The B2 flowering time locus of beet encodes a zinc finger transcription factor

Abstract

Sugar beet (Beta vulgaris) is a biennial root crop that grows vegetatively in the first year and starts shoot elongation (bolting) and flowering after exposure to cold temperatures over winter. Early bolting before winter is controlled by the dominant allele of the B locus. Recently, the BOLTING time control 1 (BTC1) gene has been cloned from this locus. BTC1 promotes early bolting through repression of the downstream bolting repressor B. vulgaris flowering locus T1 (BvFT1) and activation of the downstream floral activator BvFT2. We have identified a new bolting locus B2 acting epistatically to B. B2 houses a transcription factor which is diurnally regulated and acts like BTC1 upstream of BvFT1 and BvFT2. It was termed BvBBX19 according to its closest homolog from Arabidopsis thaliana. The encoded protein has two conserved domains with homology to zinc finger B-boxes. Ethyl methanesulfonate-induced mutations within the second B-box caused up-regulation of BvFT1 and complete down-regulation of BvFT2. In Arabidopsis, the expression of FT is promoted by the B-box containing protein CONSTANS (CO). We performed a phylogenetic analysis with B-box genes from beet and A. thaliana but only BvCOL1 clustered with CO. However, BvCOL1 had been excluded as a CO ortholog by previous studies. Therefore, a new model for flowering induction in beet is proposed in which BTC1 and BvBBX19 complement each other and thus acquire a CO function to regulate their downstream targets BvFT1 and BvFT2.

Keywords: map-based cloning; sucrose; winter beet.

Fig. 1.

Map-based cloning of the B2 gene in beet. (A) Genetic map position of the B2 locus on chromosome 9. (B) Physical map of the B2 locus and the location of the two sequence scaffolds sc00497 and sc00048, covering the B2 locus. Crossover events are given as black triangles. Black boxes indicate gene models. (C) Molecular marker CAU3784 used for mapping the B2 locus; M₁ and M₂ are the alleles from annual and biennial parents, respectively.

Fig. 2.

(A) In silico prediction of the BvBBX19 gene structure. Exons are drawn as open boxes; B-box regions (BB1, BB2) are drawn in blue; red bars: 3′-UTR and 5′-UTR; black bars: translated region; Green bars: introns. (B) Sequence variations within BvBBX19 alleles from the two EMS mutants (seed codes 056822 and 031823) and the nonmutated donor line (930190); gray triangles: SNP positions; black triangles: nucleotide derived from the nonmutated donor line; black asterisk: premature stop codon. (C) Multiple alignment of BvBBX19 protein sequences from beet and from A. thaliana (AtBBX19); +: highly conserved amino acid positions within BB2 (Conserved Domains Database, NCBI). Amino acid changes due to EMS mutations are highlighted in red; asterisks: stop codon.

Fig. 3.

Maximum likelihood phylogenetic tree of 15 BBX deduced amino acid sequences identified in the sugar beet genome. The respective domain structures of the proteins and their chromosomal locations in the sugar beet genome are given in Table S4. The bootstrap consensus tree inferred from 1,000 replicates was constructed by Mega5.2 after aligning the predicted protein sequences from 15 BvBBXs by MAFFT. The percentage bootstrap values are indicated at the branch points. Scale bar represents 0.2 amino acid substitutions per site. BvCOL1, BvCOL2, and BvCOL3 have been already published by Chia et al. (9). Best Arabidopsis homologs of uncharacterized BvBBXs are indicated in brackets.

Fig. 4.

Diurnal expression analysis of BvBBX19 in beet. Leaves were harvested 44 d after sowing. Each value is the mean of three biological and three technical replicates. The relative BvBBX19 expression is given on the left vertical axis. The bar at the bottom indicates light (open bar) and dark (black bar) phases. Error bars represent the SEM of three biological replicates.

Fig. 5.

Phenotypes of annual and biennial beets (nonvernalized and vernalized) and the expression of BvBBX19 and three putative downstream targets. (A) The annual wild beet accession 991971 (Left) carries the dominant BTC1 allele and bolts without vernalization. The biennial sugar beet accession 93161P (Right) carries the recessive btc1 allele and does not bolt without vernalization. (B) The biennial sugar beet accession 93161P does not bolt without vernalization (Left) but bolts after vernalization (Right). (C) The B2 mutant plant from the M₃ line 056822 with the dominant BTC1 allele and the mutated B2 allele does not bolt without vernalization (Left) but bolts after vernalization (Right). (D) Expression analysis of BvBBX19, BTC1, BvFT1, and BvFT2 in annual (seed codes 991971 and 001684) and biennial accessions (seed codes 056822 and 93161P). The biennial B2 mutant 056822 was obtained after EMS treatment of the annual accession 001684. All plants were grown in a greenhouse under LD conditions for 7 wk. Then, biennials were vernalized for 12 wk and grown in the greenhouse under LD conditions. Black boxes, gene expression 44 d after sowing; gray boxes, gene expression 2 wk after vernalization. Each value is the mean of three biological and three technical replicates. The relative gene expression is given on the left vertical axis. Error bars represent the SEM of three biological replicates. Statistically significant different gene expression was analyzed applying student’s t test (confidence interval 95%) between: a, the annual EMS donor 001684 and biennial accessions (seed code 056822, 93161P); b, nonvernalized and vernalized mutant 056822; c, nonvernalized biennial accessions; and d, vernalized biennial accessions.

Fig. 6.

A proposed epistatic model for bolting time control in beet with BvBBX19 and BTC1_d acting upstream of BvFT1 and BvFT2. The domain structure of BvBBX19 and BTC1_d proteins is indicated by boxes. The open box represents the mutated domain. An interaction between the two proteins BvBBX19 and BTC1_d or binding of the BvBBX19 protein to the BTC1_d promoter to acquire a CO function is anticipated. In annuals, the proteins BvBBX19 and BTC1_d act together to repress BvFT1 and activate BvFT2 to promote bolting and flowering under LD conditions, whereas a mutation in B2 mutants prevents floral transition without vernalization, because the floral activator BvFT2 is repressed by BvFT1. After vernalization and under LD conditions BvFT2 is activated to initiate bolting and flowering.