Flower transcriptional response to long term hot and cold environments in Antirrhinum majus.

Short term experiments have identified heat shock and cold response elements in many biological systems. However, the effect of long-term low or high temperatures is not well documented. To address this gap, we grew Antirrhinum majus plants from two-weeks old until maturity under control (normal) (22/16°C), cold (15/5°C), and hot (30/23°C) conditions for a period of two years. Flower size, petal anthocyanin content and pollen viability obtained higher values in cold conditions, decreasing in middle and high temperatures. Leaf chlorophyll content was higher in cold conditions and stable in control and hot temperatures, while pedicel length increased under hot conditions. The control conditions were optimal for scent emission and seed production. Scent complexity was low in cold temperatures. The transcriptomic analysis of mature flowers, followed by gene enrichment analysis and CNET plot visualization, showed two groups of genes. One group comprised genes controlling the affected traits, and a second group appeared as long-term adaptation to non-optimal temperatures. These included hypoxia, unsaturated fatty acid metabolism, ribosomal proteins, carboxylic acid, sugar and organic ion transport, or protein folding. We found a differential expression of floral organ identity functions, supporting the flower size data. Pollinator-related traits such as scent and color followed opposite trends, indicating an equilibrium for rendering the organs for pollination attractive under changing climate conditions. Prolonged heat or cold cause structural adaptations in protein synthesis and folding, membrane composition, and transport. Thus, adaptations to cope with non-optimal temperatures occur in basic cellular processes.

gcProfileMakeR: An R Package for Automatic Classification of Constitutive and Non-Constitutive Metabolites.

Metabolomes comprise constitutive and non-constitutive metabolites produced due to physiological, genetic or environmental effects. However, finding constitutive metabolites and non-constitutive metabolites in large datasets is technically challenging. We developed gcProfileMakeR, an R package using standard Excel output files from an Agilent Chemstation GC-MS for automatic data analysis using CAS numbers. gcProfileMakeR has two filters for data preprocessing removing contaminants and low-quality peaks. The first function NormalizeWithinFiles, samples assigning retention times to CAS. The second function NormalizeBetweenFiles, reaches a consensus between files where compounds in close retention times are grouped together. The third function getGroups, establishes what is considered as Constitutive Profile, Non-constitutive by Frequency i.e., not present in all samples and Non-constitutive by Quality. Results can be plotted with the plotGroup function. We used it to analyse floral scent emissions in four snapdragon genotypes. These included a wild type, Deficiens nicotianoides and compacta affecting floral identity and RNAi:AmLHY targeting a circadian clock gene. We identified differences in scent constitutive and non-constitutive profiles as well as in timing of emission. gcProfileMakeR is a very useful tool to define constitutive and non-constitutive scent profiles. It also allows to analyse genotypes and circadian datasets to identify differing metabolites.

The Effect of Post-harvest Conditions in Narcissus sp. Cut Flowers Scent Profile.

Narcissus flowers are used as cut flowers and to obtain high quality essential oils for the perfume industry. As a winter crop in the Mediterranean area, it flowers at temperatures ranging between 10 and 15°C during the day and 3-10°C during the night. Here we tested the impact of different light and temperature conditions on scent quality during post-harvest. These two types of thermoperiod and photoperiod. We also used constant darkness and constant temperatures. We found that under conditions of 12:12 Light Dark and 15-5°C, Narcissus emitted monoterpenes and phenylpropanoids. Increasing the temperature to 20°-10°C in a 12:12 LD cycle caused the loss of cinnamyl acetate and emission of indole. Under constant dark, there was a loss of scent complexity. Constant temperatures of 20°C caused a decrease of scent complexity that was more dramatic at 5°C, when the total number of compounds emitted decreased from thirteen to six. Distance analysis confirmed that 20°C constant temperature causes the most divergent scent profile. We found a set of four volatiles, benzyl acetate, eucalyptol, linalool, and ocimene that display a robust production under differing environmental conditions, while others were consistently dependent on light or thermoperiod. Scent emission changed significantly during the day and between different light and temperature treatments. Under a light:dark cycle and 15-5°C the maximum was detected during the light phase but this peak shifted toward night under 20-10°C. Moreover, under constant darkness the peak occurred at midnight and under constant temperature, at the end of night. Using Machine Learning we found that indole was the volatile with a highest ranking of discrimination followed by D-limonene. Our results indicate that light and temperature regimes play a critical role in scent quality. The richest scent profile is obtained by keeping flowers at 15°-5°C thermoperiod and a 12:12 Light Dark photoperiod.

Transcriptional Structure of Petunia Clock in Leaves and Petals.

The plant circadian clock coordinates environmental signals with internal processes including secondary metabolism, growth, flowering, and volatile emission. Plant tissues are specialized in different functions, and petals conceal the sexual organs while attracting pollinators. Here we analyzed the transcriptional structure of the petunia (Petunia x hybrida) circadian clock in leaves and petals. We recorded the expression of 13 clock genes in petunia under light:dark (LD) and constant darkness (DD). Under light:dark conditions, clock genes reached maximum expression during the light phase in leaves and the dark period in petals. Under free running conditions of constant darkness, maximum expression was delayed, especially in petals. Interestingly, the rhythmic expression pattern of PhLHY persisted in leaves and petals in LD and DD. Gene expression variability differed among leaves and petals, time of day and photoperiod. The transcriptional noise was higher especially in leaves under constant darkness. We found that PhPRR7, PhPRR5, and PhGI paralogs showed changes in gene structure including exon number and deletions of CCT domain of the PRR family. Our results revealed that petunia petals presented a specialized clock.

The Snapdragon LATE ELONGATED HYPOCOTYL Plays A Dual Role in Activating Floral Growth and Scent Emission.

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.

The Petunia CHANEL Gene is a ZEITLUPE Ortholog Coordinating Growth and Scent Profiles.

The floral perianth, comprising sepals and petals, conceals the sexual organs and attracts pollinators. The coordination of growth and scent emission is not fully understood. We have analyzed the effect of knocking down CHANEL (PhCHL), the ZEITLUPE ortholog in petunia (PhCHL) by hairpin RNAs. Plants with low PhCHL mRNA had overall decreased size. Growth evaluation using time lapse image analysis showed that early leaf movement was not affected by RNAi:PhCHL, but flower angle movement was modified, moving earlier during the day in knockdown plants than in wild types. Despite differences in stem length, growth rate was not significantly affected by loss of PhCHL. In contrast, petal growth displayed lower growth rate in RNAi:PhCHL. Decreased levels of PhCHL caused strongly modified scent profiles, including changes in composition and timing of emission resulting in volatile profiles highly divergent from the wild type. Our results show a role of PhCHL in controlling growth and development of vegetative and reproductive organs in petunia. The different effects of PhCHL on organ development indicate an organ-specific interpretation of the down regulation of PhCHL. Through the control of both timing and quantitative volatile emissions, PhCHL appears to be a major coordinator of scent profiles.

Diel pattern of circadian clock and storage protein gene expression in leaves and during seed filling in cowpea (Vigna unguiculata).

BACKGROUND: Cowpea (Vigna unguiculata) is an important source of protein supply for animal and human nutrition. The major storage globulins VICILIN and LEGUMIN (LEG) are synthesized from several genes including LEGA, LEGB, LEGJ and CVC (CONVICILIN). The current hypothesis is that the plant circadian core clock genes are conserved in a wide array of species and that primary metabolism is to a large extent controlled by the plant circadian clock. Our aim was to investigate a possible link between gene expression of storage proteins and the circadian clock. RESULTS: We identified cowpea orthologues of the core clock genes VunLHY, VunTOC1, VunGI and VunELF3, the protein storage genes VunLEG, VunLEGJ, and VunCVC as well as nine candidate reference genes used in RT-PCR. ELONGATION FACTOR 1-A (ELF1A) resulted the most suitable reference gene. The clock genes VunELF3, VunGI, VunTOC1 and VunLHY showed a rhythmic expression profile in leaves with a typical evening/night and morning/midday phased expression. The diel patterns were not completely robust and only VungGI and VungELF3 retained a rhythmic pattern under free running conditions of darkness. Under field conditions, rhythmicity and phasing apparently faded during early pod and seed development and was regained in ripening pods for VunTOC1 and VunLHY. Mature seeds showed a rhythmic expression of VunGI resembling leaf tissue under controlled growth chamber conditions. Comparing time windows during developmental stages we found that VunCVC and VunLEG were significantly down regulated during the night in mature pods as compared to intermediate ripe pods, while changes in seeds were non-significant due to high variance. The rhythmic expression under field conditions was lost under growth chamber conditions. CONCLUSIONS: The core clock gene network is conserved in cowpea leaves showing a robust diel expression pattern except VunELF3 under growth chamber conditions. There appears to be a clock transcriptional reprogramming in pods and seeds compared to leaves. Storage protein deposition may be circadian regulated under field conditions but the strong environmental signals are not met under artificial growth conditions. Diel expression pattern in field conditions may result in better usage of energy for protein storage.

Daily rhythms of toxicity and effectiveness of anesthetics (MS222 and eugenol) in zebrafish (Danio rerio).

Although the chronotoxicity of xenobiotics is relatively well known in mammals, the existence of daily rhythms of drug toxicity and effectiveness in fish has been neglected to date. The aim of this research was to investigate the influence of the time (middle of the light phase [ML] versus middle of the dark phase [MD]) of exposure to two anesthetic substances (MS-222 or clove oil) commonly used with fish on the median lethal concentration (LC(50)) and swimming activity of zebrafish (Danio rerio). To this end, adult zebrafish were kept under a 12 h:12 h light-dark (LD) cycle and exposed to different concentrations of the anesthetics for 15 min at ML or MD. LC(50) calculations were performed using the Spearman-Karber program, whereas swimming activity was video-recorded and analyzed with specialized software. Zebrafish exhibited a mostly diurnal activity pattern (77.9% of activity occurring during daytime). The acute toxicity and mortality caused by MS-222 and eugenol varied with the time of exposure. For MS-222, the LC(50) was 170.6 ± 7.4 mg/L in fish exposed at ML and 215.6 ± 3.9 mg/L at MD, whereas for eugenol the LC(50) was 70.3 ± 3.1 mg/L at ML and 104.9 ± 5.4 mg/L at MD. Exposure to sublethal concentrations of MS-222 and eugenol altered the swimming patterns of zebrafish in a different manner depending on the time of exposure. Thus, the time required for decreasing swimming activity during exposure to anesthetics was shorter at ML than at MD, whereas the recovery period was longer during the day. In conclusion, these results revealed that the toxicity and effectiveness of both anesthetic substances is highest during daytime, the active phase of fish, thus suggesting a link between the daily rhythms of behavior and toxicity.