The fact that the biosynthesis of active GAs (see Glossary) is a complex, multistepped process with diverse intermediates (Figure 1) makes it difficult to pinpoint the exact tissue or organ in which GAs are synthesized and localize to. Studies focusing on the spatial organization of the GA biosynthesis pathway, characterizing the expression patterns of different GA biosynthetic enzymes using GUS as a reporter, have led to several insights. First, GA biosynthesis genes are differentially expressed among different tissues, cell types, and developmental stages . Second, several members of the GA3ox family, which catalyze the final step in the synthesis of bioactive GAs, are expressed in growing and elongating shoot and root organs . Third, although there are several examples of tissues in which the expression of GA biosynthesis genes co-localizes with GA perception genes (e.g., in inflorescence meristem and developing leaves), there are also examples where these two groups do not overlap (e.g., GA-biosynthesis genes are not expressed in the aleurone cells of the endosperm but GA signaling genes are) . Such spatial separation between genes involved in GA biosynthesis and perception suggests the requirement for GA movement. Finally, levels of expression of genes constituting the GA biosynthetic pathway itself do not always coincide . For example, the expression of the late stage GA biosynthesis genes AtGA3ox1 and AtGA3ox2 in germinating embryos is spatially different from that of the early GA biosynthesis gene AtCPS. This and other examples suggest that the location of GA precursors could play an important role in regulating GA responses.
Figure 1. Gibberellins are Mobile Signaling Molecules in Plants. Illustration of a schematic plant (left) and gibberellin (GA) biosynthesis pathway (right). Arrows indicate documented long-distance movement of mobile GAs. The arrows are color-coded to correlate with GA forms shown in the biosynthetic pathway. Root-to-shoot and shoot-to-root movement of GA12 in Arabidopsis and GA20 in Pisum sativum (blue) . GA9 movement from the ovaries to the sepals and petals was shown in Cucumis sativus flowers (red) . Movement of GA from leaves to stem was demonstrated in tobacco and Arabidopsis and from stamens to petals in Arabidopsis and Petunia (black); in these cases, the exact form of mobile GA is not clear.
A recent study, combining mathematical and experimental approaches, compared the putative GA response, represented by the expression pattern of the SCR3 GA responsive gene (pSCR3:GUS reporter) and GA perception sites, represented by the expression pattern of GA perception proteins (GID1 and DELLA). The study demonstrated that alternating temperatures act as an instructive signal in the embryonic root tip in Arabidopsis dormant seeds . The modeling nicely showed that the process of dormancy break in the seed is defined by the distribution of the plant hormones GA and abscisic acid (ABA) . This spatial separation of ABA and GA responses suggests that crosstalk between ABA and GA is non-cell-autonomous and is controlled at the level of hormone movement between spatially separated signaling centers .
It should be noted that the observations and interpretations regarding GA localization are limited by several factors. First, the spatiotemporal resolution of the studies, using GUS reporters or mRNA expression, is relatively low. It would be constructive to increase the resolution of such studies through dynamic monitoring of fluorescent reporters. Second, only a few of the GA biosynthesis genes families, and only a few members from those families, have been analyzed so far. In order to draw a comprehensive map of the spatial distribution of GA biosynthesis, a concurrent characterization of the whole pathway will be required. Third, studies to date have usually analyzed expression of GA biosynthetic genes at the mRNA level. As it is possible that these enzymes are subjected to post-translational modifications and non-cell-autonomous movement, it will be important to examine their localization as translational fusions. The ultimate goal should be to generate specific sensors that will provide a readout for the enzyme family activity. This would allow a specific readout of the final enzymatic biochemical activity and overcome redundancies. It is reasonable to assume that GA localization is also regulated by catabolism, conjugation, and transport steps . Thus, expression patterns of GA biosynthesis genes will not necessarily enable identification of all sites of active GA localization and response.
In order to overcome several of the limitations illustrated above, a novel fluorescence resonance energy transfer-based GA biosensor (termed GPS1) was developed. The GPS1 biosensor, constructed by fusing GID1 variants to the DELLA N-termini, showed an increased emission ratio in response to nanomolar concentrations of GA4. With the exception of a few limitations such as nonreversible response to GA4, phenotypic hypersensitivity to a GA biosynthesis inhibitor, and a limited response to GA3 and GA1, this biosensor should be a useful new tool for identifying GA response sites. For example, GPS1 revealed that GA response is higher in the elongation zone compared with the root meristematic zone. GA localization correlated with cell length when GA4 was exogenously applied, suggesting that rapid transport or catabolism of GA in the root may generate local GA gradients independently of GA biosynthesis. In addition, the GPS1 sensor indicated that high levels of GA4 in the elongating hypocotyl depend on darkness . GPS1 should find broad utility in exploration of GA distribution and transport mechanisms and is expected, for example, to shed light on GA distribution in known and novel GA transporter loss-of-function and gain-of-function lines reported for the nitrate transporter 1/peptide transporter family (NPF) and SWEET families (further discussed below).
A different approach to address the question of GA distribution and accumulation sites utilized fluorescently labeled versions of GA3 and GA4 (termed GA-Fl). Combining imaging of GA-Fl localization with information on transporter expression levels and genetics showed that the TEMPRANILLO (TEM) proteins play an essential role not only in GA biosynthesis but also in regulating GA distribution in the mesophyll, which, in turn, regulates epidermal trichome formation . In roots, GA-Fl accumulated specifically in the elongating endodermal cells of Arabidopsis roots. The localization of GA-Fl in the elongating cells is consistent with GFP-RGA levels and with other studies indicating that GA activity is necessary for root elongation and gravitropic response , but only partially overlaps with GPS1 signal, which was not restricted to the endodermis. Since GA-Fl is highly specific to NPF3 , it is possible that it represents only a subset of GA forms. Alternatively, it is possible that GPS1 and GA-Fl report on GA levels on different sites within the cell; whereas GPS1 mainly responds to nuclear GA4 levels, the fluorescent GA4 reports on transport and localization of exogenously applied GA that eventually localizes to the vacuole. Since the GA response was shown to be restricted to the root endodermis cell layer, it will be important to evaluate the distribution of active GA and its precursors at a cellular resolution, as has been successfully carried out for the plant hormones auxin and cytokinin .
Cells entering the elongation zone increase in length by approximately 10-fold over 5 hours. Such a rapid expansion is expected to result in a rapid intracellular dilution of GA, practically reducing its effective concentration. Modeling of this process suggests a correlation between GA distribution and root cell growth, and the study authors posit that cellular GA levels decrease at the elongation zone due to cytosolic dilution . Independent analyses of the GPS1 sensor response and GA-Fl distribution indicate that GA levels are higher in the root elongation zone compared with the meristematic zone; therefore, GA is probably either synthesized locally or imported from surrounding tissues to compensate for dilution.
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