HEARTBREAK Controls Post-translational Modification of INDEHISCENT to Regulate Fruit Morphology in Capsella.

Morphological variation is the basis of natural diversity and adaptation. For example, angiosperms (flowering plants) evolved during the Cretaceous period more than 100 mya and quickly colonized terrestrial habitats [1]. A major reason for their astonishing success was the formation of fruits, which exist in a myriad of different shapes and sizes [2]. Evolution of organ shape is fueled by variation in expression patterns of regulatory genes causing changes in anisotropic cell expansion and division patterns [3-5]. However, the molecular mechanisms that alter the polarity of growth to generate novel shapes are largely unknown. The heart-shaped fruits produced by members of the Capsella genus comprise an anatomical novelty, making it particularly well suited for studies on morphological diversification [6-8]. Here, we show that post-translational modification of regulatory proteins provides a critical step in organ-shape formation. Our data reveal that the SUMO protease, HEARTBREAK (HTB), from Capsella rubella controls the activity of the key regulator of fruit development, INDEHISCENT (CrIND in C. rubella), via de-SUMOylation. This post-translational modification initiates a transduction pathway required to ensure precisely localized auxin biosynthesis, thereby facilitating anisotropic cell expansion to ultimately form the heart-shaped Capsella fruit. Therefore, although variation in the expression of key regulatory genes is known to be a primary driver in morphological evolution, our work demonstrates how other processes-such as post-translational modification of one such regulator-affects organ morphology.

The power of model-to-crop translation illustrated by reducing seed loss from pod shatter in oilseed rape.

KEY MESSAGE: Elucidation of key regulators in Arabidopsis fruit patterning has facilitated knowledge-translation into crop species to address yield loss caused by premature seed dispersal (pod shatter). In the 1980s, plant scientists descended on a small weed Arabidopsis thaliana (thale cress) and developed it into a powerful model system to study plant biology. The massive advances in genetics and genomics since then have allowed us to obtain incredibly detailed knowledge on specific biological processes of Arabidopsis growth and development, its genome sequence and the function of many of the individual genes. This wealth of information provides immense potential for translation into crops to improve their performance and address issues of global importance such as food security. Here, we describe how fundamental insight into the genetic mechanism by which seed dispersal occurs in members of the Brassicaceae family can be exploited to reduce seed loss in oilseed rape (Brassica napus). We demonstrate that by exploiting data on gene function in model species, it is possible to adjust the pod-opening process in oilseed rape, thereby significantly increasing yield. Specifically, we identified mutations in multiple paralogues of the INDEHISCENT and GA4 genes in B. napus and have overcome genetic redundancy by combining mutant alleles. Finally, we present novel software for the analysis of pod shatter data that is applicable to any crop for which seed dispersal is a serious problem. These findings highlight the tremendous potential of fundamental research in guiding strategies for crop improvement.

A protein complex required for polar growth of rhizobial infection threads.

During root nodule symbiosis, intracellular accommodation of rhizobia by legumes is a prerequisite for nitrogen fixation. For many legumes, rhizobial colonization initiates in root hairs through transcellular infection threads. In Medicago truncatula, VAPYRIN (VPY) and a putative E3 ligase LUMPY INFECTIONS (LIN) are required for infection thread development but their cellular and molecular roles are obscure. Here we show that LIN and its homolog LIN-LIKE interact with VPY and VPY-LIKE in a subcellular complex localized to puncta both at the tip of the growing infection thread and at the nuclear periphery in root hairs and that the punctate accumulation of VPY is positively regulated by LIN. We also show that an otherwise nuclear and cytoplasmic exocyst subunit, EXO70H4, systematically co-localizes with VPY and LIN during rhizobial infection. Genetic analysis shows that defective rhizobial infection in exo70h4 is similar to that in vpy and lin. Our results indicate that VPY, LIN and EXO70H4 are part of the symbiosis-specific machinery required for polar growth of infection threads.

Regulatory Diversification of INDEHISCENT in the Capsella Genus Directs Variation in Fruit Morphology.

Evolution of gene-regulatory sequences is considered the primary driver of morphological variation [1-3]. In animals, the diversity of body plans between distantly related phyla is due to the differential expression patterns of conserved "toolkit" genes [4]. In plants, variation in expression domains similarly underlie most of the reported diversity of organ shape both in natural evolution and in the domestication of crops [5-9]. The heart-shaped fruit from members of the Capsella genus is a morphological novelty that has evolved after Capsella diverged from Arabidopsis ∼8 mya [10]. Comparative studies of fruit growth in Capsella and Arabidopsis revealed that the difference in shape is caused by local control of anisotropic growth [11]. Here, we show that sequence variation in regulatory domains of the fruit-tissue identity gene, INDEHISCENT (IND), is responsible for expansion of its expression domain in the heart-shaped fruits from Capsella rubella. We demonstrate that expression of this CrIND gene in the apical part of the valves in Capsella contributes to the heart-shaped appearance. While studies on morphological diversity have revealed the importance of cis-regulatory sequence evolution, few examples exist where the downstream effects of such variation have been characterized in detail. We describe here how CrIND exerts its function on Capsella fruit shape by binding sequence elements of auxin biosynthesis genes to activate their expression and ensure auxin accumulation into highly localized maxima in the fruit valves. Thus, our data provide a direct link between changes in expression pattern and altered hormone homeostasis in the evolution of morphological novelty.

NIN Acts as a Network Hub Controlling a Growth Module Required for Rhizobial Infection.

The symbiotic infection of root cells by nitrogen-fixing rhizobia during nodulation requires the transcription factor Nodule Inception (NIN). Our root hair transcriptomic study extends NIN's regulon to include Rhizobium Polar Growth and genes involved in cell wall modification, gibberellin biosynthesis, and a comprehensive group of nutrient (N, P, and S) uptake and assimilation genes, suggesting that NIN's recruitment to nodulation was based on its role as a growth module, a role shared with other NIN-Like Proteins. The expression of jasmonic acid genes in nin suggests the involvement of NIN in the resolution of growth versus defense outcomes. We find that the regulation of the growth module component Nodulation Pectate Lyase by NIN, and its function in rhizobial infection, are conserved in hologalegina legumes, highlighting its recruitment as a major event in the evolution of nodulation. We find that Nodulation Pectate Lyase is secreted to the infection chamber and the lumen of the infection thread. Gene network analysis using the transcription factor mutants for ERF Required for Nodulation1 and Nuclear Factor-Y Subunit A1 confirms hierarchical control of NIN over Nuclear Factor-Y Subunit A1 and shows that ERF Required for Nodulation1 acts independently to control infection. We conclude that while NIN shares functions with other NIN-Like Proteins, the conscription of key infection genes to NIN's control has made it a central regulatory hub for rhizobial infection.

Fruit shape diversity in the Brassicaceae is generated by varying patterns of anisotropy.

Fruits exhibit a vast array of different 3D shapes, from simple spheres and cylinders to more complex curved forms; however, the mechanism by which growth is oriented and coordinated to generate this diversity of forms is unclear. Here, we compare the growth patterns and orientations for two very different fruit shapes in the Brassicaceae: the heart-shaped Capsella rubella silicle and the near-cylindrical Arabidopsis thaliana silique. We show, through a combination of clonal and morphological analyses, that the different shapes involve different patterns of anisotropic growth during three phases. These experimental data can be accounted for by a tissue-level model in which specified growth rates vary in space and time and are oriented by a proximodistal polarity field. The resulting tissue conflicts lead to deformation of the tissue as it grows. The model allows us to identify tissue-specific and temporally specific activities required to obtain the individual shapes. One such activity may be provided by the valve-identity gene FRUITFULL, which we show through comparative mutant analysis to modulate fruit shape during post-fertilisation growth of both species. Simple modulations of the model presented here can also broadly account for the variety of shapes in other Brassicaceae species, thus providing a simplified framework for fruit development and shape diversity.

Diversification of fruit shape in the Brassicaceae family.

KEY MESSAGE: Diversity in fruit shape. Angiosperms (flowering plants) evolved during the Cretaceous Period more than 100 million years ago and quickly colonized all terrestrial habitats on the planet. A major reason for their success was the formation of fruits that would protect and nurture the developing seeds. Moreover, a massive range of diversity in fruit shape occurred during a relatively short time, which allowed for the development of ingenious ways of fertilization as well as strategies for efficient seed dispersal. The Brassicaceae family more than any exemplifies the diversity in fruit morphologies, thus providing an ideal group of plants to study how specific shapes are established. Although many genes controlling fruit patterning in the model plant Arabidopsis thaliana have been identified, the processes of carpel and fruit morphogenesis are still poorly understood. Moreover, Arabidopsis fruits are relatively simple in their structure and are therefore not ideally suited for analyzing processes of morphology determination without comparison to species with differently shaped fruits. Here, we review the diversity of fruit shape within the Brassicaceae family. As an example we describe the close relative of Arabidopsis, Capsella rubella that develops flat, heart-shaped fruits showing and highlighting its potential as a model system for research into organ shape. Recent progress in genomics including fast and cheap genome sequencing and annotation as well as development of mutant populations has opened entirely new and exciting possibilities of studying the mechanisms and processes underlying fruit formation in angiosperms.

Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease.

BACKGROUND: The RNA-guided Cas9 system represents a flexible approach for genome editing in plants. This method can create specific mutations that knock-out or alter target gene function. It provides a valuable tool for plant research and offers opportunities for crop improvement. RESULTS: We investigate the use and target specificity requirements of RNA-guided Cas9 genome editing in barley (Hordeum vulgare) and Brassica oleracea by targeting multicopy genes. In barley, we target two copies of HvPM19 and observe Cas9-induced mutations in the first generation of 23 % and 10 % of the lines, respectively. In B. oleracea, targeting of BolC.GA4.a leads to Cas9-induced mutations in 10 % of first generation plants screened. In addition, a phenotypic screen identifies T0 plants with the expected dwarf phenotype associated with knock-out of the target gene. In both barley and B. oleracea stable Cas9-induced mutations are transmitted to T2 plants independently of the T-DNA construct. We observe off-target activity in both species, despite the presence of at least one mismatch between the single guide RNA and the non-target gene sequences. In barley, a transgene-free plant has concurrent mutations in the target and non-target copies of HvPM19. CONCLUSIONS: We demonstrate the use of RNA-guided Cas9 to generate mutations in target genes of both barley and B. oleracea and show stable transmission of these mutations thus establishing the potential for rapid characterisation of gene function in these species. In addition, the off-target effects reported offer both potential difficulties and specific opportunities to target members of multigene families in crops.

Identification of a core set of rhizobial infection genes using data from single cell-types.

Genome-wide expression studies on nodulation have varied in their scale from entire root systems to dissected nodules or root sections containing nodule primordia (NP). More recently efforts have focused on developing methods for isolation of root hairs from infected plants and the application of laser-capture microdissection technology to nodules. Here we analyze two published data sets to identify a core set of infection genes that are expressed in the nodule and in root hairs during infection. Among the genes identified were those encoding phenylpropanoid biosynthesis enzymes including Chalcone-O-Methyltransferase which is required for the production of the potent Nod gene inducer 4',4-dihydroxy-2-methoxychalcone. A promoter-GUS analysis in transgenic hairy roots for two genes encoding Chalcone-O-Methyltransferase isoforms revealed their expression in rhizobially infected root hairs and the nodule infection zone but not in the nitrogen fixation zone. We also describe a group of Rhizobially Induced Peroxidases whose expression overlaps with the production of superoxide in rhizobially infected root hairs and in nodules and roots. Finally, we identify a cohort of co-regulated transcription factors as candidate regulators of these processes.

The root hair "infectome" of Medicago truncatula uncovers changes in cell cycle genes and reveals a requirement for Auxin signaling in rhizobial infection.

Nitrogen-fixing rhizobia colonize legume roots via plant-made intracellular infection threads. Genetics has identified some genes involved but has not provided sufficient detail to understand requirements for infection thread development. Therefore, we transcriptionally profiled Medicago truncatula root hairs prior to and during the initial stages of infection. This revealed changes in the responses to plant hormones, most notably auxin, strigolactone, gibberellic acid, and brassinosteroids. Several auxin responsive genes, including the ortholog of Arabidopsis thaliana Auxin Response Factor 16, were induced at infection sites and in nodule primordia, and mutation of ARF16a reduced rhizobial infection. Associated with the induction of auxin signaling genes, there was increased expression of cell cycle genes including an A-type cyclin and a subunit of the anaphase promoting complex. There was also induction of several chalcone O-methyltransferases involved in the synthesis of an inducer of Sinorhizobium meliloti nod genes, as well as a gene associated with Nod factor degradation, suggesting both positive and negative feedback loops that control Nod factor levels during rhizobial infection. We conclude that the onset of infection is associated with reactivation of the cell cycle as well as increased expression of genes required for hormone and flavonoid biosynthesis and that the regulation of auxin signaling is necessary for initiation of rhizobial infection threads.

Rhizobial infection is associated with the development of peripheral vasculature in nodules of Medicago truncatula.

Nodulation in legumes involves the coordination of epidermal infection by rhizobia with cell divisions in the underlying cortex. During nodulation, rhizobia are entrapped within curled root hairs to form an infection pocket. Transcellular tubes called infection threads then develop from the pocket and become colonized by rhizobia. The infection thread grows toward the developing nodule primordia and rhizobia are taken up into the nodule cells, where they eventually fix nitrogen. The epidermal and cortical developmental programs are synchronized by a yet-to-be-identified signal that is transmitted from the outer to the inner cell layers of the root. Using a new allele of the Medicago truncatula mutant Lumpy Infections, lin-4, which forms normal infection pockets but cannot initiate infection threads, we show that infection thread initiation is required for normal nodule development. lin-4 forms nodules with centrally located vascular bundles similar to that found in lateral roots rather than the peripheral vasculature characteristic of legume nodules. The same phenomenon was observed in M. truncatula plants inoculated with the Sinorhizobium meliloti exoY mutant, and the M. truncatula vapyrin-2 mutant, all cases where infections arrest. Nodules on lin-4 have reduced expression of the nodule meristem marker MtCRE1 and do not express root-tip markers. In addition, these mutant nodules have altered patterns of gene expression for the cytokinin and auxin markers CRE1 and DR5. Our work highlights the coordinating role that bacterial infection exerts on the developing nodule and allows us to draw comparisons with primitive actinorhizal nodules and rhizobia-induced nodules on the nonlegume Parasponia andersonii.

Genetics, evolution, and adaptive significance of the selfing syndrome in the genus Capsella.

The change from outbreeding to selfing is one of the most frequent evolutionary transitions in flowering plants. It is often accompanied by characteristic morphological and functional changes to the flowers (the selfing syndrome), including reduced flower size and opening. Little is known about the developmental and genetic basis of the selfing syndrome, as well as its adaptive significance. Here, we address these issues using the two closely related species Capsella grandiflora (the ancestral outbreeder) and red shepherd's purse (Capsella rubella, the derived selfer). In C. rubella, petal size has been decreased by shortening the period of proliferative growth. Using interspecific recombinant inbred lines, we show that differences in petal size and flower opening between the two species each have a complex genetic basis involving allelic differences at multiple loci. An intraspecific cross within C. rubella suggests that flower size and opening have been decreased in the C. rubella lineage before its extensive geographical spread. Lastly, by generating plants that likely resemble the earliest ancestors of the C. rubella lineage, we provide evidence that evolution of the selfing syndrome was at least partly driven by selection for efficient self-pollination. Thus, our studies pave the way for a molecular dissection of selfing-syndrome evolution.

SLOW MOTION is required for within-plant auxin homeostasis and normal timing of lateral organ initiation at the shoot meristem in Arabidopsis.

The regular arrangement of leaves and flowers around a plant's stem is a fascinating expression of biological pattern formation. Based on current models, the spacing of lateral shoot organs is determined by transient local auxin maxima generated by polar auxin transport, with existing primordia draining auxin from their vicinity to restrict organ formation close by. It is unclear whether this mechanism encodes not only spatial information but also temporal information about the plastochron (i.e., the interval between the formation of successive primordia). Here, we identify the Arabidopsis thaliana F-box protein SLOW MOTION (SLOMO) as being required for a normal plastochron. SLOMO interacts genetically with components of polar auxin transport, and mutant shoot apices contain less free auxin. However, this reduced auxin level at the shoot apex is not due to increased polar auxin transport down the stem, suggesting that it results from reduced synthesis. Independently reducing the free auxin level in plants causes a similar lengthening of the plastochron as seen in slomo mutants, suggesting that the reduced auxin level in slomo mutant shoot apices delays the establishment of the next auxin maximum. SLOMO acts independently of other plastochron regulators, such as ALTERED MERISTEM PROGRAM1 or KLUH/CYP78A5. We propose that SLOMO contributes to auxin homeostasis in the shoot meristem, thus ensuring a normal rate of the formation of auxin maxima and organ initiation.

SUMO E3 ligase HIGH PLOIDY2 regulates endocycle onset and meristem maintenance in Arabidopsis.

Endoreduplication involves a doubling of chromosomal DNA without corresponding cell division. In plants, many cell types transit from the mitotic cycle to the endoreduplication cycle or endocycle, and this transition is often coupled with the initiation of cell expansion and differentiation. Although a number of cell cycle regulators implicated in endocycle onset have been identified, it is still largely unknown how this transition is developmentally regulated at the whole organ level. Here, we report that a nuclear-localized SUMO E3 ligase, HIGH PLOIDY2 (HPY2), functions as a repressor of endocycle onset in Arabidopsis thaliana meristems. Loss of HPY2 results in a premature transition from the mitotic cycle to the endocycle, leading to severe dwarfism with defective meristems. HPY2 possesses an SP-RING domain characteristic of MMS21-type SUMO E3 ligases, and we show that the conserved residues within this domain are required for the in vivo and in vitro function of HPY2. HPY2 is predominantly expressed in proliferating cells of root meristems and it functions downstream of meristem patterning transcription factors PLETHORA1 (PLT1) and PLT2. These results establish that HPY2-mediated sumoylation modulates the cell cycle progression and meristem development in the PLT-dependent signaling pathway.

BIN4, a novel component of the plant DNA topoisomerase VI complex, is required for endoreduplication in Arabidopsis.

How plant organs grow to reach their final size is an important but largely unanswered question. Here, we describe an Arabidopsis thaliana mutant, brassinosteroid-insensitive4 (bin4), in which the growth of various organs is dramatically reduced. Small organ size in bin4 is primarily caused by reduced cell expansion associated with defects in increasing ploidy by endoreduplication. Raising nuclear DNA content in bin4 by colchicine-induced polyploidization partially rescues the cell and organ size phenotype, indicating that BIN4 is directly and specifically required for endoreduplication rather than for subsequent cell expansion. BIN4 encodes a plant-specific, DNA binding protein that acts as a component of the plant DNA topoisomerase VI complex. Loss of BIN4 triggers an ATM- and ATR-dependent DNA damage response in postmitotic cells, and this response coincides with the upregulation of the cyclin B1;1 gene in the same cell types, suggesting a functional link between DNA damage response and endocycle control.

LITTLE NUCLEI genes affecting nuclear morphology in Arabidopsis thaliana.

Efforts to understand nuclear organization in plant cells have received little assistance from the better-studied animal nuclei, because plant proteomes do not contain recognizable counterparts to the key animal proteins involved in nuclear organization, such as lamin nuclear intermediate filament proteins. Previous studies identified a plant-specific insoluble nuclear protein in carrot (Daucus carota), called Nuclear Matrix Constituent Protein1 (NMCP1), which contains extensive coiled-coil domains and localizes to the nuclear periphery. Here, we describe a genetic characterization of two NMCP1-related nuclear proteins in Arabidopsis thaliana, LITTLE NUCLEI1 (LINC1) and LINC2. Disruption of either gene caused a reduction in nuclear size and altered nuclear morphology. Moreover, combining linc1 and linc2 mutations had an additive effect on nuclear size and morphology but a synergistic effect on chromocenter number (reduction) and whole-plant morphology (dwarfing). The reduction in nuclear size in the linc1 linc2 double mutant was not accompanied by a corresponding change in endopolyploidy. Rather, the density of DNA packaging at all endopolyploid levels in the linc1 linc2 mutants was increased significantly. Our results indicate that the LINC coiled-coil proteins are important determinants of plant nuclear structure.

Arabidopsis SPO11-2 functions with SPO11-1 in meiotic recombination.

The Spo11 protein is a eukaryotic homologue of the archaeal DNA topoisomerase VIA subunit (topo VIA). In archaea it is involved, together with its B subunit (topo VIB), in DNA replication. However, most eukaryotes, including yeasts, insects and vertebrates, instead have a single gene for Spo11/topo VIA and no homologues for topo VIB. In these organisms, Spo11 mediates DNA double-strand breaks that initiate meiotic recombination. Many plant species, in contrast to other eukaryotes, have three homologues for Spo11/topo VIA and one for topo VIB. The homologues in Arabidopsis, AtSPO11-1, AtSPO11-2 and AtSPO11-3, all share 20-30% sequence similarity with other Spo11/topo VIA proteins, but their functional relationship during meiosis or other processes is not well understood. Previous genetic evidence suggests that AtSPO11-1 is a true orthologue of Spo11 in other eukaryotes and is required for meiotic recombination, whereas AtSPO11-3 is involved in DNA endo-reduplication as a part of the topo VI complex. In this study, we show that plants homozygous for atspo11-2 exhibit a severe sterility phenotype. Both male and female meiosis are severely disrupted in the atspo11-2 mutant, and this is associated with severe defects in synapsis during the first meiotic division and reduced meiotic recombination. Further genetic analysis revealed that AtSPO11-1 and AtSPO11-2 genetically interact, i.e. plants heterozygous for both atspo11-1 and atspo11-2 are also sterile, suggesting that AtSPO11-1 and AtSPO11-2 have largely overlapping functions. Thus, the three Arabidopsis Spo11 homologues appear to function in two discrete processes, i.e. AtSPO11-1 and AtSPO11-2 in meiotic recombination and AtSPO11-3 in DNA replication.

RHL1 is an essential component of the plant DNA topoisomerase VI complex and is required for ploidy-dependent cell growth.

How cells achieve their final sizes is a pervasive biological question. One strategy to increase cell size is for the cell to amplify its chromosomal DNA content through endoreduplication cycles. Although endoreduplication is widespread in eukaryotes, we know very little about its molecular mechanisms. Successful progression of the endoreduplication cycle in Arabidopsis requires a plant homologue of archaeal DNA topoisomerase (topo) VI. To further understand how DNA is endoreduplicated and how this process is regulated, we isolated a dwarf Arabidopsis mutant, hyp7 (hypocotyl 7), in which various large cell types that in the wild type normally endoreduplicate multiple times complete only the first two rounds of endoreduplication and stall at 8C. HYP7 encodes the RHL1 (ROOT HAIRLESS 1) protein, and sequence analysis reveals that RHL1 has similarity to the C-terminal domain of mammalian DNA topo IIalpha, another type II topo that shares little sequence homology with topo VI. RHL1 shows DNA binding activity in vitro, and we present both genetic and in vivo evidence that RHL1 forms a multiprotein complex with plant topo VI. We propose that RHL1 plays an essential role in the topo VI complex to modulate its function and that the two distantly related topos, topo II and topo VI, have evolved a common domain that extends their function. Our data suggest that plant topo II and topo VI play distinct but overlapping roles during the mitotic cell cycle and endoreduplication cycle.

A RING domain gene is expressed in different cell types of leaf trace, stem, and juvenile bundles in the stem vascular system of zinnia.

The in vitro zinnia (Zinnia elegans) mesophyll cell system, in which leaf mesophyll cells are induced to transdifferentiate into tracheary elements with high synchrony, has become an established model for studying xylogenesis. The architecture of the stem vascular system of zinnia cv Envy contains three anatomically distinct vascular bundles at different stages of development. Juvenile vascular strands of the subapical region develop into mature vascular strands with leaf trace segments and stem segments. Characteristic patterns of gene expression in juvenile, leaf trace, and stem bundles are revealed by a molecular marker, a RING domain-encoding gene, ZeRH2.1, originally isolated from a zinnia cDNA library derived from differentiating in vitro cultures. Using RNA in situ hybridization, we show that ZeRH2.1 is expressed preferentially in two specific cell types in mature zinnia stems. In leaf trace bundles, ZeRH2.1 transcript is abundant in xylem parenchyma cells, while in stem bundles it is abundant in phloem companion cells. Both of these cell types show wall ingrowths characteristic of transfer cells. In addition, ZeRH2.1 transcript is abundant in some phloem cells of juvenile bundles and in leaf palisade parenchyma. The complex and developmentally regulated expression pattern of ZeRH2.1 reveals heterogeneity in the vascular anatomy of the zinnia stem. We discuss a potential function for this gene in intercellular transport processes.

The Arabidopsis WAVY GROWTH 2 protein modulates root bending in response to environmental stimuli.

To understand how the direction of root growth changes in response to obstacles, light, and gravity, we characterized an Arabidopsis thaliana mutant, wavy growth 2 (wav2), whose roots show a short-pitch pattern of wavy growth on inclined agar medium. The roots of the wav2 mutant bent with larger curvature than those of the wild-type seedlings in wavy growth and in gravitropic and phototropic responses. The cell file rotations of the root epidermis of wav2-1 in the wavy growth pattern were enhanced in both right-handed and left-handed rotations. WAV2 encodes a protein belonging to the BUD EMERGENCE 46 family with a transmembrane domain at the N terminus and an alpha/beta-hydrolase domain at the C terminus. Expression analyses showed that mRNA of WAV2 was expressed strongly in adult plant roots and seedlings, especially in the root tip, the cell elongation zone, and the stele. Our results suggest that WAV2 is not involved in sensing environmental stimuli but that it negatively regulates stimulus-induced root bending through inhibition of root tip rotation.