CENTRORADIALIS Interacts with FLOWERING LOCUS T-Like Genes to Control Floret Development and Grain Number

Abstract

CENTRORADIALIS (CEN) is a key regulator of flowering time and inflorescence architecture in plants. Natural variation in the barley (Hordeum vulgare) homolog HvCEN is important for agricultural range expansion of barley cultivation, but its effects on shoot and spike architecture and consequently yield have not yet been characterized. Here, we evaluated 23 independent hvcen, also termed mat-c, mutants to determine the pleiotropic effects of HvCEN on developmental timing and shoot and spike morphologies of barley under outdoor and controlled conditions. All hvcen mutants flowered early and showed a reduction in spikelet number per spike, tiller number, and yield in the outdoor experiments. Mutations in hvcen accelerated spikelet initiation and reduced axillary bud number in a photoperiod-independent manner but promoted floret development only under long days (LDs). The analysis of a flowering locus t3 (hvft3) hvcen double mutant showed that HvCEN interacts with HvFT3 to control spikelet initiation. Furthermore, early flowering3 (hvelf3) hvcen double mutants with high HvFT1 expression levels under short days suggested that HvCEN interacts with HvFT1 to repress floral development. Global transcriptome profiling in developing shoot apices and inflorescences of mutant and wild-type plants revealed that HvCEN controlled transcripts involved in chromatin remodeling activities, cytokinin and cell cycle regulation and cellular respiration under LDs and short days, whereas HvCEN affected floral homeotic genes only under LDs. Understanding the stage and organ-specific functions of HvCEN and downstream molecular networks will allow the manipulation of different shoot and spike traits and thereby yield.

Figure 1.

Phenotypes of hvcen (mat-c) mutants trialed under outdoor conditions over 2 consecutive years. Mutants (red) in six different genetic backgrounds are shown next to their respective parental lines (blue; Supplemental Table S1). Various traits including flowering time (A), grain number per main spike (B), number of tillers at flowering time (C), and plant height (D) were measured over years. Flowering time was scored as time from sowing to heading when the awn of the main spike emerged from the flag leaf. The line within each box denotes the median. The sample size per genotype is given in Supplemental Table S1. Error bars: sd. Significant differences between the mutants and their wild-type parents were calculated by a one-way ANOVA using a Tukey honestly significant difference as a post hoc test, ***P < 0.001, **P < 0.01, *P < 0.05. ns, Not significant.

Figure 2.

Representative shoot apices of the hvcen mutant and the wild type. Apices were scored under LD (A) and SD (B) conditions. Under SDs, the MSA did not develop beyond the stages W3.5 and W4.5 in the mutant and Bonus, respectively. DAG, days after germination; W, Waddington stage. White bar = 1 mm.

Figure 3.

Phenotyping of three hvcen mutants (mat-c.907, mat-c.94, mat-c.943) and the wild-type Bonus under controlled LD and SD conditions. Development of the MSA and number of spikelet primordia and of axillary buds at different Waddington stages under LDs (A, C, E) and SDs (B, D, F) are shown. The number of spikelet primordia refers to the number of primordia per main spike. The axillary buds scored include all the axillary buds, primary, secondary, and higher order buds. Five or six plants per genotype were dissected at each time point under LDs (16 h light/8 h night) and SDs (8 h/16 h, light/dark). Statistical differences (P < 0.05) were calculated using a polynomial regression model at 95% confidence interval (Loess smooth line) shown in gray-shaded regions.

Figure 4.

Microscopic phenotypes of hvft3, hvelf3, and hvcen single mutants and hvft3 hvcen and hvelf3 hvcen double mutants in Bonus under SDs. Development of the MSA, number of spikelet primordia, and number of axillary buds in HvFT3 or hvft3 background (A, B, C) and HvELF3 or hvelf3 background (C, D, E) at different Waddington stages under SDs (8 h/16 h, light/dark) are shown. The hvcen line was mat-c.907. The number of spikelet primordia is referring to the number of primordia per main spike. The axillary buds scored include all the axillary buds, primary, secondary, and higher order buds. Three to six plants per genotype were dissected at each time point. Statistical differences (P < 0.05) were calculated using a polynomial regression model at 95% confidence interval (Loess smooth line) shown in gray-shaded regions.

Figure 5.

Expression pattern of HvCEN under LDs. A and B, RNA in situ hybridization using the antisense probe. Red arrows indicate the HvCEN expression domains. C and D, Negative controls using the sense probe of HvCEN in the shoot apex of cv Bowman at Waddington stages 2.0 and 3.5. Scale bars = 100 µm. E, Expression of HvCEN in the pure inflorescence meristem of cv Bonus. Pure meristems, with six replicates per stage, were obtained by carefully removing leaf primordia and stem tissue beneath the inflorescence under the microscope. LDs are 16 h/8 h light/dark. W, Waddington stage. Error bars = sd.

Figure 6.

DETs regulated between two hvcen mutants (mat-c.907 and mat-c.943) and wild type (Bonus) in at least one photoperiod condition (LD/SD) and coexpression patterns of the DETs. A, Number of DETs regulated in both mutants (mat-c.907 and mat-c.943) compared with wild type (Bonus) under LDs and/or SDs. The red circles display DETs detected under LDs, and blue circles show DETs detected under SDs. B, Heatmap of coexpression clusters for 4527 DETs. Colors represent log2-fold changes (log2-FC) in expression levels relative to the mean transcript abundance across the tested conditions, i.e. apex (enriched) samples of mat-c.907, mat-c.943, and Bonus grown under LD and SD conditions and harvested at different developmental stages (W1.0, W2.0, W3.5). LDs are 16 h/8 h, light/dark; SDs are 8 h/16 h, light/dark. W, Waddington stage. Transcripts with false discovery rate (FDR) < 0.01 were considered as DETs.

Figure 7.

Expression profiles of selected DETs between the hvcen mutants (mat-c.907, mat-c.943) and wild type (Bonus) at W2.0. Genes related to chromatin modification regulation of development, and organ initiation (A); ribosomal proteins, leaf development, leaf patterning (B); regulation of hormone level and hormone response (C); and cellular respiration, sink strength, carbohydrate metabolism, and glycolytic processes (D) under both LDs and SDs. LDs and SDs are shown by white and gray colors. LDs are 16 h/8 h, light/dark; SDs are 8 h/16 h, light/dark; white, light period (16 h); gray, dark period (8 h); W, Waddington stage. Error bars = sd. Transcripts with FDR < 0.01 were considered as DETs. Ten MSAs were pooled for each of the three biological replicates for each time point and genotype.

Figure 8.

Expression profiles of selected DETs between the hvcen mutants (mat-c.907, mat-c.943) and wild type (Bonus) at W3.5 under both LDs and SDs. A, Transcripts (selected genes in Supplemental Table S8) up-regulated to a higher extent in the mutants compared with wild type only under LD. B and C, Floral homeotic genes and SWEET Suc transporter upregulated in the mutants compared with wild type under LDs and SDs. D, transcripts down-regulated over development under SDs and higher expressed in the mutants vs wild type. LDs and SDs are shown by white and gray colors. LDs are 16 h/8 h, light/dark; SDs are 8 h/16 h, light/dark; white, light period (16 h); gray, dark period (8 h); W, Waddington stage. Error bars = sd. Ten MSAs were pooled for each of the three biological replicates for each time point and genotype. FPF1, FLOWERING PROMOTING FACTOR 1; AB, ALPHA/BETA BARREL domain protein; ACB, ACYL-COA-BINDING PROTEIN 6 (AtACBP6); CNG, CYCLIC NUCLEOTIDE-GATED ION CHANNEL2 (CNGC2).