A temperature regime that disrupts clock-controlled starch mobilization induces transient carbohydrate starvation, resulting in compact growth

In nature plants are usually subjected to a light/temperature regime of warm day and cold night (referred to as +DIF). Compared to growth under +DIF, Arabidopsis plants show compact growth under the same photoperiod, but with an inverse temperature regime (cold day and warm night: -DIF). Here we show that -DIF differentially affects the phase and amplitude of core clock gene expression. Under -DIF the phase of the morning clock gene CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) is delayed, similar to that of plants grown on low sucrose. Indeed, under -DIF carbohydrate (CHO) starvation marker genes are specifically upregulated at the End of the Night (EN) in Arabidopsis rosettes. However, only in inner-rosette tissue (small sink leaves and petioles of older leaves) sucrose levels are lower under -DIF compared to under +DIF, suggesting that sucrose in source leaf blades is not sensed for CHO status and that sucrose transport from source to sink may be impaired at EN. CHO-starvation under -DIF correlated with increased starch breakdown during the night and decreased starch accumulation during the day. Moreover, we demonstrate that different ways of inducing CHO-starvation all link to reduced growth of sink leaves. Practical implications for control of plant growth in horticulture are discussed.

The Route of Sucrose Utilization by Streptococcus mutans Affects Intracellular Polysaccharide Metabolism

Streptococcus mutans converts extracellular sucrose (Suc) into exopolysaccharides (EPS) by glucosyl-transferase and fructosyl-transferase enzymes and internalizes Suc for fermentation through the phosphotransferase system (PTS). Here, we examined how altering the routes for sucrose utilization impacts intracellular polysaccharide [IPS; glycogen, (glg)] metabolism during carbohydrate starvation. Strain UA159 (WT), a mutant lacking all exo-enzymes for sucrose utilization (MMZ952), and a CcpA-deficient mutant (∆ccpA) were cultured with sucrose or a combination of glucose and fructose, followed by carbohydrate starvation. At baseline (0h), and after 4 and 24h of starvation, cells were evaluated for mRNA levels of the glg operon, IPS storage, glucose-1-phosphate (G1P) concentrations, viability, and PTS activities. A pH drop assay was performed in the absence of carbohydrates at the baseline to measure acid production. We observed glg operon activation in response to starvation (p<0.05) in all strains, however, such activation was significantly delayed and reduced in magnitude when EPS synthesis was involved (p<0.05). Enhanced acidification and greater G1P concentrations were observed in the sucrose-treated group, but mostly in strains capable of producing EPS (p<0.05). Importantly, only the WT exposed to sucrose was able to synthesize IPS during starvation. Contrary to CcpA-proficient strains, IPS was progressively degraded during starvation in ∆ccpA, which also showed increased glg operon expression and greater PTS activities at baseline. Therefore, sucrose metabolism by secreted enzymes affects the capacity of S. mutans in synthesizing IPS and converting it into organic acids, without necessarily inducing greater expression of the glg operon.

Regulatory principles and experimental approaches to the circadian control of starch turnover

In many plants, starch is synthesized during the day and degraded during the night to avoid carbohydrate starvation in darkness. The circadian clock participates in a dynamic adjustment of starch turnover to changing environmental condition through unknown mechanisms. We used mathematical modelling to explore the possible scenarios for the control of starch turnover by the molecular components of the plant circadian clock. Several classes of plausible models were capable of describing the starch dynamics observed in a range of clock mutant plants and light conditions, including discriminating circadian protocols. Three example models of these classes are studied in detail, differing in several important ways. First, the clock components directly responsible for regulating starch degradation are different in each model. Second, the intermediate species in the pathway may play either an activating or inhibiting role on starch degradation. Third, the system may include a light-dependent interaction between the clock and downstream processes. Finally, the clock may be involved in the regulation of starch synthesis. We discuss the differences among the models’ predictions for diel starch profiles and the properties of the circadian regulators. These suggest additional experiments to elucidate the pathway structure, avoid confounding results and identify the molecular components involved.

Factors governing the regulation of Sclerotinia sclerotiorum cutinase A and polygalacturonase 1 during different stages of infection

Sclerotinia sclerotiorum releases hydrolytic enzymes that sequentially degrade the plant cuticle, middle lamellae, and primary and secondary cell walls. The cuticle was found to be a barrier to S. sclerotiorum infection, as leaves stripped of epicuticular wax were more rapidly colonized. Consequently, the factors affecting the regulation of genes encoding polygalacturonase 1 (SsPG1) and a newly identified cutinase (SsCUTA) were examined. In vitro, SsCutA transcripts were detected within 1 h postinoculation of leaves, and expression was primarily governed by contact of mycelia with solid surfaces. Expression of SsPg1 was moderately induced by contact with solid surfaces including the leaf, and expression was restricted to the expanding margin of the lesion as the infection progressed. SsPg1 expression was induced by carbohydrate starvation but repressed by galacturonic acid. Glucose supported a basal level of SsPg1 expression but accentuated expression when provided to mycelia used to inoculate leaves. These observations were contrary to earlier reports indicating that glucose repressed SsPg1 expression while galacturonic acid induced expression. Pharmacological studies showed that disruption of calcium signalling affected SsCutA and SsPg1 expression and decreased S. sclerotiorum virulence, whereas elevated cAMP levels reduced virulence without affecting gene expression. The mechanisms involved in coordinating the expression of S. sclerotiorum hydrolytic enzymes throughout the various stages of the infection are discussed.

Carbohydrate Starvation Causes a Metabolically Active but Nonculturable State in Lactococcus lactis†

ABSTRACT This study characterized the ability of lactococci to become nonculturable under carbohydrate starvation while maintaining metabolic activity. We determined the changes in physiological parameters and extracellular substrate levels of multiple lactococcal strains under a number of environmental conditions along with whole-genome expression profiles. Three distinct phases were observed, logarithmic growth, sugar exhaustion, and nonculturability. Shortly after carbohydrate starvation, each lactococcal strain lost the ability to form colonies on solid media but maintained an intact cell membrane and metabolic activity for over 3.5 years. ML3, a strain that metabolized lactose rapidly, reached nonculturability within 1 week. Strains that metabolized lactose slowly (SK11) or not at all (IL1403) required 1 to 3 months to become nonculturable. In all cases, the cells contained at least 100 pM of intracellular ATP after 6 months of starvation and remained at that level for the remainder of the study. Aminopeptidase and lipase/esterase activities decreased below detection limits during the nonculturable phase. During sugar exhaustion and entry into nonculturability, serine and methionine were produced, while glutamine and arginine were depleted from the medium. The cells retained the ability to transport amino acids via proton motive force and peptides via ATP-driven translocation. The addition of branched-chain amino acids to the culture medium resulted in increased intracellular ATP levels and new metabolic products, indicating that branched-chain amino acid catabolism resulted in energy and metabolic products to support survival during starvation. Gene expression analysis showed that the genes responsible for sugar metabolism were repressed as the cells entered nonculturability. The genes responsible for cell division were repressed, while autolysis and cell wall metabolism genes were induced neither at starvation nor during nonculturability. Taken together, these observations verify that carbohydrate-starved lactococci attain a nonculturable state wherein sugar metabolism, cell division, and autolysis are repressed, allowing the cells to maintain transcription, metabolic activity, and energy production during a state that produces new metabolites not associated with logarithmic growth.