TaGAD2 is a potential downstream effector of Rht5 in controlling wheat plant height

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The GA-responsive (GAR) dwarfing gene Rht5 decreases plant height without comprise of wheat seedling vigor and is considered as a promising candidate gene for breeding wheat varieties in water-limited conditions. However, the mechanisms underlying Rht5 -meidated dwarfism is unclear. In this study, we investigated the genetic effects of Rht5 on wheat growth and development using recombinant inbred lines (RILs) and found that Rht5 reduces plant height through inhibition of cell proliferation while it promotes cell elongation. The dual functions of Rht5 on cell growth during wheat stem elongation was associated with the alteration of the homeostasis of endogenous growth-promoting phytohormones cytokine and gibberellins. Transcriptome analysis of identified TaGAD2 , encoding a functional glutamate decarboxylase localized at plasma membrane that catalyzes γ-Aminobutyric acid (GABA) biosynthesis, as a potential downstream regulator of Rht5 -mediated dwarfism. Functional assays demonstrated that overexpression of TaGAD2 could mimic the phenotypes of dwarf Rht5 RILs while TaGAD2 knockdown increased plant height and improved lodging resistance, indicating a negative role of TaGAD2 in controlling wheat plant height. We also conducted haplotype analysis of TaGAD2 in a natural wheat population and identified TaGAD2 H1 as a potential favorable allele for wheat dwarfing breeding without compromising grain number. Our study provides new insights into the molecular mechanism of Rht5 -mediated plant height regulatory pathway and valuable gene resource to the genetic improvement of wheat plant architecture. Plant height dwarfing gene Rht5 TaGAD2 GABA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Key Messages Rht5 regulates wheat plant height and yield-related traits partly through modulation of a downstream gene TaGAD2 , which controls GABA biosynthesis and influences stem elongation, lodging resistance, and photosynthetic performance. Introduction The semi-dwarf wheat developed during the "Green Revolution" exhibits lodging resistance and high-yield characteristics. The key genes underlying this revolution in wheat– Rht-B1b and Rht-D1b –encode mutant DELLA proteins that repress wheat gibberellin (GA) responses (Peng et al. 1999). While wheat varieties carrying these genes are effectively resistant to lodging, the excessively short stature results in insufficient biomass production and is often accompanied by adverse effects such as short coleoptile and impaired seedling emergence, thereby limiting their application in some environments (Peng et al. 1999; Hedden 2003). Because semi-dwarf varieties carrying Rht-B1b and Rht-D1b typically have a shorter coleoptile, they are often sown shallowly to ensure successful emergence and to maintain early seedling vigor. This shallow sowing could be fatal for wheat production under water-deficit conditions (Jatayev et al. 2020; Zhao et al. 2022). The currently available gibberellin-insensitive (GAI) dwarfing genes are unlikely to fully meet the breeding objective of synergetic improvement of plant architecture and drought tolerance, highlighting the urgent need to develop drought resilient wheat varieties with GA-responsive dwarfing genes. In this case, the GAR dwarfing genes without adverse effects on early seedling establishment and seedling vigor have been considered as desirable substitutes for Green Revolution alleles in breeding drought adaptive dwarfing wheat varieties. In the past few decades, more than 25 reduced-height loci have been described in wheat (Hao et al. 2024). These loci include both GAR and GAI dwarfing sources, offering diverse options for optimizing plant height under contrasting environments. Among GAR loci, Rht13 was considered as new gene for breeding drought-tolerant wheat under water-limited conditions. Rht13 is not associated with reduced seedling growth or coleoptile length, and most of the height-reducing effect occurs later in development, so deeper planting in water-limiting environments to overcome the adverse effects associated with Rht-B1b and Rht-D1b . It encodes a nucleotide-binding site/leucine-rich repeat (NB-LRR) protein, and a point mutation in the semi-dwarf allele at the Rht13 locus ( Rht-B13b ) causes constitutive activation of this gene, leading to remarkable reduction in plant height (Borrill et al. 2022). Rht25 was found to encode a plant-specific PLATZ-A1 transcription factor expressed mainly in the elongating stem and developing spike. The interaction between PLATZ-A1and RHT1 protein implied that PLATZ-A1 modulates DELLA-associated growth repression within the GA-DELLA pathway (Zhang et al. 2023). Tian et al. (2022) reported that Rht24 encodes the GA inactivation enzyme gibberellin 2-oxidase A9 (TaGA2ox-A9). Elevation of TaGA2ox-A9 expression in stems of Rht24b dwarf line could enhance GA catabolism, thus reducing plant height without yield penalty (Tian et al. 2022). In addition, Rht12 is likely to reduce plant height by decreasing bioactive GA levels via upregulation of gibberellin 2-oxidase A14 ( TaGA2ox-A14 ) (Sun et al. 2019). The GAR dwarfing gene Rht5 , was first reported by Ellis et al. (2004), which could reduce plant height without compromise of the coleoptile length and seedling viability, two important traits for wheat production in arid and semi-arid regions. In addition, Rht5 can also significantly increase the spike number per plant and harvest index (Daoura et al. 2014), highlighting its potential to combine height reduction with yield improvement. Although Rht5 has been mapped to a ~1 Mb interval on wheat chromosome 3B (Cui et al. 2022), the causal gene has not yet been determined. Glutamate decarboxylase (GAD) is a key enzyme that catalyzes the biosynthesis of γ-aminobutyric acid (GABA), a non-protein amino acid metabolite acting as a potential signaling molecule in plants (Bouché et al. 2004; Turano et al. 1998; Michaeli et al. 2015). Baum et al. (1993) first characterized a plant GAD and described a canonical C-terminal calmodulin-binding domain (CaMBD), a feature subsequently shown to be plant-specific (Yun and Oh 1998). This C-terminal module directly couples Ca 2+ /calmodulin signaling to regulate GAD activity. Under stress conditions, elevated cytosolic Ca 2+ binds calmodulin (CaM) to form a Ca 2+ /CaM complex that interacts with the CaMBD, thereby releasing C-terminal autoinhibition and activating GAD; in contrast, in the absence of Ca 2+ /CaM, the CaMBD maintains an autoinhibited state that suppresses enzyme activity (Snedden et al. 1995). Studies have revealed that GABA strongly influences plant growth and development (Uzma Jalil et al. 2019; Du et al. 2020; Khan et al. 2021). In Arabidopsis, accumulation of GABA could inhibit cell elongation. Consistently, the pop2 mutant, which accumulates high levels of GABA, shows defective root, hypocotyl and pollen tube elongation (Renault et al. 2011). In rice, OsGAD2 , a member of the GAD family lacking the CaMBD, has been characterized as a negative regulator of plant height and yield traits (Zhang et al. 2023). Overexpression of Os GAD2ΔC (C-terminally truncated OsGAD2 ) led to significantly elevated GABA levels in roots, shoots and leaves compared with the wild type and exhibited a pronounced dwarf phenotype characterized by pale, curled leaves and infertility, whereas rice plants overexpressing full-length OsGAD2 displayed an essentially normal phenotype (Akama and Takaiwa 2007). In wheat, GABA could regulate plant pollen tube and root by directly modulating ALMT-type anion transporter activity (e.g., TaALMT1 ) (Ramesh et al. 2015). Recent study suggested that overexpression of TaGAD1 resulted in stunted spikes and increased grain abortion, accompanied by abnormal spike development (Li et al. 2024) . Takayama and Ezura (2015) identified two tomato GAD genes, SlGAD2 and SlGAD3 , that regulate GABA accumulation and fruit maturation. Tomato fruits from SlGAD3ΔC overexpression lines driven by the E8-HSP cassette displayed an orange-ripe phenotype with reduced carotenoids and downregulated ethylene-responsive carotenogenic genes, implying a compromised ethylene sensitivity during ripening, while SlGAD3OX lines were indistinguishable from those of the wild type in appearance (Takayama et al. 2017). Further studies have demonstrated that GABA exerts dose-dependent effects on plant development (Yue et al. 2018; Xie et al. 2020). Additionally, GABA is also involved in stress tolerance (Xu et al. 2021; Li et al. 2024; Wang et al. 2025). The Rht5 dwarfing locus was originally discovered from a JM47 × Marfed M cross and represents an important GA-responsive dwarfing allele in wheat that reduces plant height and enhances lodging resistance. We have previously mapped Rht5 to an ~1 Mb interval on chromosome 3B flanked by the molecular markers Kasp-25 and Kasp-23 , while the potential regulatory mechanism remains unknown. In this study, we investigated the genetic and physiological basis of Rht5 -mediated dwarfism. We showed that Rht5 regulates plant height primarily through inhibition of cell proliferation although it promotes cell elongation which could be attribute to the altered homeostasis. Using transcriptome analysis we identified TaGAD2, a glutamate decarboxylase involved in GABA biosynthesis that localized at plasma membrane, as a potential downstream component of Rht5 -mediated wheat plant height regulatory pathway. We demonstrated that TaGAD2 as a negative regulator of wheat plant height and overexpression of TaGAD2 could phenocopy Rht5 , highlighting a potential novel regulatory mechanism of wheat plant height. Materials and methods Plant materials and growth conditions Rht5 RILs used in this study were derived from the cross JM47 × Marfed M followed by continuous selfing of the F 1 progenies for six generations to obtain genetically stable-inherited lines. Three tall lines ( rht5rht5 ) and three dwarf lines ( Rht5Rht5 ) together with the parents Jinmai47 (JM47) and Marfed M were used for analyses. The Rht5 RILs were grown at the experimental farm of Northwest A&F University (Yangling, Shaanxi, China). Transgenic wheat lines were generated in the Fielder background. TaGAD2-RNAi plants were grown at the Transgenic Experimental Farm of Northwest A&F University. Field experiments at the experimental farm and the Transgenic Experimental Farm were conducted with the same planting density: 1.5-m rows, 25-cm row spacing and 20 seeds per row. TaGAD2-OE plants were grown in a greenhouse at Northwest A&F University. For greenhouse cultivation of TaGAD2-OE plants, seedlings were transplanted into 16 × 16 × 18 cm pots (L × W × H) and grown at 20°C/16°C (day/night) under a 16-h light/8-h dark photoperiod with 60% relative humidity. Nicotiana benthamiana plants were grown in a greenhouse under a 16-h light/8-h dark photoperiod at 23°C with 70% relative humidity. Plants were cultivated in pots with substrate consisting of soil: vermiculite (2:1, v/v), and 4-week-old plants were used for subsequent analysis. Phytohormone treatment Phytohormone treatments were applied to Rht5 RILs by whole-plant spraying twice at the jointing stage and twice at the heading stage. The concentrations were 20 mg L -1 for 6-BA treatment and 35 mg L -1 for GA 3 treatment, with 0.04% (v/v) Tween-20 respectively. Agronomic traits were scored at maturity with five replicates. For each replicate, data were scored from four plants and the mean values were used for statistical analysis. Relative promotion rate (RPR) was calculated with the formula below: The development of transgenic wheat lines To generate the TaGAD2 overexpression (OE) lines, the full-length coding sequence (CDS) of TaGAD2 was amplified from Marfed M and cloned into the pCUB-3×Flag vector via homologous recombination, forming the pCUB-TaGAD2-3×Flag plasmid. To generate the TaGAD2 - RNAi plants, two 280-bp fragments of the TaGAD2 CDS were inserted in reverse tandem into the pCUB-RNAi vector, forming the pCUB-TaGAD2-RNAi . All the constructs were transformed into Agrobacterium tumefaciens strain EHA105 and introduced into wheat cultivar Fielder by Agrobacteriu m-mediated transformation (Wu et al. 2003). Primers used for vector construction are listed in Table S1. Evaluation of lodging-related traits in transgenic lines At the mid-grain-filling stage, main stems from 10 individuals were randomly selected from each line for measurement of lodging-related traits. For the second basal internode (BI2), internode length, diameter, fresh weight and dry weight were measured. In addition, whole-culm fresh weight, spike fresh weight and the height of the center of gravity of the main culm were recorded for lodging assessment. According to the method described by Murphy et al. (1958). The mechanical strength of BI2 (the second basal internode, counted from the soil surface upward) was determined using a plant stalk strength tester (YYD-1; Zhejiang Top Instrument Co., Ltd., Zhejiang, China). The tester was placed on a stable benchtop, and the second basal internode of the wheat main stem was laid horizontally across the two grooves (2.5-cm apart) at the base of the device. The culm lodging resistance index (CLRI) was calculated as mechanical strength of BI2/(height of the center of gravity × whole-culm fresh weight). Evaluation of leaf photosynthetic characteristics Leaf photosynthetic traits were assessed at the early grain-filling stage (GS69). Photosynthetic parameters of flag leaves including net photosynthetic rate (A), stomatal conductance (g s ), transpiration rate (E) and intercellular CO 2 concentration (Ci) were measured using a LI-6400XT portable photosynthesis system (Li-COR Inc., Lincoln, NE, USA). Instantaneous water use efficiency (iWUE) was determined as the ratio of photosynthesis to transpiration (A/E). The relative chlorophyll content (SPAD) was measured at the middle portion of the flag leaf with a SPAD-502 chlorophyll meter (Konica Minolta, Osaka, Japan). Histological analysis of peduncle For cell size analysis, peduncles from the Rht5 RILs were collected at the flowering stage (GS69; Zadoks et al. 1974) and paraffin sections were prepared as described by Chai et al. (2019). Briefly, one centimeter of segments from the basal, middle and uppermost regions of the peduncle were fixed in FAA, dehydrated, embedded in paraffin, and sectioned longitudinally at 10 μm (Kong et al. 2023). Images were acquired using the BX63 Olympus microscope (Evident Corporation, Tokyo, Japan) and cell length, cell width and cell number per unit area (1 mm 2 ) were quantified using ImageJ software (Schindelin et al. 2012). RNA sequencing (RNA-seq) analysis Elongating peduncle segments were collected at the heading stage from Rht5 RILs, JM47 and Marfed M. Three homozygous dwarf RILs and one tall RIL were sampled for RNA-seq with three biological replicates for each line. The parental JM47 and Marfed M plants were also subjected to RNA-seq analysis with one biological replicate for each line. Paired-end RNA-seq libraries were prepared and sequenced on the Illumina HiSeq 4000 platform (PE150) (Novogene Co., Ltd., Beijing, China) following the manufacturer’s standard procedures. Differentially expressed genes (DEGs) were identified from four pairwise comparisons (JM47 vs. Marfed M, T vs. D1, T vs. D2 and T vs. D3) using DESeq2 (Love et al. 2014). Genes with p -value ≤ 0.05 and |log 2 FoldChange| ≥ 1 were defined as DEGs. Venn diagrams illustrating overlaps among DEG sets were generated using an online Venn diagram tool (https://www.genescloud.cn/chart/VennPlot). Gene Ontology (GO) enrichment significance for each GO term was calculated using a hypergeometric test, the top five significant categories were visualized using R. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed at the Gene Denovo (OmicShare) cloud platform (https://www.omicshare.com/tools/) and the top 15 significant pathways were visualized using OmicShare. Heatmaps were generated using TBtools (Chen et al. 2023). The input matrix consisted of TPM (transcripts per million) values for the selected genes across groups. Phylogenetic analysis TaGAD2 and its homologous protein sequences from wheat ( Triticum aestivum L.), maize ( Zea mays ), rice ( Oryza sativa ), soybean ( Glycine max ) and Arabidopsis thaliana were retrieved from EnsemblPlants (https://plants.ensembl.org/). Protein sequences were aligned using ClustalW with default parameters. A phylogenetic tree was generated in MEGA 11 using the neighbor-joining (NJ) method with 1,000 bootstrap replicates to evaluate branch support (Tamura et al. 2021). Endogenous phytohormone and GABA content determination Internodes were collected from the Rht5 RILs at the heading stage. Samples were sent to Nanjing Webiolotech Testing Technology Co., Ltd., for quantification of endogenous phytohormones using an ultra-performance liquid chromatography (UPLC) system (Qsight LX50, PerkinElmer, Waltham, Massachusetts, USA) coupled with tandem mass spectrometry (QSight 420 triple quadrupole, PerkinElmer, Waltham, Massachusetts, USA). To measure GABA, tissues from Fielder and TaGAD2-RNAi plants were collected at the grain-filling stage. Approximately 0.1 g of tissue was ground in a pre-chilled mortar with 1 mL of extraction buffer on ice. The homogenate was centrifuged at 12,000 rpm for 10 min at 4°C, and the resulting supernatant was used for GABA quantification using the GABA Assay Kit (ADS-W-AJS009, ADSBio, Jiangsu, China) following the manufacturer’s protocol. Subcellular localization analysis The full-length CDS of TaGAD2 and the truncated TaGAD2ΔC were amplified from Marfed M using specific primers (Table S1) and recombinated in-frame with GFP under the control of the CaMV 35S promoter to generate the 35S pro :TaGAD2-GFP and 35S pro :TaGAD2ΔC-GFP constructs, respectively. The mCherry-OsPAD was used as a plasma membrane marker. The verified plasmids were introduced into Agrobacterium tumefaciens strain GV3101 (pSoup-P19) and infiltrated into the abaxial epidermis of Nicotiana benthamiana leaves as previously described (Liu et al. 2010). Fluorescence signals were examined 48 hours post infiltration using a confocal laser-scanning microscope (Olympus FV3000, Tokyo, Japan) with an excitation wavelength of 488 nm for GFP and 561 nm for mCherry, respectively. Prokaryotic expression and enzyme activity assay The TaGAD2 and TaGAD2ΔC coding sequences described above were amplified and cloned into the pGEX-4T-1 vector using restriction enzymes Nco I and Not I. Recombinant plasmids were transformed into Escherichia coli BL21 (DE3). Bacterial cells were grown in lysogeny broth (LB) medium at 37°C to OD 600 of 0.6. After induction with 0.5 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) at 18°C for 16 h, cells were harvested and lysed by ultrasonication on ice. The lysates were centrifuged at 7,000 g for 20 min at 4°C and GST-tagged proteins were purified using glutathione magnetic agarose beads (KTSM1355, KTSM, Shenzhen, China) following the manufacturer’s instructions. Purified proteins were analyzed by 10% SDS-PAGE. One gel was stained with Coomassie Brilliant Blue G-250 (Cat. No. P0017, Beyotime Biotechnology, Shanghai, China) to assess total protein expression and purification quality, and a parallel gel was subjected to Western blotting probed with a mouse anti-GST monoclonal antibody at a dilution of 1:5000 (AF0174, Beyotime Biotechnology, Shanghai, China) and visualized using ECL chemiluminescence (Cat. No. CW0049M, CWBIO, Jiangsu, China). GAD enzymatic activity was quantified by determining GABA production using the Berthelot colorimetric reaction. GAD activity was quantified using a Glutamate Decarboxylase Assay Kit (ADS-W-AJS010, Suzhou Mengxi Bio-Medical Technology Co., Ltd., Jiangsu, China). For GAD enzymatic activity assay, flag leaves and rachis tissues from transgenic lines and wild-type plants were collected at the heading stage. Approximately 0.1 g of tissue was ground in a pre-chilled mortar with 1 mL of extraction buffer on ice. The homogenate was centrifuged at 12,000 rpm for 10 min at 4°C, and the resulting supernatant was used for GAD activity determination. The assay is based on the phenol-hypochlorite colorimetric reaction, in which GABA produced by GAD reacts with phenol and sodium hypochlorite under alkaline conditions to generate a blue chromogenic product. The absorbance of this product at 645 nm is proportional to the amount of GABA formed, reflecting GAD enzymatic activity. GAD activity was quantified using a Glutamate Decarboxylase Assay Kit (ADS-W-AJS010, ADSBio, Jiangsu, China) following the manufacturer’s instructions. Primer sequences used for vector construction are listed in Table S1. Quantitative PCR analysis For gene expression profiling, roots, stems and leaves were collected at three-leaf stage. Total RNA was extracted using RNAiso Plus (Cat. No. 9109, Takara Bio Inc., Kusatsu, Japan) and first-strand cDNA was synthesized from 1 μg RNA using the All-in-One First-Strand Synthesis MasterMix (with dsDNase) (Cat. No. EG15133S, Yugong Biotech Co., Ltd., Jiangsu, China). Quantitative real-time PCR (qRT-PCR) was performed on a Bio-Rad CFX96 Real-Time PCR System using F488 SYBR qPCR Mix (Cat. No. EG23111L, Yugong Biotech Co, Ltd., Jiangsu, China). Gene expression levels were normalized to TaActin and relative transcript abundance was calculated using the 2 −ΔΔCt method. Primer sequences used for qRT-PCR are listed in Table S2. Results Phenotypic characterization of Rht5 recombinant inbred lines To evaluate the effects of Rht5 on wheat development, Rht5 recombinant inbred lines (RILs) were generated from a cross between JM47 ( rht5rht5 ) and Marfed M ( Rht5Rht5 ), the Rht5 donor, followed by consecutive self-pollination for six generations to obtain homozygous Rht5Rht5 (dwarf, D) and rht5rht5 (tall, T) lines. Compared with tall lines, the Rht5 dwarf lines exhibited significantly reduced plant height (Fig. 1a, c). A detailed comparison of internode lengths between the dwarf and tall lines indicated that each internode in the dwarf Rht5 RILs was shortened to varying degrees relative to the tall Rht5 RILs (Fig. 1b, Fig. S1). Compared with the tall RILs, the spike number per plant was significantly increased in the dwarf lines (Fig. 1d). In addition, Marfed M showed significant reduction in spike length, spikelet number per spike and grain number per spike relative to JM47 (Fig. 1e–g). However, in the Rht5 RILs, no significant differences were detected between tall and dwarf RILs in spike length, spikelet number per spike and grain number per spike (Fig. 1e–g). Similarly, Marfed M showed significant reductions in grain length, grain width, thousand-grain weight and grain yield per plant compared with JM47, whereas these differences were reduced or absent in the tall and dwarf Rht5 RILs (Fig. 1h–k). Rht5 inhibits cell proliferation to regulate stem elongation Because plant height is largely determined by cell proliferation and growth in the intercalary meristem in the elongation zone of the stem, we performed histological analyses to evaluate the effects of Rht5 on cell morphologies during wheat stem elongation (Fig. 2a). The basal internodes showed the strongest dwarfing effect, with the fifth and fourth internodes reduced by 34.08% and 34.39%, respectively. Peduncle length was reduced by 31.24% and accounted for 29.50% of the total reduction in plant height (Table S3). Longitudinal sections of the young peduncles showed that Marfed M exhibited longer cell length and shorter cell width, as well as significantly fewer cell numbers across the upper, middle and lower regions of the peduncles compared with JM47 (Fig. 2b–d). Consistent with the observation in the parental lines, the dwarf Rht5 RILs displayed significant increase in cell length and marked decrease in cell number across the upper, middle and lower regions of the peduncles (Fig. 2b, d). Despite the comparable cell width in the upper region of the peduncles between the T1 and D1 lines, the cell width was significantly decreased in the middle and lower regions in the D1 line (Fig. 2c). These results indicated that the Rht5 -conferred dwarfism was mainly associated with reduced cell number resulting from inhibited cell proliferation in the intercalary meristem of the stem rather than cell elongation. Responses of Rht5 RILs to exogenous phytohormone treatments To examine whether the phenotypic differences between the tall and dwarf Rht5 RILs were associated with altered endogenous phytohormone profiles, we quantified phytohormone levels in the peduncles of tall and dwarf RILs at the heading stage. Among the growth-related phytohormones, the dwarf RILs showed reduced levels of bioactive indole-3-acetic acid (IAA) but increased accumulation of auxin precursors including indole-3-acetonitrile (IAN), tryptamine (TAM), indole-3-acetamide (IAM) and indole-3-pyruvic acid (IPyA) (Fig. 3a; Fig. S2a). Measurement of cytokinin levels indicated that the dwarf lines showed marked reduction in zeatin, trans -zeatin and isopentenyladenosine (IPA) (Fig. 3b; Fig. S2b), whereas the cytokinin precursors, including trans -zeatin riboside (TZR) and isopentenyladenine (iP), were markedly elevated compared with the tall lines (Fig. S2b). The levels of bioactive gibberellins (GA 3 and GA 4 ) in dwarf RILs were significantly higher than those in the tall RILs (Fig. 3c). The levels of stress-related hormones, including salicylic acid (SA), jasmonic acid (JA) and abscisic acid (ABA), were significantly higher in the dwarf lines than those in the tall lines. In addition, JA precursor levels were also significantly higher in the dwarf lines (Fig. 3d; Fig. S2c), suggesting that Rht5 may be involved in the regulation of wheat stress responsiveness. To further evaluate the effects of Rht5 on hormonal responsiveness in wheat, exogenous gibberellic acid (GA 3 ) and 6-benzylaminopurine (6-BA) were applied twice at the jointing stage and twice at the heading stage. The results showed that the tall and dwarf RILs had differential responses to exogenous phytohormone treatments. Under mock conditions, dwarf RILs had significantly lower plant height and spike length than tall RILs (Fig. 3e, g). The 6-BA treatment showed little effect on plant height and spike length, while GA 3 treatment significantly increased both plant height and spike length of the Rht5 RILs (Fig. 3e, g). In addition, the dwarf lines exhibited stronger GA 3 -induced promotion in stem and spike elongation with significant increases in relative promotion rates (Fig. 3f, h), indicating enhanced GA 3 responsiveness in the dwarf lines. Transcriptome analysis identifies TaGAD2 as a potential downstream effector of Rht5 -mediated dwarfism To identify the downstream genes involved in the Rht5 -mediated plant height regulatory pathway, RNA-seq was performed with peduncle tissues at the heading stage to reveal transcriptional changes between tall and dwarf lines. Using DESeq2 ( p -value ≤ 0.05 and |log 2 FoldChange| ≥ 1), we identified DEGs in comparisons of Marfed M vs. JM47 and D1/D2/D3 vs. T. Compared with JM47, 15,686 upregulated DEGs and 12,941 downregulated DEGs were identified in Marfed M. There were 1,630, 2,121 and 5,648 DEGs significantly upregulated in D1, D2 and D3 lines, respectively. Compared with the tall Rht5 RILs (T), 4,220, 3,924 and 9,880 DEGs were significantly downregulated in D1, D2 and D3 lines, respectively (Fig. 4a). In total, 993 overlapping DEGs were identified across RILs and their parental lines carrying different Rht5 alleles (Fig. 4b). Gene Ontology (GO) analysis showed that the 993 common DEGs were significantly enriched in the molecular function terms “protein kinase binding” and “indole-3-butyrate beta-glucosyltransferase activity” and in the biological process terms “salicylic acid metabolic process”, “response to biotic stimulus” and “response to chitin” (Fig. 4c; Table S4). These enriched functions and processes terms are associated with plant growth, environmental adaptation and stress/disease resistance (Vanneste and Friml 2009; Yang and Guo 2018; Zhou and Zhang 2020; Waadt et al. 2022). In concordance with GO enrichment, KEGG pathway enrichment further showed that these enriched pathways (e.g., phenylalanine metabolism, biosynthesis of secondary metabolites and phenylpropanoid biosynthesis) are closely associated with secondary metabolism and stress and defense adaptation. In addition, the “MAPK signaling pathway” and “plant–pathogen interaction” were also enriched by KEGG analysis, indicating that Rht5 -associated transcriptional changes may impact secondary metabolic and stress-related processes that contribute to coordinating wheat growth and stress resistance (Dixon and Paiva 1995a; Vogt 2010a; Meng and Zhang 2013) (Fig. 4d; Table S5). Heatmap analysis showed that DEGs involved in the biosynthesis or signaling of growth-related hormones (auxin, cytokinin and gibberellins) were differentially expressed between tall and dwarf lines (Fig. 4e; Table S6). In addition, genes associated with stress-related hormone pathways (SA, JA and ABA) exhibited distinct expression patterns between tall and dwarf lines (Fig. S3; Table S7). Among these, WRKY62 and WRKY76 were expressed at significantly higher levels in the dwarf lines than in the tall lines. WRKY62 and WRKY76 are important transcription factors and have been linked to SA, JA and ABA signaling and associated immune responses and abiotic stress tolerance (Table S7) (Mao et al. 2007; Yokotani et al. 2013; Fukushima et al. 2016). Together, these expression patterns suggest that the Rht5 allele may also be involved in the regulation of wheat stress or defense responses. Among DEGs related to plant growth and development, TraesCS3B02G022900 , encoding a homolog of OsGAD1, was remarkably upregulated in dwarf RILs (Fig. 4f; Table S8), and was therefore designated as TaGAD2 . Given that GAD is a key enzyme in GABA biosynthesis that is involved in controlling rice plant architecture and plays an important role in cell elongation and stress responses (Akama et al. 2001; Ramesh et al. 2015), TaGAD2 was selected as a potential candidate gene downstream of Rht5 for subsequent functional validation. TaGAD2 encodes a functional glutamate decarboxylase (GAD) To characterize the molecular characterization of TaGAD2 , we first examined its expression in the tall and dwarf RILs. qRT-PCR analysis showed that TaGAD2 expression was significantly higher in dwarf Rht5 RILs and Marfed M compared with tall Rht5 RILs and JM47 (Fig. 5a). Expression profiling analysis with data from WheatOmics showed that TaGAD2 was ubiquitously expressed in various tissues and was highly expressed in young spikes at Zadoks stage 39 (Z39) (Fig. 5b). At the seedling stage, TaGAD2 showed the highest expression in shoots, followed by leaves and roots in Chinese Spring (CS) (Fig. 5c). Multiple sequence alignment showed that TaGAD2 shares high sequence similarity in the glutamate decarboxylase domain with its homologs from Triticum aestivum L., Glycine max , Oryza sativa , Zea mays , Arabidopsis thaliana (Fig. S4). Phylogenetic analysis showed that there were multiple GAD homologs in wheat, and TaGAD2 was closely related to OsGAD1 (Fig. 5d; Table S9). Because plant GAD proteins harbor a canonical C-terminal auto-inhibitory calmodulin-binding domain (CaMBD) (Baum et al. 1993), we tested the glutamate decarboxylase activity of TaGAD2 using purified GST-TaGAD2 (full-length) and GST-TaGAD2ΔC (C-terminal-truncated TaGAD2) in vitro (Fig. S5a–b). Enzyme activity assays revealed that TaGAD2ΔC exhibited markedly higher decarboxylase activity than the full-length TaGAD2 (Fig. 5e), supporting an autoinhibitory role of the C-terminal region of TaGAD2. Subcellular localization analysis showed that both full-length and C-terminal-truncated TaGAD2 proteins were predominantly localized at the plasma membrane and co-localized with the plasma membrane marker mCherry-OsPAD (Kurusu et al. 2012) (Fig. 5f), indicating that deletion of the C-terminal auto-inhibitory domain (AID) had little impact on TaGAD2 subcellular localization. Together, these data suggested that TaGAD2 acts as a functional glutamate decarboxylase. TaGAD2 negatively regulates wheat plant height To verify the biological function of TaGAD2 , TaGAD2 overexpression lines ( TaGAD2-OE ) were generated in the wheat cultivar Fielder background . Phenotypic analysis showed that TaGAD2-OE plants displayed severe developmental defects, including growth retardation and defective grain filling (Fig. 6a–g). A previous study demonstrated that overexpression of TaGAD1 resulted in stunted spikes and increased grain abortion, accompanied by abnormal spike development (Li et al. 2024) . We obtained four fertile independent overexpression transgenic lines with limited grain numbers (Fig. 6a). qRT-PCR analysis showed that TaGAD2 expression was markedly higher in TaGAD2-OE lines than in the wild-type Fielder (Fig. 6b). Compared with Fielder, TaGAD2-OE plants exhibited significant reduction in plant height (Fig. 6c), whereas the spike number per plant was comparable to that of Fielder (Fig. 6d). Due to severe defects in spike development, the spike length, spikelet number and grain number per spike were significantly reduced in TaGAD2-OE lines compared with those of Fielder (Fig. 6e–g). These results indicate that TaGAD2 negatively regulates wheat plant height and yield-related traits. In addition, the flag leaves of TaGAD2-OE lines were shorter than those of Fielder, with significant reduction in SPAD values (Fig. 6h, i, k). Notably, TaGAD2-OE plants exhibited altered photosynthetic characteristics: the instantaneous water-use efficiency (WUEi) and net photosynthetic rate (A) were significantly higher than the wild-type plants (Fig. 6j, l), while significantly decreased were detected in transpiration rate (E), stomatal conductance (g s ), and intercellular CO 2 concentration (Ci) in the TaGAD2-OE lines compare to Fielder (Fig. 6m–o) implicating prolonged influences of TaGAD2 on photosynthetic capacity and instantaneous water-use efficiency. Collectively, these results identified TaGAD2 as a negative regulator of wheat growth and development. Silencing TaGAD2 increases plant height and lodging resistance To test whether knockdown of TaGAD2 could increase wheat plant height, we generated TaGAD2-RNAi line in the wheat cultivar Fielder background (Fig. 7a). Quantitative analysis showed that TaGAD2 expression was markedly lower in TaGAD2- RNAi line than in the wild-type Fielder (Fig. 7b). Enzymatic activity assays showed that the GAD activity was lower in multiple tissues and organs in the TaGAD2- RNAi plants compared to that in Fielder (Fig. 7c; Fig. S6a–b). Consistently, GABA content was markedly reduced in TaGAD2-RNAi plants compared with Fielder (Fig. 7d). Agronomic trait analyses showed that TaGAD2-RNAi plants exhibited significant increase in plant height compared with Fielder (Fig. 7e–f). By contrast, no significant differences were detected in spike length, spikelet number per spike and grain number per spike (Fig. 7g–i). Other traits, including flag leaf length, spike number per plant, thousand-grain weight and grain yield per plant were also comparable between the TaGAD2-RNAi plants and Fielder (Fig. S6c–f). Lodging resistance evaluations revealed that TaGAD2-RNAi plants exhibited enhanced lodging resistance (Fig. 7j). This improvement was associated with notable reduction in fresh weight of the main culm, the second basal internode (BI2; basal internode 2) and the spike (Fig. 7l, m; Fig. S6i), along with increased mechanical strength of the BI2 (Fig. 7k) and no significant difference in the center of gravity height (Fig. 7n). Other BI2-associated morphological traits, including BI2 length, BI2 diameter and BI2 dry weight, did not differ significantly between Fielder and TaGAD2-RNAi plants (Fig. S6g, h, j). Physiological analysis revealed compromised photosynthetic performance in TaGAD2-RNAi plants compared with Fielder. TaGAD2-RNAi plants showed significantly reduced instantaneous water-use efficiency (WUEi), net photosynthetic rate (A), intercellular CO 2 concentration (Ci), stomatal conductance (g s ) and transpiration rate (E) (Fig. 7o–s), suggesting that TaGAD2 is required for optimal photosynthetic performance in wheat. Collectively, these results identified TaGAD2 as an important regulator of wheat growth, lodging resistance and photosynthetic traits. Haplotype analysis of TaGAD2 To assess the potential of TaGAD2 in wheat breeding, we performed haplotype analysis in a panel comprising 273 wheat accessions. Based on the sequence variations in the coding region of TaGAD2 , these accessions were grouped into three major haplotypes (accession number ≥ 10 for each one) among which haplotype H3 of TaGAD2 was identical to the Chinese Spring (CS) reference sequence. Among the 13 single nucleotide polymorphisms (SNPs) identified in the coding region of TaGAD2 , there were four nonsynonymous SNPs that resulted in three amino acid substitutions: Met126 (H3) to Ile126 (H1 and H2), Arg172 (H3) to Lys172 (H1 and H2) and Arg445 (H3 and H1) to Ala (H2) (Fig. 8a). Among the identified haplotypes, H1 comprised 88 wheat accessions (32.23%), H2 included 72 accessions (26.37%), and H3 contained 10 accessions (3.66%) (Fig. 8a–b). Accessions with the H1 haplotype exhibited significantly lower plant height than that of H2, while no significant difference was observed in spike length between the H1 and H3 haplotypes (Fig. 8c). Compared with H3, H1 and H2 accessions had longer spike lengths (Fig. 8d) but comparable spikelet numbers and grain numbers (Fig. 8e–f). Collectively, these results suggested that TaGAD2 was associated with variations of wheat plant height in natural population. As TaGAD2 H1 has the strongest effects in reducing plant height, it represents a potential favorable allele of TaGAD2 for wheat dwarfing breeding. Discussion The wide application of Green Revolution Rht-B1b and Rht-D1b has increased lodging resistance, while compromising seedling vigor and coleoptile length, thereby reducing their drought tolerance. Rht5 is a potential alternative reduced-height loci suitable for breeding dwarfing wheat varieties because it does not influence seedling vigor and coleoptile length. In this study, we investigated the genetic effects of Rht5 on wheat growth and development using Rht5 inbred lines and explored the physiological mechanisms by which Rht5 regulates plant height. In addition, we identified TaGAD2 as a potential downstream regulator of Rht5 -mediated plant height regulatory pathway, broadening the understanding of the molecular basis underlying wheat plant height. Rht5 plays pleotropic roles in wheat growth and stress tolerance Previous studies have characterized Rht5 as a GAR dwarf gene that could reduce plant height and multiple agronomic important traits (Ellis et al. 2004; Cui et al. 2022). In our study, we developed Rht5 RILs and systematically evaluated its genetic effects on wheat growth and development. We confirmed the pleotropic negative effects of Rht5 on wheat plant architecture and yield-related traits including plant height, grain traits but the differences in Rht5 RILs were lower or even diminished compared with their parents (Fig. 1), indicating that introduction of Rht5 in different genetic backgrounds could partially improve the yield traits of Marfed M. The pleotropic effects of Rht5 in shaping wheat plant architecture were also observed in the diverse function of Rht5 on cell growth and endogenous hormonal homeostasis. We found that Rht5 inhibits cell proliferation to decrease cell number but promotes cell elongation (Fig. 2). Hormonal profiling of Rht5 RILs revealed that the stress-related hormones JA, ABA and SA were significantly higher in the dwarf lines (Fig. 3d). RNA-seq analysis revealed that some key regulators including WRKY62 and WRKY76 which are closely associated with the signaling pathways of SA, JA, and ABA (Mao et al. 2007; Yokotani et al. 2013; Fukushima et al. 2016), had significantly higher expression in the dwarf lines (Fig. S3; Table S7), suggesting that Rht5 may also involve in plant stress responses. This notion was also supported by the significant enrichment of GO terms “response to biotic stimulus” and “response to endogenous stimulus”, “response to oxygen-containing compound”, “protein kinase binding” as well as “indole-3-butyrate beta-glucosyltransferase activity” and “salicylic acid metabolic process” (Fig. 4c; Table S4), which are associated with promoted plant growth construction and increased resistance to diseases and mechanical damage (Vanneste and Friml 2009; Yang and Guo 2018; Zhou and Zhang 2020; Waadt et al. 2022). The KEGG enrichment revealed the “MAPK signaling pathway–plant” involved in signal transduction in response to environmental stresses and plant hormones, and several pathways including “phenylpropanoid biosynthesis”, “cinnamic acid biosynthetic process”, “linoleic acid metabolism” and “diterpenoid biosynthesis”, stress-responsive metabolism that could contribute to improved resilience (Dixon and Paiva 1995b; Howe and Schilmiller 2002; Vogt 2010a; Vaughan et al. 2015) (Fig. 4d; Table S5). Collectively, our study suggested that Rht5 acts as a pleotropic regulator in wheat growth and development. Rht5 causes dwarfism through inhibiting cell proliferation Plant height is largely determined by the cell proliferation in the intercalary meristem (IM) and cell elongation in the elongation zone of crop stem (Sauter and Kende 1992). In wheat, Rht-B1b and Rht8 confer a semi-dwarf phenotype by reducing cell elongation, thereby shortening stem internodes (Gasperini et al. 2012; Xu et al. 2023), while Rht22 impairs cell proliferation and reduces internode cell number (Peng et al. 2011; Wang et al. 2022). Other reduced-height locus could decrease both cell number and cell length to inhibit wheat stem elongation (Xu et al. 2017). By paraffin section analysis, we showed that Rht5 increases cell length but remarkably reduces cell number during stem elongation (Fig. 2). Quantification of endogenous phytohormones revealed lower content of bioactive cytokinin and auxin but elevated accumulation of GA 3 and GA 4 (Fig. 3b–c). Given that GA is well known to promote cell elongation, whereas cytokinin mainly stimulates cell division (Takatsuka and Umeda 2014), the dual effects of Rht5 on cell growth during stem elongation are consistent with hormonal profiling disequilibrium. These cellular changes on cell growth conferred by Rht5 differ from that of the well-known “Green Revolution” alleles Rht-B1b and Rht-D1b , which reduce plant height mainly by inhibition of GA signaling and GA-promoted cell elongation (KEYES et al. 1989; Pearce et al. 2011). To date, multiple dwarfing loci–including several GA-responsive loci–have been reported in wheat. Among them, Rht8 and Rht12 primarily inhibit cell elongation (Gasperini et al. 2012; Sun et al. 2019), whereas Rht22 mainly reduces plant height by inhibiting cell division (Peng et al. 2011; Wang et al. 2022). Furthermore, the autoactive NB-LRR allele Rht13 elicited another distinct mechanism of dwarfism (Borrill et al. 2022). Rht25 affects plant height by regulating the expression of DELLA-associated growth genes (Zhang et al. 2023). Given that Rht5 suppressed cell proliferation while promoting cell elongation with disordered accumulation of multiple growth-promoting hormones, together with the distinct effectiveness of exogenous 6-BA and GA 3 application on plant height of the dwarf Rht5 RILs, we proposed that Rht5 may regulate stem elongation and wheat plant height through mechanisms distinct from previously characterized the dwarfing loci. Genetic manipulation of TaGAD2 mimics Rht5 dwarfing phenotype Although Rht5 -mediated wheat dwarfism is associated with abnormal accumulation of multiple phytohormones, RNA-seq analysis showed moderate transcriptional changes of genes related to the growth-promoting phytohormone pathways between the tall and dwarf lines. Notably, we found that TaGAD2 was significantly upregulated in the dwarf lines compared with tall Rht5 RILs (Fig. 4f; Fig. 5a). We characterized TaGAD2 as a functional glutamate decarboxylase (GAD) that localized to plasma membrane (Fig. 5e, f). It has been demonstrated that GAD catalyzes the decarboxylation of L-glutamate (Glu) to GABA with release of CO 2. GAD functions as an entry-point enzyme of the GABA shunt, after which GABA is sequentially metabolized by GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) to feed carbon into the TCA cycle (Ansari et al. 2021). In wheat, TaNHX2 has been reported to target the C-terminal autoinhibitory domain of TaGAD1 , enhancing GAD activity and promoting endogenous GABA accumulation to regulate drought tolerance (Li et al. 2024), implying that altered GABA content may influence wheat growth, development, and stress responses. Interestingly, genetic manipulation of GADs leads to various phenotypes across plant species. In rice, overexpressing full-length OsGAD2 displayed an essentially normal phenotype, while overexpression of OsGAD2ΔC led to significantly elevated GABA levels and exhibited a pronounced dwarf phenotype (Akama and Takaiwa 2007). In tomato, SlGAD3OX lines were similar to the wild type in appearance, while SlGAD3ΔC overexpression lines displayed an orange-ripe phenotype with reduced carotenoids (Takayama et al. 2017). GABA accumulation can severely compromise the Agrobacterium-mediated transformation process (Brencic and Winans 2005; Chevrot et al. 2006; Planamente et al. 2012; Lang et al. 2016). In wheat, a TaGAD1 overexpression transgenic line ( GOE-1 ) was reported to cause severe growth retardation, including stunted spikes with grain abortion and development abnormalities (Li et al. 2024). In our study, overexpression of full-length TaGAD2 led to wheat dwarfism with marked developmental defects including reduction of spike length, spikelet number and grain number (Fig. 6a–g) and therefore phenocopied the dwarfism conferred by Rht5 (Fig. 6a–c). Conversely, TaGAD2 knockdown could increase plant height (Fig. 7a–f), suggesting that TaGAD2 acts as a negative regulator of stem elongation. Moreover, we identified TaGAD2 H1 as an elite allele that could reduce plant height without compromising grain number in a natural wheat population (Fig. 8). As genetic manipulation of TaGAD2 could mimic the dwarfing phenotypes of Rht5 dwarf lines, our study identified TaGAD2 as a potential downstream regulator of the Rht5 -mediated plant height regulatory pathway, providing new insights into the molecular basis of wheat plant height and offering genetic resources for improving wheat lodging resistance. Declarations Authorship contribution statement Xianglan Kong designed the research, performed the experiments, analysis the data and wrote the manuscript; Chunge Cui, Yuxin Lei and LiZhe performed the experiments, analyzed the data; Xuefen Cai, Aozhe Wang, Qiumei Lu, Zhangchen Zhao, and Altyn Shayahkhmetova performed some of the experiments and data scoring; Liang Chen and Linzhou Huang designed research, analysis data and wrote the manuscript. Yin-Gang Hu, conceived the study, designed research, analysis the data and wrote the manuscript. All authors read and approved the final manuscript. Conflict of interest The authors have no competing interests to declare. Data availability Data presented in this study are available in this research article and supplementary materials. The raw data of the RNA-seq was submitted to the NCBI SRA database with accession number PRJNA1424380. Acknowledgements This work was supported by the grants from National Natural Science Foundation of China (32171991), the Natural Science Basic Research Program of Shaanxi Province (2023-JC-ZD-08). References Akama K, Akihiro T, Kitagawa M, Takaiwa F (2001) Rice ( Oryza sativa ) contains a novel isoform of glutamate decarboxylase that lacks an authentic calmodulin-binding domain at the C-terminus. 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Cell 181(5):978–989. https://doi.org/10.1016/j.cell.2020.04.028 Additional Declarations No competing interests reported. Supplementary Files SupplementalTables.pdf Supplementary tables: Table S1. Primers for plasmid construction Table S2. Primers used in qRT-PCR Table S3. Effects of Rht5 on internodes elongation and spike length Table S4. Gene Ontology (GO) enrichment analysis of the differentially expressed genes (DEGs) Table S5. KEGG pathway enrichment of the differentially expressed genes (DEGs) Table S6. Transcripts per million (TPM) values of phytohormone-related DEGs Table S7. TPM values of DEGs involving in phytohormone pathway related to stress responses Table S8. TPM values of plant growth- and development-associated DEGs Table S9. Accession numbers of the GAD homologs used for phylogenetic analysis SupplementalFigures.pdf Supplementary figures: Fig S1 Statistical analysis of internode lengths of Rht5 RILs Fig S2 Measurement of endogenous phytohormone precursors in Rht5 RILs Fig S3 Heatmap showing TPM values of stress-related phytohormone pathways Fig S4 Multiple sequence alignment of glutamate decarboxylase (GAD) proteins across plant species. Fig S5 Prokaryotic expression analysis of TaGAD2 and TaGAD2ΔC recombinant proteins Fig S6Effects of TaGAD2 silencing on agronomic traits in wheat Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 21 Apr, 2026 Reviews received at journal 19 Apr, 2026 Reviews received at journal 13 Apr, 2026 Reviewers agreed at journal 19 Mar, 2026 Reviewers agreed at journal 19 Mar, 2026 Reviewers invited by journal 18 Mar, 2026 Editor assigned by journal 28 Feb, 2026 Submission checks completed at journal 27 Feb, 2026 First submitted to journal 27 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8985940","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":608995064,"identity":"79aecb85-4284-413d-8be7-fed6c898737f","order_by":0,"name":"Xianglan Kong","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Xianglan","middleName":"","lastName":"Kong","suffix":""},{"id":608995065,"identity":"b79ee0e1-4261-4926-a01d-87a443383df4","order_by":1,"name":"Yuxin Lei","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Yuxin","middleName":"","lastName":"Lei","suffix":""},{"id":608995066,"identity":"76bee4b3-25d9-405e-801d-6fa66f6ffb23","order_by":2,"name":"Aozhe Wang","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Aozhe","middleName":"","lastName":"Wang","suffix":""},{"id":608995074,"identity":"94876b64-4c2f-4582-b150-221770cc1ac7","order_by":3,"name":"Xuefen Cai","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Xuefen","middleName":"","lastName":"Cai","suffix":""},{"id":608995076,"identity":"29ac2fc8-718b-4f9d-a0a1-4752b42bfd9a","order_by":4,"name":"Li Zhe","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Zhe","suffix":""},{"id":608995078,"identity":"d5bd5c81-42ac-4350-b78d-91ce68a78035","order_by":5,"name":"Chunge Cui","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Chunge","middleName":"","lastName":"Cui","suffix":""},{"id":608995079,"identity":"110f65fa-7297-43de-b6dc-fd709c18c1a3","order_by":6,"name":"Zhangchen Zhao","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Zhangchen","middleName":"","lastName":"Zhao","suffix":""},{"id":608995080,"identity":"fde22e36-ee38-4098-82bd-25616366a50e","order_by":7,"name":"Altyn Shayahkhmetova","email":"","orcid":"","institution":"Manash Kozybayev North Kazakhstan University","correspondingAuthor":false,"prefix":"","firstName":"Altyn","middleName":"","lastName":"Shayahkhmetova","suffix":""},{"id":608995081,"identity":"b4b38f37-1620-42f3-9265-62e36d88819b","order_by":8,"name":"Linzhou Huang","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Linzhou","middleName":"","lastName":"Huang","suffix":""},{"id":608995086,"identity":"67727e4e-a487-4f15-aa6b-ac9b1943164c","order_by":9,"name":"Liang Chen","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Chen","suffix":""},{"id":608995087,"identity":"56fb8146-2b90-4a3e-affa-32e2ce0d9488","order_by":10,"name":"Yin-gang Hu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYBACAyA+AGaxNwAJGxArgVgtPCAqjUgtECCRQKQWc4kcw8MFv+zy5CPfHpP4kXCYgZ89x4Dh5w7cWixn5BgcntmXXGx4Oy9NsgeoRbLnjQFj7xk8DrsB1MLbw5y4cXaO2Q3eH4fBIsyMbQS11CdunHnG7OYfoC32RGnh+XE4cb4Ej9ltHqAWAwlCWs48KzjM23A8cQNPjvlvmYR0HgmgyMFefFqOJ2/+zPOnOnF++xljwzcJ1nL87ckbH/zEo4WBgcOAAeQMgwMQLg+IOIBPAzChPGBg+MPAIN+AX9koGAWjYBSMYAAAAX5YAdxnUbAAAAAASUVORK5CYII=","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":true,"prefix":"","firstName":"Yin-gang","middleName":"","lastName":"Hu","suffix":""}],"badges":[],"createdAt":"2026-02-27 09:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8985940/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8985940/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105308853,"identity":"5d1cd5ab-8c3b-4ed9-b6bd-723ca8ca2f3a","added_by":"auto","created_at":"2026-03-24 15:00:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":317131,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypes of \u003cem\u003eRht5 \u003c/em\u003erecombinant inbred lines (RILs)\u003c/p\u003e\n\u003cp\u003e(a) Comparison of the \u003cem\u003eRht5 \u003c/em\u003eRILs at mature stage. T and D indicate tall RILs (\u003cem\u003erht5rht5\u003c/em\u003e) and dwarf RILs (\u003cem\u003eRht5Rht5\u003c/em\u003e) derived from a cross between JM47 (\u003cem\u003erht5rht5\u003c/em\u003e) and Marfed M (\u003cem\u003eRht5Rht5\u003c/em\u003e). Scale bar, 10 cm. (b) Comparison of internode lengths between the tall and dwarf lines. Scale bar, 10 cm. (c) Statistical analysis of the plant height of lines in (a). (d–g) Statistical analysis of spike number per plant (d), spike length (e), spikelet number per spike (f) and grain number per spike (g) among the tall lines, dwarf lines and their parents. (h–k) Statistical analysis of the grain length (h), grain width (i), thousand-grain weight (j) and gain yield per plant (k) among the tall lines, dwarf lines and the parents. Data are presented as means ± SD (\u003cem\u003en\u003c/em\u003e = 8). \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003e**P\u003c/em\u003e \u0026lt; 0.01,\u003cem\u003e ns\u003c/em\u003e, not significant. Student’s \u003cem\u003et\u003c/em\u003e test.\u003c/p\u003e","description":"","filename":"Figures1.png","url":"https://assets-eu.researchsquare.com/files/rs-8985940/v1/a56237dafa8d996fa37a151b.png"},{"id":105308855,"identity":"fa21f2a6-5a68-47db-9c03-1910b3d8e200","added_by":"auto","created_at":"2026-03-24 15:00:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":767343,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRht5 \u003c/em\u003einhibits cell proliferation but promotes cell elongation in wheat\u003c/p\u003e\n\u003cp\u003e(a) Longitudinal sections of the peduncles from JM47, Marfed M, T1 and D1 RIL lines at the flowering stage. Scale bars, 200 μm. (b–d) Statistical analysis of cell length (b), cell width (c) and cell number (d) in the upper, middle and lower parts of peduncles from JM47 (\u003cem\u003erht5rht5\u003c/em\u003e), Marfed M (\u003cem\u003eRht5Rht5\u003c/em\u003e) and \u003cem\u003eRht5\u003c/em\u003eRILs. Data are presented as means ± SD (b, c, \u003cem\u003en\u003c/em\u003e \u0026gt; 200. d,\u003cem\u003e n\u003c/em\u003e = 10). \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003e**P\u003c/em\u003e \u0026lt; 0.01,\u003cem\u003e ns\u003c/em\u003e, not significant. Student’s \u003cem\u003et\u003c/em\u003e test.\u003c/p\u003e","description":"","filename":"Figures2.png","url":"https://assets-eu.researchsquare.com/files/rs-8985940/v1/6f4a204b6296bfa72e568873.png"},{"id":105308854,"identity":"4b4c7cdc-f270-4a0a-81e4-67f342af07fe","added_by":"auto","created_at":"2026-03-24 15:00:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":105439,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRht5 \u003c/em\u003eis involved in wheat hormone homeostasis\u003c/p\u003e\n\u003cp\u003e(a–d) Contents of endogenous indoleacetic acid (IAA) (a), bioactive cytokinin (b) and gibberellins (GAs) (c) and defense-related phytohormones (d) in \u003cem\u003eRht5\u003c/em\u003e RILs. (e–h) Statistical analysis of plant height (e, f) and spike length (g, h) of \u003cem\u003eRht5 \u003c/em\u003eRILs in response to exogenous 6-BA (20 mg L\u003csup\u003e–1\u003c/sup\u003e) and GA\u003csub\u003e3\u003c/sub\u003e (35 mg L\u003csup\u003e–1\u003c/sup\u003e) treatments. Phytohormones were applied by spraying the \u003cem\u003eRht5 \u003c/em\u003eRILs twice at the jointing stage and twice at the heading stage. Agronomic trait data were recorded at maturity. Data are presented as means ± SD (\u003cem\u003en\u003c/em\u003e = 5). \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003e**P\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003ens\u003c/em\u003e, not significant. Student’s \u003cem\u003et\u003c/em\u003e test.\u003c/p\u003e","description":"","filename":"Figures3.png","url":"https://assets-eu.researchsquare.com/files/rs-8985940/v1/d42393f82b44df49699eeddb.png"},{"id":105308860,"identity":"3a24595b-6638-4baa-8cdc-f603bd098fa2","added_by":"auto","created_at":"2026-03-24 15:00:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1288630,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptome analysis of \u003cem\u003eRht5 \u003c/em\u003erecombinant inbred lines (RILs)\u003c/p\u003e\n\u003cp\u003e(a) Overview of differentially expressed genes (DEGs) in peduncles from \u003cem\u003eRht5 \u003c/em\u003eRILs and their parents. T indicates the tall RILs (\u003cem\u003erht5rht5\u003c/em\u003e). D1, D2, and D3 represent three independent dwarf RILs (\u003cem\u003eRht5Rht5\u003c/em\u003e). (b) Venn diagram showing the 993 overlapping DEGs shared by T \u003cem\u003evs. \u003c/em\u003eD1, T \u003cem\u003evs.\u003c/em\u003e D2, T \u003cem\u003evs.\u003c/em\u003e D3 and JM47 \u003cem\u003evs.\u003c/em\u003e Marfed M. (c–d) GO enrichment analysis (c) and KEGG enrichment analysis (d) of the 993 overlapping DEGs. (e) Heatmap showing TPM values (transcripts per million) of phytohormone-related DEGs identified by RNA-seq. (f) Heatmap showing TPM values of plant growth- and development-related DEGs identified by RNA-seq. (JM, \u003cem\u003en \u003c/em\u003e= 1, Marfed M, \u003cem\u003en\u003c/em\u003e = 1, T, \u003cem\u003en\u003c/em\u003e = 3, D1-D3, \u003cem\u003en\u003c/em\u003e = 3)\u003c/p\u003e","description":"","filename":"Figures4.png","url":"https://assets-eu.researchsquare.com/files/rs-8985940/v1/42f2016e3961d5f7a9b67444.png"},{"id":105308862,"identity":"fb2de4b6-afa7-46d4-9ba5-fc67c05ad01e","added_by":"auto","created_at":"2026-03-24 15:00:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":627965,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular characterization of \u003cem\u003eTaGAD2\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(a) Relative expression levels of \u003cem\u003eTaGAD2\u003c/em\u003e in \u003cem\u003eRht5\u003c/em\u003e RILs and the parents at the three-leaf stage. (b) Expression patterns of \u003cem\u003eTaGAD2 \u003c/em\u003eduring wheat development based on the TPM data from WheatOmics. (c) Relative expression levels of \u003cem\u003eTaGAD2\u003c/em\u003e in root, shoot and leaf tissues of Chinese Spring at the seedling stage. (d) Phylogenetic analysis of TaGAD2 and GAD homologs across plant species. (e) Schematic diagram illustrating the conserved domains of TaGAD2 protein and the glutamate decarboxylase (GAD) activity assay of full-length TaGAD2 and C-terminally truncated TaGAD2ΔC. (f) Subcellular localization analysis of TaGAD2-GFP and TaGAD2ΔC-GFP with mCherry-OsPAD (plasma membrane marker) in tobacco epidermal cells. Scale bars, 50 μm. Data are presented as means ± SD (\u003cem\u003en \u003c/em\u003e= 3). \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, \u003cem\u003e**P\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003ens\u003c/em\u003e, not significant. Student’s \u003cem\u003et\u003c/em\u003etest.\u003c/p\u003e","description":"","filename":"Figures5.png","url":"https://assets-eu.researchsquare.com/files/rs-8985940/v1/2596df01ca42eff48a06e901.png"},{"id":105308856,"identity":"9380471e-5e78-4d63-a5d0-348c9230e7d9","added_by":"auto","created_at":"2026-03-24 15:00:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":253163,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of \u003cem\u003eTaGAD2 \u003c/em\u003ecould reduce plant height but increase net photosynthetic rate in wheat\u003c/p\u003e\n\u003cp\u003e(a) Plant architecture of Fielder (WT) and \u003cem\u003eTaGAD2-OE\u003c/em\u003e transgenic plants. Scale bar, 10 cm. (b) Relative expression of \u003cem\u003eTaGAD2\u003c/em\u003e in leaves of Fielder and \u003cem\u003eTaGAD2-OE \u003c/em\u003eplants. (c–g) Comparison of plant height (c), spike number per plant (d), spike length (e), spikelet number per spike (f), and grain number per spike (g) between Fielder and \u003cem\u003eTaGAD2-OE\u003c/em\u003e transgenic lines. (h–j) Comparison of flag leaf length (h), Soil and Plant Analysis Development (SPAD) value (i) and instantaneous water use efficiency (WUEi, A/E) (j) between Fielder and \u003cem\u003eTaGAD2-OE\u003c/em\u003e plants. (k) Flag leaves architecture of Fielder (WT) and \u003cem\u003eTaGAD2-OE\u003c/em\u003e transgenic plants. Scale bar, 5 cm. (l–o) Comparison of net photosynthetic rate (A) (l), transpiration rate (E) (m), stomatal conductance (g\u003csub\u003es\u003c/sub\u003e) (n) and intercellular CO\u003csub\u003e2\u003c/sub\u003e concentration (Ci) (o) between Fielder and \u003cem\u003eTaGAD2-OE\u003c/em\u003e plants shown in (k). Data are presented as means ± SD (b, \u003cem\u003en \u003c/em\u003e= 3. c–g, \u003cem\u003en \u003c/em\u003e= 4. h–o, \u003cem\u003en\u003c/em\u003e = 6).\u003cem\u003e *P\u003c/em\u003e \u0026lt; 0.05,\u003cem\u003e **P \u003c/em\u003e\u0026lt; 0.01, \u003cem\u003ens\u003c/em\u003e, not significant. Student’s \u003cem\u003et\u003c/em\u003e test.\u003c/p\u003e","description":"","filename":"Figures6.png","url":"https://assets-eu.researchsquare.com/files/rs-8985940/v1/3718e8371d3d4204261127ca.png"},{"id":105308858,"identity":"cae9cb83-a03b-429d-856a-09a8cd3e781c","added_by":"auto","created_at":"2026-03-24 15:00:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":286768,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of \u003cem\u003eTaGAD2 \u003c/em\u003ecould increase plant height while reducing net photosynthetic rate in wheat\u003c/p\u003e\n\u003cp\u003e(a) Plant architecture of Fielder (WT) and \u003cem\u003eTaGAD2-RNAi \u003c/em\u003etransgenic plants. Scale bar, 10 cm. (b) Relative expression of \u003cem\u003eTaGAD2\u003c/em\u003e in stems of Fielder and \u003cem\u003eTaGAD2-RNAi \u003c/em\u003eplants. (c–d) Comparison of glutamate decarboxylase (GAD) activity (c) and GABA content (d) in stems between Fielder and \u003cem\u003eTaGAD2-RNAi \u003c/em\u003eplants. (e–i) Comparison of plant height (e), internode length (f), spike length (g), spikelet number per spike (h) and grain number per spike (i) between Fielder and \u003cem\u003eTaGAD2-RNAi \u003c/em\u003eplants shown in (a). (j–n) Comparison of culm lodging resistance index (j), mechanical strength of BI2 (the second basal internode) (k), fresh weight of the main culm (l) and spike (m), and height of the center of gravity (n) between Fielder and \u003cem\u003eTaGAD2-RNAi \u003c/em\u003etransgenic plants shown in (a). (o–s) Comparison of WUEi (o), net photosynthetic rate (A) (p), intercellular CO\u003csub\u003e2\u003c/sub\u003e concentration (Ci) (q), stomatal conductance (g\u003csub\u003es\u003c/sub\u003e) (r), and transpiration rate (E) (s) between Fielder and \u003cem\u003eTaGAD2-RNAi \u003c/em\u003eplants. Data are presented as means ± SD (b–d, \u003cem\u003en \u003c/em\u003e= 3. e–i, \u003cem\u003en\u003c/em\u003e = 20. j–n, \u003cem\u003en \u003c/em\u003e= 10. o–s,\u003cem\u003e n\u003c/em\u003e \u0026gt; 5). \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003e**P\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003ens\u003c/em\u003e, not significant. Student’s\u003cem\u003e t\u003c/em\u003e test.\u003c/p\u003e","description":"","filename":"Figures7.png","url":"https://assets-eu.researchsquare.com/files/rs-8985940/v1/aa47471239617ed91f352b49.png"},{"id":105308861,"identity":"ddfabcca-2959-4bdd-b958-77cfbf0b84a1","added_by":"auto","created_at":"2026-03-24 15:00:51","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":98072,"visible":true,"origin":"","legend":"\u003cp\u003eHaplotype analysis of \u003cem\u003eTaGAD2 \u003c/em\u003ein wheat accessions\u003c/p\u003e\n\u003cp\u003e(a) Sequence variations in the coding region of \u003cem\u003eTaGAD2 \u003c/em\u003eamong three major haplotypes (\u003cem\u003en \u003c/em\u003e\u0026gt; 10). (b) Haplotype frequencies of three major \u003cem\u003eTaGAD2 \u003c/em\u003ehaplotypes in a panel of 273 accessions. Other haplotypes with accession number less than 10 were grouped into others. (c–f) Comparison of plant height (c), spike length (d), spikelet number per spike (e) and grain number per spike (f) among accessions with different \u003cem\u003eTaGAD2 \u003c/em\u003ehaplotypes. Data are presented as means ± SD\u003cem\u003e \u003c/em\u003e(H1, \u003cem\u003en \u003c/em\u003e= 88, H2, \u003cem\u003en \u003c/em\u003e= 72, H3, \u003cem\u003en \u003c/em\u003e= 10). \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003e**P\u003c/em\u003e\u0026lt; 0.01, \u003cem\u003ens\u003c/em\u003e, not significant. Student’s\u003cem\u003e t\u003c/em\u003e test.\u003c/p\u003e","description":"","filename":"Figures8.png","url":"https://assets-eu.researchsquare.com/files/rs-8985940/v1/89e88bd557abc026479317da.png"},{"id":105564284,"identity":"4be9b3ec-c0a5-4339-8acf-d057ba3dcbfc","added_by":"auto","created_at":"2026-03-27 12:49:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4673336,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8985940/v1/97bbf9a1-caa0-452f-a642-d9adb4daf743.pdf"},{"id":105308857,"identity":"4064c400-823c-4537-87cf-a1f618a588ce","added_by":"auto","created_at":"2026-03-24 15:00:51","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":387620,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary tables:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S1.\u003c/strong\u003e Primers for plasmid construction\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S2.\u003c/strong\u003e Primers used in qRT-PCR\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S3.\u003c/strong\u003e Effects of \u003cem\u003eRht5 \u003c/em\u003eon internodes elongation and spike length\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S4.\u003c/strong\u003e Gene Ontology (GO) enrichment analysis of the differentially expressed genes (DEGs)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S5. \u003c/strong\u003eKEGG pathway enrichment of the differentially expressed genes (DEGs)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S6.\u003c/strong\u003e Transcripts per million (TPM) values of phytohormone-related DEGs\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S7.\u003c/strong\u003e TPM values of DEGs involving in phytohormone pathway related to stress responses\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S8.\u003c/strong\u003e TPM values of plant growth- and development-associated DEGs\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S9.\u003c/strong\u003e Accession numbers of the GAD homologs used for phylogenetic analysis\u003c/p\u003e","description":"","filename":"SupplementalTables.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8985940/v1/15eb143df4fdc9309e810b8f.pdf"},{"id":105308859,"identity":"96761e98-706e-4311-bbe5-021292dd0604","added_by":"auto","created_at":"2026-03-24 15:00:51","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2760592,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary figures:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig S1\u003c/strong\u003e Statistical analysis of internode lengths of \u003cem\u003eRht5\u003c/em\u003eRILs\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig S2 \u003c/strong\u003eMeasurement of endogenous phytohormone precursors in \u003cem\u003eRht5 \u003c/em\u003eRILs\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig S3 \u003c/strong\u003eHeatmap showing TPM values of stress-related phytohormone pathways\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig S4\u003c/strong\u003e Multiple sequence alignment of glutamate decarboxylase (GAD) proteins across plant species.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig S5\u003c/strong\u003e Prokaryotic expression analysis of TaGAD2 and TaGAD2ΔC recombinant proteins\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig S6\u003c/strong\u003eEffects of \u003cem\u003eTaGAD2 \u003c/em\u003esilencing on agronomic traits in wheat\u003c/p\u003e","description":"","filename":"SupplementalFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8985940/v1/0d2b8c61f1b75743ff2d0908.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"TaGAD2 is a potential downstream effector of Rht5 in controlling wheat plant height","fulltext":[{"header":"Key Messages","content":"\u003cp\u003e\u003cem\u003eRht5\u0026nbsp;\u003c/em\u003eregulates wheat plant height and yield-related traits partly through modulation of a downstream gene\u0026nbsp;\u003cem\u003eTaGAD2\u003c/em\u003e, which controls GABA biosynthesis and influences stem elongation, lodging resistance, and photosynthetic performance.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe semi-dwarf wheat developed during the \u0026quot;Green Revolution\u0026quot; exhibits lodging resistance and high-yield characteristics. The key genes underlying this revolution in wheat\u0026ndash;\u003cem\u003eRht-B1b\u003c/em\u003e and\u0026nbsp;\u003cem\u003eRht-D1b\u003c/em\u003e\u0026ndash;encode mutant DELLA proteins that repress wheat gibberellin (GA) responses (Peng et al. 1999). While wheat varieties carrying these genes are effectively resistant to lodging, the excessively short stature results in insufficient biomass production and is often accompanied by adverse effects such as short coleoptile and impaired seedling emergence, thereby limiting their application in some environments (Peng et al. 1999; Hedden 2003). Because semi-dwarf varieties carrying\u0026nbsp;\u003cem\u003eRht-B1b\u003c/em\u003e and\u0026nbsp;\u003cem\u003eRht-D1b\u003c/em\u003e typically have a shorter coleoptile, they are often sown shallowly to ensure successful emergence and to maintain early seedling vigor. This shallow sowing could be fatal for wheat production under water-deficit conditions\u0026nbsp;(Jatayev et al. 2020; Zhao et al. 2022). The currently available gibberellin-insensitive (GAI) dwarfing genes are unlikely to fully meet the breeding objective of synergetic improvement of plant architecture and drought tolerance, highlighting the urgent need to develop drought resilient wheat varieties with GA-responsive dwarfing genes. In this case, the GAR dwarfing genes without adverse effects on early seedling establishment and seedling vigor have been considered as desirable substitutes for Green Revolution alleles in breeding drought adaptive dwarfing wheat varieties.\u003c/p\u003e\n\u003cp\u003eIn the past few decades, more than 25 reduced-height loci have been described in wheat (Hao et al. 2024). These loci include both GAR and GAI dwarfing sources, offering diverse options for optimizing plant height under contrasting environments. Among GAR loci, \u003cem\u003eRht13\u003c/em\u003e was considered as new gene for breeding drought-tolerant wheat under water-limited conditions. \u003cem\u003eRht13\u003c/em\u003e is not associated with reduced seedling growth or coleoptile length, and most of the height-reducing effect occurs later in development, so deeper planting in water-limiting environments to overcome the adverse effects associated with \u003cem\u003eRht-B1b\u003c/em\u003e and \u003cem\u003eRht-D1b\u003c/em\u003e. It\u003cstrong\u003e\u0026nbsp;encodes\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ea\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003enucleotide-binding site/leucine-rich repeat (NB-LRR) protein, and a point mutation in\u003cstrong\u003e\u0026nbsp;\u003cstrong\u003ethe semi-dwarf allele at the \u003cem\u003eRht13\u003c/em\u003e locus (\u003cem\u003eRht-B13b\u003c/em\u003e)\u003c/strong\u003e\u0026nbsp;\u003c/strong\u003ecauses constitutive activation of this gene, leading to remarkable reduction in plant height (Borrill et al. 2022).\u003cem\u003e\u0026nbsp;Rht25\u003c/em\u003e was found to encode a plant-specific PLATZ-A1 transcription factor expressed mainly in the elongating stem and developing spike. The interaction between PLATZ-A1and RHT1 protein implied that PLATZ-A1\u003cem\u003e\u0026nbsp;\u003c/em\u003emodulates DELLA-associated growth repression within the GA-DELLA pathway (Zhang et al. 2023). Tian et al. (2022) reported that \u003cem\u003eRht24\u0026nbsp;\u003c/em\u003eencodes the GA inactivation enzyme gibberellin 2-oxidase A9 (TaGA2ox-A9). Elevation of \u003cem\u003eTaGA2ox-A9\u0026nbsp;\u003c/em\u003eexpression in stems of \u003cem\u003eRht24b\u0026nbsp;\u003c/em\u003edwarf line could enhance GA catabolism, thus reducing plant height without yield penalty (Tian et al. 2022). In addition, \u003cem\u003eRht12\u003c/em\u003e is likely to reduce plant height by decreasing bioactive GA levels via upregulation of \u003cem\u003egibberellin 2-oxidase A14\u0026nbsp;\u003c/em\u003e(\u003cem\u003eTaGA2ox-A14\u003c/em\u003e) (Sun et al. 2019). The GAR dwarfing gene \u003cem\u003eRht5\u003c/em\u003e,\u0026nbsp;was first reported by\u0026nbsp;Ellis et al. (2004), which could\u0026nbsp;reduce plant height without\u0026nbsp;compromise of the coleoptile length and seedling viability, two important traits for wheat production in arid and semi-arid regions. In addition,\u0026nbsp;\u003cem\u003eRht5\u003c/em\u003e can also significantly increase the spike number per plant and harvest index\u0026nbsp;(Daoura et al. 2014), highlighting its potential to combine height reduction with yield improvement. Although \u003cem\u003eRht5\u003c/em\u003e has been mapped to a ~1 Mb interval on\u0026nbsp;wheat\u0026nbsp;chromosome 3B\u0026nbsp;(Cui et al. 2022), the causal gene\u0026nbsp;has not yet been determined.\u003c/p\u003e\n\u003cp\u003eGlutamate decarboxylase (GAD) is a key enzyme that catalyzes the biosynthesis of \u0026gamma;-aminobutyric acid (GABA), a non-protein amino acid metabolite acting as a potential signaling molecule in plants (Bouch\u0026eacute; et al. 2004; Turano et al. 1998; Michaeli et al. 2015). Baum et al. (1993) first characterized a plant GAD and described a canonical C-terminal calmodulin-binding domain (CaMBD), a feature subsequently shown to be plant-specific (Yun and Oh 1998). This C-terminal module directly couples Ca\u003csup\u003e2+\u003c/sup\u003e/calmodulin signaling to regulate GAD activity. Under stress conditions, elevated cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e binds calmodulin (CaM) to form a Ca\u003csup\u003e2+\u003c/sup\u003e/CaM complex that interacts with the CaMBD, thereby releasing C-terminal autoinhibition and activating GAD; in contrast, in the absence of Ca\u003csup\u003e2+\u003c/sup\u003e/CaM, the CaMBD maintains an autoinhibited state that suppresses enzyme activity (Snedden et al. 1995).\u0026nbsp;Studies have revealed that\u0026nbsp;GABA strongly influences plant growth and development\u0026nbsp;(Uzma Jalil et al. 2019; Du et al. 2020; Khan et al. 2021). In Arabidopsis, accumulation of GABA could inhibit cell elongation.\u0026nbsp;Consistently, the \u003cem\u003epop2\u003c/em\u003e mutant, which accumulates high levels of GABA, shows defective root, hypocotyl and pollen tube elongation\u0026nbsp;(Renault et al. 2011).\u0026nbsp;In rice,\u0026nbsp;\u003cem\u003eOsGAD2\u003c/em\u003e, a member of the GAD family lacking the CaMBD, has been characterized as a negative regulator of plant height and yield traits (Zhang et al. 2023).\u0026nbsp;Overexpression of \u003cem\u003eOs\u003c/em\u003e\u003cem\u003eGAD2\u0026Delta;C\u003c/em\u003e (C-terminally truncated \u003cem\u003eOsGAD2\u003c/em\u003e) led to significantly elevated GABA levels in roots, shoots and leaves compared with the wild type and exhibited a pronounced dwarf phenotype characterized by pale, curled leaves and infertility, whereas rice plants overexpressing full-length \u003cem\u003eOsGAD2\u003c/em\u003e displayed an essentially normal phenotype\u0026nbsp;(Akama and Takaiwa 2007). In wheat, GABA could regulate plant pollen tube and root by directly modulating ALMT-type anion transporter activity (e.g., \u003cem\u003eTaALMT1\u003c/em\u003e)\u0026nbsp;(Ramesh et al. 2015). Recent study suggested that overexpression of\u0026nbsp;\u003cstrong\u003e\u003cem\u003eTaGAD1\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eresulted in stunted spikes and increased grain abortion, accompanied by abnormal spike development\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(Li et al. 2024)\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eTakayama and Ezura (2015)\u0026nbsp;identified two tomato\u0026nbsp;\u003cem\u003eGAD\u0026nbsp;\u003c/em\u003egenes,\u0026nbsp;\u003cem\u003eSlGAD2\u0026nbsp;\u003c/em\u003eand\u0026nbsp;\u003cem\u003eSlGAD3\u003c/em\u003e, that regulate GABA accumulation and fruit maturation. Tomato fruits from \u003cem\u003eSlGAD3\u0026Delta;C\u003c/em\u003e overexpression lines driven by the E8-HSP cassette displayed an orange-ripe phenotype with reduced carotenoids and downregulated ethylene-responsive carotenogenic genes, implying a compromised ethylene sensitivity during ripening, while \u003cem\u003eSlGAD3OX\u003c/em\u003e lines were indistinguishable from those of the wild type in appearance\u0026nbsp;(Takayama et al. 2017). Further studies have demonstrated that GABA exerts dose-dependent effects on plant development\u0026nbsp;(Yue et al. 2018; Xie et al. 2020). Additionally, GABA is also involved in stress tolerance\u0026nbsp;(Xu et al. 2021; Li et al. 2024; Wang et al. 2025).\u003c/p\u003e\n\u003cp\u003eThe\u003cem\u003e\u0026nbsp;Rht5\u0026nbsp;\u003c/em\u003edwarfing locus\u003cem\u003e\u0026nbsp;\u003c/em\u003ewas originally discovered from a JM47 \u0026times; Marfed M cross and represents an important GA-responsive dwarfing allele in wheat that reduces plant height and enhances lodging resistance. We have previously mapped\u0026nbsp;\u003cem\u003eRht5\u0026nbsp;\u003c/em\u003eto an ~1 Mb interval on chromosome 3B flanked by the molecular markers \u003cem\u003eKasp-25\u003c/em\u003e and \u003cem\u003eKasp-23\u003c/em\u003e, while the potential regulatory mechanism remains unknown. In this study, we investigated the genetic and physiological basis of\u0026nbsp;\u003cem\u003eRht5\u003c/em\u003e-mediated dwarfism. We showed that\u0026nbsp;\u003cem\u003eRht5\u0026nbsp;\u003c/em\u003eregulates plant height primarily through inhibition of cell proliferation although it promotes cell elongation which could be attribute to the altered homeostasis. Using transcriptome analysis we identified\u0026nbsp;TaGAD2,\u0026nbsp;a glutamate decarboxylase involved in GABA biosynthesis that localized at plasma membrane, as a potential downstream component of \u003cem\u003eRht5\u003c/em\u003e-mediated wheat plant height regulatory pathway. We demonstrated that\u0026nbsp;\u003cem\u003eTaGAD2\u0026nbsp;\u003c/em\u003eas a negative regulator of wheat plant height and overexpression of \u003cem\u003eTaGAD2\u003c/em\u003e could phenocopy \u003cem\u003eRht5\u003c/em\u003e, highlighting a potential novel regulatory mechanism of wheat plant height.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003ch3\u003ePlant materials and growth conditions\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eRht5\u0026nbsp;\u003c/em\u003eRILs used in this study were derived from the cross JM47\u0026nbsp;\u0026times;\u0026nbsp;Marfed M followed by continuous selfing of the F\u003csub\u003e1\u003c/sub\u003e progenies for six generations to obtain genetically stable-inherited lines. Three tall lines (\u003cem\u003erht5rht5\u003c/em\u003e) and three dwarf lines (\u003cem\u003eRht5Rht5\u003c/em\u003e) together with the parents Jinmai47 (JM47) and Marfed M were used for analyses. The \u003cem\u003eRht5\u0026nbsp;\u003c/em\u003eRILs were grown at the experimental farm of Northwest A\u0026amp;F University (Yangling, Shaanxi, China). Transgenic wheat lines were generated in the Fielder background. \u003cem\u003eTaGAD2-RNAi\u003c/em\u003e plants were grown at the Transgenic Experimental Farm of Northwest A\u0026amp;F University. Field experiments at the experimental farm and the Transgenic Experimental Farm were conducted with the same planting density: 1.5-m rows, 25-cm row spacing and 20 seeds per row.\u003cem\u003e\u0026nbsp;TaGAD2-OE\u003c/em\u003e plants were grown in a greenhouse at Northwest A\u0026amp;F University. For greenhouse cultivation of \u003cem\u003eTaGAD2-OE\u003c/em\u003e plants, seedlings were transplanted into 16 \u0026times; 16 \u0026times; 18 cm pots (L \u0026times; W \u0026times; H) and grown at 20\u0026deg;C/16\u0026deg;C (day/night) under a 16-h light/8-h dark photoperiod with 60% relative humidity.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNicotiana benthamiana\u003c/em\u003e plants were grown in a greenhouse under a 16-h light/8-h dark photoperiod at 23\u0026deg;C with 70% relative humidity. Plants were cultivated in pots with substrate consisting of soil: vermiculite (2:1, v/v), and 4-week-old plants were used for subsequent analysis.\u003c/p\u003e\n\u003ch3\u003ePhytohormone treatment\u003c/h3\u003e\n\u003cp\u003ePhytohormone treatments were applied to \u003cem\u003eRht5\u0026nbsp;\u003c/em\u003eRILs by whole-plant spraying twice at the jointing stage and twice at the heading stage. The concentrations were 20 mg L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003efor\u003csup\u003e\u0026nbsp;\u003c/sup\u003e6-BA treatment and 35 mg L\u003csup\u003e-1\u003c/sup\u003e for GA\u003csub\u003e3\u003c/sub\u003e treatment, with 0.04% (v/v) Tween-20 respectively. Agronomic traits were scored at maturity with five replicates. For each replicate, data were scored from four plants and the mean values were used for statistical analysis. Relative promotion rate (RPR) was calculated with the formula below:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/58895_8739fc6c57c1c19a/58895_custom_files/img1774363994.png\" width=\"673\" height=\"192\"\u003e\u003c/p\u003e\n\u003ch3\u003eThe development of transgenic wheat lines\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eTo generate the \u003cem\u003eTaGAD2\u003c/em\u003e overexpression (OE) lines, the full-length coding sequence (CDS) of \u003cem\u003eTaGAD2\u0026nbsp;\u003c/em\u003ewas amplified from Marfed M and cloned into the pCUB-3\u0026times;Flag vector via homologous recombination, forming the \u003cem\u003epCUB-TaGAD2-3\u0026times;Flag\u003c/em\u003e plasmid. To generate the \u003cem\u003eTaGAD2\u003c/em\u003e-\u003cem\u003eRNAi\u003c/em\u003e plants, two 280-bp fragments of the \u003cem\u003eTaGAD2\u003c/em\u003e CDS were inserted in reverse tandem into the pCUB-RNAi vector, forming the \u003cem\u003epCUB-TaGAD2-RNAi\u003c/em\u003e.\u003cem\u003e\u0026nbsp;\u003c/em\u003eAll the constructs were transformed into \u003cem\u003eAgrobacterium\u0026nbsp;\u003c/em\u003e\u003cem\u003etumefaciens\u003c/em\u003e strain EHA105 and introduced into wheat cultivar Fielder by\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eAgrobacteriu\u003c/em\u003e\u003cem\u003em-mediated\u003c/em\u003e transformation (Wu et al. 2003). Primers used for vector construction are listed in Table S1.\u003c/p\u003e\n\u003ch3\u003eEvaluation of lodging-related traits in transgenic lines\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eAt the mid-grain-filling stage, main stems from 10 individuals were randomly selected from each line for measurement of lodging-related traits. For the second basal internode (BI2), internode length, diameter, fresh weight and dry weight were measured. In addition, whole-culm fresh weight, spike fresh weight and the height of the center of gravity of the main culm were recorded for lodging assessment. According to the method described by Murphy et al. (1958). The mechanical strength of BI2 (the second basal internode, counted from the soil surface upward) was determined using a plant stalk strength tester (YYD-1; Zhejiang Top Instrument Co., Ltd., Zhejiang, China). The tester was placed on a stable benchtop, and the second basal internode of the wheat main stem was laid horizontally across the two grooves (2.5-cm apart) at the base of the device. The culm lodging resistance index (CLRI) was calculated as mechanical strength of BI2/(height of the center of gravity \u0026times; whole-culm fresh weight).\u003c/p\u003e\n\u003ch3\u003eEvaluation of leaf photosynthetic characteristics\u003c/h3\u003e\n\u003cp\u003eLeaf photosynthetic traits were assessed at the early grain-filling stage (GS69). Photosynthetic parameters of flag leaves including net photosynthetic rate (A), stomatal conductance (g\u003csub\u003es\u003c/sub\u003e), transpiration rate (E) and intercellular CO\u003csub\u003e2\u003c/sub\u003e concentration (Ci) were measured using a LI-6400XT portable photosynthesis system (Li-COR Inc., Lincoln, NE, USA). Instantaneous water use efficiency (iWUE) was determined as the ratio of photosynthesis to transpiration (A/E). The relative chlorophyll content (SPAD) was measured at the middle portion of the flag leaf with a SPAD-502 chlorophyll meter (Konica Minolta, Osaka, Japan).\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eHistological analysis of peduncle\u003c/h3\u003e\n\u003cp\u003eFor cell size analysis, peduncles from the \u003cem\u003eRht5\u0026nbsp;\u003c/em\u003eRILs were collected at the flowering stage (GS69; Zadoks et al. 1974) and paraffin sections were prepared as described by Chai et al. (2019). Briefly, one centimeter of segments from the basal, middle and uppermost regions of the peduncle were fixed in FAA, dehydrated, embedded in paraffin, and sectioned longitudinally at 10\u0026nbsp;\u0026mu;m\u0026nbsp;(Kong et al. 2023). Images were acquired using the BX63 Olympus microscope (Evident Corporation, Tokyo, Japan) and cell length, cell width and cell number per unit area (1 mm\u003csup\u003e2\u003c/sup\u003e) were quantified using ImageJ software\u0026nbsp;(Schindelin et al. 2012).\u003c/p\u003e\n\u003ch3\u003eRNA sequencing (RNA-seq) analysis\u003c/h3\u003e\n\u003cp\u003eElongating peduncle segments were collected at the heading stage from \u003cem\u003eRht5\u0026nbsp;\u003c/em\u003eRILs, JM47 and Marfed M. Three homozygous dwarf RILs and one tall RIL were sampled for RNA-seq with three biological replicates for each line. The parental JM47 and Marfed M plants were also subjected to RNA-seq analysis with one biological replicate for each line. Paired-end RNA-seq libraries were prepared and sequenced on the Illumina HiSeq 4000 platform (PE150) (Novogene Co., Ltd., Beijing, China) following the manufacturer\u0026rsquo;s standard procedures. Differentially expressed genes (DEGs) were identified from four pairwise comparisons (JM47 \u003cem\u003evs.\u003c/em\u003e Marfed M, T \u003cem\u003evs.\u003c/em\u003e D1, T \u003cem\u003evs.\u003c/em\u003e D2 and T \u003cem\u003evs.\u003c/em\u003e D3) using DESeq2 (Love et al. 2014). Genes with \u003cem\u003ep\u003c/em\u003e-value\u0026nbsp;\u0026le; 0.05 and\u0026nbsp;|log\u003csub\u003e2\u003c/sub\u003eFoldChange| \u0026ge; 1 were defined as DEGs. Venn diagrams illustrating overlaps among DEG sets were generated using an online Venn diagram tool (https://www.genescloud.cn/chart/VennPlot). Gene Ontology (GO) enrichment significance for each GO term was calculated using a hypergeometric test, the top five significant categories were visualized using R. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed at the Gene Denovo (OmicShare) cloud platform (https://www.omicshare.com/tools/) and the top 15 significant pathways were visualized using OmicShare. Heatmaps were generated using TBtools (Chen et al. 2023). The input matrix consisted of TPM (transcripts per million) values for the selected genes across groups.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003ePhylogenetic analysis\u003c/h3\u003e\n\u003cp\u003eTaGAD2\u003cem\u003e\u0026nbsp;\u003c/em\u003eand its homologous protein sequences from wheat (\u003cem\u003eTriticum aestivum\u0026nbsp;\u003c/em\u003eL.), maize (\u003cem\u003eZea mays\u003c/em\u003e), rice (\u003cem\u003eOryza sativa\u003c/em\u003e), soybean (\u003cem\u003eGlycine max\u003c/em\u003e) and \u003cem\u003eArabidopsis thaliana\u0026nbsp;\u003c/em\u003ewere retrieved from EnsemblPlants (https://plants.ensembl.org/). Protein sequences were aligned using ClustalW with default parameters. A phylogenetic tree was generated in MEGA 11 using the neighbor-joining (NJ) method with 1,000 bootstrap replicates to evaluate branch support (Tamura et al. 2021).\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eEndogenous phytohormone and GABA content determination\u003c/h3\u003e\n\u003cp\u003eInternodes were collected from the \u003cem\u003eRht5\u003c/em\u003e RILs at the heading stage. Samples were sent to Nanjing Webiolotech Testing Technology Co., Ltd., for quantification of endogenous phytohormones using an ultra-performance liquid chromatography (UPLC) system (Qsight LX50, PerkinElmer, Waltham, Massachusetts, USA) coupled with tandem mass spectrometry (QSight 420 triple quadrupole, PerkinElmer, Waltham, Massachusetts, USA).\u003c/p\u003e\n\u003cp\u003eTo measure GABA, tissues from Fielder and \u003cem\u003eTaGAD2-RNAi\u003c/em\u003e plants were collected at the grain-filling stage. Approximately 0.1 g of tissue was ground in a pre-chilled mortar with 1 mL of extraction buffer on ice. The homogenate was centrifuged at 12,000 rpm for 10 min at 4\u0026deg;C, and the resulting supernatant was used for GABA quantification using the GABA Assay Kit (ADS-W-AJS009, ADSBio, Jiangsu, China) following the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003ch3\u003eSubcellular localization analysis\u003c/h3\u003e\n\u003cp\u003eThe full-length CDS of \u003cem\u003eTaGAD2\u003c/em\u003e and the truncated \u003cem\u003eTaGAD2\u0026Delta;C\u003c/em\u003e were amplified from Marfed M using specific primers (Table S1) and recombinated in-frame with GFP under the control of the CaMV 35S promoter to generate the\u003cem\u003e\u0026nbsp;35S\u003csub\u003epro\u003c/sub\u003e:TaGAD2-GFP\u003c/em\u003e and \u003cem\u003e35S\u003csub\u003epro\u003c/sub\u003e:TaGAD2\u0026Delta;C-GFP\u003c/em\u003e constructs, respectively. The mCherry-OsPAD was used as a plasma membrane marker. The verified plasmids were introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101 (pSoup-P19) and infiltrated into the abaxial epidermis of \u003cem\u003eNicotiana benthamiana\u0026nbsp;\u003c/em\u003eleaves as previously described (Liu et al. 2010). Fluorescence signals were examined 48 hours post infiltration using a confocal laser-scanning microscope (Olympus FV3000, Tokyo, Japan) with an excitation wavelength of 488 nm for GFP and 561 nm for mCherry, respectively.\u003c/p\u003e\n\u003ch3\u003eProkaryotic expression and enzyme activity assay\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eTaGAD2\u0026nbsp;\u003c/em\u003eand \u003cem\u003eTaGAD2\u0026Delta;C\u0026nbsp;\u003c/em\u003ecoding sequences described above were amplified and cloned into the pGEX-4T-1 vector using restriction enzymes \u003cem\u003eNco\u003c/em\u003eI and \u003cem\u003eNot\u003c/em\u003eI. Recombinant plasmids were transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e BL21 (DE3). Bacterial cells were grown in lysogeny broth (LB) medium at 37\u0026deg;C to OD\u003csub\u003e600\u003c/sub\u003e of 0.6. After induction with 0.5 mM IPTG (Isopropyl \u0026beta;-D-1-thiogalactopyranoside) at 18\u0026deg;C for 16 h, cells were harvested and lysed by ultrasonication on ice. The lysates were centrifuged at 7,000 g for 20 min at 4\u0026deg;C and GST-tagged proteins were purified using glutathione magnetic agarose beads (KTSM1355, KTSM, Shenzhen, China) following the manufacturer\u0026rsquo;s instructions. Purified proteins were analyzed by 10% SDS-PAGE. One gel was stained with Coomassie Brilliant Blue G-250 (Cat. No. P0017, Beyotime Biotechnology, Shanghai, China) to assess total protein expression and purification quality, and a parallel gel was subjected to Western blotting probed with a mouse anti-GST monoclonal antibody at a dilution of 1:5000 (AF0174, Beyotime Biotechnology, Shanghai, China) and visualized using ECL chemiluminescence (Cat. No. CW0049M, CWBIO, Jiangsu, China). GAD enzymatic activity was quantified by determining GABA production using the Berthelot colorimetric reaction. GAD activity was quantified using a Glutamate Decarboxylase Assay Kit (ADS-W-AJS010, Suzhou Mengxi Bio-Medical Technology Co., Ltd., Jiangsu, China).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor GAD enzymatic activity assay, flag leaves and rachis tissues from transgenic lines and wild-type plants were collected at the heading stage. Approximately 0.1 g of tissue was ground in a pre-chilled mortar with 1 mL of extraction buffer on ice. The homogenate was centrifuged at 12,000 rpm for 10 min at 4\u0026deg;C, and the resulting supernatant was used for GAD activity determination. The assay is based on the phenol-hypochlorite colorimetric reaction, in which GABA produced by GAD reacts with phenol and sodium hypochlorite under alkaline conditions to generate a blue chromogenic product. The absorbance of this product at 645 nm is proportional to the amount of GABA formed, reflecting GAD enzymatic activity. GAD activity was quantified using a Glutamate Decarboxylase Assay Kit (ADS-W-AJS010, ADSBio, Jiangsu, China) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003ePrimer sequences used for vector construction are listed in Table S1.\u003c/p\u003e\n\u003ch3\u003eQuantitative PCR analysis\u003c/h3\u003e\n\u003cp\u003eFor gene expression profiling, roots, stems and leaves were collected at three-leaf stage. Total RNA was extracted using RNAiso Plus (Cat. No. 9109, Takara Bio Inc., Kusatsu, Japan) and first-strand cDNA was synthesized from 1 \u0026mu;g RNA using the All-in-One First-Strand Synthesis MasterMix (with dsDNase) (Cat. No. EG15133S, Yugong Biotech Co., Ltd., Jiangsu, China). Quantitative real-time PCR (qRT-PCR) was performed on a Bio-Rad CFX96 Real-Time PCR System using F488 SYBR qPCR Mix (Cat. No. EG23111L, Yugong Biotech Co, Ltd., Jiangsu, China). Gene expression levels were normalized to \u003cem\u003eTaActin\u003c/em\u003e and relative transcript abundance was calculated using the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method. Primer sequences used for qRT-PCR are listed in Table S2.\u003c/p\u003e"},{"header":"Results","content":"\u003ch3\u003ePhenotypic characterization of \u003cem\u003eRht5\u003c/em\u003e recombinant inbred lines\u003c/h3\u003e\n\u003cp\u003eTo evaluate the effects of \u003cem\u003eRht5\u003c/em\u003e on wheat development, \u003cem\u003eRht5\u003c/em\u003e recombinant inbred lines (RILs) were generated from a cross between JM47 (\u003cem\u003erht5rht5\u003c/em\u003e) and Marfed M (\u003cem\u003eRht5Rht5\u003c/em\u003e), the \u003cem\u003eRht5\u003c/em\u003e donor, followed by consecutive self-pollination for six generations to obtain homozygous\u0026nbsp;\u003cem\u003eRht5Rht5\u003c/em\u003e (dwarf, D) and \u003cem\u003erht5rht5\u003c/em\u003e (tall, T) lines. Compared with tall lines, the \u003cem\u003eRht5\u003c/em\u003e dwarf lines exhibited significantly reduced plant height (Fig. 1a, c). A detailed comparison of internode lengths between the dwarf and tall lines indicated that each internode in the dwarf \u003cem\u003eRht5\u0026nbsp;\u003c/em\u003eRILs was shortened to varying degrees relative to the tall \u003cem\u003eRht5\u0026nbsp;\u003c/em\u003eRILs (Fig. 1b, Fig. S1). Compared with the tall RILs, the spike number per plant was significantly increased in the dwarf lines (Fig. 1d). In addition, Marfed M showed significant reduction in spike length, spikelet number per spike and grain number per spike relative to JM47 (Fig. 1e\u0026ndash;g). However, in the \u003cem\u003eRht5\u0026nbsp;\u003c/em\u003eRILs, no significant differences were detected between tall and dwarf RILs in spike length, spikelet number per spike and grain number per spike (Fig. 1e\u0026ndash;g).\u0026nbsp;Similarly,\u0026nbsp;Marfed M showed significant reductions in grain length, grain width, thousand-grain weight and grain yield per plant compared with JM47, whereas these differences were reduced or absent in the tall and dwarf\u0026nbsp;\u003cem\u003eRht5\u0026nbsp;\u003c/em\u003eRILs (Fig. 1h\u0026ndash;k).\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003e\u003cem\u003eRht5\u003c/em\u003e inhibits cell proliferation to regulate stem elongation\u003c/h3\u003e\n\u003cp\u003eBecause plant height is largely determined by cell proliferation and growth in the intercalary meristem in the elongation zone of the stem, we performed histological analyses to evaluate the effects of \u003cem\u003eRht5\u003c/em\u003e on cell morphologies during wheat stem elongation (Fig. 2a). The basal internodes showed the strongest dwarfing effect, with the fifth and fourth internodes reduced by 34.08% and 34.39%, respectively. Peduncle length was reduced by 31.24% and accounted for 29.50% of the total reduction in plant height (Table S3). Longitudinal sections of the young peduncles showed that Marfed M exhibited longer cell length and shorter cell width, as well as significantly fewer cell numbers across the upper, middle and lower regions of the peduncles compared with JM47 (Fig. 2b\u0026ndash;d). Consistent with the observation in the parental lines, the dwarf \u003cem\u003eRht5\u003c/em\u003e RILs displayed significant increase in cell length and marked decrease in cell number across the upper, middle and lower regions of the peduncles (Fig. 2b, d). Despite the comparable cell width in the upper region of the peduncles between the T1 and D1 lines, the cell width was significantly decreased in the middle and lower regions in the D1 line (Fig. 2c). These results indicated that the \u003cem\u003eRht5\u003c/em\u003e-conferred dwarfism was mainly associated with reduced cell number resulting from inhibited cell proliferation in the intercalary meristem of the stem rather than cell elongation.\u003c/p\u003e\n\u003ch3\u003eResponses of\u0026nbsp;\u003cem\u003eRht5\u0026nbsp;\u003c/em\u003eRILs to exogenous phytohormone treatments\u003c/h3\u003e\n\u003cp\u003eTo examine whether the phenotypic differences between the tall and dwarf \u003cem\u003eRht5\u003c/em\u003e RILs were associated with altered endogenous phytohormone profiles, we quantified phytohormone levels in the peduncles of tall and dwarf RILs at the heading stage. Among the growth-related phytohormones, the dwarf RILs showed reduced levels of bioactive indole-3-acetic acid (IAA) but increased accumulation of auxin precursors including indole-3-acetonitrile (IAN), tryptamine (TAM), indole-3-acetamide (IAM) and indole-3-pyruvic acid (IPyA) (Fig. 3a; Fig. S2a). Measurement of cytokinin levels indicated that the dwarf lines showed marked reduction in zeatin, \u003cem\u003etrans\u003c/em\u003e-zeatin and isopentenyladenosine (IPA) (Fig. 3b; Fig. S2b), whereas the cytokinin precursors, including\u0026nbsp;\u003cem\u003etrans\u003c/em\u003e-zeatin riboside (TZR) and isopentenyladenine (iP), were markedly elevated compared with the tall lines (Fig. S2b). The levels of bioactive gibberellins (GA\u003csub\u003e3\u0026nbsp;\u003c/sub\u003eand GA\u003csub\u003e4\u003c/sub\u003e) in dwarf RILs were significantly higher than those in the tall RILs (Fig. 3c). The levels of stress-related hormones, including salicylic acid (SA), jasmonic acid (JA) and abscisic acid (ABA), were significantly higher in the dwarf lines than those in the tall lines. In addition, JA precursor levels were also significantly higher in the dwarf lines (Fig. 3d; Fig. S2c),\u0026nbsp;suggesting that \u003cem\u003eRht5\u0026nbsp;\u003c/em\u003emay be involved in the regulation of wheat stress responsiveness.\u0026nbsp;To further evaluate the effects of \u003cem\u003eRht5\u0026nbsp;\u003c/em\u003eon hormonal responsiveness in wheat, exogenous gibberellic acid (GA\u003csub\u003e3\u003c/sub\u003e) and 6-benzylaminopurine (6-BA) were applied twice at the jointing stage and twice at the heading stage. The results showed that the tall and dwarf RILs had differential responses to exogenous phytohormone treatments. Under mock conditions, dwarf RILs had significantly lower plant height and spike length than tall RILs (Fig. 3e, g). The 6-BA treatment showed little effect on plant height and spike length, while GA\u003csub\u003e3\u003c/sub\u003e treatment significantly increased both plant height and spike length of the \u003cem\u003eRht5\u003c/em\u003e RILs (Fig. 3e, g). In addition, the dwarf lines exhibited stronger GA\u003csub\u003e3\u003c/sub\u003e-induced promotion in stem and spike elongation with significant increases in relative promotion rates (Fig. 3f, h), indicating enhanced GA\u003csub\u003e3\u003c/sub\u003e responsiveness in the dwarf lines.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eTranscriptome analysis identifies\u0026nbsp;\u003cem\u003eTaGAD2\u003c/em\u003e as a potential downstream effector of \u003cem\u003eRht5\u003c/em\u003e-mediated dwarfism\u003c/h3\u003e\n\u003cp\u003eTo identify the downstream genes involved in the\u0026nbsp;\u003cem\u003eRht5\u003c/em\u003e-mediated plant height regulatory pathway, RNA-seq was performed with peduncle tissues at the heading stage to reveal transcriptional changes between tall and dwarf lines. Using DESeq2 (\u003cem\u003ep\u003c/em\u003e-value\u0026nbsp;\u0026le; 0.05 and\u0026nbsp;|log\u003csub\u003e2\u003c/sub\u003eFoldChange|\u0026nbsp;\u0026ge; 1), we identified DEGs in comparisons of Marfed M\u0026nbsp;\u003cem\u003evs.\u003c/em\u003e JM47\u003cem\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;\u003c/em\u003eD1/D2/D3\u0026nbsp;\u003cem\u003evs.\u003c/em\u003e T. Compared with JM47, 15,686 upregulated DEGs and 12,941 downregulated DEGs were identified in Marfed M. There were 1,630, 2,121 and 5,648 DEGs significantly upregulated in D1, D2 and D3 lines, respectively. Compared with the tall \u003cem\u003eRht5\u003c/em\u003e RILs (T), 4,220, 3,924 and 9,880 DEGs were significantly downregulated in D1, D2 and D3 lines, respectively (Fig. 4a). In total, 993 overlapping DEGs were identified across RILs and their parental lines carrying different\u0026nbsp;\u003cem\u003eRht5\u003c/em\u003e alleles (Fig. 4b). Gene Ontology (GO) analysis showed that the 993 common DEGs were significantly enriched in the molecular function terms \u0026ldquo;protein kinase binding\u0026rdquo; and \u0026ldquo;indole-3-butyrate beta-glucosyltransferase activity\u0026rdquo; and in the biological process terms \u0026ldquo;salicylic acid metabolic process\u0026rdquo;, \u0026ldquo;response to biotic stimulus\u0026rdquo; and \u0026ldquo;response to chitin\u0026rdquo; (Fig. 4c; Table S4). These enriched functions and processes terms are associated with plant growth, environmental adaptation and stress/disease resistance (Vanneste and Friml 2009; Yang and Guo 2018; Zhou and Zhang 2020; Waadt et al. 2022). In concordance with GO enrichment, KEGG pathway enrichment further showed that these enriched pathways (e.g., phenylalanine metabolism, biosynthesis of secondary metabolites and phenylpropanoid biosynthesis) are closely associated with secondary metabolism and stress and defense adaptation. In addition, the \u0026ldquo;MAPK signaling pathway\u0026rdquo; and \u0026ldquo;plant\u0026ndash;pathogen interaction\u0026rdquo; were also enriched by KEGG analysis, indicating that \u003cem\u003eRht5\u003c/em\u003e-associated transcriptional changes may impact secondary metabolic and stress-related processes that contribute to coordinating wheat growth and stress resistance (Dixon and Paiva 1995a; Vogt 2010a; Meng and Zhang 2013) (Fig. 4d; Table S5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHeatmap analysis showed that DEGs involved in the biosynthesis or signaling of growth-related hormones (auxin, cytokinin and gibberellins) were differentially expressed between tall and dwarf lines (Fig. 4e; Table S6). In addition, genes associated with stress-related hormone pathways (SA, JA and ABA) exhibited distinct expression patterns between tall and dwarf lines (Fig. S3; Table S7). Among these, \u003cem\u003eWRKY62\u003c/em\u003e and \u003cem\u003eWRKY76\u003c/em\u003e were expressed at significantly higher levels in the dwarf lines than in the tall lines. \u003cem\u003eWRKY62\u003c/em\u003e and \u003cem\u003eWRKY76\u003c/em\u003e are important transcription factors and have been linked to SA, JA and ABA signaling and associated immune responses and abiotic stress tolerance (Table S7) (Mao et al. 2007; Yokotani et al. 2013; Fukushima et al. 2016). Together, these expression patterns suggest that the \u003cem\u003eRht5\u003c/em\u003e allele may also be involved in the regulation of wheat stress or defense responses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong DEGs related to plant growth and development, \u003cem\u003eTraesCS3B02G022900\u003c/em\u003e, encoding a homolog of\u0026nbsp;OsGAD1,\u0026nbsp;was remarkably upregulated in dwarf RILs (Fig. 4f; Table S8), and was therefore designated as \u003cem\u003eTaGAD2\u003c/em\u003e. Given that\u003cem\u003e\u0026nbsp;\u003c/em\u003eGAD is a key enzyme in GABA biosynthesis that is involved in controlling rice plant architecture and plays an important role in cell elongation and stress responses\u0026nbsp;(Akama et al. 2001; Ramesh et al. 2015), \u003cem\u003eTaGAD2\u0026nbsp;\u003c/em\u003ewas selected as a potential candidate gene downstream of \u003cem\u003eRht5\u003c/em\u003e for subsequent functional validation.\u003c/p\u003e\n\u003ch3\u003e\u003cem\u003eTaGAD2\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eencodes a functional\u0026nbsp;glutamate decarboxylase (GAD)\u003c/h3\u003e\n\u003cp\u003eTo characterize the molecular characterization of \u003cem\u003eTaGAD2\u003c/em\u003e, we first examined its expression in the tall and dwarf RILs. qRT-PCR analysis showed that \u003cem\u003eTaGAD2\u003c/em\u003e expression was significantly higher in dwarf \u003cem\u003eRht5\u003c/em\u003e RILs and Marfed M compared with tall \u003cem\u003eRht5\u003c/em\u003e RILs and JM47 (Fig. 5a). Expression profiling analysis with data from WheatOmics showed that \u003cem\u003eTaGAD2\u0026nbsp;\u003c/em\u003ewas ubiquitously expressed in various tissues and was highly expressed in young spikes at Zadoks stage 39 (Z39) (Fig. 5b). At the seedling stage, \u003cem\u003eTaGAD2\u0026nbsp;\u003c/em\u003eshowed the highest expression in shoots,\u003cem\u003e\u0026nbsp;\u003c/em\u003efollowed by leaves and roots in Chinese Spring (CS) (Fig. 5c). Multiple sequence alignment showed that TaGAD2 shares high sequence similarity in the glutamate decarboxylase domain with its homologs from \u003cem\u003eTriticum aestivum\u003c/em\u003e L., \u003cem\u003eGlycine max\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eZea mays\u003c/em\u003e, \u003cem\u003eArabidopsis thaliana\u0026nbsp;\u003c/em\u003e(Fig. S4). Phylogenetic analysis showed that there were multiple GAD homologs in wheat, and TaGAD2 was closely related to OsGAD1 (Fig. 5d; Table S9).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBecause plant GAD proteins harbor a canonical C-terminal auto-inhibitory calmodulin-binding domain (CaMBD) (Baum et al. 1993), we tested the glutamate decarboxylase activity of TaGAD2 using purified GST-TaGAD2 (full-length) and GST-TaGAD2\u0026Delta;C (C-terminal-truncated TaGAD2) in vitro (Fig. S5a\u0026ndash;b). Enzyme activity assays revealed that TaGAD2\u0026Delta;C exhibited markedly higher decarboxylase activity than the full-length TaGAD2 (Fig. 5e), supporting an autoinhibitory role of the C-terminal region of TaGAD2. Subcellular localization analysis showed that both full-length and C-terminal-truncated TaGAD2 proteins were predominantly localized at the plasma membrane and co-localized with the plasma membrane marker mCherry-OsPAD (Kurusu et al. 2012) (Fig. 5f), indicating that deletion of the C-terminal auto-inhibitory domain (AID) had little impact on TaGAD2 subcellular localization. Together, these data suggested that TaGAD2 acts as a functional glutamate decarboxylase.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003e\u003cem\u003eTaGAD2\u0026nbsp;\u003c/em\u003enegatively regulates wheat plant height\u003c/h3\u003e\n\u003cp\u003eTo verify the biological function of \u003cem\u003eTaGAD2\u003c/em\u003e, \u003cem\u003eTaGAD2\u0026nbsp;\u003c/em\u003eoverexpression lines (\u003cstrong\u003e\u003cem\u003eTaGAD2-OE\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e were generated in the wheat cultivar\u0026nbsp;\u003cem\u003eFielder background\u003c/em\u003e. Phenotypic analysis showed that\u0026nbsp;\u003cstrong\u003e\u003cem\u003eTaGAD2-OE\u003c/em\u003e\u003c/strong\u003e plants displayed severe developmental defects, including growth retardation and defective grain filling (Fig. 6a\u0026ndash;g). A previous study demonstrated that overexpression of\u0026nbsp;\u003cstrong\u003e\u003cem\u003eTaGAD1\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eresulted in stunted spikes and increased grain abortion, accompanied by abnormal spike development\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(Li et al. 2024)\u003c/strong\u003e\u003cstrong\u003e.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eWe obtained four fertile independent overexpression transgenic lines with limited grain numbers (Fig. 6a). qRT-PCR analysis showed that \u003cem\u003eTaGAD2\u003c/em\u003e expression was markedly higher in\u003cem\u003e\u0026nbsp;TaGAD2-OE\u003c/em\u003e lines than in the wild-type Fielder (Fig. 6b). Compared with Fielder, \u003cem\u003eTaGAD2-OE\u003c/em\u003e plants exhibited significant reduction in plant height (Fig. 6c), whereas the spike number per plant was comparable to that of Fielder (Fig. 6d). Due to severe defects in spike development, the spike length, spikelet number and grain number per spike were significantly reduced in \u003cem\u003eTaGAD2-OE\u003c/em\u003e lines compared with those of Fielder (Fig. 6e\u0026ndash;g). These results indicate that \u003cem\u003eTaGAD2\u0026nbsp;\u003c/em\u003enegatively regulates wheat plant height and yield-related traits. In addition, the flag leaves of \u003cem\u003eTaGAD2-OE\u0026nbsp;\u003c/em\u003elines were shorter than those of Fielder, with significant reduction in SPAD values (Fig. 6h, i, k). Notably, \u003cem\u003eTaGAD2-OE\u003c/em\u003e plants exhibited altered photosynthetic characteristics: the instantaneous water-use efficiency (WUEi) and net photosynthetic rate (A) were significantly higher than the wild-type plants (Fig. 6j, l), while significantly decreased were detected in transpiration rate (E), stomatal conductance (g\u003csub\u003es\u003c/sub\u003e), and intercellular CO\u003csub\u003e2\u003c/sub\u003e concentration (Ci) in the \u003cem\u003eTaGAD2-OE\u003c/em\u003e lines compare to Fielder (Fig. 6m\u0026ndash;o) implicating prolonged influences of\u0026nbsp;\u003cem\u003eTaGAD2\u0026nbsp;\u003c/em\u003eon photosynthetic capacity and instantaneous water-use efficiency.\u003c/p\u003e\n\u003cp\u003eCollectively, these results identified \u003cem\u003eTaGAD2\u0026nbsp;\u003c/em\u003eas a negative regulator of wheat growth and development.\u003c/p\u003e\n\u003ch3\u003eSilencing\u003cem\u003e\u0026nbsp;TaGAD2\u0026nbsp;\u003c/em\u003eincreases plant height and lodging resistance\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eTo test whether knockdown of \u003cem\u003eTaGAD2\u0026nbsp;\u003c/em\u003ecould increase wheat plant height, we generated\u0026nbsp;\u003cem\u003eTaGAD2-RNAi\u003c/em\u003e line in\u0026nbsp;the wheat cultivar\u0026nbsp;\u003cem\u003eFielder\u003c/em\u003e background (Fig. 7a). Quantitative analysis showed that \u003cem\u003eTaGAD2\u003c/em\u003e expression was markedly lower in\u003cem\u003e\u0026nbsp;TaGAD2-\u003c/em\u003e\u003cem\u003eRNAi\u003c/em\u003e line than in the wild-type Fielder (Fig. 7b). Enzymatic activity assays showed that the GAD activity was lower in multiple tissues and organs in the \u003cem\u003eTaGAD2-\u003c/em\u003e\u003cem\u003eRNAi\u003c/em\u003e plants compared to that in Fielder (Fig. 7c; Fig. S6a\u0026ndash;b). Consistently, GABA content was markedly reduced in \u003cem\u003eTaGAD2-RNAi\u003c/em\u003e plants compared with Fielder (Fig. 7d). Agronomic trait analyses showed that \u003cem\u003eTaGAD2-RNAi\u003c/em\u003e plants exhibited significant increase in plant height compared with Fielder (Fig. 7e\u0026ndash;f). By contrast, no significant differences were detected in spike length, spikelet number per spike and grain number per spike (Fig. 7g\u0026ndash;i). Other traits, including flag leaf length, spike number per plant, thousand-grain weight and grain yield per plant were also comparable between the \u003cem\u003eTaGAD2-RNAi\u003c/em\u003e plants and Fielder (Fig. S6c\u0026ndash;f).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLodging resistance evaluations revealed that \u003cem\u003eTaGAD2-RNAi\u003c/em\u003e plants exhibited enhanced lodging resistance (Fig. 7j). This improvement was associated with notable reduction in fresh weight of the main culm, the second basal internode (BI2; basal internode 2) and the spike (Fig. 7l, m; Fig. S6i), along with increased mechanical strength of the BI2 (Fig. 7k) and no significant difference in the center of gravity height (Fig. 7n). Other BI2-associated morphological traits, including BI2 length, BI2 diameter and BI2 dry weight, did not differ significantly between Fielder and \u003cem\u003eTaGAD2-RNAi\u003c/em\u003e plants (Fig. S6g, h, j). Physiological analysis revealed compromised photosynthetic performance in \u003cem\u003eTaGAD2-RNAi\u003c/em\u003e plants compared with Fielder. \u003cem\u003eTaGAD2-RNAi\u003c/em\u003e plants showed significantly reduced instantaneous water-use efficiency (WUEi), net photosynthetic rate (A), intercellular CO\u003csub\u003e2\u003c/sub\u003e concentration (Ci), stomatal conductance (g\u003csub\u003es\u003c/sub\u003e) and transpiration rate (E) (Fig. 7o\u0026ndash;s), suggesting that \u003cem\u003eTaGAD2\u0026nbsp;\u003c/em\u003eis required for optimal photosynthetic performance in wheat. Collectively, these results identified \u003cem\u003eTaGAD2\u0026nbsp;\u003c/em\u003eas an important regulator of wheat growth, lodging resistance and photosynthetic traits.\u003c/p\u003e\n\u003ch3\u003eHaplotype analysis of\u0026nbsp;\u003cem\u003eTaGAD2\u0026nbsp;\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003eTo assess the potential of \u003cem\u003eTaGAD2\u003c/em\u003e in wheat breeding, we performed haplotype analysis in a panel comprising 273 wheat accessions. Based on the sequence variations in the coding region of \u003cem\u003eTaGAD2\u003c/em\u003e, these accessions were grouped into three major haplotypes (accession number \u0026ge; 10 for each one) among which haplotype H3 of\u0026nbsp;\u003cem\u003eTaGAD2\u0026nbsp;\u003c/em\u003ewas identical to the Chinese Spring (CS) reference sequence. Among the 13 single nucleotide polymorphisms (SNPs) identified in the coding region of \u003cem\u003eTaGAD2\u003c/em\u003e, there were four nonsynonymous SNPs that resulted in three amino acid substitutions: Met126 (H3) to Ile126 (H1 and H2), Arg172 (H3) to Lys172 (H1 and H2) and Arg445 (H3 and H1) to Ala (H2) (Fig. 8a). Among the identified haplotypes, H1 comprised 88 wheat accessions (32.23%), H2 included 72 accessions (26.37%), and H3 contained 10 accessions (3.66%) (Fig. 8a\u0026ndash;b). Accessions with the H1 haplotype exhibited significantly lower plant height than that of H2, while no significant difference was observed in spike length between the H1 and H3 haplotypes (Fig. 8c). Compared with H3, H1 and H2 accessions had longer spike lengths (Fig. 8d) but comparable spikelet numbers and grain numbers (Fig. 8e\u0026ndash;f). Collectively, these results suggested that \u003cem\u003eTaGAD2\u003c/em\u003e was associated with variations of wheat plant height in natural population. As \u003cem\u003eTaGAD2\u003csup\u003eH1\u003c/sup\u003e\u003c/em\u003e has the strongest effects in reducing plant height, it represents a potential favorable allele of \u003cem\u003eTaGAD2\u003c/em\u003e for wheat dwarfing breeding.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe wide application of Green Revolution \u003cem\u003eRht-B1b\u003c/em\u003e and \u003cem\u003eRht-D1b\u003c/em\u003e has increased lodging resistance, while compromising seedling vigor and coleoptile length, thereby reducing their drought tolerance. \u003cem\u003eRht5\u003c/em\u003e is a potential alternative reduced-height loci suitable for breeding dwarfing wheat varieties because it does not influence seedling vigor and coleoptile length. In this study, we investigated the genetic effects of \u003cem\u003eRht5\u003c/em\u003e on wheat growth and development using \u003cem\u003eRht5\u003c/em\u003e inbred lines and explored the physiological mechanisms by which \u003cem\u003eRht5\u003c/em\u003e regulates plant height. In addition, we identified \u003cem\u003eTaGAD2\u003c/em\u003e as a potential downstream regulator of \u003cem\u003eRht5\u003c/em\u003e-mediated plant height regulatory pathway, broadening the understanding of the molecular basis underlying wheat plant height.\u003c/p\u003e\n\u003ch3\u003e\u003cem\u003eRht5\u0026nbsp;\u003c/em\u003eplays pleotropic roles in wheat growth and stress tolerance\u003c/h3\u003e\n\u003cp\u003ePrevious studies have characterized \u003cem\u003eRht5\u003c/em\u003e as a GAR dwarf gene that could reduce plant height and multiple agronomic important traits (Ellis et al. 2004; Cui et al. 2022). In our study, we developed \u003cem\u003eRht5\u003c/em\u003e RILs and systematically evaluated its genetic effects on wheat growth and development. We confirmed the pleotropic negative effects of \u003cem\u003eRht5\u003c/em\u003e on wheat plant architecture and yield-related traits including plant height, grain traits but the differences in \u003cem\u003eRht5\u003c/em\u003e RILs were lower or even diminished compared with their parents (Fig. 1), indicating that introduction of \u003cem\u003eRht5\u003c/em\u003e in different genetic backgrounds could partially improve the yield traits of Marfed M.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe pleotropic effects of \u003cem\u003eRht5\u003c/em\u003e in shaping wheat plant architecture were also observed in the diverse function of \u003cem\u003eRht5\u003c/em\u003e on cell growth and endogenous hormonal homeostasis. We found that \u003cem\u003eRht5\u003c/em\u003e inhibits cell proliferation to decrease cell number but promotes cell elongation (Fig. 2). Hormonal profiling of \u003cem\u003eRht5\u003c/em\u003e RILs revealed that the stress-related hormones JA, ABA and SA were significantly higher in the dwarf lines (Fig. 3d). RNA-seq analysis revealed that some key regulators including\u0026nbsp;\u003cem\u003eWRKY62\u0026nbsp;\u003c/em\u003eand\u0026nbsp;\u003cem\u003eWRKY76\u003c/em\u003e which are closely associated with the signaling pathways of SA, JA, and ABA\u0026nbsp;(Mao et al. 2007; Yokotani et al. 2013; Fukushima et al. 2016),\u003cem\u003e\u0026nbsp;\u003c/em\u003ehad\u003cem\u003e\u0026nbsp;\u003c/em\u003esignificantly higher expression in the dwarf lines (Fig. S3;\u0026nbsp;Table S7), suggesting that \u003cem\u003eRht5\u003c/em\u003e may also involve in plant stress responses. This notion was also supported by the significant enrichment of GO terms \u0026ldquo;response to biotic stimulus\u0026rdquo; and \u0026ldquo;response to endogenous stimulus\u0026rdquo;, \u0026ldquo;response to oxygen-containing compound\u0026rdquo;, \u0026ldquo;protein kinase binding\u0026rdquo; as well as \u0026ldquo;indole-3-butyrate beta-glucosyltransferase activity\u0026rdquo; and \u0026ldquo;salicylic acid metabolic process\u0026rdquo; (Fig. 4c;\u0026nbsp;Table S4), which are associated with promoted plant growth construction and increased resistance to diseases and mechanical damage\u0026nbsp;(Vanneste and Friml 2009; Yang and Guo 2018; Zhou and Zhang 2020; Waadt et al. 2022). The KEGG enrichment revealed the \u0026ldquo;MAPK signaling pathway\u0026ndash;plant\u0026rdquo; involved in signal transduction in response to environmental stresses and plant hormones, and several pathways including \u0026ldquo;phenylpropanoid biosynthesis\u0026rdquo;, \u0026ldquo;cinnamic acid biosynthetic process\u0026rdquo;, \u0026ldquo;linoleic acid metabolism\u0026rdquo; and \u0026ldquo;diterpenoid biosynthesis\u0026rdquo;, stress-responsive metabolism that could contribute to improved resilience\u0026nbsp;(Dixon and Paiva 1995b; Howe and Schilmiller 2002; Vogt 2010a; Vaughan et al. 2015)\u0026nbsp;(Fig. 4d;\u0026nbsp;Table S5). Collectively, our study suggested that \u003cem\u003eRht5\u003c/em\u003e acts as a pleotropic regulator in wheat growth and development.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003e\u003cem\u003eRht5\u003c/em\u003e causes dwarfism through inhibiting cell proliferation\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003ePlant height is largely determined by the cell proliferation in the intercalary meristem (IM) and cell elongation in the elongation zone of crop stem (Sauter and Kende 1992). In wheat, \u003cem\u003eRht-B1b\u003c/em\u003e and \u003cem\u003eRht8\u003c/em\u003e confer a semi-dwarf phenotype by reducing cell elongation, thereby shortening stem internodes (Gasperini et al. 2012; Xu et al. 2023), while \u003cem\u003eRht22\u003c/em\u003e impairs cell proliferation and reduces internode cell number (Peng et al. 2011; Wang et al. 2022). Other reduced-height locus could decrease both cell number and cell length to inhibit wheat stem elongation (Xu et al. 2017). By paraffin section analysis, we showed that \u003cem\u003eRht5\u003c/em\u003e increases cell length but remarkably reduces cell number during stem elongation (Fig. 2). Quantification of endogenous phytohormones revealed lower content of bioactive cytokinin and auxin but elevated accumulation of GA\u003csub\u003e3\u003c/sub\u003e and GA\u003csub\u003e4\u003c/sub\u003e (Fig. 3b\u0026ndash;c). Given that GA is well known to promote cell elongation, whereas cytokinin mainly stimulates cell division (Takatsuka and Umeda 2014), the dual effects of \u003cem\u003eRht5\u003c/em\u003e on cell growth during stem elongation are consistent with hormonal profiling disequilibrium. These cellular changes on cell growth conferred by \u003cem\u003eRht5\u003c/em\u003e differ from that of the well-known \u0026ldquo;Green Revolution\u0026rdquo; alleles \u003cem\u003eRht-B1b\u003c/em\u003e and \u003cem\u003eRht-D1b\u003c/em\u003e, which reduce plant height mainly by inhibition of GA signaling and GA-promoted cell elongation (KEYES et al. 1989; Pearce et al. 2011).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo date, multiple dwarfing loci\u0026ndash;including several GA-responsive loci\u0026ndash;have been reported in wheat. Among them, \u003cem\u003eRht8\u003c/em\u003e and \u003cem\u003eRht12\u003c/em\u003e primarily inhibit cell elongation (Gasperini et al. 2012; Sun et al. 2019), whereas \u003cem\u003eRht22\u003c/em\u003e mainly reduces plant height by inhibiting cell division (Peng et al. 2011; Wang et al. 2022). Furthermore, the autoactive NB-LRR allele \u003cem\u003eRht13\u003c/em\u003e elicited another distinct mechanism of dwarfism (Borrill et al. 2022). \u003cem\u003eRht25\u003c/em\u003e affects plant height by regulating the expression of DELLA-associated growth genes (Zhang et al. 2023). Given that \u003cem\u003eRht5\u003c/em\u003e suppressed cell proliferation while promoting cell elongation with disordered accumulation of multiple growth-promoting hormones, together with the distinct effectiveness of exogenous 6-BA and GA\u003csub\u003e3\u003c/sub\u003e application on plant height of the dwarf \u003cem\u003eRht5\u003c/em\u003e RILs, we proposed that \u003cem\u003eRht5\u003c/em\u003e may regulate stem elongation and wheat plant height through mechanisms distinct from previously characterized the dwarfing loci.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eGenetic manipulation of \u003cem\u003eTaGAD2\u003c/em\u003e mimics \u003cem\u003eRht5\u003c/em\u003e dwarfing phenotype\u003c/h3\u003e\n\u003cp\u003eAlthough \u003cem\u003eRht5\u003c/em\u003e-mediated wheat dwarfism is associated with abnormal accumulation of multiple phytohormones, RNA-seq analysis showed moderate transcriptional changes of genes related to the growth-promoting phytohormone pathways between the tall and dwarf lines. Notably, we found that \u003cem\u003eTaGAD2\u003c/em\u003e was significantly upregulated in the dwarf lines compared with tall \u003cem\u003eRht5\u003c/em\u003e RILs (Fig. 4f; Fig. 5a). We characterized TaGAD2 as a\u0026nbsp;functional\u0026nbsp;glutamate decarboxylase (GAD) that localized to plasma membrane (Fig. 5e, f). It has been demonstrated that GAD catalyzes the decarboxylation of L-glutamate (Glu) to GABA with release of CO\u003csub\u003e2.\u0026nbsp;\u003c/sub\u003eGAD functions\u0026nbsp;as an entry-point enzyme of the GABA shunt, after which GABA is sequentially metabolized by GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) to feed carbon into the TCA cycle (Ansari et al. 2021). In wheat, \u003cem\u003eTaNHX2\u003c/em\u003e has been reported to target the C-terminal autoinhibitory domain of \u003cem\u003eTaGAD1\u003c/em\u003e, enhancing GAD activity and promoting endogenous GABA accumulation to regulate drought tolerance (Li et al. 2024), implying that altered GABA content may influence wheat growth, development, and stress responses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInterestingly, genetic manipulation of \u003cem\u003eGADs\u003c/em\u003e leads to various phenotypes across plant species. In rice, overexpressing full-length\u0026nbsp;\u003cem\u003eOsGAD2\u003c/em\u003e displayed an essentially normal phenotype, while overexpression of \u003cem\u003eOsGAD2\u0026Delta;C\u003c/em\u003e led to significantly elevated GABA levels and exhibited a pronounced dwarf phenotype (Akama and Takaiwa 2007). In tomato, \u003cem\u003eSlGAD3OX\u003c/em\u003e lines were similar to the wild type in appearance, while \u003cem\u003eSlGAD3\u0026Delta;C\u003c/em\u003e overexpression lines displayed an orange-ripe phenotype with reduced carotenoids (Takayama et al. 2017). GABA accumulation can severely compromise the Agrobacterium-mediated transformation process (Brencic and Winans 2005; Chevrot et al. 2006; Planamente et al. 2012; Lang et al. 2016). In wheat, a \u003cem\u003eTaGAD1\u003c/em\u003e overexpression transgenic line (\u003cem\u003eGOE-1\u003c/em\u003e) was reported to cause severe growth retardation, including stunted spikes with grain abortion and development abnormalities (Li et al. 2024). In our study, overexpression of full-length \u003cem\u003eTaGAD2\u003c/em\u003e led to wheat dwarfism with marked developmental defects including reduction of spike length, spikelet number and grain number (Fig. 6a\u0026ndash;g) and therefore phenocopied the dwarfism conferred by \u003cem\u003eRht5\u003c/em\u003e (Fig. 6a\u0026ndash;c). Conversely, \u003cem\u003eTaGAD2\u003c/em\u003e knockdown could increase plant height (Fig. 7a\u0026ndash;f), suggesting that \u003cem\u003eTaGAD2\u003c/em\u003e acts as a negative regulator of stem elongation. Moreover, we identified \u003cem\u003eTaGAD2\u003csup\u003eH1\u003c/sup\u003e\u003c/em\u003e as an elite allele that could reduce plant height without compromising grain number in a natural wheat population (Fig. 8). As genetic manipulation of \u003cem\u003eTaGAD2\u003c/em\u003e could mimic the dwarfing phenotypes of \u003cem\u003eRht5\u003c/em\u003e dwarf lines, our study identified \u003cem\u003eTaGAD2\u003c/em\u003e as a potential downstream regulator of the \u003cem\u003eRht5\u003c/em\u003e-mediated plant height regulatory pathway, providing new insights into the molecular basis of wheat plant height and offering genetic resources for improving wheat lodging resistance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eAuthorship contribution statement\u003c/h3\u003e\n\u003cp\u003eXianglan Kong designed the research, performed the experiments, analysis the data and wrote the manuscript; Chunge Cui, Yuxin Lei and LiZhe performed the experiments, analyzed the data; Xuefen Cai, Aozhe Wang, Qiumei Lu, Zhangchen Zhao, and Altyn Shayahkhmetova performed some of the experiments and data scoring; Liang Chen and Linzhou Huang designed research, analysis data and wrote the manuscript. Yin-Gang Hu, conceived the study, designed research, analysis the data and wrote the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare.\u003c/p\u003e\n\u003ch3\u003eData availability\u003c/h3\u003e\n\u003cp\u003eData presented in this study are available in this research article and supplementary materials. The raw data of the RNA-seq was submitted to the NCBI SRA database with accession number PRJNA1424380.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eAcknowledgements\u003c/h3\u003e\n\u003cp\u003eThis work was supported by the grants from National Natural Science Foundation of China (32171991), the Natural Science Basic Research Program of Shaanxi Province (2023-JC-ZD-08).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAkama K, Akihiro T, Kitagawa M, Takaiwa F (2001) Rice (\u003cem\u003eOryza sativa\u003c/em\u003e) contains a novel isoform of glutamate decarboxylase that lacks an authentic calmodulin-binding domain at the C-terminus. 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Cell 181(5):978\u0026ndash;989. https://doi.org/10.1016/j.cell.2020.04.028\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"theoretical-and-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"taag","sideBox":"Learn more about [Theoretical and Applied Genetics](https://www.springer.com/journal/122)","snPcode":"122","submissionUrl":"https://submission.nature.com/new-submission/122/3","title":"Theoretical and Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Plant height, dwarfing gene, Rht5, TaGAD2, GABA ","lastPublishedDoi":"10.21203/rs.3.rs-8985940/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8985940/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlant height is a key determinant of wheat plant architecture that determines lodging resistance thus grain yield. The GA-responsive (GAR) dwarfing gene\u003cem\u003e Rht5\u003c/em\u003e decreases plant height without comprise of wheat seedling vigor and is considered as a promising candidate gene for breeding wheat varieties in water-limited conditions. However, the mechanisms underlying \u003cem\u003eRht5\u003c/em\u003e-meidated dwarfism is unclear. In this study, we investigated the genetic effects of \u003cem\u003eRht5\u003c/em\u003e on wheat growth and development using recombinant inbred lines (RILs) and found that \u003cem\u003eRht5\u003c/em\u003e reduces plant height through inhibition of cell proliferation while it promotes cell elongation. The dual functions of \u003cem\u003eRht5\u003c/em\u003e on cell growth during wheat stem elongation was associated with the alteration of the homeostasis of endogenous growth-promoting phytohormones cytokine and gibberellins. Transcriptome analysis of identified \u003cem\u003eTaGAD2\u003c/em\u003e, encoding a functional glutamate decarboxylase localized at plasma membrane that catalyzes γ-Aminobutyric acid (GABA) biosynthesis, as a potential downstream regulator of \u003cem\u003eRht5\u003c/em\u003e-mediated dwarfism. Functional assays demonstrated that overexpression of \u003cem\u003eTaGAD2\u003c/em\u003e could mimic the phenotypes of dwarf \u003cem\u003eRht5 \u003c/em\u003eRILs while \u003cem\u003eTaGAD2\u003c/em\u003e knockdown\u003cem\u003e \u003c/em\u003eincreased plant height and improved lodging resistance, indicating a negative role of \u003cem\u003eTaGAD2 \u003c/em\u003ein controlling wheat plant height. We also conducted haplotype analysis of \u003cem\u003eTaGAD2\u003c/em\u003e in a natural wheat population and identified \u003cem\u003eTaGAD2\u003c/em\u003e\u003csup\u003e\u003cem\u003eH1\u003c/em\u003e\u003c/sup\u003e as a potential favorable allele for wheat dwarfing breeding without compromising grain number. Our study provides new insights into the molecular mechanism of \u003cem\u003eRht5\u003c/em\u003e-mediated plant height regulatory pathway and valuable gene resource to the genetic improvement of wheat plant architecture.\u003c/p\u003e","manuscriptTitle":"TaGAD2 is a potential downstream effector of Rht5 in controlling wheat plant height","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-24 15:00:46","doi":"10.21203/rs.3.rs-8985940/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-21T15:48:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-19T10:18:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-13T07:32:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"255740829489629065886135167010548112462","date":"2026-03-19T15:28:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"262846724702824036615631960449160351990","date":"2026-03-19T10:12:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-18T23:00:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-28T13:48:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-27T14:53:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Theoretical and Applied Genetics","date":"2026-02-27T09:05:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"theoretical-and-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"taag","sideBox":"Learn more about [Theoretical and Applied Genetics](https://www.springer.com/journal/122)","snPcode":"122","submissionUrl":"https://submission.nature.com/new-submission/122/3","title":"Theoretical and Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"00fcf7b2-9cdc-4e64-89ea-aa24c39e589a","owner":[],"postedDate":"March 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-21T15:54:42+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-24 15:00:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8985940","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8985940","identity":"rs-8985940","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}