FLC expression is down-regulated by cold treatment in Diplotaxis tenuifolia (wild rocket), but flowering time is unaffected.

Wild rocket (Diplotaxis tenuifolia) has become a very popular salad leaf due to its peppery taste. It is part of the Brassicaceae family and thus has a high level of homology at the DNA level to other Brassica species including Arabidopsis thaliana. The vernalization and photoperiodic requirements of wild rocket have not been reported to date. Photoperiodic experiments described here demonstrate that rocket is a facultative long day plant. To investigate the vernalization requirement, both seed and young plants were given vernalization treatments at 4°C for different lengths of time. A rocket homologue of FLOWERING LOCUS C (DtFLC) was isolated and shown to functionally complement the Arabidopsis FRI+flc3 null mutant. Whilst the expression of DtFLC was significantly reduced after just one week of cold treatment, cold treatments of two to eight weeks had no significant effect on bolting time of wild rocket indicating that rocket does not have a vernalization requirement. These findings illustrate that important fundamental differences can exist between model and crop plant species, such as in this case where down-regulation of DtFLC expression does not enable earlier flowering in wild rocket as it does in Arabidopsis and many other Brassica species.

Selection of reference genes for diurnal and developmental time-course real-time PCR expression analyses in lettuce.

BACKGROUND: Real-time quantitative polymerase chain reaction (RT-qPCR) analysis is a low cost and sensitive technique that is widely used to measure levels of gene expression. Selecting and validating appropriate reference genes for normalising target gene expression should be the first step in any expression study to avoid inaccurate results. RESULTS: In this study, ten candidate genes were tested for their suitability for use as reference genes in diurnal and developmental timecourse experiments in lettuce. The candidate reference genes were then used to normalise the expression pattern of the FLOWERING LOCUS T (FT) gene, one of key genes involved in the flowering time pathway whose expression is known to vary throughout the day and at different stages of development. Three reference genes, LsPP2A-1 (PROTEIN PHOSPHATASE 2A-1), LsPP2AA3 (PROTEIN PHOSPHATASE 2A REGULATORY SUBUNIT A3) and LsTIP41 (TAP42-INTERACTING PROTEIN OF 41 kDa), were the most stably expressed candidate reference genes throughout both the diurnal and developmental timecourse experiments. In the developmental experiment using just LsPP2A-1 and LsTIP41 as reference genes would be sufficient for accurate normalisation, whilst in the diurnal experiment all three reference genes, LsPP2A-1, LsPP2AA3 and LsTIP41, would be necessary. The FT expression pattern obtained demonstrates that the use of multiple and robust reference genes for RT-qPCR expression analyses results in a more accurate and reliable expression profile. CONCLUSIONS: Reference genes suitable for use in diurnal and developmental timecourse experiments in lettuce were identified and used to produce a more accurate and reliable analysis of lsFT expression levels than previously obtained in such timecourse experiments.

TEMPRANILLO is a regulator of juvenility in plants.

Many plants are incapable of flowering in inductive daylengths during the early juvenile vegetative phase (JVP). Arabidopsis mutants with reduced expression of TEMPRANILLO (TEM), a repressor of flowering locus T (FT) had a shorter JVP than wild-type plants. Reciprocal changes in mRNA expression of TEM and FT were observed in both Arabidopsis and antirrhinum, which correlated with the length of the JVP. FT expression was induced just prior to the end of the JVP and levels of TEM1 mRNA declined rapidly at the time when FT mRNA levels were shown to increase. TEM orthologs were isolated from antirrhinum (AmTEM) and olive (OeTEM) and were expressed most highly during their juvenile phase. AmTEM functionally complemented AtTEM1 in the tem1 mutant and over-expression of AmTEM prolonged the JVP through repression of FT and CONSTANS (CO). We propose that TEM may have a general role in regulating JVP in herbaceous and woody species.

Starch metabolism and antiflorigenic signals modulate the juvenile-to-adult phase transition in Arabidopsis.

The physiology and genetics underlying juvenility is poorly understood. Here, we exploit Arabidopsis as a system to understand the mechanisms that regulate floral incompetence during juvenility. Using an experimental assay that allows the length of juvenility to be estimated and mutants impaired in different pathways, we show that multiple inputs influence juvenility. Juvenile phase lengths of wild type (WT) accessions Col-0, Ler-0 and Ws-4 are shown to differ, with Col-0 having the shortest and Ws-4 the longest length. Plants defective in sugar signalling [gin1-1, gin2-1, gin6 (abi4)] and floral repressor mutants [hst1, tfl1, tfl2 (lhp1)] showed shortened juvenile phase lengths compared to their respective WTs. Mutants defective in starch anabolism (adg1-1, pgm1) and catabolism (sex1, sex4, bam3) showed prolonged juvenile phase lengths compared to Col-0. Examination of diurnal metabolite changes in adg1-1 and sex1 mutants indicates that their altered juvenile phase length may be due to lack of starch turnover, which influences carbohydrate availability. In this article, we propose a model in which a variety of signals including floral activators and repressors modulate the juvenile-to-adult phase transition. The role of carbohydrates may be in their capacity as nutrients, osmotic regulators, signalling molecules and/ or through their interaction with phytohormonal networks.

Florigenic and antiflorigenic signaling in plants.

The evidence that FLOWERING LOCUS T (FT) protein, and its paralog TWIN SISTER OF FT, act as the long-distance floral stimulus, or at least that they are part of it in diverse plant species, has attracted much attention in recent years. Studies to understand the physiological and molecular apparatuses that integrate spatial and temporal signals to regulate developmental transitions in plants have occupied countless scientists and have resulted in an unmanageably large amount of research data. Analysis of these data has helped to identify multiple systemic florigenic and antiflorigenic regulators. This study gives an overview of the recent research on gene products, phytohormones and other metabolites that have been demonstrated to have florigenic or antiflorigenic functions in plants.

Conservation of Arabidopsis thaliana photoperiodic flowering time genes in onion (Allium cepa L.).

The genetics underlying onion development are poorly understood. Here the characterization of onion homologs of Arabidopsis photoperiodic flowering pathway genes is reported with the end goal of accelerating onion breeding programs by understanding the genetic basis of adaptation to different latitudes. The expression of onion GI, FKF1 and ZTL homologs under short day (SD) and long day (LD) conditions was examined using quantitative reverse transcription-PCR (qRT-PCR). The expression of AcGI and AcFKF1 was examined in onion varieties which exhibit different daylength responses. Phylogenetic trees were constructed to confirm the identity of the homologs. AcGI and AcFKF1 showed diurnal expression patterns similar to their Arabidopsis counterparts, while AcZTL was found to be constitutively expressed. AcGI showed similar expression patterns in varieties which exhibit different daylength responses, whereas AcFKF1 showed differences. It is proposed that these differences could contribute to the different daylength responses in these varieties. Phylogenetic analyses showed that all the genes isolated are very closely related to their proposed homologs. The results presented here show that key genes controlling photoperiodic flowering in Arabidopsis are conserved in onion, and a role for these genes in the photoperiodic control of bulb initiation is predicted. This theory is supported by expression and phylogenetic data.

Characterization of FaRB7, a near root-specific gene from strawberry (Fragariaxananassa Duch.) and promoter activity analysis in homologous and heterologous hosts.

Many insect and fungal pathogens posing agronomically important threats specifically target the roots in strawberry. The use of a root-specific promoter to confer expression of resistance genes in a targeted manner has the potential appreciably to benefit the genetic improvement of commercial strawberry varieties. A novel gene, FaRB7, was isolated from strawberry (Fragariaxananassa Duch.) and found to contain motifs characteristic of tonoplast intrinsic proteins (TIPs). Phylogenetic analysis revealed that FaRB7 represents an RB7-type TIP. In strawberry, this gene is expressed predominantly in roots, with very low expression in petioles. A 2.843 kb region representing the FaRB7 gene upstream regulatory sequence was isolated and found to share a number of sequence motifs with the promoter of the Nicotiana tabacum TobRB7 root-specific RB7-type TIP. When cloned upstream of the gusA reporter gene and introduced into strawberry plants, the FaRB7 promoter was shown to direct strong, near root-specific expression with expression patterns very similar to that of the endogenous gene. Furthermore, the FaRB7 promoter was found to confer constitutive expression, comparable to that produced by the cauliflower mosaic virus (CaMV) 35S RNA promoter, in tobacco. Thus, the FaRB7 promoter may be used to achieve near-root-specific transgene expression in strawberry and also represents an alternative to the CaMV 35S promoter for producing constitutive foreign gene expression in heterologous hosts. The FaRB7 full-length genomic sequence and 5' upstream regulatory region have been submitted to the EMBL/GenBank database under accession number DQ178022.