To gain new insights into the role of GAs, we investigated hybrid aspen plants with increased rates of 2-oxidation of GA in specific parts of the vascular tissue. We demonstrated that changes in the expression patterns of specific GA2ox genes can have a significant effect on the phenotype of the transgenic trees, and that this, to some extent, is the result of differences in their auxin response.
Increased GA catabolism in the vascular tissues reduces primary and secondary growth
We planned to investigate transgenic plants transformed with different combinations of four constructs, consisting of two promoters, LMP1 and LMX5 (which are known to promote expression specifically in different parts of the vascular tissue), paired with the AtGA2ox2 or AtGA2ox8 genes. However, we were unable to obtain viable plants from two of these combinations, and therefore focused on the effects of the two viable combinations. Although the two promoters were both shown to be expressed in the vasculature of young tissues, the levels of expression differed: LMP1 induced high expression in the apex, whereas LMX5 induced a much lower level of expression (Figure 1). It is possible that this variable expression is the reason why the LMP1::AtGA2ox8 and LMX5::AtGA2ox2 constructs did not produce viable plants. LMP1::AtGA2ox8 expression may have significantly decreased GA levels by promoting the degradation of early intermediates in GA biosynthesis; the resulting GA deficiency in their young tissues may have prevented the plants from regenerating. In contrast, the LMX5 promoter induced lower levels of AtGA2ox2 expression in the young tissue, allowing the growth of some plants. However, these plants did not survive further because of their inability to produce roots. The presence of bioactive GAs in the xylem seems to be crucial for adventitious rooting. Although a connection between GA levels and KNOX genes has been established (Bolduc and Hake, 2009), Ikezaki and co-authors have shown that KNOX genes are involved in the regulation of adventitious rooting in Arabidopsis, and that in this species, this role is independent of the GA pathway (Ikezaki et al., 2009).
The plants obtained with the two other constructs, LMP1::AtGA2ox2 and LMX5::AtGA2ox8, exhibited a decreased growth rate relative to the wild-type plants (Figure 2a–d). The dwarfism obtained with GA2ox of different subgroups (AtGA2ox2 uses the C19-GAs, and AtGA2ox8 modifies the C20-GAs; Figure S1) is consistent with that observed in other species. Indeed, overexpression of AtGA2ox8 or AtGA2ox7 in Arabidopsis also causes dwarfism (Schomburg et al., 2003); in hybrid aspen (Populus tremula × Populus alba), dwarf and semi-dwarf trees were obtained by overexpression (by activation tagging) of PtaGA2ox1, a GA2ox from the same subfamily as AtGA2ox2 (Busov et al., 2003).
Hybrid aspens with activation-tagged PtaGA2ox1 have shorter, but equivalent numbers of internodes when compared with their wild-type counterparts (Busov et al., 2003). The LMP1::AtGA2ox2 and LMX5::AtGA2ox8 plants had shorter and fewer internodes (Figure 2e,f). Compared with wild-type trees, hybrid aspens overexpressing genes involved in GA biosynthesis or a GA receptor have similar numbers of internodes, but their internodes are longer than in the wild type (Eriksson et al., 2000; Mauriat and Moritz, 2009). By using specific promoters, one obtains a decreased concentration of GA in the apex (Figure 2g,h) rather than a more widespread effect. This leads not only to a change in elongation but also in cell division, causing slower development. GA is also known to be involved in cell division, for example in the control of root cell proliferation (Achard et al., 2009; Ubeda-Tomas et al., 2009).
The slower primary growth is also followed by an obvious reduction in the extent of secondary growth. The global shape of the stems provides information about the nature of the reduction. As observed in the activation-tagged PtaGA2ox1 hybrid aspen (Busov et al., 2003), the LMP1::AtGA2ox2 and LMX5::AtGA2ox8 plants presented stems that were less tapered than in the wild type (Figure 3a). The young internodes were of the same width as the corresponding wild-type internodes, whereas the older internodes were narrower. The narrow internodes appeared to result from a lower level of xylogenesis in these plants (Figure 3b). Similar observations were made in studies on tobacco overexpressing one AtGA2ox and rapeseed overexpressing AtGA2ox8, where the dwarf plants were shown to have decreased xylogenesis and lignification (Biemelt et al., 2004; Zhao et al., 2010). However, hybrid aspen and tobacco transformed with 35S::AtGA20ox1 was observed to have more extensive xylogenesis (Biemelt et al., 2004; Eriksson et al., 2000). In hybrid aspen, an increase in the production of bioactive GAs appears to be the cause of the increased xylogenesis (Eriksson et al., 2000).
In conclusion, our results indicate that the levels of bioactive GAs are important for controlling xylogenesis in hybrid aspen. Decreases in the levels of GA precursors, or of bioactive GAs in the xylem (Figure 3c,d), reduce secondary growth.
Hybrid aspen defective in GA in early vasculature produces sylleptic branches
In many tropical species, sylleptic branching appears to be correlated with high growth rates in the main shoot. However, in a study of three hybrid poplars with different growth rates and sylleptic branching aptitudes, Cline and Dong (2002) found that the smallest hybrid exhibited the highest degree of sylleptism. In our study, we also observed that the semi-dwarf LMP1::AtGA2ox2 plants exhibited a higher degree of sylleptism than the wild-type hybrid aspen (Figure 4d). Other semi-dwarf plants (LMX5::AtGA2ox8 plants) did not exhibit any branching (Figure 4d). The presence of sylleptic branches corresponds to a loss of apical dominance earlier than might normally be expected (after the first winter). Among the hybrid poplars studied by Cline and Dong (2002), the shortest tree with the highest loss of apical dominance also exhibited the weakest response to exogenous auxin applied to the apex to inhibit bud outgrowth. Similarly, we observed lower levels of endogenous IAA and decreased IAA transport in the semi-dwarf branching trees than in the semi-dwarf non-branching trees (Figure 4e,f).
A number of early studies on apical dominance in non-woody species showed that GA amplifies auxin action (Jacobs and Case, 1965; Scott et al., 1967). For example, in studies involving the application of lanolin containing auxins or a mixture of auxins and GAs to decapitated pea seedlings, auxin was transported further down the stem in the presence of GA (Scott et al., 1967), suggesting that GA enhances auxin transport. Such an increase of auxin transport resulting from the presence of GA has also previously been hypothesized by Bjorklund et al. (2007). In our study, the decreased GA levels in the LMP1::AtGA2ox2 plants could explain the loss of apical dominance. However, the LMX5::AtGA2ox8 plants also exhibited a significant decrease in bioactive GA, but did not lose their apical dominance (Figure 4). The basis of this phenotype may relate to the specific type of GA2ox or the precise localization of the expression of the transgene. The development of lateral buds in the apex (Figure 4b,c) suggests that the altered GA levels affected IAA biosynthesis or transport in this young tissue. The levels of bioactive GA were low in the apices of the two types of transgenic plants (Figure 4e,f). However, even allowing for low GA levels in a specific region of the apex of the LMX5::AtGA2ox8 plants, this does not mean that low levels of GAs exist in all parts of the apex. As the measurements were performed on complete apices, rather than on the different tissues of a dissected apex, the concentration of GA in specific apex tissue layers could not be assessed. It was possible to monitor the patterns of expression from the promoters used in this study: the LMX5 promoter promotes gene expression in mature vasculature, and at a much lower level in young tissue, whereas the LMP1 promoter primarily induces expression in the apex and young tissue (almost at the same level than in the phloem) (Figure 1). Thus, the localization of the transgene expression directed by the promoters had a greater influence on the effects on auxin than the type of GA2ox being expressed.
Precisely localized downregulation of GA levels also affects leaf development
Leaf size was reduced in the LMP1::AtGA2ox2 plants. The LMP1 promoter induces a high level of gene expression in young leaves, but the LMX5 promoter induces only a very low level. Therefore, GA levels would be expected to be lower in the young leaves of the LMP1::AtGA2ox2 plant. Similar findings were obtained in studies on hybrid aspen overexpressing genes involved in GA biosynthesis, which had larger leaves that exhibited higher levels of bioactive GA (Eriksson et al., 2000). A correlation between the level of GA and the size of the leaves has also been shown in Arabidopsis (Ross et al., 1993).
The two types of transgenic hybrid aspen also exhibited different leaf shapes. The leaf shape and its vasculature are defined very early in leaf development, in the leaf primordia, before the leaves attain 10% of their final size (Efroni et al., 2010). Thus, the spatial repartitioning of GA in the leaf primordia is important in the regulation of factors that determine the shape of the leaves (Hay et al., 2002). To our knowledge, there have been no reports of modifications of leaf morphology as a result of changes in the GA content of a whole plant. However, altered leaf shapes were observed in Arabidopsis transformed with the tissue-specific promoter construct MEA::PsGA2ox2 (Singh et al., 2009). Gene expression induced by this promoter is thought to be localized in the shoot apical meristem (SAM) and its general vicinity: it is not homogenously distributed throughout the apex. In our study, the LMP1 promoter induced gene expression in the very young vasculature (probably in the protophloem), resulting in a modified leaf form, whereas expression of a GA2ox gene under the control of the LMX5 promoter (in the xylem) did not affect leaf shape (Figure 5b). Xylem is formed at a later stage in the development of the vasculature than the (proto)phloem, and it is only partially developed in the leaf primordia. In the leaf primordia, when the main vein starts to be established, a localized decrease of bioactive GA (in the LMP1::AtGA2ox2 plants) in the young vasculature influences the future shape and vasculature of the leaf. Interestingly, however, this effect has not been noted in hybrid aspen overexpressing the same gene under the control of a 35S promoter (Busov et al., 2003). It is possible that the hormonal balance between the different parts of the primordia (as in the SAM) controls their development. In the LMX5::AtGA2ox8 plants, the decrease of GA levels occurs at a late stage and is unlikely to have a significant influence on those parts of leaf development that might affect the global form of the leaf vasculature.
An important element in leaf shape and vasculature patterning is the KNOX gene BP. The BP gene is expressed in the SAM but not in the leaves of simple leaf species such as Arabidopsis (Lincoln et al., 1994). However, expression has been noted in the leaves of a compound leaf species (Tomato) (Hareven et al., 1996). It has been shown that overexpression of this gene induces altered leaf shape in many plants, and that this only needs to occur in a specific region at a very early stage of leaf development in order to produce the effect (Kano-Murakami et al., 1993). BP and its orthologs are known to repress GA biosynthesis genes and activate GA 2-oxidation genes (Hay et al., 2002; Kusaba et al., 1998a,b). GAs are also known to regulate BP accumulation. For example in the MEA::PsGA2ox2 plants, a significant increase in expression of the class I-KNOX genes was reported (Singh et al., 2009). We observed an increase in ARK2 (hybrid aspen BP ortholog) expression of about 10-fold in the young leaves of LMP1:AtGA2ox2 compared with wild-type plants with normal leaves. However, a general increase in ARK2 expression in young leaves does not appear to directly influence leaf shape, as the gene is also upregulated in the normal-shaped leaves of LMX5::AtGA2ox8 trees (Figure 5f). It is therefore likely that upregulation of ARK2 only results in a modified leaf shape if it occurs very early in leaf development, as is the case in the LMP1:AtGA2ox2 plants. Increased expression resulting from altered GA levels occurring later on in leaf development, as observed in LMX5::AtGA2ox8 plants, does not seem to have any effect on the simple leaves of hybrid aspen.
Because auxin and its polar transport have also been reported to regulate vasculature patterning and leaf shape (Scarpella et al., 2010), we investigated whether IAA levels or transport were altered in the plants in our study. In the apex samples (containing the leaf primordia) of the LMP1::AtGA2ox2 plants the expression of PttPIN1 was significantly downregulated in comparison with the wild-type plants, and the repartitioning of auxin was found to be altered in the young leaves (Figure 5e,d). In the other transgenic hybrid aspen being tested, no significant modification of PttPIN1 expression or of auxin repartition was observed. The auxin transport therefore seems to be affected by the decreased GA levels in the early vasculature of the leaf primordia. An effect of GA on the PIN expression has also been suggested by Gou et al. (2010), but in a different developmental process: lateral root formation.
In conclusion, our results have improved our understanding of the effects of GA expression in hybrid aspen in two key areas. First, we have provided evidence that both primary and secondary growth are affected by an increase in GA catabolism, using a GA2ox gene encoding a member of one or the other GA2ox subfamily. Secondly, we showed that the precise localization of bioactive GA in the vasculature of the apex is necessary for normal plant development because of its effects on auxin transport.