Arabidopsis thaliana ecotype Columbia-0 (Col-0), proDR5::GFP (Friml et al., 2003), proPIN1::PIN1-GFP (Benková et al., 2003), proPIN3::PIN3-GFP and proPIN7::PIN7-GFP (Blilou et al., 2005) were obtained from the Arabidopsis Biological Resource Centre, Columbus, OH, USA. The vector of pSN1301:MtHB2 which was driven by CaMV 35S was constructed and generation of transgenic plants was previously described (Clough and Bent, 1998). Homozygous transgenic lines of all plants above were selected for this study. Plants with various GFP fusions were obtained by crossing proDR5::GFP, proPIN1::PIN1-GFP, proPIN3::PIN3-GFP and proPIN7::PIN7-GFP lines with MtHB2-expressing transgenic plants L4.

For all Arabidopsis experiments, seeds underwent surface sterilization by a 1-minute incubation in 75% ethanol, followed by rinsing with sterile water, treatment with 10% (v/v) sodium hypochlorite for 15 minutes, and thorough washing with sterile water. These sterilized seeds were then sown on half-strength Murashige and Skoog (1/2 MS) agar plates supplemented with 0.8% sucrose and 0.6% agar (w/v) at pH 5.8. After stratification for 2 days at 4°C, the seeds were grown in a growth chamber under a 14-hour photoperiod at 23/20°C and a photosynthetic photon flux density of 100–120 µmol m^-2 s^-1.

Seeds of Medicago truncatula ‘Jemalong A17’ were treated by soaking in concentrated sulfuric acid for approximately 6 minutes, followed by thorough rinsing with tap water, and then sown on Petri plates containing 0.8% agar. After chilling at 4°C for a day, the seeds were germinated in darkness at 25°C for 2 days. Subsequently, seedlings with approximately 2 cm radicles were transferred to plastic buckets filled with fully aerated nutrient solution and grown in a growth chamber under a 14-hour photoperiod at 25/20°C with a photosynthetic photon flux density of 200–230 µmol m^-2 s^-1. To assess MtHB2 expression in response to auxin, two-week-old seedlings were exposed to a solution containing 10 µM indole 3-acetic acid (IAA) for varying periods (0.5, 1, 2, 4, 8 hours), followed by root sampling for total RNA extraction.

To characterize root phenotypes, 6-day-old seedlings were transferred to 1/2 MS medium with 1% (w/v) agar and grown vertically for an additional 4 days. For the analysis of root responses to auxin, 6-day-old seedlings were transferred to 1/2 MS medium with 1% (w/v) agar containing 0.1 µM 1-naphthyl acetic acid (NAA), 1 µM IAA, and 0.05 µM 2,4-dichloro-phenoxyacetic acid (2,4-D) and grown for 4 days. To study the inhibition of lateral root formation by naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), 6-day-old seedlings were transferred to 1/2 MS medium with 1% (w/v) agar containing 0.5 µM NPA and 0.1 µM TIBA and grown for 6 days. Primary root length was measured using a ruler, and the number of lateral roots exceeding approximately ≥0.5 mm in length was documented. Each treatment involved at least 15 independent seedlings, and all experiments were conducted at least three times.

To quantify the number of lateral root primordia, the entire root was stained with methylene blue (Johnson et al., 1996), with slight modifications. The roots were fixed in a solution of absolute ethanol and glacial acetic acid (3:1, v/v) at 4°C for a minimum of 24 hours, rinsed with distilled water, and then stained with methylene blue (0.01% in distilled water) to visualize the lateral root primordia under a microscope (SZX12, OLYMPUS, Japan). Using this method, dome-shaped lateral root primordia can be observed at the later stages of development before emerging from the parental roots (Peret et al., 2009; Ni et al., 2014).

Roots of 2-day-old and 6-day-old seedlings carrying proDR5::GFP, proPIN1::PIN1-GFP, proPIN3::PIN3-GFP, and proPIN7::PIN7-GFP were imaged using a laser confocal scanning microscope Zeiss LSM510. The fluorescence intensity was quantified using the ImageJ program on confocal sections acquired with consistent settings on the same day for all samples within an experiment.

Total RNA was extracted from the roots of 18-day-old wild-type and MtHB2-expressing transgenic plants using TRIzol reagent. The microarrays were manufactured as previously described (Yang et al., 2012), and all labeling, hybridization, and washing processes were conducted through the Affymetrix custom service (Capitalbio) following the provided protocols. Normalization was carried out as per standard Affymetrix protocols to enable sample comparison, with genes showing more than a 2-fold change considered significantly different. The study employed two biological replicates.

Total RNA was extracted using RNAiso Plus reagent (TaKaRa) and reverse-transcribed into first-strand cDNA with the PrimeScript^® RT reagent Kit (with gDNA eraser) (TaKaRa). Real-time quantitative PCR (RT-qPCR) was conducted using an ABI StepOne Plus instrument. The gene-specific primers utilized for RT-qPCR are detailed in Supplementary Table S1 . The MtActin gene (accession No. BT141409) and AtActin11 (accession No. NM_112046) were employed as internal controls to standardize the expression levels. Each reaction mixture consisted of 5 µL of 2×UltarSYBR Mixture (with ROX) reagent (Cwbio), 2 µL of cDNA samples, and 0.6 µL of 10mM gene-specific primers in a final volume of 10 µL. The thermal cycling program included an initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The relative expression level was determined using the comparative CT method as described by Livak and Schmittgen (2001).

All data were statistically analyzed using analysis of variance with the One-way ANOVA or Independent-Samples t-test through the SPSS 17.0 statistics program. Statistical significance was denoted when P < 0.05.

Upon expressing MtHB2 in transgenic Arabidopsis under the control of the cauliflower mosaic virus (CaMV) 35S promoter, several independent transgenic lines were obtained, as confirmed by PCR analysis. The expression levels of MtHB2 in the selected lines, determined through RT-qPCR, are illustrated in Supplementary Figure S1 . Notably, the transgenic lines exhibited shorter primary roots compared to wild-type plants under normal growth conditions with full nutrient supplementation ( Figure 1A ). Furthermore, the expression of MtHB2 resulted in decreased lateral root number and density in the transgenic plants compared to wild-type plants ( Figure 1B ). The extent of these phenotypic changes appeared to be positively correlated with the level of MtHB2 expression. Considering the crucial role of auxin in root growth and development, the observed alterations in the transgenic lines suggest a potential disruption in auxin transport and distribution. Further analysis revealed an increased MtHB2 transcript in response to IAA treatment, peaking at 2 hours and declining thereafter ( Supplementary Figure S2 ). The presence of auxin-responsive elements in the promoter sequence of MtHB2 suggests its involvement in auxin response mechanisms.

To investigate the altered auxin responses in the transgenic lines, we compared the root responses of wild-type and transgenic seedlings expressing MtHB2 to various exogenous auxin analogs with distinct transport mechanisms. Six-day-old wild-type and transgenic seedlings were subjected to different auxin analog treatments, and root phenotypes were evaluated after a 4-day incubation period. In the absence of auxin analogs, the primary root length in transgenic lines was notably shorter than in wild-type plants, and the growth of primary roots in both genotypes was inhibited by the auxin analogs. While NAA induced similar reductions in primary root growth in both wild-type and transgenic seedlings, IAA and 2,4-D had a greater inhibitory effect on primary root growth in wild-type plants compared to the transgenic lines. For instance, treatment with IAA and 2,4-D inhibited PR growth of WT by 91%, while the same treatment reduced PR growth of transgenic line L11 by 79% and 74%, respectively ( Figure 2A ). Moreover, the transgenic lines exhibited a lower lateral root number than wild-type plants in the absence of auxin analogs, yet treatment with these analogs led to a more increase rate of lateral root number in transgenic lines than that of wild-type plants ( Figure 2B ). Treatment with IAA increased the number of lateral roots per seedlings of WT plants by 93%, while this percentage became 300% and 256% for transgenic line L4 and L11, respectively. This differential response indicates that the expression of MtHB2 renders the transgenic plants more sensitive to auxin. Similar trends were observed in the effects of IAA treatment on lateral root density ( Figure 2C ). IAA increased the LR density of WT plants by 357%, while this percentage was about 800% for transgenic line L4. Additionally, the investigation on lateral root primordia development revealed significant differences between wild-type and transgenic seedlings in response to IAA and 2,4-D treatments, further indicating the altered auxin response in transgenic plants ( Figure 2D ).

The contrasting responses of lateral roots to exogenously applied auxin analogs between wild-type (WT) and transgenic plants imply a potential association between MtHB2 and auxin translocation and distribution in roots. To delve deeper into whether the reduced lateral root phenotype of transgenic seedlings is linked to impaired polar auxin transport, the impact of auxin efflux inhibitors NPA and TIBA on lateral root number and density in both WT and transgenic plants was assessed.

Treatment with NPA and TIBA resulted in a significant reduction in lateral root number in both WT and transgenic plants ( Figures 2E, F ). The inhibitory effects were more pronounced in WT plants than in transgenic plants. For instance, treatment with 0.5 μM NPA decreased lateral root number in WT, L4, and L11 by 38%, 15%, and 16%, respectively ( Figure 2E ). In contrast, a greater inhibitory effect was observed in transgenic seedlings compared to WT upon application of TIBA ( Figure 2F ).

To ascertain the effects of MtHB2 expression on auxin response, accumulation, and polar auxin transport in transgenic seedlings, we introduced the auxin response reporter proDR5::GFP and auxin efflux carriers proPIN1::PIN1-GFP, proPIN3::PIN3-GFP, and proPIN7::PIN7-GFP into transgenic seedling L4 through crossing. Confocal microscopy examination of GFP fluorescence in 6-day-old seedlings revealed significant reductions in DR5 activity in root tips, including quiescent center (QC) cells, columella cells, and certain vascular cells in transgenic seedlings. This suggests that the expression of MtHB2 leads to diminished auxin accumulation or response in the root tips of transgenic lines. Similarly, the expression of PIN1 in the stele and PIN3 and PIN7 in the stele and columella was notably decreased in transgenic seedling root apices, potentially contributing to the attenuated DR5 activity ( Figure 3 ). Additionally, weaker GFP fluorescence was observed in vascular tissues of transgenic seedling roots ( Figure 4 ).

For a more in-depth investigation into whether MtHB2 expression impairs auxin response, accumulation, and polar auxin transport in transgenic seedlings at earlier developmental stages before lateral root initiation, GFP fluorescence of 2-day-old seedlings was examined. Consistently, the expression levels of proDR5::GFP, proPIN1::PIN1-GFP, proPIN3::PIN3-GFP, and proPIN7::PIN7-GFP were all significantly diminished in the root tips of transgenic seedlings ( Figure 5 ). These findings suggest that reduced auxin accumulation in roots due to impaired polar auxin transport may contribute to the decreased formation of lateral roots and slower primary root growth in transgenic seedlings.

To delve deeper into the mechanisms underlying the alteration of root phenotype by MtHB2 expression, a comparative transcriptomic analysis was conducted between Arabidopsis transgenic (35S:MtHB2) and wild-type plants. Root tissues from 18-day-old seedlings of the transgenic line and wild-type plants grown vertically on agar plates under standard conditions were used for total RNA extraction. The data was then normalized to evaluate relative changes in gene expression in the transgenic line (L4) compared to wild-type plants. Out of 22,746 genes analyzed, 250 genes were down-regulated while 243 genes were up-regulated by more than 2-fold ( Supplementary Table S1 , Figures 6A, B ).

The validity of the microarray data was confirmed via RT-qPCR analysis. Twelve genes with diverse expression profiles were selected for analysis, all of which exhibited expression patterns consistent with the microarray results ( Figures 6C, D ). A high level of agreement was observed between the findings obtained from both methods ( Figure 6E ).

Subsequently, a detailed examination of the differentially expressed genes, particularly those related to auxin, was carried out ( Table 1 ). In line with the reduced DR5 expression, several auxin-responsive genes, including Aux/IAA genes, SAUR genes, and GH3 genes, displayed decreased expression levels. Notably, genes such as IAA3, IAA14, SAUR31, SAUR59, SAUR72, SAUR76, and GH3.17 exhibited marked reductions in expression ( Table 1 ). Furthermore, alterations were observed in genes involved in auxin homeostasis. The down-regulation of auxin biosynthesis genes (TAA1, YUC6, CYP79B2/B3) and up-regulation of IAA-amino acid hydrolase ILL6 and MeIAA methyl esterases AtMES1, 16 ( Table 1 ) hinted at potential impairment of auxin accumulation in the roots of transgenic lines. Additionally, three auxin-responsive genes from the LATERAL ORGAN BOUNDARIES (LBD) family (LBD4, LBD16, and LBD25) showed reduced expression in the transgenic lines ( Table 1 ). Conversely, genes that are typically up-regulated by auxin, such as PLDζ2, PBP, ACS6, and STZ, were found to be down-regulated ( Table 1 ). Surprisingly, the expression of auxin transport genes in the transgenic lines remained unaffected ( Table 1 ), contrasting with the observed down-regulation of PIN1, PIN3, and PIN7 in the root apices of the transgenic lines as visualized by GFP fluorescence.

Moreover, several genes known to act as repressors of jasmonate (JA) response were found to be up-regulated in the transgenic lines. The elevated transcript levels of CYP94B3, responsible for encoding a JA-Ile-12-Hydroxylase, and five JAZ (JASMONATE-ZIM DOMAIN) family genes suggested potential attenuation of JA signaling in the transgenic lines (Kazan and Manners, 2012). Additionally, there were indications of diminished cytokinin signaling, as evidenced by the down-regulation of three cytokinin signaling genes, including CRE1 (CYTOKININ RESPONSE 1), ARR7 (RESPONSE REGULATOR 7), and ARR11 (Fukaki and Tasaka, 2009) ( Table 2 ). The transcript levels of Arabidopsis HD-Zip II members homologous to MtHB2 were notably reduced, particularly the HD-ZIP II β genes (HAT9 and HAT22) and γ genes (HAT1, HAT2, and ATHB2) within the transgenic plants ( Table 3 ).