GAMYB-like genes, flowering, and gibberellin signaling in Arabidopsis.

We have identified three Arabidopsis genes with GAMYB-like activity, AtMYB33, AtMYB65, and AtMYB101, which can substitute for barley (Hordeum vulgare) GAMYB in transactivating the barley alpha-amylase promoter. We have investigated the relationships between gibberellins (GAs), these GAMYB-like genes, and petiole elongation and flowering of Arabidopsis. Within 1 to 2 d of transferring plants from short- to long-day photoperiods, growth rate and erectness of petioles increased, and there were morphological changes at the shoot apex associated with the transition to flowering. These responses were accompanied by accumulation of GAs in the petioles (GA(1) by 11-fold and GA(4) by 3-fold), and an increase in expression of AtMYB33 at the shoot apex. Inhibition of GA biosynthesis using paclobutrazol blocked the petiole elongation induced by long days. Causality was suggested by the finding that, with GA treatment, plants flowered in short days, AtMYB33 expression increased at the shoot apex, and the petioles elongated and grew erect. That AtMYB33 may mediate a GA signaling role in flowering was supported by its ability to bind to a specific 8-bp sequence in the promoter of the floral meristem-identity gene, LEAFY, this same sequence being important in the GA response of the LEAFY promoter. One or more of these AtMYB genes may also play a role in the root tip during germination and, later, in stem tissue. These findings extend our earlier studies of GA signaling in the Gramineae to include a dicot species, Arabidopsis, and indicate that GAMYB-like genes may mediate GA signaling in growth and flowering responses.

Control of flowering time by FLC orthologues in Brassica napus.

FLOWERING LOCUS C (FLC) in Arabidopsis encodes a dosage dependent repressor of flowering. We isolated five FLC-related sequences from Brassica napus (BnFLC1-5). Expression of each of the five sequences in Arabidopsis delayed flowering significantly, with the delay in flowering time ranging from 3 weeks to more than 7 months, relative to the flowering time of 3 weeks in untransformed Ler. In the reciprocal experiment, expression of Arabidopsis FLC (AtFLC) in an early flowering B. napus cultivar delayed flowering by 2-6 weeks, confirming the requirement of this gene for floral repression. In B. napus, we show that late flowering and responsiveness to vernalization correlate with the level of BnFLC mRNA expression. The different BnFLC genes show differential expression in leaves, stems and shoot tips, but expression is not detectable in roots. Vernalization dramatically reduces the level of BnFLC transcript and restores early flowering in the winter cultivar Colombus. We conclude that BnFLC genes confer winter requirement in B. napus and account for the major vernalization-responsive flowering time differences in the different cultivars of B. napus in a manner analogous to that of AtFLC in Arabidopsis ecotypes.

The control of flowering by vernalization.

The process by which vernalization, the exposure of a germinating seed or a juvenile plant to a prolonged period of low temperature, promotes flowering in the adult plant has remained a mystery for many years. The recent isolation of one of the key genes involved in vernalization, FLOWERING LOCUS C, has now provided an insight into the molecular mechanism involved, including the role of DNA methylation.

The molecular basis of vernalization: the central role of FLOWERING LOCUS C (FLC).

In Arabidopsis, the MADS-box protein encoded by FLOWERING LOCUS C (FLC) is a repressor of flowering. Vernalization, which promotes flowering in the late-flowering ecotypes and many late-flowering mutants, decreases the level of FLC transcript and protein in the plant. This vernalization-induced reduction in FLC transcript levels is mitotically stable and occurs in all tissues. FLC activity is restored in each generation, as is the requirement of a low-temperature exposure for the promotion of flowering. The level of FLC determines the extent of the vernalization response in the promotion of flowering, and there is a quantitative relationship between the duration of cold treatment and the extent of down-regulation of FLC activity. We conclude that FLC is the central regulator of the induction of flowering by vernalization. Other vernalization-responsive late-flowering mutants, which are disrupted in genes that encode regulators of FLC, are late-flowering as a consequence of their elevated levels of FLC.

The FLF MADS box gene: a repressor of flowering in Arabidopsis regulated by vernalization and methylation.

A MADS box gene, FLF (for FLOWERING LOCUS F ), isolated from a late-flowering, T-DNA-tagged Arabidopsis mutant, is a semidominant gene encoding a repressor of flowering. The FLF gene appears to integrate the vernalization-dependent and autonomous flowering pathways because its expression is regulated by genes in both pathways. The level of FLF mRNA is downregulated by vernalization and by a decrease in genomic DNA methylation, which is consistent with our previous suggestion that vernalization acts to induce flowering through changes in gene activity that are mediated through a reduction in DNA methylation. The flf-1 mutant requires a greater than normal amount of an exogenous gibberellin (GA3) to decrease flowering time compared with the wild type or with vernalization-responsive late-flowering mutants, suggesting that the FLF gene product may block the promotion of flowering by GAs. FLF maps to a region on chromosome 5 near the FLOWERING LOCUS C gene, which is a semidominant repressor of flowering in late-flowering ecotypes of Arabidopsis.

Cloning of the Arabidopsis ent-kaurene oxidase gene GA3.

The ga3 mutant of Arabidopsis is a gibberellin-responsive dwarf. We present data showing that the ga3-1 mutant is deficient in ent-kaurene oxidase activity, the first cytochrome P450-mediated step in the gibberellin biosynthetic pathway. By using a combination of conventional map-based cloning and random sequencing we identified a putative cytochrome P450 gene mapping to the same location as GA3. Relative to the progenitor line, two ga3 mutant alleles contained single base changes generating in-frame stop codons in the predicted amino acid sequence of the P450. A genomic clone spanning the P450 locus complemented the ga3-2 mutant. The deduced GA3 protein defines an additional class of cytochrome P450 enzymes. The GA3 gene was expressed in all tissues examined, RNA abundance being highest in inflorescence tissue.

Methylation controls the low temperature induction of flowering in Arabidopsis.

Control of the transition to flowering is critical for reproductive success of a plant. Studies in Arabidopsis have led us to suggest how this species has harnessed the environmental cue of a period of low temperature to ensure flowering occurs at an appropriate time. We propose that Arabidopsis has both vernalization-independent and vernalization-dependent pathways for the initiation of inflorescence development in the shoot apex. The vernalization-independent pathway may be concerned with the supply of carbohydrate to the shoot apex. In late flowering ecotypes which respond to vernalization the vernalization-independent pathway is blocked by the action of two dominant repressors of flowering, FRI and FLC, which interact to produce very late flowering plants which respond strongly to vernalization. We have isolated a gene which may correspond to FLC. We suggest the vernalization-dependent pathway, which may be concerned with apical GA biosynthesis, is blocked by methylation of a gene critical for flowering. This gene may correspond to that encoding kaurenoic acid hydroxylase (KAH), an enzyme catalysing a step in the GA biosynthetic pathway. Under this scheme vernalization causes unblocking of this pathway by demethylation possibly of the KAH gene and consequent biosynthesis of active GAs in the apex.

Is hammerhead self-cleavage involved in the replication of a virusoid in vivo?

The hammerhead self-cleavage reaction is generally considered to be involved in the in vivo production of (+) and (-) monomeric RNAs of the viroid-like, satellite RNA or virusoid of lucerne transient streak virus (vLTSV) from multimeric replicative intermediates, as part of the symmetrical rolling circle mechanism. To test this, three cDNA clones of vLTSV RNA, mutated at sites that inactivate in vitro self-cleavage of (-) RNA, were inoculated as excised plasmid cDNA inserts, together with helper virus LTSV, on Nicotiana clevelandii plants. As was predicted if hammerhead self-cleavage is involved in in vivo cleavage of (-) RNAs, high molecular weight (-) vLTSV RNAs were present in total RNA extracts from these mutant inoculated plants, but not in wild-type inoculated plants. Surprisingly however, the mutated virusoids also produced monomeric (-) RNAs in vivo. Sequence analysis of cDNA clones prepared from progeny virusoid RNA revealed 8-20% of progeny contained reversions and pseudo-reversions of the introduced mutations. Hence, monomeric (-) RNAs were most likely produced by restoration of in vivo self-cleavage activity in the (-) RNA. Overall, the results indicate that mutations which disrupted self-cleavage in vitro also abolished the production of monomeric (-) RNAs in vivo and that hammerhead self-cleavage is involved in the cleavage of multimeric (-) RNAs to monomers. The observation that greater than 50% of the progeny cDNA clones generated from plants inoculated with mutant vLTSV contained base changes, compared to 20% from wild-type inoculated plants, may reflect an interesting adaptive response on the part of the mutated virusoids.

Alternative hammerhead structures in the self-cleavage of avocado sunblotch viroid RNAs.

The plus and minus RNAs of the 247 nt avocado sunblotch viroid (ASBV) undergo site specific RNA self-cleavage reactions in vitro. As with several other self-cleaving RNAs, we proposed hammerhead secondary structures for the sequence around the site of self-cleavage of both RNAs. We have shown previously that, during transcription of a dimeric plus ASBV RNA, a double-hammerhead structure formed and was necessary for self-cleavage. Here, we show that the purified full-length dimeric plus RNA, when incubated under our standard self-cleavage conditions, also self-cleaved by a double-hammerhead structure. In contrast, a dimeric minus ASBV RNA self-cleaved by a double-hammerhead structure during transcription, but by a single-hammerhead structure after purification. This illustrates the importance of the pathway of folding of the RNA in determining which active self-cleaving structure is formed.

RNA stem stability in the formation of a self-cleaving hammerhead structure.

The proposed single-hammerhead structure of the self-cleaving newt RNA is unstable due to a weak stem III and therefore is unable to mediate self-cleavage. A double-hammerhead structure with greater theoretical stability has been shown to mediate the self-cleavage of this RNA (Forster et al., 1988, Nature 334, 265). We have found that the double-hammerhead mediated self-cleavage reaction of a 40 base RNA containing the newt sequence (termed nCG) can be converted to a single-hammerhead reaction by increasing the size of stem III and/or of its loop, thereby enabling a single-hammerhead structure to form. In addition, the 5'-self-cleavage fragment of the nCG RNA can act in trans to mediate the self-cleavage of a full-length RNA by the formation of a partial double-hammerhead structure.

Mutagenesis analysis of a self-cleaving RNA.

The hammerhead structural model proposed for sequences that mediate self-cleavage of certain RNAs contains base-paired three stems and 13 conserved bases. Insertion, deletion and base substitution mutations were carried out on a 58 base RNA containing the sequence of the single-hammerhead structure of the plus RNA of the virusoid of lucerne transient streak virus, and the effects on self-cleavage assessed. Results showed that there is flexibility in the sequence requirements for self-cleavage in vitro, but alterations of the conserved sequence or predicted secondary structure generally reduced the efficiency of self-cleavage.

Self-cleaving viroid and newt RNAs may only be active as dimers.

Avocado sunblotch viroid (ASBV) is a 247-nucleotide, single-stranded, circular RNA. It is considered to replicate via a rolling-circle mechanism in which circular, monomeric plus and minus RNAs act as templates for the synthesis of longer-than-unit-length precursor RNAs. Processing of these RNAs in vivo may occur by a self-cleavage reaction, as indicated by ability of dimeric, linear plus and minus ASBV RNAs to specifically self-cleave in vitro with the excision of a monomeric RNA with 5'-hydroxyl and 2',3'-cyclic phosphodiester termini. A similar self-cleavage reaction has also been reported to occur in an RNA transcript containing a dimeric copy of a tandemly repeated, 330-base-pair sequence of the newt genome. Based on comparisons with self-cleaving plant viral satellite RNAs, hammerhead-shaped active structures, each containing one self-cleavage site, were proposed for the plus and minus ASBV RNAs and the newt RNA, but the stability of these hammerheads has been questioned. Here, more stable active structures that contain two self-cleavage sites are proposed and data supporting these models are presented.