Gravity profoundly influences plant growth and development. Plants respond to changes in orientation by using gravitropic responses to modify their growth. Cholodny and Went hypothesized over 80 years ago that plants bend in response to a gravity stimulus by generating a lateral gradient of a growth regulator at an organ's apex, later found to be auxin. Auxin regulates root growth by targeting Aux/IAA repressor proteins for degradation. We used an Aux/IAAbased reporter, domain II (DII)-VENUS, in conjunction with a mathematical model to quantify auxin redistribution following a gravity stimulus. Our multidisciplinary approach revealed that auxin is rapidly redistributed to the lower side of the root within minutes of a 908 gravity stimulus. Unexpectedly, auxin asymmetry was rapidly lost as bending root tips reached an angle of 408 to the horizontal. We hypothesize roots use a "tipping point" mechanism that operates to reverse the asymmetric auxin flow at the midpoint of root bending. These mechanistic insights illustrate the scientific value of developing quantitative reporters such as DII-VENUS in conjunction with parameterized mathematical models to provide high-resolution kinetics of hormone redistribution.environmental sensing | systems biology R oot gravitropism has fascinated researchers since Knight (1) and Darwin (2). More recently, reorientation of Arabidopsis seedlings has been shown to trigger the asymmetric release of the growth regulator auxin from gravity-sensing columella cells at the root apex (Fig. 1A) (3-5). The resulting lateral auxin gradient is hypothesized to drive a differential growth response, where cell expansion on the lower side of the elongation zone is reduced relative to the upper side, causing the root to bend downward (6-8). Despite representing one of the oldest hypotheses in plant biology, key questions about auxin-regulated root gravitropism remain to be experimentally determined. How rapidly does the lateral auxin gradient form? Is this timescale consistent with the theory that auxin redistribution drives root bending? How long does the lateral auxin gradient persist? What triggers auxin redistribution to return to equal levels?Our understanding of gravity-induced auxin redistribution has been limited by the tools available to monitor auxin concentrations at high spatiotemporal resolution. Currently, the most widely used tools to follow auxin distribution in tissues are auxin-inducible reporters such as DR5::GFP (3, 4). However, as an output of the auxin response pathway (Fig. 1B), the activity of the DR5 reporter does not directly relate to endogenous auxin abundance, but also depends on additional parameters including local auxin signaling capacities and rates of transcription and translation (Fig. 1B). In practice, these intermediate processes confer a time delay of ∼1.5-2 h between changes in auxin abundance and DR5 reporter activity (9, 4), making it difficult to quantify the speed and magnitude of fold changes in auxin distribution during a root gravitropic response.Auxi...

Auxin represents a key signal in plants, regulating almost every aspect of their growth and development. Major breakthroughs have been made dissecting the molecular basis of auxin transport, perception, and response. In contrast, how plants control the metabolism and homeostasis of the major form of auxin in plants, indole-3-acetic acid (IAA), remains unclear. In this paper, we initially describe the function of the Arabidopsis thaliana gene DIOXYGENASE FOR AUXIN OXIDATION 1 (AtDAO1). Transcriptional and translational reporter lines revealed that AtDAO1 encodes a highly root-expressed, cytoplasmically localized IAA oxidase. Stable isotope-labeled IAA feeding studies of loss and gain of function AtDAO1 lines showed that this oxidase represents the major regulator of auxin degradation to 2-oxoindole-3-acetic acid (oxIAA) in Arabidopsis. Surprisingly, AtDAO1 loss and gain of function lines exhibited relatively subtle auxin-related phenotypes, such as altered root hair length. Metabolite profiling of mutant lines revealed that disrupting AtDAO1 regulation resulted in major changes in steady-state levels of oxIAA and IAA conjugates but not IAA. Hence, IAA conjugation and catabolism seem to regulate auxin levels in Arabidopsis in a highly redundant manner. We observed that transcripts of AtDOA1 IAA oxidase and GH3 IAA-conjugating enzymes are auxin-inducible, providing a molecular basis for their observed functional redundancy. We conclude that the AtDAO1 gene plays a key role regulating auxin homeostasis in Arabidopsis, acting in concert with GH3 genes, to maintain auxin concentration at optimal levels for plant growth and development.Arabidopsis thaliana | IAA degradation | oxidase | dioxygenase | root hair elongation D istinct indole-3-acetic acid (IAA) conjugation and degradation pathways operate to maintain optimal auxin concentrations for plant growth and developmental processes. There are three major forms of auxin conjugates identified in diverse plants: ester-linked IAA-sugar conjugates, amide-linked IAA-amino acid conjugates, and amide-linked IAA peptide and protein conjugates (reviewed in ref.

We have developed a method for the colocalization of xyloglucan endotransglycosylase (XET) activity and the donor substrates to which it has access in situ and in vivo. Sulforhodamine conjugates of xyloglucan oligosaccharides (XGOSRs), infiltrated into the tissue, act as acceptor substrate for the enzyme; endogenous xyloglucan acts as donor substrate. Incorporation of the XGO-SRs into polymeric products in the cell wall yields an orange fluorescence indicative of the simultaneous colocalization, in the same compartment, of active XET and donor xyloglucan chains. The method is specific for XET, as shown by competition experiments with nonfluorescent acceptor oligosaccharides, by negligible reaction with cello-oligosaccharide-SR conjugates that are not XET acceptor substrates, by heat lability, and by pH optimum. Thin-layer chromatographic analysis of remaining unincorporated XGO-SRs showed that these substrates are not extensively hydrolyzed during the assays. A characteristic distribution pattern was found in Arabidopsis and tobacco roots: in both species, fluorescence was most prominent in the cell elongation zone of the root. Proposed roles of XET that include cell wall loosening and integration of newly synthesized xyloglucans could thus be supported. INTRODUCTIONThe primary cell walls of flowering plants consist fundamentally of a framework of cellulose microfibrils embedded in a matrix of hemicellulose, pectins, and structural proteins (Carpita and Gibeaut, 1993; Brett and Waldron, 1996). Xyloglucan, the major hemicellulosic polysaccharide in the primary cell wall matrix of dicots, consists of a backbone of ␤ -(1 → 4)-linked D -glucose residues, the majority of which are ␣ -D -xylosylated at O-6. Some xylose residues are further substituted by galactosyl and fucosyl-galactosyl groups. Other, minor carbohydrate side chains and O-acetyl groups are also present (Fry, 1989a; Hayashi, 1989). Because xyloglucans can form tight hydrogen bonds with cellulose microfibrils (Valent and Albersheim, 1974; Hayashi et al., 1987 Hayashi et al., , 1994a Hayashi et al., , 1994b Hayashi, 1989), they may thereby tether adjacent microfibrils (Fry, 1989b). A proportion of the xyloglucan molecules are covalently attached to acidic pectins (Thompson and Fry, 2000). Xyloglucans also serve as storage polysaccharides in some seeds (Edwards et al., 1985).For plant cells to expand, cellulose microfibrils in parallel alignment need to move apart or past one another, and this movement may create the possibility for newly synthesized xyloglucan molecules to become hydrogen-bonded (Fry, 1989b). Because xyloglucan tethers are thought to be the principal tension-bearing molecules in the cell wall, breaking of the tethers has been proposed as a mechanism for achieving reversible cell wall loosening in elongating tissue without compromising strength (Fry, 1989b; Hayashi, 1989; Hoson et al., 1991). Although the cell wall contains numerous enzymes that can modify polysaccharides (Fry, 1995), xyloglucan endotransglycosylases (XETs) seem well...

In the growing apex of Arabidopsis thaliana primary roots, cells proceed through four distinct phases of cellular activities. These zones and their boundaries can be well defined based on their characteristic cellular activities. The meristematic zone comprises, and is limited to, all cells that undergo mitotic divisions. Detailed in vivo analysis of transgenic lines reveals that, in the Columbia-0 ecotype, the meristem stretches up to 200 microm away from the junction between root and root cap (RCJ). In the transition zone, 200 to about 520 microm away from the RCJ, cells undergo physiological changes as they prepare for their fast elongation. Upon entering the transition zone, they progressively develop a central vacuole, polarize the cytoskeleton and remodel their cell walls. Cells grow slowly during this transition: it takes ten hours to triplicate cell length from 8.5 to about 35 microm in the trichoblast cell files. In the fast elongation zone, which covers the zone from 520 to about 850 microm from the RCJ, cell length quadruplicates to about 140 microm in only two hours. This is accompanied by drastic and specific cell wall alterations. Finally, root hairs fully develop in the growth terminating zone, where root cells undergo a minor elongation to reach their mature lengths.

The results provide evidence that XTHs can act as cell wall-loosening enzymes.

Summary We focused on a developmentally regulated growth acceleration in the dark‐grown Arabidopsis hypocotyl to study the role of changes in cell wall metabolism in the control of cell elongation. To this end, precise transcriptome analysis on dissected dark‐grown hypocotyls, Fourier transform infrared (FT‐IR) microspectroscopy and kinematic analysis were used. Using a cellulose synthesis inhibitor, we showed that the growth acceleration marks a developmental transition during which growth becomes uncoupled from cellulose synthesis. We next investigated the cellular changes that take place during this transition. FT‐IR microspectroscopy revealed significant changes in cell wall composition during, but not after, the growth acceleration. Transcriptome analysis suggested a role for cell wall remodeling, in particular pectin modification, in this growth acceleration. This was confirmed by the overexpression of a pectin methylesterase inhibitor, which caused a delay in the growth acceleration. This study shows that the acceleration of cell elongation marks a developmental transition in dark‐grown hypocotyl cells and supports a role for pectin de‐methylesterification in the timing of this event.

Xyloglucan endotransglucosylase/hydrolases (XTHs) are a class of enzymes that are capable of splitting and reconnecting xyloglucan molecules, and are implicated in the construction and restructuring of the cellulose/xyloglucan framework. Thirty-three members of the XTH gene family are found in the genome of Arabidopsis thaliana, but their roles remain unclear. Here, we describe the tissue-specific and growth stage-dependent expression profiles of promoter::GUS fusion constructs for four Arabidopsis XTH genes, AtXTH17, AtXTH18, AtXTH19 and AtXTH20, which are phylogenetically closely related to one another. AtXTH17 and AtXTH18 were expressed in all cell types in the elongating and differentiating region of the root, while AtXTH19 was expressed in the apical dividing and elongating regions, as well as in the differentiation zone, and was up-regulated by auxin. In contrast, AtXTH20 was expressed specifically in vascular tissues in the basal mature region of the root. This expression analysis also disclosed cis-regulatory sequences that are conserved among the four genes, and are responsible for the root-specific expression profile. These results indicate that the four XTH genes, which were generated by gene duplication, have diversified their expression profile within the root in such a way as to take responsibility for particular physiological roles in the cell wall dynamics.

Plants are sessile organisms rooted in one place. The soil resources that plants require are often distributed in a highly heterogeneous pattern. To aid foraging, plants have evolved roots whose growth and development are highly responsive to soil signals. As a result, 3D root architecture is shaped by myriad environmental signals to ensure resource capture is optimised and unfavourable environments are avoided. The first signals sensed by newly germinating seeds - gravity and light - direct root growth into the soil to aid seedling establishment. Heterogeneous soil resources, such as water, nitrogen and phosphate, also act as signals that shape 3D root growth to optimise uptake. Root architecture is also modified through biotic interactions that include soil fungi and neighbouring plants. This developmental plasticity results in a 'custom-made' 3D root system that is best adapted to forage for resources in each soil environment that a plant colonises.