Article

Ectopic Expression of WINDING 1 Leads to Asymmetrical Distribution of Auxin and a Spiral Phenotype in Rice

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Abstract

Ectopic expression of the rice WINDING 1 (WIN1) gene leads to a spiral phenotype only in shoots but not in roots. Rice WIN1 belongs to a specific class of proteins in cereal plants containing a Bric-a-Brac/Tramtrack/Broad (BTB) complex, a non-phototropic hypocotyl 3 (NPH3) domain and a coiled-coil motif. The WIN1 protein is predominantly localized to the plasma membrane, but is also co-localized to plasmodesmata, where it exhibits a punctate pattern. It is observed that WIN1 is normally expressed in roots and the shoot-root junction, but not in the rest of shoots. In roots, WIN1 is largely localized to the apical and basal sides of cells. However, upon ectopic expression, WIN1 appears on the longitudinal sides of leaf sheath cells, correlated with the appearance of a spiral phenotype in shoots. Despite the spiral phenotype, WIN1-overexpressing plants exhibit a normal phototropic response. Although treatments with exogenous auxins or a polar auxin transport inhibitor do not alter the spiral phenotype, the excurvature side has a higher auxin concentration than the incurvature side. Furthermore, actin filaments are more prominent in the excurvature side than in the incurvature side, which correlates with cell size differences between these two sides. Interestingly, ectopic expression of WIN1 does not cause either unequal auxin distribution or actin filament differences in roots, so a spiral phenotype is not observed in roots. The action of WIN1 appears to be different from that of other proteins causing a spiral phenotype, and it is likely that WIN1 is involved in 1-N-naphthylphthalamic acid-insensitive plasmodesmata-mediated auxin transport.

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... After overexpression of WIN1 (WINDING 1) gene in rice, the helical phenotype and auxin were unevenly distributed in seedlings. Exogenous application of auxin polar transport inhibitor did not affect the helical phenotype, and overexpression of WIN1 did not affect the phototropism (Cheng et al., 2017). SlBTB5, a member of NRL gene family in tomato cultivar M82, is highly expressed in roots and flowers and down-regulated under environmental stress. ...
... Only two members of NRL gene family, CPT1 and WIN1, were identified in rice. CPT1 has been proved to be involved in the phototropism of coleoptile and the lateral transport of auxin, while WIN1 is related to auxin transport, but does not participate in the phototropism of coleoptile and root (Haga et al., 2005, Cheng et al., 2017. ...
... On the other hand, the seed size of the two lines was smaller than that of wild type seeds. However, OsNRL7 (OsWIN1) was expressed in many rice tissues including seeds and embryos, indicating that OsNRL7 (OsWIN1) may be involved in the regulation of rice seed size (Cheng et al., 2017). Combined with tissue expression analysis and evolutionary relationship analysis, it was found that genes similar to OsNRL3 (OsCPT1), namely OsNRL1 and OsNRL15, and OsNRL7 (OsWIN1), which are OsNRL6, OsNRL7, OsNRL22, OsNRL23, OsNRL24 and OsNRL25 in rice NRL family genes may play a role in physiological activities such as phototropism and auxin transport in plants. ...
... focusing on leaf development also uncovered genes involved in controlling growth direction (Fig. 1e, f; (Buschmann et al., 2004)). Eventually helical growth was observed in rice (Cheng et al., 2017;Sunohara et al., 2009) and the analysis of herbicide resistance revealed helical growth in the monocot Lolium rigidum (Chu et al., 2018). Table 1 lists detailed information concerning plants showing helical growth as mentioned in this review. ...
... Bric-a-Brac/NPH3 Oryza sativa plasma membrane L/R alt. leaves oe (Cheng et al., 2017) ccepted Article ...
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Many plant mutants are known that exhibit some degree of helical growth. This “twisted” phenotype has arisen frequently in mutant screens of model organisms but it is also found in cultivars of ornamental plants including trees. The phenomenon, in many cases, is based on defects in cell expansion symmetry. Any complete model to explain the anisotropy of plant cell growth must ultimately explain how helical cell expansion comes into existence – and how it is normally avoided. While the mutations observed in model plants mainly point to the microtubule system, additional affected components involve cell wall functions, auxin transport and more. Evaluation of published data suggests a two‐way mechanism to underlie the helical growth phenomenon: there is, apparently, a microtubular component that determines handedness, but there is also an influence arising in the cell wall that feeds back into the cytoplasm and affects cellular handedness. This is supported by recent reports demonstrating the involvement of the cell wall integrity pathway. In addition there is mounting evidence that calcium is an important relayer of signals relating to the symmetry of cell expansion. These concepts suggest experimental approaches to untangle the phenomenon of helical cell expansion in plant mutants. This article is protected by copyright. All rights reserved.
... Double mutants of Auxin Response Factor 3 (ARF3)/ETT and the closely ETT-related gene ARF4 in Arabidopsis exhibit leaf up-curling [38]. Ectopic overexpression of the WINDING 1 (WIN1) gene in rice leads to a higher auxin concentration distribution in leaf sheath excurvature side cells, and a leaf spiral phenotype [39]. Mutation of YUCCA6, encoding putative flavin monooxygenase enzymes, leads to excessive production of auxin and curled leaves phenotype of Arabidopsis [40,41]. ...
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... But other morphological screens focusing, for example, on leaf development, also uncovered genes involved in controlling growth direction (Fig. 1e,f;Buschmann et al., 2004). Eventually helical growth was observed in rice (Sunohara et al., 2009;Cheng et al., 2017) and the analysis of herbicide resistance revealed helical growth in the monocot Lolium rigidum (Chu et al., 2018). Table 1 lists detailed information concerning plants showing helical growth as mentioned in this review. ...
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This work is concerned with the rules determining the place of joining of two vascular strands. Auxin can induce the differentiation of vascular tissue, and this fact is used here for an experimental study of the spatial interactions of vascular strands. Differentiated vascular tissue whose source of auxin has been removed attracts newly induced vascular strands. This attraction is expressed in the joining of the new strands to the pre-existing vascular tissue. Differentiated vascular tissue which is well supplied with auxin inhibits rather than attracts the formation of new vascular strands in its vicinity. Experiments on pea apices have extended these results to naturally induced vascular strands. It is shown that when a leaf primordium is damaged at an early age its vascular strands are joined by the strands induced by new leaves, and the contacts may be formed across the leaf gap. The joining of the vascular strands is, therefore, much closer to the leaf than is normal and this is probably due to the reduction in the supply of auxin from the damaged leaf to its vascular traces. It is also shown that when a lateral bud grows its vascular traces join preferentially the vascular system leading to organs which have been removed. These vascular traces of a bud specifically avoid the vascular system of a leaf or a shoot which is still growing and producing auxin. These results are discussed in reference to the relation between the vascular system and phyllotaxis and to the existence of leaf gaps.
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Polar transport of auxin has been identified as a central element of pattern formation. To address the underlying cellular mechanisms, we use the tobacco cell line (Nicotiana tabacum L. cv. Bright Yellow 2; BY-2) as model. We showed previously that cell divisions within a cell file are synchronized by polar auxin flow, linked to the organization of actin filaments (AF) which, in turn, is modified via actin-binding proteins (ABPs). From a preparatory study for disturbed division synchrony in cell lines overexpressing different ABPs, we identified the actin depolymerizing factor 2 (ADF2). A cell line overexpressing GFP-NtADF2 was specifically affected in division synchrony. The cell division pattern could be rescued by addition of Phosphatidylinositol 4,5-bisphosphate (PIP2) or by phalloidin. These observations allow to draw first conclusions on the pathway linking auxin signalling via actin reorganization to synchronized cell division placing the regulation of cortical actin turnover by ADF2 into the focus.
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Methods for alignment of protein sequences typically measure similarity by using a substitution matrix with scores for all possible exchanges of one amino acid with another. The most widely used matrices are based on the Dayhoff model of evolutionary rates. Using a different approach, we have derived substitution matrices from about 2000 blocks of aligned sequence segments characterizing more than 500 groups of related proteins. This led to marked improvements in alignments and in searches using queries from each of the groups.
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At only 50 nm in diameter, plasmodesmata (PD) are below the limit of resolution of conventional light microscopy. Consequently, much of our current interpretation of the substructure of PD is derived from transmission electron microscopy. However, PD can be imaged with alternative techniques, including field emission scanning electron microscopy and 'super-resolution' imaging approaches such as 3D-structured illumination microscopy. This review considers the methods currently available for studying PD and focuses on the boundary between light- and electron-based imaging approaches.
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The existence of auxin as a mobile growth regulator was famously inferred by Charles and Frances Darwin, as described in their 1880 book, The Power of Movement in Plants (Darwin and Darwin, 1880). However, auxin was not isolated until much later by Went (1926). Its first ever mention in Plant Physiology was in Went’s obituary, written by George Peirce, which credits Went with having “introduced into plant physiology the general conception of regulators or coordinators” (Peirce, 1936, p. 222), exemplified by the enduring value of the Cholodny-Went hypothesis, which posits that dynamic redistribution of auxin directs tropic growth (Whippo and Hangarter, 2006). Peirce continues, “ The conception of hormones is now common, and some day it may become clear!” (p. 222). Three-quarters of a century later, the picture is still rather hazy, an enticing destination shimmering indistinctly on the horizon, but it is at least in view. Perhaps it is the vanity of every generation to imagine enlightenment is just around the corner. But I think there is no better time to be an auxin biologist. The past few decades of auxin biology have been primarily occupied with nuts and bolts and other component parts. A formidable cast of players has been assembled: receptors, transporters, synthesizers, inactivators (for recent reviews, see Chapman and Estelle, 2009; Lokerse and Weijers, 2009; Petrasek and Friml, 2009; Zhao, 2010). There are surely still some important components missing, but nonetheless, a noticeable shift in the main focus has now begun, toward understanding how the parts act and interact to “regulate and co-ordinate,” as envisaged by Went (Peirce, 1936, p. 222). How do whole plant-level patterns and behaviors emerge from the actions and interactions of the molecular components of the auxin machinery? Striking and pervasive features of the answer to this question are the extraordinary self-organizing and self-regulating properties of auxin biology, which characterize auxin action at every level (Benjamins and Scheres, 2008; Jaillais and Chory, 2010).
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The classic phytohormones cytokinin and auxin play essential roles in the maintenance of stem-cell systems embedded in shoot and root meristems, and exhibit complex functional interactions. Here we show that the activity of both hormones directly converges on the promoters of two A-type ARABIDOPSIS RESPONSE REGULATOR (ARR) genes, ARR7 and ARR15, which are negative regulators of cytokinin signalling and have important meristematic functions. Whereas ARR7 and ARR15 expression in the shoot apical meristem (SAM) is induced by cytokinin, auxin has a negative effect, which is, at least in part, mediated by the AUXIN RESPONSE FACTOR5/MONOPTEROS (MP) transcription factor. Our results provide a mechanistic framework for hormonal control of the apical stem-cell niche and demonstrate how root and shoot stem-cell systems differ in their response to phytohormones.
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The directional transport of the plant hormone auxin depends on transcellular gradients of auxin-efflux carriers that continuously cycle between plasma membrane and intracellular compartments. This cycling has been proposed to depend on actin filaments. However, the role of actin for the polarity of auxin transport has been disputed. To get insight into this question, actin bundling was induced by overexpression of the actin-binding domain of talin in tobacco BY-2 cells and in rice plants. This bundling can be reverted by addition of auxins, which allows to address the role of actin organization on the flux of auxin. In both systems, the reversion of a normal actin configuration can be restored by addition of exogenous auxins and this fully restores the respective auxin-dependent functions. These findings lead to a model of a self-referring regulatory circuit between polar auxin transport and actin organization. To further dissect the actin-auxin oscillator, we used photoactivated release of caged auxin in tobacco cells to demonstrate that auxin gradients can be manipulated at a subcellular level.
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The directional transport of the plant hormone auxin has been identified as central element of axis formation and patterning in plants. This directionality of transport depends on gradients, across the cell, of auxin-efflux carriers that continuously cycle between plasma membrane and intracellular compartments. This cycling has been proposed to depend on actin filaments. However, the role of actin for the polarity of auxin transport has been disputed. The organization of actin, in turn, has been shown to be under control of auxin. By overexpression of the actin-binding protein talin, we have generated transgenic rice (Oryza sativa) lines, where actin filaments are bundled to variable extent and, in consequence, display a reduced dynamics. We show that this bundling of actin filaments correlates with impaired gravitropism and reduced longitudinal transport of auxin. We can restore a normal actin configuration by addition of exogenous auxins and restore gravitropism as well as polar auxin transport. This rescue is mediated by indole-3-acetic acid and 1-naphthyl acetic acid but not by 2,4-dichlorophenoxyacetic acid. We interpret these findings in the context of a self-referring regulatory circuit between polar auxin transport and actin organization. This circuit might contribute to the self-amplification of auxin transport that is a central element in current models of auxin-dependent patterning.
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We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.
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We have constructed a library in Escherichia coli of mutant gfp genes (encoding green fluorescent protein, GFP) expressed from a tightly regulated inducible promoter. We introduced random amino acid (aa) substitutions in the twenty aa flanking the chromophore Ser-Tyr-Gly sequence at aa 65-67. We then used fluorescence-activated cell sorting (FACS) to select variants of GFP that fluoresce between 20-and 35-fold more intensely than wild type (wt), when excited at 488 nm. Sequence analysis reveals three classes of aa substitutions in GFP. All three classes of mutant proteins have highly shifted excitation maxima. In addition, when produced in E. coli, the folding of the mutant proteins is more efficient than folding of wt GFP. These two properties contribute to a greatly increased (100-fold) fluorescence intensity, making the mutants useful for a number of applications.
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A highly active synthetic auxin response element (AuxRE), referred to as DR5, was created by performing site-directed mutations in a natural composite AuxRE found in the soybean GH3 promoter. DR5 consisted of tandem direct repeats of 11 bp that included the auxin-responsive TGTCTC element. The DR5 AuxRE showed greater auxin responsiveness than a natural composite AuxRE and the GH3 promoter when assayed by transient expression in carrot protoplasts or in stably transformed Arabidopsis seedlings, and it provides a useful reporter gene for studying auxin-responsive transcription in wild-type plants and mutants. An auxin response transcription factor, ARF1, bound with specificity to the DR5 AuxRE in vitro and interacted with Aux/IAA proteins in a yeast two-hybrid system. Cotransfection experiments with natural and synthetic AuxRE reporter genes and effector genes encoding Aux/IAA proteins showed that overexpression of Aux/IAA proteins in carrot protoplasts resulted in specific repression of TGTCTC AuxRE reporter gene expression.
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New plant genes are being discovered at a rapid pace. Yet, in most cases, their precise function remains elusive. The recent advent of recombinational cloning techniques has significantly improved our ability to investigate gene functions systematically. For example, proteins fused with diverse fluorescent tags can be expressed at will using versatile cloning cassettes. In addition, novel binary T-DNA vectors are now available to assemble multiple DNA fragments simultaneously, which greatly facilitate plant cell and protein engineering.
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The question of how many angels can dance on the point of a pin has been a touchstone in the long-running theological debate on the nature of angels. The plant hormone auxin is a less supernatural messenger, but there is an equally interesting debate about the nature of its extraordinary powers in regulating plant development. The debate about auxin also centers on PINs, in this case a family of membrane-spanning proteins that are required for auxin efflux from cells (reviewed in Paponov et al. [2005]).
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Polar transport of auxin is essential for normal plant growth and development. On a cellular level, directional auxin transport is primarily controlled by an efflux carrier complex that is characterized by the PIN-FORMED (PIN) family of proteins. Detailed developmental studies of PIN distribution and subcellular localization have been combined with the analysis of changes in localized auxin levels to map PIN-mediated auxin movement throughout Arabidopsis tissues. Plant orthologs of mammalian multidrug-resistance/P-glycoproteins (MDR/PGPs) also function in auxin efflux. MDR/PGPs appear to stabilize efflux complexes on the plasma membrane and to function as ATP-dependent auxin transporters, with the specificity and directionality of transport being provided by interacting PIN proteins.
Plasmodesmata as a supracellular control network in plants
  • W.J. Lucas
  • J.Y. Lee
Lucas, W.J. and Lee, J.Y. (2004) Plasmodesmata as a supracellular control network in plants. Nat. Rev. Mol. Cell Biol. 5: 712-726.
A matter of size: developmental control of organ size in plants
  • Y. Mizukami
Mizukami, Y. (2001) A matter of size: developmental control of organ size in plants. Curr. Opin. Plant Biol. 4: 533-539.
  • A W Robards
Robards, A.W. (1975) Plasmodesmata. Annu. Rev. Plant Physiol. 26: 17.