CONSTANS-LIKE 5 facilitates flower opening and scent biosynthesis in Solanaceae.

BackgroundA plant’s endogenous clock (circadian clock) entrains physiological processes to light/dark and temperature cycles. Forward and reverse genetic approaches in Arabidopsis have revealed the mechanisms of the circadian clock and its components in the genome. Similar approaches have been used to characterize conserved clock elements in several plant species. A wild tobacco, Nicotiana attenuata has been studied extensively to understand responses to biotic or abiotic stress in the glasshouse and also in their native habitat. During two decades of field experiment, we observed several diurnal rhythmic traits of N. attenuata in nature. To expand our knowledge of circadian clock function into the entrainment of traits important for ecological processes, we here report three core clock components in N. attenuata.ResultsProtein similarity and transcript accumulation allowed us to isolate orthologous genes of the core circadian clock components, LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1/PSEUDO-RESPONSE REGULATOR 1 (TOC1/PRR1), and ZEITLUPE (ZTL). Transcript accumulation of NaLHY peaked at dawn and NaTOC1 peaked at dusk in plants grown under long day conditions. Ectopic expression of NaLHY and NaZTL in Arabidopsis resulted in elongated hypocotyl and late-flowering phenotypes. Protein interactions between NaTOC1 and NaZTL were confirmed by yeast two-hybrid assays. Finally, when NaTOC1 was silenced in N. attenuata, late-flowering phenotypes under long day conditions were clearly observed.ConclusionsWe identified three core circadian clock genes in N. attenuata and demonstrated the functional and biochemical conservation of NaLHY, NaTOC1, and NaZTL.

Being shaded is a common environmental stress for plants, especially for densely planted crops. Shade decreases red: far-red (R:FR) ratios that inactivate phytochrome B (PHYB) and subsequently release p̱hytochrome i̱nteraction f̱actors (PIFs). Shaded plants display elongated hypocotyls, internodes, and petioles, hyponastic leaves, early flowering and are inhibited in branching: traits collectively called the shade avoidance syndrome (SAS). ZEITLUPE (ZTL) is a circadian clock component and blue light photoreceptor, which is also involved in floral rhythms and plant defense in Nicotiana attenuata. ztl mutants are hypersensitive to red light and ZTL physically interacts with PHYB, suggesting the involvement of ZTL in R:FR light signaling. Here, we show that N. attenuata ZTL-silenced plants display a phenotype opposite to that of the SAS under normal light. After simulated shade, the normally induced transcript levels of the SAS marker gene, ATHB2 are attenuated in ZTL-silenced plants. The auxin signaling pathway, known to be involved in SAS, was also significantly attenuated. Furthermore, NaZTL directly interacts with NaPHYBs, and regulates the transcript levels of PHYBs, PIF3a, PIF7 and PIF8 under shade. Our results suggest that ZTL may regulate PHYB- and the auxin-mediated signaling pathway, which functions in the SAS of N. attenuata.

To successfully recruit pollinators, plants often release attractive floral scents at specific times of day to coincide with pollinator foraging. This timing of scent emission is thought to be evolutionarily beneficial to maximize resource efficiency while attracting only useful pollinators. Temporal regulation of scent emission is tied to the activity of the specific metabolic pathways responsible for scent production. Although floral volatile profiling in various plants indicated a contribution by the circadian clock, the mechanisms by which the circadian clock regulates timing of floral scent emission remained elusive. Recent studies using two species in the Solanaceae family provided initial insight into molecular clock regulation of scent emission timing. In Petunia hybrida, the floral volatile benzenoid/phenylpropanoid (FVBP) pathway is the major metabolic pathway that produces floral volatiles. Three MYB-type transcription factors, ODORANT 1 (ODO1), EMISSION OF BENZENOIDS I (EOBI), and EOBII, all of which show diurnal rhythms in mRNA expression, act as positive regulators for several enzyme genes in the FVBP pathway. Recently, in P. hybrida and Nicotiana attenuata, homologs of the Arabidopsis clock gene LATE ELONGATED HYPOCOTYL (LHY) have been shown to have a similar role in the circadian clock in these plants, and to also determine the timing of scent emission. In addition, in P. hybrida, PhLHY directly represses ODO1 and several enzyme genes in the FVBP pathway during the morning as an important negative regulator of scent emission. These findings facilitate our understanding of the relationship between a molecular timekeeper and the timing of scent emission, which may influence reproductive success.

Tulip (Tulipa gesneriana L.) cultivars exhibit diverse floral scents, the emissions of which are generally influenced by temperature. To fully benefit from these scents and add value to cut tulips, it is necessary to evaluate the scent emission response to temperature. This study investigated the daily emission changes of 82 volatiles, including the main scent compounds, at different temperatures (13, 18, and 23°C), in the cut tulips from eight tulip cultivars with different scents. At 23 and 18°C, the total scent emissions of each cultivar increased with flower opening and subsequently decreased with visible senescence. Floral senescence progressed more rapidly at 23°C, and the decrease in total scent emission occurred most rapidly at 23°C. Scent emissions at 18°C tended to be lower than at 23°C. The vase life of the cut tulips was most prolonged at 13°C owing to the slow senescence; however, scent emissions remained low after the flower opening. These results indicate that improving both tulip scent emission and vase life under constant temperature conditions is difficult. It was observed that scent composition changed before and after flower opening, and with floral senescence. In contrast, temperature had little effect on scent composition. Cut tulips stored at a low temperature (10 or 15°C) over four and a half days and then transferred to room temperature (20°C) had a longer vase life than tulips maintained at room temperature, due to delayed senescence. In addition, scent emissions were low at low temperatures, but increased rapidly upon transfer to room temperature. Therefore, storage of cut tulips at low temperatures may lead to increased scent emissions upon transfer to room temperature. These findings imply that low-temperature storage of cut tulips before sale contributes to both vase life and a rich scent after sale. The results reported here demonstrate the improvement of cut tulip scent emissions by appropriate temperature management.

Regulation of protein turnover mediated by ZEITLUPE (ZTL) constitutes an important mechanism of the circadian clock in Arabidopsis thaliana. Here, we report that FLAVIN BINDING, KELCH REPEAT, F-BOX1 (FKF1) and LOV KELCH PROTEIN2 (LKP2) play similar roles to ZTL in the circadian clock when ZTL is absent. In contrast with subtle circadian clock defects in fkf1, the clock in ztl fkf1 has a considerably longer period than in ztl. In ztl fkf1 lkp2, several clock parameters were even more severely affected than in ztl fkf1. Although LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1) expression levels are lower in ztl than in the wild type, introducing both fkf1 and lkp2 mutations into the ztl mutant dramatically diminished LHY expression without further affecting CCA1 expression. This demonstrates different contributions of ZTL, FKF1, and LKP2 in the regulation of LHY and CCA1 expression. In addition, FKF1 and LKP2 also interacted with TIMING OF CAB EXPRESSION1 (TOC1) and PSEUDO-RESPONSE REGULATOR5 (PRR5), and both proteins were further stabilized in ztl fkf1 and ztl fkf1 lkp2 compared with in ztl. Our results indicate that ZTL, FKF1, and LKP2 together regulate TOC1 and PRR5 degradation and are major contributors to determining the period of circadian oscillation and enhancing robustness.

Scent emissions and expression of scent emission-related genes: A comparison between cut and intact carnation flowers

One of the best-characterized physiological rhythms in plants is the circadian rhythm of CO(2) metabolism in Crassulacean acid metabolism (CAM) plants, which is the focus here. The central components of the plant circadian clock have been studied in detail only in Arabidopsis (Arabidopsis thaliana). Full-length cDNAs have been obtained encoding orthologs of CIRCADIAN CLOCK-ASSOCIATED1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION1 (TOC1), EARLY FLOWERING4 (ELF4), ZEITLUPE (ZTL), FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1), EARLY FLOWERING3 (ELF3), and a partial cDNA encoding GIGANTEA in the model stress-inducible CAM plant, Mesembryanthemum crystallinum (Common Ice Plant). TOC1 and LHY/CCA1 are under reciprocal circadian control in a manner similar to their regulation in Arabidopsis. ELF4, FKF1, ZTL, GIGANTEA, and ELF3 are under circadian control in C(3) and CAM leaves. ELF4 transcripts peak in the evening and are unaffected by CAM induction. FKF1 shows an abrupt transcript peak 3 h before subjective dusk. ELF3 transcripts appear in the evening, consistent with their role in gating light input to the circadian clock. Intriguingly, ZTL transcripts do not oscillate in Arabidopsis, but do in M. crystallinum. The transcript abundance of the clock-associated genes in M. crystallinum is largely unaffected by development and salt stress, revealing compensation of the central circadian clock against development and abiotic stress in addition to the well-known temperature compensation. Importantly, the clock in M. crystallinum is very similar to that in Arabidopsis, indicating that such a clock could control CAM without requiring additional components of the central oscillator or a novel CAM oscillator.

The rhythmic opening/closing and volatile emissions of flowers are known to attract pollinators at specific times. That these rhythms are maintained under constant light or dark conditions suggests a circadian clock involvement. Although a forward and reverse genetic approach has led to the identification of core circadian clock components in Arabidopsis thaliana, the involvement of these clock components in floral rhythms has remained untested, probably because of the weak diurnal rhythms in A.thaliana flowers. Here, we addressed the role of these core clock components in the flowers of the wild tobacco Nicotiana attenuata, whose flowers open at night, emit benzyl acetone (BA) scents and move vertically through a 140° arc. We first measured N.attenuata floral rhythms under constant light conditions. The results suggest that the circadian clock controls flower opening, BA emission and pedicel movement, but not flower closing. We generated transgenic N.attenuata lines silenced in the homologous genes of Arabidopsis LATE ELONGATED HYPOCOTYL (LHY) and ZEITLUPE (ZTL), which are known to be core clock components. Silencing NaLHY and NaZTL strongly altered floral rhythms in different ways, indicating that conserved clock components in N.attenuata coordinate these floral rhythms.

Mol Syst Biol. 2: 60 In the 18th Century, the Swedish botanist Karl von Linne designed a ‘Flower‐Clock’ by arranging a series of various plant species according to the respective time their flowers open or close every day. Watching this ‘Flower‐Clock’, one can then estimate the time of the day by noting the pattern of flower opening and closing. It has been a well‐known fact since Linne's early times that plants can open or close their flowers at a precise time of the day. However, we still do not fully understand the design principles of the molecular network that underlies the cellular circadian clock, which achieves to predict, often with exquisite precision, the cyclic changes in the environment due to the rotation of earth. In two articles currently published in Molecular Systems Biology , Millar and co‐workers (Locke et al , 2006) and Doyle and co‐workers (Zeilinger et al , 2006) propose a plausible design for the plant circadian clock. In previous work, Millar and co‐workers extended an initial ‘one‐loop model’ of the plant circadian clock into a ‘two‐loop model’ (Figure 1) (Locke et al , 2005). In the simple ‘one‐loop model’ (Figure 1 …

The plant circadian clock controls a large number of internal processes, including growth and metabolism. Scent emission displays a circadian pattern in many species such as the snapdragon. Here we show that knocking down LATE ELONGATED HYPOCOTYL in Antirrhinum majus affects growth and scent emission. In order to gain an understanding of the growth kinetics, we took a phenomic approach using in-house artificial vision systems, obtaining time-lapse videos. Wild type flowers showed a higher growth speed than knockdown plants. The maximal growth rate was decreased by 22% in plants with lower LHY expression. Floral volatiles were differentially affected as RNAi plants showed advanced emission of compounds synthesized from cinnamic acid and delayed emission of metabolites of benzoic acid. The monoterpenes myrcene and ocimene were delayed, whereas the sesquiterpene farnesene was advanced. Overall, transgenic lines showed an altered volatile emission pattern and displayed a modified scent profile. Our results show that AmLHY plays an important role in the quantitative and qualitative control of floral growth and scent emission.

Members of the R2R3-MYB transcription factor subgroup 19 (SG19) have been extensively studied in multiple plant species using different silenced or mutated lines. Some studies have proposed a function in flower opening, others in floral organ development/maturation, or specialized metabolism production. While SG19 members are clearly key players during flower development and maturation, the resulting picture is complex, confusing our understanding in how SG19 genes function. To clarify the function of the SG19 transcription factors, we used a single system, Petunia axillaris, and targeted its two SG19 members (EOB1 and EOB2) by CRISPR-Cas9. Although EOB1 and EOB2 are highly similar, they display radically different mutant phenotypes. EOB1 has a specific role in scent emission while EOB2 has pleiotropic functions during flower development. The eob2 knockout mutants reveal that EOB2 is a repressor of flower bud senescence by inhibiting ethylene production. Moreover, partial loss-of-function mutants (transcriptional activation domain missing) show that EOB2 is also involved in both petal and pistil maturation through regulation of primary and secondary metabolism. Here, we provide new insights into the genetic regulation of flower maturation and senescence. It also emphasizes the function of EOB2 in the adaptation of plants to specific guilds of pollinators.

The circadian clock can entrain to forced light-dark cycles by adjusting the phases and periods of flower opening and closing in ephemeral flowers. The responses of circadian rhythms to the same light conditions differ from species. However, the differences in internal genetic mechanisms underlying the different responses between species remain unclear. Iris domestica and I. dichotoma have ephemeral flowers and significantly divergent flower opening and closing times. The effects of different photoperiods (continuous darkness, 4L20D, 8L16D, 12L12D, 16L8D, 20L4D and continuous white light) on flower opening and closing, and expression patterns of seven genes (CRYPTOCHROME 1, PHYTOCHROME B, LATE ELONGATED HYPOCOTYL, PSEUDO RESPONSE REGULATOR 95, PHYTOCHROME INTERACTING FACTOR 4-like, SMUX AUXIN UP RNA 64-like and senescence-associated gene 39-like) involved in the circadian regulation of flower opening and closing were compared between I. domestica and I. dichotoma. Flower opening and closing in the two species exhibited circadian rhythms under continuous darkness (DD), but showed arrhythmia in continuous white light (LL). In the two species, keeping robust rhythms, strong synchronicity, rapid progressions of flower opening and closing and reaching full opening stage required a dark period longer than 4h. In light-dark cycles with dark periods longer than 4h, flower opening and closing times of the two species delayed with the delay of dawn, and the degree to which flower opening time varies with the time of dawn was greater in I. dichotoma than in I. domestica. The arrhythmia of flower opening and closing under 20L4D and LL would result from the arrhythmic output signals rather than arrhythmia of oscillators and photoreceptors. The different responses of the two species to the change of photoperiods would be caused by the transcriptional differences of genes in the output pathway of circadian clock system rather than in the input pathway or oscillators.

Blue light (BL) is a key environmental signal influencing plant flowering, yet its role in floral development beyond the transition phase remains underexplored. This review provides a comprehensive synthesis of current research on BL-mediated floral development, with a particular emphasis on horticultural crops grown in a controlled environment. Unlike prior reviews that focus primarily on floral induction, this article systematically examines BL’s effects on later stages of flowering, including floral organ morphogenesis, sex expression, bud abortion, flower opening, scent emission, coloration, pollination, and senescence. Drawing on evidence from both model plants (e.g., Arabidopsis thaliana) and crop species, this review identifies key photoreceptors, hormonal regulators, and signaling components involved in BL responses. It also highlights species-specific and context-dependent outcomes of BL manipulation, proposes mechanistic hypotheses to explain conflicting findings, and outlines critical knowledge gaps. By integrating molecular, physiological, and environmental perspectives, this review offers a framework for optimizing BL applications to improve flowering traits and postharvest quality in horticultural production systems.

Anatomical features related with pollination of Neottia ovata (L.) Bluff & Fingerh. (Orchidaceae)

Genome-wide identification of clock-associated genes and circadian rhythms in Fragaria × ananassa seedlings