The circadian clock mutant lhy cca1 elf3 paces starch mobilization to dawn despite severely disrupted circadian clock function.

Many plants, including Arabidopsis (Arabidopsis thaliana), accumulate starch in the daytime and remobilize it to support maintenance and growth at night. Starch accumulation is increased when carbon is in short supply, for example, in short photoperiods. Mobilization is paced to exhaust starch around dawn, as anticipated by the circadian clock. This diel pattern of turnover is largely robust against loss of day, dawn, dusk, or evening clock components. Here, we investigated diel starch turnover in the triple circadian clock mutant lhy cca1 elf3, which lacks the LATE ELONGATED HYPOCOTYL and the CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) dawn components and the EARLY FLOWERING3 (ELF3) evening components of the circadian clock. The diel oscillations of transcripts for the remaining clock components and related genes like REVEILLE and PHYTOCHROME-INTERACING FACTOR family members exhibited attenuated amplitudes and altered peak time, weakened dawn dominance, and decreased robustness against changes in the external light-dark cycle. The triple mutant was unable to increase starch accumulation in short photoperiods. However, it was still able to pace starch mobilization to around dawn in different photoperiods and growth irradiances and to around 24 h after the previous dawn in T17 and T28 cycles. The triple mutant was able to slow down starch mobilization after a sudden low-light day or a sudden early dusk, although in the latter case it did not fully compensate for the lengthened night. Overall, there was a slight trend to less linear mobilization of starch. Thus, starch mobilization can be paced rather robustly to dawn despite a major disruption of the transcriptional clock. It is proposed that temporal information can be delivered from clock components or a semi-autonomous oscillator.

Response of the Circadian Clock and Diel Starch Turnover to One Day of Low Light or Low CO2.

Diel starch turnover responds rapidly to changes in the light regime. We investigated if these responses require changes in the temporal dynamics of the circadian clock. Arabidopsis (Arabidopsis thaliana) was grown in a 12-h photoperiod for 19 d, shifted to three different reduced light levels or to low CO2 for one light period, and returned to growth conditions. The treatments produced widespread changes in clock transcript abundance. However, almost all of the changes were restricted to extreme treatments that led to carbon starvation and were small compared to the magnitude of the circadian oscillation. Changes included repression of EARLY FLOWERNG 4, slower decay of dusk components, and a slight phase delay at the next dawn, possibly due to abrogated Evening Complex function and sustained expression of PHYTOCHROME INTERACTING FACTORs and REVEILLEs during the night. Mobilization of starch in the night occurred in a linear manner and was paced to dawn, both in moderate treatments that did not alter clock transcripts and in extreme treatments that led to severe carbon starvation. We conclude that pacing of starch mobilization to dawn does not require retrograde carbon signaling to the transcriptional clock. On the following day, growth decreased, sugars rose, and starch accumulation was stimulated in low-light-treated plants compared to controls. This adaptive response was marked after moderate treatments and occurred independently of changes in the transcriptional clock. It is probably a time-delayed response to low-C signaling in the preceding 24-h cycle, possibly including changes in PHYTOCHROME INTERACTING FACTOR and REVEILLE expression.

Multiple circadian clock outputs regulate diel turnover of carbon and nitrogen reserves.

Plants accumulate reserves in the daytime to support growth at night. Circadian regulation of diel reserve turnover was investigated by profiling starch, sugars, glucose 6-phosphate, organic acids, and amino acids during a light-dark cycle and after transfer to continuous light in Arabidopsis wild types and in mutants lacking dawn (lhy cca1), morning (prr7 prr9), dusk (toc1, gi), or evening (elf3) clock components. The metabolite time series were integrated with published time series for circadian clock transcripts to identify circadian outputs that regulate central metabolism. (a) Starch accumulation was slower in elf3 and prr7 prr9. It is proposed that ELF3 positively regulates starch accumulation. (b) Reducing sugars were high early in the T-cycle in elf3, revealing that ELF3 negatively regulates sucrose recycling. (c) The pattern of starch mobilization was modified in all five mutants. A model is proposed in which dawn and dusk/evening components interact to pace degradation to anticipated dawn. (d) An endogenous oscillation of glucose 6-phosphate revealed that the clock buffers metabolism against the large influx of carbon from photosynthesis. (e) Low levels of organic and amino acids in lhy cca1 and high levels in prr7 prr9 provide evidence that the dawn components positively regulate the accumulation of amino acid reserves.

Trehalose 6-phosphate coordinates organic and amino acid metabolism with carbon availability.

Trehalose 6-phosphate (Tre6P) is an essential signal metabolite in plants, linking growth and development to carbon metabolism. The sucrose-Tre6P nexus model postulates that Tre6P acts as both a signal and negative feedback regulator of sucrose levels. To test this model, short-term metabolic responses to induced increases in Tre6P levels were investigated in Arabidopsis thaliana plants expressing the Escherichia coli Tre6P synthase gene (otsA) under the control of an ethanol-inducible promoter. Increased Tre6P levels led to a transient decrease in sucrose content, post-translational activation of nitrate reductase and phosphoenolpyruvate carboxylase, and increased levels of organic and amino acids. Radio-isotope ((14)CO2) and stable isotope ((13)CO2) labelling experiments showed no change in the rates of photoassimilate export in plants with elevated Tre6P, but increased labelling of organic acids. We conclude that high Tre6P levels decrease sucrose levels by stimulating nitrate assimilation and anaplerotic synthesis of organic acids, thereby diverting photoassimilates away from sucrose to generate carbon skeletons and fixed nitrogen for amino acid synthesis. These results are consistent with the sucrose-Tre6P nexus model, and implicate Tre6P in coordinating carbon and nitrogen metabolism in plants.

The Interplay between Carbon Availability and Growth in Different Zones of the Growing Maize Leaf.

Plants assimilate carbon in their photosynthetic tissues in the light. However, carbon is required during the night and in nonphotosynthetic organs. It is therefore essential that plants manage their carbon resources spatially and temporally and coordinate growth with carbon availability. In growing maize (Zea mays) leaf blades, a defined developmental gradient facilitates analyses in the cell division, elongation, and mature zones. We investigated the responses of the metabolome and transcriptome and polysome loading, as a qualitative proxy for protein synthesis, at dusk, dawn, and 6, 14, and 24 h into an extended night, and tracked whole-leaf elongation over this time course. Starch and sugars are depleted by dawn in the mature zone, but only after an extension of the night in the elongation and division zones. Sucrose (Suc) recovers partially between 14 and 24 h into the extended night in the growth zones, but not the mature zone. The global metabolome and transcriptome track these zone-specific changes in Suc. Leaf elongation and polysome loading in the growth zones also remain high at dawn, decrease between 6 and 14 h into the extended night, and then partially recover, indicating that growth processes are determined by local carbon status. The level of Suc-signaling metabolite trehalose-6-phosphate, and the trehalose-6-phosphate:Suc ratio are much higher in growth than mature zones at dusk and dawn but fall in the extended night. Candidate genes were identified by searching for transcripts that show characteristic temporal response patterns or contrasting responses to carbon starvation in growth and mature zones.

Photosynthate partitioning to starch in Arabidopsis thaliana is insensitive to light intensity but sensitive to photoperiod due to a restriction on growth in the light in short photoperiods.

Photoperiod duration can be predicted from previous days, but irradiance fluctuates in an unpredictable manner. To investigate how allocation to starch responds to changes in these two environmental variables, Arabidopsis Col-0 was grown in a 6 h and a 12 h photoperiod at three different irradiances. The absolute rate of starch accumulation increased when photoperiod duration was shortened and when irradiance was increased. The proportion of photosynthate allocated to starch increased strongly when photoperiod duration was decreased but only slightly when irradiance was decreased. There was a small increase in the daytime level of sucrose and twofold increases in glucose, fructose and glucose 6-phosphate at a given irradiance in short photoperiods compared to long photoperiods. The rate of starch accumulation correlated strongly with sucrose and glucose levels in the light, irrespective of whether these sugars were responding to a change in photoperiod or irradiance. Whole plant carbon budget modelling revealed a selective restriction of growth in the light period in short photoperiods. It is proposed that photoperiod sensing, possibly related to the duration of the night, restricts growth in the light period in short photoperiods, increasing allocation to starch and providing more carbon reserves to support metabolism and growth in the long night.

Dissecting the subcellular compartmentation of proteins and metabolites in arabidopsis leaves using non-aqueous fractionation.

Non-aqueous fractionation is a technique for the enrichment of different subcellular compartments derived from lyophilized material. It was developed to study the subcellular distribution of metabolites. Here we analyzed the distribution of about 1,000 proteins and 70 metabolites, including 22 phosphorylated intermediates in wild-type Arabidopsis rosette leaves, using non-aqueous gradients divided into 12 fractions. Good separation of plastidial, cytosolic, and vacuolar metabolites and proteins was achieved, but cytosolic, mitochondrial, and peroxisomal proteins clustered together. There was considerable heterogeneity in the fractional distribution of transcription factors, ribosomal proteins, and subunits of the vacuolar-ATPase, indicating diverse compartmental location. Within the plastid, sub-organellar separation of thylakoids and stromal proteins was observed. Metabolites from the Calvin-Benson cycle, photorespiration, starch and sucrose synthesis, glycolysis, and the tricarboxylic acid cycle grouped with their associated proteins of the respective compartment. Non-aqueous fractionation thus proved to be a powerful method for the study of the organellar, and in some cases sub-organellar, distribution of proteins and their association with metabolites. It remains the technique of choice for the assignment of subcellular location to metabolites in intact plant tissues, and thus the technique of choice for doing combined metabolite-protein analysis on a single tissue sample.

Regulatory properties of ADP glucose pyrophosphorylase are required for adjustment of leaf starch synthesis in different photoperiods.

Arabidopsis (Arabidopsis thaliana) leaves synthesize starch faster in short days than in long days, but the mechanism that adjusts the rate of starch synthesis to daylength is unknown. To understand this mechanism, we first investigated whether adjustment occurs in mutants lacking components of the circadian clock or clock output pathways. Most mutants adjusted starch synthesis to daylength, but adjustment was compromised in plants lacking the GIGANTEA or FLAVIN-BINDING, KELCH REPEAT, F BOX1 components of the photoperiod-signaling pathway involved in flowering. We then examined whether the properties of the starch synthesis enzyme adenosine 5'-diphosphate-glucose pyrophosphorylase (AGPase) are important for adjustment of starch synthesis to daylength. Modulation of AGPase activity is known to bring about short-term adjustments of photosynthate partitioning between starch and sucrose (Suc) synthesis. We found that adjustment of starch synthesis to daylength was compromised in plants expressing a deregulated bacterial AGPase in place of the endogenous AGPase and in plants containing mutant forms of the endogenous AGPase with altered allosteric regulatory properties. We suggest that the rate of starch synthesis is in part determined by growth rate at the end of the preceding night. If growth at night is low, as in short days, there is a delay before growth recovers during the next day, leading to accumulation of Suc and stimulation of starch synthesis via activation of AGPase. If growth at night is fast, photosynthate is used for growth at the start of the day, Suc does not accumulate, and starch synthesis is not up-regulated.

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.

Low levels of ribosomal RNA partly account for the very high photosynthetic phosphorus-use efficiency of Proteaceae species.

Proteaceae species in south-western Australia occur on phosphorus- (P) impoverished soils. Their leaves contain very low P levels, but have relatively high rates of photosynthesis. We measured ribosomal RNA (rRNA) abundance, soluble protein, activities of several enzymes and glucose 6-phosphate (Glc6P) levels in expanding and mature leaves of six Proteaceae species in their natural habitat. The results were compared with those for Arabidopsis thaliana. Compared with A. thaliana, immature leaves of Proteaceae species contained very low levels of rRNA, especially plastidic rRNA. Proteaceae species showed slow development of the photosynthetic apparatus ('delayed greening'), with young leaves having very low levels of chlorophyll and Calvin-Benson cycle enzymes. In mature leaves, soluble protein and Calvin-Benson cycle enzyme activities were low, but Glc6P levels were similar to those in A. thaliana. We propose that low ribosome abundance contributes to the high P efficiency of these Proteaceae species in three ways: (1) less P is invested in ribosomes; (2) the rate of growth and, hence, demand for P is low; and (3) the especially low plastidic ribosome abundance in young leaves delays formation of the photosynthetic machinery, spreading investment of P in rRNA. Although Calvin-Benson cycle enzyme activities are low, Glc6P levels are maintained, allowing their effective use.

Source-Sink Dynamics in Field-Grown Durum Wheat Under Contrasting Nitrogen Supplies: Key Role of Non-Foliar Organs During Grain Filling.

The integration of high-throughput phenotyping and metabolic approaches is a suitable strategy to study the genotype-by-environment interaction and identify novel traits for crop improvement from canopy to an organ level. Our aims were to study the phenotypic and metabolic traits that are related to grain yield and quality at canopy and organ levels, with a special focus on source-sink coordination under contrasting N supplies. Four modern durum wheat varieties with contrasting grain yield were grown in field conditions under two N fertilization levels in north-eastern Spain. We evaluated canopy vegetation indices taken throughout the growing season, physiological and metabolic traits in different photosynthetic organs (flag leaf blade, sheath, peduncle, awn, glume, and lemma) at anthesis and mid-grain filling stages, and agronomic and grain quality traits at harvest. Low N supply triggered an imbalance of C and N coordination at the whole plant level, leading to a reduction of grain yield and nutrient composition. The activities of key enzymes in C and N metabolism as well as the levels of photoassimilates showed that each organ plays an important role during grain filling, some with a higher photosynthetic capacity, others for nutrient storage for later stages of grain filling, or N assimilation and recycling. Interestingly, the enzyme activities and sucrose content of the ear organs were positively associated with grain yield and quality, suggesting, together with the regression models using isotope signatures, the potential contribution of these organs during grain filling. This study highlights the use of holistic approaches to the identification of novel targets to improve grain yield and quality in C3 cereals and the key role of non-foliar organs at late-growth stages.

Arabidopsis coordinates the diurnal regulation of carbon allocation and growth across a wide range of photoperiods.

In short photoperiods, plants accumulate starch more rapidly in the light and degrade it more slowly at night, ensuring that their starch reserves last until dawn. To investigate the accompanying changes in the timing of growth, Arabidopsis was grown in a range of photoperiods and analyzed for rosette biomass, photosynthesis, respiration, ribosome abundance, polysome loading, starch, and over 40 metabolites at dawn and dusk. The data set was used to model growth rates in the daytime and night, and to identify metabolites that correlate with growth. Modeled growth rates and polysome loading were high in the daytime and at night in long photoperiods, but decreased at night in short photoperiods. Ribosome abundance was similar in all photoperiods. It is discussed how the amount of starch accumulated in the light period, the length of the night, and maintenance costs interact to constrain growth at night in short photoperiods, and alter the strategy for optimizing ribosome use. Significant correlations were found in the daytime and the night between growth rates and the levels of the sugar-signal trehalose 6-phosphate and the amino acid biosynthesis intermediate shikimate, identifying these metabolites as hubs in a network that coordinates growth with diurnal changes in the carbon supply.