The conserved brassinosteroid-related transcription factor BIM1a negatively regulates fruit growth in tomato.

Brassinosteroids (BRs) are steroid hormones that play key roles in plant development and defense. Our goal is to harness the extensive knowledge of the Arabidopsis BR signaling network to improve productivity in crop species. This first requires identifying components of the conserved network and their function in the target species. Here, we investigated the function of SlBIM1a, the closest tomato homolog of AtBIM1, which is highly expressed in fruit. SlBIM1a-overexpressing lines displayed severe plant and fruit dwarfism, and histological characterization of different transgenic lines revealed that SlBIM1a expression negatively correlated with fruit pericarp cell size, resulting in fruit size modifications. These growth phenotypes were in contrast to those found in Arabidopsis, and this was confirmed by the reciprocal ectopic expression of SlBIM1a/b in Arabidopsis and of AtBIM1 in tomato. These results determined that BIM1 function depends more on the recipient species than on its primary sequence. Yeast two-hybrid interaction studies and transcriptomic analyses of SlBIM1a-overexpressing fruit further suggested that SlBIM1a acts through its interaction with SlBZH1 to govern the transcriptional regulation of growth-related BR target genes. Together, these results suggest that SlBIM1a is a negative regulator of pericarp cell expansion, possibly at the crossroads with auxin and light signaling.

Ubiquitin carboxyl-terminal hydrolases are required for period maintenance of the circadian clock at high temperature in Arabidopsis.

Protein ubiquitylation participates in a number of essential cellular processes including signal transduction and transcription, often by initiating the degradation of specific substrates through the 26S proteasome. Within the ubiquitin-proteasome system, deubiquitylating enzymes (DUBs) not only help generate and maintain the supply of free ubiquitin monomers, they also directly control functions and activities of specific target proteins by modulating the pool of ubiquitylated species. Ubiquitin carboxyl-terminal hydrolases (UCHs) belong to an enzymatic subclass of DUBs, and are represented by three members in Arabidopsis, UCH1, UCH2 and UCH3. UCH1 and UCH2 influence auxin-dependent developmental pathways in Arabidopsis through their deubiquitylation activities, whereas biological and enzymatic functions of UCH3 remain unclear. Here, we demonstrate that Arabidopsis UCH3 acts to maintain the period of the circadian clock at high temperatures redundantly with UCH1 and UCH2. Whereas single uch1, uch2 and uch3 mutants have weak circadian phenotypes, the triple uch mutant displays a drastic lengthening of period at high temperatures that is more extreme than the uch1 uch2 double mutant. UCH3 also possesses a broad deubiquitylation activity against a range of substrates that link ubiquitin via peptide and isopeptide linkages. While the protein target(s) of UCH1-3 are not yet known, we propose that these DUBs act on one or more factors that control period length of the circadian clock through removal of their bound ubiquitin moieties, thus ensuring that the clock oscillates with a proper period even at elevated temperatures.

Temporal restriction of salt inducibility in expression of salinity-stress related gene by the circadian clock in Solanum lycopersicum.

Exposure to salinity causes plants to trigger transcriptional induction of a particular set of genes for initiating salinity-stress responses. Recent transcriptome analyses reveal that expression of a population of salinity-inducible genes also exhibits circadian rhythms. However, since the analyses were performed independently from those with salinity stress, it is unclear whether the observed circadian rhythms simply represent their basal expression levels independently from their induction by salinity, or these rhythms demonstrate the function of the circadian clock to actively limit the timing of occurrence of the salinity induction to particular times in the day. Here, by using tomato, we demonstrate that salt inducibility in expression of particular salinity-stress related genes is temporally controlled in the day. Occurrence of salinity induction in expression of SlSOS2 and P5CS, encoding a sodium/hydrogen antiporter and an enzyme for proline biosynthesis, is limited specifically to the morning, whereas that of SlDREB2, which encodes a transcription factor involved in tomato responses to several abiotic stresses such as salinity and drought, is restricted specifically to the evening. Our findings not only demonstrate potential importance in further investigating the basis and significance of circadian gated salinity stress responses under fluctuating day/night conditions, but also provide the potential to exploit an effective way for improving performance of salinity resistance in tomato.

Evaluation of internal control genes for quantitative realtime PCR analyses for studying fruit development of dwarf tomato cultivar 'Micro-Tom'.

Quantitative real-time PCR (qRT-PCR) is widely used to analyze the expression profiles of the genes of interest. In order to obtain accurate quantification data, normalization by using reliable internal control genes is essential. In this study, we evaluated the stability and applicability of eight internal control gene candidates for analyzing gene expression during fruit development in dwarf tomato cultivar Micro-Tom. We collected seventeen different samples from flowers and fruits at different developmental stages, and estimated the expression stability of the candidate genes by two statistical algorithms, geNorm and NormFinder. The combined ranking order and qRT-PCR analyses for expression profiles of SlYABBY2a, SlYABBY1a, FRUITFULL1 and APETALA2c suggested that EXPRESSED was the most stable and reliable internal control gene among the candidates. Our analysis also suggested that RPL8 was also suitable if the sample group is limited to fruits at different maturation stages. In addition to EXPRESSED, GAPDH was also applicable for relative quantitation to monitor gene expression profiles through fruit development from pistil to pericarp.

The FT-like gene PehFT in petunia responds to photoperiod and light quality but is not the main gene promoting light quality-associated flowering.

In Arabidopsis, flowering is delayed under red light and induced under far red light and blue light. Studies suggest that the florigen, FLOWERING LOCUS T, is involved in the control of light quality-associated flowering in Arabidopsis. In petunia, similar to Arabidopsis, flowering is delayed under red light and induced under blue light, however its mechanism still remains unknown. Here we isolated a gene which has 75% amino acid sequence similarity with Arabidopsis FT (AtFT), named PehFT. By overexpressing PehFT in Arbidopsis and petunia, we tested its ability to induce flowering. Also, by conducting expression analyses of PehFT under different light quality treatments, we tested its response to light quality. We concluded that PehFT, like AtFT, is a gene which responds to photoperiod and light quality, but unlike AtFT, is not the main gene controlling the light quality-associated flowering.

A loss-of-function mutation in the DWARF4/PETANKO5 gene enhances the late-flowering and semi-dwarf phenotypes of the Arabidopsis clock mutant lhy-12;cca1-101 under continuous light without affecting FLC expression.

The circadian clock plays important roles in the control of photoperiodic flowering in Arabidopsis. Mutations in the LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) genes (lhy;cca1) accelerate flowering under short days, whereas lhy;cca1 delays flowering under continuous light (LL). The lhy;cca1 mutant also exhibits short hypocotyls and petioles under LL. However, the molecular mechanisms underlying the regulation of both flowering time and organ lengths in the LHY/CCA1-dependent pathway are not fully understood. To address these questions, we performed EMS mutagenesis of the lhy-12;cca1-101 line and screened for mutations that enhance the lhy;cca1 phenotypes under LL. In this screen, we identified a novel allele of dwarf4 (dwf4) and named it petanko 5 (pta5). A similar level of enhancement of the delay in flowering was observed in these two dwf4 mutants when combined with the lhy;cca1 mutations. The lhy;cca1 and dwf4 mutations did not significantly affect the expression level of the floral repressor gene FLC under LL. Our results suggest that a defect in brassinosteroid (BR) signaling delayed flowering independent of the FLC expression level, at least in plants with the lhy;cca1 mutation grown under LL. The dwf4/pta5 mutation did not enhance the late-flowering phenotype of plants overexpressing SVP under LL, suggesting that SVP and BR function in a common pathway that controls flowering time. Our results suggest that the lhy;cca1 mutant exhibits delayed flowering due to both the BR signaling-dependent and -independent pathways under LL.

Density effects on late flowering mutants of Arabidopsis thaliana under continuous light.

In general, plant growth is inhibited under high-density conditions, while it is promoted under low-density conditions. This is known as the "density effect". Growing plants at high densities is often associated with an accelerated flowering time. Three major pathways [the long day (LD), gibberellic acid (GA), and autonomous/vernalization pathways] are known to play important roles in the control of flowering time. Circadian clock genes, namely, LHY, CCA1, GI, and ELF3, regulate the LD pathway. GAI and FCA control flowering via GA and autonomous pathways, respectively. The density effect on plant size is caused by specific factors such as the amount of nutrition obtained from the soil and touch frequency among plants. However, the molecular mechanism underlying the acceleration of flowering time due to density effects remains unclear. Here, we show the density effects on three Brassicaceae plants, namely, Brassica rapa var. nipposinica, Brassica napus, and Brassica chinensis f. honsaitai. They showed shorter stems and leaves when grown at high densities on soil under continuous light (LL). Shorter stems and leaves, as well as accelerated flowering times, were observed when a model plant, Arabidopsis thaliana, was grown under the same conditions. Unexpectedly, ethylene insensitive 2 (ein2) showed no differences in density effects in our experiments. The acceleration of flowering at higher densities was largely suppressed by gai, but not by gi, lhy;cca1, or fca. These results suggest that the promotion of flowering (as a density effect) is likely dependent on the GA pathway, but not the LD or autonomous pathways.

Density effects on semi-dwarf and early flowering mutants of Arabidopsis thaliana under continuous light.

Plant growth promotion and inhibition under low- and high-density conditions (referred to as the density effect) has been studied extensively. Here, we show that such density effects were unaffected by the position of wild-type (WT) and gibberellic acid insensitive (gai) strains of Arabidopsis thaliana (Arabidopsis) within pots. Additionally, petanko 1 (pta1) and pta5 were newly discovered alleles of the ROTUNDIFOLIA 3 (ROT3) and DWARF 4 (DWF4) genes that are involved in brassinosteroid biosynthesis. Unlike gai, the semi-dwarf mutants of pta1 and pta5 exhibited normal flowering times and a shortening of rosette leaves at high densities. Moreover, the pta1 and pta5 variants suppressed flowering stem shortening at high densities. pta5, but not pta1 suppressed the reduction in silique number at intermediate densities. SPINDLY (SPY) is a negative regulator of GA signaling, while PHYTOCHROME B (PHYB) is a red-light photoreceptor. High-density growth did not reduce the flowering time of phyB mutants, but did affect that of spy mutants. Neither spy nor phyB suppressed the shortening of rosette leaves at high densities; however, spy suppressed flowering stem shortening. Moreover, spy suppressed the reduction of silique number at high densities, while and phyB promoted the decrease. These data suggest that GA, BR, and light signaling pathways play important roles in the density effect.

The unique function of the Arabidopsis circadian clock gene PRR5 in the regulation of shade avoidance response.

Shade avoidance response (S.A.R) is regulated by light and circadian clock. Circadian clock controls S.A.R by the transcriptional regulation of positive regulators of S.A.R, PIF4 and PIF5, to prevent plants from responding to 'light' of dark period. Thus, in many cases, deficits in circadian clock appear in abnormalities of hypocotyl and/or petiole elongation. Previously, interesting phenomena were reported that the triple mutants of PSEUDO RESPONSE REGULATORS9, 7 and 5, which are clock components, show longer petioles and smaller leaves under light/dark cycle than those under continuous lighting. These S.A.R-like phenotypes cannot be explained by their hyposensitivity to red light. We demonstrated detailed analyses of this mutant to reveal the leaf-specific S.A.R regulated by circadian clock. Expression analyses of S.A.R-related genes suggested that PRR5 functions as a repressor of S.A.R. Morphological analyses of leaves under different light condition revealed that PRR5 is involved in the inhibition of leaf expansion in S.A.R.

Tomato TILLING technology: development of a reverse genetics tool for the efficient isolation of mutants from Micro-Tom mutant libraries.

To accelerate functional genomic research in tomato, we developed a Micro-Tom TILLING (Targeting Induced Local Lesions In Genomes) platform. DNA pools were constructed from 3,052 ethyl methanesulfonate (EMS) mutant lines treated with 0.5 or 1.0% EMS. The mutation frequency was calculated by screening 10 genes. The 0.5% EMS population had a mild mutation frequency of one mutation per 1,710 kb, whereas the 1.0% EMS population had a frequency of one mutation per 737 kb, a frequency suitable for producing an allelic series of mutations in the target genes. The overall mutation frequency was one mutation per 1,237 kb, which affected an average of three alleles per kilobase screened. To assess whether a Micro-Tom TILLING platform could be used for efficient mutant isolation, six ethylene receptor genes in tomato (SlETR1-SlETR6) were screened. Two allelic mutants of SlETR1 (Sletr1-1 and Sletr1-2) that resulted in reduced ethylene responses were identified, indicating that our Micro-Tom TILLING platform provides a powerful tool for the rapid detection of mutations in an EMS mutant library. This work provides a practical and publicly accessible tool for the study of fruit biology and for obtaining novel genetic material that can be used to improve important agronomic traits in tomato.

Suppression of late-flowering and semi-dwarf phenotypes in the Arabidopsis clock mutant lhy-12;cca1-101 by phyB under continuous light.

Photoperiodic flowering in Arabidopsis is controlled not only by floral activators such as GI, CO, and FT, but also by repressors such as SVP and FLC. Double mutations in LHY and CCA1 (lhy;cca1) accelerated flowering under short days, mainly by the GI-CO dependent pathway. In contrast, lhy;cca1 showed delayed flowering under continuous light (LL), probably due to the GI-CO independent pathway. This late-flowering phenotype was suppressed by svp, flc, and elf3. However, how SVP, FLC, and ELF3 mediate LHY/CCA1 and flowering time is not fully understood. We found that lhy;cca1 exhibited short hypocotyls and petioles under LL, but the molecular mechanism for these effects has not been elucidated. To address these questions, we performed a screen for mutations that suppress either or both of the lhy;cca1 phenotypes under LL, using two different approaches. We identified two novel mutations, a dominant (del1) and a recessive (phyB-2511) allele of phyB. The flowering times of single mutants of three phyB alleles, hy3-1, del1, and phyB-2511, are almost the same and earlier than those of wild-type plants. A similar level of acceleration of flowering time was observed in all three phyB mutants tested when combined with the late-flowering mutations co-2 and SVPox. However, the effect of phyB-2511 on lhy;cca1 was different from those by hy3-1 or del1. svp-3 did not strongly enhance the early-flowering phenotypes of phyB-2511 or del1. These results suggest that light signaling via PhyB may affect factors downstream of the clock proteins, controlling flowering time and organ elongation. phyB mutations with different levels of effects on lhy;cca1-dependent late flowering would be useful to determine a specific role for PHYB in the flowering pathway controlled by lhy;cca1 under LL.

A trial of production of the plant-derived high-value protein in a plant factory: photosynthetic photon fluxes affect the accumulation of recombinant miraculin in transgenic tomato fruits.

One of the ultimate goals of plant science is to test a hypothesis obtained by basic science and to apply it to agriculture and industry. A plant factory is one of the ideal systems for this trial. Environmental factors affect both plant yield and the accumulation of recombinant proteins for industrial applications within transgenic plants. However, there have been few reports studying plant productivity for recombinant protein in closed cultivation systems called plant factories. To investigate the effects of photosynthetic photon flux (PPF) on tomato fruit yield and the accumulation of recombinant miraculin, a taste-modifying glycoprotein, in transgenic tomato fruits, plants were cultivated at various PPFs from 100 to 400 (µmol m(-2) s(-)1) in a plant factory. Miraculin production per unit of energy used was highest at PPF100, although miraculin production per unit area was highest at PPF300. The commercial productivity of recombinant miraculin in transgenic tomato fruits largely depended on light conditions in the plant factory. Our trial will be useful to consider the trade-offs between the profits from production of high-value materials in plants and the costs of electricity.

Calmodulin-dependent activation of MAP kinase for ROS homeostasis in Arabidopsis.

Rapid recognition and signal transduction of mechanical wounding through various signaling molecules, including calcium (Ca²+), protein phosphorylation, and reactive oxygen species (ROS), are necessary early events leading to stress resistance in plants. Here we report that an Arabidopsis mitogen-activated protein kinase 8 (MPK8) connects protein phosphorylation, Ca²+, and ROS in the wound-signaling pathway. MPK8 is activated through mechanical wounding, and this activation requires direct binding of calmodulins (CaMs) in a Ca²+-dependent manner. MPK8 is also phosphorylated and activated by a MAPKK MKK3 in the prototypic kinase cascade, and full activation of MPK8 needs both CaMs and MKK3 in planta. The MPK8 pathway negatively regulates ROS accumulation through controlling expression of the Rboh D gene. These findings suggest that two major activation modes in eukaryotes, Ca²+/CaMs and the MAP kinase phosphorylation cascade, converge at MPK8 to monitor or maintain an essential part of ROS homeostasis.

Double loss-of-function mutation in EARLY FLOWERING 3 and CRYPTOCHROME 2 genes delays flowering under continuous light but accelerates it under long days and short days: an important role for Arabidopsis CRY2 to accelerate flowering time in continuous light.

The photoperiodic response is one of the adaptation mechanisms to seasonal changes of lengths of day and night. The circadian clock plays pivotal roles in this process. In Arabidopsis, LHY, CCA1, ELF3, and other clock proteins play major roles in maintaining circadian rhythms. lhy;cca1 double mutants with severe defects in circadian rhythms showed accelerated flowering under short days (SDs), but delayed flowering under continuous light (LL). The protein level of the floral repressor SVP increased in lhy;cca1 mutants under LL, and the late-flowering phenotype of lhy;cca1 mutants was partially suppressed by svp, flc, or elf3. ELF3 interacted with both CCA1 and SVP, and elf3 suppressed the SVP accumulation in lhy;cca1 under LL. These results suggest that the unique mechanism of the inversion of the flowering response of lhy;cca1 under LL may involve both the ELF3-SVP/FLC-dependent and -independent pathways. In this work, elf3-1 seeds were mutagenized with heavy-ion beams and used to identify mutation(s) that delayed flowering under LL but not long days (LDs) or SDs even without ELF3. In this screening, seven candidate lines named suppressor of elf3 1 (self1), sel3, sel5, sel7, sel14, sel15, and sel20 were identified. Genetic analysis indicated that sel20 was a new deletion allele of a mutation in the blue light receptor, CRY2. A late-flowering phenotype and decrease of FT expression in the elf3;sel20 double mutant was obvious under LL but not under SDs or LDs. These results indicated that the late-flowering phenotype in the double mutant elf3;sel20 as well as in lhy;cca1 was affected by the presence of darkness. The results suggest that CRY2 may play more essential roles in the acceleration of flowering under LL than LDs or SDs.

TOMATOMA: a novel tomato mutant database distributing Micro-Tom mutant collections.

The tomato is an excellent model for studies of plants bearing berry-type fruits and for experimental studies of the Solanaceae family of plants due to its conserved genetic organization. In this study, a comprehensive mutant tomato population was generated in the background of Micro-Tom, a dwarf, rapid-growth variety. In this and previous studies, a family including 8,598 and 6,422 M(2) mutagenized lines was produced by ethylmethane sulfonate (EMS) mutagenesis and γ-ray irradiation, and this study developed and investigated these M(2) plants for alteration of visible phenotypes. A total of 9,183 independent M(2) families comprising 91,830 M(2) plants were inspected for phenotypic alteration, and 1,048 individual mutants were isolated. Subsequently, the observed mutant phenotypes were classified into 15 major categories and 48 subcategories. Overall, 1,819 phenotypic categories were found in 1,048 mutants. Of these mutants, 549 were pleiotropic, whereas 499 were non-pleiotropic. Multiple different mutant alleles per locus were found in the mutant libraries, suggesting that the mutagenized populations were nearly saturated. Additionally, genetic analysis of backcrosses indicated the successful inheritance of the mutations in BC(1)F(2) populations, confirming the reproducibility in the morphological phenotyping of the M(2) plants. To integrate and manage the visible phenotypes of mutants and other associated data, we developed the in silico database TOMATOMA, a relational system interfacing modules between mutant line names and phenotypic categories. TOMATOMA is a freely accessible database, and these mutant recourses are available through the TOMATOMA (http://tomatoma.nbrp.jp/index.jsp).

Molecular breeding of tomato lines for mass production of miraculin in a plant factory.

A transgenic tomato line (56B, "Moneymaker") that expresses the miraculin gene driven by the CaMV 35S promoter was crossed with a dwarf tomato ("Micro-Tom") for the molecular breeding of cultivars that are suitable for miraculin production in a closed cultivation system. Plant size, miraculin accumulation, and self-pruning growth were used as selection indicators for F2 plants. Two lines were chosen for further analysis, bred to the F6 or F7 generation and cultivated in a closed cultivation system. In 56B and the two crossed lines, the concentrations of miraculin in the pericarp were 140, 367, and 343 microg/g FW, respectively. We also estimated that 26.2, 73.6, and 45.9 kg FW/m2 of tomatoes and 2.2, 16.6, and 9.8 mg/m2 of miraculin in the pericarp, respectively, could be harvested per year. These two crossed lines will be useful for the mass production of miraculin, especially in a closed cultivation system.

Punctual coordination: switching on and off for flowering during a day.

The photoperiodic flowering of Arabidopsis is shown to be explained in part by the Bünning's external coincidence model in which clock-controlled expression of CO and stabilization of CO protein by light have important roles. The floral activators, GI and CO, together with ZTL, FKF and CDF1 have been shown to be central for the induction of FT expression during evening to promote the photoperiodic flowering of Arabidopsis. Here we discuss a role of diurnal accumulation of a floral repressor SVP protein in the repression of the FT and SOC1 expression during daytime. A punctual coordination of the diurnal regulation of both positive and negative regulators by circadian clock appears to be important for the photoperiodic flowering in Arabidopsis.

Possible role of early flowering 3 (ELF3) in clock-dependent floral regulation by short vegetative phase (SVP) in Arabidopsis thaliana.

Circadian clock proteins play key roles in adaptations of plants to diurnal environmental conditions. The photoperiodic flowering response is one of the mechanisms of adaptation to seasonal changes in the lengths of day and night. Double mutations in two clock genes, late elongated hypocotyl (LHY) and circadian clock associated 1 (CCA1), accelerated flowering under short days (SDs) but delayed flowering under continuous light (LL) in Arabidopsis thaliana. The mechanism underlying the late flowering of lhy;cca1 mutants under LL was investigated here. Late flowering of plants with overexpression of short vegetative phase (SVP) was much more pronounced under SDs and enhanced by constans 2 (co-2) under long days (LDs), suggesting that SVP and CO act independently in the photoperiodic flowering pathway. However, how SVP and flowering locus C (FLC) mediated the effects of LHY/CCA1 and thus influenced flowering time was not completely clear. A mutant line lhy;cca1 in the Landsberg erecta (Ler) background was established, ethyl methanesulfonate (EMS)-mutagenized and used to screen suppressors of late flowering of lhy;cca1 under LL. Mutations in the clock gene early flowering 3 (ELF3) were identified as suppressors. Overexpression and loss-of-function of ELF3 influenced SVP protein accumulation. Therefore, we propose that, as well as the classical GIGANTEA (GI)-CO pathway, LHY/CCA1 regulates a pathway negatively controlling flowering locus T (FT), possibly via ELF3-SVP/FLC.

Circadian clock proteins LHY and CCA1 regulate SVP protein accumulation to control flowering in Arabidopsis.

The floral regulators GIGANTEA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) play key roles in the photoperiodic flowering responses of the long-day plant Arabidopsis thaliana. The GI-CO-FT pathway is highly conserved in plants. Here, we demonstrate that the circadian clock proteins LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) not only repressed the floral transition under short-day and long-day conditions but also accelerated flowering when the plants were grown under continuous light (LL). LHY and CCA1 accelerated flowering in LL by promoting FT expression through a genetic pathway that appears to be independent of the canonical photoperiodic pathway involving GI and CO proteins. A genetic screen revealed that the late-flowering phenotype of the lhy;cca1 double mutant under LL was suppressed through mutations in SHORT VEGETATIVE PHASE (SVP), a MADS box transcription factor. Yeast two-hybrid analysis demonstrated an interaction between SVP and FLOWERING LOCUS C, and genetic analysis indicated that these two proteins act as partially redundant repressors of flowering time. SVP protein accumulated in lhy;cca1 plants under LL. We propose a model in which LHY and CCA1 accelerate flowering in part by reducing the abundance of SVP and thereby antagonizing its capacity to repress FT expression under LL.