Sleep in everyday life - relationship to mood and performance in young and older adults: a study protocol.

Laboratory based sleep deprivation studies demonstrate that lack of sleep impairs well-being and performance ability, but suggest that these effects are mitigated in older adults. Yet, much less is known whether day-to-day variations of sleep have similar consequences in the context of everyday life. This project uses an intensive longitudinal design to investigate the occurrence of day-to-day variations in sleep and their impact on mood and performance in everyday life and to examine whether effects differ between young and older adults. We aim to include 160 young (18-30 years) and 160 older adults (55-75 years) to complete a 21-day experience sampling method (ESM) protocol. During the ESM period, participants are asked to fill in (i) a brief morning questionnaire, (ii) 8 short daytime questionnaires addressing momentary well-being, sleepiness, stress, and mind wandering, followed by a 1 min cognitive task and (iii) a brief evening questionnaire, all delivered via a mobile phone application. Sleep will be measured using self-reports (daily questions) and objectively with wrist actigraphy. The impact of adult age on mean levels and intraindividual variability of sleep will be analyzed using mixed-effects location scale models. The impact of sleep on daily cognitive performance will be analyzed using multilevel linear mixed models. The relationship of sleep to mean values and variability of positive and negative affect in young and older adults will be analyzed using mixed-effects location scale modeling. The overarching purpose of the project is improving the current knowledge on the occurrence of day-to-day variations in sleep and their relationship to performance as well as positive and negative affect in young and older adults.

Working environment and fatigue among fishers in the north Atlantic: a field study.

BACKGROUND: This study investigates how Faroese deep-sea fishers' exposure to work-related stressors affects their sleep, sleepiness, and levels of fatigue. Being constantly exposed to the unpredictable and harsh North Atlantic Ocean, having long work hours and split sleep for up to 40 days consecutively, they will arguably suffer from fatigue. MATERIALS AND METHODS: One hundred and fifty seven fishers participated in this study, and data was gathered throughout 202 days at sea. Subjective data was collected at the start and end of trips via questionnaires, sleep and sleepiness diaries and supplemented by objective sleep data through actigraphs. Ship movements were logged with a gyroscope connected to a laptop. A noise metre measured each work station and resting area, and noise exposure profiles were calculated based on each participant's activity and location. Linear mixed-effect models investigated the effects of work exposure variables on sleep efficiency, and cumulative link mixed models measured effects on the Karolinska Sleepiness Scale and physical fatigue scale. RESULTS: Time of day followed by ship movement were the exposure variables with the highest impact on the outcome variables of sleep efficiency, sleepiness and physical fatigue. The number of days at sea revealed correlations to outcome variables either by itself or interacting with the sleep periods per day. Crew size, shift system or noise did not impact outcome variables when in the model with other variables. Larger catches improved sleep efficiency but did not affect sleepiness and physical fatigue ratings. CONCLUSIONS: The findings indicate a chronically fatigued fisher population, and recommends urgent attention being paid to improving the structure of vessels and installing stabilators for greater stability at sea; work schedules being evaluated for protection of health; and work environments being designed that fulfill human physiological requirements in order to ensure the wellbeing and safety of those at sea.

Stress-induced phase separation of ERES components into Sec bodies precedes ER exit inhibition in mammalian cells.

Phase separation of components of ER exit sites (ERES) into membraneless compartments, the Sec bodies, occurs in Drosophila cells upon exposure to specific cellular stressors, namely, salt stress and amino acid starvation, and their formation is linked to the early secretory pathway inhibition. Here, we show Sec bodies also form in secretory mammalian cells upon the same stress. These reversible and membraneless structures are positive for ERES components, including both Sec16A and Sec16B isoforms and COPII subunits. We find that Sec16A, but not Sec16B, is a driver for Sec body formation, and that the coalescence of ERES components into Sec bodies occurs by fusion. Finally, we show that the stress-induced coalescence of ERES components into Sec bodies precedes ER exit inhibition, leading to their progressive depletion from ERES that become non-functional. Stress relief causes an immediate dissolution of Sec bodies and the concomitant restoration of ER exit. We propose that the dynamic conversion between ERES and Sec body assembly, driven by Sec16A, regulates protein exit from the ER during stress and upon stress relief in mammalian cells, thus providing a conserved pro-survival mechanism in response to stress.

Impact of work exposure on cognitive performance in Faroese deep-sea fishers: a field study.

BACKGROUND: This study examines the impact of work-related exposure on the cognitive performance of Faroese deep-sea fishers. Faroese fishing crews work long hours in demanding and noisy environments amidst highly uncertain and challenging weather conditions. These factors, together with compromised patterns of rest and sleep, are known to increase fatigue. Our aim was to study if changes could be measured in fishers' cognitive performance at the end of the trip when compared with the baseline measure at the beginning. MATERIALS AND METHODS: Data was collected over 15 months (May 2017 to July 2018) from 157 fishers on 18 fishing trips which involved 202 investigative days on board. Questionnaires and six computerised cognitive tests: Simple Reaction Time, Numeric Working Memory, Corsi Blocks, Rapid Visual Information Processing, Digit Vigilance, and Card Sorting Test were used for data collection at the beginning and end of the trip. Differences between the outcomes on the two test points were analysed with one-way ANOVA comparing the performances at the beginning and end of the voyage, and two-way ANOVA to examine the interactive effect of chronotype and test occasions on the outcomes. Mixed models were used to test for the effects of predictor variables. RESULTS: Significant declines in cognitive performance were observed from the beginning to the end of the trip, with decreases in visuospatial memory and reaction times, and increases in cognitive lapses. Furthermore, slowing in response times was observed in the second half of the Digit Vigilance test when comparing the halves. CONCLUSIONS: Declines in performance were observed from the start to the end of the trip. Furthermore, fishers performed significantly worse in the second half of some parted tests, and evening types seem less influenced by irregular work hours. These findings call for improving the safety of the vessels and their crew.

Identification of the stress granule transcriptome via RNA-editing in single cells and in vivo.

Stress granules are phase-separated assemblies formed around RNAs. So far, the techniques available to identify these RNAs are not suitable for single cells and small tissues displaying cell heterogeneity. Here, we used TRIBE (target of RNA-binding proteins identified by editing) to profile stress granule RNAs. We used an RNA-binding protein (FMR1) fused to the catalytic domain of an RNA-editing enzyme (ADAR), which coalesces into stress granules upon oxidative stress. RNAs colocalized with this fusion are edited, producing mutations that are detectable by VASA sequencing. Using single-molecule FISH, we validated that this purification-free method can reliably identify stress granule RNAs in bulk and single S2 cells and in Drosophila neurons. Similar to mammalian cells, we find that stress granule mRNAs encode ATP binding, cell cycle, and transcription factors. This method opens the possibility to identify stress granule RNAs and other RNA-based assemblies in other single cells and tissues.

Sleep, Sleepiness, and Fatigue on Board Faroese Fishing Vessels.

Purpose: Faroese fishers have four times more accidents than workers on land. The aim was to understand fishers' fatigue better and how their work and sleep patterns influenced their sleepiness levels and cognitive performance. Materials and Methods: A total of 157 Faroese fishers wore wrist-worn actigraphs at sea and one week on land and filled in sleep and sleepiness diaries during the trip. Furthermore, a 3-minute simple reaction time (SRT) test was completed at the beginning and end of the trip. The ship's movement and noise were also logged. The actiwatch results were analysed with mixed methods repeated measures. The sleepiness registrations and performance on the SRT-test were analysed with paired t-test. The ship movements (Pitch and roll) were divided into approximately three same-sized groups (lowest 1/3, medium 1/3, and highest 1/3) and compared against the Karolinska Sleepiness Scores (KSS ranging from 1-9) ≥7 and physical tiredness (ranging from 1-9) scores ≥7. Chi-square tests were used to determine the significance of these differences. Mean sleepiness scores at sea, and the proportion of sleepiness scores ≥7 were calculated, as well as sleepiness scores as a function of the time of day. Results: While at sea, fishers had more split sleep, slept less, and had lower sleep efficiency than onshore. Sleepiness was higher at the end of the trip, and cognitive decline was found. The number of major lapses was higher at the end of the trip, but with no significant difference between the median reaction times. Conclusion: The crew on-board the freezer longliner, who worked 8-8 shifts, slept the most, had the longest continuous sleep periods, the highest sleep efficiency, the lowest sleepiness levels, and the highest noise exposure during their time off.

Activation of IRE1, PERK and salt-inducible kinases leads to Sec body formation in Drosophila S2 cells.

The phase separation of the non-membrane bound Sec bodies occurs in Drosophila S2 cells by coalescence of components of the endoplasmic reticulum (ER) exit sites under the stress of amino acid starvation. Here, we address which signaling pathways cause Sec body formation and find that two pathways are critical. The first is the activation of the salt-inducible kinases (SIKs; SIK2 and SIK3) by Na+ stress, which, when it is strong, is sufficient. The second is activation of IRE1 and PERK (also known as PEK in flies) downstream of ER stress induced by the absence of amino acids, which needs to be combined with moderate salt stress to induce Sec body formation. SIK, and IRE1 and PERK activation appear to potentiate each other through the stimulation of the unfolded protein response, a key parameter in Sec body formation. This work shows the role of SIKs in phase transition and re-enforces the role of IRE1 and PERK as a metabolic sensor for the level of circulating amino acids and salt. This article has an associated First Person interview with the first author of the paper.

Cellular stress leads to the formation of membraneless stress assemblies in eukaryotic cells.

In cells at steady state, two forms of cell compartmentalization coexist: membrane-bound organelles and phase-separated membraneless organelles that are present in both the nucleus and the cytoplasm. Strikingly, cellular stress is a strong inducer of the reversible membraneless compartments referred to as stress assemblies. Stress assemblies play key roles in survival during cell stress and in thriving of cells upon stress relief. The two best studied stress assemblies are the RNA-based processing-bodies (P-bodies) and stress granules that form in response to oxidative, endoplasmic reticulum (ER), osmotic and nutrient stress as well as many others. Interestingly, P-bodies and stress granules are heterogeneous with respect to both the pathways that lead to their formation and their protein and RNA content. Furthermore, in yeast and Drosophila, nutrient stress also leads to the formation of many other types of prosurvival cytoplasmic stress assemblies, such as metabolic enzymes foci, proteasome storage granules, EIF2B bodies, U-bodies and Sec bodies, some of which are not RNA-based. Nutrient stress leads to a drop in cytoplasmic pH, which combined with posttranslational modifications of granule contents, induces phase separation.

Fluorescently-labelled CPD and 6-4PP photolyases: new tools for live-cell DNA damage quantification and laser-assisted repair.

UV light induces cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PPs), which can result in carcinogenesis and aging, if not properly repaired by nucleotide excision repair (NER). Assays to determine DNA damage load and repair rates are invaluable tools for fundamental and clinical NER research. However, most current assays to quantify DNA damage and repair cannot be performed in real time. To overcome this limitation, we made use of the damage recognition characteristics of CPD and 6-4PP photolyases (PLs). Fluorescently-tagged PLs efficiently recognize UV-induced DNA damage without blocking NER activity, and therefore can be used as sensitive live-cell damage sensors. Importantly, FRAP-based assays showed that PLs bind to damaged DNA in a highly sensitive and dose-dependent manner, and can be used to quantify DNA damage load and to determine repair kinetics in real time. Additionally, PLs can instantly reverse DNA damage by 405 nm laser-assisted photo-reactivation during live-cell imaging, opening new possibilities to study lesion-specific NER dynamics and cellular responses to damage removal. Our results show that fluorescently-tagged PLs can be used as a versatile tool to sense, quantify and repair DNA damage, and to study NER kinetics and UV-induced DNA damage response in living cells.

Does sleep deprivation increase the vulnerability to acute psychosocial stress in young and older adults?

Sleep loss and psychosocial stress often co-occur in today's society, but there is limited knowledge on the combined effects. Therefore, this experimental study investigated whether one night of sleep deprivation affects the response to a psychosocial challenge. A second aim was to examine if older adults, who may be less affected by both sleep deprivation and stress, react differently than young adults. 124 young (18-30 years) and 94 older (60-72 years) healthy adults participated in one of four conditions: i. normal night sleep & Placebo-Trier Social Stress Test (TSST), ii. normal night sleep & Trier Social Stress Test, iii. sleep deprivation & Placebo-TSST, iv. sleep deprivation & TSST. Subjective stress ratings, heart rate variability (HRV), salivary alpha amylase (sAA) and cortisol were measured throughout the protocol. At the baseline pre-stress measurement, salivary cortisol and subjective stress values were higher in sleep deprived than in rested participants. However, the reactivity to and recovery from the TSST was not significantly different after sleep deprivation for any of the outcome measures. Older adults showed higher subjective stress, higher sAA and lower HRV at baseline, indicating increased basal autonomic activity. Cortisol trajectories and HRV slightly differed in older adults compared with younger adults (regardless of the TSST). Moreover, age did not moderate the effect of sleep deprivation. Taken together, the results show increased stress levels after sleep deprivation, but do not confirm the assumption that one night of sleep deprivation increases the responsivity to an acute psychosocial challenge.

Modulation of the secretory pathway by amino-acid starvation.

As a major anabolic pathway, the secretory pathway needs to adapt to the demands of the surrounding environment and responds to different exogenous signals and stimuli. In this context, the transport in the early secretory pathway from the endoplasmic reticulum (ER) to the Golgi apparatus appears particularly regulated. For instance, protein export from the ER is critically stimulated by growth factors. Conversely, nutrient starvation also modulates functions of the early secretory pathway in multiple ways. In this review, we focus on amino-acid starvation and how the function of the early secretory pathway is redirected to fuel autophagy, how the ER exit sites are remodeled into novel cytoprotective stress assemblies, and how secretion is modulated in vivo in starving organisms. With the increasingly exciting knowledge on mechanistic target of rapamycin complex 1 (mTORC1), the major nutrient sensor, it is also a good moment to establish how the modulation of the secretory pathway by amino-acid restriction intersects with this major signaling hub.

Physiological and autonomic stress responses after prolonged sleep restriction and subsequent recovery sleep in healthy young men.

PURPOSE: Sleep restriction is increasingly common and associated with the development of health problems. We investigated how the neuroendocrine stress systems respond to prolonged sleep restriction and subsequent recovery sleep in healthy young men. METHODS: After two baseline (BL) nights of 8 h time in bed (TIB), TIB was restricted to 4 h per night for five nights (sleep restriction, SR, n = 15), followed by three recovery nights (REC) of 8 h TIB, representing a busy workweek and a recovery weekend. The control group (n = 8) had 8 h TIB throughout the experiment. A variety of autonomic cardiovascular parameters, together with salivary neuropeptide Y (NPY) and cortisol levels, were assessed. RESULTS: In the control group, none of the parameters changed. In the experimental group, heart rate increased from 60 ± 1.8 beats per minute (bpm) at BL, to 63 ± 1.1 bpm after SR and further to 65 ± 1.8 bpm after REC. In addition, whole day low-frequency to-high frequency (LF/HF) power ratio of heart rate variability increased from 4.6 ± 0.4 at BL to 6.0 ± 0.6 after SR. Other parameters, including salivary NPY and cortisol levels, remained unaffected. CONCLUSIONS: Increased heart rate and LF/HF power ratio are early signs of an increased sympathetic activity after prolonged sleep restriction. To reliably interpret the clinical significance of these early signs of physiological stress, a follow-up study would be needed to evaluate if the stress responses escalate and lead to more unfavourable reactions, such as elevated blood pressure and a subsequent elevated risk for cardiovascular health problems.

Seafarer fatigue: a review of risk factors, consequences for seafarers' health and safety and options for mitigation.

BACKGROUND: The consequences of fatigue for the health and safety of seafarers has caused concern in the industry and among academics, and indicates the importance of further research into risk factors and preventive interventions at sea. This review gives an overview of the key issues relating to seafarer fatigue. MATERIALS AND METHODS: A literature study was conducted aiming to collect publications that address risk factors for fatigue, short-term and long-term consequences for health and safety, and options for fatigue mitigation at sea. Due to the limited number of publications that deals with seafarers, experiences from other populations sharing the same exposures (e.g. shift work) were also included when appropriate. RESULTS: Work at sea involves multiple risk factors for fatigue, which in addition to acute effects (e.g., impaired cognition, accidents) contributes through autonomic, immunologic and metabolic pathways to the development of chronic diseases that are particularly prevalent in seafarers. CONCLUSIONS: Taking into account the frequency of seafarer fatigue and the severity of its consequences, one should look into the efficacy of the current legislative framework and the industry's compliance, the manning of the international merchant fleet, and optimised working, living and sleeping conditions at sea. Considering circumstances at sea, e.g. working in shifts and crossing time zones, that cannot be altered, further assessment of the potentials of preventive interventions including fatigue prediction tools and individual fatigue mitigation management systems is recommended.

Validating and extending the three process model of alertness in airline operations.

Sleepiness and fatigue are important risk factors in the transport sector and bio-mathematical sleepiness, sleep and fatigue modeling is increasingly becoming a valuable tool for assessing safety of work schedules and rosters in Fatigue Risk Management Systems (FRMS). The present study sought to validate the inner workings of one such model, Three Process Model (TPM), on aircrews and extend the model with functions to model jetlag and to directly assess the risk of any sleepiness level in any shift schedule or roster with and without knowledge of sleep timings. We collected sleep and sleepiness data from 136 aircrews in a real life situation by means of an application running on a handheld touch screen computer device (iPhone, iPod or iPad) and used the TPM to predict sleepiness with varying level of complexity of model equations and data. The results based on multilevel linear and non-linear mixed effects models showed that the TPM predictions correlated with observed ratings of sleepiness, but explorative analyses suggest that the default model can be improved and reduced to include only two-processes (S+C), with adjusted phases of the circadian process based on a single question of circadian type. We also extended the model with a function to model jetlag acclimatization and with estimates of individual differences including reference limits accounting for 50%, 75% and 90% of the population as well as functions for predicting the probability of any level of sleepiness for ecological assessment of absolute and relative risk of sleepiness in shift systems for safety applications.

Partial sleep restriction activates immune response-related gene expression pathways: experimental and epidemiological studies in humans.

Epidemiological studies have shown that short or insufficient sleep is associated with increased risk for metabolic diseases and mortality. To elucidate mechanisms behind this connection, we aimed to identify genes and pathways affected by experimentally induced, partial sleep restriction and to verify their connection to insufficient sleep at population level. The experimental design simulated sleep restriction during a working week: sleep of healthy men (N = 9) was restricted to 4 h/night for five nights. The control subjects (N = 4) spent 8 h/night in bed. Leukocyte RNA expression was analyzed at baseline, after sleep restriction, and after recovery using whole genome microarrays complemented with pathway and transcription factor analysis. Expression levels of the ten most up-regulated and ten most down-regulated transcripts were correlated with subjective assessment of insufficient sleep in a population cohort (N = 472). Experimental sleep restriction altered the expression of 117 genes. Eight of the 25 most up-regulated transcripts were related to immune function. Accordingly, fifteen of the 25 most up-regulated Gene Ontology pathways were also related to immune function, including those for B cell activation, interleukin 8 production, and NF-κB signaling (P<0.005). Of the ten most up-regulated genes, expression of STX16 correlated negatively with self-reported insufficient sleep in a population sample, while three other genes showed tendency for positive correlation. Of the ten most down-regulated genes, TBX21 and LGR6 correlated negatively and TGFBR3 positively with insufficient sleep. Partial sleep restriction affects the regulation of signaling pathways related to the immune system. Some of these changes appear to be long-lasting and may at least partly explain how prolonged sleep restriction can contribute to inflammation-associated pathological states, such as cardiometabolic diseases.

Sleep, sleepiness, and neurobehavioral performance while on watch in a simulated 4 hours on/8 hours off maritime watch system.

Seafarer sleepiness jeopardizes safety at sea and has been documented as a direct or contributing factor in many maritime accidents. This study investigates sleep, sleepiness, and neurobehavioral performance in a simulated 4 h on/8 h off watch system as well as the effects of a single free watch disturbance, simulating a condition of overtime work, resulting in 16 h of work in a row and a missed sleep opportunity. Thirty bridge officers (age 30 ± 6 yrs; 29 men) participated in bridge simulator trials on an identical 1-wk voyage in the North Sea and English Channel. The three watch teams started respectively with the 00-04, the 04-08, and the 08-12 watches. Participants rated their sleepiness every hour (Karolinska Sleepiness Scale [KSS]) and carried out a 5-min psychomotor vigilance test (PVT) test at the start and end of every watch. Polysomnography (PSG) was recorded during 6 watches in the first and the second half of the week. KSS was higher during the first (mean ± SD: 4.0 ± 0.2) compared with the second (3.3 ± 0.2) watch of the day (p < 0.001). In addition, it increased with hours on watch (p < 0.001), peaking at the end of watch (4.1 ± 0.2). The free watch disturbance increased KSS profoundly (p < 0.001): from 4.2 ± 0.2 to 6.5 ± 0.3. PVT reaction times were slower during the first (290 ± 6 ms) compared with the second (280 ± 6 ms) watch of the day (p < 0.001) as well as at the end of the watch (289 ± 6 ms) compared with the start (281 ± 6 ms; p = 0.001). The free watch disturbance increased reaction times (p < 0.001) from 283 ± 5 to 306 ± 7 ms. Similar effects were observed for PVT lapses. One third of all participants slept during at least one of the PSG watches. Sleep on watch was most abundant in the team working 00-04 and it increased following the free watch disturbance. This study reveals that-within a 4 h on/8 h off shift system-subjective and objective sleepiness peak during the night and early morning watches, coinciding with a time frame in which relatively many maritime accidents occur. In addition, we showed that overtime work strongly increases sleepiness. Finally, a striking amount of participants fell asleep while on duty.

Prolonged sleep restriction affects glucose metabolism in healthy young men.

This study identifies the effects of sleep restriction and subsequent recovery sleep on glucose homeostasis, serum leptin levels, and feelings of subjective satiety. Twenty-three healthy young men were allocated to a control group (CON) or an experimental (EXP) group. After two nights of 8 h in bed (baseline, BL), EXP spent 4 h in bed for five days (sleep restriction, SR), followed by two nights of 8 h (recovery, REC). CON spent 8 h in bed throughout the study. Blood samples were taken after the BL, SR, and REC period. In EXP, insulin and insulin-to-glucose ratio increased after SR. IGF-1 levels increased after REC. Leptin levels were elevated after both SR and REC; subjective satiety remained unaffected. No changes were observed in CON. The observed increase of serum IGF-1 and insulin-to-glucose ratio indicates that sleep restriction may result in an increased risk to develop type 2 diabetes.

Sleep restriction increases the risk of developing cardiovascular diseases by augmenting proinflammatory responses through IL-17 and CRP.

BACKGROUND: Sleep restriction, leading to deprivation of sleep, is common in modern 24-h societies and is associated with the development of health problems including cardiovascular diseases. Our objective was to investigate the immunological effects of prolonged sleep restriction and subsequent recovery sleep, by simulating a working week and following recovery weekend in a laboratory environment. METHODS AND FINDINGS: After 2 baseline nights of 8 hours time in bed (TIB), 13 healthy young men had only 4 hours TIB per night for 5 nights, followed by 2 recovery nights with 8 hours TIB. 6 control subjects had 8 hours TIB per night throughout the experiment. Heart rate, blood pressure, salivary cortisol and serum C-reactive protein (CRP) were measured after the baseline (BL), sleep restriction (SR) and recovery (REC) period. Peripheral blood mononuclear cells (PBMC) were collected at these time points, counted and stimulated with PHA. Cell proliferation was analyzed by thymidine incorporation and cytokine production by ELISA and RT-PCR. CRP was increased after SR (145% of BL; p<0.05), and continued to increase after REC (231% of BL; p<0.05). Heart rate was increased after REC (108% of BL; p<0.05). The amount of circulating NK-cells decreased (65% of BL; p<0.005) and the amount of B-cells increased (121% of BL; p<0.005) after SR, but these cell numbers recovered almost completely during REC. Proliferation of stimulated PBMC increased after SR (233% of BL; p<0.05), accompanied by increased production of IL-1beta (137% of BL; p<0.05), IL-6 (163% of BL; p<0.05) and IL-17 (138% of BL; p<0.05) at mRNA level. After REC, IL-17 was still increased at the protein level (119% of BL; p<0.05). CONCLUSIONS: 5 nights of sleep restriction increased lymphocyte activation and the production of proinflammatory cytokines including IL-1beta IL-6 and IL-17; they remained elevated after 2 nights of recovery sleep, accompanied by increased heart rate and serum CRP, 2 important risk factors for cardiovascular diseases. Therefore, long-term sleep restriction may lead to persistent changes in the immune system and the increased production of IL-17 together with CRP may increase the risk of developing cardiovascular diseases.

The impact of the absence of aliphatic glucosinolates on insect herbivory in Arabidopsis.

Aliphatic glucosinolates are compounds which occur in high concentrations in Arabidopsis thaliana and other Brassicaceae species. They are important for the resistance of the plant to pest insects. Previously, the biosynthesis of these compounds was shown to be regulated by transcription factors MYB28 and MYB29. We now show that MYB28 and MYB29 are partially redundant, but in the absence of both, the synthesis of all aliphatic glucosinolates is blocked. Untargeted and targeted biochemical analyses of leaf metabolites showed that differences between single and double knock-out mutants and wild type plants were restricted to glucosinolates. Biosynthesis of long-chain aliphatic glucosinolates was blocked by the myb28 mutation, while short-chain aliphatic glucosinolates were reduced by about 50% in both the myb28 and the myb29 single mutants. Most remarkably, all aliphatic glucosinolates were completely absent in the double mutant. Expression of glucosinolate biosynthetic genes was slightly but significantly reduced by the single myb mutations, while the double mutation resulted in a drastic decrease in expression of these genes. Since the myb28myb29 double mutant is the first Arabidopsis genotype without any aliphatic glucosinolates, we used it to establish the relevance of aliphatic glucosinolate biosynthesis to herbivory by larvae of the lepidopteran insect Mamestra brassicae. Plant damage correlated inversely to the levels of aliphatic glucosinolates observed in those plants: Larval weight gain was 2.6 fold higher on the double myb28myb29 mutant completely lacking aliphatic glucosinolates and 1.8 higher on the single mutants with intermediate levels of aliphatic glucosinolates compared to wild type plants.

Visualization of phosphatidylinositol 4,5-bisphosphate in the plasma membrane of suspension-cultured tobacco BY-2 cells and whole Arabidopsis seedlings.

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] is an important signalling lipid in mammalian cells, where it functions as a second-messenger precursor in response to agonist-dependent activation of phospholipase C (PLC) but also operates as a signalling molecule on its own. Much of the recent knowledge about it has come from a new technique to visualize PtdIns(4,5)P(2)in vivo, by expressing a green or yellow fluorescent protein (GFP or YFP) fused to the pleckstrin homology (PH) domain of human PLCdelta1 that specifically binds PtdIns(4,5)P(2). In this way, YFP-PH(PLCdelta1) has been shown to predominantly label the plasma membrane and to transiently translocate into the cytoplasm upon PLC activation in a variety of mammalian cell systems. In plants, biochemical studies have shown that PtdIns(4,5)P(2) is present in very small quantities, but knowledge of its localization and function is still very limited. In this study, we have used YFP-PH(PLCdelta1) to try monitoring PtdIns(4,5)P(2)/PLC signalling in stably-transformed tobacco Bright Yellow-2 (BY-2) cells and Arabidopsis seedlings. In both plant systems, no detrimental effects were observed, indicating that overexpression of the biosensor did not interfere with the function of PtdIns(4,5)P(2). Confocal imaging revealed that most of the YFP-PH(PLCdelta1) fluorescence was present in the cytoplasm, and not in the plasma membrane as in mammalian cells. Nonetheless, four conditions were found in which YFP-PH(PLCdelta1) was concentrated at the plasma membrane: (i) upon treatment with the PLC inhibitor U73122; (ii) in response to salt stress; (iii) as a gradient at the tip of growing root hairs; (iv) during the final stage of a BY-2 cell division. We conclude that PtdIns(4,5)P(2), as in animals, is present in the plasma membrane of plants, but that its concentration in most cells is too low to be detected by YFP-PH(PLCdelta1). Hence, the reporter remains unbound in the cytosol, making it unsuitable to monitor PLC signalling. Nonetheless, YFP-PH(PLCdelta1) is a valuable plant PtdIns(4,5)P(2) reporter, for it highlights specific cells and conditions where this lipid becomes abnormally concentrated in membranes, raising the question of what it is doing there. New roles for PtdIns(4,5)P(2) in plant cell signalling are discussed.