We have isolated a dominant, auxin-insensitive mutant of Arabidopsis thaliana, massugu2 (msg2), that displays neither hypocotyl gravitropism nor phototropism, fails to maintain an apical hook as an etiolated seedling, and is defective in lateral root formation. Yet other aspects of growth and development of msg2 plants are almost normal. These characteristics of msg2 are similar to those of another auxin-insensitive mutant, non-phototropic hypocotyl4 (nph4), which is a loss-of-function mutant of AUXIN RESPONSE FACTOR7 (ARF7) (Harper et al., 2000). Map-based cloning of the MSG2 locus reveals that all four mutant alleles result in amino acid substitutions in the conserved domain II of an Auxin/Indole-3-Acetic Acid protein, IAA19. Interestingly, auxin inducibility of MSG2/IAA19 gene expression is reduced by 65% in nph4/arf7. Moreover, MSG2/IAA19 protein binds to the C-terminal domain of NPH4/ARF7 in a Saccharomyces cerevisiae (yeast) twohybrid assay and to the whole latter protein in vitro by pull-down assay. These results suggest that MSG2/IAA19 and NPH4/ ARF7 may constitute a negative feedback loop to regulate differential growth responses of hypocotyls and lateral root formation.

Organ bending through differential growth represents a major mechanism by which plants are able to adaptively alter their morphology in response to local changes in the environment. Two plant hormones, auxin and ethylene, have been implicated as regulators of differential growth responses; however, the mechanisms by which they elicit their effects remain largely unknown. Here, we describe isolation of the NPH4 gene of Arabidopsis, which is conditionally required for differential growth responses of aerial tissues, and we report that NPH4 encodes the auxin-regulated transcriptional activator ARF7. The phenotypes of nph4 mutants, which include multiple differential growth defects associated with reduced auxin responsiveness, including impaired auxin-induced gene expression, are consistent with the predicted loss of function of a transcriptional activator, and these phenotypes indicate that auxin-dependent changes in gene transcription are prerequisite for proper organ bending responses. Although NPH4/ARF7 appears to be a major regulator of differential growth, it is not the sole regulator because phenotypes of nph4 null mutants were suppressed by application of ethylene. This latter finding illustrates the intimate connection between auxin and ethylene in the control of growth in higher plants. INTRODUCTIONPlants have evolved movement strategies that involve organ bending to respond adaptively to environmental signals. Dramatic and rapid changes in plant morphology can result from differential growth, that is, unequal cellular growth in one position of an organ relative to an opposing position. Examples of differential growth responses include stem and root tropisms, modification of apical hook structures, and nastic movements of leaves (reviewed in Darwin and Darwin, 1896;Palmer, 1985). Each of these examples of stimulusdriven organ bending represents a process by which plants maximize the positive attributes of their environment while minimizing the negatives.Two plant hormones, auxin and ethylene, have been implicated as regulators of differential growth responses (Went and Thimann, 1937; Davies, 1987; Kaufman et al., 1995). Although each of these hormones is capable of modulating growth when applied externally, the relative contribution of each in response to changes in their endogenous concentrations, and the sensitivities to either, has been difficult to reconcile (Davies, 1987). Much of this ambiguity stems from functional overlap between the auxin and ethylene signal and response, as well as their biosynthetic, pathways. For example, auxin stimulates ethylene production (Yang and Hoffman, 1984), which in turn stimulates the expression of genes, such as HOOKLESS 1 ( HLS1 ; Lehman et al., 1996), that are involved in auxin homeostatic processes. Results from recent genetic and molecular studies suggest that auxin may be the major regulator of differential growth responses, with ethylene modifying the auxin responses (Romano et al., 1993; Lehman et al., 1996; Chen et al., 1998;Luschnig et al., 1998;Madlung ...

SummaryAuxin response factor (ARF) family genes play a central role in controlling sensitivity to the plant hormone auxin. We characterized the function of ARF8 in Arabidopsis by investigating a T-DNA insertion line (arf8-1) and overexpression lines (ARF8 OX) of ARF8. arf8-1 showed a long-hypocotyl phenotype in either white, blue, red or far-red light conditions, in contrast to ARF8 OX that displayed short hypocotyls in the light. Stronger and weaker apical dominance, and promotion and inhibition of lateral root formation were observed in arf8-1 and ARF8 OX respectively. Sensitivity to auxin was unaltered in arf8-1 hypocotyls with respect to growth inhibition caused by exogenously applied auxin and growth promotion induced by higher temperatures. ARF8 expression was observed constitutively in shoot and root apexes, and was induced in the light condition in hypocotyls. Free IAA contents were approximately 30% reduced in light-grown hypocotyls of ARF8 OX, but were similar between those of arf8-1 and wild type. Expression of the three GH3 genes was reduced in arf8-1 and increased in ARF8 OX, indicating that they are targets of ARF8 transcriptional control. Because the three GH3 proteins may be involved in the conjugation of IAA as suggested by Staswick et al. (2002), and because two of the three GH3 genes are auxin inducible, ARF8 may control the free IAA level in a negative feedback fashion by regulating GH3 gene expression. ARF family genes seem to control both auxin sensitivity and homeostasis in Arabidopsis.

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