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Cloning and sequence analysis of a FLO/LFY homologue isolated from cauliflower
Abstract
A cDNA library has been constructed from cauliflower curd in which floral development had been initiated. Two cDNAs (pBOFH3 and pBOFH8) have been isolated using the Antirrhinum flo gene as a heterologous probe. The two clones were sequenced and found to contain introns. Comparison of the deduced cDNA sequence of bofh with flo and the Arabidopsis homologue lfy reveals extensive homology. An mRNA transcript of 1.6 kb appears on northern RNA blots. This transcript can be detected, at low levels, before any obvious signs of floral differentiation, reflecting the role bofh plays in determining floral meristem identity.
... Thus, the presence of two homologs of LFY in gymnosperms and of only one in most angiosperms (probably due to the loss of the NLY-like lineage) would be Figure 2 -Alignment of deduced amino acid LFY-like complete sequences with PcLFY. Sequences include: NEEDLY and PRFLL from radiata pine (Mouradov et al., 1998; Mellerowicz et al., 1998); PbLEAFY: LFY-like protein of Populus balsamifera (GenBank accession number U93196); NtLFY from Nicotiana tabacum (Kelly et al., 1995); Leafy from Arabidopsis thaliana (Weigel et al., 1992); FLO from Antirrhinum majus (Coen et al., 1990), BOFH from Brassica oleracea (Anthony et al., 1993). Identical residues are marked by asterisks, similar residues are marked by dots, gaps are marked by dashes. ...
... A single band corresponding to the transcript size of about 1.5 kb was observed, predominantly in developing female cones. This size corresponded to the cloned cDNA size and the transcript size of angiosperm LFY orthologs (Coen et al., 1990; Weigel et al., 1992; Kelly et al., 1995; Anthony et al., 1993). ...
... As in angiosperm species, PcLFY was preferentially expressed in reproductive tissues and was not detected in fully differentiated vegetative tissues (Figures 4B and 5; Kelly et al., 1995; Anthony et al., 1993) Mouradov et al. (1998) have shown that NLY was highly expressed only in the very early developing female reproductive structures, before any specifically female attributes have arisen. In contrast, Mellerowicz et al. (1998) reported the expression of PRFLL in vegetative buds and in the early stages of the male reproductive cone. ...
In angiosperms, flower formation is controlled by meristem identity genes, one of which, FLORICAULA (FLO)/LEAFY (LFY), plays a central role. It is not known if the formation of reproductive organs of pre-angiosperm species is similarly regulated. Here, we report the cloning of a conifer (Pinus caribaea var. caribaea) FLO/LFY homolog, named PcLFY. This gene has a large C-terminal region of high similarity to angiosperm FLO/LFY orthologs and shorter regions of local similarity. In contrast to angiosperms, conifers have two divergent genes resembling LFY. Gymnosperm FLO/LFY proteins constitute a separate clade, that can be divided into two divergent groups. Phylogenetic analysis of deduced protein sequences has shown that PcLFY belongs to the LFY-like clade. Northern hybridization analysis has revealed that PcLFY is preferentially expressed in developing female cones but not in developing male cones. This expression pattern was confirmed by in situ hybridization and is consistent with the hypothesis of PcLFY being involved in the determination of the female cone identity. Additionally, mutant complementation experiments have shown that the expression of the PcLFY coding region, driven by the Arabidopsis LFY promoter, can confer the wild-type phenotype to lfy-26 transgenic mutants, suggesting that both gymnosperm and angiosperm LFY homologs share the same biological role.
... For this reason, the curd is considered as an early stage of inflorescence development and represents an interesting source of plant material to identify genes specifically expressed in reproductive meristems. Recently, the orthologues of LFY and AP1 have been cloned in cauliflower and their mRNAs detected in the cauliflower curd, suggesting that the curd contains a combination of inflorescence and floral meristems [2,3,4,5,9]. Another form of B. oleracea, the variety geminifera (Brussels sprouts), presenting proliferation of lateral vegetative meristems, was used as a source of vegetative meristem cDNAs for the enrichment. ...
... DNA used as template for the synthesis of the BoREM1 probe was obtained after linearization of CM6.2 clone (287 bp, 1456-1743 of the cDNA BoREM1). The synthesis of the LFY probe was accomplished from the BoLFY clone isolated from the subtracted cauliflower meristem cDNA library constructed in this work (276 bp, corresponding to 232-324; [2]). Hybridization was carried out on 8 µm tissue sections that had been fixed in 2% formaldehyde, 5% acetic acid, 60% ethanol, dehydrated in ethanolic series, and embedded in Paraplast Plus (Sigma). ...
Using the meristems of the cauliflower curd as a source of tissue and a series of subtractive hybridizations and amplification reactions, we have constructed a cDNA library highly enriched in cDNAs expressed in reproductive meristems. The analysis of a sample of 250 clones from this library identified 22 cDNA clones corresponding to genes specifically expressed in these cauliflower meristems. Apart from two clones that corresponded to APETALA1, and two other ones showing similarity to different aminoacyl-tRNA synthetases, the remaining clones showed no similarity to any sequence in the databases and may correspond to novel genes. One of these clones, BoREM1, was further characterized and found to correspond to a gene encoding a protein with features of regulatory proteins that follows a expression pattern very similar to the LEAFY transcripts.
... The APETALA1 gene in A. thaliana is part of the regulatory pathway that controls the transition from inflorescence to floral meristems, as well as the specification of sepals and petals in developing flowers ( Yanofsky 1995). Orthologues to the AP1 locus have been isolated in other Brassicaceae species, including S. alba (Menzel et al. 1995) and B. oleracea (Anthony et al. 1993Anthony et al. , 1996 Carr and Irish 1997). BoAP1 is the B. oleracea orthologue of AP1 and is expressed in developing B. oleracea inflorescence structures, including the cauliflower curd found in B. oleracea ssp. ...
... botrytis requires mutations in both the BoCAL and BoAP1 loci (Anthony et al. 1996; Kempin et al. 1995). Previous studies have already identified a unique nonsense mutation in exon 5 of the BoCAL allele in domesticated cauliflower ( Kempin et al. 1995 ), but it remains unclear whether mutations in BoAP1 are also present to condition the inflorescence phenotype in subspecies botrytis (Anthony et al. 1993Anthony et al. , 1996). We have shown that BoAP1 is present in duplicate copies, BoAP1-A and BoAP1-B, within B. oleracea and its close relative B. insularis. ...
Development of the cauliflower phenotype in Arabidopsis thaliana requires mutations at both the CAULIFLOWER and APETALA1 loci. BoAP1 is the Brassica oleracea orthologue to the Arabidopsis AP1 gene, and is present in two copies in Brassica genomes. The BoAP1-A gene appears to encode a full-length protein, but BoAP1-B alleles in B. oleracea contain insertions that lead to premature translation termination. The BoAP1-B allele found in most B. oleracea subspecies, including B. oleracea ssp. botrytis (domesticated cauliflower) contains a 9 bp insertion in exon 4. This insertion leads to the formation of an in-frame translation termination codon, and these alleles can encode a protein that is truncated at the K domain of this MADS-box transcriptional activator. The allele in B. oleracea ssp. oleracea (wild cabbage) lacks this insertion and instead contains a downstream 4 bp frameshift mutation resulting in the formation of a nonsense mutation. The structure of the BoAP1-B alleles suggests that they are impaired in their ability to perform their floral meristem identity function. These mutations, in conjunction with mutations at the BoCAULIFLOWER (BoCAL) locus, may be associated with the evolution of domesticated cauliflower.
... However, LFY homologues from some plants (FLO gene from snapdragon, FaLFY gene from strawberry, BOFH gene from cauliflower and AFL1 gene from apple) have not been reported to be expressed in the leaves. [13,21,33,34] This variation in the pattern and expression levels of this gene in different species may indicate the existence of a functional divergence in the FLO/LFY homologues. Many reports [35,36] have indicated that LFY and its homologues are multi-functional genes, because they can be expressed not only in the meristem. ...
LEAFY is a floral meristem identity gene in Arabidopsis thaliana, which plays a key role during flowering. BrcLEAFY (BrcLFY), a FLO/LFY homologue, was cloned from Pak Choi by reverse transcription-polymerase chain reaction (RT-PCR), and its expression patterns in different organs and the apex at different developmental stages, as well as during vernalization, were analysed by quantitative real-time PCR (qPCR). The full cDNA of BrcLFY was 1353 bp in length with an open reading frame of 1260 bp encoding 419 amino acids. qPCR results showed that BrcLFY expression gradually increased during the vegetative growth stage and increased sharply at flower differentiation stage 1, with the highest expression level at flower differentiation stage 5. When comparing BrcLFY expression in different organs, we concluded that the level in young and mature leaves was significantly higher than in the apex and other tissues at flower differentiation stage 3 and visible flower bud stage and fully-bloom stage. The BrcLFY expression in germinated seeds and seedlings with five leaves was analysed during cold treatment for 0 d, 10 d and 20 d at 4 ºC and the results showed that BrcLFY gene expression rose gradually with prolonged low temperature.
... This is similar to the expression pattern observed in A. thaliana and A. majus [6,16]. oleracea, ELF gene from Eucalyptus, AFL1 gene from apple and NFL genes from tobacco) have not been reported to express in leaf and stem [17,18,23,34]. ...
A LEAFY cDNA was cloned from the short-day (SD) plant Brassica juncea cv Varuna. (BjLFY) consists of 1261 bp encoding for a protein of 420 amino acids and an estimated isoelectric point of 6.8. The deduced amino acid showed 99% and 86% identity to Arabi-dopsis thaliana and Brassica oleracea LFY cDNA respectively. The LFY transcript was detected throughout the vegetative and reproductive phase but an increase in transcript level was observed during transition. The earlier induction of BjLFY was observed in early flowering variety of B. juncea as compared to late flowering varieties further proving the critical role LEAFY plays in floral transition.
... The UNI and FA genes are homologs of the floral meristem identity genes FLORICAULA ( FLO ; Coen et al., 1990) and LEAFY ( LFY ; Weigel et al., 1992) from Antirrhinum and Arabidopsis, respectively (Hofer et al., 1997;Molinero-Rosales et al., 1999). Other potential homologs of FLO / LFY have been identified in monocotyledonous (Colombo et al., 1998;Kyozuka et al., 1998) and dicotyledonous (Anthony et al., 1993;Rottman et al., 1993;Kelly et al., 1995;Pouteau et al., 1997;Souer et al., 1998;Molinero-Rosales et al., 1999) angiosperm species, in basal angiosperms and gnetales (Frohlich and Meyerowitz, 1997), and in the gymnosperm pine (Mouradov et al., 1998). Of the dicots studied to date, FLO/LFY transcripts were detected in the leaf primordia of tobacco, Arabidopsis, pea, Impatiens, tomato, and petunia (Kelly et al., 1995;Blázquez et al., 1997;Hofer et al., 1997;Pouteau et al., 1997;Pnueli et al., 1998;Souer et al., 1998;Molinero-Rosales et al., 1999), but mutant leaf phenotypes have been described only for those species with compound leaves: pea and tomato. ...
The compound leaf primordium of pea represents a marginal blastozone that initiates organ primordia, in an acropetal manner, from its growing distal region. The UNIFOLIATA (UNI) gene is important in marginal blastozone maintenance because loss or reduction of its function results in uni mutant leaves of reduced complexity. In this study, we show that UNI is expressed in the leaf blastozone over the period in which organ primordia are initiated and is downregulated at the time of leaf primordium determination. Prolonged UNI expression was associated with increased blastozone activity in the complex leaves of afila (af), cochleata (coch), and afila tendril-less (af tl) mutant plants. Our analysis suggests that UNI expression is negatively regulated by COCH in stipule primordia, by AF in proximal leaflet primordia, and by AF and TL in distal and terminal tendril primordia. We propose that the control of UNI expression by AF, TL, and COCH is important in the regulation of blastozone activity and pattern formation in the compound leaf primordium of the pea.
... BOFH. Anthony et al., 1993;Nicotiana tabacum. NFL. Kelly et al., 1995;Oraza sativa. ...
The FLORICAULA/LEAFY (FLO/LFY) homologues' genes are necessary for normal flower development and play a key role in diverse angiosperm species. In this paper, an orthologue of FLORICAULA/LEAFY, CmLFY (chestnut FLO/LFY), was isolated from the inflorescence of chestnut trees. Its expression was detected in various tissues. Furthermore, the flowering effectiveness of the gene was assessed with transgenic Arabidopsis. CmLFY protein showed a high degree of similarity to PEAFLO (78%), which is a homologue of FLO/LFY from pea. RT-PCR analysis showed that, CmLFY expressed at high levels in inflorescences, but not in young leaves, fruits or stems. The transgenic Arabidopsis with over-expressed CmLFY showed accelerated flowering, which supports that CmLFY encodes a functional orthologue of the FLORICAULA/LEAFY genes of angiosperms despite its sequence divergence. These results suggest that CmLFY is involved in inflorescence development in chestnut.
... This is similar to the expression pattern observed in A. thaliana and A. majus [6,16]. oleracea, ELF gene from Eucalyptus, AFL1 gene from apple and NFL genes from tobacco) have not been reported to express in leaf and stem [17,18,23,34]. ...
... This is similar to the expression pattern observed in A. thaliana and A. majus [6,16]. oleracea, ELF gene from Eucalyptus, AFL1 gene from apple and NFL genes from tobacco) have not been reported to express in leaf and stem [17,18,23,34]. ...
A LEAFY cDNA was cloned from the short-day (SD) plant Brassica juncea cv Varuna. (BjLFY) consists of 1261 bp encoding for a protein of 420 amino acids and an estimated isoelectric point of 6.8. The deduced amino acid showed 99% and 86% identity to Arabi-dopsis thaliana and Brassica oleracea LFY cDNA respectively. The LFY transcript was detected throughout the vegetative and reproductive phase but an increase in transcript level was observed during transition. The earlier induction of BjLFY was observed in early flowering variety of B. juncea as compared to late flowering varieties further proving the critical role LEAFY plays in floral transition.
... AP1 mutation determines loss of sepals and petals with leaf-like structures, whereas CAL participates in specification of flower meristem identity, and the genotypes which are mutant for both genes are arrested in development at the inflorescence meristem stage (Kempin et al., 1995). The orthologues genes, named BoAP1 (Anthony et al., 1993;Anthony et al., 1996;Carr and Irish, 1997) and BoCAL (Kempin et al., 1995), were found in B. oleracea to segregate in both wild and cultivated varieties carrying nonsense mutations and are fixed in the var. botrytis and italica (Purugganan et al., 2000). ...
Origin and Domestication of Cole Crops (
Brassica oleracea
L.): Linguistic and Literary Considerations. Various attempts have been made to locate the area of domestication of Brassica oleracea crops (i.e., cole crops). Contrasting hypotheses suggest either a North Atlantic or a Mediterranean origin. In the absence
of archaeological proof, linguistic and literary considerations can offer some insight into this issue. Expressions indicating
a deep-rooted knowledge and use of these crops are present in early works of ancient Greek and Latin literature, while no
trace of cole crops has been found in documents from ancient Egyptian or other Fertile Crescent civilizations. Most cole crop
terminology used in modern European languages can etymologically be traced to ancient Latin or Greek roots, particularly those
terms indicating the most obvious morphological feature of the primitive domesticated forms, i.e., the solid upright stem
(kaulos, caulis). Celtic tradition is not documented earlier than the Christian era, other than in stone inscriptions, and there is no clear
evidence of a “cole tradition” among the Celts. This paper gathers information from the linguistic, literary, and historical
points of view that are compatible with the domestication of B. oleracea in the ancient Greek-speaking area of Central and East Mediterranean.
... The genotypes which are mutant for both genes stop their development at the inflorescence meristem stage (Kempin et al., 1995). The orthologues genes, named BoAP1 (Anthony et al., 1993;Anthony et al., 1996;Carr and Irish, 1997) and BoCAL (Kempin et al., 1995), were found in B. oleracea to segregate in both wild and cultivated varieties carrying nonsense mutations and are fixed in the var. botrytis and italica (Purugganan et al., 2000). ...
The interest in cauliflower and broccoli cultivation has grown in recent years due to the genetic improvement programs carried out in several countries, mainly in Asia, Europe and the USA, and due to the new opportunities offered by the food industry in exploiting traditional and new phenotypes in new transformation processes (IV and V gamma). Also, the healthy compounds contained in the produce of several brassicas, that allows them to be defined as functional foods, are important for increasing the consumption of cauliflower and broccoli. The great diversity still present for Cauliflower and Broccoli in Germplasm banks, which could be exploited to provide new horticultural items, are important for breeding programmes aimed at satisfying new consumer requirements. In this context, recent scientific results, in terms of knowledge and understanding of the genetic resources available for Brassicaceae, the traditional and new breeding techniques, and the current breeding tasks are summarized here.
... The genotypes which are mutant for both genes stop their development at the inflorescence meristem stage (Kempin et al., 1995). The orthologues genes, named BoAP1 (Anthony et al., 1993;Anthony et al., 1996;Carr and Irish, 1997) and BoCAL (Kempin et al., 1995), were found in B. oleracea to segregate in both wild and cultivated varieties carrying nonsense mutations and are fixed in the var. botrytis and italica (Purugganan et al., 2000). ...
Cauliflower (Brassica oleracea var. botrytis) and broccoli (Brassica oleracea var. italica) are traditional European crops that have become widespread in Asia in recent decades whereas their presence in Europe has
been quite stable. Statistical data on cauliflower are available, whereas for broccoli they are merged with those of cauliflower
and with cabbage, and so its trends are not easy to determine.
... Predicted amino-acid sequence of PEAFLO aligned with the coding sequences of FLORICAULA (FLO; [9]) from Antirrhinum, and its homologues from tobacco (NFL1 and NFL2; [15]), cauliflower (BOFH; [33]) and Arabidopsis (LFY; [10]). Identical amino acids are boxed. ...
The vegetative phenotype of the pea mutant unifoliata (uni) is a simplification of the wild-type compound leaf to a single leaflet. Mutant uni plants are also self-sterile and the flowers resemble known floral meristem and organ identity mutants. In Antirrhinum and Arabidopsis, mutations in the floral meristem identity gene FLORICAULA/LEAFY (FLO/LFY) affect flower development alone, whereas the tobacco FLO/LFY homologue, NFL, is expressed in vegetative tissues, suggesting that NFL specifies determinacy in the progenitor cells for both flowers and leaves. In this paper, we characterised the pea homologue of FLO/LFY.
The pea cDNA homologue of FLO/LFY, PEAFLO, mapped to the uni locus in recombinant-inbred mapping populations and markers based on PEAFLO cosegregated with uni in segregating sibling populations. The characterisation of two spontaneous uni mutant alleles, one containing a deletion and the other a point mutation in the PEAFLO coding sequences, predicted that PEAFLO corresponds to UNI and that the mutant vegetative phenotype was conferred by the defective PEAFLO gene.
The uni mutant demonstrates that there are shared regulatory processes in the morphogenesis of leaves and flowers and that floral meristem identity genes have an extended role in plant development. Pleiotropic regulatory genes such as UNI support the hypothesis that leaves and flowers derive from a common ancestral sporophyll-like structure. The regulation of indeterminancy during leaf and flower morphogenesis by UNI may reflect a primitive function for the gene in the pre-angiosperm era.
... Likewise, plants overexpressing LFY genes produce ectopic flowers (Weigel and Nilsson, 1995;Souer et al., 1998). Thus, despite species-to-species variation in their expression (see Coen et al., 1990;Weigel et al., 1992;Anthony, James, and Jordan, 1993;Kelly, Bonnlander, and Meeks-Wagner, 1995;Weigel and Nilsson, 1995;Hofer et al., 1997;Pouteau et al., 1997;Kyozuka et al., 1998;Souer et al., 1998), LFY homologs appear to play a conserved role in the regulation of flower meristem formation (Weigel and Nilsson, 1995). Additionally, LFY has been implicated in the suppression of bracts in Arabidopsis (Coen and Nugent, 1994). ...
Arabidopsis and most other Brassicaceae produce an elongated inflorescence of mainly ebracteate flowers. However, the early-flowering species violet cress (Jonopsidium acaule) and a handful of other species produce flowers singly in the axils of rosette leaves. In Arabidopsis the gene LEAFY (LFY) is implicated in both the determination of flower meristem identity and in the suppression of leaves (bracts) that would otherwise subtend the flowers. In this study we examined the role of LFY homologs in the evolution of rosette flowering in violet cress. We cloned two LFY homologs, vcLFY1 and vcLFY2, from violet cress. Their exon sequences show ∼90% nucleotide similarity with Arabidopsis LFY and 99% similarity to each other. We used in situ hybridization to study vcLFY expression in violet cress. The patterns were very similar to LFY in Arabidopsis except for stronger expression in the shoot apical meristem outside of the region of flower meristem initiation. It is possible that the relatively diffuse expression of vcLFY contributes to the lack of bract suppression in violet cress. Additionally, the earliest flowers produced by violet cress express vcLFY, suggesting that accelerated flowering in violet cress could also result from changes in the regulation of vcLFY.
... The UNI and FA genes are homologs of the floral meristem identity genes FLORICAULA ( FLO ; Coen et al., 1990) and LEAFY ( LFY ; Weigel et al., 1992) from Antirrhinum and Arabidopsis, respectively (Hofer et al., 1997; MolineroRosales et al., 1999). Other potential homologs of FLO / LFY have been identified in monocotyledonous (Colombo et al., 1998; Kyozuka et al., 1998) and dicotyledonous (Anthony et al., 1993; Rottman et al., 1993; Kelly et al., 1995; Pouteau et al., 1997; Souer et al., 1998; Molinero-Rosales et al., 1999) angiosperm species, in basal angiosperms and gnetales (Frohlich and Meyerowitz, 1997), and in the gymnosperm pine (Mouradov et al., 1998). Of the dicots studied to date, FLO/LFY transcripts were detected in the leaf primordia of tobacco, Arabidopsis, pea, Impatiens, tomato, and petunia (Kelly et al., 1995; Blázquez et al., 1997; Hofer et al., 1997; Pouteau et al., 1997; Pnueli et al., 1998; Souer et al., 1998; Molinero-Rosales et al., 1999), but mutant leaf phenotypes have been described only for those species with compound leaves: pea and tomato. ...
The compound leaf primordium of pea represents a marginal blastozone that initiates organ primordia, in an acropetal manner, from its growing distal region. The UNIFOLIATA (UNI) gene is important in marginal blastozone maintenance because loss or reduction of its function results in uni mutant leaves of reduced complexity. In this study, we show that UNI is expressed in the leaf blastozone over the period in which organ primordia are initiated and is downregulated at the time of leaf primordium determination. Prolonged UNI expression was associated with increased blastozone activity in the complex leaves of afila (af), cochleata (coch), and afila tendril-less (af tl) mutant plants. Our analysis suggests that UNI expression is negatively regulated by COCH in stipule primordia, by AF in proximal leaflet primordia, and by AF and TL in distal and terminal tendril primordia. We propose that the control of UNI expression by AF, TL, and COCH is important in the regulation of blastozone activity and pattern formation in the compound leaf primordium of the pea.
... The homologues in B. oleracea are not consistently expressed in a manner that directly parallels their Arabidopsis functions. The onset of BoLFY expression does not correlate with the initiation of the floral primordium in cauliflower (Anthony et al., 1993;Jordan et al., 1994). Several lines of evidence support a model for developmental arrest in which BoAP1 and BoCAL are primary regulators. ...
The regulation of reproductive development in cauliflower (Brassica oleracea var. botrytis DC) and broccoli (B. oleracea L. var. italica Plenck) is unusual in that most enlargement occurs while development is arrested at a distinct stage. Cauliflower and broccoli
curds are composed of inflorescence meristems and flower buds, respectively. To determine whether this arrest is maintained
by altered expression of the genes that specify these steps in Arabidopsis, the expression of each copy of their homologues (MADS-box genes BoAP1-a, BoAP1-c, BoCAL, BoFUL-a, BoFUL-b, BoFUL-c, and BoFUL-d; and non-MADS-box genes BoLFY, AP2, UFO, and BoTFL1) and the cauliflower curd-specific genes CCE1 and BoREM1 were measured simultaneously in heads that were arrested at different developmental stages by varying temperature, but had
a common genotype. Transcript abundance of BoFUL paralogues and BoLFY was highest at the cauliflower stage of arrest, consistent with these genes initiating inflorescence meristems. The expression
of other genes was the same regardless of the developmental stage of arrest. The expected models can therefore be excluded,
wherein maintenance of arrest at the inflorescence meristem is a consequence of suppression of BoCAL, BoAP1-a, or BoLFY, or failure to suppress BoTFL1. Floral primordia and floral buds were normal in boap1-a boap1-c bocal triple mutants; therefore, other meristem identity genes can specify floral initiation (A-function) in B. oleracea. BoTFL1, a strong repressor of flowering in Arabidopsis, did not suppress the formation of the floral primordium in B. oleracea. Initiation of floral primordia and enlargement of floral buds in broccoli and cauliflower is not controlled solely by homologues
of the genes that do so in Arabidopsis.
Many genes are known that, upon mutation, significantly change plant development and architecture in a coordinated fashion. Of particular interest are the floral meristem identity genes, which specify the floral fate of meristems that appear at the flanks of inflorescence meristems. Several genes of this type have been cloned during recent years. Important representatives are FLORICAULA (FLO) and LEAFY (LFY), two orthologous genes from Antirrhinum majus (snapdragon) and Arabidopsis thaliana (thale cress), respectively, which encode members of a novel family of transcription factors. The expression of these genes strongly influences the time to flowering and the number of flowers formed. Studying the origin and evolution of FLO-like meristem identity genes may thus provide new insights into the evolutionary origin of flower development. Moreover, the time to flowering is a critical parameter that significantly contributes to the agronomic value of crop and forest plants. FLO-like genes may thus also be used as suitable molecular tools to design such plants according to our desires. The past few years have seen important progress in our understanding of both aspects of FLO-like genes, their evolution, and their suitability as genetic tools for the agronomic improvement of crop and forest plants.
Flowering is critical to the growth and development of plants, and LFY gene homologues play a major role in flowering initiation. To understand the genetic and molecular mechanisms underlying floral initiation and development in Brassica rapa subsp. pekinensis, BrpLFY, a homologue of LFY, was cloned using RT-PCR. Sequence analysis showed that the cDNA sequence of BrpLFY is 1,341 bp in length, with an ORF of 1,245 bp encoding a predicted protein of 415 amino acids. The predicted protein showed a high degree of identity with LFY homologues from other angiosperm species. Real-time PCR analysis showed that BrpLFY mRNA was detected in all tissues during plant development from the vegetative state to fully differentiated flowers, and its expression was highest in the cotyledon and lowest in the root. BrpLFY expression in the shoot apex increased gradually during vegetative growth and increased dramatically at stage 1 of flower bud differentiation. The relative expression peaked at stage 5 and then decreased in later stages. Moreover, the trend in BrpLFY expression level change in the shoot apex was similar regardless of variety or vernalization method. The relative expression of BrpLFY in leaves gradually decreased with leaf development. We overexpressed the gene in Arabidopsis thaliana using the floral dip method, and examined flowering time in wild-type and transgenic plants. Overexpression of BrpLFY specifically caused early flowering; the transgenic plants flowered 10-14 d earlier than did wild-type plants, and leaf number decreased by 0.5-1 when the plants bolted. Real-time PCR analysis showed that the expression of BrpLFY in transgenic Arabidopsis was higher than in wild-type plants. These results indicate that BrpLFY plays a role in promoting flowering in Chinese cabbage.
Models to simulate the induction of the "bracting" and "riciness" defects in cauliflower were estimated from field experiments. Bracting, where small cauline leaves develop and penetrate the curd surface, is caused by high temperatures - a kind of de-vernalization. Riciness, on the other hand, where small flower buds develop on the curd surface, is caused by low temperatures, especially after a preceding period of high temperature - a kind of strong vernalization. Both bracting and riciness can be induced only at certain stages during plant development. Plots of cauliflower were given periods of different temperature treatments in the field by portable compartments with both cooling and heating units. The treatments were both constant high or low temperatures (24, 18, 13 and 8°C) in 10 d periods, and alternating high and low temperatures (23/23, 23/15, 23/10 and 23/5°C) in 7 d periods. The treatments were started both before and after curd induction. The incidences of bracting and riciness in harvested curds were recorded in 84 plots with different temperature regimes. These data were used to estimate the combined effect of plant development and temperature on quality defects. The responsive developmental stages in the plants were described by a parabolic function, which depends on the apex/curd diameter. The curd diameter with the highest risk of induction of bracting was estimated to be around 12 mm (range 1-23 mm). A combined model of the diameter function and the summation of daily average temperature with a base temperature of 15°C explained 66% of the variation in the data of observed bracting. The apex diameter with the highest risk of induction of riciness was estimated to be around 0.35 mm (range 0.2-0.5 mm). A combined model of sensitive apex diameter and the preceding temperatures (average daily temperature of 10 d), during a temperature drop (average minimum temperature of 10 d), and after (average daily temperature of 6 d) explained 71% of the variation in the data of observed riciness. These models may be used to set up test equipment and to test susceptibility of new cultivars in breeding programmes. On a farm level, the models may be used to forecast risk of quality defects in cauliflower production. Alternatively, the knowledge of how the combination of developmental stage and temperature scenario induce either bracting or riciness may be used to study flower induction by temperature.
In the last decade, the study of mutants defective in floral development has contributed significantly to our understanding of floral evocation and morphogenesis. Genes in Arabidopsis thaliana and Antirrhinum majus that play key roles in (i) the transition from the vegetative to reproductive phase, (ii) the activation of floral development in specific shoots, and (iii) the unique arrangement of floral organs have been identified genetically and in many cases cloned. Many of the genes appear to encode transcription factors that act to select specific developmental programs of division and differentiation for groups of primordial cells. Other genes may be involved in detecting environmental conditions and transducing the signal to the developing meristems. Key questions remaining include how the regulatory proteins are produced in specific temporal and spatial patterns, interact with each other and initiate specific morphological programs. Although current research on floral morphogenesis has been limited to only a few species there is growing evidence that the basic processes are common to all flowering plants.Thus the information and tools currently being generated should be useful for studying a wide variety of flowering species. It seems reasonable to predict that within the next decade, we should have a fairly complete understanding of the basic mechanisms underlying floral morphogenesis and its evolution among the angiosperms. Key words: Arabidopsis thaliana, floral morphogenesis, molecular genetics.
Brassica crops are unique as various plant parts have been modified during domestication for use-for example, roots, leaves, stems, and inflorescences in various vegetables and seeds in edible oils and condiments. The genus Brassica comprises six crop species: B. nigra (2n=16), B. oleracea (2n=18), B. rapa (2n=20), B. carinata (2n=34), B. juncea (2n=36), and B. napus (2n=38). Of these, B. oleracea, B. rapa, and B. juncea (2n=36) are highly polymorphic, displaying a range of morphotypes. Cytogenetic evidence point to an archetype with a basic chromosome number of x=6. However, molecular markers strongly suggest the involvement of two evolutionary pathways: B. nigra in one direction and B. oleracea/B. rapa together in the other. Comparison of chromosome collinearity and comparative chromosome painting (CCP) suggested the paleopolyploid nature of the three basic genomes, which are composed of three variants of an ancestral genome originating through an ancient hexaploid event, nowreferred to as the triplication theory. The Brassicaceae paleoarchetype had the chromosome constitution 2n=8; following another cycle of genome duplication it produced a tetraploid genome of 2n=4x=16 referred to as the ancestral crucifer karyotype (ACK). Another cycle of genome duplication resulted into a hexaploid (2n=6x=24). This hexaploid ancestor gave rise to three diploid basic genomes following reduction in chromosome number. An alternative view proposes that a reduction in chromosome number in ACK resulted into a smaller Brassica genome, known as the ancestral Brasssicaceae karyotpe with 6 haploid chromosomes. This subsequently diverged into nigra and rapa/oleracea lineages 7.3 to 4 million years ago. Brassicas are believed to have originated in the countries surrounding the Mediterranean basin and further extension into southwest and central Asia encompassing mainly Mediterranean, Irano-Turanian, and Saharo- Sindian phytogeographical regions. It is now believed that B. rapa was the first species to be domesticated followed by B. nigra and B. juncea; B. oleracea entered into cultivation later. The history of the domestication of B. carinata and B. napus is relatively recent. Ancient Indian, Chinese, Greek, and Roman literature is extremely rich in detailing information concerning brassica crops. Based on information from these sources and genetical and molecular evidence, possible domestication centers have been constructed. Brassicas first entered into domestication as vegetables and later as edible oil crop.
Cauliflower homologues to the Arabidopsis genes leafy (Ify) and apetalal (ap1) have been isolated and designated bofh (Ify) and boap1 (ap1). A non-isotopic digoxigenin labelling system was used to visualize these homologues in the developing cauliflower curd.
Strong hybridization to antisense riboprobes indicate expression of both bofh and boap1 in all developmental stages, from curd initiation through to petal formation but not in vegetative meristems. The in situ results were supported by Northern and PCR analysis. The data indicate that these two genes are transcribed in the curd meristems,
suggesting that cauliflower curd proliferation is not due to bofh or boap1 gene inactivation, but is probably due to negative regulation by a factor/s, indicated by an apparent transcript threshold
requirement for each gene prior to floral primordia development.
Bofh reaches a peak level at curd maturity. However, boap1 reaches a peak level at pedicel elongation, suggesting that boap1 plays a primary role in specifying conversion of reiterating curd meristems to floral primordia. The differences in the pattern
of expression between Arabidopsis and cauliflower is discussed. It is shown that bofh and boap1 transcripts are detectable in apices of photoinduced Brassica campestris L. cv. Ceres, but not in apices of non-induced plants, demonstrating that they are stage-specific in both cauliflower and
cv. Ceres.
In addition, bofh and boap1 gene expression are affected by temperature, with both genes being switched off during vegetative reversion at high temperatures.
Two Eucalyptus homologues of the Arabidopsis floral homeotic gene AP1 (EAP1 and EAP2) show 60-65% homology to AP1. EAP1 and EAP2 are expressed predominantly in flower buds. EAP2 produces two different polypeptides arising from differential splicing at an intron, the shorter EAP2 protein diverging from the longer sequence after amino acid 197 and having a translation stop after residue 206. This truncated protein includes both MADS- and K-box amino acid sequences. Ectopic expression of the EAP1 or either of the two EAP2 polypeptides in Arabidopsis driven by the 35S promoter produces effects similar to the corresponding AP1 construct, causing plants to flower earlier, have shorter bolts and resemble the terminal flower mutant (tfl).
In angiosperms, the formation of the flower meristem is controlled by partially redundant flower meristem identity genes of which FLORICAULA (FLO)/LEAFY (LFY ) plays a central role. It is not known whether formation of reproductive organs of pre-angiosperm species is similarly regulated. Recently, a FLO/LFY-like cDNA, NEEDLY (NLY ), has been cloned in a conifer species Pinus radiata (D. Don). Here we report cloning of a different P
inus
radiata F
LO/L
FY-l ike cDNA, PRFLL. PRFLL had two large regions of high similarity to angiosperm FLO/LFY orthologues: amino acids 61–126 and 247–406 (50% and 81% identity, and 75% and 88% similarity, respectively, to LFY) and shorter regions of local similarity. Overall identity was 53% to LFY and 61% to NLY. Phylogenetic analysis of deduced protein sequences including partial LFY-like sequences from Pseudotsuga menziesii indicated that conifer proteins constituted a separate clade that could be divided into two groups represented by NLY and PRFLL. In contrast to angiosperms, both conifers had two paralogous proteins resembling LFY. Northern hybridisation analysis revealed expression of PRFLL in vegetative buds of juvenile, adolescent and mature trees. The transcript was not detected in vascular cambium, roots or secondary needles. To follow PRFLL expression during the early stages of cone development we analysed a temporal series of buds containing cone primordia, and developing cones, using Northern hybridisation and confocal microscopy in parallel. PRFLL mRNA was detected in buds from dominant and subordinate branches, in which cone and shoot primordia develop, and in developing male cones but not in developing female cones. Expression was particularly high in buds containing axillary primordia prior to their differentiation as male cone primordia. This is consistent with PRFLL being involved in determination of the male cone primordium identity.
A method for the development of consensus genetic markers between species of the same taxonomic family is described in this paper. It is based on the conservation of the peptide sequences and on the potential polymorphism within non-coding sequences. Six loci sequenced from Arabidopsis thaliana, AG, LFY3, AP3, FAD7, FAD3, and ADH, were analysed for one ecotype of A. thaliana, four lines of Brassica napus, and one line for each parental species, Brassica oleracea and Brassica rapa. Positive amplifications with the degenerate primers showed one band for A. thaliana, two to four bands in rapeseed, and one to two bands in the parental species. Direct sequencing of the PCR products confirms their peptide similarity with the "mother" sequence. By comparison of intron sequences, the correspondence between each rapeseed gene and its homologue in one of the parental species can be determined without ambiguity. Another important result is the presence of a polymorphism inside these fragments between the rapeseed lines. This variability could generally be detected by differences of electrophoretic migration on long non-denaturing polyacrylamide gels. This method enables a quick and easy shuttle between A. thaliana and Brassica species without cloning.
The short day plant (SDP) Chenopodium rubrum L. (ecotype 374) has been a model plant for physiological studies on photoperiodic flower initiation for many years. Using reverse transcription-polymerase chain reaction (RT-PCR) we identified a C. rubrum putative orthologue of the FLORICAULA/LEAFY genes from Antirrhinum majus and Arabidopsis thaliana, referred to as CrFL. Kinetics of the expression of CrFL in the apical part of C. rubrum during flower induction was followed using semi-quantitative RT-PCR. Expression of CrFL in vegetative apices was relatively high and started to decrease after 6 h of darkness (critical photoperiod). It reached its minimum between the 9th and the 12th hour of the 12-h inductive dark span, stayed at low levels for the next 6 h and increased again after the flower induction was completed. Our results indicate that expression of CrFL is regulated by photoperiod and that it is important both in the vegetative state and during flower development.
LEAFY (LFY) is a DNA-binding transcription factor that regulates floral meristem identity. LFY is unusual among angiosperm developmental regulators because it is not part of an extended gene family. Recent expression studies and transgenic experiments have suggested that changes at the LFY locus might have played a role in the evolution of rosette flowering, a modified plant architecture that has evolved at least three times in Brassicaceae. Here we examined the sequences of LFY genes from 16 species of Brassicaceae to evaluate whether gene duplication and/or the shift to rosette flowering correlate with changes in the molecular evolution of LFY. We found evidence of gene duplication in four taxa, but phylogenetic analysis suggested that duplicate genes have generally not persisted through multiple speciation events. This result can be explained if LFY is prone to be lost by drift due to a low probability of subfunctionalization or neofunctionalization. Despite great heterogeneity in dN/dS ratios, duplicate genes show a significant tendency to have elevated dN/dS ratios. Rosette-flowering lineages also show elevated dN/dS ratios and two of the rosette-flowering taxa, Idahoa and Leavenworthia, have some radical amino acid substitutions that are candidates for having played a causal role in the evolution of rosette flowering.
The first step in flower development is the generation of a floral meristem by the inflorescence meristem. We have analyzed how this process is affected by mutant alleles of the Arabidopsis gene LEAFY. We show that LEAFY interacts with another floral control gene, APETALA1, to promote the transition from inflorescence to floral meristem. We have cloned the LEAFY gene, and, consistent with the mutant phenotype, we find that LEAFY RNA is expressed strongly in young flower primordia. LEAFY expression procedes expression of the homeotic genes AGAMOUS and APETALA3, which specify organ identify within the flower. Furthermore, we demonstrate that LEAFY is the Arabidopsis homolog of the FLORICAULA gene, which controls floral meristem identity in the distantly related species Antirrhinum majus.
Yeast GCN4 belongs to the class of eukaryotic transcription factors whose bZIP DNA-binding domains dimerize via a leucine zipper motif that structurally resembles a coiled coil. The leucine zipper contains 4-5 highly conserved leucine residues spaced exactly 7 residues apart that are located within the alpha-helical hydrophobic interface between protein monomers. Here, we investigate the role of the four canonical leucines in the GCN4 leucine zipper by analyzing a series of mutated derivatives for their ability to activate transcription in vivo and to bind DNA in vitro. The GCN4 leucine zipper is surprisingly tolerant of mutations, with a wide variety of single substitutions at any of the four leucines including basic and acidic amino acids behaving indistinguishably from wild-type GCN4. Moreover, some derivatives containing two leucine substitutions display detectable though reduced function. These results indicate that other residues within the coiled coil are crucial for efficient dimerization, and they suggest that some eukaryotic transcriptional regulatory proteins lacking the conserved leucine repeat will dimerize through a structurally homologous motif. Interestingly, our results differ in several respects from those obtained by analyzing mutations in the GCN4 leucine zipper in the context of a lambda repressor-GCN4 zipper hybrid protein. These apparent differences may reflect a functional interrelationship between the leucine zipper and basic region subdomains for DNA-binding by bZIP proteins.
The analysis of mutations affecting flower structure has led to the identification of some of the genes that direct flower development. Cloning of these genes has allowed the formulation of molecular models of how floral meristem and organ identity may be specified, and has shown that the distantly related flowering plants Arabidopsis thaliana and Antirrhinum majus use homologous mechanisms in floral pattern formation.
Mutations in the APETALA3 (AP3) gene of A. thaliana result in homeotic transformations of petals to sepals and stamens to carpels. We have cloned the AP3 gene from Arabidopsis based on its homology to the homeotic flower gene deficiens (DEFA) from the distantly related plant Antirrhinum majus. The sequence of four ap3 mutant alleles and genetic mapping analysis prove that the DEFA homolog is AP3. Like several other plant homeotic genes, the AP3 gene contains a MADS box and likely acts as a transcription factor. The region-specific spatial expression pattern of AP3 rules out certain types of sequential models of flower development and argues in favor of a spatial model based on positional information. Since DEFA and AP3 have very similar protein products, mutant phenotypes, and spatial expression patterns, it is likely that these genes are cognate homologs.
The first step in flower development is the transition of an inflorescence meristem into a floral meristem. Each floral meristem differentiates into a flower consisting of four organ types that occupy precisely defined positions within four concentric whorls. Genetic studies in Arabidopsis thaliana and Antirrhinum majus have identified early-acting genes that determine the identify of the floral meristem, and late-acting genes that determine floral organ identity. In Arabidopsis, at least two genes, APETALA1 and LEAFY, are required for the transition of an influorescence meristem into a floral meristem. We have cloned the APETALA1 gene and here we show that it encodes a putative transcription factor that contains a MADS-domain. APETALA1 RNA is uniformly expressed in young flower primordia, and later becomes localized to sepals and petals. Our results suggest that APETALA1 acts locally to specify the identity of the floral meristem, and to determine sepal and petal development.
Arabidopsis flowers develop from groups of undifferentiated cells on the flank of an inflorescence meristem. The cells in these flower primordia must somehow assess their position within the primordium and differentiate accordingly to produce a flower with a precisely defined pattern of organ types and positions. The molecular mechanisms by which this is accomplished are largely unknown. We are studying a set of genes whose mutations give homeotic phenotypes in Arabidopsis flowers. A genetic model to explain the specification of organ identity by combinatorial action of the products of these homeotic genes is presented, along with several aspects that are not readily addressed by the model. The recent cloning of one of the Arabidopsis homeotic genes, and an additional homeotic gene from Antirrhinum, has provided an opportunity for molecular tests of our genetic model. So far, the molecular data are in accord with the genetic model.
Plants carrying the floricaula (flo) mutation cannot make the transition from inflorescence to floral meristems and have indeterminate shoots in place of flowers. The flo-613 allele carries a Tam3 transposon insertion, which allowed the isolation of the flo locus. The flo gene encodes a putative protein (FLO) containing a proline-rich N-terminus and a highly acidic region. In situ hybridization shows that the flo gene is transiently expressed in the very early stages of flower development. The earliest expression seen is in bract primordia, followed by sepal, petal, and carpel primordia, but no expression is detected in stamen primordia. This pattern of expression has implications for how flo affects phyllotaxis, organ identity, and determinacy. We propose that flo interacts in a sequential manner with other homeotic genes affecting floral organ identity.
The University of Wisconsin Genetics Computer Group (UWGCG) has been organized to develop computational tools for the analysis
and publication of biological sequence data. A group of programs that will interact with each research-article has been developed
for the Digital Equipment Corporation VAX computer using the VMS operating system. The programs available and the conditions
for transfer are described.
Making cauliflower out ofArabidopsis: The specification of floral meristem identity
- J L Bowman
- JL Bowman
Genetic control of pattern formation during flower development in Arabidopsis Molecular Biology of Plant Development, 45th Symposium of the Society of Experi-mental Biology
- Bowman Jl
- Meyerowitz
- Em
Bowman JL, Meyerowitz EM: Genetic control of pattern formation during flower development in Arabidopsis. In: Jenkins GI, Schuch W (eds) Molecular Biology of Plant Development, 45th Symposium of the Society of Experi-mental Biology, pp 89-115. The Company of Biologists, Cambridge (1992).
Floral homeotic genes: isola-tion, characterization, and expression during floral devel-opment The Molecular Biology of Flowering
- Jordan Br Anthony
Jordan BR, Anthony RG: Floral homeotic genes: isola-tion, characterization, and expression during floral devel-opment. In: Jordan BR (ed.) The Molecular Biology of Flowering, pp. 93-116. C.A.B. International (1993).
Floral homeotic genes: isolation, characterization, and expression during floral development
- B R Jordan
- R G Anthony