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Embryonic Hopes PINned on Auxin Distribution

neckardt{at}aspb.org

The hormone auxin plays a central role in regulating patterns of cell division and differentiation at critical stages of plant growth and development, such as during embryogenesis, organogenesis, and tropic growth responses. In all of these processes, cell-to-cell polar transport of auxin and differential distribution are key aspects of its function. This has led to the idea (Friml, 2003; Barlow, 2005) that auxin might function in a similar manner to morphogens, substances that influence morphogenesis and embryogenesis by forming a concentration gradient. The term was originally used in reference to plant development (Turing, 1952), where it was given the rather vague definition, a "form producer...not intended to have any very exact meaning." It has since become widely used in animal biology, as a number of proteins, notably Bicoid, Hunchback, Wingless, and Hedgehog in Drososphila, were discovered to form gradients and influence embryogenesis and morphogenesis in a concentration-dependent manner. Such gradients act at a distance, provide positional information to cells, and influence gene expression, resulting in the establishment of appropriate asymmetric patterns of cell differentiation in the developing embryo or organ (reviewed in Tabata and Takei, 2004).

The polar transport of auxin in plants depends on the activity of members of the PIN-FORMED (PIN) family of auxin efflux carriers (reviewed in Leyser, 2005; Paponov et al., 2005). Arabidopsis contains eight PIN genes numbered PIN1 to PIN8, which may have diverged from a single ancestral gene sequence. PIN proteins are similar to bacterial transporters and contain a membrane spanning domains at both the N and C termini and a central hydrophilic region (Paponov et al., 2005). Genes homologous to Arabidopsis PINs can be found throughout the plant kingdom. PIN expression analysis and studies of pin mutants suggest that PIN proteins have distinct but overlapping functions intimately associated with polar auxin transport (Blilou et al., 2005), but it remains to be determined whether they act directly as auxin efflux carriers or rather as auxin efflux facilitators that regulate the activity of other proteins and/or participate in a larger complex (Paponov et al., 2005). In this issue of The Plant Cell, Weijers et al. (pages 2517–2526) show that PIN-dependent polar auxin transport is crucial for the control of proper patterning during embryogenesis and, surprisingly, that this differential distribution of auxin appears to be maintained or buffered despite alterations in auxin biosynthesis or conjugation.

The authors adopted a unique approach of utilizing targeted expression of bacterial auxin biosynthesis and conjugation enzymes to manipulate active auxin levels in regions of the developing Arabidopsis embryo. The bacterial genes iaaM and iaaL, which encode enzymes for auxin biosynthesis and conjugation, respectively, were introduced into a GAL4/UAS two-component transactivation gene expression system optimized for Arabidopsis (Weijers et al., 2003). The system makes use of two plant lines: one that expresses the heterologous transcription factor GAL4 under the control of a domain-specific plant promoter and another that contains a gene of interest under the control of the silent promoter UAS, which is only activated by GAL4. Crossing of the two lines leads to domain-specific expression of the gene of interest and is of particular use for investigating and propagating genes that may have embryo-lethal effects. In this case, UAS was linked to iaaM or iaaL and to the reporter genes GUS or GFP to monitor expression. As expected, no expression was detected in lines expressing these constructs (lines EF iaaM and EF iaaL) in the absence of GAL4. The EF iaaM and EF iaaL lines were then crossed with a range of lines expressing GAL4 in a subset of embryonic cells to drive iaaM or iaaL expression in the embryo. Lines carrying the iaaM and iaaL transgenes were further transformed to express GFP under the control of the auxin-responsive DR5 promoter to monitor the auxin response.

Linking iaaM and iaaL to GUS or GFP showed that the genes were correctly expressed in various regions of the embryo in response to GAL4 expression. Surprisingly, however, and in contrast with strong effects on postembryonic development when iaaM or iaaL are expressed in postembryonic tissue, expression of iaaM or iaaL in the embryo did not result in abnormal effects on embryo patterning. This left open the possibility that, despite expression of iaaM and iaaL genes, the iaaM and iaaL proteins might not be able to function in the embryo to cause alterations in auxin concentration. The technology does not yet exist to measure auxin concentration directly in the embryo, and this represents a limitation of the study that the authors acknowledge. Instead, they employed a number of indirect tests that, taken together, suggested that active auxin levels were in fact increased or decreased, respectively, by iaaM and iaaL expression in the embryo but that PIN activity provides a buffering capacity that maintains essential auxin gradients despite fluctuations in auxin biosynthesis or conjugation.

First, in the case of iaaM activity, it was shown that expression of the auxin-responsive DR5:GFP reporter was enhanced in embryos expressing iaaM. Although it is important to recognize that DR5:GFP expression only indicates auxin responsiveness and not auxin levels, the data suggest that auxin levels were correspondingly increased in embryos expressing iaaM. For corroboration, it was shown that DR5:GFP expression was also enhanced in embryos by treatment with the synthetic auxin 2,4-D. Second, it was shown that treatment with the auxin transport inhibitor NPA disrupted wild-type embryo patterning causing fusion of cotyledons at a concentration of 20 µM but not at the lower concentration of 5 µM, whereas the 5 µM concentration led to complete fusion of cotyledons in lines expressing iaaM in the embryo. This result indicated that pattern formation in iaaM-expressing embryos was more sensitive to inhibition of polar auxin transport. As for iaaL activity, DR5:GFP expression was not observed to be reduced in iaaL-expressing embryos, so it could not be shown by this test that iaaL was active in embryos.

The strongest evidence substantiating an effect of iaaM and iaaL expression on embryonic auxin levels and indicating involvement of PIN proteins in buffering changes in embryonic auxin levels came from experiments conducted with pin mutants. Four PIN proteins, PIN1, PIN3, PIN4, and PIN7, are expressed in the Arabidopsis embryo (Friml et al., 2003; Blilou et al., 2005). Expression patterns suggested to Weijers et al. that PIN1 and PIN4 were most likely to be involved in maintenance of differential auxin distribution important for embryo patterning. First, it was shown that PIN-dependent polar auxin transport to maintain differential distribution of auxin is important for embryo patterning and for buffering alterations in auxin levels because pin1 and pin4 mutants, but not wild-type controls, exhibited embryo defects when seed was cultured in the presence of the auxin naphthalene acetic acid. Next, the authors crossed the transgenic lines expressing iaaM and iaaL in embryonic tissue into a pin4 mutant. The pin4 mutant was used because, in contrast with the pin1 mutant, it exhibits only weak embryo defects of its own; therefore, defects caused by iaaM or iaaL expression would be more easily observable. Similar to the results from culturing pin mutants in exogenously applied auxin, a significant percentage of the pin4 mutants that expressed iaaM or iaaL showed embryo patterning defects in establishment of the apical-basal axis. These results provide evidence that both iaaM and iaaL are active in altering auxin levels in the embryo of the transgenic plants and that PIN4 activity functions to buffer these changes and maintain an auxin distribution that is required for normal patterning during embryogenesis.

This work extends previous observations on the function of PIN proteins in setting up an apical-basal auxin activity gradient in the embryo that establishes the apical-basal axis in Arabidopsis (Friml et al., 2003). In addition, it provides good support for the importance of polar auxin transport and the maintenance of PIN-dependent differential distribution of auxin in the embryo, relative to auxin biosynthesis and conjugation. Whether a gradient of auxin instructs different developmental outcomes, part of commonly accepted morphogen definitions, or whether auxin accumulation thresholds act as instructive switches remains to be proven.

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Maintenance of Embryonic Auxin Distribution for Apical-Basal Patterning by PIN-FORMED–Dependent Auxin Transport in Arabidopsis

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