Grazing intensity drives plant diversity but does not affect forage production in a natural grassland dominated by the tussock-forming grass Andropogon lateralis Nees.

Andropogon lateralis is a tall and highly plastic tussock-forming grass native from southern South America. It is a frequent component of Campos and Subtropical highland grasslands that often becomes dominant under lax grazing regimes. The aim of this work was to analyze the response of species diversity and forage production of a natural grassland dominated by A. lateralis to a wide range of grazing intensity. We hypothesized that species diversity and forage production would both peak at the intermediate canopy heights determined by grazing regimes of moderate intensity. A grazing experiment was conducted in a highland grassland with mesothermal humid climate at 922 masl (Atlantic Forest biome, Santa Catarina state, Brazil) that comprised 87 species from 20 families but had 50% of its standing biomass accounted by A. lateralis. Four pre-/post-grazing canopy heights-12/7, 20/12, 28/17, and 36/22 cm (measured on A. lateralis)-were arranged in a complete randomized block design with four replications, and intermittently stocked with beef heifers from October 2015 to October 2017. Andropogon lateralis cover decreased (from 75 to 50%), and species richness increased (15-25 species m-2) as canopy height decreased. Grazing intensity did not affect annual forage production (4.2 Mg DM ha-1). This natural grassland dominated by A. lateralis had a high capacity to adjust to grazing regimes of contrasting intensity, maintaining forage production stable over a wide range of canopy heights. However, to prevent losses in floristic diversity, such grassland should not be grazed at canopy heights higher than 28 cm.

Time Course of Root Axis Elongation and Lateral Root Formation in Perennial Ryegrass (Lolium perenne L.).

Grasses have a segmental morphology. Compared to leaf development, data on root development at the phytomer level are scarce. Leaf appearance interval was recorded over time to allow inference about the age of segmental sites that later form roots. Hydroponically grown Lolium perenne cv. Aberdart tillers were studied in both spring and autumn in increasing and decreasing day length conditions, respectively, and dissected to define the development status of roots of known age on successive phytomers basipetally on the tiller axis. Over a 90-day observation period spring and autumn tillers produced 10.4 and 18.1 root bearing phytomers (Pr), respectively. Four stages of root development were identified: (0) main axis elongation (~0-10 days), (1) primary branching (~10-18 days), (2) secondary branching (~18-25 days), and (3) tertiary and quaternary branching without further increase in root dry weight. The individual spring roots achieved significantly greater dry weight (35%) than autumn roots, and a mechanism for seasonal shift in substrate supply to roots is proposed. Our data define a root turnover pattern likely also occurring in field swards and provide insight for modelling the turnover of grass root systems for developing nutrient efficient or stress tolerant ryegrass swards.

Reciprocal Effects of Silicon Supply and Endophytes on Silicon Accumulation and Epichloë Colonization in Grasses.

Cool season grasses associate asymptomatically with foliar Epichloë endophytic fungi in a symbiosis where Epichloë spp. protects the plant from a number of biotic and abiotic stresses. Furthermore, many grass species can accumulate large quantities of silicon (Si), which also alleviates a similar range of stresses. While Epichloë endophytes may improve uptake of minerals and nutrients, their impact on Si is largely unknown. Likewise, the effect of Si availability on Epichloë colonization remains untested. To assess the bidirectional relationship, we grew tall fescue (Festuca arundinacea) and perennial ryegrass (Lolium perenne) hydroponically with or without Si. Grasses were associated with five different Epichloë endophyte strains [tall fescue: AR584 or wild type (WT); perennial ryegrass: AR37, AR1, or WT] or as Epichloë-free controls. Reciprocally beneficial effects were observed for tall fescue associations. Specifically, Epichloë presence increased Si concentration in the foliage of tall fescue by at least 31%, regardless of endophyte strain. In perennial ryegrass, an increase in foliar Si was observed only for plants associated with the AR37. Epichloë promotion of Si was (i) independent of responses in plant growth, and (ii) positively correlated with endophyte colonization, which lends support to an endophyte effect independent of their impacts on root growth. Moreover, Epichloë colonization in tall fescue increased by more than 60% in the presence of silicon; however, this was not observed in perennial ryegrass. The reciprocal benefits of Epichloë-endophytes and foliar Si accumulation reported here, especially for tall fescue, might further increase grass tolerance to stress.

The allocation of assimilated carbon to shoot growth: in situ assessment in natural grasslands reveals nitrogen effects and interspecific differences.

In grasslands, sustained nitrogen loading would increase the proportion of assimilated carbon allocated to shoot growth (A shoot), because it would decrease allocation to roots and also encourage the contribution of species with inherently high A shoot. However, in situ measurements of carbon allocation are scarce. Therefore, it is unclear to what extent species that coexist in grasslands actually differ in their allocation strategy or in their response to nitrogen. We used a mobile facility to perform steady-state (13)C-labeling of field stands to quantify, in winter and autumn, the daily relative photosynthesis rate (RPR~tracer assimilated over one light-period) and A shoot (~tracer remaining in shoots after a 100 degree days chase period) in four individual species with contrasting morpho-physiological characteristics coexisting in a temperate grassland of Argentina, either fertilized or not with nitrogen, and either cut intermittently or grazed continuously. Plasticity in response to nitrogen was substantial in most species, as indicated by positive correlations between A shoot and shoot nitrogen concentration. There was a notable interspecific difference: productive species with higher RPR, enhanced by fertilization and characterized by faster leaf turnover rate, allocated ~20% less of the assimilated carbon to shoot growth than species of lower productivity (and quality) characterized by longer leaf life spans and phyllochrons. These results imply that, opposite to the expected response, sustained nitrogen loading would change little the A shoot of grassland communities if increases at the species-level are offset by decreases associated with replacement of 'low RPR-high A shoot' species by 'high RPR-low A shoot' species.

Pulse-labelling trees to study carbon allocation dynamics: a review of methods, current knowledge and future prospects.

Pulse-labelling of trees with stable or radioactive carbon (C) isotopes offers the unique opportunity to trace the fate of labelled CO(2) into the tree and its release to the soil and the atmosphere. Thus, pulse-labelling enables the quantification of C partitioning in forests and the assessment of the role of partitioning in tree growth, resource acquisition and C sequestration. However, this is associated with challenges as regards the choice of a tracer, the methods of tracing labelled C in tree and soil compartments and the quantitative analysis of C dynamics. Based on data from 47 studies, the rate of transfer differs between broadleaved and coniferous species and decreases as temperature and soil water content decrease. Labelled C is rapidly transferred belowground-within a few days or less-and this transfer is slowed down by drought. Half-lives of labelled C in phloem sap (transfer pool) and in mature leaves (source organs) are short, while those of sink organs (growing tissues, seasonal storage) are longer. (13)C measurements in respiratory efflux at high temporal resolution provide the best estimate of the mean residence times of C in respiratory substrate pools, and the best basis for compartmental modelling. Seasonal C dynamics and allocation patterns indicate that sink strength variations are important drivers for C fluxes. We propose a conceptual model for temperate and boreal trees, which considers the use of recently assimilated C versus stored C. We recommend best practices for designing and analysing pulse-labelling experiments, and identify several topics which we consider of prime importance for future research on C allocation in trees: (i) whole-tree C source-sink relations, (ii) C allocation to secondary metabolism, (iii) responses to environmental change, (iv) effects of seasonality versus phenology in and across biomes, and (v) carbon-nitrogen interactions. Substantial progress is expected from emerging technologies, but the largest challenge remains to carry out in situ whole-tree labelling experiments on mature trees to improve our understanding of the environmental and physiological controls on C allocation.

13C-labeling shows the effect of hierarchy on the carbon gain of individuals and functional groups in dense field stands.

Measurements of resource capture by individuals, species, or functional groups coexisting in field stands improve our ability to investigate the ecophysiological basis of plant competition. But methodological and technical difficulties have limited the use of such measurements. Carbon capture, in particular, is difficult to asses in heterogeneous, dense field stands. Here we present a new approach to measure in situ daily gross carbon gain of individuals. It is based on measuring the 13C content of shoots after a few hours of continuous labeling of all assimilated CO2. The technique is simple and has few assumptions. A new, fully mobile facility was developed, capable of providing a labeling environment with a CO2 concentration close to atmospheric air and known, constant 13C-enrichment, while maintaining temperature and relative humidity within ambient values. This facility was used in seminatural grasslands of Germany and Argentina to explore the relationship between size and carbon gain of individuals of coexisting species growing in contrasting hierarchical positions, and to analyze the carbon gain of functional groups. In general, carbon gain per unit shoot mass increased with increasing size among small individuals, but it became independent of size among the largest ones. In consequence, competition appeared to be size asymmetric between subordinate individuals but size symmetric between dominant individuals. When comparing functional groups, the carbon gain per unit shoot mass of rosette dicots vs. grasses reflected not their relative contribution to stand biomass, but their hierarchical position: irrespectively of mass or growth form, being taller than neighbors was most important in determining carbon gain per unit shoot mass. We believe these results show that in situ measurements of carbon gain can provide valuable insight in field studies of plant competition.

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.

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.

Nitrogen deficiency inhibits leaf blade growth in Lolium perenne by increasing cell cycle duration and decreasing mitotic and post-mitotic growth rates.

Nitrogen deficiency severely inhibits leaf growth. This response was analysed at the cellular level by growing Lolium perenne L. under 7.5 mM (high) or 1 mM (low) nitrate supply, and performing a kinematic analysis to assess the effect of nitrogen status on cell proliferation and cell growth in the leaf blade epidermis. Low nitrogen supply reduced leaf elongation rate (LER) by 43% through a similar decrease in the cell production rate and final cell length. The former was entirely because of a decreased average cell division rate (0.023 versus 0.032 h(-1)) and thus longer cell cycle duration (30 versus 22 h). Nitrogen status did not affect the number of division cycles of the initial cell's progeny (5.7), and accordingly the meristematic cell number (53). Meristematic cell length was unaffected by nitrogen deficiency, implying that the division and mitotic growth rates were equally impaired. The shorter mature cell length arose from a considerably reduced post-mitotic growth rate (0.033 versus 0.049 h(-1)). But, nitrogen stress did not affect the position where elongation stopped, and increased cell elongation duration. In conclusion, nitrogen deficiency limited leaf growth by increasing the cell cycle duration and decreasing mitotic and post-mitotic elongation rates, delaying cell maturation.

Phosphorus deficiency decreases cell division and elongation in grass leaves.

Leaf growth in monocotyledons results from the flux of newly born cells out of the division zone and into the adjacent elongation-only zone, where cells reach their final length. We used a kinematic method to analyze the effect of phosphorus nutrition status on cell division and elongation parameters in the epidermis of Lolium perenne. Phosphorus deficiency reduced the leaf elongation rate by 39% due to decreases in the cell production rate (-19%) and final cell length (-20%). The former was solely due to a lower average cell division rate (0.028 versus 0.046 cell cell(-1) h(-1)) and, thus, a lengthened average cell cycle duration (25 versus 15 h). The number of division cycles of the initial cell progeny (five to six) and, as a result, the number of meristematic cells (32-64) and division zone length were independent of phosphorus status. Accordingly, low-phosphorus cells maintained meristematic activity longer. Lack of effect of phosphorus deficiency on meristematic cell length implies that a lower division rate was matched to a lower elongation rate. Phosphorus deficiency did not affect the elongation-only zone length, thus leading to longer cell elongation duration (99 versus 75 h). However, the substantially reduced postmitotic average relative elongation rate (0.045 versus 0.064 mm mm(-1) h(-1)) resulted in shorter mature cells. In summary, phosphorus deficiency did not affect the general controls of cell morphogenesis, but, by slowing down the rates of cell division and expansion, it slowed down its pace.

The sources of carbon and nitrogen supplying leaf growth. Assessment of the role of stores with compartmental models.

Patterns of synthesis and breakdown of carbon (C) and nitrogen (N) stores are relatively well known. But the role of mobilized stores as substrates for growth remains less clear. In this article, a novel approach to estimate C and N import into leaf growth zones was coupled with steady-state labeling of photosynthesis ((13)CO(2)/(12)CO(2)) and N uptake ((15)NO(3)(-)/(14)NO(3)(-)) and compartmental modeling of tracer fluxes. The contributions of current C assimilation/N uptake and mobilization from stores to the substrate pool supplying leaf growth were then quantified in plants of a C(3) (Lolium perenne) and C(4) grass (Paspalum dilatatum Poir.) manipulated thus to have contrasting C assimilation and N uptake rates. In all cases, leaf growth relied largely on photoassimilates delivered either directly after fixation or short-term storage (turnover rate = 1.6-3.3 d(-1)). Long-term C stores (turnover rate < 0.09 d(-1)) were generally of limited relevance. Hence, no link was found between the role of stores and C acquisition rate. Short-term (turnover rate = 0.29-0.90 d(-1)) and long-term (turnover rate < 0.04 d(-1)) stores supplied most N used in leaf growth. Compared to dominant (well-lit) plants, subordinate (shaded) plants relied more on mobilization from long-term N stores to support leaf growth. These differences correlated well with the C-to-N ratio of growth substrates and were associated with responses in N uptake. Based on this, we argue that internal regulation of N uptake acts as a main determinant of the importance of mobilized long-term stores as a source of N for leaf growth.