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

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.

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

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.

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.

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.

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.

Fluxes in central carbohydrate metabolism of source leaves in a fructan-storing C3 grass: rapid turnover and futile cycling of sucrose in continuous light under contrasted nitrogen nutrition status.

This work assessed the central carbohydrate metabolism of actively photosynthesizing leaf blades of a C3 grass (Lolium perenne L.). The study used dynamic (13)C labelling of plants growing in continuous light with contrasting supplies of nitrogen ('low N' and 'high N') and mathematical analysis of the tracer data with a four-pool compartmental model to estimate rates of: (i) sucrose synthesis from current assimilation; (ii) sucrose export/use; (iii) sucrose hydrolysis (to glucose and fructose) and resynthesis; and (iv) fructan synthesis and sucrose resynthesis from fructan metabolism. The contents of sucrose, fructan, glucose, and fructose were almost constant in both treatments. Labelling demonstrated that all carbohydrate pools were turned over. This indicated a system in metabolic steady state with equal rates of synthesis and degradation/consumption of the individual pools. Fructan content was enhanced by nitrogen deficiency (55 and 26% of dry mass at low and high N, respectively). Sucrose content was lower in nitrogen-deficient leaves (2.7 versus 6.7%). Glucose and fructose contents were always low (<1.5%). Interconversions between sucrose, glucose, and fructose were rapid (with half-lives of individual pools ranging between 0.3 and 0.8 h). Futile cycling of sucrose through sucrose hydrolysis (67 and 56% of sucrose at low and high N, respectively) and fructan metabolism (19 and 20%, respectively) was substantial but seemed to have no detrimental effect on the relative growth rate and carbon-use efficiency of these plants. The main effect of nitrogen deficiency on carbohydrate metabolism was to increase the half-life of the fructan pool from 27 to 62 h and to effectively double its size.

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.

Nitrogen deficiency increases the residence time of respiratory carbon in the respiratory substrate supply system of perennial ryegrass.

Plant respiration draws on substrate pools of different functional/biochemical identity. Little is known about the effect of nitrogen deficiency on those pools' sizes, half-lives and relative contribution to respiration, and consequently, of carbon residence time in respiratory metabolism. Here we studied how nitrogen fertilization affects the respiratory carbon supply system of shoots and roots of Lolium perenne, a perennial grass. Plants grown at two nitrogen supply levels in continuous light were labelled with (13)CO(2)/(12)CO(2) for intervals ranging from 1 h to 1 month. The rate and isotopic composition of shoot, root and plant respiration were measured, and the time-courses of tracer incorporation into respired CO(2) were analysed by compartmental modelling. Nitrogen deficiency reduced specific respiration rate by 30%, but increased the size of the respiratory supply system by 30%. In consequence, mean residence time of respiratory carbon increased with nitrogen deficiency (4.6 d at high nitrogen and 9.2 d at low nitrogen supply). To a large extent, this was due to a greater involvement of stores with a long half-life in respiratory carbon metabolism of nitrogen-deficient plants. At both nitrogen supply levels, stores supplying root respiration were primarily located in the shoot, probably in the form of fructans.

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.

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.

Root and shoot respiration of perennial ryegrass are supplied by the same substrate pools: assessment by dynamic 13C labeling and compartmental analysis of tracer kinetics.

The substrate supply system for respiration of the shoot and root of perennial ryegrass (Lolium perenne) was characterized in terms of component pools and the pools' functional properties: size, half-life, and contribution to respiration of the root and shoot. These investigations were performed with perennial ryegrass growing in constant conditions with continuous light. Plants were labeled with (13)CO(2)/(12)CO(2) for periods ranging from 1 to 600 h, followed by measurements of the rates and (13)C/(12)C ratios of CO(2) respired by shoots and roots in the dark. Label appearance in roots was delayed by approximately 1 h relative to shoots; otherwise, the tracer time course was very similar in both organs. Compartmental analysis of respiratory tracer kinetics indicated that, in both organs, three pools supplied 95% of all respired carbon (a very slow pool whose kinetics could not be characterized provided the remaining 5%). The pools' half-lives and relative sizes were also nearly identical in shoot and root (half-life < 15 min, approximately 3 h, and 33 h). An important role of short-term storage in supplying respiration was apparent in both organs: only 43% of respiration was supplied by current photosynthate (fixed carbon transferred directly to centers of respiration via the two fastest pools). The residence time of carbon in the respiratory supply system was practically the same in shoot and root. From this and other evidence, we argue that both organs were supplied by the same pools and that the residence time was controlled by the shoot via current photosynthate and storage deposition/mobilization fluxes.

Allocation of reserve-derived and currently assimilated carbon and nitrogen in seedlings of Helianthus annuus under sub-ambient and elevated CO growth conditions.

Here, we analysed the transition from heterotrophic to autotrophic growth of the epigeal species sunflower (Helianthus annuus), and how transition is affected by CO(2). Growth analysis and steady-state (13)CO(2)/(12)CO(2) and (15)NO(3) (-)/(14)NO(3) (-) labelling were used to quantify reserve- and current assimilation-derived carbon (C) and nitrogen (N) allocation to shoots and roots in the presence of 200 and 1,000 micromol CO(2) mol(-1) air. Growth was not influenced by CO(2) until cotyledons unfolded. Then, C accumulation at elevated CO(2) increased to a rate 2-2.5 times higher than in sub-ambient CO(2) due to increased unit leaf rate (+120%) and leaf expansion (+60%). CO(2) had no effect on mobilization and allocation of reserve-derived C and N, even during the transition period. Export of autotrophic C from cotyledons began immediately following the onset of photosynthetic activity, serving roots and shoots near-simultaneously. Allocation of autotrophic C to shoots was increased at sub-ambient CO(2). The synchrony in transition from heterotrophic to autotrophic supply for different sinks in sunflower contrasts with the sequential transition reported for species with hypogeal germination.