Genetic approaches to auxin action

Answers to long-standing questions concerning the molecular mechanism of auxin action and auxin's exact functions in plant growth and development are beginning to be uncovered through studies using mutant and transgenic plants. We review recent work in this area in vascular plants. A number of conclusions can be drawn from these studies. First, auxin appears essential for cell division and viability, as auxin auxotrophs isolated in tissue culture are dependent on auxin for growth and cannot be regenerated into plants even when auxin is supplied exogenously. Secondly, plants with transgenes that alter auxin levels are able to regulate cellular auxin concentrations by synthesis and conjugation; wild-type plants are probably also capable of such regulation. Thirdly, the phenotypes of transgenic plants with altered auxin levels and of mutant plants with altered sensitivity to auxin confirm earlier physiological studies which indicated a role for auxin in regulation of apical dominance, in development of roots and vascular tissue, and in the gravitropic response. Finally, the cloning of a mutationally identified gene important for auxin action, along with accumulating biochemical evidence, hints at a major role for protein degradation in the auxin response pathway.     

Growth and development of the axr1 mutants of Arabidopsis.

We have recovered eight new auxin-resistant lines of Arabidopsis that carry mutations in the AXR1 gene. These eight lines, together with the 12 lines described in a previous report, define at least five different axr1 alleles. All of the mutant lines have a similar phenotype. Defects include decreases in plant height, root gravitropism, hypocotyl elongation, and fertility. Mutant line axr1-3 is less resistant to auxin than the other mutant lines and has less severe morphological abnormalities. This correlation suggests that the morphological defects are a consequence of a defect in auxin action. To determine whether the altered morphology of mutant plants is associated with changes in cell size or tissue organization, tissue sections were examined using scanning electron microscopy. No clear differences in cell size were observed between wild-type and mutant tissues. However, the vascular bundles of mutant stems were found to be less well differentiated than those in wild-type stems. The auxin sensitivity of rosette-stage plants was determined by spraying plants with auxin solutions. Mutant rosettes were found to be significantly less sensitive to exogenously applied auxin than wild-type rosettes, indicating that the AXR1 gene functions in aerial portions of the plant. Our studies suggest that the AXR1 gene is required for auxin action in most, if not all, tissues of the plant and plays an important role in plant development. Linkage studies indicate that the gene is located on chromosome 1 approximately 2 centiMorgans from the closest restriction fragment length polymorphism.     

Oligosaccharins, brassinolides, and jasmonates: nontraditional regulators of plant growth, development, and gene expression.

Each of the nontraditional plant hormones reviewed in this article, oligosaccharins, brassinolides, and JA, can exert major effects on plant growth and development. However, in many cases, the mechanisms by which these compounds are involved in the endogenous regulation of morphogenesis remain to be established. Nevertheless, the use of mutant or transgenic plants with altered levels or perception of these hormones is leading to phenomenal increases in our understanding of the roles they play in the life cycle of plants. It is likely that in the future, novel modulators of plant growth and development will be identified; some will perhaps be related to the peptide encoded by ENOD40 (Van de Sande et al., 1996), which modifies the action of auxin.     

The plant hormone auxin: insight in sight.

Physiological experiments conducted over the last 60 years indicate that the plant hormone auxin regulates a diverse set of developmental processes via changes in cell division, cell elongation and cell differentiation. Recent studies using transgenic plants with altered auxin levels support these conclusions and promise to provide more detailed information on the role of auxin during plant development. Although it is possible that all auxin responses are mediated by the same primary biochemical events, the studies described in this review are more consistent with multiple modes of auxin action. The development of molecular and genetic approaches to the study of hormone action should resolve this issue. The accelerated rate of progress in this field suggests that real insight into the mechanism of auxin action may be forthcoming.     

Auxin acts in xylem-associated or medullary cells to mediate apical dominance

A role for auxin in the regulation of shoot branching was described originally in the Thimann and Skoog model, which proposes that apically derived auxin is transported basipetally directly into the axillary buds, where it inhibits their growth. Subsequent observations in several species have shown that auxin does not enter axillary buds directly. We have found similar results in Arabidopsis. Grafting studies indicated that auxin acts in the aerial tissue; hence, the principal site of auxin action is the shoot. To delineate the site of auxin action, the wild-type AXR1 coding sequence, which is required for normal auxin sensitivity, was expressed under the control of several tissue-specific promoters in the auxin-resistant, highly branched axr1-12 mutant background. AXR1 expression in the xylem and interfascicular schlerenchyma was found to restore the mutant branching to wild-type levels in both intact plants and isolated nodes, whereas expression in the phloem did not. Therefore, apically derived auxin can suppress branching by acting in the xylem and interfascicular schlerenchyma, or in a subset of these cells.     

An auxin-auxotrophic mutant of Nicotiana plumbaginifolia

Summary Auxin (indole-3-acetic acid) is considered to be an important signalling molecule in the regulation of plant growth and development but neither auxin synthesis nor its mode of action is clearly understood. To identify genes involved in these processes, mutations were sought that altered the auxin requirement of plant tissues for growth. For the first time mutant plants were obtained that carry a recessive mutation at a single nuclear locus (auxl) which results in an absolute requirement for exogenous auxin for normal growth. In the absence of auxin treatment, mutant plants undergo premature senescence and die.Abbreviations BAP 6-benzylaminopurine - BUdR 5-bromodeoxyuridine - 2,4-D 2,4-dichlorophenoxyacetic acid - FUdR 5-fluorodeoxyuridine - IAA-EE indole-3-acetic acid ethyl ester - IMS indole-3-methanesulfonic acid     

Auxin inhibition of decapitation-induced branching is dependent on graft-transmissible signals regulated by genes Rms1 and Rms2

Decapitation-induced axillary bud outgrowth is a vital mechanism whereby shoots are able to continue normal growth and development. In many plants, including wild-type garden pea (Pisum sativum L.), this process can be inhibited by exogenous auxin. Using the ramosus (rms) increased branching mutants of pea, we present evidence that this response to auxin is dependent on graft-transmissible substance(s) regulated by the genes Rms1 and Rms2. The response to exogenous auxin is massively diminished in decapitated rms1 and rms2 mutant plants. However, basipetal auxin transport is not reduced in intact or decapitated mutants. Grafting rms1 or rms2 shoots onto wild-type rootstocks restored the auxin response, indicating that Rms1 and Rms2 gene action in the rootstock is sufficient to enable an auxin response in mutant shoots. We conclude that Rms1 and Rms2 act in the rootstock and shoot to control levels of mobile substance(s) that interact with exogenous auxin in the inhibition of bud outgrowth after decapitation. At least for rms1, the reduced auxin response is unlikely to be due to an inability of auxin to decrease xylem sap cytokinin content, as this is already low in intact rms1 plants. Consequently, we have genetic evidence that auxin action in decapitated plants depends on at least one novel long-distance signal.     

The Interactions among DWARF10, Auxin and Cytokinin Underlie Lateral Bud Outgrowth in Rice

Previous studies have shown that DWARF10 (D10) is a rice ortholog of MAX4/RMS1/DAD1, encoding a carotenoid cleavage dioxygenase and functioning in strigolactones/strigolactone-derivatives (SL)biosynthesis. Here we use D10- RNA interference (RNAi) transgenic plants similar to d10 mutant in phenotypes to investigate the interactions among D10, auxin and cytokinin in regulating rice shoot branching. Auxin levels in node 1 of both decapitated D10-RNAi and wild type plants decreased significantly, showing that decapitation does reduce endogenous auxin concentration, but decapitation has no clear effects on auxin levels in node 2 of the same plants. This implies that node 1 may be the location where a possible interaction between auxin and D10 gene would be detected. D10 expression in node 1 is inhibited by decapitation, and this inhibition can be restored by exogenous auxin application,indicating that D10 may play an important role in auxin regulation of SL. The decreased expression of most OsPINs in shoot nodes of D10- RNAi plants may cause a reduced auxin transport capacity.Furthermore, effects of auxin treatment of decapitated plants on the expression of cytokinin biosynthetic genes suggest that D10 promotes cytokinin biosynthesis by reducing auxin levels. Besides, in D10- RNAi plants, decreased storage cytokinin levels in the shoot node may partly account for the increased active cytokinin contents, resulting in more tillering phenotypes.     

Inception of maleness: auxin contribution to flower masculinization in the dioecious cactus Opuntia stenopetala

In Opuntia stenopetala, flowers initiate as hermaphrodite; however, at maturity, only the stamens in male flowers and the gynoecium in female flowers become functional. At early developmental stages, growth and morphogenesis of the gynoecium in male flowers cease, forming a short style lacking stigmatic tissue at maturity. Here, an analysis of the masculinization process of this species and its relationship with auxin metabolism during gynoecium morphogenesis is presented. Histological analysis and scanning electron microscopy were performed; auxin levels were immunoanalyzed and exogenous auxin was applied to developing gynoecia. Male flower style-tissue patterning revealed morphological defects in the vascular bundles, stylar canal, and transmitting tissue. These features are similar to those observed in Arabidopsis thaliana mutant plants affected in auxin transport, metabolism, or signaling. Notably, when comparing auxin levels between male and female gynoecia from O. stenopetala at an early developmental stage, we found that they were particularly low in the male gynoecium. Consequently, exogenous auxin application on male gynoecia partially restored the defects of gynoecium development. We therefore hypothesize that, the arrest in male flower gynoecia patterning could be related to altered auxin homeostasis; alternatively, the addition of auxin could compensate for the lack of another unknown factor affecting male flower gynoecium development.     

Auxin Response Factor2 (ARF2) and its regulated homeodomain gene HB33 mediate abscisic acid response in Arabidopsis

The phytohormone abscisic acid (ABA) is an important regulator of plant development and response to environmental stresses. In this study, we identified two ABA overly sensitive mutant alleles in a gene encoding Auxin Response Factor2 (ARF2). The expression of ARF2 was induced by ABA treatment. The arf2 mutants showed enhanced ABA sensitivity in seed germination and primary root growth. In contrast, the primary root growth and seed germination of transgenic plants over-expressing ARF2 are less inhibited by ABA than that of the wild type. ARF2 negatively regulates the expression of a homeodomain gene HB33, the expression of which is reduced by ABA. Transgenic plants over-expressing HB33 are more sensitive, while transgenic plants reducing HB33 by RNAi are more resistant to ABA in the seed germination and primary root growth than the wild type. ABA treatment altered auxin distribution in the primary root tips and made the relative, but not absolute, auxin accumulation or auxin signal around quiescent centre cells and their surrounding columella stem cells to other cells stronger in arf2-101 than in the wild type. These results indicate that ARF2 and HB33 are novel regulators in the ABA signal pathway, which has crosstalk with auxin signal pathway in regulating plant growth.     

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.     

Isolation of COV1, a gene involved in the regulation of vascular patterning in the stem of Arabidopsis

The molecular mechanisms that control the ordered patterning of vascular tissue development in plants are not well understood. Several models propose a two-component system for vascular differentiation. These components include an inducer of vascular tissue development and an inhibitor that prevents the formation of vascular bundles near pre-existing bundles. We have identified two recessive allelic mutants in Arabidopsis, designated continuous vascular ring (cov1), that display a dramatic increase in vascular tissue development in the stem in place of the interfascicular region that normally separates the vascular bundles. The mutant plants exhibited relatively normal vascular patterning in leaves and cotyledons. Analysis of the interaction of cov1 with a known auxin signalling mutant and direct analysis of auxin concentrations suggests that cov1 affects vascular pattering by some mechanism that is independent of auxin. The COV1 protein is predicted to be an integral membrane protein of unknown function, highly conserved between plants and bacteria. In plants, COV1 is likely to be involved in a mechanism that negatively regulates the differentiation of vascular tissue in the stem.     

The role of hormones in apical dominance. New approaches to an old problem in plant development

The role of hormones in apical dominance has been under investigation with traditional 'spray and weigh' methods for nearly 5 decades. Even though the precision of hormone content analyses in tissue has greatly improved in recent years, there have been no significant breakthroughs in our understanding of the action mechanism of this classical developmental response. Auxin appears to inhibit axillary bud outgrowth whereas cytokinins will often promote it. Conclusive evidence for a direct role of these or other hormones in apical dominance has not been forthcoming. However, promising new tools and approaches recently have begun to be utilized. The manipulation of endogenous hormone levels via the use of transgenic plants transformed with bacterial genes ( iaaM and ipt from Agrobacterium tumefaciens and iaaL from Pseudomonas syringae pv. savastanoi ) has demonstrated powerful effects of auxin and cytokinin on axillary bud outgrowth. Also, possible auxin and cytokinin involvement of rolB and C genes from Agrobacterium rhizogenes whose activity is associated with reduced apical dominance in dicotyledons has received considerable attention. The characterization of unique mRNAs and proteins in non-growing and growing lateral buds before and after apical dominance release is helping to lay the groundwork for the elucidation of signal transduction and cell cycle regulation in this response. The use of auxin-deficient, and auxin/ethylene-resistant mutants has provided another approach for analyzing the role of these hormones. The presumed eventual employment of molecular assay systems (SAUR/GH3 promoters fused with GUS reporter gene) which are presently being developed for analyzing auxin localized in lateral buds will hopefully provide a critical test for the direct auxin inhibition hypothesis.     

Shade avoidance responses are mediated by the ATHB-2 HD-zip protein, a negative regulator of gene expression.

The ATHB-2 gene encoding an homeodomain-leucine zipper protein is rapidly and strongly induced by changes in the ratio of red to far-red light which naturally occur during the daytime under the canopy and induce in many plants the shade avoidance response. Here, we show that elevated ATHB-2 levels inhibit cotyledon expansion by restricting cell elongation in the cotyledon-length and -width direction. We also show that elevated ATHB-2 levels enhance longitudinal cell expansion in the hypocotyl. Interestingly, we found that ATHB-2-induced, as well as shade-induced, elongation of the hypocotyl is dependent on the auxin transport system. In the root and hypocotyl, elevated ATHB-2 levels also inhibit specific cell proliferation such as secondary growth of the vascular system and lateral root formation. Consistent with the key role of auxin in these processes, we found that auxin is able to rescue the ATHB-2 lateral root phenotype. We also show that reduced levels of ATHB-2 result in reciprocal phenotypes. Moreover, we demonstrate that ATHB-2 functions as a negative regulator of gene expression in a transient assay. Remarkably, the expression in transgenic plants of a derivative of ATHB-2 with the same DNA binding specificity but opposite regulatory properties results in a shift in the orientation of hypocotyl cell expansion toward radial expansion, and in an increase in hypocotyl secondary cell proliferation. A model of ATHB-2 function in the regulation of shade-induced growth responses is proposed.