Abstract

   Wheat is an important global food crop. The peduncle significantly
   impacts the plant’s height, architecture, and yield, and understanding
   its genetic mechanisms is crucial not only for improving wheat’s
   architecture but also for enhancing its yield. In this study, we
   identified an elongated uppermost internode (eui) mutant in the
   EMS-induced progeny of Jinmai 90 (JM90). We conducted phenotypic
   identification, genetic analysis, and cytological observation combined
   with transcriptome and targeted hormone metabolism analysis and
   compared the differences between the eui mutant and the wild-type (WT).
   The results indicated that an incompletely dominant gene mutation
   caused the eui mutant to display significant elongation of the peduncle
   and an increase in the plant height. This was attributed to the
   considerable elongation of parenchyma cells, while no significant
   differences were noted in other internodes. These traits were
   accompanied by an increase in the spikelets per spike and grains per
   spike. Subsequently, transcriptome and targeted hormone metabolome
   sequencing were performed and identified 15,969 differentially
   expressed genes (DEGs) and 27 hormone-related differentially
   accumulated metabolites (DAMs). KEGG enrichment analysis indicated that
   the DEGs in MS1_VS_WS1 were significantly enriched in two pathways:
   those related to tryptophan metabolism and diterpenoid biosynthesis.
   Analysis indicated that the peduncle elongation caused by the eui
   mutant is primarily regulated by auxin. This study offers a foundation
   for the exploration and cloning of genes associated with the peduncle,
   establishing a theoretical basis for understanding the molecular
   mechanisms behind wheat peduncle elongation and for developing ideal
   plant types and breeding high-yield varieties.

   Keywords: wheat, peduncle, eui mutant, transcriptomics,
   hormone-targeted metabolomics

1. Introduction

   Wheat (Triticum aestivum L.) is a vital global food crop, supplying 55%
   of human energy consumption [[40]1,[41]2,[42]3]. The peduncle is the
   distance from the base of the wheat ear to the first internode, which
   primarily serves as a conduit for transporting photosynthetic products
   from the leaves to the ears and is a significant trait as it influences
   both the height and type of the wheat plant. Additionally, it is an
   important source of energy during the filling stage, which can
   significantly impact the wheat yield [[43]4]. Furthermore, longer
   peduncles may enhance air circulation, reduce the spread of pathogens,
   and improve the plant’s resistance to diseases [[44]5]. However,
   varieties with longer peduncles often exhibit an increased plant height
   [[45]6], which can easily lead to lodging. Therefore, breeding
   varieties with longer peduncles but a relatively low plant height will
   improve the internode configuration and benefit crop production
   practices. The relationship between the peduncle length and plant
   height is complex; in some cases, the peduncle changes in tandem with
   the plant height [[46]7], while in others, it appears to be independent
   of it [[47]8]. The associated regulatory mechanisms remain unclear.
   Therefore, elucidating the genetic regulatory networks that control
   peduncle development is crucial for enhancing the wheat yield and
   optimizing the plant architecture.

   The use of transcriptomics allows for the sequence and expression
   information of nearly all transcripts from a specific cell or tissue in
   a given state to be comprehensively and rapidly obtained. For instance,
   transcriptomes have been utilized to uncover genes associated with stem
   elongation [[48]9,[49]10,[50]11,[51]12]. Metabolomics involves the
   simultaneous qualitative and quantitative analysis of all the
   low-molecular-weight metabolites within an organism or cell during a
   specific physiological period. Integrating these analyses enables a
   concurrent understanding of gene regulatory mechanisms and changes in
   the metabolite abundance within cells, providing comprehensive insights
   into the systemic alterations within organisms. For instance,
   transcriptomics and metabolomics have been used to elucidate the stem
   development mechanisms of crops [[52]13,[53]14].

   Studies have shown that plant hormones are integral to regulating the
   elongation of crop stems [[54]15]. Auxin plays a crucial role in plant
   growth by influencing cell elongation, division, and differentiation,
   promoting cell elongation by facilitating cell wall loosening and
   stimulating the synthesis of proteins and nucleic acids
   [[55]16,[56]17]. For example, the overexpression of OsYUCCA2 promotes
   rice (Oryza sativa L.) growth [[57]18]. Auxin response factors (ARFs)
   also impact plant stem growth [[58]19,[59]20]. The VT2 mutation in corn
   substantially impacts both nutritional and reproductive growth
   processes [[60]21]. The brachytic2 (Br2) gene mutant can reduce the
   plant height and ear height in maize (Zea mays L.) and encodes an
   ATP-binding cassette auxin transporter [[61]22]. Furthermore, auxin can
   synergize with other hormones to enhance stem elongation [[62]23].
   Auxin induces an increase in the gibberellin content in deep rice,
   thereby promoting stem elongation [[63]24]. Gibberellin exerts a direct
   influence on plant stem elongation [[64]25]. The loss-of-function
   mutation of the “Green Revolution gene” OsGA20ox2 causes semi-dwarfism
   [[65]26]. Mutations in the GA13ox gene result in elongated peduncles in
   rice [[66]27]. Some studies have also suggested that brassinosteroids
   play an important role in plant stem elongation. Mutations in the BR
   receptor gene OsBRI1 typically result in the second internode being
   significantly shorter than the first, leading to dwarfism in rice
   [[67]28]. However, the regulation of wheat peduncle elongation by these
   hormones remains poorly understood.

   In this study, we subjected the eui mutant, derived from the EMS
   mutagenesis of JM90 and characterized by an elongated uppermost
   internode, to phenotypic identification and cytological observations,
   comparing it with the WT. Moreover, we analyzed the transcriptome and
   hormone-targeted metabolome of peduncles at two stages of elongation:
   S1 (prior to full spike emergence) and S2 (ten days post-S1, when the
   spikes fully emerged). Our objective was to investigate the mechanisms
   responsible for the morphological changes in these mutants, with a
   focus on metabolic pathways closely associated with peduncle
   elongation, in order to identify key genes and metabolites. This
   research aimed to establish a theoretical foundation for understanding
   the molecular mechanisms behind wheat peduncle elongation and ensuring
   the ideal plant type in wheat.

2. Results

2.1. The Manifestations of the Elongated Uppermost Internode Mutant (eui)

   The eui mutant was derived from the common wheat JM90 through EMS
   mutagenesis. At the booting stage, there were no significant
   differences in the plant height (PH) between eui and JM90 plants
   ([68]Figure 1A,F), while at the maturity stage, the PH of eui plants
   was significantly higher than that of JM90 plants ([69]Figure 1B,G).
   Further analysis revealed that both had five internodes, with the
   differences primarily present in the peduncle. The peduncle of eui
   plants was significantly longer than that of JM90 plants, while the
   lengths of the other internodes were not significantly different
   ([70]Figure 1C,H). A comparison of the yield traits showed that the
   number of spikelets per spike in eui plants was greater than in JM90
   plants ([71]Figure 1D), resulting in a significant increase in the
   number of grains per spike ([72]Figure 1I). The grain length was also
   significantly longer than that of JM90 plants ([73]Figure 1E,J), but
   there were no significant differences in other agronomic traits
   ([74]Supplementary Figure S1). Thus, eui is characterized by a
   significant plant height increase, primarily due to a longer peduncle,
   and a significant increase in the grains per spike.

Figure 1.

   [75]Figure 1
   [76]Open in a new tab

   Phenotypic characteristics of the wild-type (JM90) and the mutant
   (eui): (A) the plant height at the booting stage; (B) the plant height
   at the mature stage; (C) the internodes at the mature stage; (D) the
   spike; (E) the grain length; (F–J) histograms of the corresponding
   indices. The data represent the average from 10 plants, with an *
   indicating a significant difference between the wild-type JM90 and the
   mutant (p < 0.05, t-test).

2.2. Genetic Analysis of eui

   To elucidate the genetic basis underlying the elongated peduncle
   phenotype, the eui mutant was crossed with AK58, a dwarf variety, and
   the peduncle length approached that of JM90. The sequence was
   determined, which will facilitate the localization of the mutant gene
   in the future. All F[1] individuals exhibited a phenotype characterized
   by a tall height with a long peduncle, indicating the dominant nature
   of the elongated peduncle trait. Subsequently, the F[1] individuals
   were self-pollinated to generate an F[2] population. In the AK58×eui
   cross, 521 plants were similar to the wild-type, 476 plants were tall
   with long peduncles, and 1035 exhibited an intermediate phenotype,
   fitting a 1:2:1 ratio (χ2 = 2.704 < χ2 (0.05,1) = 3.84) ([77]Table 1).
   These results suggested that the elongated peduncle phenotype of eui
   plants was caused by an incompletely dominant mutation at a single
   locus.

Table 1.

   Inheritance of eui in wheat crosses with AK58.
   Cross Phenotype of F[2] Observed Count (O) Expected Count (E) (O-E)2/E
   χ2 Probable Ratio
   AK58/eui Mutation type 476 508 2.01575 2.704 1
   Intermediate type 1035 1016 0.35531 2
   Wild-type 521 508 0.33268 1
   Sum 2032 2032 2.70374
   [78]Open in a new tab

2.3. Observation of Cell Structure

   To understand the cytological basis of the significant eui-caused
   peduncle elongation, the middle parts of the peduncles from the eui
   mutant and WT plants were cut into 1 cm segments at the S1 and S2
   stages to prepare paraffin sections. The sizes of the parenchyma cells
   were then measured. At the S1 stage, there was no significant
   difference in the peduncle elongation ([79]Figure 2A,D). The cell
   microstructure revealed that the cells in eui plants were longer than
   those in the WT, but the difference did not reach statistical
   significance ([80]Figure 2B,C,E). However, at the S2 stage, the
   peduncle of eui plants was significantly longer than that of WT plants
   ([81]Figure 2F,I) and the cell length in eui plants was 19.08% greater
   than in WT plants ([82]Figure 2G,H,J). This indicated that the growth
   of the eui peduncle was due to the elongated internode cell length,
   with cells elongating rapidly during the heading stage. When there was
   a significant difference in the plant height, the cell length also
   showed a significant difference.

Figure 2.

   [83]Figure 2
   [84]Open in a new tab

   The elongation state of the peduncle and cell microstructure in the WT
   and eui plants at two stages. (A) Spike extension state of the WT and
   eui plants at the S1 stage. (B,C) Longitudinal section microstructures
   of the peduncles of WT and eui plants at the S1 stage, respectively.
   (D) Histogram of the peduncle length at the S1 stage. (E) Histogram of
   the cell length at the S1 stage. (F–J) S2 stage. The data are the
   average from 10 cells; * indicates a significant difference between the
   mutant and the WT (p < 0.05, t-test).

2.4. Dynamic Transcriptional Variations in eui and WT Wheat Peduncles

   A total of 12 samples from two materials and two periods yielded 188.33
   Gb of Clean Data, with each sample producing over 14.36 Gb. Of this,
   93.67–94.56% aligned with the Chinese Spring reference genome
   (IWGSC_RefSeq_v2.1) ([85]Supplementary Table S1). The correlation
   between the biological replicates was strong, with correlation
   coefficients ranging from 0.993 to 1.00. Samples within each group and
   between groups showed significant separation, indicating good
   consistency within groups and notable differences between the two
   varieties ([86]Figure 3A). Six genes were randomly selected for RT-qPCR
   validation, and the majority of the expression trends matched the
   RNA-seq results ([87]Supplementary Figure S2). These findings suggest
   that the test sampling was appropriate and that the RNA-seq data
   quality was reliable.

Figure 3.

   [88]Figure 3
   [89]Open in a new tab

   An overview of the eui and WT peduncle transcriptomes. (A) Correlation
   coefficient heatmaps for 12 samples: On the right and lower sides are
   the sample names, on the left and upper sides are the sample clusters,
   and the squares of different colors represent the correlation between
   the two samples. The correlation coefficient is shown by a color
   gradient ranging from low (blue) to high (red). (B) Volcano plot of the
   differentially expressed genes (DEGs), in which red represents
   upregulation and blue represents downregulation. (C) Venn diagram of
   the DEGs: the circles of different colors represent different gene
   sets, and the values represent the number of common and unique genes
   between different gene sets. (D) Top twenty pathways enriched according
   to the KEGG for the DEGs: The vertical axis represents the pathway
   name, and the horizontal axis represents the ratio of the number of
   genes enriched in the pathway (sample number) to the number of
   annotated genes (background number). The larger the rich factor, the
   greater the degree of enrichment. The size of the point indicates the
   number of genes in that pathway, and its color corresponds to different
   p-adjust ranges.

   The comparisons MS1_vs_WS1, MS2_vs_WS2, MS2_vs_MS1, and WS2_vs_WS1
   revealed 16,007 (8444 upregulated and 7563 downregulated), 16,851 (9901
   upregulated and 6950 downregulated), 38,277 (15,879 upregulated and
   22,398 downregulated), and 35,636 (14,259 upregulated and 21,377
   downregulated) differentially expressed genes (DEGs) ([90]Figure 3B,C).
   Subsequently, the Kyoto Encyclopedia of Genes and Genomes (KEGG)
   pathway enrichment analysis of the eui and WT samples indicated that
   the DEGs were primarily enriched in several pathways, including those
   related to “Phenylpropanoid biosynthesis”, “Flavonoid biosynthesis”,
   “Biosynthesis of various plant secondary metabolites”, and
   “Phenylalanine metabolism”. In the MS1_vs_WS1 comparison, the DEGs were
   also notably enriched in two hormone signaling pathways, namely those
   related to “tryptophan metabolism” and “diterpenoid biosynthesis”,
   which corresponded to the auxin and gibberellin synthesis pathways,
   respectively ([91]Figure 3D, [92]Supplementary Table S2).

2.5. Dynamic Metabolomic Changes in eui and WT Peduncles

   Considering the significant role of plant hormones in plant stems, the
   DEGs of eui plants and the WT were notably enriched in hormone-related
   pathways. A targeted hormone metabolomics analysis of the peduncles
   from eui and WT plants at the S1 and S2 stages was conducted using
   liquid chromatography–mass spectrometry (LC-MS) techniques. We measured
   41 hormone metabolites, and a total of 27 differentially accumulated
   metabolites (DAMs) were observed, encompassing auxins, gibberellins,
   cytokinins, jasmonic acids, salicylic acid, and abscisic acid (refer to
   [93]Supplementary Table S3). Cluster analysis demonstrated that the
   three replicates within each group clustered, and significant
   differences were observed between the four groups ([94]Figure 4A). This
   suggests that the metabolomics data exhibited good repeatability and
   high reliability. The comparisons MS1_vs_WS1, MS2_vs_WS2, MS2_vs_MS1,
   and WS2_vs_WS1 revealed 27 (16 upregulated and 11 downregulated), 22
   (10 upregulated and 12 downregulated), 27 (7 upregulated and 20
   downregulated), and 26 (11 upregulated and 15 downregulated) DAMs
   ([95]Figure 4B,C). KEGG enrichment analysis indicated that these DAMs
   were predominantly enriched in 11 metabolic pathways, including those
   corresponding to tryptophan metabolism (map00380), zeatin biosynthesis
   (map00908), carotenoid biosynthesis (map00906), alpha-linolenic acid
   metabolism (map00592), the biosynthesis of terpenoids and steroids
   (map01062), the biosynthesis of plant hormones (map01070), plant
   hormone signal transduction (map04075), diterpenoid biosynthesis
   (map00904), the biosynthesis of plant secondary metabolites (map01060),
   the biosynthesis of secondary metabolites (map01110), and metabolic
   pathways (map01100) ([96]Figure 4D).

Figure 4.

   [97]Figure 4
   [98]Open in a new tab

   An overview of the eui and WT peduncle metabolomes. (A) Heatmap of the
   DAMs detected and related to hormones; on the right side are the
   metabolites’ names, on the lower side are the sample names, on the left
   and upper sides are the sample clusters, and the relative level of
   accumulation is shown by a color gradient ranging from low (blue) to
   high (red). (B) Column chart of the DAMs, in which red represents
   upregulation and blue represents downregulation. (C) Venn diagram of
   the DAMs; the circles of different colors represent different
   metabolite sets, and the values represent the number of common and
   unique metabolites between different sets. (D) Correlation network
   diagram of the DAMs and metabolic pathways; the dots represent
   metabolites, the squares represent metabolic pathways, and the lines
   represent the correlation between them. The darker the line color is,
   the greater the correlation is. Green represents a negative
   correlation, red represents a positive correlation, and the thicker the
   line is, the smaller the p-value is.

   Considering the pivotal roles of auxin and gibberellin in plant stem
   elongation and the significant enrichment of DEGs in biosynthesis
   pathways related to these two hormones, we conducted a detailed
   analysis of the DEGs and DAMs associated with auxin and gibberellin.

2.6. Differences in Detection of Auxin Between eui and WT

   The transcriptomic results indicated an abundance of DEGs in the
   tryptophan metabolism pathway, which is the primary pathway for auxin
   biosynthesis. Some DAMs related to auxins were also identified. Thus,
   we analyzed the DEGs and DAMs in this pathway ([99]Figure 5,
   [100]Supplementary Table S4). Significant changes were observed in the
   MS1_vs_WS1 comparison, particularly for genes that were upregulated in
   the mutant, such as the tryptophan decarboxylase (DDC/TDC),
   N-acetylserotonin O-methyltransferase (ASMT), CYP71P1, and E3.5.1.4
   genes. For instance, TraesCS7D03G1269200, TraesCS4B03G0980700,
   TraesCS6B03G0312400, and TraesCS2A03G1357400 were upregulated 8.20-,
   16.23-, 7.83-, and 1.89-fold, respectively, implying they have positive
   regulatory roles during the peduncle elongation process. By contrast,
   some genes were downregulated, such as the aldehyde dehydrogenase
   (ALDH) and flavin monooxygenase (YUCCA) genes. For example,
   TraesCS6A03G0768200LC and TraesCS3A03G0706200 were downregulated 3.09-
   and 5.68-fold, respectively. In addition, some auxin-related genes
   related to lignin synthesis, such as caffeic acid O-methyltransferase
   (OMT/COMT), TraesCS2B03G0142800, and TraesCS5D03G1074800, were
   significantly upregulated by 6.50- and 5.15-fold, respectively.
   Furthermore, the growth-promoting compounds IBA, 4-CPA, and IAAsp were
   significantly accumulated, whereas the growth-inhibiting substances
   PPP333 and MH were significantly reduced in MS1. In the MS2_vs_WS2
   comparison, most DDC/TDC, ASMT, and OMT/COMT genes were upregulated,
   and some exhibited greater than 6-fold upregulation. The metabolomics
   results showed that IAAla and PP333 significantly increased in the
   mutants, while IAA significantly decreased in MS2. This indicates that
   the elongation process of the mutant peduncles consumed more auxin.

Figure 5.

   [101]Figure 5
   [102]Open in a new tab

   Schematic representation of the tryptophan biosynthesis pathway, which
   is involved in auxin synthesis. Note: Squares and circles denote genes
   and metabolites, respectively. Red squares indicate upregulated genes,
   blue squares indicate downregulated genes, and squares with both colors
   represent genes that were upregulated and downregulated at the S1
   stage. DDC/TDC: tryptophan decarboxylase; CYP71P1: cytochrome P450
   monooxygenase, family 71, subfamily P1; ALDH: aldehyde dehydrogenase;
   ASMT: N-acetylserotonin O-methyltransferase; YUCCA: flavin
   monooxygenase; AAO1: aldehyde oxidase 1. The inset bar plots display
   the abundance of the corresponding metabolites. MS1 and WS1 represent
   the mutant and WT at the S1 stage, respectively, while MS2 and WS2
   represent the mutant and WT at the S2 stage. Statistically significant
   metabolites were identified using Student’s t-test (p < 0.05) and
   corrected for multiple comparisons. Different letters represent a
   significant difference between the mutant and the wild-type in the same
   stage. IBA: 3-indolebutyric acid; 4-CPA: 4-chlorophenoxyacetic acid;
   IAAsp: indole-3-acetyl-L-aspartic acid; IAA: indole-3-acetic acid;
   IAAla: N-(3-indolylacetyl)-L-alanine; PP333: paclobutrazol; MH: maleic
   hydrazide; IATrp: indole-3-acetyl-L-tryptophan; UCZ: uniconazole.

2.7. Differences in Detection of Gibberellin Between eui and WT

   The transcriptomic results indicated an abundance of DEGs in the
   diterpenoid biosynthesis pathway, which is upstream of the gibberellin
   biosynthesis pathway. Additionally, four gibberellin-derived DAMs were
   also identified in the metabolome. Consequently, we constructed a
   regulatory network that incorporated both the DEGs and DAMs within this
   pathway ([103]Figure 6, [104]Supplementary Table S5). Significant
   changes were noted in the MS1_vs_WS1 comparison, with certain genes
   demonstrating downregulation, such as copalyl diphosphate synthase
   (CPS), cytochrome P450 714B1-like (CYP714B), ent-kaurenoic acid oxidase
   (KAO), and most GA[20OX] and GA[3OX] genes. However, more genes were
   upregulated, including copalyl diphosphate synthase 4 (CPS4),
   cytochrome P450 99A2-like (CYP99A2_3), ent-kaurene synthase (CPS-KS),
   ent-kaurene synthase-like (KSL), E1.14.14.86 (GA[3]), and most GA[2OX]
   genes. Furthermore, the levels of GA[3], GA[7,] and GA[8], known for
   their roles in stem elongation, were significantly downregulated,
   whereas GA[4,] which promotes flowering, was slightly upregulated at
   the S1 stage. In the MS2_vs_WS2 comparison, the expression level of
   GA[20OX] remained lower, while that of GA[2OX] remained higher.
   Notably, at the S2 stage, the mutant GA[4], GA[8], and GA[3] contents
   were higher than those of the WT; however, this difference did not
   reach statistical significance, and the GA[7] content in the mutant
   plants remained significantly lower than that in the WT.

Figure 6.

   [105]Figure 6
   [106]Open in a new tab

   Schematic representation of the diterpenoid biosynthesis pathways
   involved in gibberellin synthesis. Note: Refer to [107]Figure 5.
   Different letters represent a significant difference between the mutant
   and the wild-type in the same stage. CPS: copalyl diphosphate synthase;
   KSL: ent-kaurene synthase-like; CPS-KS: ent-kaurene synthase; CYP99A:
   cytochrome P450 99A-like; GA[3]: ent-kaurene oxidase; KAO:
   ent-kaurenoic acid oxidase; CYP714B: cytochrome P450 714B-like;
   GA[2]OX: gibberellin 2-beta-dioxygenase; GA[3]OX: gibberellin
   3-beta-dioxygenase; GA[20]OX: gibberellin 20 oxidase; GA: gibberellin
   A.

2.8. Differences in Detection of Other Hormones Between eui and WT

   The DEGs and DAMs of other hormones in the eui mutant and WT peduncles
   at the two developmental stages were also compared ([108]Supplementary
   Figure S3, [109]Supplementary Table S6). Brassinosteroids (BRs) are
   essential for regulatory processes in plant stems. We identified
   several BR-related DEGs, but no corresponding metabolites were
   detected. Three deetiolated 2 (DET2) genes were upregulated in the eui
   mutant at both the S1 and S2 stages, whereas two brassinazole-resistant
   1_2 (BZR1_2) genes and one brassinosteroid-insensitive 1 (BRI1) gene
   exhibited upregulated expression at the S1 stage. By contrast, the
   expression of brassinosteroid-insensitive 2 (BIN2) was significantly
   downregulated at the S2 stage. Cytokinins (CYTs) also play a role in
   stem elongation. In the eui mutant, most of the genes involved in the
   CYT signaling pathway, including cis-zeatin-O-glucosyltransferase
   (CISZOG), UDP glycosyltransferase (UGT73C), arabidopsis
   histidine-phosphotransfer proteins (AHPs), and cytokinin
   oxidase/dehydrogenase (CKX), were significantly upregulated at stage
   S1. Correspondingly, the metabolites tZ, tZR, cz, DZ, and DPU also
   showed significant accumulation, whereas the accumulation of iP, iPR,
   and mT was significantly reduced. Additionally, most genes in the
   jasmonic acid (JA) signaling pathway were significantly upregulated,
   resulting in significant JA, MeJA, and OPDA accumulation.
   Pathogenesis-related protein (PR1) genes associated with the SA
   signaling pathway were also significantly upregulated, with significant
   salicylic acid (SA) accumulation. By contrast, the protein phosphatase
   2C (PP2C) and SNF1-related protein kinase 1 (SnRK2) genes, which are
   involved in the abscisic acid (ABA) signaling pathway, were
   downregulated to varying degrees at both stages, with a significant
   reduction in ABA accumulation observed at stage S2.

3. Discussion

3.1. eui Is a Mutant Causing Significant Peduncle Elongation and a
Significant GNS Increase

   The peduncle of gramineous crops plays a crucial role in providing
   mechanical support and supplying nutrients to the panicles. It is also
   a key indicator for enhancing plant varieties. In this study, we
   observed that the peduncle of the eui mutant was significantly
   elongated—a phenotype consistent with the known effects of the eui gene
   on the uppermost internode in rice. However, the other internodes did
   not show significant differences compared with the WT. Therefore, the
   eui mutant is a valuable tool for investigating the regulatory
   mechanisms governing the wheat peduncle.

   To date, research on the wheat peduncle has primarily focused on
   Quantitative Trait Loci (QTLs) distributed across 21 chromosomes, most
   of which are associated with the pH [[110]29,[111]30,[112]31], while
   only a few remain independent of the PH [[113]32]. Research has
   indicated that the peduncle length plays a significant role in
   determining the yield of wheat crops. For instance, Wang proposed that
   photosynthesis in the exposed peduncle and flag leaf sheath contributes
   significantly to the grain dry mass, with estimates suggesting that its
   contribution accounts for about 9–12% of the total [[114]33]. This is
   supported by research indicating that the photosynthetic activity in
   these plant parts is crucial for dry matter accumulation and the grain
   yield, as seen in studies on rice leaf sheaths and wheat flag leaves.
   Selection for an increased peduncle length in later generations may
   indirectly improve the yield [[115]34]. In this study, the mutant eui
   exhibited more grains per spike (GNS), consistent with previous
   research. Varieties with longer peduncles not only enhance the plant
   architecture and boost the photosynthetic efficiency of the lower
   leaves but also continue to synthesize photosynthetic products as a
   “source” after the flag leaf begins to senesce during the filling
   stage, ultimately leading to an increased yield. Notably, an
   excessively long peduncle can also negatively impact the yield, as
   longer peduncles are often accompanied by a greater plant height
   [[116]35], which may lead to lodging. Therefore, breeding varieties
   with longer peduncles is only beneficial for production within a
   certain plant height range.

   The mutant identified in this study exhibited significant peduncle
   elongation without notable changes in other internodes. We also
   utilized this mutant to conduct a population-based preliminary genetic
   analysis of the mutant gene and found that this mutant trait may be
   controlled by an incompletely dominant gene. The inheritance pattern
   was consistent with that of barley HvSS1 [[117]36] but differed from
   the recessive inheritance of OsEui [[118]37]. The further mapping and
   cloning of this mutant gene will enable the precise regulation of wheat
   plants’ height, thus aiding in enhancing the plant architecture and
   increasing the yield.

3.2. eui Causes the Elongation of the Peduncle Through Cell Elongation

   The length of the peduncle is primarily determined by internode
   meristem division and cell elongation. The former primarily leads to
   changes in the number of cells, while the latter causes changes in the
   cell size. Previous studies have suggested that alterations in the cell
   size and number may result in changes in the plant height
   [[119]38,[120]39,[121]40]. In this study, we found that eui promoted
   peduncle elongation mainly through internode cell elongation rather
   than through an increased number of cells, which is consistent with the
   findings of Wang [[122]40].

3.3. The Elongation of eui Plants’ Peduncle May Be Mainly Regulated by Auxin

   Integrating transcriptome and metabolome data facilitates a systematic
   analysis of biological systems, examining states and functions from the
   perspectives of gene expression and metabolite accumulation. Some
   studies have utilized multi-omics strategies to explore the mechanisms
   behind early stem thickness phenotypes in wheat, identifying the
   effects of the growth stage and allele on the expression of metabolites
   and transcripts related to stem solidity, cell wall development, and
   programmed cell death [[123]41]. A multi-omics analysis of lignin
   biosynthesis and accumulation in wheat has offered insights into the
   variations in lodging resistance among different hybrid wheat varieties
   [[124]13]. Our study also revealed the potential mechanisms involving
   auxin in peduncle elongation using multi-omics approaches. In the
   mutant, the auxin-related genes DDC/TDC and ASMT, which are part of the
   tryptophan biosynthesis pathway, were significantly upregulated.
   Tryptophan is a precursor for auxin synthesis and can be produced via
   various pathways, including the tryptamine, indole pyruvate, and indole
   acetonitrile pathways [[125]42]. DDC/TDC is also involved in the
   tryptamine pathway. Through its action, tryptophan is converted into
   tryptamine, which subsequently transforms into IAA via the tryptamine
   pathway [[126]43,[127]44]. Both TDC and ASMT play roles in the
   biosynthesis of melatonin, a plant hormone that is crucial for
   controlling growth and various physiological responses [[128]45]. The
   increased expression of these genes may lead to the accumulation of
   auxin metabolites. Our metabolome analysis indicated a significant
   elevation in auxin metabolite levels, including 4-CPA and IBA. Studies
   have suggested that the TaPRR9 mutant exhibits phenotypes similar to
   those of the eui mutant, where the specific expression of
   photosynthetic genes and auxin-related genes in the peduncle promotes
   significant peduncle elongation during the heading stage [[129]46]. br2
   mutants display a classic brachytic phenotype in maize, characterized
   by shortened stalk internodes without a reduction in the size of other
   plant parts, due to defects in the transport of the plant hormone auxin
   [[130]47]. In this study, the elongation of the mutant peduncle may
   also have been related to changes in auxin levels. However, how auxin
   specifically regulates the elongation of the peduncle remains to be
   further investigated.

   Numerous reports have indicated that gibberellin significantly enhances
   plant stem elongation [[131]48,[132]49,[133]50]. However, although we
   observed upregulation in the expression of several gibberellin
   synthesis-related genes, such as CPS and KS, the majority of the
   GA[20OX] and GA[3OX] genes were downregulated, significantly decreasing
   the levels of active GA[7] and GA[3] in the mutant, with only a slight
   increase in GA[4] at the S1 stage. It has been suggested that GA[4]
   specifically regulates the elongation of the rice uppermost internode
   [[134]51,[135]52]. In this study, the eui mutant exhibited significant
   peduncle elongation, while other internodes showed little difference,
   which may have been related to this phenomenon. Brassinosteroids have
   also been reported to participate in the regulation of plant stem
   elongation [[136]53,[137]54]. In Arabidopsis, loss-of-function mutants
   of DET2 exhibit growth inhibition phenotypes such as dwarfism
   [[138]55]. The BR signaling-related genes BZR and BIN2 also regulate
   stem elongation [[139]56]. In this study, the upregulated expression of
   DET2, BRI1, and BZR1_2 indicates their positive regulatory roles in
   peduncle elongation, while the downregulated expression of the BIN2
   gene further confirms its negative regulatory effect. Other plant
   hormones also regulate plant stem elongation [[140]57,[141]58], and we
   detected the differential expression of various other hormones. For
   instance, several CYT DEGs and DAMs were upregulated in the eui mutant,
   whereas genes related to the ABA levels were downregulated. Notably,
   genes and metabolites associated with JAs and SA in the mutant were
   also upregulated. Studies have reported that JAs and SA are the main
   plant hormones involved in regulating disease resistance [[142]59].
   This suggests that the presence of the mutant might enhance disease
   resistance due to peduncle elongation; however, further experimentation
   is required to confirm this.

   In conclusion, the elongation of the peduncle in this mutant appears to
   be mostly regulated by auxin, which may interact with other hormones
   during the early heading stage. Considering that EMS mutagenesis can
   generate high-density point mutations, the DEGs and DAMs identified in
   this study may have been influenced by mutations at other loci. Further
   population development, including the creation and utilization of
   backcross populations, will assist in minimizing the background
   differences and aid in the mapping and cloning of mutant genes that are
   directly related to peduncle elongation. Nonetheless, multi-omics
   analysis can still provide valuable references for the mining of mutant