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Article

LAZY2 and LAZY3 Regulate Rice Root Gravitropism by Affecting Starch Accumulation

by
Haoran Song
1,
Linzhou Huang
2,
Jiaze Chen
1 and
Wenguang Wang
1,*
1
State Key Laboratory of Wheat Improvement, College of Life Sciences, Shandong Agricultural University, Tai’an 271018, China
2
State Key Laboratory for Crop Stress Resistance and High-Efficiency Production, College of Agronomy, Northwest A&F University, Yangling 712100, China
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(11), 2639; https://doi.org/10.3390/agronomy15112639
Submission received: 17 September 2025 / Revised: 11 November 2025 / Accepted: 14 November 2025 / Published: 18 November 2025
(This article belongs to the Special Issue Recent Advances in Crops Genome-Wide Assisted Selection Breeding)
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Abstract

Root gravitropism is essential for plants to establish proper root system architecture, enabling efficient soil exploration and optimal acquisition of water and nutrients. However, the molecular mechanisms underlying rice root gravitropism remain poorly understood. Here, we demonstrate that LAZY2 (LA2) and LA3, previously identified regulators for shoot gravitropism control, play critical roles in root gravitropism by maintaining starch accumulation in root columella cells. Loss-of-function mutants (CR-la2, CR-la3) exhibited impaired root gravitropism, resulting in a shallow root architecture. Further analysis revealed a significant reduction in starch accumulation within root statocytes of CR-la2 and CR-la3 mutants, causing reduced root gravity sensing. The complementation lines of LA2 or LA3 can rescue the defect in starch accumulation and root gravitropism. Haplotype analysis linked natural LA2 variation to distinct root architectures, with Hap5 accessions showing steeper angles than Hap1. Additionally, we confirmed that LA2 forms homodimers both in vivo and in vitro. Our findings establish LA2 and LA3 as key regulators coupling starch metabolism with root gravitropism, providing both fundamental insights into gravity sensing and genetic targets for optimizing root architecture to increase grain yield in cereal crops.

1. Introduction

Root system architecture represents a critical adaptive strategy for plants to optimize soil resource acquisition under varying environmental conditions [1]. Among root architectural traits, growth angle plays a particularly pivotal role in determining nutrient and water uptake efficiency. Shallower root angles facilitate phosphorus capture from nutrient-rich topsoil layers, while steeper angles enable access to deeper water reserves, enhancing drought tolerance and nitrogen acquisition [2,3,4]. This plasticity makes root angle an important target trait for developing climate-resilient crops, particularly as recent evidence demonstrates its impact on yield stability under saline stress [5].
Great progress has been made in illustrating the molecular mechanism underlying root angle by studying Arabidopsis, and root angle is mainly regulated by root gravitropism [6,7,8,9,10]. Gravitropism, the directional growth response to gravity, underlies root angle determination through a conserved three-phase process: gravity perception in statocytes, signal transduction, and organ curvature response [11]. The root gravitropic response is initiated when columella cells in the root tip perceive changes in orientation, triggering a lateral auxin gradient and ultimately causing root bending [12,13]. In Arabidopsis, the LAZY1 (LA1) gene family consists of six members. While the functions of AtLA5 and AtLA6 in gravitropism are still unknown, the other members exhibit functional divergence across various organs. AtLA1 controls gravitropism in inflorescence stems, AtLA3/LA4 mediates root gravitropism, and both AtLA2 and AtLA4/LA3 participate in gravitropism in both shoots and roots [7]. In Arabidopsis, simultaneous mutation of LA2, LA3, and LA4 genes results in roots losing their positive gravitropic response [9,10,14,15]. Genetic complementation experiments confirm that these genes are functionally redundant in root gravitropism [9]. The starch statolith hypothesis posits that gravity sensing occurs through sedimentation of starch-filled amyloplasts in specialized columella cells [16]. Recently, two research groups significantly advanced our understanding of gravity sensing in plants. Both studies demonstrated that amyloplast sedimentation triggers the relocation of LA proteins from statoliths to the plasma membrane, facilitating gravitropic response [8,9]. Investigations of gravitropism in starchless and starch-deficient mutants support a role for amyloplast sedimentation in gravity sensing [17,18,19]. By disrupting starch biosynthesis, mutations in Arabidopsis phosphoglucomutase 1 (PGM1) produce starchless plastids that fail to sediment properly, leading to a delayed gravitropic response in both roots and shoots [20].
Despite significant progress in dissecting the genetic basis of gravity sensing using Arabidopsis as a model, the regulatory network that controls root gravity sensing in crops still needs to be explored. In rice, DEEPER ROOTING 1 (DRO1) promotes steeper crown root angles to enhance drought tolerance [2], while LARGE ROOT ANGLE 1 (LRA1/OsPIN2) modulates shallow growth for improved topsoil foraging [21]. Beyond these root-specific factors, developmental regulators like VERNALIZATION 1 (VRN1) mediate root architectural variation between winter and spring cereal varieties [22]. Auxin transport proteins, including auxin influx carrier OsAUX1 and auxin efflux carrier protein OsPIN1a/OsPIN1b, play a central role in root gravitropic responses and root angle determination in rice [23,24]. Additionally, the E3 ubiquitin ligase soil-surface rooting 1 (SOR1/MHZ2) controls root-specific ethylene responses by modulating the stability of Aux/IAA protein to regulate root gravity response and root growth angle [25]. The actin-binding protein rice morphology determinant (RMD) modulates root growth angle by anchoring statoliths to actin filaments, with its phosphate-responsive regulation slowing gravitropism by restricting statolith movement [4]. Recent studies have identified ENHANCED GRAVITROPISM 1 (EGT1) and EGT2 as critical regulators of root angle in wheat and barley [1,26]. It was reported that EGT1 modulates tissue stiffness to fine-tune gravitropic responses [1], while EGT2, a STERILE ALPHA MOTIF domain-containing protein, promotes steeper seminal and lateral root growth via an auxin-independent mechanism [26]. Collectively, these findings highlight the genetic complexity underlying root growth angle and its significance in agricultural production. A deeper understanding of these regulatory networks could enable the development of crop varieties with optimized root systems for improving nutrient and water acquisition under challenging environmental conditions.
Our previous works found starch granule-binding protein LAZY3 (LA3) can interact with the starch biosynthesis regulator LA2 to regulate starch granule formation in shoot gravity-sensing tissue, which modulates the shoot gravitropism and tiller angle in rice [27,28]. Whereas Arabidopsis LA2 and LA3 belong to the LA1 family, rice LA2 and LA3 encode YbaB family protein and chloroplast-localized tryptophan-rich protein, respectively. However, the potential roles of rice LA2 and LA3 in root gravitropism and root system architecture have remained unexplored.
Here, we demonstrate that the LA2 and LA3 critically regulate starch accumulation in root statocytes, thereby governing root gravitropism and overall root architecture. Our findings reveal the novel functions of these genes in shaping root system architecture and provide novel targets for optimizing rice root traits.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

The rice (Oryza sativa L.) lines used in this study were as previously described: wild-type (WT) cultivars Zhonghua 11 (ZH11), 9311, and Guanghui 998 (GH998); the loss-of-function mutants la2 (9311 background) and la3 (GH998 background); the CRISPR-Cas9-generated loss-of-function mutants CR-la2 and CR-la3 (ZH11 background); and the transgenic lines Ubipro:LA2-3×Flag/la2 (9311 background), LA2pro:LA2/la2 (9311 background), and LA3:LA3/la3 (GH998 background) [27,28]. For field experiments, the planting spacing for rice was 30 cm × 30 cm, and plants were cultivated under standard paddy field conditions at the Shandong Agricultural University experimental station.

2.2. Analysis of Root Gravitropism

Analysis of root gravitropism was performed according to methods described previously [22] with minor modifications. To investigate the root gravitropic response of transgenic rice lines, the seeds of cultivars Zhonghua 11 (ZH11), 9311, and Guanghui 998 (GH998); the loss-of-function mutants la2 and la3; the CRISPR-Cas9-generated loss-of-function mutants CR-la2 and CR-la3; and the transgenic lines Ubipro:LA2-3×Flag/la2, LA2pro:LA2/la2, and LA3:LA3/la3 were germinated in water for 2 days. The germinated rice seeds were transferred to the plates (13 cm × 13 cm × 3 cm) with 0.4% agar. The seedlings were grown under dark environments at 28 °C. Three-day-old seedlings grown on agar plates were reoriented by 90° to assess gravitropic responses. Root curvature was measured using ImageJ software (1.53K WIN).

2.3. Starch Granules Staining Assay

For visualization of starch granules in roots of CR-la2 and CR-la3 mutants, root tips of four-day-old rice seedlings were cut with a blade. Starch granules were stained with 1% KI/I2 solution for 10 s and then washed with anhydrous alcohol for 1 min to remove the KI/I2 solution, before being rinsed with water 6 times. The root tips were treated with chloral hydrate, which was used as the clearing agent in a ratio of 8:3:1 for chloral hydrate/water/glycerol before imaging. The images were taken using the OLYMPUS SZX16 (OLYMPUS, Tokyo, Japan).

2.4. Root System Architecture Analysis

Field-grown plants at the tillering stage were carefully uprooted, and root systems were washed thoroughly to remove the soil. To clearly visualize the root growth and root architecture, we also planted the plants in a rhizobox. The imaging of root architecture was performed using a rhizobox system. Briefly, rhizoboxes (dimensions: 25 cm × 25 cm × 3 cm) were filled with potting soil. The germinated rice seeds were placed at the top of the rhizobox. The rhizoboxes were placed on a standardized imaging platform with a high-resolution camera. The root angle was defined geometrically as the angle between the vertical line and the side roots with maximum inclination. Angles were measured using the ImageJ software (1.53K WIN).

2.5. RNA Extraction and qRT-PCR

TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate total RNA from rice tissues. The total RNA was treated with DNase I (Ambion, Austin, TX, USA) and then reverse transcribed into cDNA using the iScriptTM cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). RT-qPCR was conducted using the CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA) with the SsoFast EvaGreen Supermix Kit (Bio-Rad, Hercules, CA, USA). Rice UBIQUITIN (LOC_Os03g13170) was used as the endogenous control. Primers used for RT-qPCR are listed in Table S1.

2.6. Haplotype Analysis of LA2 and LA3

We used the high-density SNP set of rice based on high-density whole-genome resequencing data of both wild and cultivated rice germplasms (acquired from the National Genomics Data Center, China National Center for Bioinformation) for the haplotype analysis. Haplotype blocks were calculated using Plink v1.90b6.13 with options “-ld-window-kb 500-blocks-min-maf0.05-blocks-strong-lowci 0.7-blocks-strong-highci 0.98”. Variants in the haplotype blocks containing the flanking regions of the LA2 and LA3 genes were extracted for downstream analyses. The pairwise Euclidean distances were then calculated based on the recoded genotypes, and the hierarchical cluster analysis was performed (hclust in R version 4.3.1, with method “ward.D2”). The number of clusters was determined based on the silhouette analysis of k-means clustering using R/cluster. The haplotype-phenotype association analyses were performed with an MLM Q+K model using Genome-wide Complex Trait Analysis (GCTA) version v1.94.1. The top three principal components of PCA analyses and the genetic relationship matrix were used as Q and K.

2.7. Luciferase Complementation Imaging (LCI) Assay

To generate the 35Spro:LA2-cLUC plasmid, the full-length CDS (without stop codon) of LA2 was amplified from ZH11 cDNA with primer pair LA2-LCI-cF/LA2-LCI-cR (Table S1) and cloned into the BamHI/SalI digested 35Spro:cLUC vector. The 35Spro:LA2-nLUC plasmid was documented previously [27]. The nLUC and cLUC fusion vectors were transformed into A. tumefaciens EHA105 and then co-infiltrated into N. benthamiana leaves. The images were taken by the NightShade LB 985 In Vivo Plant Imaging System (IndiGO; Berthold Technologies Co., Bad Wildbad, Germany) using beetle luciferin as the substrate (E1603; Promega, Madison, WI, USA). Primers used for vector construction are listed in Table S1.

2.8. Bimolecular Fluorescence Complementation (BiFC) Assay

The full-length CDS of LA2 without the stop codon was amplified from ZH11 cDNA with the primer pair LA2-CE-F/LA2-CE-R (Table S1) and cloned into KpnI/SalI digested pSCYCE (SCC) vector, forming the 35Spro:LA2-cCFP plasmid. The 35Spro:LA2-nCFP (SCN-LA2) was reported previously [27]. The combinations of the constructs were co-transformed into rice protoplasts. After 12 h, CFP fluorescence and chlorophyll autofluorescence were observed at excitation wavelengths of 405 and 647 nm with confocal microscopy (FluoView 1000; Olympus, Tokyo, Japan), respectively.

2.9. Pull-Down Assay

The GST pull-down assay was performed using recombinant proteins purified from the E. coli prokaryotic expression system [27]. and plant protein supernatants extracted from the shoot bases of the Ubipro:LA2-3×Flag/la2 transgenic line. The rice protein extraction protocol was as follows: The basal segments (3 cm) of seedlings from the Ubipro:LA2-3×Flag/la2 transgenic line were collected, thoroughly ground in liquid nitrogen, and lysed with two volumes of IP buffer. The mixture was incubated on ice for 20 min, with vortexing every 5 min. The lysate was then centrifuged at 14,000 rpm for 20 min at 4 °C, and the supernatant was collected for the GST pull-down assay. The GST pull-down procedure was as follows: Beads containing 2 μg of GST or GST-LA2 fusion protein were incubated with 1 mL of total plant protein extracts at 4 °C with gentle rotation for 3 h. The beads were then pelleted by centrifugation at 500× g for 5 min at 4 °C and washed four times with 1 × PBS containing 2 mM DTT. An equal volume of 2 × SDS loading buffer was added to the beads, followed by denaturation at 100 °C for 5 min. After cooling to room temperature, the samples were centrifuged, and 15 μL of the supernatant was subjected to Western blot analysis.

2.10. Co-Immunoprecipitation Assay

The In-Fusion HD cloning system was used to recombine the full-length LA2 CDS (without the stop codon) amplified with LA2-SCC-Flag-F/LA2-SCC-Flag-R (Table S1) into the BamHI/KpnI digested pSCC-3×Flag vector, generating the pSCC-LA2-3×Flag transient expression construct. For the co-transfection experiments, the pSCC-LA2-3×Flag plasmid was co-transfected with the 35Spro:LA2-GFP plasmid [27] into protoplasts derived from wild-type rice (ZH11). The pSCC-LA2-3×Flag plasmid was co-transfected with the 35Spro:GFP vector [27] into ZH11 protoplasts. After transfection, the protoplasts were cultured in darkness at 28 °C for 12 h, then centrifuged at 150× g for 3 min to pellet the cells and immediately flash-frozen in liquid nitrogen for storage. Total protein was extracted from the transformed protoplasts using IP buffer, and 1 mL of the protein supernatant was incubated with 20 μL of GFP antibody-coupled beads (pre-equilibrated three times with IP buffer) at 4 °C for 2 h. The beads were washed three times with IP buffer, and the bound proteins were eluted with an equal volume of 2 × SDS loading buffer, followed by denaturation at 100 °C for 5 min. After cooling to room temperature, the samples were centrifuged, and 15 μL of the supernatant was analyzed by immunoblotting.

3. Results

3.1. LA2 and LA3 Regulate Root Gravitropism in Rice

Previous studies have shown that both LA2 and LA3 positively regulate shoot gravitropism and, thus, tiller angle in rice [27,28]. Our expression analysis revealed that LA2 and LA3 are both expressed not only in aerial tissues but also in roots (Figures S1 and S2), implying their potential roles in root development.
To verify their involvement in root gravitropism, we examined the gravitropic response of the loss-of-function mutants CR-la2-1 and CR-la3-1. We grew the CR-la2-1 and CR-la3-1 mutants on 0.4% agar plates for 3 days and then rotated the plates upside down by 90 degrees for time-course gravity stimulation. Morphological observation indicated that the root curvature was less in the CR-la2-1 and CR-la3-1 mutants than that in the wild-type ZH11 (Figure 1A). We further fitted the root curvature data using a GLM model with ZH11, CR-la2-1, and CR-la3-1 as independent variables, in addition to the gravity stimulation time. Results showed that CR-la2-1 and CR-la3-1 mutants showed defective root gravitropism (Figure 1B). The coefficients summary table is provided in Table S2. These findings show that the LA2 and LA3 are also involved in regulating root gravitropism in rice.

3.2. LA2 and LA3 Regulate Starch Biosynthesis in Rice Root Tips

To further elucidate the mechanism by which LA2 and LA3 modulate root gravitropism, we investigated starch accumulation in root tips using the I2-KI starch-staining assay. Starch granules, which serve as statoliths in plant gravity-sensing cells, are critical for proper gravitropic responses. Remarkably, both the CR-la2 and CR-la3 mutants exhibited significant reductions in starch granule accumulation within the columella cells of the root caps (Figure 2), the primary sites of gravity perception in plants. While starch accumulation was markedly diminished in the mutants, it was not entirely abolished, unlike the complete absence of starch granules observed in their shoots [27,28].
To further confirm the function of LA2 and LA3 in regulating starch accumulation and root gravitropism, we analyzed the root curvature and root-tip starch not only in Ubi:LA2-3×Flag/la2 line but also in the LA2pro:LA2/la2 and LA3pro:LA3/la3 complementation lines, and the results showed that both could rescue root curvature and root-tip starch of the la2 or la3 mutant (Figure 3 and Figure S3).
These findings demonstrate that LA2 and LA3 are key regulators of starch biosynthesis in root columella cells, thereby influencing root gravitropism through modulating gravity perception in rice roots.

3.3. LA2 and LA3 Regulate Root Architecture in Rice

Given the observed gravitropic defects in CR-la2 and CR-la3 mutants, we investigated their root system architectures under field conditions. Phenotypic and statistical analysis revealed that both CR-la2 and CR-la3 mutants grown in the field displayed significantly shallower root systems compared to wild-type ZH11 (Figure 4A,C).
In order to further confirm that LA2 and LA3 regulate root architecture, we also planted the CR-la2 and CR-la3 mutants in the rhizobox and observed their root architecture. Results showed that both the CR-la2 and CR-la3 mutants showed larger root angles and also exhibited shallower root systems compared to ZH11 (Figure 4B,D). These results demonstrate that LA2 and LA3 regulate root architecture in rice.
To explore the natural variation in LA2- and LA3-mediated root architecture regulation, we performed haplotype analysis across diverse rice accessions. LA2 polymorphisms segregated into seven haplotype clusters (HCs), while LA3 showed more limited variations with two major HCs (Figure 5A and Figure S4A, Table S3). Notably, rice accessions carrying LA2HC5 displayed larger root growth angles than those with LA2HC1 (Figure 5B,C), whereas the two LA3 haplotypes showed comparable root growth angles (Figure S4B,C). These findings show that LA2 contributes to natural variation in rice root architecture, demonstrating the novel functions of LA2 and LA3 in the establishment of root architecture beyond rice tiller angle.

3.4. LA2 Can Form Homodimers In Vivo and In Vitro

Our previous study has shown that LA2 is homologous to YbaB in Escherichia coli (Ec-YbaB) and Haemophilus influenzae (Hi-YbaB) [27]. It was demonstrated that bacterial YbaB proteins adopt a unique tweezer-like conformation and function as DNA-binding homodimers [29]. To test whether LA2 exhibits similar oligomerization properties, we performed multiple biochemical assays. First, luciferase complementation imaging (LCI) assay in Nicotiana benthamiana leaves revealed strong self-interaction of LA2, as evidenced by significant luminescence signals (Figure 6A). This interaction was further confirmed in planta using bimolecular fluorescence complementation (BiFC) assay, which showed the dimerization of LA2 specifically occurred in rice chloroplasts (Figure 6B). Furthermore, we further confirmed that LA2 can form homodimers both in vitro and in vivo through pull-down assay and co-immunoprecipitation analysis (Figure 6C,D). Taken together, our study shows that LA2 can physically form homodimers.

4. Discussion

In this study, we demonstrate that the LA2 and LA3 play a crucial role in root gravitropism and root system architecture by maintaining starch accumulation in root columella cells. Our findings reveal that loss-of-function mutations in LA2 or LA3 lead to defective root gravitropic responses and a shallower root architecture resulting from a dramatic reduction in starch granules in root statocytes. These findings unravel a novel function of LA2 and LA3 in regulating root gravity perception and root architecture establishment.
The starch statolith hypothesis has long been proposed as the primary mechanism for gravity sensing in plants, where the sedimentation of starch-filled amyloplasts in columella cells triggers downstream signaling [11,30]. Our results demonstrate that CR-la2 and CR-la3 mutants exhibit dramatically reduced starch content in root columella cells (Figure 2), mirroring our previous findings in shoots, where LA3 was found to bind to starch granules and interact with LA2 to regulate starch granule formation [27,28]. However, we observed an intriguing tissue-specific difference in starch accumulation. Unlike the shoot gravity-sensing cell, where LA2 and LA3 mutations completely abolish starch accumulation [27,28], root statocytes seem to retain residual starch granules in both CR-la2 and CR-la3 mutants (Figure 2). The differential contributions of LA2/LA3 to starch biosynthesis between shoot and roots suggest the existence of either tissue-specific starch biosynthesis pathways with varying dependence on LA2/LA3 activity or differential compensatory mechanisms of starch accumulation in root columella cells. The partial starch retention in root statocytes of CR-la2 and CR-la3 mutants is also consistent with weaker deficits in root gravitropism than in shoots. Our previous work in shoots demonstrated that LA3 is a starch granule-binding protein and that LA2 interacts with the key starch synthesis enzyme osplastidic phosphoglucomutase (OspPGM) [27,28]. OspPGM catalyzes the conversion of glucose-6-phosphate to glucose-1-phosphate, a critical step in starch biosynthesis. The severe reduction in starch in la2 and la3 mutants strongly implies that the LA2-LA3 complex is essential for OspPGM activity or for the metabolic channeling of substrates within the plastid, thereby directly linking these proteins to the core starch metabolic pathway.
The shallow root architecture phenotype observed in CR-la2 and CR-la3 mutants (Figure 4) has important agronomic implications. Such root morphological changes are known to enhance topsoil foraging capacity, particularly for immobile nutrients like phosphorus [4,31,32]. This phenotype aligns with previous reports of improved phosphorus acquisition in shallow-rooted mutants such as lra1/ospin2 and rmd-1 [4,21]. Notably, our haplotype analysis revealed significant natural variation in root growth angles associated with different LA2 alleles (Figure 5), suggesting that LA2 polymorphisms may serve as genetic donors for breeding programs aimed at optimizing root architecture for specific soil conditions. Structural studies of bacterial YbaB proteins revealed a tweezer-like homodimeric structure that binds DNA [29], which prompted us to investigate the oligomerization stage of LA2. As a homolog of bacterial YbaB proteins [27], LA2 can form homodimers in vitro and in vivo (Figure 6A–D). A key question arising from this finding is whether this dimerization is functionally required for LA2’s role in gravitropism. While our current study demonstrates dimerization occurs, it remains a limitation that we have not yet directly tested its functional necessity in planta. We propose two non-mutually exclusive functional models for the LA2 dimer. First, the homodimer could act as a structural scaffold within the starch biosynthesis complex. In this capacity, it might stabilize the protein complex LA3-LA2-OspPGM, thereby ensuring efficient starch synthesis. Second, given that its bacterial homolog possesses DNA-binding capability, the LA2 dimer might function in transcriptional or plastid gene regulation, potentially influencing the expression of starch biosynthesis genes.
To unequivocally determine the functional significance of dimerization, future work should involve creating structure-based interface mutants of LA2 that are deficient in dimerization. Introducing these mutants into the la2 background and assessing their ability to rescue the mutant’s starch deficiency and gravitropic defects would provide direct genetic evidence. Furthermore, investigating whether the dimerization interface is necessary for its interactions with known partners like LA3 or OspPGM would clarify its primary molecular role. The conservation of homodimerization from prokaryotic YbaB to rice LA2 suggests an ancient and evolutionarily conserved functional mechanism. Understanding the precise molecular architecture of the LA2 dimer and its interface could also open avenues for targeted protein engineering to modulate its function, potentially leading to fine-tuning of root architecture for agricultural improvement.

5. Conclusions

In conclusion, our findings identified LA2 and LA3 as critical modulators coupling starch metabolism with root gravitropism, not only providing insights into the molecular basis of gravity sensing and root gravitropism in rice but also unraveling novel roles of LA2 and LA3 in root system plasticity. Future studies should explore whether modulating LA2 and LA3 expression or activity can enhance nutrient use efficiency without compromising other agronomic traits.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15112639/s1, Figure S1: Expression pattern of LA2 in different rice tissues; Figure S2: Expression pattern of LA3 in different rice tissues; Figure S3: The starch accumulation and root gravitropism in the LA3 complemented lines; Figure S4: Haplotype analysis of LA3 and their contributions to root angle; Table S1: Primers used for RT-qPCR and constructions in rice; Table S2: The coefficients summary table of the mixed model of gravitropism time-course assay; Table S3: The list of accessions and haplotypes.

Author Contributions

H.S.: investigation, writing—original draft, writing—review and editing; L.H.: investigation, formal analysis, writing—original draft, writing—review and editing; J.C.: investigation, formal analysis, writing—original draft, writing—review and editing; W.W.: investigation, formal analysis, methodology, supervision, writing—original draft, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China (32472117 to W.W.; 32372075 to L.H.) and the Taishan Scholar Project of Shandong Province of China (NO.tsqn202408117).

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Materials; further inquiries can be directed to the corresponding author.

Acknowledgments

We would like to thank Dali Zeng (Zhejiang A&F University) and Guosheng Xiong (Nanjing Agricultural University) for supplying rice cultivars and information relating to them.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. LA2 and LA3 are both involved in root gravitropism. (A) Root gravitropism of wild-type ZH11, CR-la2-1, and CR-la3-1 after a time-course gravistimulation (0 h, 2 h, 4 h, 6 h, 8 h). Bars = 1 cm. (B) Statistical analysis of root curvature of the lines in (A). Values are means ± SD (n = 30).
Figure 1. LA2 and LA3 are both involved in root gravitropism. (A) Root gravitropism of wild-type ZH11, CR-la2-1, and CR-la3-1 after a time-course gravistimulation (0 h, 2 h, 4 h, 6 h, 8 h). Bars = 1 cm. (B) Statistical analysis of root curvature of the lines in (A). Values are means ± SD (n = 30).
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Figure 2. LA2 and LA3 are both involved in starch biosynthesis in rice root tips. Starch granule staining of the root tips of wild-type ZH11, CR-la2-1, CR-la2-2, CR-la3-1, and CR-la3-6 at the seedling stage. Bars = 200 μm.
Figure 2. LA2 and LA3 are both involved in starch biosynthesis in rice root tips. Starch granule staining of the root tips of wild-type ZH11, CR-la2-1, CR-la2-2, CR-la3-1, and CR-la3-6 at the seedling stage. Bars = 200 μm.
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Figure 3. Starch accumulation and root gravitropism in LA2 complemented lines. (A) Starch granule staining of the root tips of wild-type 9311, la2, and LA2pro:LA2/la2 lines and the Ubi:LA2-3×Flag/la2 line at the seedling stage. Bars = 200 μm. (B) Root gravitropism of wild-type 9311, la2, and LA2pro:LA2/la2 lines and the Ubi:LA2-3×Flag/la2 line after gravistimulation for 4 h. Bars = 1 cm. (C) Statistical analysis of root curvature of the lines in (A). Values are means ± SD (n = 30). Different letters above the column represent statistically significant differences at p < 0.05; one-way ANOVA, Tukey’s honestly significant difference.
Figure 3. Starch accumulation and root gravitropism in LA2 complemented lines. (A) Starch granule staining of the root tips of wild-type 9311, la2, and LA2pro:LA2/la2 lines and the Ubi:LA2-3×Flag/la2 line at the seedling stage. Bars = 200 μm. (B) Root gravitropism of wild-type 9311, la2, and LA2pro:LA2/la2 lines and the Ubi:LA2-3×Flag/la2 line after gravistimulation for 4 h. Bars = 1 cm. (C) Statistical analysis of root curvature of the lines in (A). Values are means ± SD (n = 30). Different letters above the column represent statistically significant differences at p < 0.05; one-way ANOVA, Tukey’s honestly significant difference.
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Figure 4. LA2 and LA3 are both involved in root architecture. (A) The gross phenotypes of wild-type ZH11, CR-la2-1, CR-la2-2, CR-la3-1, and CR-la3-6 grown in the field. Bar = 5 cm. (B) The gross phenotypes of wild-type ZH11, CR-la2-1, CR-la2-2, CR-la3-1, and CR-la3-6 grown in a rhizobox. Bar = 5 cm. (C) Statistical analysis of the root angle of the lines in (A). (D) Statistical analysis of the root angle of the lines in (B). Values are means ± SEM (n = 10). Different letters above the column represent statistically significant differences at p < 0.05; one-way ANOVA, Tukey’s honestly significant difference.
Figure 4. LA2 and LA3 are both involved in root architecture. (A) The gross phenotypes of wild-type ZH11, CR-la2-1, CR-la2-2, CR-la3-1, and CR-la3-6 grown in the field. Bar = 5 cm. (B) The gross phenotypes of wild-type ZH11, CR-la2-1, CR-la2-2, CR-la3-1, and CR-la3-6 grown in a rhizobox. Bar = 5 cm. (C) Statistical analysis of the root angle of the lines in (A). (D) Statistical analysis of the root angle of the lines in (B). Values are means ± SEM (n = 10). Different letters above the column represent statistically significant differences at p < 0.05; one-way ANOVA, Tukey’s honestly significant difference.
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Figure 5. Haplotype analysis of LA2 and its contributions to root angle. (A) Haplotype clusters of LA2. The different colors on the top of the heat map indicated variant type. HC1~HC7 indicated different haplotype clusters of LA2. (B) The representative root architecture of HC1 and HC5 of LA2. Scale bars = 5 cm. (C) Statistical analysis of the root angle of HC1 and HC5 of LA2. n = 33 (HC1) and 30 (HC5). * p ≤ 0.05; linear mixed model regression.
Figure 5. Haplotype analysis of LA2 and its contributions to root angle. (A) Haplotype clusters of LA2. The different colors on the top of the heat map indicated variant type. HC1~HC7 indicated different haplotype clusters of LA2. (B) The representative root architecture of HC1 and HC5 of LA2. Scale bars = 5 cm. (C) Statistical analysis of the root angle of HC1 and HC5 of LA2. n = 33 (HC1) and 30 (HC5). * p ≤ 0.05; linear mixed model regression.
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Figure 6. LA2 can form homodimers in vivo and in vitro. (A) LA2 could form homodimers in the tobacco leaves in the LUC assay. The pseudocolor bar shows the luminescence intensity in the image. (B) LA2 formed homodimers in the rice chloroplast. Scale bars = 10 µm. (C) Pull-down assay using recombinant GST-LA2 and lysates prepared from the Ubi:LA2-3×Flag/la2 transgenic line. Immunoblotting was conducted with an anti-Flag M2 monoclonal antibody and an anti-GST monoclonal antibody. (D) LA2 could form homodimers in the co-immunoprecipitation assay. Immunoprecipitation was conducted with an anti-GFP mAb-agarose, and immunoblotting was performed with an anti-Flag M2 monoclonal antibody and an anti-GFP monoclonal antibody.
Figure 6. LA2 can form homodimers in vivo and in vitro. (A) LA2 could form homodimers in the tobacco leaves in the LUC assay. The pseudocolor bar shows the luminescence intensity in the image. (B) LA2 formed homodimers in the rice chloroplast. Scale bars = 10 µm. (C) Pull-down assay using recombinant GST-LA2 and lysates prepared from the Ubi:LA2-3×Flag/la2 transgenic line. Immunoblotting was conducted with an anti-Flag M2 monoclonal antibody and an anti-GST monoclonal antibody. (D) LA2 could form homodimers in the co-immunoprecipitation assay. Immunoprecipitation was conducted with an anti-GFP mAb-agarose, and immunoblotting was performed with an anti-Flag M2 monoclonal antibody and an anti-GFP monoclonal antibody.
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Song, H.; Huang, L.; Chen, J.; Wang, W. LAZY2 and LAZY3 Regulate Rice Root Gravitropism by Affecting Starch Accumulation. Agronomy 2025, 15, 2639. https://doi.org/10.3390/agronomy15112639

AMA Style

Song H, Huang L, Chen J, Wang W. LAZY2 and LAZY3 Regulate Rice Root Gravitropism by Affecting Starch Accumulation. Agronomy. 2025; 15(11):2639. https://doi.org/10.3390/agronomy15112639

Chicago/Turabian Style

Song, Haoran, Linzhou Huang, Jiaze Chen, and Wenguang Wang. 2025. "LAZY2 and LAZY3 Regulate Rice Root Gravitropism by Affecting Starch Accumulation" Agronomy 15, no. 11: 2639. https://doi.org/10.3390/agronomy15112639

APA Style

Song, H., Huang, L., Chen, J., & Wang, W. (2025). LAZY2 and LAZY3 Regulate Rice Root Gravitropism by Affecting Starch Accumulation. Agronomy, 15(11), 2639. https://doi.org/10.3390/agronomy15112639

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