MADS box genes control vernalization-induced flowering in cereals

Abstract

By comparing expression levels of MADS box transcription factor genes between near-isogenic winter and spring lines of bread wheat, Triticum aestivum, we have identified WAP1 as the probable candidate for the Vrn-1 gene, the major locus controlling the vernalization flowering response in wheat. WAP1 is strongly expressed in spring wheats and moderately expressed in semispring wheats, but is not expressed in winter wheat plants that have not been exposed to vernalization treatment. Vernalization promotes flowering in winter wheats and strongly induces expression of WAP1. WAP1 is located on chromosome 5 in wheat and, by synteny with other cereal genomes, is likely to be collocated with Vrn-1. These results in hexaploid bread wheat cultivars extend the conclusion made by Yan et al. [Yan, L., Loukoianov, A., Tranquilli, G., Helguera, M., Fahima, T. & Dubcovsky, J. (2003) Proc. Natl. Acad. Sci. USA 100, 6263-6268] in the diploid wheat progenitor Triticum monococcum that WAP1 (TmAP1) corresponds to the Vrn-1 gene. The barley homologue of WAP1, BM5, shows a similar pattern of expression to WAP1 and TmAP1. BM5 is not expressed in winter barleys that have not been vernalized, but as with WAP1, expression of BM5 is strongly induced by vernalization treatment. In spring barleys, the level of BM5 expression is determined by interactions between the Vrn-H1 locus and a second locus for spring habit, Vrn-H2. There is now evidence that AP1-like genes determine the time of flowering in a range of cereal and grass species.

Fig. 1.

RT-PCR analysis of wheat MADS box gene expression in 2-week-old plants. Shown are RT-PCR results for the positive control gene Ubiquitin and four of the MADS box genes expressed in wheat plants before floral transition, TaMX10, TaMX17, TaMX21, TaMX23, and WAP1. Control samples were water as template (-T) or without addition reverse transcriptase (-RT). Test samples included first-strand cDNA from 14- or 21-day old TDA (a strong spring wheat with the genotype Vrn-A1/Vrn-B1/vrn-d1) and 14- or 21-day-old TDC (a vernalization-responsive winter wheat, genotype vrn-a1/vrn-b1/vrn-d1). None of the plants were vernalized. The products were all ≈150 bp and match the sizes predicted on the basis of sequence.

Fig. 2.

Expression of WAP1 compared with head emergence time for plants from the Triple Dirk and Festival cultivar series. (A) Days until head emergence for nonvernalized plants from each of the following lines: Triple Dirk (TD), Triple Dirk A (TDA), Triple Dirk D (TDD), Triple Dirk B (TDB), Triple Dirk E (TDE), Triple Dirk C^* (TDC), Spring Festival (SF), Semispring Festival (SSF), and Winter Festival^* (WF). The winter lines (^*) did not show head emergence within the 120-day time period assayed. (B) Expression of WAP1 in 2-week-old, nonvernalized plants from each line. WAP1 expression was assayed by high stringency hybridization of RNA gel blots with a WAP1-specific riboprobe. Ethidium bromide staining of ribosomal RNA is shown in C as a loading comparison. The genotype of each line is included underneath each line with uppercase letters indicating dominant spring Vrn-1 alleles. Lowercase letters represent recessive winter vrn-1 alleles.

Fig. 3.

Expression of WAP1 compared with head emergence dates for vernalized and nonvernalized spring and winter wheat cultivars. (A–D) Data from nonvernalized and vernalized plants from the lines TDA, TDD, Triple Dirk C (TDC)^*, and Cheyenne^* (CH, a winter wheat). (E–G) Data from nonvernalized plants from three lines of the winter wheat Blackhull [Blackhull (B)^*, Early Blackhull (EB)^*, and Extra Early Blackhull (EEB)^*], and then vernalized plants from each of the same three lines. Days until heading are shown for each combination of line and treatment in A and E. This does not include the 60-day vernalization period for the vernalized plants. Without vernalization, winter lines (^*) showed no head emergence within the 120-day time period assayed. RNA gel blot analysis of WAP1 expression in 2-week-old plants from each combination of line and treatment is shown in B and F.(C) Expression of TaMX23 in 2-week-old vernalized or nonvernalized plants from the Triple Dirk series and winter wheat Cheyenne, analyzed by hybridization of RNA blots with a TaMX23 specific riboprobe. Ethidium bromide staining of ribosomal RNA is shown in D and G as a loading comparison. The genotype of each line is included (a capital letter indicates dominant spring Vrn-1 allele from the respective genome), as is vernalization status by + for vernalized and - for nonvernalized.

Fig. 4.

Expression of BM5 in vernalized and nonvernalized seedlings of 12 barley cultivars. Days until heading for nonvernalized or vernalized plants are shown in A. Lane 1, Morgenrot; lane 2, vernalized Morgenrot (+V); lane 3, Randolph; lane 4, Randolph +V; lane 5, Malta; lane 6, Malta +V; lane 7, Sonja^*; lane 8, Sonja +V; lane 9, Hudson^*; lane 10, Hudson +V; lane 11, Mirra^*; lane 12, Mirra +V; lane 13, Perga; lane 14, Perga +V; lane 15, Dunja; lane 16, Dunja +V; lane 17, Will; lane 18, Will +V; lane 19, Hydra^*; lane 20 Hydra +V; lane 21, Bollo^*; lane 22, Bollo +V; lane 23 Igri^*; lane 24, Igri +V. This does not include the 60-day vernalization period for the vernalized plants. Winter lines (^*) did not show head emergence within the 120-day time period assayed when not vernalized. (B) Expression of BM5 determined by RNA gel blot analysis for 2-week-old plants from the same combinations of lines and treatments. Ethidium bromide staining of ribosomal RNA is shown in C as a loading comparison. Vernalization status (-, nonvernalized; +, vernalized) and spring habit phenotype are shown beneath each cultivar/treatment combination.

Fig. 5.

Expression of BM5 in seedlings of barleys of known spring habit genotype. Expression of BM5 in nonvernalized barley cultivars was determined by hybridizing RNA gel blots with a BM5-specific riboprobe. Cha., Chame 11 (Vrn-H1 spring); Sik, Sikangense Type 15 (Vrn-H1 spring); Ton., Tongyeong Covered (vrn-H2 spring); Ich, Icheon Naked (Vrn-H1/vrn-H2 spring); Him., Himalayense Type 5 (Vrn-H1/Vrn-H3 spring); Oll, Olli (Vrn-H1/ vrn-H2/Vrn-H3 spring); Mir., Mirra (winter); Igr, Igri (winter). Numbers below each lane indicate locus or loci where spring alleles are present: 1, Vrn-H1; 2, vrn-H2;or3, Vrn-H3. Ethidium bromide staining of ribosomal RNA is shown in B as a loading comparison.

Fig. 6.

RT-PCR assay of BM5 expression in spring and winter barley lines. RT-PCR was performed with 12 spring and 3 winter barley cultivars, and the resulting fragments were separated by gel electrophoresis. Lane 1, No template; lane 2, Chame 11; lane 3, Sikangense Type 15; lane 4, Tongyeong Covered; lane 5, Icheon Naked; lane 6, Himalayense Type 5; lane 7, Olli; lane 8, Morgenrot; lane 9, Randolph; lane 10, Malta; lane 11, Perga; lane 12, Dunja; lane 13, Will; lane 14, Hudson; lane 15, Mirra; lane 16, Hydra. For spring barleys of known genotype, the presence of spring alleles is shown beneath each sample as 1 (Vrn-H1), 2 (vrn-H2), or 3 (Vrn-H3).

Fig. 7.

A model for the regulation of BM5 expression by vernalization and spring habit genotypes. Vernalization controls two genetic systems that regulate BM5 transcription through the BM5 promoter. One represses BM5 expression in plants that have not been vernalized. Extended cold treatment counteracts this repression and activates a second regulatory mechanism that activates BM5 transcription. Known flowering time genes such as the barley equivalent of SOC1 are likely to be involved in this activation pathway. Recessive spring habit alleles of the Vrn-H2 locus abolish the repression pathway and allow some expression of BM5. Dominant alleles of Vrn-H1 activate expression regardless of vernalization status, but do not completely counteract repression, leading to some BM5 activity. Combining both spring habit genotypes results in BM5 expression levels that approach those in vernalized plants, through simultaneous activation and de-repression of the BM5 promoter.