Andreas E. Müller

Flowering time control and applications in plant breeding

Shifting the seasonal timing of reproduction is a major goal of plant breeding efforts to produce novel varieties that are better adapted to local environments and changing climatic conditions. The key regulators of floral transition have been studied extensively in model species, and in recent years a growing number of related genes have been identified in crop species, with some notable exceptions. These sequences and variants thereof, as well as several major genes which were only identified in crop species, can now be used by breeders as molecular markers and for targeted genetic modification of flowering time. This article reviews the major floral regulatory pathways and discusses current and novel strategies for altering bolting and flowering behavior in crop plants.

Sugar beet contains a large CONSTANS-LIKE gene family including a CO homologue that is independent of the early-bolting (B) gene locus

Floral transition in the obligate long-day (LD) plant sugar beet (Beta vulgaris ssp. vulgaris) is tightly linked to the B gene, a dominant early-bolting quantitative trait locus, the expression of which is positively regulated by LD photoperiod. Thus, photoperiod regulators like CONSTANS (CO) and CONSTANS-LIKE (COL) genes identified in many LD and short-day (SD)-responsive plants have long been considered constituents and/or candidates for the B gene. Until now, the photoperiod response pathway of sugar beet (a Caryophyllid), diverged from the Rosids and Asterids has not been identified. Here, evidence supporting the existence of a COL gene family is provided and the presence of Group I, II, and III COL genes in sugar beet, as characterized by different zinc-finger (B-box) and CCT (CO, CO-like, TOC) domains is demonstrated. BvCOL1 is identified as a close-homologue of Group 1a (AtCO, AtCOL1, AtCOL2) COL genes, hence a good candidate for flowering time control and it is shown that it maps to chromosome II but distant from the B gene locus. The late-flowering phenotype of A. thaliana co-2 mutants was rescued by over-expression of BvCOL1 thereby suggesting functional equivalence with AtCO, and it is shown that BvCOL1 interacts appropriately with the endogenous downstream genes, AtFT and AtSOC1 in the transgenic plants. Curiously, BvCOL1 has a dawn-phased diurnal pattern of transcription, mimicking that of AtCOL1 and AtCOL2 while contrasting with AtCO. Taken together, these data suggest that BvCOL1 plays an important role in the photoperiod response of sugar beet.

The role of a pseudo-response regulator gene in life cycle adaptation and domestication of beet

Life cycle adaptation to latitudinal and seasonal variation in photoperiod and temperature is a major determinant of evolutionary success in flowering plants. Whereas the life cycle of the dicotyledonous model species Arabidopsis thaliana is controlled by two epistatic genes, FLOWERING LOCUS C and FRIGIDA, three unrelated loci (VERNALIZATION) determine the spring and winter habits of monocotyledonous plants such as temperate cereals. In the core eudicot species Beta vulgaris, whose lineage diverged from that leading to Arabidopsis shortly after the monocot-dicot split 140 million years ago, the bolting locus B is a master switch distinguishing annuals from biennials. Here, we isolated B and show that the pseudo-response regulator gene BOLTING TIME CONTROL 1 (BvBTC1), through regulation of the FLOWERING LOCUS T genes, is absolutely necessary for flowering and mediates the response to both long days and vernalization. Our results suggest that domestication of beets involved the selection of a rare partial loss-of-function BvBTC1 allele that imparts reduced sensitivity to photoperiod that is restored by vernalization, thus conferring bienniality, and illustrate how evolutionary plasticity at a key regulatory point can enable new life cycle strategies.

Flowering time variation in oilseed rape (Brassica napus L.) is associated with allelic variation in the FRIGIDA homologue BnaA.FRI.a

Oilseed rape (Brassica napus L.) is a major oil crop which is grown worldwide. Adaptation to different environments and regional climatic conditions involves variation in the regulation of flowering time. Winter types have a strong vernalization requirement whereas semi-winter and spring types have a low vernalization requirement or flower without exposure to cold, respectively. In Arabidopsis thaliana, FRIGIDA (FRI) is a key regulator which inhibits floral transition through activation of FLOWERING LOCUS C (FLC), a central repressor of flowering which controls vernalization requirement and response. Here, four FRI homologues in B. napus were identified by BAC library screening and PCR-based cloning. While all homologues are expressed, two genes were found to be differentially expressed in aerial plant organs. One of these, BnaA.FRI.a, was mapped to a region on chromosome A03 which co-localizes with a major flowering time quantitative trait locus in multiple environments in a doubled-haploid mapping population. Association analysis of BnaA.FRI.a revealed that six SNPs, including at least one at a putative functional site, and one haplotype block, respectively, are associated with flowering time variation in 248 accessions, with flowering times differing by 13–19 d between extreme haplotypes. The results from both linkage analysis and association mapping indicate that BnaA.FRI.a is a major determinant of flowering time in oilseed rape, and suggest further that this gene also contributes to the differentiation between growth types. The putative functional polymorphisms identified here may facilitate adaptation of this crop to specific environments through marker-assisted breeding.

A survey of EMS-induced biennial Beta vulgaris mutants reveals a novel bolting locus which is unlinked to the bolting gene B

Beta vulgaris is a facultative perennial species which exhibits large intraspecific variation in vernalization requirement and includes cultivated biennial forms such as the sugar beet. Vernalization requirement is under the genetic control of the bolting locus B on chromosome II. Previously, ethyl methanesulfonate (EMS) mutagenesis of an annual accession had yielded several mutants which require vernalization to bolt and behave as biennials. Here, five F2 populations derived from crosses between biennial mutants and annual beets were tested for co-segregation of bolting phenotypes with genotypic markers located at the B locus. One mutant appears to be mutated at the B locus, suggesting that an EMS-induced mutation of B can be sufficient to abolish annual bolting. Co-segregation analysis in four populations indicates that the genetic control of bolting also involves previously unknown major loci not linked to B, one of which also affects bolting time and was genetically mapped to chromosome IX.

Conservation and divergence of autonomous pathway genes in the flowering regulatory network of Beta vulgaris

The transition from vegetative growth to reproductive development is a complex process that requires an integrated response to multiple environmental cues and endogenous signals. In Arabidopsis thaliana, which has a facultative requirement for vernalization and long days, the genes of the autonomous pathway function as floral promoters by repressing the central repressor and vernalization-regulatory gene FLC. Environmental regulation by seasonal changes in daylength is under control of the photoperiod pathway and its key gene CO. The root and leaf crop species Beta vulgaris in the caryophyllid clade of core eudicots, which is only very distantly related to Arabidopsis, is an obligate long-day plant and includes forms with or without vernalization requirement. FLC and CO homologues with related functions in beet have been identified, but the presence of autonomous pathway genes which function in parallel to the vernalization and photoperiod pathways has not yet been reported. Here, this begins to be addressed by the identification and genetic mapping of full-length homologues of the RNA-regulatory gene FLK and the chromatin-regulatory genes FVE, LD, and LDL1. When overexpressed in A. thaliana, BvFLK accelerates bolting in the Col-0 background and fully complements the late-bolting phenotype of an flk mutant through repression of FLC. In contrast, complementation analysis of BvFVE1 and the presence of a putative paralogue in beet suggest evolutionary divergence of FVE homologues. It is further shown that BvFVE1, unlike FVE in Arabidopsis, is under circadian clock control. Together, the data provide first evidence for evolutionary conservation of components of the autonomous pathway in B. vulgaris, while also suggesting divergence or subfunctionalization of one gene. The results are likely to be of broader relevance because B. vulgaris expands the spectrum of evolutionarily diverse species which are subject to differential developmental and/or environmental regulation of floral transition.

EcoTILLING in Beta vulgaris reveals polymorphisms in the FLC-like gene BvFL1 that are associated with annuality and winter hardiness

BackgroundSugar beet (Beta vulgaris ssp. vulgaris L.) is an important crop for sugar and biomass production in temperate climate regions. Currently sugar beets are sown in spring and harvested in autumn. Autumn-sown sugar beets that are grown for a full year have been regarded as a cropping system to increase the productivity of sugar beet cultivation. However, for the development of these “winter beets” sufficient winter hardiness and a system for bolting control is needed. Both require a thorough understanding of the underlying genetics and its natural variation.ResultsWe screened a diversity panel of 268 B. vulgaris accessions for three flowering time genes via EcoTILLING. This panel had been tested in the field for bolting behaviour and winter hardiness. EcoTILLING identified 20 silent SNPs and one non-synonymous SNP within the genes BTC1, BvFL1 and BvFT1, resulting in 55 haplotypes. Further, we detected associations of nucleotide polymorphisms in BvFL1 with bolting before winter as well as winter hardiness.ConclusionsThese data provide the first genetic indication for the function of the FLC homolog BvFL1 in beet. Further, it demonstrates for the first time that EcoTILLING is a powerful method for exploring genetic diversity and allele mining in B. vulgaris.

Bolting and flowering control in sugar beet: relationships and effects of gibberellin, the bolting gene B and vernalization

Using a co-dominant genotypic PCR marker we show for the first time that, in sugar beet, the GA and B-gene pathways are independent for bolt initiation. We show that vernalization permits GA-dependant stem elongation and that the B-allele influences subsequent flowering.

The FLC-like gene BvFL1 is not a major regulator of vernalization response in biennial beets.

Many plant species in temperate climate regions require vernalization over winter to initiate flowering. Flowering Locus C (FLC) and FLC-like genes are key regulators of vernalization requirement and growth habit in winter-annual and perennial Brassicaceae. In the biennial crop species Beta vulgaris ssp. vulgaris in the evolutionarily distant Caryophyllales clade of core eudicots growth habit and bolting time are controlled by the vernalization and photoperiod response gene BTC1 and the downstream BvFT1-BvFT2 module. B. vulgaris also contains a vernalization-responsive FLC homolog (BvFL1). Here, to further elucidate the regulation of vernalization response and growth habit in beet, we functionally characterized BvFL1 by RNAi and over-expression in transgenic plants. BvFL1 RNAi neither eliminated the requirement for vernalization of biennial beets nor had a major effect on bolting time after vernalization. Over-expression of BvFL1 resulted in a moderate late-bolting phenotype, with bolting after vernalization being delayed by approximately 1 week. By contrast, RNAi-induced down-regulation of the BvFT1-BvFT2 module led to a strong delay in bolting after vernalization by several weeks. The data demonstrate for the first time that an FLC homolog does not play a major role in the control of vernalization response in a dicot species outside the Brassicaceae.

BvPRR7 is a cold responsive gene with a clock function in beet

The life cycle of flowering plants is partially defined by environmental cues like day length and temperature. In the model plant Arabidopsis thaliana and temperate cereals, such as barley (Hordeum vulgare) and wheat (Triticum spp.), differences in life cycle control have been associated with a natural variation in FLOWERING LOCUS C (FLC) and VERNALIZATION 1-3 (VRN1-3). In sugar beet (Beta vulgaris L.), variation in vernalization requirement and life cycle is determined by a major gene at the B locus. This gene has recently been identified as a pseudo-response regulator (PRR) gene BOLTING TIME CONTROL 1 (BTC1). A second gene in beet with homology to BTC1 and ARABIDOPSIS PSEUDO RESPONSE REGULATOR 7 (APRR7) in Arabidopsis was identified and termed Beta vulgaris PSEUDO RESPONSE REGULATOR 7 (BvPRR7). We functionally characterized BvPRR7 by transgenic analysis in Arabidopsis and expression profiling during development in beet. We show that BvPRR7 was diurnally regulated and responded to cold. Constitutive expression of BvPRR7 distorted diurnal rhythms and caused late flowering in Arabidopsis suggesting a conserved function of BvPRR7 in clock regulation. Conceivably, the retention of a functional role of BvPRR7 in clock regulation may have facilitated the evolution of a distinct role as major floral regulator of the second PRR7 homolog in beet, BTC1.