Effects of LAZY family genes on shoot gravitropism in Lotus japonicus.

Plant architecture is an important agronomic trait to determine the biomass and sward structure of forage grass. The IGT family plays a pivotal role in plant gravitropism, encompassing both the gravitropic response and the modulation of plant architecture. We have previously shown that LjLAZY3, one of the IGT genes, plays a distinct role in root gravitropism in L. japonicus. However, the function of LAZY proteins on shoot gravitropism in this species is poorly understood. In this study, we identified nine IGT genes in the L. japonicus genome, which have been categorized into four clades based on the phylogenetic relationships of IGT proteins from 18 legumes: LAZY1, NGR (NEGATIVE GRAVITROPIC RESPONSE OF ROOTS), IGT-LIKE, and TAC1. We found that LAZY genes in the first three clades have demonstrated distinct role for modulating plant gravitropism in L. japonicus with specific impacts as follows. Mutation of the LAZY1 gene, LjLAZY1, defected the gravitropic response of hypocotyl without impacting the main stem's branch angle. In contrast, the overexpression of the NGR gene, LjLAZY3, substantially modulated the shoot's gravitropism, leading to narrower lateral branch angles. Additionally, it enhanced the shoots' gravitropic response. The overexpression of another NGR gene, LjLAZY4, specifically reduced the main stem's branch angle and decreased plant stature without affecting the shoot gravitropic response. The phenotype of IGT-LIKE gene LjLAZY2 overexpression is identical to that of LjLAZY4. While overexpression of the IGT-LIKE gene LjLAZY5 did not induce any observable changes in branch angle, plant height, or gravitropic response. Furthermore, the LjLAZYs were selectively interacted with different BRXL and RLD proteins, which should the important factor to determine their different functions in controlling organ architecture in L. japonicus. Our results deepen understanding of the LjLAZY family and its potential for plant architecture improvement in L. japonicus.

PagKNAT2/6b regulates tension wood formation and gravitropism by targeting cytokinin metabolism.

Tension wood is a specialized xylem tissue associated with gravitropism in angiosperm trees. However, few regulators of tension wood formation have been identified. The molecular mechanisms underpinning tension wood formation remain elusive. Here, we report that a Populus KNOTTED-like homeobox gene, PagKNAT2/6b, is involved in tension wood formation and gravity response. Transgenic poplar plants overexpressing PagKNAT2/6b displayed more sensitive gravitropism than controls, as indicated by increased stem curvature. Microscopic examination revealed greater abundance of fibre cells with a gelatinous cell wall layer (G-layer) and asymmetric growth of secondary xylem in PagKNAT2/6b overexpression lines. Conversely, PagKNAT2/6b dominant repression plants exhibited decreased tension wood formation and reduced response to gravity stimulation. Moreover, sensitivity to gravity stimulation showed a negative relationship with development stage. Expression of genes related to growth and senescence was affected in PagKNAT2/6b transgenic plants. More importantly, transcription activation and electrophoretic mobility shift assays suggested that PagKNAT2/6b promotes the expression of cytokinin metabolism genes. Consistently, cytokinin content was increased in PagKNAT2/6b overexpression plants. Therefore, PagKNAT2/6b is involved in gravitropism and tension wood formation, likely via modulation of cytokinin metabolism.

Ethylene regulates auxin-mediated root gravitropic machinery and controls root angle in cereal crops.

Root angle is a critical factor in optimizing the acquisition of essential resources from different soil depths. The regulation of root angle relies on the auxin-mediated root gravitropism machinery. While the influence of ethylene on auxin levels is known, its specific role in governing root gravitropism and angle remains uncertain, particularly when Arabidopsis (Arabidopsis thaliana) core ethylene signaling mutants show no gravitropic defects. Our research, focusing on rice (Oryza sativa L.) and maize (Zea mays), clearly reveals the involvement of ethylene in root angle regulation in cereal crops through the modulation of auxin biosynthesis and the root gravitropism machinery. We elucidated the molecular components by which ethylene exerts its regulatory effect on auxin biosynthesis to control root gravitropism machinery. The ethylene-insensitive mutants ethylene insensitive2 (osein2) and ethylene insensitive like1 (oseil1), exhibited substantially shallower crown root angle compared to the wild type. Gravitropism assays revealed reduced root gravitropic response in these mutants. Hormone profiling analysis confirmed decreased auxin levels in the root tips of the osein2 mutant, and exogenous auxin (NAA) application rescued root gravitropism in both ethylene-insensitive mutants. Additionally, the auxin biosynthetic mutant mao hu zi10 (mhz10)/tryptophan aminotransferase2 (ostar2) showed impaired gravitropic response and shallow crown root angle phenotypes. Similarly, maize ethylene-insensitive mutants (zmein2) exhibited defective gravitropism and root angle phenotypes. In conclusion, our study highlights that ethylene controls the auxin-dependent root gravitropism machinery to regulate root angle in rice and maize, revealing a functional divergence in ethylene signaling between Arabidopsis and cereal crops. These findings contribute to a better understanding of root angle regulation and have implications for improving resource acquisition in agricultural systems.

Transcription factor OsWRKY72 controls rice leaf angle by regulating LAZY1-mediated shoot gravitropism.

Leaf angle is a major trait of ideal architecture, which is considered to influence rice (Oryza sativa) cultivation and grain yield. Although a few mutants with altered rice leaf inclination angles have been reported, the underlying molecular mechanism remains unclear. In this study, we showed that a WRKY transcription factor gene, OsWRKY72, was highly expressed in the leaf sheath and lamina joint. Phenotypic analyses showed that oswrky72 mutants had smaller leaf angles than the wild type, while OsWRKY72 overexpression lines exhibited an increased leaf angle. This observation suggests that OsWRKY72 functions as a positive regulator, promoting the enlargement of the leaf angle. Our bioinformatics analysis identified LAZY1 as the downstream gene of OsWRKY72. Electrophoretic mobility shift assays and dual-luciferase analysis revealed that OsWRKY72 directly inhibited LAZY1 by binding to its promoter. Moreover, knocking out OsWRKY72 enhanced shoot gravitropism, which contrasted with the phenotype of lazy1 plants. These results imply that OsWRKY72 regulates the leaf angle through gravitropism by reducing the expression of LAZY1. In addition, OsWRKY72 could directly regulate the expression of other leaf angle-related genes such as FLOWERING LOCUS T-LIKE 12 (OsFTL12) and WALL-ASSOCIATED KINASE 11 (OsWAK11). Our study indicates that OsWRKY72 contributes positively to the expansion of the leaf angle by interfering with shoot gravitropism in rice.

Soft Robots with Plant-Inspired Gravitropism Based on Fluidic Liquid Metal.

Plants can autonomously adjust their growth direction based on the gravitropic response to maximize energy acquisition, despite lacking nerves and muscles. Endowing soft robots with gravitropism may facilitate the development of self-regulating systems free of electronics, but remains elusive. Herein, acceleration-regulated soft actuators are described that can respond to the gravitational field by leveraging the unique fluidity of liquid metal in its self-limiting oxide skin. The soft actuator is obtained by magnetic printing of the fluidic liquid metal heater circuit on a thermoresponsive liquid crystal elastomer. The Joule heat of the liquid metal circuit with gravity-regulated resistance can be programmed by changing the actuator's pose to induce the flow of liquid metal. The actuator can autonomously adjust its bending degree by the dynamic interaction between its thermomechanical response and gravity. A gravity-interactive soft gripper is also created with controllable grasping and releasing by rotating the actuator. Moreover, it is demonstrated that self-regulated oscillation motion can be achieved by interfacing the actuator with a monostable tape spring, allowing the electronics-free control of a bionic walker. This work paves the avenue for the development of liquid metal-based reconfigurable electronics and electronics-free soft robots that can perceive gravity or acceleration.

Defects in the cell wall and its deposition caused by loss-of-function of three RLKs alter root hydrotropism in Arabidopsis thaliana.

Root tips can sense moisture gradients and grow into environments with higher water potential. This process is called root hydrotropism. Here, we report three closely related receptor-like kinases (RLKs) that play critical roles in root hydrotropism: ALTERED ROOT HYDROTROPIC RESPONSE 1 (ARH1), FEI1, and FEI2. Overexpression of these RLKs strongly reduce root hydrotropism, but corresponding loss-of-function mutants exhibit an increased hydrotropic response in their roots. All these RLKs show polar localization at the plasma membrane regions in root tips. The biosynthesis of the cell wall, cutin, and wax (CCW) is significantly impaired in root tips of arh1-2 fei1-C fei2-C. A series of known CCW mutants also exhibit increased root hydrotropism and reduced osmotic tolerance, similar to the characteristics of the triple mutant. Our results demonstrat that the integrity of the cell wall, cutin, and root cap wax mediate a trade-off between root hydrotropism and osmotic tolerance.

Hydrotropism mechanisms and their interplay with gravitropism.

Plants partly optimize their water recruitment from the growth medium by directing root growth toward a moisture source, a phenomenon termed hydrotropism. The default mechanism of downward growth, termed gravitropism, often functions to counteract hydrotropism when the water-potential gradient deviates from the gravity vector. This review addresses the identity of the root sites in which hydrotropism-regulating factors function to attenuate gravitropism and the interplay between these various factors. In this context, the function of hormones, including auxin, abscisic acid, and cytokinins, as well as secondary messengers, calcium ions, and reactive oxygen species in the conflict between these two opposing tropisms is discussed. We have assembled the available data on the effects of various chemicals and genetic backgrounds on both gravitropism and hydrotropism, to provide an up-to-date perspective on the interactions that dictate the orientation of root tip growth. We specify the relevant open questions for future research. Broadening our understanding of root mechanisms of water recruitment holds great potential for providing advanced approaches and technologies that can improve crop plant performance under less-than-optimal conditions, in light of predicted frequent and prolonged drought periods due to global climate change.

Defying gravity: WEEP promotes negative gravitropism in peach trees by establishing asymmetric auxin gradients.

Trees with weeping shoot architectures are valued for their beauty and are a resource for understanding how plants regulate posture control. The peach (Prunus persica) weeping phenotype, which has elliptical downward arching branches, is caused by a homozygous mutation in the WEEP gene. Little is known about the function of WEEP despite its high conservation throughout Plantae. Here, we present the results of anatomical, biochemical, biomechanical, physiological, and molecular experiments that provide insight into WEEP function. Our data suggest that weeping peach trees do not have defects in branch structure. Rather, transcriptomes from the adaxial (upper) and abaxial (lower) sides of standard and weeping branch shoot tips revealed flipped expression patterns for genes associated with early auxin response, tissue patterning, cell elongation, and tension wood development. This suggests that WEEP promotes polar auxin transport toward the lower side during shoot gravitropic response, leading to cell elongation and tension wood development. In addition, weeping peach trees exhibited steeper root systems and faster lateral root gravitropic response. This suggests that WEEP moderates root gravitropism and is essential to establishing the set-point angle of lateral roots from the gravity vector. Additionally, size exclusion chromatography indicated that WEEP proteins self-oligomerize, like other proteins with sterile alpha motif domains. Collectively, our results from weeping peach provide insight into polar auxin transport mechanisms associated with gravitropism and lateral shoot and root orientation.

A quantitative model for spatio-temporal dynamics of root gravitropism.

Plant organs adapt their morphology according to environmental signals through growth-driven processes called tropisms. While much effort has been directed towards the development of mathematical models describing the tropic dynamics of aerial organs, these cannot provide a good description of roots due to intrinsic physiological differences. Here we present a mathematical model informed by gravitropic experiments on Arabidopsis thaliana roots, assuming a subapical growth profile and apical sensing. The model quantitatively recovers the full spatio-temporal dynamics observed in experiments. An analytical solution of the model enables us to evaluate the gravitropic and proprioceptive sensitivities of roots, while also allowing us to corroborate the requirement for proprioception in describing root dynamics. Lastly, we find that the dynamics are analogous to a damped harmonic oscillator, providing intuition regarding the source of the observed oscillatory behavior and the importance of proprioception for efficient gravitropic control. In all, the model provides not only a quantitative description of root tropic dynamics, but also a mathematical framework for the future investigation of roots in complex media.

Long-term root electrotropism reveals habituation and hysteresis.

Plant roots sense many physical and chemical cues in soil, such as gravity, humidity, light, and chemical gradients, and respond by redirecting their growth toward or away from the source of the stimulus. This process is called tropism. While gravitropism is the tendency to follow the gravitational field downwards, electrotropism is the alignment of growth with external electric fields and the induced ionic currents. Although root tropisms are at the core of their ability to explore large volumes of soil in search of water and nutrients, the molecular and physical mechanisms underlying most of them remain poorly understood. We have previously provided a quantitative characterization of root electrotropism in Arabidopsis (Arabidopsis thaliana) primary roots exposed for 5 h to weak electric fields, showing that auxin asymmetric distribution is not necessary for root electrotropism but that cytokinin biosynthesis is. Here, we extend that study showing that long-term electrotropism is characterized by a complex behavior. We describe overshoot and habituation as key traits of long-term root electrotropism in Arabidopsis and provide quantitative data about the role of past exposures in the response to electric fields (hysteresis). On the molecular side, we show that cytokinin, although necessary for root electrotropism, is not asymmetrically distributed during the bending. Overall, the data presented here represent a step forward toward a better understanding of the complexity of root behavior and provide a quantitative platform for future studies on the molecular mechanisms of electrotropism.

Gravitropism: The LAZY way of intracellular hitchhiking.

Plant gravitropism has fascinated scientists for centuries. A new study provides a major mechanistic update of the so-called starch/statolith hypothesis, revealing how gravity perception is converted into a physiological response.

Plants sum and subtract stimuli over different timescales.

Mounting evidence suggests that plants engage complex computational processes to quantify and integrate sensory information over time, enabling remarkable adaptive growth strategies. However, quantitative understanding of these computational processes is limited. We report experiments probing the dependence of gravitropic responses of wheat coleoptiles on previous stimuli. First, building on a mathematical model that identifies this dependence as a form of memory, or a filter, we use experimental observations to reveal the mathematical principles of how coleoptiles integrate multiple stimuli over time. Next, we perform two-stimulus experiments, informed by model predictions, to reveal fundamental computational processes. We quantitatively show that coleoptiles respond not only to sums but also to differences between stimuli over different timescales, constituting evidence that plants can compare stimuli-crucial for search and regulation processes. These timescales also coincide with oscillations observed in gravitropic responses of wheat coleoptiles, suggesting shoots may combine memory and movement in order to enhance posture control and sensing capabilities.

Shoot gravitropism and organ straightening cooperate to arrive at a mechanically favorable shape in Arabidopsis.

Gravitropism is the plant organ bending in response to gravity, while a straightening mechanism prevents bending beyond the gravitropic set-point angle. The promotion and prevention of bending occur simultaneously around the inflorescence stem tip. How these two opposing forces work together and what part of the stem they affect are unknown. To understand the mechanical forces involved, we rotated wild type and organ-straightening-deficient mutant (myosin xif xik) Arabidopsis plants to a horizontal position to initiate bending. The mutant stems started to bend before the wild-type stems, which led us to hypothesize that the force preventing bending was weaker in mutant. We modeled the wild-type and mutant stems as elastic rods, and evaluated two parameters: an organ-angle-dependent gravitropic-responsive parameter (ß) and an organ-curvature-dependent proprioceptive-responsive parameter (γ). Our model showed that these two parameters were lower in mutant than in wild type, implying that, unexpectedly, both promotion and prevention of bending are weak in mutant. Subsequently, finite element method simulations revealed that the compressive stress in the middle of the stem was significantly lower in wild type than in mutant. The results of this study show that myosin-XIk-and-XIf-dependent organ straightening adjusts the stress distribution to achieve a mechanically favorable shape.

Exploring the Dual Functionality of Plant Pulvini Using a Physical Modeling Approach.

Pulvini are plant motor organs that fulfill two conflicting mechanical roles. At rest, pulvini function as rigid beams that support the cantilevered weight of leafy appendages. During thigmonastic (touch-induced) or nyctinastic ("sleep"-induced) plant movements, however, pulvini function as flexible joints capable of active bending. I hypothesized that the ability to alternate between these roles emerges from the interaction of two structural features of pulvini: anisotropically reinforced parenchyma cells comprising the body of the pulvinus and a longitudinally stiff but flexurally pliant vascular bundle running through the pulvinus core. To investigate how these two components might interact within biological pulvini, I built a set of pulvinus-inspired physical models with varying combinations of these elements present. I compared the abilities of the models to (1) resist imposed bending deformation (i.e., act as rigid beams) and (2) exhibit bending deformation when asymmetrically pressurized (i.e., act as actively deformable joints). Pulvinus models displayed the greatest ability to resist bending deformation when both an anisotropically reinforced parenchyma and a vasculature-like core were present. Disruption of either element reduced hydrostatic fluid pressures developed within the models, resulting in a decreased ability to resist externally applied forces. When differentially pressurized to induce active bending, the degree of bending achieved varied widely between models with and without adequately reinforced parenchyma elements. Bending, however, was not influenced by the presence of a vasculature-like core. These findings suggest that biological pulvini achieve their dual functionality by pairing anisotropically reinforced parenchyma tissues with a longitudinally stiff but flexurally pliant vascular core. Together, these elements compose a hydrostatic skeleton within the pulvinus that strongly resists external deformation when pressurized, but that bends easily when the balance of fluid pressures within it is altered. These results illustrate the emergent nature of pulvinus motor abilities and highlight structural specialization as an important aspect of pulvinus physiology.

Design and synthesis of strong root gravitropism inhibitors with no concomitant growth inhibition.

Herein, we describe a highly potent gravitropic bending inhibitor with no concomitant growth inhibition. Previously, we reported that (2Z,4E)-5-phenylpenta-2,4-dienoic acid (ku-76) selectively inhibits root gravitropic bending of lettuce radicles at 5 µM. Based on the structure-activity relationship study of ku-76 as a lead compound, we designed and synthesized various C4-substituted analogs of ku-76. Among the analogs, 4-phenylethynyl analog exhibited the highest potency for gravitropic bending inhibition, which was effective at only 0.01 µM. Remarkably, 4-phenylethynyl analog is much more potent than the known inhibitor, NPA. Substitution in the para position on the aromatic ring of 4-phenylethynyl group was tolerated without diminished activity. In addition, evaluation using Arabidopsis indicated that 4-phenylethynyl analog inhibits gravitropism by affecting auxin distribution in the root tips. Based on the effects on Arabidopsis phenotypes, 4-phenylethynyl analog may be a novel inhibitor that differs in action from the previously reported auxin transport inhibitors.

Maize root behavior as three-inputs-three-outputs logical gates due to positive gravitropism and nutritropism.

Root growth and their interactions can provide valuable information for the development of asynchronous logic systems. Here, maize root behavior due to positive gravitropism and nutritropism is evaluated as three-inputs-three-outputs logical gates. Using plant roots as the element for unconventional computing, the Boolean functions of each root tropism were constructed through arithmetic-logical operations. One gravity gate (rGG) and two nutrient gates (rNG1 and rNG2) were fabricated using additive manufacturing. The rGG platform was oriented with roots directly pulled down by gravity which computes (x, y, z) = (xz + yz, x + y¯z+yz¯, xy + yz), whereas specific output channels in rNG1 and rNG2 were fertigated with high phosphorus concentration resulting in (x, y, z) = (x + y + z, xy + xz, xyz) for rNG1 and (x, y, z) = (xyz, xy¯z+xyz¯, x + y + z) for rNG2. For rGG, rNG1, and rNG2, the symbols x, y, and z pertain to "root presence" in the related channel, whereas top bar on the symbols indicates "root absence". Anatomical traits of roots were evaluated to assess possible differences in vascular tissues due to gravitropic and nutritropic responses. Overall, maize primary roots showed prominent positive gravitropism and nutritropism, and the roots that were most attracted by gravitational or nutritional stimuli showed an increase in the diameter of phloem and xylem. The logic exhibited by roots was dependent on the gravitropic and nutritropic stimuli to which they were exposed in the different logic gates. The responsiveness of maize roots to environmental stimuli such as gravity and nutrients provided valuable information to be used in computational bioelectronics.

Dynamic GOLVEN-ROOT GROWTH FACTOR 1 INSENSITIVE signaling in the root cap mediates root gravitropism.

Throughout the exploration of the soil, roots interact with their environment and adapt to different conditions. Directional root growth is guided by asymmetric molecular patterns but how these become established or are dynamically regulated is poorly understood. Asymmetric gradients of the phytohormone auxin are established during root gravitropism, mainly through directional transport mediated by polarized auxin transporters. Upon gravistimulation, PIN-FORMED2 (PIN2) is differentially distributed and accumulates at the lower root side to facilitate asymmetric auxin transport up to the elongation zone where it inhibits cell elongation. GOLVEN (GLV) peptides function in gravitropism by affecting PIN2 abundance in epidermal cells. In addition, GLV signaling through ROOT GROWTH FACTOR 1 INSENSITIVE (RGI) receptors regulates root apical meristem maintenance. Here, we show that GLV-RGI signaling in these 2 processes in Arabidopsis (Arabidopsis thaliana) can be mapped to different cells in the root tip and that, in the case of gravitropism, it operates mainly in the lateral root cap (LRC) to maintain PIN2 levels at the plasma membrane (PM). Furthermore, we found that GLV signaling upregulates the phosphorylation level of PIN2 in an RGI-dependent manner. In addition, we demonstrated that the RGI5 receptor is asymmetrically distributed in the LRC and accumulates in the lower side of the LRC after gravistimulation. Asymmetric GLV-RGI signaling in the root cap likely accounts for differential PIN2 abundance at the PM to temporarily support auxin transport up to the elongation zone, thereby representing an additional level of control on the asymmetrical auxin flux to mediate differential growth of the root.

Local auxin biosynthesis regulates brace root angle and lodging resistance in maize.

Root lodging poses a major threat to maize production, resulting in reduced grain yield and quality, and increased harvest costs. Here, we combined expressional, genetic, and cytological studies to demonstrate a role of ZmYUC2 and ZmYUC4 in regulating gravitropic response of the brace root and lodging resistance in maize. We show that both ZmYUC2 and ZmYUC4 are preferentially expressed in root tips with partially overlapping expression patterns, and the protein products of ZmYUC2 and ZmYUC4 are localized in the cytoplasm and endoplasmic reticulum, respectively. The Zmyuc4 single mutant and Zmyuc2/4 double mutant exhibit enlarged brace root angle compared with the wild-type plants, with larger brace root angle being observed in the Zmyuc2/4 double mutant. Consistently, the brace root tips of the Zmyuc4 single mutant and Zmyuc2/4 double mutant accumulate less auxin and are defective in proper reallocation of auxin in response to gravi-stimuli. Furthermore, we show that the Zmyuc4 single mutant and the Zmyuc2/4 double mutant display obviously enhanced root lodging resistance. Our combined results demonstrate that ZmYUC2- and ZmYUC4-mediated local auxin biosynthesis is required for normal gravity response of the brace roots and provide effective targets for breeding root lodging resistant maize cultivars.

Understanding the role of starch sheath layer in graviception of Alternanthera philoxeroides: a biophysical and microscopical study.

Plants' ability to sense and respond to gravity is a unique and fundamental process. When a plant organ is tilted, it adjusts its growth orientation relative to gravity direction, which is achieved by a curvature of the organ. In higher, multicellular plants, it is thought that the relative directional change of gravity is detected by starch-filled organelles that occur inside specialized cells called statocytes, and this is followed by signal conversion from physical information to physiological information within the statocytes. The classic starch statolith hypothesis, i.e., the starch accumulating amyloplasts movement along the gravity vector within gravity-sensing cells (statocytes) is the probable trigger of subsequent intracellular signaling, is widely accepted. Acharya Jagadish Chandra Bose through his pioneering research had investigated whether the fundamental reaction of geocurvature is contractile or expansive and whether the geo-sensing cells are diffusedly distributed in the organ or are present in the form of a definite layer. In this backdrop, a microscopy based experimental study was undertaken to understand the distribution pattern of the gravisensing layer, along the length (node-node) of the model plant Alternanthera philoxeroides and to study the microrheological property of the mobile starch-filled statocytes following inclination-induced graviception in the stem of the model plant. The study indicated a prominent difference in the pattern of distribution of the gravisensing layer along the length of the model plant. The study also indicated that upon changing the orientation of the plant from vertical position to horizontal position there was a characteristic change in orientation of the mobile starch granules within the statocytes. In the present study for the analysis of the microscopic images of the stem tissue cross sections, a specialized and modified microscopic illumination setup was developed in the laboratory in order to enhance the resolution and contrast of the starch granules.