Relationship between starch degradation and carbon demand for maintenance and growth in Arabidopsis thaliana in different irradiance and temperature regimes

Experiments were designed to compare the relationship between starch degradation and the use of carbon for maintenance and growth in Arabidopsis in source‐limited and sink‐limited conditions. It is known that starch degradation is regulated by the clock in source‐limited plants, which degrade their starch in a linear manner such that it is almost but not completely exhausted at dawn. We asked whether this response is maintained under an extreme carbon deficit. Arabidopsis was subjected to a sudden combination of a day of low irradiance, to decrease starch at dusk, and a warm night. Starch was degraded in a linear manner through the night, even though the plants became acutely carbon starved. We conclude that starch degradation is not increased to meet demand in carbon‐limited plants. This network property will allow stringent control of starch turnover in a fluctuating environment. In contrast, in sink‐limited plants, which do not completely mobilize their starch during the night, starch degradation was accelerated in warm nights to meet the increased demand for maintenance and growth. Across all conditions, the rate of growth at night depends on the rate of starch degradation, whereas the rate of maintenance respiration decreases only when starch degradation is very slow.     

Aberrant temporal growth pattern and morphology of root and shoot caused by a defective circadian clock in Arabidopsis thaliana

Circadian clocks synchronized with the environment allow plants to anticipate recurring daily changes and give a fitness advantage. Here, we mapped the dynamic growth phenotype of leaves and roots in two lines of Arabidopsis thaliana with a disrupted circadian clock: the CCA1 over‐expressing line (CCA1ox) and the prr9 prr7 prr5 (prr975) mutant. We demonstrate leaf growth defects due to a disrupted circadian clock over a 24 h time scale. Both lines showed enhanced leaf growth compared with the wild‐type during the diurnal period, suggesting increased partitioning of photosynthates for leaf growth. Nocturnal leaf growth was reduced and growth inhibition occurred by dawn, which may be explained by ineffective starch degradation in the leaves of the mutants. However, this growth inhibition was not caused by starch exhaustion. Overall, these results are consistent with the notion that the defective clock affects carbon and energy allocation, thereby reducing growth capacity during the night. Furthermore, rosette morphology and size as well as root architecture were strikingly altered by the defective clock control. Separate analysis of the primary root and lateral roots revealed strong suppression of lateral root formation in both CCA1ox and prr975, accompanied by unusual changes in lateral root growth direction under light–dark cycles and increased lateral extension of the root system. We conclude that growth of the whole plant is severely affected by improper clock regulation in A. thaliana, resulting not only in altered timing and capacity for growth but also aberrant development of shoot and root architecture.     

Mutations in leaf starch metabolism modulate the diurnal root growth profiles of Arabidopsis thaliana

Roots of Arabidopsis thaliana exhibit stable diurnal growth profiles that are controlled by the circadian clock. Here we describe the effects of mutations in leaf starch metabolism on the diurnal root growth characteristics of Arabidopsis thaliana. High temporal and spatial resolution video imaging was performed to quantify the growth kinetics of Arabidopsis wild-type as well as pgm, sex1, mex1, dpe1 and dpe2 starch metabolism mutants grown in three different photoperiods. As a result, root growth patterns of all genotypes displayed characteristic modifications in their diurnal kinetics that were also affected by the photoperiod. To further investigate the role of starch derived substrate deficiency on root growth, the effect of 0.05% extracellular sucrose was studied in 12 h-12 h light-dark cycles.Key words: diurnal root growth kinetics, dpe1, dpe2, mex1, pgm, sex1, starch metabolism, video imagingRoot growth of Arabidopsis thaliana is highly rhythmic with respect to the time of the day.^1–3 In general, root growth rates increase at night while most of the light period is characterized by declining elongation rates. Since a slow oscillation in root growth rate with a periodicity of approximately 24 h persists in free running conditions it was demonstrated that the circadian clock mediates these daily fluctuations.¹ Root growth at night is fueled by the degradation of starch within the leaves. Thus, a correspondence between the time taken to degrade starch reserves and the length of the night is important to optimize growth in C-limiting conditions. Gibon et al. observed a strong correlation between the rate of starch degradation and the relative growth rate when Arabidopsis Col-0 was grown in a range of different photoperiods.⁴ Therefore, to avoid periods of C starvation at the end of the night the circadian clock was postulated to function as a timer that adjusts degradation of starch to the prevailing length of the night.^1,5Root growth strongly depends on the supply of sucrose from the leaves. To investigate the effects of substrate depletion on root elongation at night, 12-day-old seedlings of Col-0, pgm and sex1 growing in a 16 h photoperiod were previously investigated by digital time resolved video imaging.¹ As a result, the diel growth response was strongly modified in pgm and sex1 as compared to the wild-type. Both mutants showed a pronounced inhibition of growth during the night and a gradual recovery of growth during the light period. To substantiate these findings, we here report on the root elongation patterns of additional mutants in starch metabolism, e.g., mex1, dpe1 and dpe2 detected at different photoperiods and elevated external sucrose supply.     

Rising rates of starch degradation during daytime and trehalose 6-phosphate optimize carbon availability

Many plants, including Arabidopsis (Arabidopsis thaliana), accumulate starch in the light and remobilize it to support maintenance and growth at night. Starch synthesis and degradation are usually viewed as temporally separate processes. Recently, we reported that starch is also degraded in the light. Degradation rates are generally low early in the day but rise with time. Here, we show that the rate of degradation in the light depends on time relative to dawn rather than dusk. We also show that degradation in the light is inhibited by trehalose 6-phosphate, a signal for sucrose availability. The observed responses of degradation in the light can be simulated by a skeletal model in which the rate of degradation is a function of starch content divided by time remaining until dawn. The fit is improved by extension to include feedback inhibition of starch degradation by trehalose 6-phosphate. We also investigate possible functions of simultaneous starch synthesis and degradation in the light, using empirically parameterized models and experimental approaches. The idea that this cycle buffers growth against falling rates of photosynthesis at twilight is supported by data showing that rates of protein and cell wall synthesis remain high during a simulated dusk twilight. Degradation of starch in the light may also counter over-accumulation of starch in long photoperiods and stabilize signaling around dusk. We conclude that starch degradation in the light is regulated by mechanisms similar to those that operate at night and is important for stabilizing carbon availability and signaling, thus optimizing growth in natural light conditions.

Starch degradation in the light is regulated by similar mechanisms to those operating at night, stabilizing carbon availability, and thereby optimizing growth in natural light conditions     

Diurnal patterns of growth and transient reserves of sink and source tissues are affected by cold nights in barley

Barley is described to mostly use sucrose for night carbon requirements. To understand how the transient carbon is accumulated and utilized in response to cold, barley plants were grown in a combination of cold days and/or nights. Both daytime and night cold reduced growth. Sucrose was the main carbohydrate supplying growth at night, representing 50–60% of the carbon consumed. Under warm days and nights, starch was the second contributor with 26% and malate the third with 15%. Under cold nights, the contribution of starch was severely reduced, due to an inhibition of its synthesis, including under warm days, and malate was the second contributor to C requirements with 24–28% of the total amount of carbon consumed. We propose that malate plays a critical role as an alternative carbon source to sucrose and starch in barley. Hexoses, malate, and sucrose mobilization and starch accumulation were affected in barley elf3 clock mutants, suggesting a clock regulation of their metabolism, without affecting growth and photosynthesis however. Altogether, our data suggest that the mobilization of sucrose and malate and/or barley growth machinery are sensitive to cold.     

Diel growth patterns of young soybean (Glycine max) leaflets are synchronous throughout different positions on a plant

Leaf growth is controlled by various internal and external factors. Leaves of dicotyledonous plants show pronounced diel (24 h) growth patterns that are controlled by the circadian clock. To date, it is still uncertain whether diel leaf growth patterns remain constant throughout the development of a plant. In this study, we followed growth from the primary leaves to leaflets of the seventh trifoliate leaf of soybean (Glycine max) on the same plants with a recently developed imaging‐based method under controlled conditions and at a high temporal resolution. We found that all leaflets displayed a consistent diel growth pattern with maximum growth towards the end of the night. In some leaves, growth maxima occurred somewhat later – at dawn – as long as the leaves were still in a very early developmental stage. Yet, overall, diel growth patterns of leaves from different positions within the canopy were highly synchronous. Therefore, the diel growth pattern of any leaf at a given point in time is representative for the overall diel growth pattern of the plant leaf canopy and a deviation from the normal diel growth pattern can indicate that the plant is currently facing stress.     

Carbon partitioning in Arabidopsis thaliana is a dynamic process controlled by the plants metabolic status and its circadian clock

Plant growth involves the coordinated distribution of carbon resources both towards structural components and towards storage compounds that assure a steady carbon supply over the complete diurnal cycle. We used ¹⁴CO₂ labelling to track assimilated carbon in both source and sink tissues. Source tissues exhibit large variations in carbon allocation throughout the light period. The most prominent change was detected in partitioning towards starch, being low in the morning and more than double later in the day. Export into sink tissues showed reciprocal changes. Fewer and smaller changes in carbon allocation occurred in sink tissues where, in most respects, carbon was partitioned similarly, whether the sink leaf assimilated it through photosynthesis or imported it from source leaves. Mutants deficient in the production or remobilization of leaf starch exhibited major alterations in carbon allocation. Low‐starch mutants that suffer from carbon starvation at night allocated much more carbon into neutral sugars and had higher rates of export than the wild type, partly because of the reduced allocation into starch, but also because of reduced allocation into structural components. Moreover, mutants deficient in the plant's circadian system showed considerable changes in their carbon partitioning pattern suggesting control by the circadian clock.     

Circadian clock component,LHY, tells a plant when to respond photosynthetically to light in nature

The circadian clock is known to increase plant growth and fitness, and is thought to prepare plants for photosynthesis at dawn and dusk; whether this happens in nature was unknown. We transformed the native tobacco, Nicotiana attenuata to silence two core clock components, NaLHY (irLHY) and NaTOC1 (irTOC1). We characterized growth and light‐ and dark‐adapted photosynthetic rates (A_c) throughout a 24 h day in empty vector‐transformed (EV), irLHY, and irTOC1 plants in the field, and in NaPhyA‐ and NaPhyB1‐silenced plants in the glasshouse. The growth rates of irLHY plants were lower than those of EV plants in the field. While irLHY plants reduced A_c earlier at dusk, no differences between irLHY and EV plants were observed at dawn in the field. irLHY, but not EV plants, responded to light in the night by rapidly increasing A_c. Under controlled conditions, EV plants rapidly increased A_c in the day compared to dark‐adapted plants at night; irLHY plants lost these time‐dependent responses. The role of NaLHY in gating photosynthesis is independent of the light‐dependent reactions and red light perceived by NaPhyA, but not NaPhyB1. In summary, the circadian clock allows plants not to respond photosynthetically to light at night by anticipating and gating red light‐mediated in native tobacco.     

Starch and the clock: the dark side of plant productivity

Efforts to improve photosynthetic efficiency should result in increased rates of carbon assimilation in crop plants in the next few decades. Translation of increased assimilation into higher productivity will require a greater understanding of the relationship between assimilation and growth. In this review, we discuss new progress in understanding how carbon is provided for metabolism and growth at night. In Arabidopsis leaves, the circadian clock controls the rate of degradation of starch to ensure an optimal carbon supply and hence continued growth during the night. These discoveries shed new light on the integration of carbon assimilation and growth over the light-dark cycle. They reveal the importance of considering the carbon economy of the whole plant in attempting to increase crop productivity.     

Diel changes in the particulate environment,feeding activity and digestive enzyme concentration in neritic zooplankton

Diel changes in the particulate environment and nutritional metabolism of neritic zooplankton were studied in the Bay of Villefranche-sur-mer (France) for 30 h during February. Although the survey was carried out over a period of negligible wind speed, the changes in the particle size spectra and species composition of the Zooplankton sampled suggested an abrupt and temporary oscillation in the water mass at the second sampling time. The rest of the 30-h period was characterized by gradual changes which usually returned to conditions close to, but different from, the starting ones. A clear diel rhythm was recorded for the biochemical composition of the phytoplankton, probably related to the periodicity of the photosynthetic metabolism.The feeding rate of Clausocalanus arcuicornis Giesbrecht, 1888, measured by gut fluorescence, also showed diel rhythmicity with maximum rate at night. Digestive enzyme activities were measured on the total Zooplankton population composed of five major species: Clausocalanus arcuicornis, Paracalanus sp., Calanus helgolandicus Claus, 1863, Acartia clausi Giesbrecht, 1889, and Oikopleura dioica Fol, 1859. The activities of the carbohydrases showed varying patterns of periodicity with one or two night maxima. The proteases showed a different pattern with acidic proteases peaking just before dawn and decreasing gradually during the day and no periodicity for trypsin-like activity. No association could be established between the enzyme changes and those of either the food supply or the species composition of the Zooplankton sampled, suggesting that both feeding and digestion diel rhythms are controlled by an internal clock. Influences of internal, external and experimental factors on the digestive enzyme activity rhythm are discussed. The resulting scheme of short term regulation was compared with that proposed earlier for medium term changes.     

Spatio-temporal leaf growth patterns of Arabidopsis thaliana and evidence for sugar control of the diel leaf growth cycle

Leaf growth dynamics are driven by diel rhythms. The analysis of spatio-temporal leaf growth patterns in Arabidopsis thaliana wild type and mutants of interest is a promising approach to elucidate molecular mechanisms controlling growth. The diel availability of carbohydrates is thought to affect diel growth. A digital image sequence processing (DISP)-based noninvasive technique for visualizing and quantifying highly resolved spatio-temporal leaf growth was adapted for the model plant A. thaliana. Diel growth patterns were analysed for the wild type and for a mutant with altered diel carbohydrate metabolism. A. thaliana leaves showed highest relative growth rates (RGRs) at dawn and lowest RGRs at the beginning of the night. Along the lamina, a clear basipetal gradient of growth rate distribution was found, similar to that in many other dicotyledonous species. The starch-free 1 (stf1) mutant revealed changed temporal growth patterns with reduced nocturnal, and increased afternoon, growth activity. The established DISP technique is presented as a valuable tool to detect altered temporal growth patterns in A. thaliana mutants. Endogenous changes in the diel carbohydrate availability of the starch-free mutant clearly affected its diel growth rhythms.     

DIEL PATTERNS IN LIGHT ABSORPTION AND ABSORPTION EFFICIENCY FACTORS OF ISOCHRYSIS GALBANA (PRYMNESIOPHYCEAE)1

The chl a specific absorption coefficients [a* (λ), m²·mg chl a ^{? 1}] were examined in chemostat culture of the Prymnesiophyceae Isochrysis galbana (Parke) under a 12:12‐h light:dark cycle at low light (75 μmol photons·m ^{? 2}·s ^{? 1}) and high light (500 μmol photons· m ^{? 2}·s ^{? 1}) conditions. Other associated measurements such as pigment composition, cell density, and diameter as the measure of cell size were also made at the two light regimes every 2 h for 2 days to confirm the periodicity. A distinct diel variability was observed for the a* (λ) with maxima near dawn and minima near dusk. The magnitude of diel variation in a* (440) was 15% at low light and 22% at high light. Pronounced diel patterns were observed for cell size with minima near dawn and maxima near dusk. The magnitude of diel variation in cell size was 9.3% at low light and 21% at high light. The absorption efficiency factors [Q _a (440)] were determined by reconstruction using intracellular concentrations of pigments and cell size. The Q _a (440) also showed a distinct diel variability, with minima near dawn and maxima near dusk. The diel variation in a* (λ) and Q _a (λ) was primarily caused by changes in cell size due to growth, although there was some influence from diel variations in the intracellular pigment concentrations. The results presented here indicated that diel variation in a* (λ) was an important component of the optical characterization of phytoplankton.     

Diel variation of the cellular carbon to nitrogen ratio of Chlorella autotrophica (Chlorophyta) growing in phosphorus‐ and nitrogen‐limited continuous cultures

We investigated the relationship between daily growth rates and diel variation of carbon (C) metabolism and C to nitrogen (N) ratio under P‐ and N‐limitation in the green algae Chlorella autotrophica. To do this, continuous cultures of C. autotrophica were maintained in a cyclostat culture system under 14:10 light:dark cycle over a series of P‐ and N‐limited growth rates. Cell abundance, together with cell size, as reflected by side scatter signal from flow cytometric analysis demonstrated a synchronized diel pattern with cell division occurring at night. Under either type of nutrient limitation, the cellular C:N ratio increased through the light period and decreased through the dark period over all growth rates, indicating a higher diel variation of C metabolism than that of N. Daily average cellular C:N ratios were higher at lower dilution rates under both types of nutrient limitation but cell enlargement was only observed at lower dilution rates under P‐limitation. Carbon specific growth rates during the dark period positively correlated with cellular daily growth rates (dilution rates), with net loss of C during night at the lowest growth rates under N‐limitation. Under P‐limitation, dark C specific growth rates were close to zero at low dilution rates but also exhibited an increasing trend at high dilution rates. In general, diel variations of cellular C:N were low when dark C specific growth rates were high. This result indicated that the fast growing cells performed dark C assimilation at high rates, hence diminished the uncoupling of C and N metabolism at night.     

The Nocturnal Activity of Fruit Flies Exposed to Artificial Moonlight Is Partly Caused by Direct Light Effects on the Activity Level That Bypass the Endogenous Clock

Artificial moonlight was recently shown to shift the endogenous clock of fruit flies and make them nocturnal. To test whether this nocturnal activity is partly due to masking effects of light, we exposed the clock‐mutants per⁰¹, tim⁰¹, per⁰¹;tim⁰¹, cyc⁰¹, and Clk^JRK to light/dark and light/dim‐light cycles and determined the activity level during the day and night. We found that under moonlit nights, all clock mutants shifted their activity significantly into the night, suggesting that this effect is independent of the clock. We also recorded the flies under continuous artificial moonlight and darkness to judge the effect of dim constant light on the activity level. All mutants, except Clk^JRK flies, were significantly more active under artificial moonlight conditions than under complete darkness. Unexpectedly, we found residual rhythmicity of per⁰¹ and especially tim⁰¹ mutants under these conditions, suggesting that TIM and especially PER retained some activity in the absence of its respective partner. Nevertheless, as even the double mutants and the cyc⁰¹ and Clk^JRK mutants shifted their activity into the night, we conclude that dim light stimulates the activity of fruit flies in a clock‐independent manner. Thus, nocturnal light has a twofold influence on flies: it shifts the circadian clock, and it increases nocturnal activity independently of the clock. The latter was also observed in some primates by others and might therefore be of a more general validity.