypocotyl elongation in dark-grown seedlings (Gendreau et al., 1999). When seedlings are grown on low nutrient medium (LNM) in the light, hypocotyl elongation can also be induced by ethylene and auxins (Smalle et al., 1997). However, auxin shows the opposite effect when seedlings are grown on a nutrient-rich medium (Collett et al., 2000). Both GA and ethylene promote hypocotyl growth via cell elongation (Cowling and Harberd, 1999; Smalle et al., 1997) and control endoreduplication levels in the hypocotyl of A. thaliana (Gendreau et al., 1999). Ethylene increases the level of endoreduplication, whereas GA is strictly required for it (Gendreau et al., 1999). Altogether, the data suggest that interactions can occur between these hormone signalling pathways controlling hypocotyl growth in the developing seedling.
Many hormone biosynthesis and signalling mutants are available to study the hormonal control of hypocotyl growth. Short hypocotyls were found in GA deficient1 (ga1) which has reduced levels of GAs due to a mutation in the gene that encodes the ent-kaurene synthetase A, a protein involved in the synthesis of a GA precursor (Sun et al., 1992). Long hypocotyls were found in constitutive triple response1 (ctr1; Kieber et al., 1993), a mutant that shows a constitutive ethylene response. These mutants demonstrate stimulating effects of GA and ethylene on hypocotyl elongation. However, the ethylene-insensitive mutants, ethylene resistant1 (etr1; Chang and Stadler, 2001; Hua and Meyerowitz, 1998) and ethylene insensitive2 (ein2; Alonso et al., 1999), have a hypocotyl length comparable with that of wild type (Hall et al., 1999; Smalle et al., 1997), suggesting that ethylene is not strictly required for hypocotyl elongation. The auxin-resistant mutant axr2 has shorter hypocotyls than wild type. Auxin-mediated cell elongation is induced by AXR2 (Timpte et al., 1992), which is a member of the Aux/IAA protein family (Nagpal et al., 2000) known to regulate auxin-induced gene expression (Gray et al., 2001; Reed, 2001).
Despite the fact that GA, ethylene and auxin have all been implicated in the control of hypocotyl growth, few data are available concerning interactions of these hormones in the process. In Arabidopsis seedlings grown on a rich medium, it has been suggested that ethylene, GAs and auxins each regulate hypocotyl elongation independently (Collett et al., 2000). Here, the interactions between GAs and ethylene or auxins are studied in the hypocotyl of seedlings grown on LNM in the light. In these conditions, all three hormones stimulate hypocotyl growth. Treatments with combinations of GA and either the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) or auxin strongly affected hypocotyl elongation. We analysed their influence on the kinetics of hypocotyl elongation, their role in cell division and their effect on endoreduplication. In addition, we demonstrated that hypocotyls treated with a combination of hormones formed an increased number of stomata, the development of which is strictly GA dependent. A model for the hormonal interactions governing development of stomata in the hypocotyl is presented.
Hypocotyl growth kinetics of seedlings grown on different hormone combinations
Ethylene can stimulate hypocotyl elongation in the light when seedlings are grown on LNM. This response is also induced when ACC, the precursor of ethylene, is applied to the growth medium with a maximal response at 50 µm ACC (Smalle et al., 1997). Seedlings grown on LNM, supplemented either with 6 µm indole-3-acetic acid (IAA) or 10 µm gibberellic acid (GA3), also exhibit longer hypocotyls than those grown on plain LNM (Figure 1a,b). To investigate the kinetics of these hormonal effects, the hypocotyl length of seedlings treated with ACC, IAA, GA3, ACC + GA3 and IAA + GA3 was followed for 9 days after imbibition. On LNM, most (approximately 80%) of the hypocotyl elongation occurred during the first 3 days after imbibition (Figure 1a). As a result, in the presence of ACC this period of rapid growth continued for an additional day with, on average, an approximately 60% increase in final hypocotyl length. In contrast, GA3 did not affect the duration of rapid growth but increased the growth rate between days 2 and 3, resulting in an approximately 55% increase in hypocotyl length at day 9. When both ACC and GA3 were added to the growth medium, their combined effect on hypocotyl elongation was at least additive, in most cases even synergistic, resulting in a final increase of 150% in the hypocotyl length. The combination of ACC and GA3 enhanced the growth rate as observed with GA3 alone, and extended the period of rapid growth as observed in seedlings grown on LNM with ACC alone.
Indole-3-acetic acid inhibited hypocotyl elongation during the first 2 days after imbibition, but between days 2 and 3 the growth rate was comparable to that of LNM-grown seedlings. Thereafter, the presence of IAA extended the period of rapid growth at least until day 6, resulting in 25% longer hypocotyls (Figure 1b). The combination of GA3 and IAA also revealed at least an additive effect on hypocotyl length with an inhibition of growth during the first 2 days followed by a stimulation during the next 4 days as seen with auxin alone, and a general increase in growth rate as observed upon application of GA3 (Figure 1a).
Hypocotyl elongation is thought to result from longitudinal cell expansion (Gendreau et al., 1997). We investigated cortical cell length in response to hormonal treatments. Indeed, GA3 alone increased cortical cell length, as did ACC and IAA (Figure 1c). The effect of GA3 on cell elongation was enhanced by either ACC or IAA. In addition to cell elongation, ACC also induced radial expansion of the hypocotyl. When seedlings were grown on LNM supplemented with ACC or ACC + GA3, hypocotyls were 20% thicker than those grown on LNM or LNM + GA3 or IAA (Figure 1c). The presence of 0.5 µm paclobutrazol (PAC), a GA biosynthesis inhibitor, did not alter the thickness of hypocotyls of seedlings grown either on LNM or LNM supplemented with ACC (data not shown). Therefore, besides being ethylene specific, this effect was GA independent. In addition, hypocotyls grown in the presence of IAA were often more curled than those grown with ACC or GA3.
Figure 2 shows that 9-day-old seedlings grown in the presence of the 0.5 µm PAC had hypocotyls significantly shorter than those of non-treated seedlings. The reduction of hypocotyl growth on PAC-containing medium was almost completely recovered by the addition of GA3, supporting a specific effect of PAC in inhibiting GA biosynthesis. Treatment with PAC slightly reduced the effect of ACC on hypocotyl elongation but completely abolished the promotive effect of auxin. These data suggest that GA plays a major role in hypocotyl growth on LNM.
Gibberellins, ethylene and auxin modulate endoreduplication levels in the hypocotyl
Mature cell size is often correlated with endoreduplication (Galbraith et al., 1991; Melaragno et al., 1993). To investigate the relationship between hormone-stimulated cell elongation and endoreduplication, we measured the nuclear DNA content of hypocotyl cells extracted from seedlings treated with different hormones and hormonal combinations.
Figure 3(a) shows that hypocotyl cells from seedlings grown on LNM contained nuclei with three ploidy levels: 2C, 4C and 8C (C = haploid DNA content). The 2C and 8C nuclei were approximately equally represented, whereas 4C was the most abundant (51%). A small fraction of cells in the hypocotyls grown on LNM supplemented with ACC underwent an additional round of endoreduplication (2% of 16C nuclei) and an increase in the ratio 8C:4C was observed. Likewise, treatment with GA3 caused an increase of the ratio 8C:4C; however, no 16C nuclei were detected. The combination of ACC and GA3 caused the strongest effect on endoreduplication reflected by an increase in the ratio 8C:4C (8C:4C = 1.4 for LNM + ACC + GA3 as compared to 0.5 for LNM) and one extra round of DNA synthesis (16C) in a small fraction (4%) of the nuclei. The percentage of 16C nuclei with this hormonal combination was, in three independent experiments, always higher than that observed for seedlings treated with ACC only. Hypocotyls treated with IAA had a ploidy profile indistinguishable from that obtained with GA3 treatment (Figure 3a, compare LNM + GA3 and LNM + IAA). Indole-3-acetic acid and GA3 together had a clear effect on endoreduplication, the ratio 8C:4C was increased and some hypocotyl cells showed an extra round of endoreduplication (2% of 16C nuclei), which was not observed when each hormone was applied separately.
Flow cytometry enabled us to measure the ploidy levels in whole hypocotyls but did not provide information concerning cell or cell file-specific localization of the nuclei with enhanced ploidy levels. The hypocotyl epidermis contains two different cell types: those in non-protruding cell files from which stomata can originate and those in the protruding cell files that do not develop stomata (Figure 5f). In order to investigate where in the hypocotyl epidermis endoreduplication takes place, we used confocal microscopy to visualize the nuclei of epidermal cells in propidium iodide-stained hypocotyls (Figure 3b). Taking the small nuclei of guard cells (indicated by an arrow on Figure 3(b), LNM + ACC) as a reference, which is assumed to have a 2C ploidy level (Melaragno et al., 1993), we observed that most cells underwent endoreduplication independent of their localization, as reflected by their larger nuclei. In ACC-treated seedlings, some nuclei in the hypocotyl epidermis were bigger than those observed in non-treated samples. In addition, these nuclei appeared elongated. To investigate whether protruding and non-protruding epidermal cell files had different nuclear DNA content, we used image analysis to measure the relative amount of DNA in individual cells (Figure 3c). Low nutrient medium- and LNM + ACC-grown seedlings showed ploidy levels in the hypocotyl epidermis (protruding and non-protruding cells together) consistent with those found for the whole hypocotyl (Figure 3a). The shift caused by ACC treatment is similar in whole hypocotyl samples and in epidermis only, indicating that the ACC effect also occurs in inner layers. In addition, the effect of ACC in inducing endoreduplication was fairly similar in both epidermal cell types. Interestingly, both LNM- and LNM + ACC-grown seedlings revealed a differential regulation of endoreduplication in the epidermis (compare on Figure 3c, non-protruding versus protruding cells). Protruding cell files showed higher DNA levels than the non-protruding ones. The smallest nuclei (2C) were hardly observed in protruding cell files, whereas the biggest nuclei (8C on LNM and 16C on LNM + ACC) were mostly limited to these cells.
Cortical cell number is enhanced by ethylene and gibberellins in the hypocotyl
Figure 1(c) shows that the increase in hypocotyl length could be attributed primarily to cell elongation. However, it could not be ruled out that cell division also took place under the influence of the different hormones. Therefore, the number of cortical cells in the hypocotyls was determined. For seedlings grown in the presence of IAA or IAA and GA3 together, the cortical cell number could not be counted reliably because these treatments caused twisting and curling of the basal part of the hypocotyl. The number of cells in two single cortical cell files per hypocotyl was counted, starting from the first cell above root hair cells until the bifurcation of the vascular bundle (Figure 4a). Control hypocotyls contained on average 23.5 cortical cells per file. 1-aminocyclopropane-1-carboxylic acid and GA3 treatments individually increased this number by 1.4 and 1.3 (i.e. ≈6%), respectively, and the combination of ACC + GA3 showed an additive effect resulting in 2.5 (i.e. ≈11%) additional cells (Figure 4b). To investigate whether lower levels of GA could affect cortical cell number in the hypocotyl, we treated seedlings with 0.5 µm PAC. Although a slight decrease was observed (0.9 cells, i.e. ≈4%), this effect was not significant (Student's t-test: P = 0.08, when LNM + PAC is compared with LNM) (Figure 4b).
To analyse where and when these cell divisions took place in the development, the expression of the mitotic cyclin genes, CycB1;1 and CycB1;2, was used as a marker for cell division. Both genes are excellent markers for mitosis (Donnelly et al., 1999; Ferreira et al., 1994). Therefore, two different transgenic lines were used in this study. One line produced the standard β-glucuronidase (GUS) protein under the control of the CycB1;1 promoter (Ferreira et al., 1994). The maximum activity occurs during mitosis (Ferreira et al., 1994) and is thereafter gradually decreased over a period of at least 2 days (data not shown). The second line produces a fusion protein containing the first 150 amino acids of CycB1;2, which includes the cyclin destruction box (CDB), fused with the GUS protein under the control of the CycB1;2 promoter (Donnelly et al., 1999). The CDB mediates a rapid breakdown comparable to that of the native protein, which is degraded at the end of the mitosis. Seeds from both transformants were grown on LNM and LNM supplemented with ACC, GA3, ACC + GA3, IAA and IAA + GA3, and hypocotyls of seedlings aged between 0 and 9 days were analysed for GUS activity. The seed coat of 0- and 1-day-old seedlings was removed immediately before the GUS assay. Cell division events in the cortical layers were, however, rarely noticed (approximately one cortical cell division for every 10 seedlings).
Epidermal cell divisions are induced by hormonal treatment during hypocotyl growth
In contrast to the absence of cell divisions in the cortical layers, GUS activity was often observed in the epidermal layer of the hypocotyl and more so in seedlings treated with a combination of GA3 and either ACC or IAA (Figure 5a,b). It has been suggested that cell divisions occurring in the epidermal layers of the Arabidopsis hypocotyl contribute to the development of stomata (Gendreau et al., 1997). Our observations support this suggestion because most of the observed dividing cells in the epidermal layer of the hypocotyl resulted in the formation of new stomata. This was evidenced by 3- and 4-day-old seedlings, producing the stable GUS protein (Figure 5c,d). In these seedlings, most of the stained cells that underwent mitosis some time before could be identified as developing (Figure 5c) or fully developed guard cells (Figure 5d). The activity of the CDB-GUS fusion protein was used to follow the cell division kinetics in the hypocotyls. Cell divisions occurring in the hypocotyl epidermis of seedlings treated with different hormones started between days 1 and 2 after imbibition, and the maximum number of stained cells was observed on day 2 or 3 depending on the hormone(s) applied (Figure 5e). Treatment with either ACC or GA3 slightly increased the number of dividing epidermal cells, whereas IAA treatment did not significantly affect the number of GUS-stained cells. Interestingly, 2-day-old seedlings expressing CDB-GUS, grown in the presence of 0.5 µm PAC, did not show dividing cells in the hypocotyl epidermis in any of the following treatments: LNM, LNM + ACC and LNM + IAA (data not shown). The PAC effect was recovered by addition of GA3, indicating that GAs are required for cell division leading to stomata formation. The strongest stimulus for epidermal cell division was found in seedlings treated with the combinations ACC + GA3 or IAA + GA3, respectively (Figure 5e). Under these treatments, the number of cells producing the CDB-GUS protein remained high until day 3 or 4, respectively.
Stomata formation in the hypocotyl is strongly induced by gibberellins combined with either ethylene or auxin
As epidermal cell divisions in the hypocotyl occur mainly to form stomata and because hormones seem to play a role in that process, we decided to analyse the hormonal influence on stomata development. CDB-GUS activity kinetics (Figure 5e) revealed that cell division in the hypocotyl epidermis stopped at day 6 after imbibition. In addition, a maximal density of stomata per cell file is reached at day 9 (Berger et al., 1998). Therefore, 9-day-old seedlings were used for counting stomata, which are mainly formed on the upper part of the hypocotyl, close to the cotyledons. In our experiments, the number of stomata in the cotyledons did not change upon exogenous application of hormones (data not shown). The number of stomata in the hypocotyl was increased by 33 and 21% by ACC and GA3, respectively (Table 1), and the combination of both hormones revealed an additive effect resulting in a 55% increase. The ctr1-1 mutant (Kieber et al., 1993) also showed an increased number of stomata when grown on LNM or LNM + GA3, comparable with that of wild type grown on LNM + ACC or on LNM + ACC and GA3 together, respectively. This supports the assumption that the stimulatory ACC effect on wild type was indeed an ethylene effect. Although IAA had no significant effect on stomata number, the IAA + GA3 combination resulted in a 65% increase, much higher than upon treatment with GA3 alone (21%). In addition, IAA + GA3 also induced stomata formation in the middle part of the hypocotyl. Moreover, all treatments involving GA3 yielded an increased incidence of stomata developed from the same epidermal cell (Figure 5f). In order to investigate whether the increased number of stomata resulted from ectopic formation in the protruding cell files, we used scanning electron microscopy to analyse the hypocotyls of seedlings grown on media supplemented with the different hormones and their combinations. Ectopic stomata were seldom found either in treated or non-treated seedlings (approximately 1–2% of the total number of stomata). This result is consistent with previous observations (Berger et al., 1998).
Table 1. Number of stomata in the hypocotyl of 9-day-old seedlings of wild-type and hormone mutants treated with different hormones
Col-0 (0.5 µm PAC)
Data represent mean ± SD (n≥20).
N.D. = not determined.
Student's t-test: *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001 versus low nutrient medium (LNM)-grown seedlings; a0.01 < P < 0.05; bP < 0.001 versus Col-0 on LNM.
The hypocotyl epidermis of 9-day-old wild-type seedlings grown on LNM + ACC showed a few cells that had divided but had not differentiated into stomata in non-protruding cell files (Figure 5h), an effect that was found to be enhanced by GA3 (data not shown). The same observation was made for 9-day-old ctr1-1 seedlings (Figure 5i).
Gibberellins are specifically required for stomata formation in the hypocotyl
The observation that hormones play an important role in stomata formation in the hypocotyl led us to analyse the effect of impaired hormone signals in this process. Table 1 shows that 0.5 µm PAC reduced the number of stomata to 20% of the value observed in untreated seedlings. Higher concentrations of PAC completely inhibited stomata development (data not shown). This inhibition could be counteracted by addition of GA3, indicating a PAC-specific effect on GA biosynthesis. Surprisingly, GA requirement appears to be specific for stomata formation in the hypocotyl, as in the cotyledons the number of stomata was not significantly altered even upon treatment with 5 µm PAC (data not shown). These results were confirmed by analysis of the GA-deficient mutant, ga1-3 (Sun et al., 1992), which had only stomata in the cotyledons. The formation of stomata in the hypocotyl of ga1-3 seedlings was recovered by treatment with exogenous GA3 (data not shown). The number of stomata in hypocotyls of wild-type seedlings treated with PAC was higher in the presence of ACC; however, their normal frequency was only reached in the presence of exogenously applied GA3 (Table 1). This result strengthens the obligatory need of GAs for stomata formation and suggests that both hormone signalling pathways might interact in this process. The ethylene-resistant mutant, etr1-1 (Bleecker et al., 1988), showed a number of stomata in the hypocotyl comparable with wild type, either in untreated or in GA3-treated seedlings (Table 1), indicating that ethylene is not required for either normal or GA-induced stomata formation. On the other hand, etr1-1 did not show the combined effect of IAA + GA3. The ethylene-insensitive mutant ein2-1 (Alonso et al., 1999) showed effects comparable with etr1-1 in all treatments (data not shown). The auxin-resistant mutant, axr2-1 (Wilson et al., 1990) appeared to be more responsive to ACC and ACC + GA3 than wild type, concerning stomata formation. The IAA treatment, however, had a negative effect on stomata formation and the effect of IAA + GA3 was completely abolished in axr2-1.
Gibberellin, ethylene and auxin can promote hypocotyl elongation of Arabidopsis when seedlings are grown in the light (Cowling and Harberd, 1999; Smalle et al., 1997). Here, we describe the combined effect of these hormones on hypocotyl elongation, cell expansion, endoreduplication, cell division and stomata formation in the hypocotyl of seedlings grown on LNM in the light. Gibberellin seems to play a major role controlling elongation and stomata formation in the hypocotyl of Arabidopsis, and its signal can be enhanced by ethylene and auxins.
On LNM, GA3, ACC and IAA each promote hypocotyl elongation and their effects seem mutually dependent. The analysis of the growth kinetics of hypocotyls indicated that GA enhances the hypocotyl growth rate while ACC extends the duration of the rapid growth phase (Figure 1a). The later induction of growth by ACC implicates that GA3 probably does not induce growth via an ethylene signal. Conversely, ethylene might exert its effect on hypocotyl elongation via the GA signal. PAC reduces the ethylene effect in promoting hypocotyl elongation and completely abolishes the auxin effect (Figure 2), suggesting that the main signal stimulating hypocotyl growth is transduced via the GA signalling pathway. Moreover, combinations of GA with either ACC or IAA induce stronger effects than the sum of the effects of each hormone individually. Interactions between hormones can take place on the level of biosynthesis, as has been described for ethylene and auxin (Abel et al., 1995), but a synergistic effect cannot solely be explained by this mechanism. The way through which ethylene and auxin influence the GA signalling pathway resulting in this synergy remains to be elucidated.
Gibberellin and ethylene are known to induce hypocotyl elongation as a result of cell elongation (Cowling and Harberd, 1999; Smalle et al., 1997) and the combined action of other hormones on cell elongation has been observed previously (Ephritikhine et al., 1999). Auxin is known to inhibit cell elongation in hypocotyls (Collett et al., 2000); however, it stimulates cell length on LNM-grown seedlings indicating a nutrient-dependent action of this hormone. Auxin seems to influence hypocotyl length independent of ethylene, as the growth kinetics of hypocotyls treated with IAA differs strongly from that in the presence of ACC. In addition, ACC causes thickening of the hypocotyl independent of GA, an effect that was not observed in IAA-treated seedlings.
Endoreduplication plays a role in hormone-induced cell elongation
Endoreduplication constitutes an effective strategy of cell growth. The potential cell size for a given cell type is generally correlated with the amount of nuclear DNA (Edgar and Orr-Weaver, 2001; Gendreau et al., 1998), although exceptions have been observed (De Veylder et al., 2002). In Arabidopsis, trichomes, leaf epidermal cells, root tips and hypocotyl cells reveal polyploidy (Galbraith et al., 1991; Melaragno et al., 1993). Previous studies indicated that GA is required for normal endoreduplication (Gendreau et al., 1999). In this study, it was demonstrated that the highest levels of endoreduplication were observed upon treatment with those hormone combinations that also result in the strongest effect on hypocotyl and cell elongation. As stated above, higher DNA levels might contribute to the ability of the hypocotyl to elongate. Nevertheless, the contribution of endoreduplication to hormone-induced cell elongation is difficult to estimate because endogenous hormones already induce endoreduplication. Gendreau et al. (1998) showed that endoreduplication in the hypocotyl is regulated in a tissue-specific way. Epidermal and cortical cells have higher DNA levels than cells from the central cylinder and the endodermis. This study illustrates that endoreduplication is differentially regulated within the hypocotyl epidermis. Higher DNA levels were found in the protruding cell files compared to the non-protruding cell files where the stomata are located. This is an interesting parallel to the situation in leaves where there appears to be one population of cells that remains 2C and serves as a pool for future guard cells and a second population that endoreduplicates and can never become guard cells (Melaragno et al., 1993).
Can the hypocotyl cell number increase without cell division?
The Arabidopsis hypocotyl is known as an organ of embryonic origin (Scheres et al., 1994) and cell division is absent or insignificant during hypocotyl growth after germination (Gendreau et al., 1997; Raz and Koornneef, 2001). We have found that ethylene and GA together promote an 11% increase of the total number of cortical cells. However, no significant cortical cell divisions take place in the hypocotyl after seed imbibition. The paradox of additional cortical cells in hypocotyls of seedlings treated with GA and ethylene together without cell division taking place might be explained by a conversion in cell fate. Specification of cell lineage for root or hypocotyl fate is not defined at the early stages of embryogenesis (Scheres et al., 1994). Thus, upon hormonal treatment, cells from root/hypocotyl or hypocotyl/cotyledon junction might become hypocotyl cells during germination.
Gibberellins are strictly required for cell division and stomata formation in the hypocotyl
Although cell division was not induced by GA3 or ACC in the hypocotyl cortex, it was promoted in epidermal cells to form stomata. Little is known about the role of hormones in stomata formation. Induction of stomata differentiation by ethylene has been reported in Arabidopsis leaves (Serna and Fenoll, 1996). Stomata formation in the hypocotyl has been described as a process that normally requires three cell divisions preceding guard cell formation (Berger et al., 1998). Our results show that cell division in the hypocotyl epidermis is regulated by at least three hormones and is correlated with stomata differentiation. Cyclin B promoter activity revealed that both ethylene and GA induce epidermal cell division, particularly in the upper part of the hypocotyl. Interestingly, IAA only induced cell division when applied together with GA3, implicating a synergistic effect of these two hormones. Two-day-old seedlings with a high number of dividing cells also exhibit a high number of guard cells differentiating after day 3 and have a higher number of stomata at day 9, supporting the idea that at least part of these cell divisions took place to form stomata. However, combined hormonal treatments consistently induced more cell divisions than the final number of stomata formed. Hypocotyls of 9-day-old wild-type seedlings treated with ACC, as well as those of ctr1-1, have cells that divided but did not differentiate into guard cells (Figure 5h,i), in addition to a higher number of stomata (Table 1). This observation revealed not only that ethylene can induce cell division but also that not all induced divisions lead to stomata formation. Ethylene is not essential for a normal stomata formation as the ethylene-insensitive mutant etr1-1 has a number of stomata very similar to that in wild type (Table 1). Gibberellin stimulated stomata formation in the absence of ethylene signalling, as demonstrated in etr1-1. However, the strong effect exerted by auxin + GA on stomata formation was not observed in etr1-1, indicating that an intact ethylene signalling pathway is required for the synergistic effect observed in wild type (Table 1). These results imply that the auxin signal acts through ethylene, possibly by enhancing ethylene biosynthesis. This is confirmed by the strong response of the axr2-1 to the treatments including ACC (Table 1). The results obtained with axr2-1 carrying a gain-of-function mutation in the AXR2/IAA7 gene, which encodes a repressor of the auxin response (Tiwari et al., 2001), suggested that auxin can modulate stomata formation through AXR2 and other Aux/IAA proteins. The negative effect exerted by auxin on stomata formation in axr2-1 was also observed for hypocotyl elongation (NS, unpublished data) and is probably due to an impaired balance of inhibitory and stimulatory Aux/IAA proteins, whose activity is regulated by auxin (for a review, see Reed, 2001). In the axr2-1 mutant, auxin does not induce AXR2 degradation but it might induce degradation of stimulatory Aux/IAA proteins, causing a negative effect.
Gibberellin is strictly required for stomata development, as revealed by PAC-treated seedlings. In addition, stomata formation is restricted to non-protruding cell files in the upper part of the hypocotyl. Thus, GA might alter cell fate, but only in competent, non-protruding cell files as compared to Transparent Testa Glabra (TTG) and Glabra2 (GL2), which act on protruding cell files (Berger et al., 1998; Hung et al., 1998). We propose a model where GA is the main hormone in charge of stomata differentiation in the hypocotyl whereas ethylene can strengthen this signal. Likewise, auxin might play a role in stomata formation mainly by enhancing the GA effect as ethylene does. Figure 6 presents a model for the possible hormone interactions involved in the control of stomata formation in hypocotyls. The fact that PAC-treated seedlings showed a normal number of stomata in the cotyledons suggests the existence of different pathways leading to stomata formation in hypocotyl and cotyledons. This is in agreement with earlier observations that TTG and GL2 prevent stomata differentiation in protruding hypocotyl cells (Berger et al., 1998; Hung et al., 1998), but do not seem to play a role in cotyledon and leaf stomatal development (Berger et al., 1998). The organ-specific control of stomata development is also demonstrated by the too many mouths (tmm) mutant, which shows an increased precursor cell formation in cotyledons and an absence of stomata in the inflorescence stem (Yang et al., 1995).
The model in Figure 6 also represents the major hormonal interactions occurring in the hypocotyl during post-embryonic development and will serve as a basis for further research. Although GA remains the major signal, an inverse relationship between ethylene and auxin appears to govern hypocotyl elongation on LNM, with a stimulatory effect of ethylene on the auxin signal (Vandenbussche et al., 2003). Hormonal interactions are only part of the complex framework regulating plant growth. The often opposite effects of the same hormone depending on external factors show that phytohormones strictly regulate plant morphogenesis in order to reach the phenotype that is best fit to the environment and consequently improve the capacity to adapt and survive under changing environmental conditions.
Plant material and growth conditions
Arabidopsis thaliana (L) Heynh. (ecotype Columbia) seeds were purchased from Lehle Seeds (Round Rock, TX). The Arabidopsis mutants etr1-1, ein2-1, ctr1-1 and axr2-1, all in Columbia, and ga1-3 in Landsberg background were obtained from the Arabidopsis Biological Resource Center at Ohio State University. Both transgenic lines containing the GUS reporter are in Columbia background (Donnelly et al., 1999; Ferreira et al., 1994). Gibberellic acid, IAA and PAC were purchased from Sigma–Aldrich (St Louis, MO), and ACC was obtained from ICN Biomedicals Inc. (Aurora, OH, USA). Arabidopsis seeds were surface-sterilized by incubation for 15 min in 5% (v/v) sodium hypochlorite + 0.1% (v/v) Tween 20 and subsequently washed with sterile, distilled water. Sterile seeds were sown on LNM (Smalle et al., 1997) supplemented with hormones (50 µm ACC, 10 µm GA3 and 6 µm IAA) and allowed to imbibe at 4°C for 2 days before incubation in a growth chamber at 22°C and 60% relative humidity under white fluorescent light (photosynthetic photon flux density (PPFD): 75 µmol m−2 sec−1) and long-day conditions (16-h light/8-h dark).
Hypocotyl measurements, cell and stomata counting
Seedlings for hypocotyl length measurements were grown on vertical plates for 9 days after imbibition. The increase of hypocotyl length was followed for individual seedlings by taking pictures daily at a given time using a colour video camera (JVC, TK-1280E) mounted on a binocular (Stemi SV 11, Carl Zeiss, Germany). Images were analysed using the public domain image analysis software imagej 1.20 (http://rsb.info.nih.gov/ij/). Hypocotyl length was determined by measuring the distance from the most basal root hairs until the ‘V’ made by the cotyledon shoulders (Figure 4). For cell and stomata counting, 9-day-old seedlings were cleared overnight in chlorallactophenol (CLP; Beeckman and Engler, 1994) at room temperature, followed by 24–48 h incubation in 100% lactic acid at room temperature. Seedlings were then mounted in lactic acid and examined by differential interference contrast (DIC) microscopy, with a LEICA DM LB microscope (20×/0.5 HC PL Fluotar and 40×/0.75 HCS PL Fluotar). Two cortical cell files were counted per seedling (n ≥ 20), starting from the first cell above root hairs to the cell that reached the level of the hypocotyl vascular bundle separation. Stomata were counted by means of optical sectioning throughout the hypocotyl.
Flow cytometry analysis
Flow cytometry was carried out on 9-day-old hypocotyls after removal of apex, cotyledons and roots. Hypocotyls of 9-day-old seedlings (30–40 per sample) were chopped with a razor blade in 500 µl of extraction buffer at pH 7.0, containing 45 mm magnesium chloride, 30 mm sodium citrate, 20 mm 4-morpholinopropane sulfonate and 0.1% (v/w) Triton X-100 (Galbraith et al., 1983). The suspension was filtered through a nylon filter (30 µm) and after 2.5 µg ml−1 of 4′, 6-diamidino-2-phenylindole (DAPI; Sigma–Aldrich) had been added, 1500 nuclei per sample were analysed on the BRYTE HS flow cytometer and winbryte™ software (Bio-Rad, Hercules, CA, USA) with laser excitation. Each result represents at least three independent experiments.
DNA staining and nuclei measurements
DNA staining in intact nuclei was performed in 9-day-old seedlings fixed overnight in a solution containing 4% paraformaldehyde (freshly prepared) and 0.5% Triton X-100 in buffer (0.02 m Pipes + 1 mm CaCl2; pH 7.2. Subsequently, seedlings were rinsed in the same buffer and incubated for 1 h at 37°C in 10 mm Tris–HCl (pH 7.4) with 1 µg ml−1 RNAse. After rinsing with incubation buffer, seedlings were incubated overnight in 6.5 mm Tris–HCl (pH 7.4) + 1 mm EDTA containing 2.5 µg ml−1 propidium iodide. Seedlings were washed and mounted in 10 mm Tris–HCl (pH 7.4). The analysis of propidium iodide-stained nuclei was done with an inverted confocal microscope LSM510 (Zeiss) fitted with a ×20 plan-apochromat objective. Optical sections were projected to generate Z-stacks (6 µm thick for seedlings grown on LNM and 8 µm for those grown on LNM + ACC), using the lsm510 software. The corresponding pictures were used to measure the integrated fluorescence of individual nuclei, using the image analysis software (imagej 1.20) mentioned above. Integrated fluorescence was calculated for each nucleus by multiplying the mean pixel intensity (background subtracted) by the number of pixels corresponding to the area. The logarithmic values of the integrated fluorescence were analysed. cDNA content was calculated based on the mean integrated fluorescence of 10 guard cell nuclei, which are 2C (Melaragno et al., 1993).
Histochemical GUS staining
For histochemical analysis of GUS expression, 0–9-day-old seedlings were submerged in 90% acetone for 30 min at 4°C, washed twice with 1 m phosphate buffer (pH 7.0) for 15 min at room temperature and then incubated at 37°C for at least 24 h in 0.1 m phosphate buffer (pH 7.0) containing 0.5 mm Fe(CN)2, 0.5 mm Fe(CN)3 and 2 mm 5-bromo-4-chloro-3-indolyl-beta-d-glucuronide (X-gluc; ImmunoSource, Antwerp, Belgium). Thereafter, seedlings were incubated overnight in 70% ethanol at 4°C and subsequently cleared overnight in CLP (Beeckman and Engler, 1994) at room temperature. After CLP treatment, seedlings were incubated overnight and subsequently mounted in lactic acid. Analysis of the seedlings was carried out using DIC microscopy (LEICA DM LB microscope).
Scanning electron microscopy analysis
Light-grown 9-day-old seedlings were fixed overnight in 4% paraformaldehyde and 1% glutaraldehyde in buffer (0.1 m phosphate buffer, pH 7.2), followed by a post-fixation in 2% osmium tetroxide. After dehydration in a graded ethanol series, seedlings were dried to critical point in carbon dioxide, sputter-coated with gold and subsequently analysed with a scanning electron microscope (JSM-840; JEOL, Ltd., Tokyo).
We thank Tom Beeckman for advice with microscopy techniques and helpful discussions, Rita Van Driessche and Mira Haegman for technical assistance with SEM and plant material, respectively, Dirk Inzé (Gent University-VIB) and John Celenza (Boston University) for providing the CycB1;1::GUS and CycB1;2::CDB-GUS lines, respectively and Martine De Cock for help in preparing the layout of the manuscript. This work was supported by a grant from the European Union (RTN1 – 1999-00086). N.S. is indebted for PhD fellowships to the Fundação para a Ciência e Tecnologia (PRAXIS BD/13760/97) and to Ghent University (BOF 2001-144).