A hormonal regulatory module that provides flexibility to tropic responses

Plants orient their growth depending on directional stimuli such as light and gravity, in a process known as tropic response. Tropisms result from asymmetrical accumulation of auxin across the responding organ relative to the direction of the stimulus, which causes differential growth rates on both sides of the organ. Here, we show that gibberellins (GAs) attenuate the gravitropic reorientation of stimulated hypocotyls of dark-grown Arabidopsis (Arabidopsis thaliana) seedlings. We show that the modulation occurs through induction of the expression of the negative regulator of auxin signaling INDOLE-3-ACETIC ACID INDUCIBLE19/MASSUGU2. The biological significance of this regulatory mechanism involving GAs and auxin seems to be the maintenance of a high degree of flexibility in tropic responses. This notion is further supported by observations that GA-deficient seedlings showed a much lower variance in the response to gravity compared to wild-type seedlings and that the attenuation of gravitropism by GAs resulted in an increased phototropic response. This suggests that the interplay between auxin and GAs may be particularly important for plant orientation under competing tropic stimuli.     

Molecular genetic analysis of phototropism in Arabidopsis

Plant life is strongly dependent on the environment, and plants regulate their growth and development in response to many different environmental stimuli. One of the regulatory mechanisms involved in these responses is phototropism, which allows plants to change their growth direction in response to the location of the light source. Since the study of phototropism by Darwin, many physiological studies of this phenomenon have been published. Recently, molecular genetic analyses of Arabidopsis have begun to shed light on the molecular mechanisms underlying this response system, including phototropin blue light photoreceptors, phototropin signaling components, auxin transporters, auxin action mechanisms and others. This review highlights some of the recent progress that has been made in further elucidating the phototropic response, with particular emphasis on mutant phenotypes.     

The far side of auxin signaling: fundamental cellular activities and their contribution to a defined growth response in plants

Recent years have provided us with spectacular insights into the biology of the plant hormone auxin, leaving the impression of a highly versatile molecule involved in virtually every aspect of plant development. A combination of genetics, biochemistry, and cell biology has established auxin signaling pathways, leading to the identification of two distinct modes of auxin perception and downstream regulatory cascades. Major targets of these signaling modules are components of the polar auxin transport machinery, mediating directional distribution of the phytohormone throughout the plant body, and decisively affecting plant development. Alterations in auxin transport, metabolism, or signaling that occur as a result of intrinsic as well as environmental stimuli, control adjustments in morphogenetic programs, giving rise to defined growth responses attributed to the activity of the phytohormone. Some of the results obtained from the analysis of auxin, however, do not fit coherently into a picture of highly specific signaling events, but rather suggest mutual interactions between auxin and fundamental cellular pathways, like the control of intracellular protein sorting or translation. Crosstalk between auxin and these basic determinants of cellular activity and how they might shape auxin effects in the control of morphogenesis are the subject of this review.     

Reduced phototropism in pks mutants may be due to altered auxin‐regulated gene expression or reduced lateral auxin transport

Phototropism allows plants to orient their photosynthetic organs towards the light. In Arabidopsis, phototropins 1 and 2 sense directional blue light such that phot1 triggers phototropism in response to low fluence rates, while both phot1 and phot2 mediate this response under higher light conditions. Phototropism results from asymmetric growth in the hypocotyl elongation zone that depends on an auxin gradient across the embryonic stem. How phototropin activation leads to this growth response is still poorly understood. Members of the phytochrome kinase substrate (PKS) family may act early in this pathway, because PKS1, PKS2 and PKS4 are needed for a normal phototropic response and they associate with phot1 in vivo. Here we show that PKS proteins are needed both for phot1‐ and phot2‐mediated phototropism. The phototropic response is conditioned by the developmental asymmetry of dicotyledonous seedlings, such that there is a faster growth reorientation when cotyledons face away from the light compared with seedlings whose cotyledons face the light. The molecular basis for this developmental effect on phototropism is unknown; here we show that PKS proteins play a role at the interface between development and phototropism. Moreover, we present evidence for a role of PKS genes in hypocotyl gravi‐reorientation that is independent of photoreceptors. pks mutants have normal levels of auxin and normal polar auxin transport, however they show altered expression patterns of auxin marker genes. This situation suggests that PKS proteins are involved in auxin signaling and/or lateral auxin redistribution.     

Recent progress in auxin biology

Like animals, plants have evolved into complex organisms. Developmental cohesion between tissues and cells is possible due to signaling molecules (messengers) like hormones. The first hormone discovered in plants was auxin. This phytohormone was first noticed because of its involvement in the response to directional light. Nowadays, auxin has been established as a central key player in the regulation of plant growth and development and in responses to environmental changes. At the cellular level, auxin controls division, elongation, and differentiation as well as the polarity of the cell. Auxin, to integrate so many different signals, needs to be regulated at many different levels. A tight regulation of auxin synthesis, activity, degradation as well as transport has been demonstrated. Another possibility to modulate auxin signaling is to modify the capacity of response of the cells by expressing differentially the signaling components. In this review, we provide an overview of the present knowledge in auxin biology, with emphasis on root development.     

Auxins and tropisms

Differential growth of plants in response to the changes in the light and gravity vectors requires a complex signal transduction cascade. Although many of the details of the mechanisms by which these differential growth responses are induced are as yet unknown, auxin has been implicated in both gravitropism and phototropism. Specifically, the redistribution of auxin across gravity or light-stimulated tissues has been detected and shown to be required for this process. The approaches by which auxin has been implicated in tropisms include isolation of mutants altered in auxin transport or response with altered gravitropic or phototropic response, identification of auxin gradients with radiolabeled auxin and auxin-inducible gene reporter systems, and by use of inhibitors of auxin transport that block gravitropism and phototropism. Proteins that transport auxin have been identified and the mechanisms which determine auxin transport polarity have been explored. In addition, recent evidence that reversible protein phosphorylation controls this process is summarized. Finally, the data in support of several hypotheses for mechanisms by which auxin transport could be differentially regulated during gravitropism are examined. Although many details of the mechanisms by which plants respond to gravity and light are not yet clear, numerous recent studies demonstrate the role of auxin in these processes.     

RPT2. A signal transducer of the phototropic response in Arabidopsis

The blue light receptor NPH1 (for nonphototropic hypocotyl) has been considered to be the only UV-A/blue light receptor that induces a phototropic response by the hypocotyl and root of Arabidopsis. By analysis of root phototropism (rpt) mutants, we show, however, the involvement of another blue light receptor as well as the existence of two separate signaling pathways working downstream of these receptors in the phototropic response. A newly isolated gene, RPT2, controls one of these pathways. The RPT2 gene is light inducible; encodes a novel protein with putative phosphorylation sites, a nuclear localization signal, a BTB/POZ domain, and a coiled-coil domain; and belongs to a large gene family that includes the recently isolated NPH3 gene. From genetic, physiological, and biochemical evidence, we propose a genetic model of the signaling pathways that induce the phototropic response in Arabidopsis.     

phot1 inhibition of ABCB19 primes lateral auxin fluxes in the shoot apex required for phototropism

It is well accepted that lateral redistribution of the phytohormone auxin underlies the bending of plant organs towards light. In monocots, photoreception occurs at the shoot tip above the region of differential growth. Despite more than a century of research, it is still unresolved how light regulates auxin distribution and where this occurs in dicots. Here, we establish a system in Arabidopsis thaliana to study hypocotyl phototropism in the absence of developmental events associated with seedling photomorphogenesis. We show that auxin redistribution to the epidermal sites of action occurs at and above the hypocotyl apex, not at the elongation zone. Within this region, we identify the auxin efflux transporter ATP-BINDING CASSETTE B19 (ABCB19) as a substrate target for the photoreceptor kinase PHOTOTROPIN 1 (phot1). Heterologous expression and physiological analyses indicate that phosphorylation of ABCB19 by phot1 inhibits its efflux activity, thereby increasing auxin levels in and above the hypocotyl apex to halt vertical growth and prime lateral fluxes that are subsequently channeled to the elongation zone by PIN-FORMED 3 (PIN3). Together, these results provide new insights into the roles of ABCB19 and PIN3 in establishing phototropic curvatures and demonstrate that the proximity of light perception and differential phototropic growth is conserved in angiosperms.     

Dynamics of auxin-dependent Ca2+ and pH signaling in root growth revealed by integrating high-resolution imaging with automated computer vision-based analysis

Plants adapt to a changing environment by entraining their growth and development to prevailing conditions. Such 'plastic' development requires a highly dynamic integration of growth phenomena with signal perception and transduction systems, such as occurs during tropic growth. The plant hormone auxin has been shown to play a key role in regulating these directional growth responses of plant organs to environmental cues. However, we are still lacking a cellular and molecular understanding of how auxin-dependent signaling cascades link stimulus perception to the rapid modulation of growth patterns. Here, we report that in root gravitropism of Arabidopsis thaliana, auxin regulates root curvature and associated apoplastic, growth-related pH changes through a Ca2+-dependent signaling pathway. Using an approach that integrates confocal microscopy and automated computer vision-based image analysis, we demonstrate highly dynamic root surface pH patterns during vertical growth and after gravistimulation. These pH dynamics are shown to be dependent on auxin, and specifically on auxin transport mediated by the auxin influx carrier AUX1 in cells of the lateral root cap and root epidermis. Our results further indicate that these pH responses require auxin-dependent changes in cytosolic Ca2+ levels that operate independently of the TIR1 auxin perception system. These results demonstrate a methodology that can be used to visualize vectorial auxin responses in a manner that can be integrated with the rapid plant growth responses to environmental stimuli.     

Auxin induces mitogenic activated protein kinase (MAPK) activation in roots of Arabidopsis seedlings

Genome analyses have shown that plants contain gene families encoding various components of mitogen-activated protein kinase (MAPK) signaling pathways. Previous reports have described the involvement of MAPK pathways in stress and pathogen responses of leaves and suspension-cultured cells. Here we show that auxin treatment of Arabidopsis roots transiently induced increases in protein kinase activity with characteristics of mammalian ERK-like MAPKs. The MAPK response we monitored was the result of hormonal action of biologically active auxin, rather than a stress response provoked by auxin-like compounds. Auxin-induced MAPK pathway signaling was distinguished genetically in the Arabidopsis auxin response mutant axr4, in which MAPK activation by auxin, but not by salt stress, was significantly impaired. Perturbation of MAPK signaling in roots using inhibitors of a mammalian MAPKK blocked auxin-activated transgene expression in BA3-GUS seedlings, while potentiating higher than normal levels of MAPK activation in response to auxin. Data presented here indicate that MAPK pathway signaling is positively involved in auxin response, and further suggest that interactions among MAPK signaling pathways in plants influence plant responses to auxin.     

Unilateral reorientation of microtubules at the outer epidermal wall during photo- and gravitropic curvature of maize coleoptiles and sunflower hypocotyls

Auxin (indole-3-acetic acid) controls the orientation of cortical microtubes (MT) at the outer wall of the outer epidermis of growing maize coleoptiles (Bergfeld, R., Speth, V., Schopfer, P., 1988, Bot. Acta 101, 57-67). A detailed time course of MT reorientation, determined by labeling MT with fluorescent antibodies, revealed that the auxin-mediated movement of MT from the longitudinal to the transverse direction starts after less than 15 min and is completed after 60 min. This response was used for a critical test of the functional involvement of auxin in tropic curvature. It was found that phototropic (first phototropic curvature) as well as gravitropic bending are correlated with a change of MT orientation from transverse to longitudinal at the slower-growing organ flank whereas the transverse MT orientation is maintained (or even augmented) at the faster-growing organ flank. These directional changes are confined to the MT subjacent to the outer epidermal wall. The same basic results were obtained with sunflower hypocotyls subjected to phototropic or gravitropic stimulation. It is concluded that auxin is, in fact, involved in asymmetric growth leading to tropic curvature. However, our results do not allow us to discriminate between an uneven distribution of endogenous auxin or an even distribution of auxin, the activity of which is modulated by an unevenly distributed inhibitor of auxin action.     

D6PK AGCVIII Kinases Are Required for Auxin Transport and Phototropic Hypocotyl Bending in Arabidopsis

Phototropic hypocotyl bending in response to blue light excitation is an important adaptive process that helps plants to optimize their exposure to light. In Arabidopsis thaliana, phototropic hypocotyl bending is initiated by the blue light receptors and protein kinases phototropin1 (phot1) and phot2. Phototropic responses also require auxin transport and were shown to be partially compromised in mutants of the PIN-FORMED (PIN) auxin efflux facilitators. We previously described the D6 PROTEIN KINASE (D6PK) subfamily of AGCVIII kinases, which we proposed to directly regulate PIN-mediated auxin transport. Here, we show that phototropic hypocotyl bending is strongly dependent on the activity of D6PKs and the PIN proteins PIN3, PIN4, and PIN7. While early blue light and phot-dependent signaling events are not affected by the loss of D6PKs, we detect a gradual loss of PIN3 phosphorylation in d6pk mutants of increasing complexity that is most severe in the d6pk d6pkl1 d6pkl2 d6pkl3 quadruple mutant. This is accompanied by a reduction of basipetal auxin transport in the hypocotyls of d6pk as well as in pin mutants. Based on our data, we propose that D6PK-dependent PIN regulation promotes auxin transport and that auxin transport in the hypocotyl is a prerequisite for phot1-dependent hypocotyl bending.     

Physical strain-mediated microtubule reorientation in the epidermis of gravitropically or phototropically stimulated maize coleoptiles

During gravitropic and phototropic curvature of the maize coleoptile, the cortical microtubules (MTs) adjacent to the outer epidermal cell wall assume opposite orientations at the two sides of the organ. Starting from a uniformly random pattern during straight growth in darkness, the MTs reorientate perpendicularly to the organ axis at the outer (faster growing) side and parallel to the organ axis at the inner (slower growing) side. As similar reorientations can be induced during straight growth by increasing or decreasing the effective auxin concentration, it has been proposed that these reorientations may be used as a diagnostic test for assessing the auxin status of the epidermal cells during tropic curvature. This idea was tested by determining the MT orientations in the coleoptile of intact maize seedlings in which the gravitropic or phototropic curvature was prevented or inversed by an appropriate mechanical counterforce. Forces that just prevented the coleoptile from curving in a gravity or light field prevented reorientations of the MTs. Forces strong enough to overcompensate the tropic stimuli by enforcing curvature in the opposite direction induced reorientations of the MTs opposite to those produced by tropic stimulation. These results show that the MTs at the outer surface of the coleoptile respond to changes in mechanical tissue strain rather than to gravitropic or phototropic stimuli and associated changes at the level of auxin or any other element in the signal transduction chain between perception of tropic stimuli and asymmetric growth response. It is proposed that cortical MTs can act as strain gauges in a positive feed-back regulatory circle utilized for amplification and stabilization of environmentally induced changes in the direction of elongation growth.