LEAFY and WAPO1 jointly regulate spikelet number per spike and floret development in wheat

ABSTRACT In wheat, the transition of the inflorescence meristem to a terminal spikelet (IM→TS) determines the spikelet number per spike (SNS), an important yield component. In this study, we demonstrate that the plant-specific transcription factor LEAFY (LFY) physically and genetically interacts with WHEAT ORTHOLOG OF APO1 (WAPO1) to regulate SNS and floret development. Loss-of-function mutations in either or both genes result in significant and similar reductions in SNS, as a result of a reduction in the rate of spikelet meristem formation per day. SNS is also modulated by significant genetic interactions between LFY and the SQUAMOSA MADS-box genes VRN1 and FUL2, which promote the IM→TS transition. Single-molecule fluorescence in situ hybridization revealed a downregulation of LFY and upregulation of the SQUAMOSA MADS-box genes in the distal part of the developing spike during the IM→TS transition, supporting their opposite roles in the regulation of SNS in wheat. Concurrently, the overlap of LFY and WAPO1 transcription domains in the developing spikelets contributes to normal floret development. Understanding the genetic network regulating SNS is a necessary first step to engineer this important agronomic trait.

. TPM= transcripts per million.Averages are based on four biological replications per stage in each experiment and error bars are SEM.P values are from t-tests between homoeologs.ns= not significant, * = P < 0.05 and ** = P < 0.01.Raw data and statistics are available in Table S7.S8.S10.S8) and error bars are s.e.m. ns = not significant, * = P < 0.05 and *** = P < 0.001.Raw data and statistics are available in Table S8.S9.S11.(Glenn et al., 2023), the effect of SPL17 on SNS in wheat is based on (Liu et al., 2023).The positive effects of FT1 and FT2 on the regulation of VRN1 are based on (Li and Dubcovsky, 2008, Lv et al., 2014, Li et al., 2019, Shaw et al., 2019), and the positive effect of PPD1 in the regulation of FT1 and FT2 on (Shaw et al., 2013).Development: doi:10.1242/dev.202803: Supplementary information

Development • Supplementary information
Table S1.Primers used in this study.Table S3.Supporting data for Fig. S1.Effect of individual and combined LFY mutation on SNS.Table S4.Supporting Data for Fig. S2.Transcript levels of UBI:LFY-HA in leaves reltive to non-transgenic sister lines and Kronos.The LFY-A homeolog was used in these transgenic lines.Table S5.Supporting data for Fig. 2. Effect of UBI:LFY-HA on spikelet number per spike (SNS) in Kronos wildtype (WT) and lfy mutant.

Fig. S2 .
Fig. S2.Transgenic plants expressing LFY under the UBIQUITIN promoter.(A) LFY transcript levels in the leaves of transgenic plants overexpressing LFY.The LFY-A coding region fused with an HA tag was driven by the maize UBIQUITIN promoter (UBI:LFY-HA).Five independent events were evaluated.Note the absence of expression of LFY in the negative transgenic sister lines (Neg.) and the wildtype Kronos control.P values are two-tail ttests of each transgenic event (n = 4) relative to the combined Neg. and wildtype Kronos control (n = 8).* P = 0.05, ** P = 0.01, *** P = 0.001.Error bars are SEM.Raw data and statistics are available in Table S4.(B) Floral defects in UBI:LFY-HA included reduced number or fused lodicules, fused anther filaments and extra stigmas.(C) Frequency of floral defects in 14 florets.Note the higher proportion of normal plants compared to the lfy mutants in Fig. S1B.

Fig. S3 .
Fig. S3.Expression analysis of LFY and WAPO1.(A) LFY RNA-seq data for Chinese Spring across five tissues and three developmental stages (Choulet et al., 2014).(B) LFY and (C) WAPO1 transcript levels during early stages of Kronos spike development (VanGessel et al., 2022).(D) Examples of developing spikes at the different spike development stages collected in the Kronos RNA-seq study (VanGessel et al., 2022).W numbers indicate values in the Waddington scale of wheat spike development(Waddington et al., 1983).TPM= transcripts Development: doi:10.1242/dev.202803:Supplementary information Development • Supplementary information

Fig. S4 .
Fig. S4.Single-molecule fluorescence in situ hybridization (smFISH) for FRIZZY PANICLE (FZP).FZP (TraesCS2A02G116900) is used as a marker for the initiation of spikelet development.FZP transcripts are indicated by yellow dots and LFY by purple dots.Cell walls stained with calcofluor are in dark blue.(A) Elongated shoot apical meristem transitioning to the reproductive stage (W1.5).(B) Late double ridge (DR, W2.5).(C) Lemma primordia (LP, W3.25).Yellow arrows indicate FZP expression in the youngest lateral meristems, suggesting a transition to glumes and the start of the IM transition to a terminal spikelet.Quantification of the FZP signal is available in TableS8.

Fig. S6 .
Fig. S6.Single-molecule fluorescence in situ hybridization (smFISH) showing detail of the overlap between LFY and WAPO1 in spikelet meristems.Spikelet meristems at the lemma primordia stage at W3.25.WAPO1 transcripts are indicated with yellow dots and LFY transcript with purple dos.Cell walls stained with calcofluor are presented in dark blue.LP= lemma primordium.Scale bars are 100 μm.

Fig. S11 .
Fig. S11.Effect of lfy mutations on the expression of floral organ identity genes.Expression of class-B, -C, and -E MADS-box genes was characterized in wheat developing spikes at the stamen primordia stage (W4.0) using qRT-PCR with ACTIN as endogenous control.(A) class-B genes AP3-1 and PI1, (B) class-C genes AG1 and AG2, (C) class-E genes SEP1-2, SEP1-4, SEP1-6, SEP3-1, SEP3-2.(D) Representative wheat developing spikes at W4.0 in WT Kronos and lfy mutants (note the reduced SNS).Bars are averages of four biological replicates and error bars are standard errors of the means (SEM).Each biological replicate represents one RNA extraction from a pool of four apices from Kronos wildtype (WT) and lfy mutant.ns = not significant, ** P < 0.01, *** P < 0.001.Raw data and statistics are available in TableS11.

Fig. S12 .
Fig. S12.Simplified working model of the regulation of SNS in wheat.The negative effect of bZIPC1 in the regulation of the transition between the inflorescence meristem to a terminal spikelet (IM>TS), and its interaction with FT2 is based on (Glenn et al., 2023), the effect of