Leaf green-white variegation is advantageous under N deprivation in Pelargonium×hortorum.

Variegation (patchy surface area with different colours) is a common trait of plant leaves. In green-white variegated leaves, two tissues with contrasted primary carbon metabolisms (autotrophic in green and heterotrophic in white tissues) are juxtaposed. It is generally believed that variegation is detrimental to growth due to the lower photosynthetic surface area. However, the common occurrence of leaf variegation in nature raises the question of a possible advantage under certain circumstances. Here, we examined growth and metabolism of variegated Pelargonium×hortorum L.H.Bailey using metabolomics techniques under N deprivation. Our results showed that variegated plants tolerate N deficiency much better, i.e. do not stop leaf biomass production after 9 weeks of N deprivation, even though the growth of green plants is eventually arrested and leaf senescence is triggered. Metabolic analysis indicates that white areas are naturally enriched in arginine, which decreases a lot upon N deprivation, probably to feed green areas. This process may compensate for the lower proteolysis enhancement in green areas and thus contribute to maintaining photosynthetic activity. We conclude that under our experimental conditions, leaf variegation was advantageous under prolonged N deprivation.

Corrigendum to: Metabolomic characterisation of the functional division of nitrogen metabolism in variegated leaves.

Many horticultural and natural plant species have variegated leaves, that is, patchy leaves with green and non-green or white areas. Specific studies on the metabolism of variegated leaves are scarce and although white (non-green) areas have been assumed to play the role of a 'nitrogen store', there is no specific studies showing the analysis of nitrogenous metabolites and the dynamics of nitrogen assimilation. Here, we examined the metabolism of variegated leaves of Pelargonium × hortorum. We show that white areas have a larger N : C ratio, more amino acids, with a clear accumulation of arginine. Metabolomic analyses revealed clear differences in the chemical composition, suggesting contrasted metabolic commitments such as an enhancement of alkaloid biosynthesis in white areas. Using isotopic labelling followed by nuclear magnetic resonance or liquid chromatography/mass spectrometry, we further showed that in addition to glutamine, tyrosine and tryptophan, N metabolism forms ornithine in green area and huge amounts of arginine in white areas. Fine isotopic measurements with isotope ratio mass spectrometry indicated that white and green areas exchange nitrogenous molecules but nitrogen export from green areas is quantitatively much more important. The biological significance of the metabolic exchange between leaf areas is briefly discussed.

Metabolic origin of δ15 N values in nitrogenous compounds from Brassica napus L. leaves.

Nitrogen isotope composition (δ(15) N) in plant organic matter is currently used as a natural tracer of nitrogen acquisition efficiency. However, the δ(15) N value of whole leaf material does not properly reflect the way in which N is assimilated because isotope fractionations along metabolic reactions may cause substantial differences among leaf compounds. In other words, any change in metabolic composition or allocation pattern may cause undesirable variability in leaf δ(15) N. Here, we investigated the δ(15) N in different leaf fractions and individual metabolites from rapeseed (Brassica napus) leaves. We show that there were substantial differences in δ(15) N between nitrogenous compounds (up to 30‰) and the content in ((15) N enriched) nitrate had a clear influence on leaf δ(15) N. Using a simple steady-state model of day metabolism, we suggest that the δ(15) N value in major amino acids was mostly explained by isotope fractionation associated with isotope effects on enzyme-catalysed reactions in primary nitrogen metabolism. δ(15) N values were further influenced by light versus dark conditions and the probable occurrence of alternative biosynthetic pathways. We conclude that both biochemical pathways (that fractionate between isotopes) and nitrogen sources (used for amino acid production) should be considered when interpreting the δ(15) N value of leaf nitrogenous compounds.

Short-term effects of CO(2) and O(2) on citrate metabolism in illuminated leaves.

Although there is now a considerable literature on the inhibition of leaf respiration (CO(2) evolution) by light, little is known about the effect of other environmental conditions on day respiratory metabolism. In particular, CO(2) and O(2) mole fractions are assumed to cause changes in the tricarboxylic acid pathway (TCAP) but the amplitude and even the direction of such changes are still a matter of debate. Here, we took advantage of isotopic techniques, new simple equations and instant freeze sampling to follow respiratory metabolism in illuminated cocklebur leaves (Xanthium strumarium L.) under different CO(2) /O(2) conditions. Gas exchange coupled to online isotopic analysis showed that CO(2) evolved by leaves in the light came from 'old' carbon skeletons and there was a slight decrease in (13) C natural abundance when [CO(2) ] increased. This suggested the involvement of enzymatic steps fractionating more strongly against (13) C and thus increasingly limiting for the metabolic respiratory flux as [CO(2) ] increased. Isotopic labelling with (13) C(2) -2,4-citrate lead to (13) C-enriched Glu and 2-oxoglutarate (2OG), clearly demonstrating poor metabolism of citrate by the TCAP. There was a clear relationship between the ribulose-1,5-bisphosphate oxygenation-to-carboxylation ratio (v(o) /v(c) ) and the (13) C commitment to 2OG, demonstrating that 2OG and Glu synthesis via the TCAP is positively influenced by photorespiration.

δ(13) C of leaf-respired CO(2) reflects intrinsic water-use efficiency in barley.

Leaf intrinsic water-use efficiency (WUE), the ratio of photosynthetic rate to stomatal conductance (A/g(s) ), is a key plant trait linking terrestrial carbon and water cycles. A rapid, integrative proxy for A/g(s) is of benefit to crop breeding programmes aiming to improve WUE, but also for ecologists interested in plant carbon-water balance in natural systems. We hypothesize that the carbon isotope composition of leaf-respired CO(2) (δ(13) C(Rl) ), two hours after leaves are transferred to the dark, records photosynthetic carbon isotope discrimination and so provides a proxy for A/g(s) . To test this hypothesis, δ(13) C(Rl) was measured in four barley cultivars grown in the field at two levels of water availability and compared to leaf-level gas exchange (the ratio of leaf intercellular to ambient CO(2) partial pressure, C(i) /C(a) , and A/g(s) ). Leaf-respired CO(2) was more (13) C-depleted in plants grown at higher water availability, varied between days as environmental conditions changed, and was significantly different between cultivars. A strong relationship between δ(13) C(Rl) and δ(13) C of sucrose was observed. δ(13) C(Rl) was converted into apparent photosynthetic discrimination (Δ(13) C(Rl) ) revealing strong relationships between Δ(13) C(Rl) and C(i) /C(a) and A/g(s) during the vegetative stage of growth. We therefore conclude that δ(13) C(Rl) may provide a rapid, integrative proxy for A/g(s) in barley.

The 13C/12C isotopic signal of day-respired CO2 in variegated leaves of Pelargonium × hortorum.

In leaves, although it is accepted that CO(2) evolved by dark respiration after illumination is naturally (13) C-enriched compared to organic matter or substrate sucrose, much uncertainty remains on whether day respiration produces (13) C-depleted or (13) C-enriched CO(2). Here, we applied equations described previously for mesocosm CO(2) exchange to investigate the carbon isotope composition of CO(2) respired by autotrophic and heterotrophic tissues of Pelargonium × hortorum leaves, taking advantage of leaf variegation. Day-respired CO(2) was slightly (13) C-depleted compared to organic matter both under 21% O(2) and 2% O(2). Furthermore, most, if not all CO(2) molecules evolved in the light came from carbon atoms that had been fixed previously before the experiments, in both variegated and green leaves. We conclude that the usual definition of day respiratory fractionation, that assumes carbon fixed by current net photosynthesis is the respiratory substrate, is not valid in Pelargonium leaves under our conditions. In variegated leaves, total organic matter was slightly (13) C-depleted in white areas and so were most primary metabolites. This small isotopic difference between white and green areas probably came from the small contribution of photosynthetic CO(2) refixation and the specific nitrogen metabolism in white leaf areas.