Adenylate cyclase activity of TIR1/AFB auxin receptors in plants.

The phytohormone auxin is the major coordinative signal in plant development1, mediating transcriptional reprogramming by a well-established canonical signalling pathway. TRANSPORT INHIBITOR RESPONSE 1 (TIR1)/AUXIN-SIGNALING F-BOX (AFB) auxin receptors are F-box subunits of ubiquitin ligase complexes. In response to auxin, they associate with Aux/IAA transcriptional repressors and target them for degradation via ubiquitination2,3. Here we identify adenylate cyclase (AC) activity as an additional function of TIR1/AFB receptors across land plants. Auxin, together with Aux/IAAs, stimulates cAMP production. Three separate mutations in the AC motif of the TIR1 C-terminal region, all of which abolish the AC activity, each render TIR1 ineffective in mediating gravitropism and sustained auxin-induced root growth inhibition, and also affect auxin-induced transcriptional regulation. These results highlight the importance of TIR1/AFB AC activity in canonical auxin signalling. They also identify a unique phytohormone receptor cassette combining F-box and AC motifs, and the role of cAMP as a second messenger in plants.

Nonspecific phospholipases C3 and C4 interact with PIN-FORMED2 to regulate growth and tropic responses in Arabidopsis.

The dynamic changes in membrane phospholipids affect membrane biophysical properties and cell signaling, thereby influencing numerous biological processes. Nonspecific phospholipase C (NPC) enzymes hydrolyze common phospholipids to release diacylglycerol (DAG), which is converted to phosphatidic acid (PA) and other lipids. In this study, 2 Arabidopsis (Arabidopsis thaliana) tandemly arrayed genes, NPC3 and NPC4, were identified as critical factors modulating auxin-controlled plant growth and tropic responses. Moreover, NPC3 and NPC4 were shown to interact with the auxin efflux transporter PIN-FORMED2 (PIN2). The loss of NPC3 and NPC4 enhanced the endocytosis and vacuolar degradation of PIN2, which disrupted auxin gradients and slowed gravitropic and halotropic responses. Furthermore, auxin-triggered activation of NPC3 and NPC4 is required for the asymmetric PA distribution that controls PIN2 trafficking dynamics and auxin-dependent tropic responses. Collectively, our study reveals an NPC-derived PA signaling pathway in Arabidopsis auxin fluxes that is essential for fine-tuning the balance between root growth and environmental responses.

The Quantitative Biotinylproteomics Studies Reveal a WInd-Related Kinase 1 (Raf-Like Kinase 36) Functioning as an Early Signaling Component in Wind-Induced Thigmomorphogenesis and Gravitropism.

Wind is one of the most prevalent environmental forces entraining plants to develop various mechano-responses, collectively called thigmomorphogenesis. Largely unknown is how plants transduce these versatile wind force signals downstream to nuclear events and to the development of thigmomorphogenic phenotype or anemotropic response. To identify molecular components at the early steps of the wind force signaling, two mechanical signaling-related phosphoproteins, identified from our previous phosphoproteomic study of Arabidopsis touch response, mitogen-activated protein kinase kinase 1 (MKK1) and 2 (MKK2), were selected for performing in planta TurboID (ID)-based quantitative proximity-labeling (PL) proteomics. This quantitative biotinylproteomics was separately performed on MKK1-ID and MKK2-ID transgenic plants, respectively, using the genetically engineered TurboID biotin ligase expression transgenics as a universal control. This unique PTM proteomics successfully identified 11 and 71 MKK1 and MKK2 putative interactors, respectively. Biotin occupancy ratio (BOR) was found to be an alternative parameter to measure the extent of proximity and specificity between the proximal target proteins and the bait fusion protein. Bioinformatics analysis of these biotinylprotein data also found that TurboID biotin ligase favorably labels the loop region of target proteins. A WInd-Related Kinase 1 (WIRK1), previously known as rapidly accelerated fibrosarcoma (Raf)-like kinase 36 (RAF36), was found to be a putative common interactor for both MKK1 and MKK2 and preferentially interacts with MKK2. Further molecular biology studies of the Arabidopsis RAF36 kinase found that it plays a role in wind regulation of the touch-responsive TCH3 and CML38 gene expression and the phosphorylation of a touch-regulated PATL3 phosphoprotein. Measurement of leaf morphology and shoot gravitropic response of wirk1 (raf36) mutant revealed that the WIRK1 gene is involved in both wind-triggered rosette thigmomorphogenesis and gravitropism of Arabidopsis stems, suggesting that the WIRK1 (RAF36) protein probably functioning upstream of both MKK1 and MKK2 and that it may serve as the crosstalk point among multiple mechano-signal transduction pathways mediating both wind mechano-response and gravitropism.

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.

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.

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.

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.

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.

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.

Leveraging orthology within maize and Arabidopsis QTL to identify genes affecting natural variation in gravitropism.

Plants typically orient their organs with respect to the Earth's gravity field by a dynamic process called gravitropism. To discover conserved genetic elements affecting seedling root gravitropism, we measured the process in a set of Zea mays (maize) recombinant inbred lines with machine vision and compared the results with those obtained in a similar study of Arabidopsis thaliana. Each of the several quantitative trait loci that we mapped in both species spanned many hundreds of genes, too many to test individually for causality. We reasoned that orthologous genes may be responsible for natural variation in monocot and dicot root gravitropism. If so, pairs of orthologous genes affecting gravitropism may be present within the maize and Arabidopsis QTL intervals. A reciprocal comparison of sequences within the QTL intervals identified seven pairs of such one-to-one orthologs. Analysis of knockout mutants demonstrated a role in gravitropism for four of the seven: CCT2 functions in phosphatidylcholine biosynthesis, ATG5 functions in membrane remodeling during autophagy, UGP2 produces the substrate for cellulose and callose polymer extension, and FAMA is a transcription factor. Automated phenotyping enabled this discovery of four naturally varying components of a conserved process (gravitropism) by making it feasible to conduct the same large-scale experiment in two species.

Root angle is controlled by EGT1 in cereal crops employing an antigravitropic mechanism.

Root angle in crops represents a key trait for efficient capture of soil resources. Root angle is determined by competing gravitropic versus antigravitropic offset (AGO) mechanisms. Here we report a root angle regulatory gene termed ENHANCED GRAVITROPISM1 (EGT1) that encodes a putative AGO component, whose loss-of-function enhances root gravitropism. Mutations in barley and wheat EGT1 genes confer a striking root phenotype, where every root class adopts a steeper growth angle. EGT1 encodes an F-box and Tubby domain-containing protein that is highly conserved across plant species. Haplotype analysis found that natural allelic variation at the barley EGT1 locus impacts root angle. Gravitropic assays indicated that Hvegt1 roots bend more rapidly than wild-type. Transcript profiling revealed Hvegt1 roots deregulate reactive oxygen species (ROS) homeostasis and cell wall-loosening enzymes and cofactors. ROS imaging shows that Hvegt1 root basal meristem and elongation zone tissues have reduced levels. Atomic force microscopy measurements detected elongating Hvegt1 root cortical cell walls are significantly less stiff than wild-type. In situ analysis identified HvEGT1 is expressed in elongating cortical and stele tissues, which are distinct from known root gravitropic perception and response tissues in the columella and epidermis, respectively. We propose that EGT1 controls root angle by regulating cell wall stiffness in elongating root cortical tissue, counteracting the gravitropic machinery's known ability to bend the root via its outermost tissues. We conclude that root angle is controlled by EGT1 in cereal crops employing an antigravitropic mechanism.

BRXL4-LAZY1 interaction at the plasma membrane controls Arabidopsis branch angle and gravitropism.

Gravitropism guides growth to shape plant architecture above and below ground. Mutations in LAZY1 impair stem gravitropism and cause less upright inflorescence branches (wider angles). The LAZY1 protein resides at the plasma membrane and in the nucleus. The plasma membrane pool is necessary and sufficient for setting branch angles. To investigate the molecular mechanism of LAZY1 function, we screened for LAZY1-interacting proteins in yeast. We identified BRXL4, a shoot-specific protein related to BREVIS RADIX. The BRXL4-LAZY1 interaction occurred at the plasma membrane in plant cells, and not detectably in the nucleus. Mutations in the C-terminus of LAZY1, but not other conserved regions, prevented the interaction. Opposite to lazy1, brxl4 mutants displayed faster gravitropism and more upright branches. Overexpressing BRXL4 produced strong lazy1 phenotypes. The apparent negative regulation of LAZY1 function is consistent with BRXL4 reducing LAZY1 expression or the amount of LAZY1 at the plasma membrane. Measurements indicated that both are true. LAZY1 mRNA was three-fold more abundant in brxl4 mutants and almost undetectable in BRXL4 overexpressors. Plasma membrane LAZY1 was higher and nuclear LAZY1 lower in brxl4 mutants compared with the wild type. To explain these results, we suggest that BRXL4 reduces the amount of LAZY1 at the plasma membrane where it functions in gravity signaling and promotes LAZY1 accumulation in the nucleus where it reduces LAZY1 expression, possibly by suppressing its own transcription. This explanation of how BRXL4 negatively regulates LAZY1 suggests ways to modify shoot system architecture for practical purposes.

Molecular mechanism of brassinosteroids involved in root gravity response based on transcriptome analysis.

BACKGROUND: Brassinosteroids (BRs) are a class of phytohormones that regulate a wide range of developmental processes in plants. BR-associated mutants display impaired growth and response to developmental and environmental stimuli. RESULTS: Here, we found that a BR-deficient mutant det2-1 displayed abnormal root gravitropic growth in Arabidopsis, which was not present in other BR mutants. To further elucidate the role of DET2 in gravity, we performed transcriptome sequencing and analysis of det2-1 and bri1-116, bri1 null mutant allele. Expression levels of auxin, gibberellin, cytokinin, and other related genes in the two mutants of det2-1 and bri1-116 were basically the same. However, we only found that a large number of JAZ (JASMONATE ZIM-domain) genes and jasmonate synthesis-related genes were upregulated in det2-1 mutant, suggesting increased levels of endogenous JA. CONCLUSIONS: Our results also suggested that DET2 not only plays a role in BR synthesis but may also be involved in JA regulation. Our study provides a new insight into the molecular mechanism of BRs on the root gravitropism.

A single amino acid substitution in MdLAZY1A dominantly impairs shoot gravitropism in Malus.

Plant architecture is 1 of the most important factors that determines crop yield potential and productivity. In apple (Malus domestica), genetic improvement of tree architecture has been challenging due to a long juvenile phase and growth as complex trees composed of a distinct scion and a rootstock. To better understand the genetic control of apple tree architecture, the dominant weeping growth phenotype was investigated. We report the identification of MdLAZY1A (MD13G1122400) as the genetic determinant underpinning the Weeping (W) locus that largely controls weeping growth in Malus. MdLAZY1A is 1 of the 4 paralogs in apple that are most closely related to AtLAZY1 involved in gravitropism in Arabidopsis (Arabidopsis thaliana). The weeping allele (MdLAZY1A-W) contains a single nucleotide mutation c.584T>C that leads to a leucine to proline (L195P) substitution within a predicted transmembrane domain that colocalizes with Region III, 1 of the 5 conserved regions in LAZY1-like proteins. Subcellular localization revealed that MdLAZY1A localizes to the plasma membrane and nucleus in plant cells. Overexpressing the weeping allele in apple cultivar Royal Gala (RG) with standard growth habit impaired its gravitropic response and altered the growth to weeping-like. Suppressing the standard allele (MdLAZY1A-S) by RNA interference (RNAi) in RG similarly changed the branch growth direction to downward. Overall, the L195P mutation in MdLAZY1A is genetically causal for weeping growth, underscoring not only the crucial roles of residue L195 and Region III in MdLAZY1A-mediated gravitropic response but also a potential DNA base editing target for tree architecture improvement in Malus and other crops.

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.

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.

Adaptive divergence in shoot gravitropism creates hybrid sterility in an Australian wildflower.

Natural selection is responsible for much of the diversity we see in nature. Just as it drives the evolution of new traits, it can also lead to new species. However, it is unclear whether natural selection conferring adaptation to local environments can drive speciation through the evolution of hybrid sterility between populations. Here, we show that adaptive divergence in shoot gravitropism, the ability of a plant's shoot to bend upwards in response to the downward pull of gravity, contributes to the evolution of hybrid sterility in an Australian wildflower, Senecio lautus We find that shoot gravitropism has evolved multiple times in association with plant height between adjacent populations inhabiting contrasting environments, suggesting that these traits have evolved by natural selection. We directly tested this prediction using a hybrid population subjected to eight rounds of recombination and three rounds of selection in the field. Our experiments revealed that shoot gravitropism responds to natural selection in the expected direction of the locally adapted population. Using the advanced hybrid population, we discovered that individuals with extreme differences in gravitropism had more sterile crosses than individuals with similar gravitropic responses, which were largely fertile, indicating that this adaptive trait is genetically correlated with hybrid sterility. Our results suggest that natural selection can drive the evolution of locally adaptive traits that also create hybrid sterility, thus revealing an evolutionary connection between local adaptation and the origin of new species.

ENHANCED GRAVITROPISM 2 encodes a STERILE ALPHA MOTIF-containing protein that controls root growth angle in barley and wheat.

The root growth angle defines how roots grow toward the gravity vector and is among the most important determinants of root system architecture. It controls water uptake capacity, nutrient use efficiency, stress resilience, and, as a consequence, yield of crop plants. We demonstrated that the egt2 (enhanced gravitropism 2) mutant of barley exhibits steeper root growth of seminal and lateral roots and an auxin-independent higher responsiveness to gravity compared to wild-type plants. We cloned the EGT2 gene by a combination of bulked-segregant analysis and whole genome sequencing. Subsequent validation experiments by an independent CRISPR/Cas9 mutant allele demonstrated that egt2 encodes a STERILE ALPHA MOTIF domain-containing protein. In situ hybridization experiments illustrated that EGT2 is expressed from the root cap to the elongation zone. We demonstrated the evolutionary conserved role of EGT2 in root growth angle control between barley and wheat by knocking out the EGT2 orthologs in the A and B genomes of tetraploid durum wheat. By combining laser capture microdissection with RNA sequencing, we observed that seven expansin genes were transcriptionally down-regulated in the elongation zone. This is consistent with a role of EGT2 in this region of the root where the effect of gravity sensing is executed by differential cell elongation. Our findings suggest that EGT2 is an evolutionary conserved regulator of root growth angle in barley and wheat that could be a valuable target for root-based crop improvement strategies in cereals.

MIZ1 acts downstream of PGM1 in regulating root hydrotropism.

The MIZ1 play an important role in root hydrotropism. However, the relationship between MIZ1-regulated hydrotropism and amyloplast-mediated gravitropism remain largely unclear. Here, we generated the miz1/pgm1 double mutants by crossing the non-hydrotropic miz1 mutant with the amyloplast-defective pgm1 mutant, which lacks gravitropic response. Our results showed that the miz1/pgm1 mutants exhibited a significant reduction in amyloplast and gravitropic bending, while maintaining a similar ahydrotropic phenotype as the miz1 single mutant. These findings suggest that MIZ1 plays a role in hydrotropism downstream of PGM1. Understanding the mechanisms of interaction between hydrotropism and gravitropism is crucial for comprehending the rooting patterns of plants in natural conditions. The counteracting relationship between root hydrotropism and gravitropism in the miz1 mutant should receive attention in this field, particularly considering the interference from gravitropism on Earth.