Wheat VRN1, FUL2 and FUL3 play critical and redundant roles in spikelet development and spike determinacy

Abstract

The spikelet is the basic unit of the grass inflorescence. In this study, we show that wheat MADS-box genes VRN1, FUL2 and FUL3 play critical and redundant roles in spikelet and spike development, and also affect flowering time and plant height. In the vrn1ful2ful3-null triple mutant, the inflorescence meristem formed a normal double-ridge structure, but then the lateral meristems generated vegetative tillers subtended by leaves instead of spikelets. These results suggest an essential role of these three genes in the fate of the upper spikelet ridge and the suppression of the lower leaf ridge. Inflorescence meristems of vrn1ful2ful3-null and vrn1ful2-null remained indeterminate and single vrn1-null and ful2-null mutants showed delayed formation of the terminal spikelet and increased number of spikelets per spike. Moreover, the ful2-null mutant showed more florets per spikelet, which together with a higher number of spikelets, resulted in a significant increase in the number of grains per spike in the field. Our results suggest that a better understanding of the mechanisms underlying wheat spikelet and spike development can inform future strategies to improve grain yield in wheat.

Keywords: MADS-box; Meristem identity; Spike development; Spikelet; Wheat.

Fig. 1.

Effect of VRN1, FUL2 and FUL3 on stem length, leaf number and heading time. Kronos plants (vrn2-null background) grown under long-day photoperiod. Stem length was determined from the base of the plant to the base of the spike. (A) Stem length in cm (n=6-12). (B) Number of true leaves (n=6-12). Alleles in red indicate homozygous null mutants and alleles in black homozygous wild-type alleles. (C) P-values from three-way ANOVAs for stem length and leaf number including all eight homozygous VRN1, FUL2 and FUL3 allele combinations (n=59). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; NS, P>0.05. (D) Heading time of vrn1-null (n=6) versus control (n=6). (E) Heading time of ful2ful3-null (n=15) vs control (n=10) in a Vrn1 background. D and E are separate experiments. Error bars are s.e.m. ***P<0.001; NS, P>0.05, calculated by unpaired, two-tailed t-tests.

Fig. 2.

Phenotypic characterization of the vrn1ful2 and vrn1ful2ful3 mutants. (A) Stems and heads of vrn1-null, vrn1ful2-null and vrn1ful2ful3-null mutants (leaves were removed before photography). (B) Rachises of the different mutants. Arrows indicate the position of the first spikelet before removal. (C-G) vrn1ful2-null mutant. (C) Spike-like structure. Arrow points to the bract subtending a basal inflorescence tiller. (D) Spike-like structure after removal of the inflorescence tillers to show subtending bracts (arrows). (E) Dissection of an inflorescence tiller showing two glumes and one lemma partially transformed into leaves, followed by four leaves. The inset with yellow border shows the meristem transition into an IM with lateral VMs. (F) Detail of white rectangle in E revealing an ovary, two anthers, and leafy-lemma and palea. (G) Leafy palea and lodicules subtending one anther and two ovaries. (H-J) vrn1ful2ful3-null mutant. (H) Normal leaves L11 to L18 with no axillary buds. L19 marks the beginning of the spike-like structure in which spikelets have been replaced by tillers subtended by leaves (L19 and L20) or bracts. (I,J) Detail of the tillers subtended by L19 (I) and L20 (J). Insets in white rectangles are the SAM of these tillers (transitioning into IM with lateral VM) and the yellow rectangle presents the exhausted IM.

Fig. 3.

Scanning electron microscopy images. Early double-ridge stage (A-C) and later stage (D-I) showing the fate of the lateral meristems. (A) Kronos control. (D,G) vrn1-null control. Red arrows indicate the repressed lower leaf ridge, and red dots the upper ridges that develop into normal spikelets (D,G). (B,E,H) vrn1ful2-null mutants. Yellow arrows indicate the partially repressed lower leaf ridges that develop into bracts (see Fig. 2D) and yellow dots indicate the upper ridges that develop into intermediate meristems that generate tiller-like structures with altered floral organs (see Fig. 2E-G). (C,F,I) vrn1ful2ful3-null mutants. Green arrows indicate basal lower leaf ridges that develop into normal leaves (see Fig. 2H) and green dots indicate upper ridges that produce lateral vegetative meristems that generate vegetative tillers with no floral organs (see Fig. 2I-J). Scale bars: 200 µm.

Fig. 4.

VRN1 and FUL2 play redundant roles in the control of spike determinacy and spikelet number. (A) Scanning electron microscopy of a normal wheat spike with a terminal spikelet in the vrn1-null mutant. (B) vrn1ful2-null mutant spike with indeterminate apical meristem. (C-E) Number of spikelets per spike in a growth chamber experiment (n=6). (C) vrn1-null (58% increase over control), (D) ful2-null (10% increase) and (E) ful3-null (no significant increase). Bars represent mean±s.e.m. and asterisks indicate statistically significant difference to the control line. **P<0.01; ***P<0.001; NS, P>0.05, calculated by unpaired, two-tailed t-test. (F) ANOVA averages and P-values for spike traits in ful2-null and sister control lines in the field (randomized complete block design with 8 blocks).