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.

Impaired Malate and Fumarate Accumulation Due to the Mutation of the Tonoplast Dicarboxylate Transporter Has Little Effects on Stomatal Behavior.

Malate is a central metabolite involved in a multiplicity of plant metabolic pathways, being associated with mitochondrial metabolism and playing significant roles in stomatal movements. Vacuolar malate transport has been characterized at the molecular level and is performed by at least one carrier protein and two channels in Arabidopsis (Arabidopsis thaliana) vacuoles. The absence of the Arabidopsis tonoplast Dicarboxylate Transporter (tDT) in the tdt knockout mutant was associated previously with an impaired accumulation of malate and fumarate in leaves. Here, we investigated the consequences of this lower accumulation on stomatal behavior and photosynthetic capacity as well as its putative metabolic impacts. Neither the stomatal conductance nor the kinetic responses to dark, light, or high CO2 were highly affected in tdt plants. In addition, we did not observe any impact on stomatal aperture following incubation with abscisic acid, malate, or citrate. Furthermore, an effect on photosynthetic capacity was not observed in the mutant lines. However, leaf mitochondrial metabolism was affected in the tdt plants. Levels of the intermediates of the tricarboxylic acid cycle were altered, and increases in both light and dark respiration were observed. We conclude that manipulation of the tonoplastic organic acid transporter impacted mitochondrial metabolism, while the overall stomatal and photosynthetic capacity were unaffected.

The influence of alternative pathways of respiration that utilize branched-chain amino acids following water shortage in Arabidopsis.

During dark-induced senescence isovaleryl-CoA dehydrogenase (IVDH) and D-2-hydroxyglutarate dehydrogenase (D-2HGDH) act as alternate electron donors to the ubiquinol pool via the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) pathway. However, the role of this pathway in response to other stresses still remains unclear. Here, we demonstrated that this alternative pathway is associated with tolerance to drought in Arabidopsis. In comparison with wild type (WT) and lines overexpressing D-2GHDH, loss-of-function etfqo-1, d2hgdh-2 and ivdh-1 mutants displayed compromised respiration rates and were more sensitive to drought. Our results demonstrated that an operational ETF/ETFQO pathway is associated with plants' ability to withstand drought and to recover growth once water becomes replete. Drought-induced metabolic reprogramming resulted in an increase in tricarboxylic acid (TCA) cycle intermediates and total amino acid levels, as well as decreases in protein, starch and nitrate contents. The enhanced levels of the branched-chain amino acids in loss-of-function mutants appear to be related to their increased utilization as substrates for the TCA cycle under water stress. Our results thus show that mitochondrial metabolism is highly active during drought stress responses and provide support for a role of alternative respiratory pathways within this response.

High-Throughput Extraction and Enzymatic Determination of Sugars and Fructans in Fructan-Accumulating Plants.

Fructans are carbohydrates present in more than 15% of flowering plants. They represent the major pool of carbohydrates in some species, especially when facing cold or drought. However, the functions of fructans with high or low degrees of polymerization (DP), their diurnal use, and the regulation of their synthesis and degradation in response to stresses still remain unclear. Here we present an enzymatic protocol adapted to 96-well microplates that simultaneously allows the determination of fructans and glucose, fructose, and sucrose. Moreover, the protocol allows to estimate the average DP of the fructans in the samples. The protocol is based on the enzymatic degradation of fructans into glucose and fructose and their subsequent conversion into gluconate 6-phosphate concomitant with the formation of NADH in the presence of ATP.

Transient Carbon Reserves in Barley: Malate, Sucrose and Starch Are the Main Players, Their Quantitative Involvement Being Light Intensity Dependant.

Under natural environment plants experience different light intensities which can affect photosynthesis and consequently the availability of carbohydrates for daytime growth and their transient storage to supply night growth. We grew a spring barley cultivar, Propino, under three different light intensities under warm days and nights, and evaluated the spatial and diurnal adjustments occurring in the transient carbon stores. Leaves under high light at the end of the day accumulated mainly sucrose (30%) and malate (35%), with lower content of hexoses (5%), starch (15%) and fructans (15%). Under low light, plants presented reduced photosynthesis, with lower metabolite contents at end of day. The malate represented 51% of the total carbon accumulated at end of the day, at the expense of sucrose (12%), other metabolite contributions remaining similar to high light. The percentage of metabolites consumed at night was similar for all light intensities with around 75% of the sucrose and starch being mobilized whilst malate and fructans were only partially mobilized with 56 and 44%, respectively. Altogether, sucrose and malate were the main contributors of the total carbon used at night by barley plants, sucrose being predominant under high light (35% vs. 27%), but malate being the major metabolite used under low light with 40% of the total carbon consumed. Interestingly, light intensity also influenced the location of the C transient stores, the plants under low light prioritizing the accumulation of the metabolites, mostly malate, in the youngest tissues. Therefore, light influences quantitatively, but also qualitatively and spatially the carbon stores in the spring barley cv. Propino, suggesting a tight regulation of the primary metabolism.