Christoph A. Lehmeier

Day‐length effects on carbon stores for respiration of perennial ryegrass

• The mechanism controlling the use of stored carbon in respiration is poorly understood. Here, we explore if the reliance on stores as respiratory substrate depends on day length. • Lolium perenne (perennial ryegrass) was grown in continuous light (275 μmol photons m(-2) s(-1) ) or in a 16 : 8 h day : night regime (425 μmol m(-2) s(-1) during the photoperiod), with the same daily photosynthetic photon flux density (PPFD). Plants in stands were labelled with (13)CO(2) : (12)CO(2) for various time intervals. The rates and isotopic signatures of shoot- and root-respired CO(2) were measured after labelling, and water-soluble carbohydrates were determined in biomass. The tracer kinetics in respired CO(2) was analysed with compartmental models to infer the sizes, half-lives and contributions of respiratory substrate pools. • Stores were the main source for respiration in both treatments (c. 60% of all respired carbon). But, continuous light slowed the turnover (+270%) and increased the size (+160%) of the store relative to the 16 : 8 h day : night regime. This effect corresponded with a greatly elevated fructan content. Yet, day length had no effect on sizes and half-lives of other pools serving respiration. • We suggest that the residence time of respiratory carbon was strongly influenced by partitioning of carbon to fructan stores.

Increased leaf mesophyll porosity following transient retinoblastoma‐related protein silencing is revealed by microcomputed tomography imaging and leads to a system‐level physiological response to the altered cell division pattern

The causal relationship between cell division and growth in plants is complex. Although altered expression of cell-cycle genes frequently leads to altered organ growth, there are many examples where manipulation of the division machinery leads to a limited outcome at the level of organ form, despite changes in constituent cell size. One possibility, which has been under-explored, is that altered division patterns resulting from manipulation of cell-cycle gene expression alter the physiology of the organ, and that this has an effect on growth. We performed a series of experiments on retinoblastoma-related protein (RBR), a well characterized regulator of the cell cycle, to investigate the outcome of altered cell division on leaf physiology. Our approach involved combination of high-resolution microCT imaging and physiological analysis with a transient gene induction system, providing a powerful approach for the study of developmental physiology. Our investigation identifies a new role for RBR in mesophyll differentiation that affects tissue porosity and the distribution of air space within the leaf. The data demonstrate the importance of RBR in early leaf development and the extent to which physiology adapts to modified cellular architecture resulting from altered cell-cycle gene expression.

Nitrogen Stress Affects the Turnover and Size of Nitrogen Pools Supplying Leaf Growth in a Grass

Nitrogen stress has strong effects on the size and turnover of nitrogen pools supplying leaf growth of a grass but does not alter the relative contributions of currently assimilated and remobilized nitrogen for leaf growth. The effect of nitrogen (N) stress on the pool system supplying currently assimilated and (re)mobilized N for leaf growth of a grass was explored by dynamic 15N labeling, assessment of total and labeled N import into leaf growth zones, and compartmental analysis of the label import data. Perennial ryegrass (Lolium perenne) plants, grown with low or high levels of N fertilization, were labeled with 15NO3−/14NO3− from 2 h to more than 20 d. In both treatments, the tracer time course in N imported into the growth zones fitted a two-pool model (r2 > 0.99). This consisted of a “substrate pool,” which received N from current uptake and supplied the growth zone, and a recycling/mobilizing “store,” which exchanged with the substrate pool. N deficiency halved the leaf elongation rate, decreased N import into the growth zone, lengthened the delay between tracer uptake and its arrival in the growth zone (2.2 h versus 0.9 h), slowed the turnover of the substrate pool (half-life of 3.2 h versus 0.6 h), and increased its size (12.4 μg versus 5.9 μg). The store contained the equivalent of approximately 10 times (low N) and approximately five times (high N) the total daily N import into the growth zone. Its turnover agreed with that of protein turnover. Remarkably, the relative contribution of mobilization to leaf growth was large and similar (approximately 45%) in both treatments. We conclude that turnover and size of the substrate pool are related to the sink strength of the growth zone, whereas the contribution of the store is influenced by partitioning between sinks.

Combined Chlorophyll Fluorescence and Transcriptomic Analysis Identifies the P3/P4 Transition as a Key Stage in Rice Leaf Photosynthetic Development

A combined transcriptomic and physiological analysis of rice leaf development identifies the stage (P3/P4 transition) when photosynthetic competence is first established. Leaves are derived from heterotrophic meristem tissue that, at some point, must make the transition to autotrophy via the initiation of photosynthesis. However, the timing and spatial coordination of the molecular and cellular processes underpinning this switch are poorly characterized. Here, we report on the identification of a specific stage in rice (Oryza sativa) leaf development (P3/P4 transition) when photosynthetic competence is first established. Using a combined physiological and molecular approach, we show that elements of stomatal and vascular differentiation are coordinated with the onset of measurable light absorption for photosynthesis. Moreover, by exploring the response of the system to environmental perturbation, we show that the earliest stages of rice leaf development have significant plasticity with respect to elements of cellular differentiation of relevance for mature leaf photosynthetic performance. Finally, by performing an RNA sequencing analysis targeted at the early stages of rice leaf development, we uncover a palette of genes whose expression likely underpins the acquisition of photosynthetic capability. Our results identify the P3/P4 transition as a highly dynamic stage in rice leaf development when several processes for the initiation of photosynthetic competence are coordinated. As well as identifying gene targets for future manipulation of rice leaf structure/function, our data highlight a developmental window during which such manipulations are likely to be most effective.

Tracing Carbon Fluxes: Resolving Complexity Using Isotopes

Cells, organisms and ecosystems are interconnected and interdependent metabolic networks, which are operated by carbon substrate fluxes. Isotope methodologies are useful tools for tracing these fluxes. A large diversity of tracer approaches is available for such investigations, ranging from uses of position-labelled 13C substrates in steady-state systems to tracing of natural alterations of isotopic signals in natural conditions. We discuss general principles of different carbon isotope tracer methodologies and specifics of their use in studies of processes at various time frames and scales of biological complexity. Furthermore, we show how “compartmental modelling” can help to characterise the structure and kinetic features of metabolic systems.

Cell density and airspace patterning in the leaf can be manipulated to increase leaf photosynthetic capacity

Summary The pattern of cell division, growth and separation during leaf development determines the pattern and volume of airspace in a leaf. The resulting balance of cellular material and airspace is expected to significantly influence the primary function of the leaf, photosynthesis, and yet the manner and degree to which cell division patterns affect airspace networks and photosynthesis remains largely unexplored. In this paper we investigate the relationship of cell size and patterning, airspace and photosynthesis by promoting and repressing the expression of cell cycle genes in the leaf mesophyll. Using microCT imaging to quantify leaf cellular architecture and fluorescence/gas exchange analysis to measure leaf function, we show that increased cell density in the mesophyll of Arabidopsis can be used to increase leaf photosynthetic capacity. Our analysis suggests that this occurs both by increasing tissue density (decreasing the relative volume of airspace) and by altering the pattern of airspace distribution within the leaf. Our results indicate that cell division patterns influence the photosynthetic performance of a leaf, and that it is possible to engineer improved photosynthesis via this approach.

Contrasting carbon allocation responses of juvenile European beech (Fagus sylvatica) and Norway spruce (Picea abies) to competition and ozone.

Allocation of recent photoassimilates of juvenile beech and spruce in response to twice-ambient ozone (2 × O(3)) and plant competition (i.e. intra vs. inter-specific) was examined in a phytotron study. To this end, we employed continuous (13)CO(2)/(12)CO(2) labeling during late summer and pursued tracer kinetics in CO(2) released from stems. In beech, allocation of recent photoassimilates to stems was significantly lowered under 2 × O(3) and increased in spruce when grown in mixed culture. As total tree biomass was not yet affected by the treatments, C allocation reflected incipient tree responses providing the mechanistic basis for biomass partitioning as observed in longer experiments. Compartmental modeling characterized functional properties of substrate pools supplying respiratory C demand. Respiration of spruce appeared to be exclusively supplied by recent photoassimilates. In beech, older C, putatively located in stem parenchyma cells, was a major source of respiratory substrate, reflecting the fundamental anatomical disparity between angiosperm beech and gymnosperm spruce.

Atmospheric CO2 mole fraction affects stand‐scale carbon use efficiency of sunflower by stimulating respiration in light

Plant carbon-use-efficiency (CUE), a key parameter in carbon cycle and plant growth models, quantifies the fraction of fixed carbon that is converted into net primary production rather than respired. CUE has not been directly measured, partly because of the difficulty of measuring respiration in light. Here, we explore if CUE is affected by atmospheric CO2 . Sunflower stands were grown at low (200 μmol mol-1 ) or high CO2 (1000 μmol mol-1 ) in controlled environment mesocosms. CUE of stands was measured by dynamic stand-scale 13 C labelling and partitioning of photosynthesis and respiration. At the same plant age, growth at high CO2 (compared with low CO2 ) led to 91% higher rates of apparent photosynthesis, 97% higher respiration in the dark, yet 143% higher respiration in light. Thus, CUE was significantly lower at high (0.65) than at low CO2 (0.71). Compartmental analysis of isotopic tracer kinetics demonstrated a greater commitment of carbon reserves in stand-scale respiratory metabolism at high CO2 . Two main processes contributed to the reduction of CUE at high CO2 : a reduced inhibition of leaf respiration by light and a diminished leaf mass ratio. This work highlights the relevance of measuring respiration in light and assessment of the CUE response to environment conditions.

Carbon Availability Modifies Temperature Responses of Heterotrophic Microbial Respiration, Carbon Uptake Affinity, and Stable Carbon Isotope Discrimination

Microbial transformations of organic carbon (OC) generate a large flux of CO2 into the atmosphere and influence the C balance of terrestrial and aquatic ecosystems. Yet, inherent heterogeneity in natural environments precludes direct quantification of multiple microbial C fluxes that underlie CO2 production. Here we used a continuous flow bioreactor coupled with a stable C isotope analyzer to determine the effects of temperature and C availability (cellobiose concentration) on C fluxes and 13C discrimination of a microbial population growing at steady-state in a homogeneous, well-mixed environment. We estimated C uptake affinity and C use efficiency (CUE) to characterize the physiological responses of microbes to changing environmental conditions. Temperature increased biomass-C specific respiration rate and C uptake affinity at lower C availability, but did not influence those parameters at higher C availability. CUE decreased non-linearly with increasing temperature. The non-linear, negative relationship between CUE and temperature was more pronounced under lower C availability than under relatively high C availability. We observed stable isotope fractionation between C substrate and microbial biomass C (7~12‰ depletion), and between microbial biomass and respired CO2 (4~10‰ depletion). Microbial discrimination against 13C-containing cellobiose during C uptake was influenced by temperature and C availability, while discrimination during respiration was only influenced by C availability. Shifts in C uptake affinity with temperature and C availability may have modified uptake-induced 13C fractionation. By stressing the importance of C availability on temperature responses of microbial C fluxes, C uptake affinity, CUE, and isotopic fractionation, this study contributes to a fundamental understanding of C flow through microbes. This will help guide parameterization of microbial responses to varying temperature and C availability within Earth-system models.

Respiratory Turn-Over and Metabolic Compartments: From the Design of Tracer Experiments to the Characterization of Respiratory Substrate-Supply Systems

Maintenance, defense and growth in plants – and hence their ability to survive and propagate despite stress and competition – are strictly dependent on the availability of respiratory substrate as an energy source. Quantitative labeling of photosynthetic products, in conjunction with monitoring of tracer appearance in respiratory CO2 and compartmental modeling of tracer kinetics, are powerful tools to assess key features of the metabolic system supplying substrate for respiration. Such features include the number, the size and the turn-over of kinetically distinct pools that compose the system. Biological knowledge is essential for deriving a meaningful topology/architecture of respiratory substrate pools. Here, we describe basic characteristics and requirements of quantitative labeling techniques and principles of compartmental modeling for the study of the respiratory substrate supply system at both ecosystem and plant levels. Dynamic labeling associated with compartmental analysis has been used successfully to partition autotrophic and heterotrophic components of grassland ecosystem respiration. This combination of methodologies has also shown that the substrates feeding root and shoot respiration of a perennial grass are located in the shoot and sustain most of the respiratory activity of shoots and roots even during undisturbed growth. And it has provided strong support for the fructan pool in the shoot being the main storage compartment supporting respiration in this grass species. Finally, we show how a compartmental analysis of the respiratory substrate supply system can be combined with a compartmental analysis of carbohydrate metabolism in the same plant to investigate the potential identity of pools sustaining respiration.