Natural variation in metabolism of the Calvin-Benson cycle.

The Calvin-Benson cycle (CBC) evolved over 2 billion years ago but has been subject to massive selection due to falling atmospheric carbon dioxide, rising atmospheric oxygen and changing nutrient and water availability. In addition, large groups of organisms have evolved carbon-concentrating mechanisms (CCMs) that operate upstream of the CBC. Most previous studies of CBC diversity focused on Rubisco kinetics and regulation. Quantitative metabolite profiling provides a top-down strategy to uncover inter-species diversity in CBC operation. CBC profiles were recently published for twenty species including terrestrial C3 species, terrestrial C4 species that operate a biochemical CCM, and cyanobacteria and green algae that operate different types of biophysical CCM. Distinctive profiles were found for species with different modes of photosynthesis, revealing that evolution of the various CCMs was accompanied by co-evolution of the CBC. Diversity was also found between species that share the same mode of photosynthesis, reflecting lineage-dependent diversity of the CBC. Connectivity analysis uncovers constraints due to pathway and thermodynamic topology, and reveals that cross-species diversity in the CBC is driven by changes in the balance between regulated enzymes and in the balance between the CBC and the light reactions or end-product synthesis.

Direct and indirect responses of the Arabidopsis transcriptome to an induced increase in trehalose 6-phosphate.

Trehalose 6-phosphate (Tre6P) is an essential signal metabolite that regulates the level of sucrose, linking growth and development to the metabolic status. We hypothesized that Tre6P plays a role in mediating the regulation of gene expression by sucrose. To test this, we performed transcriptomic profiling on Arabidopsis (Arabidopsis thaliana) plants that expressed a bacterial TREHALOSE 6-PHOSPHATE SYNTHASE (TPS) under the control of an ethanol-inducible promoter. Induction led to a 4-fold rise in Tre6P levels, a concomitant decrease in sucrose, significant changes (FDR ≤ 0.05) of over 13,000 transcripts, and 2-fold or larger changes of over 5,000 transcripts. Comparison with nine published responses to sugar availability allowed some of these changes to be linked to the rise in Tre6P, while others were probably due to lower sucrose or other indirect effects. Changes linked to Tre6P included repression of photosynthesis-related gene expression and induction of many growth-related processes including ribosome biogenesis. About 500 starvation-related genes are known to be induced by SUCROSE-NON-FERMENTING-1-RELATED KINASE 1 (SnRK1). They were largely repressed by Tre6P in a manner consistent with SnRK1 inhibition by Tre6P. SnRK1 also represses many genes that are involved in biosynthesis and growth. These responded to Tre6P in a more complex manner, pointing toward Tre6P interacting with other C-signaling pathways. Additionally, elevated Tre6P modified the expression of genes encoding regulatory subunits of the SnRK1 complex and TPS class II and FCS-LIKE ZINC FINGER proteins that are thought to modulate SnRK1 function and genes involved in circadian, TARGET OF RAPAMYCIN, light, abscisic acid, and other hormone signaling.

In vivo protein kinase activity of SnRK1 fluctuates in Arabidopsis rosettes during light-dark cycles.

Sucrose-nonfermenting 1 (SNF1)-related kinase 1 (SnRK1) is a central hub in carbon and energy signaling in plants, and is orthologous with SNF1 in yeast and the AMP-activated protein kinase (AMPK) in animals. Previous studies of SnRK1 relied on in vitro activity assays or monitoring of putative marker gene expression. Neither approach gives unambiguous information about in vivo SnRK1 activity. We have monitored in vivo SnRK1 activity using Arabidopsis (Arabidopsis thaliana) reporter lines that express a chimeric polypeptide with an SNF1/SnRK1/AMPK-specific phosphorylation site. We investigated responses during an equinoctial diel cycle and after perturbing this cycle. As expected, in vivo SnRK1 activity rose toward the end of the night and rose even further when the night was extended. Unexpectedly, although sugars rose after dawn, SnRK1 activity did not decline until about 12 h into the light period. The sucrose signal metabolite, trehalose 6-phosphate (Tre6P), has been shown to inhibit SnRK1 in vitro. We introduced the SnRK1 reporter into lines that harbored an inducible trehalose-6-phosphate synthase construct. Elevated Tre6P decreased in vivo SnRK1 activity in the light period, but not at the end of the night. Reporter polypeptide phosphorylation was sometimes negatively correlated with Tre6P, but a stronger and more widespread negative correlation was observed with glucose-6-phosphate. We propose that SnRK1 operates within a network that controls carbon utilization and maintains diel sugar homeostasis, that SnRK1 activity is regulated in a context-dependent manner by Tre6P, probably interacting with further inputs including hexose phosphates and the circadian clock, and that SnRK1 signaling is modulated by factors that act downstream of SnRK1.

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.

13CO2 labeling kinetics in maize reveal impaired efficiency of C4 photosynthesis under low irradiance.

C4 photosynthesis allows faster photosynthetic rates and higher water and nitrogen use efficiency than C3 photosynthesis, but at the cost of lower quantum yield due to the energy requirement of its biochemical carbon concentration mechanism. It has also been suspected that its operation may be impaired in low irradiance. To investigate fluxes under moderate and low irradiance, maize (Zea mays) was grown at 550 µmol photons m-2 s-l and 13CO2 pulse-labeling was performed at growth irradiance or several hours after transfer to 160 µmol photons m-2 s-1. Analysis by liquid chromatography/tandem mass spectrometry or gas chromatography/mass spectrometry provided information about pool size and labeling kinetics for 32 metabolites and allowed estimation of flux at many steps in C4 photosynthesis. The results highlighted several sources of inefficiency in low light. These included excess flux at phosphoenolpyruvate carboxylase, restriction of decarboxylation by NADP-malic enzyme, and a shift to increased CO2 incorporation into aspartate, less effective use of metabolite pools to drive intercellular shuttles, and higher relative and absolute rates of photorespiration. The latter provides evidence for a lower bundle sheath CO2 concentration in low irradiance, implying that operation of the CO2 concentration mechanism is impaired in this condition. The analyses also revealed rapid exchange of carbon between the Calvin-Benson cycle and the CO2-concentration shuttle, which allows rapid adjustment of the balance between CO2 concentration and assimilation, and accumulation of large amounts of photorespiratory intermediates in low light that provides a major carbon reservoir to build up C4 metabolite pools when irradiance increases.

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.

Functional Features of TREHALOSE-6-PHOSPHATE SYNTHASE1, an Essential Enzyme in Arabidopsis.

In Arabidopsis (Arabidopsis thaliana), TREHALOSE-6-PHOSPHATE SYNTHASE1 (TPS1) catalyzes the synthesis of the sucrose-signaling metabolite trehalose 6-phosphate (Tre6P) and is essential for embryogenesis and normal postembryonic growth and development. To understand its molecular functions, we transformed the embryo-lethal tps1-1 null mutant with various forms of TPS1 and with a heterologous TPS (OtsA) from Escherichia coli, under the control of the TPS1 promoter, and tested for complementation. TPS1 protein localized predominantly in the phloem-loading zone and guard cells in leaves, root vasculature, and shoot apical meristem, implicating it in both local and systemic signaling of Suc status. The protein is targeted mainly to the nucleus. Restoring Tre6P synthesis was both necessary and sufficient to rescue the tps1-1 mutant through embryogenesis. However, postembryonic growth and the sucrose-Tre6P relationship were disrupted in some complementation lines. A point mutation (A119W) in the catalytic domain or truncating the C-terminal domain of TPS1 severely compromised growth. Despite having high Tre6P levels, these plants never flowered, possibly because Tre6P signaling was disrupted by two unidentified disaccharide-monophosphates that appeared in these plants. The noncatalytic domains of TPS1 ensure its targeting to the correct subcellular compartment and its catalytic fidelity and are required for appropriate signaling of Suc status by Tre6P.

Impact of the SnRK1 protein kinase on sucrose homeostasis and the transcriptome during the diel cycle.

SNF1-related Kinase 1 (SnRK1) is an evolutionarily conserved protein kinase with key functions in energy management during stress responses in plants. To address a potential role of SnRK1 under favorable conditions, we performed a metabolomic and transcriptomic characterization of rosettes of 20-d-old Arabidopsis (Arabidopsis thaliana) plants of SnRK1 gain- and loss-of-function mutants during the regular diel cycle. Our results show that SnRK1 manipulation alters the sucrose and trehalose 6-phosphate (Tre6P) relationship, influencing how the sucrose content is translated into Tre6P accumulation and modulating the flux of carbon to the tricarboxylic acid cycle downstream of Tre6P signaling. On the other hand, daily cycles of Tre6P accumulation were accompanied by changes in SnRK1 signaling, leading to a maximum in the expression of SnRK1-induced genes at the end of the night, when Tre6P levels are lowest, and to a minimum at the end of the day, when Tre6P levels peak. The expression of SnRK1-induced genes was strongly reduced by transient Tre6P accumulation in an inducible Tre6P synthase (otsA) line, further suggesting the involvement of Tre6P in the diel oscillations in SnRK1 signaling. Transcriptional profiling of wild-type plants and SnRK1 mutants also uncovered defects that are suggestive of an iron sufficiency response and of a matching induction of sulfur acquisition and assimilation when SnRK1 is depleted. In conclusion, under favorable growth conditions, SnRK1 plays a role in sucrose homeostasis and transcriptome remodeling in autotrophic tissues and its activity is influenced by diel fluctuations in Tre6P levels.

Metabolic profiles in C3, C3-C4 intermediate, C4-like, and C4 species in the genus Flaveria.

C4 photosynthesis concentrates CO2 around Rubisco in the bundle sheath, favouring carboxylation over oxygenation and decreasing photorespiration. This complex trait evolved independently in >60 angiosperm lineages. Its evolution can be investigated in genera such as Flaveria (Asteraceae) that contain species representing intermediate stages between C3 and C4 photosynthesis. Previous studies have indicated that the first major change in metabolism probably involved relocation of glycine decarboxylase and photorespiratory CO2 release to the bundle sheath and establishment of intercellular shuttles to maintain nitrogen stoichiometry. This was followed by selection for a CO2-concentrating cycle between phosphoenolpyruvate carboxylase in the mesophyll and decarboxylases in the bundle sheath, and relocation of Rubisco to the latter. We have profiled 52 metabolites in nine Flaveria species and analysed 13CO2 labelling patterns for four species. Our results point to operation of multiple shuttles, including movement of aspartate in C3-C4 intermediates and a switch towards a malate/pyruvate shuttle in C4-like species. The malate/pyruvate shuttle increases from C4-like to complete C4 species, accompanied by a rise in ancillary organic acid pools. Our findings support current models and uncover further modifications of metabolism along the evolutionary path to C4 photosynthesis in the genus Flaveria.

Engineering Strategies to Boost Crop Productivity by Cutting Respiratory Carbon Loss.

Roughly half the carbon that crop plants fix by photosynthesis is subsequently lost by respiration. Nonessential respiratory activity leading to unnecessary CO2 release is unlikely to have been minimized by natural selection or crop breeding, and cutting this large loss could complement and reinforce the currently dominant yield-enhancement strategy of increasing carbon fixation. Until now, however, respiratory carbon losses have generally been overlooked by metabolic engineers and synthetic biologists because specific target genes have been elusive. We argue that recent advances are at last pinpointing individual enzyme and transporter genes that can be engineered to (1) slow unnecessary protein turnover, (2) replace, relocate, or reschedule metabolic activities, (3) suppress futile cycles, and (4) make ion transport more efficient, all of which can reduce respiratory costs. We identify a set of engineering strategies to reduce respiratory carbon loss that are now feasible and model how implementing these strategies singly or in tandem could lead to substantial gains in crop productivity.