The radish genome “NAU-LB” (BioProject number: PRJCA011486.) (Xu et al., 2023) was used to identify RsAHL family members in radish. Twenty-nine Arabidopsis and twenty-six rice AHL family protein sequences were used to construct a Hidden Markov Model (HMM) of known AHL protein family sequences using HMMER 3.0 software. The HMM was generated by computational analysis of known homologous gene sequences and used to predict whether the homologous gene sequences exist in other species. All potential RsAHL family sequences were identified in the radish protein sequences. In contrast, BLASTP (Altschul et al., 1990) (v2.10.1+) was used to compare all radish protein sequences with the obtained AHL family reference sequences (e-value=1*10^–5), and the matched sequences were used as potential RsAHL family sequences. After synthesizing these candidate sequences, the PfamScan (Bateman et al., 2004) (v1.6) and Pfam A (Finn et al., 2014) (v33.1) databases were used to annotate the domain of the target sequence, and the sequence containing the PF03479 domain was determined as the final RsAHL protein sequence.

The RsAHL protein family sequences of radish, Arabidopsis, and rice were used to construct a neighbor-joining tree. First, MAFFT (v7.427) was used to perform multiple sequence comparisons, and MEGA10 software (Kumar et al., 2008) was used to build the neighbor-joining tree (the bootstrap was set to 1000), and then iTOL v6 (https://itol.embl.de/) was used to annotate the evolutionary tree.

“NAU-LB” genome sequence and general feature format (GFF) files were used to extract chromosome length and location information of RsAHL family members through the TBtools software. Subsequently, further visualization analysis was performed (Chen et al., 2020a).

Collinearity analysis was performed using MCScanX software (match score, 50; match size, 5; gap penalty, -1; overlap window, 5; e-value, 1e^-5; max gaps, 25). Segment and tandem duplications caused by gene duplication events were analyzed.

The CDS and genome sequences of radish RsAHL family members were used to analyze the intron-exon structure using the GSDS website (http://gsds.cbi.pku.edu.cn/) for visualization. The MEME website was used to predict the conserved motif of RsAHLs, and TBtools software was used to visualize it.

The upstream 2 kb sequences of the RsAHLs promoter were extracted from the “NAU-LB” genome and then submitted to the PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) database to predict the distribution of cis-elements on the promoter. Subsequently, all cis-elements were classified, and those related to stress and hormone responses were extracted for visual analysis using the TBtools software.

Radish (Raphanus sativus) was cultivated at 25°C with 16 h of light and 8 h of darkness per day. Radish materials with consistent growth were selected for the stress treatment. Radish leaf samples were collected after cold treatment (4°C), high temperature treatment (42°C), PEG6000 treatment and hormone treatment (methyl jasmonate, abscisic acid) at 0 h, 1 h, 3 h, 6 h, 9 h, 12 h and 24 h.

The CDS of RsAHL14 was amplified using radish cDNA with specific amplification primers ( Supplementary Table 4 ) and then cloned into pHELLSGATE8 (Chen et al., 2020b) to generate CaMV35S::AHL14 overexpression vector. This vector was transferred into Agrobacterium strain GV3101. Subsequently, genetic transformation was performed using MicroTom tomatoes as the background material. Transgenic plants were detected using PCR, and the primer sequences are shown in Supplementary Table 4 .

Total RNA was extracted using TRIzol reagent (Vazyme, Nanjing, China) and reverse-transcribed into cDNA using the HiScript II 1st Strand cDNA Synthesis Kit (+ gDNA wiper) (Vazyme). Real-time fluorescence quantitative PCR (RT-qPCR) was performed using CFX384 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) in conjunction with ChamQ Universal SYBR^® qPCR Master Mix (Vazyme). The relative expression levels of RsAHLs in radish tissues, abiotic stress samples, and overexpression plants were measured. The primer sequences are listed in Supplementary Table 4 . Three biological replicates were used for each experiment. The radish PRII (RNA polymerase-II transcription factor) gene was used as the internal control (Xu et al., 2012) and the results were calculated using the 2^−ΔΔCt method.

International Business Machine-Statistical Package for Social Sciences (IBM-SPSS) and Microsoft excel software were used for statistical analyses. All data were based on three independent biological replicates and expressed as the mean ± sd. Student’s t-test was used to calculate significant differences.

To identify AHLs in radish, 29 AtAHL and 26 OsAHL protein sequences were used as queries for BlastP and HMM searches against the radish genome. A total of 52 AHL proteins were identified and named RsAHL1–RsAHL52 ( Supplementary Table 1 ), and then their physicochemical properties were analyzed. The RsAHLs ranged from 195 to 852 amino acids in length, with a relative molecular weight (MV) spanning between 20.86 and 94.62 kDa. Their isoelectric point (pI) was between 4.81and 9.83, while their aliphatic index ranged from 49.59 to 88.53. The grand average hydropathy (GRAVY) values of all RsAHLs were less than zero, indicating that RsAHL are strongly hydrophilic. Subcellular localization prediction showed that most of the RsAHL genes were located in the nucleus, followed by the chloroplast ( Supplementary Table 1 ).

To further infer the evolutionary relationships between RsAHL and other AHL proteins, we constructed phylogenetic trees using multiple sequence alignments of AHL proteins from radish (Raphanus sativus), Arabidopsis, and rice (Oryza sativa L.). The results showed that the AHL genes of these three species were divided into two clades: clades A and B. Within clade A, radish, Arabidopsis, and rice accounted for 25, 15, and 15 proteins, respectively, whereas within clade B, they accounted for 27, 14, and 11 proteins, respectively. Furthermore, the two independent clades A and B could be further divided into three types, with Type I belonging to clade A and Types II and III belonging to clade B ( Figure 1 ). This result implies a high degree of phylogenetic consistency among AHL genes across different species and confirms the homology between the different species, suggesting that family members of the same branch may have similar functions.

Based on the location information of each RsAHL gene, we arranged the positions of the 52 RsAHL genes across nine chromosomes of the radish genome, and chromosome mapping indicated that they were 52 RsAHL genes were unevenly distributed across the chromosomes ( Figure 2 ). RsAHLs were mainly located on RsChr2, RsChr4, and RsChr9, which contained 9, 14, and 7 genes, respectively. Notably, only two RsAHL genes were found on RsChr8, namely RsAHL44 and RsAHL45.

Gene duplication was the most important cause of gene family expansion during plant evolution. To further study the evolutionary relationship of RsAHL in radish, gene duplication events were analyzed. And 49 duplications events were identified in 41 RsAHL genes. Among them, RsAHL4 had the most collinearity relationship with other RsAHLs, including RsAHL5, RsAHL38, RsAHL41, RsAHL42 and RsAHL44. However, 10 RsAHLs (RsAHL1, RsAHL2, RsAHL3, RsAHL14, RsAHL15, RsAHL23, RsAHL25, RsAHL27, RsAHL29 and RsAHL48) had no collinearity relationship with the other RsAHLs ( Figure 3 ; Supplementary Table 2 ). These results suggest that segment repeats play a crucial role in the expansion of the RsAHL family.

To infer the evolutionary relationship between RsAHL genes, MEGA 10 was used to construct a phylogenetic tree of the 52 RsAHL protein sequences in radish ( Figure 4A ). We predicted the conserved protein motifs using the Multiple Expectation maximizations for Motif Elicitation (MEME) website ( Figure 4B ). We identified a total of 10 conserved motifs among the RsAHL proteins ( Supplementary Figure 1 ; Table 1 ). These motifs ranged in length from 15 to 38 amino acids, with sites ranging from 5 to 51. Motifs 5 and 6 contained a conserved Arg-Gly-Arg core and belonged to the AT-hook motif family. In addition, motifs 5 and 6 were identified as Type I and Type II AT-hook motifs, respectively. Notably, we also identified the PPC domain (motif 2), which contains conserved Gly-Arg-Phe-Glu-Ile-Leu residues (Zhao et al., 2014). This motif has also been identified in maize, but not in soybean (Bishop et al., 2020; Wang et al., 2021b). Interestingly, almost all of the RsAHL genes contained motifs 1, 2, and 3, suggesting the consistency of the RsAHL protein sequences. Overall, the gene structure and motif prediction results indicated the consistency and evolutionary diversity of AHL genes in terrestrial plants.

Subsequently, we further analyzed the distribution of introns and exons in the RsAHL genes to explore the gene structure in radish ( Figure 4C ). The length of the RsAHL genes was between 824 and 6172 bp, among which the Type I genes were generally shorter than the Type II and III genes ( Supplementary Table 1 ). Moreover, the numbers of introns and exons were diverse. We found that the genetic structure was similar for each type of gene. For example, Type I genes contained only one or two exons, and Types II and III contained more exons and introns than Type I, suggesting a diverse genetic structure of this subgroup. Therefore, we believe that Types II and III evolved from Type I. This result is consistent with that of the AHL gene family reported in maize (Bishop et al., 2020).

Cis-elements in promoters can be bound by transcription factors to regulate plant growth, development, defense, and stress responses by regulating gene expression. To infer the function of the RsAHL gene family, we used the PlantCARE database to perform cis-element analysis with a 2 kb promoter region of RsAHL. The functions of these cis-elements have also been identified ( Supplementary Table 3 ). As shown in Figure 5 , we found that all RsAHL promoters contained light response elements, including Box4, GT1-motif, G-box, TCT-motif, GATA-motif, I-box, AE-box, MRE, chs-CMA1a/2a, Sp1, ATCT-motif, AT1-motif, GA-motif, TCCC-motif, Box II, ACE, ATC-motif, LAMP-element, Gap-box, GTGGC-motif, chs-Unit 1, 3-AF1 binding site, 4 cl-CMA2b, ACA-motif, and LS7. The Box4 cis element exists in almost all RsAHL genes, with RsAHL46 containing the highest number. This suggests that RsAHL genes are likely regulated by light. Approximately 86.5% of RsAHL family members contained anaerobic induction elements (ARE), with RsAHL6 containing the highest number. Seven cis-elements were associated with hormonal responses: abscisic acid (ABRE and TCA-element), methyl jasmonate or MeJA (CGTCA-motif, TGACG-motif), and auxin (AuxRR-core, TGA-element, and AuxRE). Additionally, we found that 26.9%, 44.2%, and 48.1% of RsAHL family members contained low-temperature elements (LTR), drought induction elements (MBS), and defense and stress responsiveness (TC-rich repeats), respectively. These results indicate that the RsAHL gene family plays an important role in regulating plant growth, hormone responses, and abiotic stress responses.

To study the expression of RsAHLs in various radish tissues, we extracted RNA from the taproots, stems, leaves, petals, calyx, filaments, and anthers of radish and detected the relative expression levels of RsAHLs among them ( Figure 6 ). The results showed that RsAHLs had different expression patterns in different tissues of the radish plant. The relatively highest expression of RsAHL genes was observed in the anthers, petals, and taproots. In particular, RsAHL44 expression was the highest in petals, RsAHL33 expression was the highest in anthers, and RsAHL25 expression was the highest in taproots and calyxes. On the contrary, RsAHL11, RsAHL17, RsAHL18, RsAHL20, RsAHL32, RsAHL34, RsAHL46 and RsAHL49 exhibited relatively low expression across various organs. In addition, the expression levels of the RsAHL family in filaments were lower than those in other flower organs. These results indicate that RsAHL family is mainly expressed in the taproots and floral organs, suggesting the potential function of RsAHL genes in taproots and flowers.

To further explore the expression pattern of the RsAHL family in radish under hormonal stress, qRT-PCR was performed to analyze the 52 RsAHL genes under different stress treatments. Based on the analysis of cis-acting elements in the promoter, MeJA and ABA were predicted to induce the strongest hormone responses. Subsequently, MeJA (200 μM) and ABA (10 μM) were further used to treat radish seedlings ( Figure 7 ). These results showed that RsAHL genes presented varying responses to the MeJA and ABA treatments. Under ABA treatment, 14 RsAHL genes were significantly upregulated after 6 h, with RsAHL4, RsAHL8, and RsAHL24 showing a gradual increase in expression. RsAHL5, RsAHL17 and RsAHL22 were upregulated at 3 h and reached their highest expression levels at 12, 24, and 6 h, respectively. In contrast, RsAHL21, RsAHL31, RsAHL33 and RsAHL39 were down-regulated after ABA treatment, whereas RsAHL15 and RsAHL43 were down-regulated after 6 h ( Figure 7A ). Under MeJA treatment, the expression of RsAHL13, RsAHL21, RsAHL28, RsAHL32 and RsAHL45 were continuously upregulated at 1 h. RsAHL22 and RsAHL52 were upregulated at 3 h, RsAHL22 reached its highest level at 6 h and then decreased, while RsAHL52 reached its highest value at 12 h and then decreased. RsAHL24 expression was upregulated at 6 h, reached its highest level, and then decreased thereafter. In contrast, the expression of RsAHL4 was downregulated immediately after treatment, whereas that of RsAHL10, RsAHL15 and RsAHL46 began to decline after 3 h. Notably, RsAHL51 did not change significantly after treatment, indicating that it did not respond to MeJA induction ( Figure 7B ). Therefore, the expression of RsAHLs in most radish samples were significantly upregulated by the ABA and MeJA treatments. Particularly, the expression of RsAHLs was significantly upregulated 6 and 1 h after ABA and MeJA induction, respectively.

To investigate the roles of the RsAHL family under abiotic stress, the expression levels of RsAHLs were determined after cold (4°C), hot (42°C) and polyethylene glycol PEG6000 (20%) treatment. Under the 4°C treatment, the expression of most RsAHL genes was significantly upregulated after 3 h, and reached the highest level at 24 h. The expression of RsAHL20 reached its highest level at 3 h, while that of RsAHL52 reached its highest level at 12 h ( Figure 8A ). At 42 °C treatment, most of the RsAHL genes were significantly upregulated and reached their highest expression at 12 h. The expression levels of RsAHL11, RsAHL16, and RsAHL51 gradually increased after 6 h of treatment, whereas the expression levels of RsAHL21 and RsAHL35 increased after 1 h. In contrast, the expression levels of RsAHL18, RsAHL31 and RsAHL32 were downregulated after treatment ( Figure 8B ). Most RsAHLs were significantly upregulated after PEG6000 treatment. The expression levels of RsAHL8, RsAHL11, RsAHL21, RsAHL33, RsAHL40, RsAHL41, and RsAHL50 were consistently upregulated after treatment. Notably, 10 RsAHL genes were consistently upregulated after 3 h of treatment and RsAHL35 and RsAHL36 were gradually upregulated after 6 h of treatment. However, RsAHL3, RsAHL17, and RsAHL23 were downregulated after treatment ( Figure 8C ). These results suggest that RsAHL plays an important role in abiotic stress.

To identify the biological function of the RsAHL family, we constructed overexpression vectors of several RsAHL genes and used MicroTom as the background material for genetic transformation. Using vector forward and gene reverse primers to detect the positive of transgenic materials, we obtained RsAHL14-overexpression positive plants ( Supplementary Figure 2 ), with the RsAHL14-OE-1/-2/-5/-6/-12 lines used for further experiments. In the observation of the phenotype of the positive transgenic tomato plants, it was found that plants with RsAHL14 overexpression were dwarfed during the developmental process compared to the control (CK) ( Figure 9A ). Further, the plant height was significantly lower in RsAHL14-overexpression lines compared with CK ( Figure 9C ). To analyze the relative expression of RsAHL14 in the overexpression lines, RsAHL14-OE-4 (which showed low expression of RsAHL14 in positive materials) was used as the control. The results showed that the relative expression levels of RsAHL14 in the leaves of the RsAHL14-OE-1/-2/-5/-6/-12 lines were 5.0-, 4.4-, 5.9-, 23.6-, and 48.0-fold higher than that in the level of RsAHL14-OE-4, respectively ( Figure 9B ). Notably, previous studies have shown that changes in the gibberellin (GA) content in plants can cause dwarfing.

To determine whether changes in GA lead to dwarfing, the expression of genes involved in GA biosynthesis and degradation pathways was detected in RsAHL14 overexpression plants. Among these, ent-Copalyl diphosphatase (CPS), ent-Kaurene synthase (KS), ent-Kaurene oxidase (KO), GA20-oxidase (GA20ox), and GA3-oxidase (GA3ox) are key enzymes in the GA biosynthetic pathway, while GA2-oxidase (GA2ox) is a key enzyme in the degradation pathway. As expected, compared with the CK, the relative expression levels of GA2ox-1 and GA2ox-3 were significantly increased in the RsAHL14-OE plants ( Figures 9D, E ), while those of GA20ox-1, GA20ox-2, GA20ox-3, GA3ox-3, GA3ox-4, GA3ox-5 and CPS were significantly decreased ( Figures 9F-L ). However, the expression levels of other GA-related genes were irregular in the overexpression lines ( Supplementary Figure 3 ). This suggests that GA synthesis was hindered in RsAHL14-overexpression lines, the degradation rate was accelerated, and the resulting plants were dwarfed.