Knockdown of microRNA390 Enhances Maize Brace Root Growth

Brace root architecture is a critical determinant of maize’s stalk anchorage and nutrition uptake, influencing root lodging resistance, stress tolerance, and plant growth. To identify the key microRNAs (miRNAs) in control of maize brace root growth, we performed small RNA sequencing using brace root samples at emergence and growth stages. We focused on the genetic modulation of brace root development in maize through manipulation of miR390 and its downstream regulated auxin response factors (ARFs). In the present study, miR167, miR166, miR172, and miR390 were identified to be involved in maize brace root growth in inbred line B73. Utilizing short tandem target mimic (STTM) technology, we further developed maize lines with reduced miR390 expression and analyzed their root architecture compared to wild-type controls. Our findings show that STTM390 maize lines exhibit enhanced brace root length and increased whorl numbers. Gene expression analyses revealed that the suppression of miR390 leads to upregulation of its downstream regulated ARF genes, specifically ZmARF11 and ZmARF26, which may significantly alter root architecture. Additionally, loss-of-function mutants for ZmARF11 and ZmARF26 were characterized to further confirm the role of these genes in brace root growth. These results demonstrate that miR390, ZmARF11, and ZmARF26 play crucial roles in regulating maize brace root growth; the involved complicated molecular mechanisms need to be further explored. This study provides a genetic basis for breeding maize varieties with improved lodging resistance and adaptability to diverse agricultural environments.


Introduction
Maize stands as one of the foremost vital crops, serving as a staple for human food and animal feed [1].Over recent decades, significant enhancements in grain yield have been achieved through advanced breeding techniques and improved management practices, notably increasing plant density [2,3].However, these improvements have also led to elevated risks of lodging, adversely impacting grain yield, quality, and escalating harvest costs [4].Consequently, the development of lodging-resistant maize cultivars has emerged as a crucial objective among breeders.An optimal root system architecture is essential for augmenting resistance to lodging [5].Lodging in maize is primarily categorized into stalk and root lodging.Stalk lodging typically manifests at late growth stages with internodes beneath the ear buckle, while root lodging can occur at any growth stage if the root system fails to anchor the plant upright [5][6][7][8][9].The maize yield loss due to root lodging can be up to 30% [10].Studies increasingly show that resistance to root lodging substantially depends on the root system's size and extent [5,11].The maize root architecture comprises embryonic primary, seminal, lateral, and nodal roots [8,9].Among these, nodal roots, including subterranean crown and aerial brace roots, play pivotal roles in anchorage and water uptake, predominantly expressing diverse water and nutrient transporters [8,12].The architecture of brace roots is intimately linked with their capacity to resist lodging [5,9,11,13].Of the developmental phases of brace roots-induction, initiation, emergence, and growth-emergence and growth phases are critical for establishing the root's angle and length, which are crucial for plant anchorage [8,12,14].Thus, deciphering the molecular mechanisms underlying brace root emergence and growth stages is imperative for a deeper understanding of maize root architecture.
MicroRNAs (miRNAs), comprising 20-24 nucleotides, are endogenous small RNAs that play critical roles in gene silencing through transcript cleavage or translational repression [23].These molecules are fundamental in regulating plant growth, reproduction, and biotic and abiotic stress responses.Recent studies have highlighted the influence of specific miRNAs, such as miR160, miR165/166, miR396, miR167, miR390, miR857, and miR169, in shaping plant root systems [24][25][26][27][28].For instance, miR390 is particularly notable, regulating root growth and structure by triggering the production of TAS3-transacting siRNAs (tasiRNAs), which target auxin response factors (ARFs), influencing various developmental processes [24,26,[29][30][31].However, the specific roles of miR390 in maize brace root development remain unclear.In maize, the miR390 family has two members, miR390a and miR390b, which cleave TAS3 precursors and generate tasiRNAs [26].TAS3-tasiRNAs further target several ARFs, including ZmARF11, ZmARF12, ZmARF23, ZmARF24, and ZmARF26 [32].In the present study, we perform deep sequencing of small RNAs to identify miRNAs involved in brace root growth.By constructing miR390 knockdown vectors through short tandem target mimic (STTM) technology, we generated transgenic mutants to validate the functional roles of the identified miRNAs.Our findings suggest that miR390 significantly influences brace root development via the TAS3-ZmARF11/ZmARF26 pathway, underscoring its potential as a target for genetic enhancements aimed at improving root system architecture and developing lodging-resistant maize varieties.

Identification of Key miRNAs in Brace Root Growth
To identify the key miRNAs involved in regulating maize brace root growth, brace root samples from the inbred line B73 were collected at emergence and growth stages and subjected to small RNA deep sequencing and transcriptome analysis.This comprehensive approach led to the identification of 22 differentially expressed miRNAs, split into 12 upregulated and 10 down-regulated miRNAs.Notably, seven known miRNAs, including zma-miR167b-3p, zma-miR172d-5p, zma-miR390b-5p, zma-miR390a-5p, zma-miR166m-3p, zma-miR172b-5p, and zma-miR166l-3p, were found to be up-regulated (Figure 1, Table 1).All ten down-regulated miRNAs were novel, suggesting they might play previously unrecognized roles in maize brace root development.

Enhancement of Brace Root Growth in Maize via STTM-Mediated Knockdown of miR390
Previous research has suggested that established short tandem target mimic (STTM) technology is highly effective for investigating miRNA function [35,36].To explore the functions of miRNAs identified as crucial for brace root growth, miR390 was specifically targeted for knockdown using an STTM vector constructed with a 48nt linker sequence and two un-cleavable miR390 binding sites.Each binding site was designed to include a three-base insertion (CTA) between the 10th and 11th bases, ensuring resistance to miR390 cleavage (Figure 2A).The obtained STTM390 transgenic mutant plants exhibited notable phenotypic changes, including reduced plant height, narrower leaf angles above the uppermost ear, and broader leaf angles below the uppermost ear compared to wild-type plants, which typically show narrow leaf angles below the uppermost ear (Figure 2B).The relative expression levels of miR390 in these STTM390 mutants ranged from 21.2% to 55.1% compared to the control line C01, demonstrating effective knockdown (Figure 2C).

Contrasting Roles of ZmARF11 and ZmARF26 in Enhancing Brace Root Growth and Impeding Soil Penetration in ZmARF EMS Mutants
In the STTM390 transgenic maize, the expression levels of miR390 downstream genes, ZmARF11 and ZmARF26, were significantly up-regulated in brace roots, suggesting their active roles in brace root development.To further investigate these roles, EMS The 1st whorl indicates the uppermost brace root whorl, the 2nd whorl is the second brace root whorl from the uppermost to the bottom, and the 3rd whorl is the third brace root whorl.U6 small nuclear RNA and Actin were used as the internal control of miR390 and target/downstream regulated genes in qRT-PCR analysis, respectively.*, ** represents that the corresponding miR390 and gene expression levels of STTM390 plants are significantly and very significantly different from the wild type, respectively.Bars show standard error of tested values.
Analysis of TAS3 precursor expressions revealed a significant decrease in TAS3c expression in STTM390 plants, whereas TAS3g expression was upregulated (Figure 2D).This differential expression pattern suggests a complex regulation by miR390 on different TAS3 precursors.The downstream target genes of TAS3-tasiRNAs, including ZmARF11 and ZmARF26, displayed up-regulated expression, aligning with the observed phenotypic alterations.Remarkably, the STTM390 plants displayed a narrow brace root angle and increased brace root whorls (Figure 2E-G).When comparing with the length of the first layer brace root, those of STTM390 mutants were more than twice as long as those in C01.These findings strongly suggest that miR390 plays a critical role in regulating both brace root growth and the gravimetric response in maize, potentially offering new avenues for enhancing root architecture through targeted miRNA manipulation.

Contrasting Roles of ZmARF11 and ZmARF26 in Enhancing Brace Root Growth and Impeding Soil Penetration in ZmARF EMS Mutants
In the STTM390 transgenic maize, the expression levels of miR390 downstream genes, ZmARF11 and ZmARF26, were significantly up-regulated in brace roots, suggesting their active roles in brace root development.To further investigate these roles, EMS mutants arf11 (EMS4-10a34d) and arf26 (EMS4-1c4e8c) were analyzed under field conditions.Compared to the wild type (B73), both arf11 and arf26 mutants exhibited a slight increase in plant height, with no notable difference in leaf shape and stalk thickness (Figure 3).Phenotypically, the arf11 mutant plants demonstrated an increase in the number and length of brace roots; however, the penetration of these brace roots into the soil was reduced.Conversely, the arf26 mutant plants had increased brace root length and diameter but a decreased number of brace roots that enter the soil.These observations suggest that ZmARF11 and ZmARF26 play distinct roles in modifying brace root architecture.While these genes clearly contribute to physical changes in root traits, the precise molecular mechanisms by which they influence brace root growth require further elucidation.This continued investigation is essential to fully understand the regulatory pathways involved in brace root development mediated by miR390 and its downstream regulated genes.mutants arf11 (EMS4-10a34d) and arf26 (EMS4-1c4e8c) were analyzed under field conditions.Compared to the wild type (B73), both arf11 and arf26 mutants exhibited a slight increase in plant height, with no notable difference in leaf shape and stalk thickness (Figure 3).Phenotypically, the arf11 mutant plants demonstrated an increase in the number and length of brace roots; however, the penetration of these brace roots into the soil was reduced.Conversely, the arf26 mutant plants had increased brace root length and diameter but a decreased number of brace roots that enter the soil.These observations suggest that ZmARF11 and ZmARF26 play distinct roles in modifying brace root architecture.While these genes clearly contribute to physical changes in root traits, the precise molecular mechanisms by which they influence brace root growth require further elucidation.This continued investigation is essential to fully understand the regulatory pathways involved in brace root development mediated by miR390 and its downstream regulated genes.

Alterations of Brace Root Microstructure and Phytohormone Levels in STTM390
The vascular bundle, a crucial transport system of vascular plants, mainly comprises phloem and xylem, serving essential roles in nutrition transport and structural support for the aerial parts of the plant [37,38].To investigate the microstructural changes in brace roots of maize STTM390 mutants, histological analyses were conducted on the cross sections from the elongation zone of brace roots in both C01 and STTM390 plants (Figure 4).

Alterations of Brace Root Microstructure and Phytohormone Levels in STTM390
The vascular bundle, a crucial transport system of vascular plants, mainly comprises phloem and xylem, serving essential roles in nutrition transport and structural support for the aerial parts of the plant [37,38].To investigate the microstructural changes in brace roots of maize STTM390 mutants, histological analyses were conducted on the cross sections from the elongation zone of brace roots in both C01 and STTM390 plants (Figure 4).The analysis revealed a significant reduction in the number of xylem vessels in maize STTM390 plants compared to C01, with the remaining vessels exhibiting uneven distribution.Additionally, there was a noticeable decrease in the thickness of the brace root phloem in STTM390 plants, suggesting that the STTM-mediated knockdown of miR390 led to substantial alterations in the microstructure of maize brace roots.The analysis revealed a significant reduction in the number of xylem vessels in maize STTM390 plants compared to C01, with the remaining vessels exhibiting uneven distribution.Additionally, there was a noticeable decrease in the thickness of the brace root phloem in STTM390 plants, suggesting that the STTM-mediated knockdown of miR390 led to substantial alterations in the microstructure of maize brace roots.
Phytohormones such as auxin are known to play pivotal roles in brace root development, and miR390 has been implicated in auxin signaling pathways.Accordingly, the levels of indole-3-acetic acid (IAA) and abscisic acid (ABA) were measured in both STTM390 and C01 plants (Figure 4).The results indicated a significant increase in ABA content and a decrease in IAA levels in STTM390 plants.These changes in phytohormone levels are indicative of the regulatory influence of miR390 on hormone synthesis or signaling pathways, further affecting root development and plant response to environmental stimuli.Phytohormones such as auxin are known to play pivotal roles in brace root development, and miR390 has been implicated in auxin signaling pathways.Accordingly, the levels of indole-3-acetic acid (IAA) and abscisic acid (ABA) were measured in both STTM390 and C01 plants (Figure 4).The results indicated a significant increase in ABA content and a decrease in IAA levels in STTM390 plants.These changes in phytohormone levels are indicative of the regulatory influence of miR390 on hormone synthesis or signaling pathways, further affecting root development and plant response to environmental stimuli.

Unexpected Regulatory Interactions: Downregulation of Both miR166 and Its Targets following miR390 Knockdown in Maize
Previously studies established an interaction between miR166 and miR390 through the TAS3-tasiRNA pathway in maize [39,40].To further explore the impact of the miR390 knockdown on miR166 and its target genes, we conducted RT-qPCR analysis on several key genes regulated by miR166.These genes include Zm00001d031061 (Homeobox-transcription factor 119, ZmHB119), Zm00001d041489 (ZmHB25), Zm00001d048527 (Rolled leaf1, Rld1), and Zm00001d027317 (Rld2) (Figure 5).The RT-qPCR results indicated a down-regulation in the expression of miR166 as well as all its analyzed target genes in STTM390 plants compared to controls.This down-regulation suggests that the knockdown of miR390 may indirectly influence the expression patterns of miR166 and its associated regulatory network, impacting developmental and morphological processes governed by these genes.These findings contribute to our understanding of the complex regulatory relationships between miRNAs in maize and underscore the broader implications of miR390 manipulation on the maize transcriptome, particularly in how it influences other significant miRNAs and their downstream effects.

Unexpected Regulatory Interactions: Downregulation of Both miR166 and Its Targets following miR390 Knockdown in Maize
Previously studies established an interaction between miR166 and miR390 through the TAS3-tasiRNA pathway in maize [39,40].To further explore the impact of the miR390 knockdown on miR166 and its target genes, we conducted RT-qPCR analysis on several key genes regulated by miR166.These genes include Zm00001d031061 (Homeobox-transcription factor 119, ZmHB119), Zm00001d041489 (ZmHB25), Zm00001d048527 (Rolled leaf1, Rld1), and Zm00001d027317 (Rld2) (Figure 5).The RT-qPCR results indicated a down-regulation in the expression of miR166 as well as all its analyzed target genes in STTM390 plants compared to controls.This down-regulation suggests that the knockdown of miR390 may indirectly influence the expression patterns of miR166 and its associated regulatory network, impacting developmental and morphological processes governed by these genes.These findings contribute to our understanding of the complex regulatory relationships between miRNAs in maize and underscore the broader implications of miR390 manipulation on the maize transcriptome, particularly in how it influences other significant miRNAs and their downstream effects.

Transcriptomic Profiling in miR390 Knockdown Maize Reveals Dysregulation in Senescence, Cell Wall Biogenesis, and Phytohormone Signaling Pathways
To elucidate the regulatory pathway influence by miR390 in maize brace root growth, transcriptome analysis was conducted on brace root samples from both STTM390 and C01 (Table 3).The transcriptome analysis identified a total of 294 differentially expressed genes (DEGs), with 244 genes showing up-regulation and 50 genes showing down-regulation in the STTM390 mutants compared to controls.Validation through RT-qPCR confirmed the expression trends observed in the transcriptome data for selected genes (Figure 6, Table 3).
Among the up-regulated DEGs, several were identified as key players in various biological processes, such as wounding response, senescence and phytohormone response,

Transcriptomic Profiling in miR390 Knockdown Maize Reveals Dysregulation in Senescence, Cell Wall Biogenesis, and Phytohormone Signaling Pathways
To elucidate the regulatory pathway influence by miR390 in maize brace root growth, transcriptome analysis was conducted on brace root samples from both STTM390 and C01 (Table 3).The transcriptome analysis identified a total of 294 differentially expressed genes (DEGs), with 244 genes showing up-regulation and 50 genes showing down-regulation in the STTM390 mutants compared to controls.Validation through RT-qPCR confirmed the expression trends observed in the transcriptome data for selected genes (Figure 6, Table 3).Among the up-regulated DEGs, several were identified as key players in various bio-logical processes, such as wounding response, senescence and phytohormone response, lignin catabolism and microtubule function, and cell death.Wounding response: genes such as Zm00001d053934, Zm00001d053933, and Zm00001d015077, which are implicated in the plant's response to physical damage, showed increased expression in STTM390 plants.Senescence and phytohormone response: genes such as Zm00001d004355, Zm00001d037303, Zm00001d045711, and Zm00001d045708 also showed up-regulated expression in STTM390 plants.Lignin catabolism and microtubule function: A gene involved in lignin breakdown (Zm00001d049085) and two genes related to microtubule-based movement (Zm00001d011129 and Zm00001d021253) exhibited increased expression levels.Cell death: three genes linked to cell death processes (Zm00001d047256, Zm00001d004355, and Zm00001d027486) were identified to display up-regulated expression in STTM390.
auxin-responsive protein encoding gene (Zm00001d021457) were among those that showed reduced expression levels in STTM390.Significant changes were also observed in auxin signaling-related genes between the STTM390 and C01 samples, highlighting genes like Zm0001d030310 (lax1), Zm0001d016277 (IAA7), Zm0001d045203 (IAA29), and Zm0001d018024 (PIN2), along with ZmARF11 and ZmARF26, which showed notable expression changes.Gene Ontology (GO) analysis indicated that most DEGs were associated with molecular functions, with fewer genes enriched in cellular components or biological processes (Figure 7).The down-regulated DEGs were mainly linked to purine ribonucleoside triphosphate binding, metabolic processes involving purine-containing compounds, and various aspects of cell wall macromolecule metabolism and biosynthesis.Up-regulated DEGs were predominantly enriched in activities involving catalysis, hydrolase and peptidase activity, proteolysis, and responses to ethylene, highlighting their roles in the cell periphery, plasma membrane, and extracellular regions.The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis further revealed that down-regulated DEGs were enriched in metabolic pathways related to secondary metabolites, purine metabolism, and plant hormone signal transduction, while up-regulated DEGs were more frequently associated with pathways involved in amino acid biosynthesis and pyrimidine metabolism (Figure 8).These findings provide valuable insights into the complex genetic and biochemical landscape altered in maize due to the knockdown of miR390, suggesting a broad impact on developmental and stress response pathways.Furthermore, genes involved in jasmonic acid biosynthetic processes (Zm00001d052096), gibberellin biosynthetic processes (Zm00001d047830), and plant-type cell wall organization (Zm00001d034551) also displayed up-regulation in STTM390 plants.Conversely, two cell wall biogenesis-related genes (Zm00001d039231 and Zm00001d027525), an ethylene biosynthesis-related gene (Zm00001d049823), and an auxin-responsive protein encoding gene (Zm00001d021457) were among those that showed reduced expression levels in STTM390.Significant changes were also observed in auxin signaling-related genes between the STTM390 and C01 samples, highlighting genes like Zm0001d030310 (lax1), Zm0001d016277 (IAA7), Zm0001d045203 (IAA29), and Zm0001d018024 (PIN2), along with ZmARF11 and ZmARF26, which showed notable expression changes.
Gene Ontology (GO) analysis indicated that most DEGs were associated with molecular functions, with fewer genes enriched in cellular components or biological processes (Figure 7).The down-regulated DEGs were mainly linked to purine ribonucleoside triphosphate binding, metabolic processes involving purine-containing compounds, and various aspects of cell wall macromolecule metabolism and biosynthesis.Up-regulated DEGs were predominantly enriched in activities involving catalysis, hydrolase and peptidase activity, proteolysis, and responses to ethylene, highlighting their roles in the cell periphery, plasma membrane, and extracellular regions.The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis further revealed that down-regulated DEGs were enriched in metabolic pathways related to secondary metabolites, purine metabolism, and plant hormone signal transduction, while up-regulated DEGs were more frequently associated with pathways involved in amino acid biosynthesis and pyrimidine metabolism (Figure 8).These findings provide valuable insights into the complex genetic and biochemical landscape altered in maize due to the knockdown of miR390, suggesting a broad impact on developmental and stress response pathways.

Discussion
Auxin plays a pivotal role in controlling plant morphology and environmental responses through its interaction with AUXIN RESPONSE FACTORS (ARFs) [41,42].These factors are crucial in translating the auxin signal into developmental changes.Three miRNA families, miR160, miR167 and miR390, have been identified to confer plant growth via targeting or regulating different ARFs.In Arabidopsis, miR160 targets three ARFs genes, ARF10, ARF16 and ARF17 [43]; miR167 regulates ARF6 and ARF8 by mediating gene silencing [44]; miR390 negatively regulates the expression of ARF2, ARF3, and ARF4 by triggering TAS3-tasiRNAs [45][46][47].Additionally, miR165/166 and miR172 were also found to be involved in other phytohormone signaling, interacting with auxin signaling.miR165/166 was proved to play critical roles in ABA homeostasis [48] and auxin signaling [49].miR172 was found to be involved in ethylene signaling [50].A recent study revealed that the crosstalk of ethylene and auxin fine-tuned root gravitropic response [51].In Arabidopsis, miR160 and miR167 regulate adventitious rooting by targeting ARF17, ARF6, and ARF8 [52]; miR390 confers lateral root development through the TAS3-tasiRNA-ARF pathway [47]; the balance of miR165/166 and its targets play important roles in root development [53]; miR172 regulates plant de novo root regeneration through the ethylene signaling pathway [51].Moreover, miRNAs such as miR444 [54], miR171 [55], miR393, and miR390 [56] have been identified to regulate root development in rice.In maize, several miRNAs were also identified in controlling maize root development, including miR172 [57], miR166 [25,26], and miR390 [26].miR172 regulates maize brace root by suppressing the expression of its target gene Zm-RAP2.7 [57].In this study, we found that the expression of miR167, miR172, miR166, and miR390 display significant alterations between the emergence and growth stages of maize brace root development.Together with these previous studies, our results indicate that the four miRNAs possibly play essential roles in regulating maize brace root growth.
The miR390-TAS3-tasiRNA-ARF regulatory module was found to play a pivotal role in regulating root development [26].The underlying mechanism of miR390 in control of brace root development remains unclear.In the present study, the miR390 inactivation mutant, STTM390, displayed similar phenotypic alterations to the loss-of-function mutants of ZmARF11 and ZmARF26 (two downstream regulated genes of miR390) [32], which indicated that miR390 and the two ARFs are involved in regulating brace root growth.Such phenotypic alterations are probably controlled by the changes in gene expression.The knockdown of miR390 mediates the two ARFs and TAS3g up-regulation; mediates TAS3c, miR166, and its target genes down-regulation.These results suggested that the miR390-TAS3-ZmARF11/26 regulatory module confers brace root growth through a complicated molecular mechanism.Transcriptome analysis revealed miR390 to be associated with the extracellular region, cell wall macromolecule metabolic process, cell wall macromolecule biosynthetic process, cell wall polysaccharide biosynthetic process, cell periphery, cell death, response to ethylene, and plant hormone signal transduction.These pathways are probably at the downstream of miR390, mediating the phenotypic alterations in maize brace root.
In modern breeding of maize, the planting density has increased from 30,000 plants per hectare in the 1930s to >80,000 plants per hectare in the 2010s [2,58,59].High-density planting increases the competition between plants for nutrients and water, leading to remodeling of the root system architecture for improving water and nitrogen use efficiency [60][61][62].The modern hybrid and inbred lines have the steep root system [15,61,63].The brace root number, whorls growing into the soil, and spread width are tightly associated with maize root lodging resistance [9,64,65].However, steeper root angle is favorable for accessing subsoil water, enhancing drought tolerance, and improving nitrogen capture, while shallow root angle improves the capture of phosphorus from topsoil [66,67].Thus, an ideal root system architecture must reach a balance between lodging resistance and nutrition uptake.In this study, four known miRNAs were identified to be involved in the regulation of maize brace root growth, including miR390, miR166, miR167, and miR390.The obtained STTM390, arf11, and arf26 mutants all showed increased brace root number and brace root length, which are beneficial for enhancing root lodging resistance and nutrition uptake.These discoveries provide valuable genetic targets for enhancing maize traits, such as lodging resistance and optimized root architecture, which are vital for improved water and nutrient acquisition.

Vector Construction and Maize Transformation
In alignment with our previous study [36,68], the STTM390 binary expression vector was constructed following the established methods for STTM vector assembly [69].Initially, STTM390 was cloned into the intermediate vector pOT2-poly-cis, yielding the recombinant vector pOT2-STTM390.Subsequently, the binary expression vector pTF101.1,with an additional PacI cleavage site, was employed for maize STTM transformation.The STTM390 insert from pOT2-STTM390 was subsequently cloned into the modified binary expression vector pTF101.1/PacI,resulting in the construct pTF101.1-STTM390.The final construct was confirmed through Spectinomycin resistance and DNA sequencing using the STTM common real primer (Table S1).The constructed pTF101.1-STTM390binary expression vector was transformed in maize inbred line C01 (Life Science and Technology Center of China Seed Group Co., Wuhan, China).

Transgenic Plant Screening, Genotyping, and Phenotyping
The transgenic maize plants harboring the STTM390 construct were initially screened by basta resistance and subsequently genotyped using the STTM common real primer (Table S1).Quantitative RT-PCR (qRT-PCR) was utilized to determine the expression levels of miR390, its target TAS3 precursors, and downstream ARFs (all the primers are listed in Table S1).To ensure genetic consistency, STTM390 plants were self-pollinated over three generations to obtain homozygous lines.Phenotypic evaluations were performed in three distinct environments.During the flowering stage, brace root samples from STTM390 and control C01 plants were collected at the growth stage for qRT-PCR analysis; each sample consisted of three biological replicates, with each replicate comprising three plants from the same transformation event.Samples were immediately frozen in liquid nitrogen and stored at −80 • C for subsequent transcriptomic analysis.

Small RNA Deep Sequencing, Transcriptome Sequencing Analysis, and qRT-PCR
Field cultivation of the inbred line B73 was conducted up to the flowering stage.Brace root samples were collected from the emergence and growth stages, with each set comprising three replicates, each including three randomly selected plants.These samples underwent small RNA deep sequencing and transcriptome sequencing analysis.Total RNA from the brace root samples was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and assessed by 1% agarose gel electrophoresis to ensure quality.RNA samples were then submitted to Berry Genomics (Beijing, China) for high throughput sequencing using the Illumina NovaSeq 6000 platform.
For small RNA library construction, RNA fragments ranging from 18 to 30 nt were isolated using 15% polyacrylamide gel electrophoresis.These fragments were subsequently ligated with 5 ′ and 3 ′ adaptors and used as templates for cDNA synthesis.PCR amplification was carried out for about 18 cycles to generate sufficient products for sequencing.Six small RNA libraries (across two developmental stages and three biological replicates) were prepared using the TruSeq Small RNA Sample Prep Kit (Illumina Technologies, San Diego, CA, USA).The raw reads were processed to trim adaptors and remove low-quality reads and poly(N) sequences.The quality of the cleaned reads was assessed by evaluating the Q20, Q30, and GC content.All clean reads, ranging from 18 to 40 nt, were mapped on the maize reference genome (B73 RefGen_V4.42)using HISAT2 V2.1.0(default parameters setting) [71].miRNA expression levels were normalized using the parameter transcripts per million (TPM).The differentially expressed miRNA was defined as |log2 fold change|> 1 and used a p-value < 0.05.
Transcriptomic sequencing involved preparing cDNA libraries from brace root samples of B73 at both developmental stages, as well as from C01 and STTM390 during the growth stage, using the TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, USA).These libraries were sequenced on the Illumina NovaSeq 6000 platform.Transcriptomic sequencing data commenced with the trimming of adapters and low-quality bases using Trimmomatic V0.36 (FSU, Tallahassee, FL, USA) [72].The clean reads were aligned to the maize reference genome (B73 RefGen_V4.42)using the HISAT2 V2.1.0(default parameters setting) [71].Transcript abundance was further calculated as fragments per kilobase of exon per million fragments mapped (FPKM).DEGs were identified using DESeq2 software V1.22.2 [73], with the threshold settings of |log2 fold change| ≥ 1 and p-values < 0.05.Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted using the maize profile database (org.Zeamays.eg.sqlite) in clus-terProfiler software V3.10.1 [74] and Annotation Hub (V2.14.5)R package.The enrichment analyses used the ensemble database to convert the gene number (maizegbdId) to the corresponding Entrez ID.
To validate the sequencing results and measure the expression levels of miR390, miR166, their target genes, and downstream regulated genes, qRT-PCR analysis was conducted.Expression levels of miR390 and miR166 were quantified using the Mir-X™ miRNA qRT-PCR SYBR ® Kit (Takara, Dalian, China), with U6 small nuclear RNA serving as the internal control.For the protein-encoding genes, total RNA of the tested samples was reverse transcribed using the PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real Time) (Takara, Dalian, China).The resulting cDNA was used for qRT-PCR with the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and the SYBR ® Premix EX Taq™ II (Tli RNaseH Plus) Kit (Takara, Dalian, China).Actin was used as an internal control, and the relative gene expression levels were calculated using the 2−∆∆Ct method [75].All assays included at least three biological replicates.The qRT-PCR primers are listed in Table S1.

Histological Analysis and Plant Hormone Content Measurement
For histological analysis, brace root samples from C01 and STTM390 maize plants were collected during the flowering stage.The elongation zones of these samples, approximately 1 cm in length, were excised and immediately submerged in a pre-chilled 70% FAA (formalin-acetic acid-alcohol) solution.These samples were then vacuum-sealed to remove air and fixed overnight at 4 • C in FAA.Following fixation, the samples underwent a graded ethanol dehydration series, were cleared, and then were embedded in paraffin wax using standard histological techniques (Sigma-Aldrich, St. Louis, MA, USA).The embedded tissues were sectioned into 4 µm slices using a Leica RM 2265 programmable rotary microtome (Leica Microsystems, Wetzlar, Hessian, Germany) and stained with 0.05% toluidine blue.The stained sections were imaged with an Olympus IX73 microscope (Olympus, Tokyo, Japan).
Additionally, the dried brace root samples from C01 and STTM390 plants, previously prepared for qRT-PCR, were analyzed to quantify plant hormone levels.The indoleacetic acid (IAA) and abscisic acid (ABA) contents were determined using high-performance liquid chromatography (HPLC).These measurements were carried out for three biological replicates and four technical replicates, with hormone concentrations calculated based on the dry weight of the samples (Suzhou Comin Biotechnology Co. Ltd., Suzhou, China).

Figure 1 .
Figure 1.Small RNA-seq and transcriptome analysis of brace root during emergence and growth stage in B73.(A) Expression classification of differentially expressed miRNAs.Red dots indicate up-regulated miRNAs, and blue dots are down-regulated miRNAs (significance level: p-value < 0.05, |log2 fold change| > 1).(B) Gene expression classification of DEGs.Red dots indicate up-regulated genes, and blue dots are down-regulated genes (significance level: p-value < 0.05, |log2 fold change| > 1).

21 Figure 2 .
Figure 2. miR390 inactivation by STTM technology and phenotypic alterations in maize.(A) Diagram of STTM390 structure showing a 48 nt length spacer with two uncleavable miR390 binding sites, designed with three-base insertion (CTA) to resist cleavage by miR390.(B) Comparative plant phenotypes of STTM390 and C01 plants, displaying noticeable differences in overall plant morphology.Bar = 20 cm.(C) qRT-PCR analysis of miR390 expression levels in STTM390 and C01 plants.(D) Expression levels of miR390 target genes and downstream regulated genes in STTM390 and C01 plants.(E-G)Phenotypic analysis of root systems in STTM390 and C01 plants.Bar = 10 cm.The 1st whorl indicates the uppermost brace root whorl, the 2nd whorl is the second brace root whorl from the uppermost to the bottom, and the 3rd whorl is the third brace root whorl.U6 small nuclear RNA and Actin were used as the internal control of miR390 and target/downstream regulated genes in qRT-PCR analysis, respectively.*, ** represents that the corresponding miR390 and gene expression levels of STTM390 plants are significantly and very significantly different from the wild type, respectively.Bars show standard error of tested values.

Figure 2 .
Figure 2. miR390 inactivation by STTM technology and phenotypic alterations in maize.(A) Diagram of STTM390 structure showing a 48 nt length spacer with two uncleavable miR390 binding sites, designed with three-base insertion (CTA) to resist cleavage by miR390.(B) Comparative plant phenotypes of STTM390 and C01 plants, displaying noticeable differences in overall plant morphology.Bar = 20 cm.(C) qRT-PCR analysis of miR390 expression levels in STTM390 and C01 plants.(D) Expression levels of miR390 target genes and downstream regulated genes in STTM390 and C01 plants.(E-G) Phenotypic analysis of root systems in STTM390 and C01 plants.Bar = 10 cm.The 1st whorl indicates the uppermost brace root whorl, the 2nd whorl is the second brace root whorl from the uppermost to the bottom, and the 3rd whorl is the third brace root whorl.U6 small nuclear RNA and Actin were used as the internal control of miR390 and target/downstream regulated genes in qRT-PCR analysis, respectively.*, ** represents that the corresponding miR390 and gene expression levels of STTM390 plants are significantly and very significantly different from the wild type, respectively.Bars show standard error of tested values.

Figure 3 .
Figure 3. Comparative phenotypic analysis of arf11 and arf26 mutants in maize.(A) Phenotypic comparison between arf26 mutant and WT.(B,C) Phenotypic difference of brace root between arf26 mutant and WT.Bar = 10 cm.(D) Phenotypic comparison of plant traits between arf11 mutant and WT.(E,F) Root system comparison between arf11 mutant and WT maize.Bar = 10 cm.

Figure 3 .
Figure 3. Comparative phenotypic analysis of arf11 and arf26 mutants in maize.(A) Phenotypic comparison between arf26 mutant and WT.(B,C) Phenotypic difference of brace root between arf26 mutant and WT.Bar = 10 cm.(D) Phenotypic comparison of plant traits between arf11 mutant and WT.(E,F) Root system comparison between arf11 mutant and WT maize.Bar = 10 cm.

Figure 4 .
Figure 4. Histological analysis and IAA and ABA content measurement in STTM390 and C01 brace roots.(A-D) Cross section views of plant brace root in STTM390 and C01 (10× and 20×).(E,F) Quantitative comparison of Indole-3-acetic acid (IAA) and Abscisic acid (ABA) contents between STTM390 and C01.**, *** represent a significant difference at p < 0.01 and p < 0.001 levels, respectively.Bars show standard error of tested values.

Figure 4 .
Figure 4. Histological analysis and IAA and ABA content measurement in STTM390 and C01 brace roots.(A-D) Cross section views of plant brace root in STTM390 and C01 (10× and 20×).(E,F) Quantitative comparison of Indole-3-acetic acid (IAA) and Abscisic acid (ABA) contents between STTM390 and C01.**, *** represent a significant difference at p < 0.01 and p < 0.001 levels, respectively.Bars show standard error of tested values.

Figure 5 .
Figure 5. Relative expression level of miR166 and its target genes in STTM390 brace roots.(A) The expression of miR166 in STTM390 and C01 brace root.(B) The expression levels of four miR166 target genes in STTM390 and C01 brace root.U6 and Actin were used as the internal control for miR166 and its target genes, respectively.** and *** represents that the corresponding gene expression levels in STTM390 plants are significantly different from the wild type at p < 0.01 and p < 0.001 levels, respectively.Bars show standard error of the tested values.

Figure 5 .
Figure 5. Relative expression level of miR166 and its target genes in STTM390 brace roots.(A) The expression of miR166 in STTM390 and C01 brace root.(B) The expression levels of four miR166 target genes in STTM390 and C01 brace root.U6 and Actin were used as the internal control for miR166 and its target genes, respectively.** and *** represents that the corresponding gene expression levels in STTM390 plants are significantly different from the wild type at p < 0.01 and p < 0.001 levels, respectively.Bars show standard error of the tested values.

Figure 6 .
Figure 6.The expression analysis of DEGs between STTM390 and C01 by RT-qPCR.(A) The expression of down-regulated genes.(B) The expression of up-regulated genes.Actin was used as an internal control.** and *** represent that the corresponding gene expression levels in STTM390 plants are significantly different from the wild type at p < 0.01 and p < 0.001 levels, respectively.Bars show standard error of the tested values.

Figure 6 .
Figure 6.The expression analysis of DEGs between STTM390 and C01 by RT-qPCR.(A) The expression of down-regulated genes.(B) The expression of up-regulated genes.Actin was used as an internal control.** and *** represent that the corresponding gene expression levels in STTM390 plants are significantly different from the wild type at p < 0.01 and p < 0.001 levels, respectively.Bars show standard error of the tested values.

Figure 7 .
Figure 7. Gene ontology analysis of the screened DEGs between STTM390 and C01 plants.(A) This panel displays the gene ontology (GO) categories for up-regulated DEGs.(B) This panel shows the GO categories for down-regulated DEGs.BP, biological process; CC, cell component; MF, molecular function.

Figure 8 .Figure 8 .
Figure 8. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of DEGs between STTM390 and C01 plants.(A) Enrichment of down-regulated genes: This panel presents the KEGG Figure 8. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of DEGs between STTM390 and C01 plants.(A) Enrichment of down-regulated genes: This panel presents the KEGG pathway analysis for genes that are down-regulated in STTM390 compared to the control.(B) Enrichment of up-regulated genes: This panel illustrates the KEGG pathway enrichment for up-regulated genes in STTM390 plants.

Table 1 .
The screened brace root growth-related miRNAs by sRNA-seq analysis in B73.

Table 1 .
The screened brace root growth-related miRNAs by sRNA-seq analysis in B73.

Table 2 .
The screened key DEGs between brace root emergence and growth stages by transcriptomic analysis in B73.

Table 3 .
The screened key DEGs between STTM390 and C01 in the transcriptomic analysis in brace roots.