Summary The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA

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Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana

Many plants, including Arabidopsis, increase in freezing tolerance in response to low, nonfreezing temperatures, a phenomenon known as cold acclimation. Previous studies established that cold acclimation involves rapid expression of the CBF transcriptional activators (also known as DREB1 proteins) in response to low temperature followed by induction of the CBF regulon (CBF-targeted genes), which contributes to an increase in freezing tolerance. Here, we present the results of transcriptome-profiling experiments indicating the existence of multiple low-temperature regulatory pathways in addition to the CBF cold response pathway. The transcript levels of ∼8000 genes were determined at multiple times after plants were transferred from warm to cold temperature and in warm-grown plants that constitutively expressed CBF1, CBF2, or CBF3. A total of 306 genes were identified as being cold responsive, with transcripts for 218 genes increasing and those for 88 genes decreasing threefold or more at one or more time points during the 7-day experiment. These results indicate that extensive downregulation of gene expression occurs during cold acclimation. Of the cold-responsive genes, 48 encode known or putative transcription factors. Two of these, RAP2.1 and RAP2.6, were activated by CBF expression and thus presumably control subregulons of the CBF regulon. Transcriptome comparisons indicated that only 12% of the cold-responsive genes are certain members of the CBF regulon. Moreover, at least 28% of the cold-responsive genes were not regulated by the CBF transcription factors, including 15 encoding known or putative transcription factors, indicating that these cold-responsive genes are members of different low-temperature regulons. Significantly, CBF expression at warm temperatures repressed the expression of eight genes that also were downregulated by low temperature, indicating that in addition to gene induction, gene repression is likely to play an integral role in cold acclimation.

https://bioinformatics.cs.vt.edu/~easychair/FowlerThomashow_PlantCell_2002.pdf

Arabidopsis Transcriptome Profiling Indicates That Multiple Regulatory Pathways Are Activated during Cold Acclimation in Addition to the CBF Cold Response Pathway

A major quantitative trait locus (QTL) controlling response to photoperiod, Hd1, was identified by means of a map-based cloning strategy. High-resolution mapping using 1505 segregants enabled us to define a genomic region of ∼12 kb as a candidate for Hd1. Further analysis revealed that the Hd1 QTL corresponds to a gene that is a homolog of CONSTANS in Arabidopsis. Sequencing analysis revealed a 43-bp deletion in the first exon of the photoperiod sensitivity 1 (se1) mutant HS66 and a 433-bp insertion in the intron in mutant HS110. Se1 is allelic to the Hd1 QTL, as determined by analysis of two se1 mutants, HS66 and HS110. Genetic complementation analysis proved the function of the candidate gene. The amount of Hd1 mRNA was not greatly affected by a change in length of the photoperiod. We suggest that Hd1 functions in the promotion of heading under short-day conditions and in inhibition under long-day conditions.

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Hd1, a Major Photoperiod Sensitivity Quantitative Trait Locus in Rice, Is Closely Related to the Arabidopsis Flowering Time Gene CONSTANS

Flowering is often triggered by exposing plants to appropriate day lengths. This response requires an endogenous timer called the circadian clock to measure the duration of the day or night. This timer also controls daily rhythms in gene expression and behavioural patterns such as leaf movements. Several Arabidopsis mutations affect both circadian processes and flowering time; but how the effect of these mutations on the circadian clock is related to their influence on flowering remains unknown. Here we show that expression of CONSTANS (CO), a gene that accelerates flowering in response to long days, is modulated by the circadian clock and day length. Expression of a CO target gene, called FLOWERING LOCUS T (FT), is restricted to a similar time of day as expression of CO. Three mutations that affect circadian rhythms and flowering time alter CO and FT expression in ways that are consistent with their effects on flowering. In addition, the late flowering phenotype of such mutants is corrected by overexpressing CO. Thus, CO acts between the circadian clock and the control of flowering, suggesting mechanisms by which day length regulates flowering time.

CONSTANS mediates between the circadian clock and the control of flowering in Arabidopsis

Plants respond to the changing seasons to initiate developmental programmes precisely at particular times of year. Flowering is the best characterized of these seasonal responses, and in temperate climates it often occurs in spring. Genetic approaches in Arabidopsis thaliana have shown how the underlying responses to changes in day length (photoperiod) or winter temperature (vernalization) are conferred and how these converge to create a robust seasonal response. Recent advances in plant genome analysis have demonstrated the diversity in these regulatory systems in many plant species, including several crops and perennials, such as poplar trees. Here, we report progress in defining the diverse genetic mechanisms that enable plants to recognize winter, spring and autumn to initiate flower development.

The genetic basis of flowering responses to seasonal cues

Flowering of Arabidopsis is promoted by long days and delayed by short days. Mutations in the GIGANTEA (GI) gene delay flowering under long days but have little or no effect under short days. We have now isolated the GI gene and show that it encodes a novel, putative membrane protein. By comparing the sequence of the Arabidopsis gene with that of a likely rice orthologue and by sequencing mutant alleles, we identify regions of the GI protein that are likely to be important for its function. We show that GI expression is regulated by the circadian clock with a peak in transcript levels 8-10 h after dawn. The timing, height and duration of this peak are influenced by daylength. We analysed the interactions between GI and the LHY, CCA1 and ELF3 genes, previously shown to affect daylength responses; we show that the rhythmic pattern of GI expression is altered in the elf3, CCA1-OX and lhy genotypes, and that CCA1 and LHY expression are reduced by gi mutations. Our results are consistent with the idea that GI plays an important role in regulating the expression of flowering time genes during the promotion of flowering by photoperiod.

/pdf/gigantea-a-circadian-clock-controlled-gene-that-regulates-4ekkjv3apn.pdf

GIGANTEA: a circadian clock-controlled gene that regulates photoperiodic flowering in Arabidopsis and encodes a protein with several possible membrane-spanning domains.

The circadian clock acts as the timekeeping mechanism in photoperiodism. In Arabidopsis thaliana, a circadian clock–controlled flowering pathway comprising the genes GIGANTEA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) promotes flowering specifically under long days. Within this pathway, GI regulates circadian rhythms and flowering and acts earlier in the hierarchy than CO and FT, suggesting that GI might regulate flowering indirectly by affecting the control of circadian rhythms. We studied the relationship between the roles of GI in flowering and the circadian clock using late elongated hypocotyl circadian clock associated1 double mutants, which are impaired in circadian clock function, plants overexpressing GI (35S:GI), and gi mutants. These experiments demonstrated that GI acts between the circadian oscillator and CO to promote flowering by increasing CO and FT mRNA abundance. In addition, circadian rhythms in expression of genes that do not control flowering are altered in 35S:GI and gi mutant plants under continuous light and continuous darkness, and the phase of expression of these genes is changed under diurnal cycles. Therefore, GI plays a general role in controlling circadian rhythms, and this is different from its effect on the amplitude of expression of CO and FT. Functional GI:green fluorescent protein is localized to the nucleus in transgenic Arabidopsis plants, supporting the idea that GI regulates flowering in the nucleus. We propose that the effect of GI on flowering is not an indirect effect of its role in circadian clock regulation, but rather that GI also acts in the nucleus to more directly promote the expression of flowering-time genes.

/pdf/distinct-roles-of-gigantea-in-promoting-flowering-and-1xheavo21s.pdf

Distinct Roles of GIGANTEA in Promoting Flowering and Regulating Circadian Rhythms in Arabidopsis

Gene tagging with insertional mutagens greatly facilitates the isolation of novel genes. A new collection of Arabidopsis T-DNA tag insertion lines (n=2165) was generated by in planta transformation. Whole plants were vacuum-infiltrated in a suspension of Agrobacterium carrying the pGKB5 tagging vector. The efficiency of transformation increased with addition of the surfactant Silwet L-77 (0.005% v/v) to the Agrobacterium suspension. Visual screens of the T-DNA lines identified two mutants with floral defects. Allelism tests suggested that a mutation in the GIGANTEA gene was responsible for the late-flowering phenotype of one of the mutants. Linkage analysis indicated that the GIGANTEA gene was tagged in this mutant.

T-DNA tagging of a flowering-time gene and improved gene transfer by in planta transformation of Arabidopsis

L'invention porte sur des isolats d'acide nucleique comportant des sequences nucleotidiques codant pour des polypeptides a fonction GI, ou complementaires desdites sequences. Des produits d'assemblage renfermant lesdits acides nucleiques peuvent servir a transformer certaines plantes pour reguler la floraison ou l'accumulation d'amidon dans les plantes transformees.

Sarah Fowler

Papers

GIGANTEA: a circadian clock-controlled gene that regulates photoperiodic flowering in Arabidopsis and encodes a protein with several possible membrane-spanning domains.

Distinct Roles of GIGANTEA in Promoting Flowering and Regulating Circadian Rhythms in Arabidopsis

T-DNA tagging of a flowering-time gene and improved gene transfer by in planta transformation of Arabidopsis

Genes de regulation de plantes