Regulation of spikelet number during wheat spike development

Wheat produces unbranched inflorescences (spikes) composed of smaller inflorescences (spikelets) as their fundamental building units. The spikelet number per spike (SNS) is a major determinant of grain yield and the gene networks that regulate this trait are the focus of this review. Spikelet development starts with the transition of the shoot apical meristem into an inflorescence meristem (IM) that produces lateral spikelet meristems (SMs). The rate at which SMs are produced and the timing of the IM transition to a terminal spikelet (IM→TS) determine the final SNS. These two traits are regulated by genes expressed in the IM (e.g. meristem identity genes), as well as by the amount of FLOWERING LOCUS T1 (florigen) transported from leaves to developing spikes. The SNS can be also increased by the production of spikes with supernumerary spikelets (SS) or branch-like structures that resemble small spikes. Mutations that delay the acquisition of SM identity or that promote a reversion from SM to IM identity can induce the formation of SS. Initial efforts to incorporate these mutations into commercial wheat varieties have faced trade-offs in fertility and grain weight, which will require additional research and breeding efforts. Meanwhile, genes and allele combinations that increase SNS without affecting the number of spikelets per node have been identified and are being deployed in wheat breeding programs. Recent spatial transcriptomics, single-cell analyses, and multi-omics studies of wheat spike development are accelerating the discovery of new genes affecting SNS and enhancing our ability to engineer more productive wheat spikes. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.07.698073doi: bioRxiv preprint 3

Inflorescences bear flowers and fruits, and their architectures affect yield in crop species. In the grasses, flowers are replaced by small inflorescences called spikelets (Fig. 1a-c), resulting in compound inflorescences or synflorescences [1]. Both spikelets and flowers are self-contained developmental modules that are expressed at different positions of the inflorescence and are commonly regulated by MADS-box genes of the SQUAMOSA clade [2,3]. Given these shared developmental characteristics, in this review we extend the simpler nomenclature used for inflorescences bearing flowers to the grass inflorescences bearing spikelets. In many grasses, spikelets are produced on inflorescences called panicles that have branches of decreasing complexity from the base to the top (e.g. rice) [1]. In the Triticeae, however, genuine branches are absent, and spikelets sit directly on the rachis forming a spike (Fig. 1a). Spikelet development proceeds faster at the central part of the spike resulting in the characteristic lanceolate shape of the wheat spike (Fig. 1a, d-e). By the time the central spikelets begin forming floret meristems (FMs), the inflorescence meristem (IM) transitions into a spikelet meristem (SM) and forms a terminal spikelet (henceforth IM→TS). The terminal spikelet has different orientation from the lateral spikelets (Fig. 1a). The timing of formation and the identity of meristems shape the inflorescence architecture. In wheat, both vegetative shoot apical meristems (vSAM) and IM produce lateral meristems in an alternate-distichous pattern, but these meristems differ in activity and identity. In the vSAM, bract meristems develop rapidly into leaves whereas their axillary meristems (AxM) are temporarily repressed before initiating tillers. By contrast, in lateral meristems generated by the IM, the lower ridges (bract meristems) are repressed, while the upper ridges (equivalent to AxM) rapidly develop into spikelets (Fig. 1d). This review focuses on gene networks that regulate wheat spike development, from the initial vSAM→ IM transition to the IM→TS transition (Fig. 1d). It includes genes that affect the rate at which SMs are produced, the timing of the IM→TS transition, and the number of spikelets in each node. Due to space limitations, the regulation of grain number per spikelet is not covered, although its agronomic importance is recognized. This review integrates recent advances in spatial transcriptomics and single-cell expression analyses and discusses the potential contributions of this knowledge to the improvement of wheat productivity. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.07.698073doi: bioRxiv preprint 4 Fig.1. (a) Wheat spike showing a terminal spikelet with a different orientation relative to the lateral spikelets. (b) Wheat spikelet showing two basal sterile bracts (glumes) and multiple florets. (c) Each floret includes a bract (lemma) subtending the floral organs: the palea, two lodicules, three stamens, and a pistil. (d) Waddington scale of wheat spike development: W1.0= vegetative shoot apical meristem (vSAM), W1.5= initial transition of the vSAM to an inflorescence meristem (IM) that produces lateral spikelet meristems (SM). W2.5= double ridge stage. The lower ridge (LR) corresponds to a repressed bract and the upper ridge (UR) to the AxM of the repressed bract. W3.0= glume primordium (GP). W3.5= floret primordia (FM) subtended by the lemma primordium (LP). This stage is also characterized by the transition of the IM into a SM that generates a terminal spikelet (IM→TS). (e-f) Developing spikes at W3.0 for vrn1 vrn2-null mutant (e) and svp1 vrt2-null mutants (f) showing a higher number of SMs. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.07.698073doi: bioRxiv preprint 5 1. The critical role of MADS-box genes in the initial stages of wheat spike development Wheat spike development starts with the vSAM→IM transition, which is driven by the upregulation of the VERNALIZATION1 (VRN1) gene (Fig. 2a and b). However, VRN1 upregulation in the vSAM is not sufficient to complete spike development and stem elongation, which requires long-day (LD) induction of FLOWERING LOCUS T1 (FT1) in the leaves. The FT1 protein (florigen) is transported to the developing spike, where it simultaneously up- regulates VRN1 and gibberellin (GA) biosynthetic genes [4] (Table S1). Both GA and VRN1 are required for the transcriptional up-regulation of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) and LEAFY (LFY), two important genes in wheat spike development [4]. Another important role of VRN1 in the leaves is to repress the LD flowering repressor VRN2, which negatively regulates FT1 [5]. VRN1 and its closest paralogues FUL2 and FUL3 are MADS-box genes of the SQUAMOSA clade that are widely expressed during early spike developing (Fig. 2a-b). Spikes of plants carrying knockout mutations in all VRN1 homeologs (vrn1-null) are normal [5], but further combinations with ful2-null and ful3-null mutations (SQUAMOSA-null) result in abnormal spikes where spikelets are replaced by vegetative tillers subtended by leaves [3]. These SQUAMOSA- null plants (combined with vrn2-null mutations to avoid extremely late heading) still undergo a vSAM→IM transition followed by a double-ridge stage, elongated stems, and AxM that develop rapidly in the “spike” region [3]. These results indicate that the SQUAMOSA genes play essential roles in suppressing the lower ridge and conferring spikelet meristem identity to the upper ridge, but that they are not essential for inflorescence initiation [3]. The genes responsible for the initial stages of inflorescence development in the wheat SQUAMOSA-null mutants are currently unknown, but in Arabidopsis MADS-box genes of the SOC1 and SHORT VEGETATIVE PHASE (SVP) clades contribute to this initial inflorescence development [6-8]. A recent study showed that the wheat SOC1-3 protein can compete with VEGETATIVE TO REPRODUCTIVE TRANSITION 2 (VRT2) for interactions with VRN1 and can also regulate heading time [9]. In wheat, there are five SOC1 paralogs [10] with different expression profiles (Fig. 2c) and possible different functions based on studies of the SOC1 orthologs in rice [11]. Therefore, a more comprehensive study of the wheat SOC1 genes would be required to elucidate their roles in heading time and spike development. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.07.698073doi: bioRxiv preprint 6 The wheat SVP transcription factors, which include SVP1, VRT2, and SVP3, physically interact with the SQUAMOSA proteins [12]. Combined loss-of-function mutations in SVP1 and VRT2 genes delay heading time, increase spikelet number per spike (SNS, Fig. 1e-f), and reduce plant height in a similar way as the vrn1-null mutants. The similar mutant effects and physical protein interactions suggest that SQUAMOSA and SVP proteins may work cooperatively to regulate early spike development [12]. Fig. 2. Dynamic changes in MADS- box gene expression during wheat early spike development. (a) Curves are based on RNA-seq data of wheat spike development in tetraploid wheat Kronos [13] and individual points are the sum of transcripts per million of all homeologs and paralogs within each MADS-box group (Table S1). (b-f) Spatial transcriptomics of MADS-box gene families across three spike developmental stages [14]. (b) SQUAMOSA. VRN1 is expressed earlier than the other two SQUAMOSA genes and all three genes are expressed at relatively lower levels at the FMs. (c) SOC1. SOC1-2 expression is higher at W1.5 and then decreases, whereas expression of other SOC1 genes increases during early spike development. (d) SVP. Genes are expressed in the IM at W1.5 and at the base of the spike at W2.5 and W3.5. SVP1 is also expressed in developing anthers. (e) SEP1. SEP1- 6 is the earliest expressed gene in this clade, SEP1-4 is enriched in the distal part of the spike and glume primordia, and SEP1-2 is enriched in lemma primordia. (f) SEP3. SEP3-1 and SEP3-2 are expressed in FMs together with other floral homeotic genes (Table S1). .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.07.698073doi: bioRxiv preprint 7 The wheat SVP genes are co-expressed with the SQUAMOSA genes in the early IM at W1.5, but their expression is restricted to the base of the spike and to the leaves at later stages (Fig. 2d). The transcriptional downregulation of the SVP genes by the SQUAMOSA genes is important for the normal progression of spike development, as SVP proteins can interfere with the SQUAMOSA-SEPALLATA1 (SEP1) protein interactions [12]. Plants carrying high-expressing natural VRT2 alleles or transgenic plants overexpressing this gene exhibit more vegetative traits such as larger glumes and lemmas [12,15]. By contrast, the vegetative characteristics of the vrn1 ful2 spikes are mitigated when combined with vrt2 mutations [12], highlighting the importance of the downregulation of the SVP genes for normal spike development. High VRT2 levels are also associated with a higher number of basal rudimentary spikelets [16], suggesting that VRT2 can delay the development of basal spikelets and contribute to the lanceolate shape of the wheat spikes [16]. This hypothesis is supported by the triangular shape of the svp1 vrt2 mutant spike at W3.0 (Fig. 1f), where basal spikelet development is not delayed relative to the central spikelets as in the wildtype (Fig. 1d). The SEP1 and SVP genes show opposite expression profiles (Fig. 2a). Among the SEP1 genes, SEP1-6 expression is initiated the earliest (Fig. 2e), and its overexpression in wheat results in reduced SNS [17]. Combined mutations in the rice homologs of SEP1-6 (OsMADS34) and SEP1-4 (OsMADS5) lead to enhanced branching in rice panicles [18], but have limited effects on barley spikes [19,20]. Mutations in the SEP1-2 ortholog in rice (OsMADS1) result in floral organs with leaf-like characteristics [21], whereas mutations in the barley ortholog lead to smaller lemmas and awns at room temperature [22] and branched spikes at high temperatures [19]. Given the different effects of the SEP1 mutants in rice and barley, it is not possible to infer their function in wheat without dedicated research efforts. The SEP3 genes are co-expressed in the FM (Fig. 2f) with other floral homeotic genes [14] and contribute to floral organ identity [23]. MADS-box proteins form quaternary complexes of varied compositions, each with specific DNA binding specificities [24]. Therefore, the early upregulation of VRN1 in the IM of wheat (Fig. 2b) and the gradual changes in the relative expression of other MADS-box genes (Fig. 2a) likely affect the composition of these quaternary complexes and the sequential morphological changes observed during the early stages of wheat spike development. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.07.698073doi: bioRxiv preprint 8 2 Regulation of wheat spike determinacy and spikelet number per spike (SNS) Both wheat and barley produce spikes with a genetically regulated number of spikelets, but they differ in the fate of the IM. In the determinate wheat spike, the IM transitions into a SM, and forms a terminal spikelet (Fig. 1a), whereas in the indeterminate barley spike the IM is exhausted and degenerates. In barley, loss-of-function mutations in GRASSY TILLERS1 (GT1) delay IM degeneration and increase SNS [25], whereas mutations in APETAL2-LIKE5 (ap2l5-null) result in a determinate spike with extra florets per spikelet [26]. In wheat, mutations in AP2L5 also lead to extra florets per spikelet and are associated with a 21% reduction in SNS [27]. Increased expression of AP2L5 in miR172 resistant alleles increases SNS by 5-10% [28], but neither the mutants nor the AP2L5 overexpressing alleles affect wheat spike determinacy. By contrast, wheat vrn1 ful2-null and vrn1 ful2 ful3-null mutants have indeterminate spikes, demonstrating overlapping and essential roles of VRN1 and FUL2 in the IM→TS transition [3]. Individual loss-of-function mutations in VRN1 or FUL2 significantly increase SNS, but the effects are more pronounced in vrn1-null (58%) than in ful2-null mutants (10%) [3]. Both vrn1- null and svp1 vrt2-null mutants show delayed spike and spikelet development associated with a reduced rate of SM production and a significantly delayed IM→TS transition (Fig. 3a-b). The rate of SM production is also reduced in lfy and wapo1 mutants relative to the wildtype, but the timing of the IM→TS transition is not significantly altered [29] (Fig. 3c). Loss-of-function mutations in LFY, WAPO1, or both result in similar reductions in SNS, suggesting that they work cooperatively to regulate this trait. This hypothesis is supported by the physical interaction between their encoded proteins and their co-expression in the early IM at W1.5 [29]. Natural alleles with positive effects on SNS have been identified for WAPO1 [30] and LFY and both show evidence of positive selection and a significant genetic interaction for SNS [31]. The IM→TS transition at W3.5 is characterized by the upregulation of SQUAMOSA genes VRN1 and FUL2 and the downregulation of both LFY [29] and the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE gene SPL14 in the IM region [14]. Loss-of-function mutations in SPL14 result in a premature IM→TS transition and a 42% reduction in SNS, demonstrating an important role of this gene in spike development [14]. This hypothesis is further supported by the discovery of natural alleles for SPL14 and its close paralog SPL17 with positive effects on wheat SNS [32,33]. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.07.698073doi: bioRxiv preprint 9 Another group of genes with significant effects on wheat SNS includes the PHOTOPERIOD1 (PPD1) gene and its downstream targets FT1 and FT2. Plants carrying the PPD1 photoperiod- sensitive (PS) allele have lower FT1 expression and higher SNS than those with photoperiod- insensitive (PI) alleles. By contrast, wheat plants with loss-of-function mutations in PPD1 show reduced FT1 expression and higher SNS [34,35]. Shorter days also reduce FT1 transcript levels, delay spike and spikelet development, and increase SNS (Fig. 3d) [35]. These effects are attenuated in the vrn2-null mutants, which exhibit higher FT1 expression and a faster rate of SM formation (Fig. 3d). The dominant role of florigen proteins in the regulation of SNS is also supported by the low SNS observed in plants overexpressing FT1 [36], FT2 [37], or FT3 [38]. Based on these results, we hypothesize that the reduced rate of SM-production and delayed IM→TS transition in the vrn1 vrn2-null and svp1 vrt2-null mutants (Fig. 3a-b) could be associated with the downregulation of FT1 observed in the leaves of these mutants [3,12]. Fig. 3. Rate of spikelet meristem (SM) formation and IM→TS transition across mutant backgrounds and photoperiods. (a) vrn1 vrn2-null mutant versus vrn2-null control. (b) svp1 vrt2-null mutant versus Kronos control. (c) lfy-null and wapo1-null mutants versus Kronos control [29]. (d) Effect of photoperiod on SM production rate: 16h light /8 h darkness (blue) vs. 12h light / 12h darkness (orange) in Kronos (dotted lines) and Kronos vrn2-null mutant (solid line). Longer photoperiods [35] and absence of vrn2 [5] both result in increased transcript levels of FT1 and a faster rate of SM production. Arrows indicate the approximate time of the IM→TS transition. Rates are expressed as number of SM formed per day. Data points represent the averages of three dissected apices and error bars indicate standard errors of the means (SEM). Data is available on Table S3. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.07.698073doi: bioRxiv preprint 10 The FT2 gene also affects SNS, and its encoded protein physically interacts with bZIPC1, a homeodomain-leucine zipper transcription factor [35,39]. The two genes affect SNS in opposite directions, with loss-of-function mutations in FT2 increasing SNS and those in bZIPC1 reducing SNS. Natural alleles with positive effects on SNS have been identified for both genes, which show evidence of positive selection and significant genetic interactions for SNS [39,40]. The genetic interactions for SNS discovered for FT2-bZIPC1 and WAPO1-LFY have both led to the identification of optimal allele combinations that maximize SNS [31,40]. These results illustrate the practical value of understanding the gene networks that regulate spike development. An increase in SNS does not guarantee an increase in grain number per spike (GNS) because floret degeneration can offset the gains in SNS. Fortunately, a natural allele of the GRAIN NUMBER INCREASE 1 gene (GNI1, or VRS1 in barley) provides a tool to increase spikelet fertility and reduce premature floret abortion [41]. Even when increases in SNS are translated into more GNS, reduced grain weight can offset the gains in grain yield. When plants cannot produce sufficient resources to fill the extra grains, a negative correlation is usually observed between GNS and grain weight. For example, the favorable WAPO1 allele for increased SNS showed positive effects on grain yield only when introgressed into high-biomass genotypes and when the lines were grown under well-fertilized and well-watered conditions [30,42]. These experiments suggest that balanced increases in both sink and source, together with favorable environmental conditions, are necessary to translate increases in SNS into higher grain yields. 3. Regulation of branching in the wheat spike An alternative strategy to improve wheat SNS is to increase the number of spikelets produced per node, generating supernumerary spikelets (SS). In mutants such as ‘Miracle-Wheat’ and ‘Compositum-Barley’ (com2), some basal spikelets are replaced by branch-like structures resembling smaller secondary spikes [43,44]. In these mutants, the basal branch-like structures resemble small spikes rather than panicle branches, suggesting that those SMs acquired IM identity rather than branch meristem (BM) identity. Branched-like phenotypes and SS have been associated with hypomorphic mutations in the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) domain transcription factor .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.07.698073doi: bioRxiv preprint 11 FRIZZY P ANICLE (FZP) [43-45]. However, in more severe FZP mutants in rice [46] and durum wheat [14], secondary axes emerge from the axils of the glumes forming new bracts/glumes that produce tertiary axes and repeat the process. These recurrent glume structures suggest that, in addition to its effect on the conversion of basal SMs into secondary IMs, an important function of FZP is to suppress the glume’s AxM. This result is consistent with the restricted expression of FZP in the axils of wheat glume primordia (Fig. 4a-b). Studies in wheat and barley suggest that FZP transcript levels are positively regulated by two transcription factors: wheat MULTI-FLORET SPIKELET1 (TaMFS1, referred to as DUO in [47]) and barley RAMOSA2 (HvRA2, synonymous VRS4) [44]. In wheat, MFS1 directly promotes the expression of FZP [47] and the expression domains of these two genes show significant overlap at early stages of spikelet development (Fig. 4b). Consistent with its effect on FZP, the wheat mfs-B1 mutant also produces spikes with SS [47]. In barley, loss-of-function mutations in HvRA2 (VRS4) are also associated with reduced FZP transcript levels. These mutants also promote lateral spikelets, occasional branch-like formation [44], and reduced expression of barley VRS1 and SISTER OF RAMOSA3 [48]. In the wheat spike, RA2 is first induced in the abaxial part of initiating SMs, then surrounds the growing SM (Fig. 4c), and later delimits the base of the developing spikelet, overlapping with the FZP expression domain in the axil of the second glume (Fig. 4d). Wheat MFS1 [47] and maize RA2 [49] are also positive regulators of TCP24, which corresponds to COMPOSITUM1 (COM1) in barley and BRANCH ANGLE DEFECTIVE 1 (BAD1) in maize [49] (Fig. 4e). Loss-of-function mutations in COM1 result in branched barley spikes, suggesting that the active COM1 promotes SM identity and contributes to branch suppression [50,51]. The interactions among MFS1, RA2, and TCP24 in wheat are also supported by significant correlations among their expression profiles in a single-cell RNA-seq (scRNA-seq) study of early wheat spike development [14] and by their overlapping expression domains (Fig. 4d). Moreover, increased expression of all three TCP24 homeologs is observed in the wheat mfs-B1 mutant [47]. A similar interaction has been observed in maize, where RA2 operates upstream of BAD1 [49]. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.07.698073doi: bioRxiv preprint 12 Fig. 4. Genes and gene network regulating branching. (a-d) Single-molecule fluorescence in situ hybridization of Kronos developing spikes at W2.5 (a and c) and W3.5 (b and d). Cell walls are stained with calcofluor white. gl= glume, le= lemma, br= suppressed bract, SM= spikelet meristem, IM= inflorescence meristem. (a-b) Expression profiles of FZP , TCP24, TB1 and MFS1 based on [14] (c-d) Expression profiles of LAX1, RA2, TCP24 and MFS1 in Kronos spikes based on a 10X-Xenium spatial transcriptomics study. (e) Working model of a gene network regulating branching in wheat. Blue arrows indicate promotion, red T-shaped symbols indicate repression, and green double arrows represent protein-protein interactions. Solid lines are supported by wheat or barley studies, whereas dotted lines are supported by studies in the more distantly related rice or maize. Gene names and IDs are included in Table S1. Numbers in parenthesis indicate references: 1:[47], 2:[49], 3:[44], 4:[45], 5:[50], 6:[51], 7:[53], 8:[55], 9:[56], 10:[57], 11:[54], 12:[56]. (f) Left: Spikes of wildtype Kronos and tb1-null CRISPR mutant showing SS. Right: nodes bearing two or three spikelets. Note the different orientation of the terminal and lateral spikelets, their similar development, and the higher frequency of triplets at the center of the spike. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.07.698073doi: bioRxiv preprint 13 TCP24 belongs to the CYC/TB1 clade of TCP transcription factors, which also includes TCP22, TEOSINTE BRANCHED1 (TB1), and TB2. In barley, TB1 is known as INTERMEDIUM SPIKE-C (INT-C or VRS5), and its mutant contributes to the six-rowed phenotype [52]. In wheat, TB1 expression is detected in the SMs at W2.5 and becomes restricted to the base of the spikelets at W3.5. At both stages it is more abundant at the base of the spike (Fig. 4a-b). Knockout mutations of both TB1 homeologs in tetraploid wheat (tb1-null) result in frequent SS (Fig. 4f, Table S2), as reported previously [53]. These results suggest that TB1/INT-C modulates SM timing or identity, thereby contributing to the repression of SS in both barley and wheat. Surprisingly, TB1 duplications or transgenic overexpression were also associated with SS [54], suggesting that balanced levels of TB1 may be required to prevent SS formation. In maize, TB1 transcription is regulated by BARREN STALK1 (BA1), the ortholog of rice LAX P ANICLE1 (LAX1) [56]. BA1 acts downstream of auxin signaling to position boundary regions for AxM formation [58]. Loss-of-function mutations in BA1 or LAX1 result in drastic reductions in panicle branching and lateral spikelet formation in both maize and rice [55,56], whereas mutations in the wheat ortholog have milder effects [59]. Wheat plants with mutations in the three LAX1 homeologs have a more compact spike [59] that resembles gain-of-function mutations in the domestication gene Q (AP2L5) [28,60]. By contrast, overexpression of wheat LAX1 reduces spike threshability and spikelet density, similar to the pre-domestication q allele [59]. LAX1 and Q proteins physically interact with each other, providing a potential mechanism for their opposite effects on these domestication traits [59]. In wheat, FZP has been reported to directly repress LAX1 transcription by binding to its regulatory regions [45]. However, the functional significance of this interaction remains unclear, given that FZP is expressed later than LAX1 in developing wheat spikes (Fig. 4a-c). From the early stages of wheat spike development, LAX1 is expressed at the adaxial boundary layer of the SMs (Fig. 4c), similar to observations in rice [55] and maize [56], suggesting a conserved function. In hexaploid wheat, a particular class of SS, designated as paired spikelets (PS), is characterized by the formation of a secondary spikelet immediately adjacent to and below (abaxial) the more advanced main spikelet. At maturity, PSs appear parallel to each other and are more frequent at the central nodes of the spike. PS are affected by genes in the photoperiod pathway, with shorter .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.07.698073doi: bioRxiv preprint 14 photoperiods, mutations in PPD1, and reduced expression of FT1 all increasing PS frequency [57]. Since these changes are associated with reduced expression of meristem identity genes (VRN1, FUL2, and FUL3), the authors of [57] hypothesized that this reduction may contribute to the delayed initiation of the SM, providing the time for PS formation. In summary, none of the known mutants in barley and wheat form genuine branches as observed in the panicles of other grasses, suggesting that these species have lost the ability to generate functional branch meristems. However, multiple mutants generate SS or branch-like structures by modifying the timing or identity of the SMs, providing the time for the formation of one or more lateral spikelets. It remains unclear why some mutations induce higher SS frequencies at the center of the spike (e.g. Tb1), whereas others result in branch-like structures with an elongated secondary rachis (e.g. FZP). Studying branching in the un-branched Triticeae spikes can provide valuable insights into our understanding of grass inflorescence development and evolution.

and perspectives Increasing spikelet number through SS or branching has been an attractive strategy to increase grain yield, but initial efforts have been limited by trade-offs in fertility and grain size. The GNI1 allele for high fertility [41] may help mitigate the reductions in SS fertility, whereas high- biomass varieties with higher source resources may limit negative correlations between grain number and grain size. A recent study using a cross between a spike-branching landrace and the elite durum cultivar CIRNO showed that a better balance of source–sink traits can reduce yield penalties associated with branched phenotype [61]. However, developing higher yielding wheat varieties with SS or branched spikes will require a dedicated breeding effort to fine-tune the multiple epistatic interactions within the complex gene network regulating branching. Meanwhile, rapid progress has been made in the identification of genes that control grain number by modifying the rate of SM production, the timing of the IM →TS transition, and/or the number of fertile florets. Favorable alleles and allele combinations are being identified and incorporated into breeding programs through marker-assisted selection or by integrating functional SNPs into high-throughput marker platforms used for genomic selection. The impressive number of .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.07.698073doi: bioRxiv preprint 15 spikelets and grains produced by high yielding triticale varieties suggest that there is still room for improvement of the SNS trait in wheat. The identification and functional characterization of genes regulating wheat spike development have been greatly accelerated in recent years, fueled by new tools including CRISPR, sequenced mutant populations, improved wheat transformation efficiency, and sequenced genomes. These resources have been complemented by single-cell RNA-seq and spatial transcriptomic studies, which provide cellular resolution to the rapid changes in gene expression that occur during spike development [14,62]. In parallel, multi-omics studies are integrating expression profiles, chromatin accessibility, binding motifs, GWAS, and evolutionary information into comprehensive networks that will help prioritize the functional characterization of novel regulators of wheat spike development [63,64]. The knowledge generated by these new tools can enhance our ability to engineer changes in spike development. Each year, more than 200 trillion wheat spikes are harvested globally, providing one fifth of the calories and proteins consumed by the human population. Therefore, every small improvement in wheat spike productivity is worth the effort.

The authors appreciate the comments and suggestions provided by Dr. Thorsen Schnurbusch (Leibniz Institute of Plant Genetics and Crop Plant Research, IPK) and Dr. Scott Boden (The University of Adelaide) on the branching section of this review. The authors also thank Dr. Francois Parcy (Universite Grenoble Alpes) for valuable discussions on inflorescence development. Funding statement This project was supported by the Agriculture and Food Research Initiative Competitive Grant 2022-68013-36439 (WheatCAP) from the USDA National Institute of Food and Agriculture, and by the Howard Hughes Medical Institute. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.07.698073doi: bioRxiv preprint 16

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This study presents a genome-wide wheat integrative gene regulatory network (wGRN) that combines an updated genome annotation, large-scale gene expression profiles, transcription factor (TF) binding motifs (DAP-seq), chromatin accessibility data, and evolutionary conserved regulatory regions. Using these datasets, the study built seven independent regulatory networks and integrated them into a single large- scale network that comprises ~7.2 million TF–target interactions, linking 5,947 TFs to 127,439 target genes. The authors demonstrated the applicability of wGRN in uncovering previously unknown gene functions and pathways, interpreting new datasets, and prioritizing candidate genes from GWAS and map- based cloning studies. By integrating the wGRN with a spike transcriptome time-series dataset, this study constructed high-resolution gene networks that enabled the identification of novel regulators of SNS and enhanced the power of spike phenotypic trait prediction. An interactive webserver (http://wheat.cau.edu.cn/wGRN) is provided to explore gene regulation and discover trait-associated genes. 64. Lin X, Xu Y , Wang D, Yang Y , Zhang X, Bie X, Gui L, Chen Z, Ding Y , Mao L, et al.: Systematic identification of wheat spike developmental regulators by integrated multi-omics, transcriptional network, GWAS, and genetic analyses. Molecular Plant 2024, 17:438-459. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.07.698073doi: bioRxiv preprint 26 The study used a multi-omics approach to analyze and integrate the transcriptome and epigenome profiles at eight early stages of the spike development in winter wheat. It combines RNA-seq, ATAC-seq, and histone modification data to construct a transcriptional regulatory network (TRN) of spike development. Further integration of this TRN with genome-wide association studies (GWAS) identified numerous transcription factors potentially involved in spike development. Mutant analysis confirmed spike phenotypes for 36 of these transcription factors and identified MYB30 as a direct target of FZP . The datasets generated in this study are available in the open access Wheat Spike Multi-Omic Database (http://39.98.48.156:8800/#/).

and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: * of special interest and ** of outstanding interest .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.07.698073doi: bioRxiv preprint