Metabolic responses to waterlogging differ between roots and shoots and reflect phloem transport alteration in medicago truncatula

Root oxygen deficiency that is induced by flooding (waterlogging) is a common situation in many agricultural areas, causing considerable loss in yield and productivity. Physiological and metabolic acclimation to hypoxia has mostly been studied on roots or whole seedlings under full submergence. The metabolic difference between shoots and roots during waterlogging, and how roots and shoots communicate in such a situation is much less known. In particular, the metabolic acclimation in shoots and how this, in turn, impacts on roots metabolism is not well documented. Here, we monitored changes in the metabolome of roots and shoots of barrel clover (Medicago truncatula), growth, and gas-exchange, and analyzed phloem sap exudate composition. Roots exhibited a typical response to hypoxia, such as γ-aminobutyrate and alanine accumulation, as well as a strong decline in raffinose, sucrose, hexoses, and pentoses. Leaves exhibited a strong increase in starch, sugars, sugar derivatives, and phenolics (tyrosine, tryptophan, phenylalanine, benzoate, ferulate), suggesting an inhibition of sugar export and their alternative utilization by aromatic compounds production via pentose phosphates and phosphoenolpyruvate. Accordingly, there was an enrichment in sugars and a decline in organic acids in phloem sap exudates under waterlogging. Mass-balance calculations further suggest an increased imbalance between loading by shoots and unloading by roots under waterlogging. Taken as a whole, our results are consistent with the inhibition of sugar import by waterlogged roots, leading to an increase in phloem sugar pool, which, in turn, exert negative feedback on sugar metabolism and utilization in shoots.This research was funded by the Région Pays de la Loire and Angers Loire Métropole, via the grant Connect Talent Isosee

Mitochondrial complex I dysfunction increases CO₂ efflux and reconfigures metabolic fluxes of day respiration in tobacco leaves

Mutants affected in complex I are useful to understand the role played by mitochondrial electron transport and redox metabolism in cellular homeostasis and signaling. However, their respiratory phenotype is incompletely described and a specific examination of day respiration (Rd) is lacking. Here, we used isotopic methods and metabolomics to investigate the impact of complex I dysfunction on Rd in two respiratory mutants of forest tobacco (Nicotiana sylvestris): cytoplasmic male sterile II (CMSII) and nuclear male sterile 1 (NMS1), previously characterized for complex I disruption. Rd was higher in mutants and the inhibition of leaf respiration by light was lower. Higher Rd values were caused by increased (phosphoenol)pyruvate (PEP) metabolism at the expense of anaplerotic (PEP carboxylase (PEPc) ‐catalyzed) activity. De novo synthesis of Krebs cycle intermediates in the light was larger in mutants than in the wild‐type, although numerically small in all genotypes. Carbon metabolism in mutants involved alternative pathways, such as alanine synthesis, and an increase in amino acid production with the notable exception of aspartate. Our results show that the alteration of NADH re‐oxidation activity by complex I does not cause a general inhibition of catabolism, but rather a re‐orchestration of fluxes in day respiratory metabolism, leading to an increased CO2 efflux

Impact of defoliation frequency on regrowth and carbohydrate metabolism in contrasting varieties of Lolium perenne

International audienceThe aims of the study were to gain a better understanding of fructan metabolism regulation during regrowth of Lolium perenne, and to evaluate the role of fructans of remaining tissues as well as carbon assimilation of new leaf tissues in refoliation. Two varieties that contrast for carbohydrate metabolism, Aurora and Perma, were subject to severe and frequent or infrequent defoliations before regrowth. Aurora, which had a greater content of fructans in leaf sheaths than Perma before defoliation, produced more leaf biomass within the 4 days following the. first cut. At the end of the regrowth period, Aurora produced more leaf biomass than Perma. Photosynthetic parameters, which were barely affected by defoliation frequency, could not explain these differences. Fructan synthesising activities [sucrose: sucrose 1-fructosyltransferase (1-SST) and fructan: fructan 6G-fructosyltransferase (6G-FFT)], declined after defoliation. In elongating leaf bases, corresponding transcript levels did not decline concomitantly, suggesting a post-transcriptional regulation of expression, while in leaf sheaths the gene expression pattern mostly followed the time-course of the enzyme activities. Regulation of Lp1-SST and Lp6G-FFT gene expression depends, therefore, on the sink-source status of the tissue after defoliation. During the phase of reserve accumulation, fructosyltransferase activities together with corresponding transcripts increased more in frequently defoliated plants than in infrequently defoliated plants

Carbon metabolism in grass leaf meristems

International audienceIn Lolium perenne, fructan polymers represent the main storage carbohydrates. Even if leaf meristems, located in elongating leaf bases, act as strong sink for imported assimilates, they also synthesize fructans in substantial amounts. Fructans are generally not evenly distributed. Their highest content is found in the growth zone (0-30 mm from the leaf base) and decreased strongly in the differentiation zone (30-60 mm). Lp1-SST, Lp6G-FFT/1-FFT and Lp6-SFT, encoding the three main fructan synthesizing activities in Lolium perenne, were also predominantly expressed in the growth zone. Their expression declined along the leaf axis, in parallel with the spatial occurrence of fructans, sucrose and enzyme activities. As a response to defoliation, the decline in fructan content occurred not only in the differentiation zone, but also in the growth zone. Before defoliation, the activity of fructan exohydrolase (FEH) was maximal in the differentiation zone. After defoliation, it increased in all segments, but peaked in the growth zone. These data strongly indicate that fructans stored in the leaf growth zone were hydrolyzed and recycled in that zone to sustain refoliation immediately after defoliation. Leaf sheaths represent the other source of fructans. When the product of fructan degradation, fructose, was supplied as 13C-fructose to leaf sheaths at the time of defoliation, its fate showed that the relative supply of C to roots was transiently reduced for the benefit of the growth zone where 13C was allocated first to the proximal part (0-10 mm). This preferential allocation of C could be at least partly explained by a strong and specific increase of the SuSy activity in that zone after defoliation.The results will be discussed in relation to plant development and defoliation tolerance

Lolium perenne, backbone of sustainable development, source of fructans for grazing animals and potential source of novel enzymes for biotechnology

Chapitre 12[i]Lolium perenne[/i], backbone of sustainable development, source of fructans for grazing animals and potential source of novel enzymes for biotechnolog

Low-amplitude, high-frequency electromagnetic field exposure causes delayed and reduced growth in <em>Rosa hybrida</em>

International audienceIt is now accepted that plants perceive high-frequency electromagnetic field (HF-EMF). We wondered if the HF-EMF signal is integrated further in planta as a chain of reactions leading to a modification of plant growth. We exposed whole small ligneous plants (rose bush) whose growth could be studied for several weeks. We performed exposures at two different development stages (rooted cuttings bearing an axillary bud and 5-leaf stage plants), using two high frequency (900 MHz) field amplitudes (5 and 200 V m(-1)). We achieved a tight control on the experimental conditions using a state-of-the-art stimulation device (Mode Stirred Reverberation Chamber) and specialized culture-chambers. After the exposure, we followed the shoot growth for over a one-month period. We observed no growth modification whatsoever exposure was performed on the 5-leaf stage plants. When the exposure was performed on the rooted cuttings, no growth modification was observed on Axis I (produced from the elongation of the axillary bud). Likewise, no significant modification was noted on Axis II produced at the base of Axis I, that came from pre-formed secondary axillary buds. In contrast, Axis II produced at the top of Axis I, that came from post-formed secondary buds consistently displayed a delayed and significant reduced growth (45%). The measurements of plant energy uptake from HF-EMF in this exposure condition (SAR of 7.2 10(-4) W kg(-1)) indicated that this biological response is likely not due to thermal effect. These results suggest that exposure to electromagnetic field only affected development of post-formed organs

Cloning, gene mapping, and functional analysis of a fructan 1-exohydrolase (1-FEH) from Lolium perenne implicated in fructan synthesis rather than in fructan mobilization

International audienceFructans, which are beta-(2,1) and/or beta-(2,6) linked polymers of fructose, are important storage carbohydrates in many plants. They are mobilized via fructan exohydrolases (FEHs). The cloning, mapping, and functional analysis of the first 1-FEH (EC 3.2.1.153) from Lolium perenne L. var. Bravo is described here. By screening a perennial ryegrass cDNA library, a 1-FEH cDNA named Lp1-FEHa was cloned. The Lp1-FEHa deduced protein has a low iso-electric point (5.22) and it groups together with plant FEHs and cell-wall type invertases. The deduced amino acid sequence shows 75% identity to wheat 1-FEH w2. The Lpl-FEHa gene was mapped at a distal position on the linkage group 3 (LG3). Functional characterization of the recombinant protein in Pichia pastoris demonstrated that it had high FEH activity towards 1-kestotriose, 1,1-kestotetraose, and inulin, but low activity against 6-kestotriose and levan. Like other fructan-plant FEHs, no hydrolase activity could be detected towards sucrose, convincingly demonstrating that the enzyme is not a classic invertase. The expression pattern analysis of Lpl-FEHa revealed transcript accumulation in leaf tissues accumulating fructans while transcript level was low in the photosynthetic tissues. The high expression level of this 1-FEH in conditions of active fructan synthesis, together with its low expression level when fructan contents are low, suggest that it might play a role as a beta-(2,1) trimming enzyme acting during fructan synthesis in concert with fructan synthesis enzymes

Cellular pathways of carbon and nitrogen assimilation in kleptoplastic foraminiferal species

Kleptoplasty is the process by which an organism sequesters algal chloroplasts while discarding or digesting other algal components. This ability is encountered in different organisms, including benthic foraminifera. Among kleptoplastic foraminiferal species, Haynesina germanica and Elphidium williamsoni are two ubiquitous species found in intertidal mudflats. Albeit significant differences, their kleptoplast functionality has been clearly shown in terms of photosynthetic efficiency and inorganic carbon uptake. However, the intracellular pathways involved in the inorganic C-assimilation remained unknown and the kleptoplast role in N-uptake, although often suggested, still needs to be clarified. Here, we correlate transmission electron microscope (TEM) observations, NanoSIMS (nanoscale secondary ion mass spectrometry) analysis and isotopic labelling experiments (H13CO3- and 15NH4-) to investigate N and C-assimilation pathways at a sub-cellular level. Additionally, we compare the results observed on E. williamsoni and H. germanica with those observed on, Ammonia cf. tepida, a non-kleptoplastic species. TEM-NanoSIMS observations suggest two different fates for the C assimilated via the kleptoplasts, involving either fatty acids in H. germanica, and probably soluble molecules such as carbohydrates in E. williamsoni. Both kleptoplastic and non-kleptoplastic species were labelled in 15NH4-. The few 15N-enriched kleptoplasts in the kleptoplastic species suggest that they play a role in the N-assimilation; whereas, in the non-kleptoplastic species our results suggest the existence of alternative N-assimilation pathway(s) in benthic foraminifera. Furthermore, the different metabolites quantified in the foraminiferal cell (sugars, organic acids, fatty acids and amino acids) provide new insights and clarify metabolic pathways involved in C and N-uptake in benthic foraminifera

Molecular and functional characterization of cDNA encoding fructan:fructan 6G-fructosyltransferase (6G-FFT)/ fructan:fructan 1-fructosyltransferase (1-FFT) from perennial ryegrass (Lolium perenne L.).

International audienceFructans are the main storage compound in Lolium perenne. To account for the prevailing neokestose-based fructan synthesis in this species, a cDNA library of L. perenne was screened by using the onion (Allium cepa) fructan:fructan 6G-fructosyltransferase (6G-FFT) as a probe. A full length Lp6G-FFT clone was isolated with significant homologies to vacuolar type fructosyltransferases and invertases. The functionality of the cDNA was tested by heterologous expression in Pichia pastoris. The recombinant protein demonstrated both 6G-FFT and fructan:fructan 1-fructosyltransferase activities (1-FFT) with a maximum 6G-FFT/1-FFT ratio of two. The activity of 6G-FFT was investigated with respect to developmental stage, tissue distribution, and alterations in carbohydrate status expression and compared to sucrose:sucrose 1-fructosyltransferase (1-SST). Lp6G-FFT and Lp1-SST were predominantly expressed in the basal part of elongating leaves and leaf sheaths. Expression of both genes declined along the leaf axis, in parallel with the spatial occurrence of fructan and fructosyltransferase activities. Surprisingly, Lp6G-FFT was highly expressed in photosynthetically active tissues where very low extractable fructosyltransferase activity and fructan amounts were detected, suggesting a post-transcriptional regulation of expression. Lp6G-FFT gene expression increased only in elongating leaves following similar increases of sucrose content in blades, sheaths, and elongating leaf bases. Regulation of Lp6G-FFT gene expression depends on the tissue according to its sink-source status