The cotyledons produce sufficient FT protein to induce flowering: evidence from cotyledon micrografting in Arabidopsis.

In Arabidopsis, long-distance movement of FLOWERING LOCUS T (FT) protein from the leaf to the shoot apex triggers flower development. In wild-type Arabidopsis plants under long-day conditions, FT is mainly expressed in the cotyledon but is weakly expressed in the first true leaf prior to floral induction. To test the importance of the cotyledon in floral induction, we developed a cotyledon micrografting (Cot-grafting) method that, unlike other grafting methods, allows the FT protein from the graft to be transported via its native route from leaves to the shoot apex. By using Cot-grafting, we found that grafting a single wild-type cotyledon onto an ft-10 mutant strongly suppressed the ft-10 late flowering phenotype. Neither Y-grafting wild-type shoots nor butt-grafting wild-type roots to ft-10 plants resulted in comparably accelerated flowering in the ft-10 recipient plants. ft-10 mutants grafted with a 35S::FT cotyledon flowered as early as wild-type plants. When phloem-specific tracers were applied to a donor cotyledon, the tracers were detected in the vein of the true leaf of recipient plants 6 d after Cot-grafting. Also, macromolecule trafficking of an FT:yellow fluorescent protein:hemagglutinin fusion occurred across the graft junction 6 d after Cot-grafting. These results suggest that Cot-grafting, which allows protein movement in a manner consistent with the natural flow of FT protein from the leaf to the shoot apex, can efficiently suppress the late flowering of ft-10 mutants. Our results further suggest that in Arabidopsis, the cotyledon is an important organ for producing FT protein to induce flowering.

A genetic screen for leaf movement mutants identifies a potential role for AGAMOUS-LIKE 6 (AGL6) in circadian-clock control.

The circadian clock in plants regulates many important physiological and biological processes, including leaf movement. We have used an imaging system to genetically screen Arabidopsis seedlings for altered leaf movement with the aim of identifying a circadian clock gene. A total of 285 genes were selected from publicly available microarrays that showed an expression pattern similar to those of the Arabidopsis core oscillator genes. We subsequently isolated 42 homozygous recessive mutants and analyzed their leaf movements. We also analyzed leaf movements of activation tagging mutants that showed altered flowering time. We found that agl6-1D plants, in which AGAMOUS-LIKE 6 (AGL6) was activated by the 35S enhancer, showed a shortened period of leaf movement as well as a high level of ZEITLUPE (ZTL) expression, reduced amplitude of LATE ELONGATED HYPOCOTYL (LHY) expression, and arrhythmic TIMING OF CAB EXPRESSION1 (TOC1)/CIRCADIAN CLOCK ASSOCIATED1 (CCA1) expression. A shortened period of leaf movement was also seen in 35S-AGL6-myc plants, although 35S-amiRAGL6 plants, transgenic plants overexpressing an artificial miRNA (amiR) targeting AGL6, showed unaltered leaf movement. The amplitude of CHLOROPHYLL A/B BINDING PROTEIN 2 (CAB2) expression, a circadian output gene, was also reduced in agl6-1D plants. Taken together, these results suggest that AGL6 plays a potential role in the regulation of the circadian clock by regulating ZTL mRNA level in Arabidopsis.

Identification and testing of superior reference genes for a starting pool of transcript normalization in Arabidopsis.

Genes that are stably expressed during development or in response to environmental changes are essential for accurate normalization in qRT-PCR experiments. To prevent possible misinterpretation caused by the use of unstable housekeeping genes, such as UBQ10, ACT, TUB and EF-1α, as a reference, the use of 20 stably expressed genes identified from microarray analyses was proposed. Furthermore, it was recommended that at least four genes among them be tested to identify suitable reference genes under different experimental conditions. However, testing the 20 potential reference genes under any condition is inefficient. Furthermore, since their stability still varies, there is a need to identify a subset of genes that are more stable than others, which can be used as a starting pool for testing. Here, we validated the expression stability of the potential candidate genes together with the above-mentioned conventional reference genes under six experimental conditions commonly used in plant developmental biology. To increase fidelity, three independent validation experiments were carried out for each experimental condition. A hypothetical normalization factor, which is the geometric mean of genes that were identified as stably expressed genes in each experiment, was used to exclude unstable genes under a given condition. We identified a subset of genes showing higher expression stability under specific experimental conditions. We recommend the use of these genes as a starting pool for the identification of suitable reference genes under given experimental conditions to ensure accurate normalization in qRT-PCR analysis.

Isolation of 151 mutants that have developmental defects from T-DNA tagging.

In order to understand the mechanisms underlying plant development, a necessary first step involves the elucidation of the functions of the genes, via the analysis of mutants that exhibit developmental defects. In this study, an activation tagging mutant library harboring 80,650 independent Arabidopsis transformants was generated in order to screen for developmental mutants. A total of 129 mutants manifesting dominant developmental abnormalities were isolated, and their T-DNA insertion loci were mapped. The activation of one or more genes adjacent to a T-DNA insertion locus was confirmed in eight dominant mutants. A gene adjacent to the right border was usually activated by the 35S enhancers. Interestingly, the transcriptional activation of multiple genes within a broad range was observed in one of the mutants, which raises the possibility that activation by the 35S enhancers was not limited strictly to a single gene. In order to gain a better understanding of sexual reproduction in higher plants, we isolated 22 mutants exhibiting defects in female gametophyte development, and determined their T-DNA insertion loci. We propose that this mutant population may prove useful in the further determination of the functions of genes that play important roles in plant development.

CONSTANS activates SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 through FLOWERING LOCUS T to promote flowering in Arabidopsis.

CONSTANS (CO) regulates flowering time by positively regulating expression of two floral integrators, FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), in Arabidopsis (Arabidopsis thaliana). FT and SOC1 have been proposed to act in parallel pathways downstream of CO based on genetic analysis using weak ft alleles, since ft soc1 double mutants showed an additive effect in suppressing the early flowering of CO overexpressor plants. However, this genetic analysis was inconsistent with the sequential induction pattern of FT and SOC1 found in inducible CO overexpressor plants. Hence, to identify genetic interactions of CO, FT, and SOC1, we carried out genetic and expression analyses with a newly isolated T-DNA allele of FT, ft-10. We found that ft-10 almost completely suppressed the early flowering phenotype of CO overexpressor plants, whereas soc1-2 partially suppressed the phenotype, suggesting that FT is the major output of CO. Expression of SOC1 was altered in gain- or loss-of-function mutants of FT, whereas expression of FT remained unchanged in gain- or loss-of-function mutants of SOC1, suggesting that FT positively regulates SOC1 to promote flowering. In addition, inactivation of FT caused down-regulation of SOC1 even in plants overexpressing CO, indicating that FT is required for SOC1 induction by CO. Taken together, these data suggest that CO activates SOC1 through FT to promote flowering in Arabidopsis.