Knock-Out of ACY-1 Like Gene in Spodoptera litura Supports the Notion that FACs Improve Nitrogen Metabolism.

Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine were originally identified in the regurgitant of Spodoptera exigua larvae. These fatty acid amino acid conjugates (FACs) are known to be elicitors that induce plants to release volatile compounds which in turn attract natural enemies of the larvae such as parasitic wasps. FAC concentrations are regulated by enzymatic biosynthesis and hydrolysis in the intestine of Lepidoptera larvae. It has been proposed that FAC metabolism activates glutamine synthetase and plays an important role in nitrogen metabolism in larvae. In this study, we identified candidate genes encoding a FACs hydrolase in Spodoptera litura using genomic information of various related lepidopteran species in which FACs hydrolases have been reported. We analyzed the importance of FAC hydrolysis on caterpillar performance with CRISPR/Cas9 knock outs. Larvae of strains with an inactive FACs hydrolase excreted FACs in their feces. They absorbed 30% less nitrogen from the diet compared to WT caterpillars resulting in a reduction of their body weight of up to 40% compared to wild type caterpillars. These results suggest that the hydrolysis of FACs is an important metabolism for insects and that FACs are important for larval growth.

Sources of carbon supporting the fast growth of developing immature moso bamboo (Phyllostachys edulis) culms: inference from carbon isotopes and anatomy.

Phyllostachys edulis is a spectacularly fast-growing species that completes its height growth within 2 months after the shoot emerges without producing leaves (fast-growing period, FGP). This phase was considered heterotrophic, with the carbon necessary for the growth being transferred from the mature culms via the rhizomes, although previous studies observed key enzymes and anatomical features related to C4-carbon fixation in developing culms. We tested whether C4-photosynthesis or dark-CO2 fixation through anaplerotic reactions significantly contributes to the FGP, resulting in differences in the natural abundance of δ13C in bulk organic matter and organic compounds. Further, pulse-13CO2-labelling was performed on developing culms, either from the surface or from the internal hollow, to ascertain whether significant CO2 fixation occurs in developing culms. δ13C of young shoots and developing culms were higher (-26.3 to -26.9 ‰) compared to all organs of mature bamboos (-28.4 to -30.1 ‰). Developing culms contained chlorophylls, most observed in the skin tissues. After pulse-13CO2-labelling, the polar fraction extracted from the skin tissues was slightly enriched in 13C, and only a weak 13C enrichment was observed in inner tissues. Main carbon source sustaining the FGP was not assimilated by the developing culm, while a limited anaplerotic fixation of respired CO2 cannot be excluded and is more likely than C4-photosynthetic carbon fixation.

Estimating divergent forest carbon stocks and sinks via a knife set approach.

Forest carbon stocks and sinks (CSSs) have been widely estimated using climate classification tables and linear regression (LR) models with common independent variables (IVs) such as the average diameter at breast height (DBH) of stems and root shoot ratio. However, this approach is relatively ineffective when the explanatory power of IVs is lower than that of unobservable variables. Various environmental and anthropogenic factors affect target variables that cause the correlation between them to be chaotic. Here, we designed a knife set (KS) approach combining LR models and the wandering through random forests (WTF) algorithm and applied it in a specific case of Phyllostachys edulis (Carrière) J. Houz. (P. edulis) forests, which have an irregular relationship between their belowground carbon (BGC) stocks and average DBH. We then validated the KS approach performed by cluster computing to estimate the aboveground carbon (AGC) and BGC stocks and the total net primary production (TNPP). The estimated CSSs were compared to the benchmark of the methodology that applied Tier 1 in the Intergovernmental Panel on Climate Change (IPCC) Guidelines for National Greenhouse Gas Inventories via 10-fold cross validation, and the KS approach significantly increased precision and accuracy of estimations. Our approach provides general insights to accurately estimate forest CSSs relying on evidence-based field data, even if some target variables are divergent in specific forest types. We also pointed out the reason why current fancy models containing machine learning (ML) or deep learning algorithms are not effective in predicting the target variables of certain chaotic systems is perhaps that the total explanatory power of observable variables is less than that of the total unobservable variables. Quantifying unobservable variables into observable variables is a linchpin of future works related to chaotic system estimation.

Drought affects the fate of non-structural carbohydrates in hinoki cypress.

Tree species that close stomata early in response to drought are likely to suffer from an imbalance between limited carbohydrate supply due to reduced photosynthesis and metabolic demand. Our objective was to clarify the dynamic responses of non-structural carbohydrates to drought in a water-saving species, the hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.). To this end, we pulse-labeled young trees with 13CO2 10 days after the beginning of the drought treatment. Trees were harvested 7 days later, early during drought progression, and 86 days later when they had suffered from a long and severe drought. The labeled carbon (C) was traced in phloem extract, in the organic matter and starch of all the organs, and in the soluble sugars (sucrose, glucose and fructose) of the most metabolically active organs (foliage, green branches and fine roots). No drought-related changes in labeled C partitioning between belowground and aboveground organs were observed. The C allocation between non-structural carbohydrates was altered early during drought progression: starch concentration was lower by half in the photosynthetic organs, while the concentration of almost all soluble sugars tended to increase. The preferential allocation of labeled C to glucose and fructose reflected an increased demand for soluble sugars for osmotic adjustment. After 3 months of a lethal drought, the concentrations of soluble sugars and starch were admittedly lower in drought-stressed trees than in the controls, but the pool of non-structural carbohydrates was far from completely depleted. However, the allocation to storage had been impaired by drought; photosynthesis and the sugar translocation rate had also been reduced by drought. Failure to maintain cell turgor through osmoregulation and to refill embolized xylem due to the depletion in soluble sugars in the roots could have resulted in tree mortality in hinoki cypress, though the total pool of carbohydrate was not completely depleted.

Diurnal variations in the thickness of the inner bark of tree trunks in relation to xylem water potential and phloem turgor.

The inner bark plays important roles in tree stems, including radial exchange of water with the xylem and translocation of carbohydrates. Both processes affect the water content and the thickness of the inner bark on a diurnal basis. For the first time, we simultaneously measured the diurnal variations in the inner bark thickness of hinoki cypress (Chamaecyparis obtusa) by using point dendrometers and those of local xylem potential by using stem psychrometers located next to the dendrometers to determine how these variations were related to each other, to phloem turgor and carbohydrate transport. We also estimated the axial hydrostatic pressure gradient by measuring the osmolality of the sap extracted from the inner bark. The inner bark shrunk during the day and swelled during the night with an amplitude related to day-to-day and seasonal variations in climate. The relationship between changes in xylem water potential and inner bark thickness exhibited a hysteresis loop during the day with a median lag of 2 h. A phloem turgor-related signal can be retrieved from the diurnal variations in the inner bark thickness, which was higher at the upper than at the lower position along the trunk. However, a downward hydrostatic pressure gradient was only observed at dawn, suggesting diurnal variations in the phloem sap flow velocity.

Using 13C to Quantify Phloem Transport on Tall Plants in the Field.

The difference in time lags between a labeling pulse of 13CO2 of the foliage and the appearance of labeled C in the respiration at different locations along the stem of a tall plant is used to estimate at which velocities the isotope tracer, i.e., the labeled carbohydrates, are transported in the phloem sap. Here we describe a method for pulse-labeling tall plants in the field and subsequently tracing 13C in the respiratory efflux of CO2.

Drought impacts on tree phloem: from cell-level responses to ecological significance.

On-going climate change is increasing the risk of drought stress across large areas worldwide. Such drought events decrease ecosystem productivity and have been increasingly linked to tree mortality. Understanding how trees respond to water shortage is key to predicting the future of ecosystem functions. Phloem is at the core of the tree functions, moving resources such as non-structural carbohydrates, nutrients, and defence and information molecules across the whole plant. Phloem function and ability to transport resources is tightly controlled by the balance of carbon and water fluxes within the tree. As such, drought is expected to impact phloem function by decreasing the amount of available water and new photoassimilates. Yet, the effect of drought on the phloem has received surprisingly little attention in the last decades. Here we review existing knowledge on drought impacts on phloem transport from loading and unloading processes at cellular level to possible effects on long-distance transport and consequences to ecosystems via ecophysiological feedbacks. We also point to new research frontiers that need to be explored to improve our understanding of phloem function under drought. In particular, we show how phloem transport is affected differently by increasing drought intensity, from no response to a slowdown, and explore how severe drought might actually disrupt the phloem transport enough to threaten tree survival. Because transport of resources affects other organisms interacting with the tree, we also review the ecological consequences of phloem response to drought and especially predatory, mutualistic and competitive relations. Finally, as phloem is the main path for carbon from sources to sink, we show how drought can affect biogeochemical cycles through changes in phloem transport. Overall, existing knowledge is consistent with the hypotheses that phloem response to drought matters for understanding tree and ecosystem function. However, future research on a large range of species and ecosystems is urgently needed to gain a comprehensive understanding of the question.

Initial hydraulic failure followed by late-stage carbon starvation leads to drought-induced death in the tree Trema orientalis.

Drought-induced tree death has become a serious problem in global forest ecosystems. Two nonexclusive hypotheses, hydraulic failure and carbon starvation, have been proposed to explain tree die-offs. To clarify the mechanisms, we investigated the physiological processes of drought-induced tree death in saplings with contrasting Huber values (sapwood area/total leaf area). First, hydraulic failure and reduced respiration were found in the initial process of tree decline, and in the last stage carbon starvation led to tree death. The carbohydrate reserves at the stem bases, low in healthy trees, accumulated at the beginning of the declining process due to phloem transport failure, and then decreased just before dying. The concentrations of non-structural carbohydrates at the stem bases are a good indicator of tree damage. The physiological processes and carbon sink-source dynamics that occur during lethal drought provide important insights into the adaptive measures underlying forest die-offs under global warming conditions.

The impact of prolonged drought on phloem anatomy and phloem transport in young beech trees.

Phloem failure has recently been recognized as one of the mechanisms causing tree mortality under drought, though direct evidence is still lacking. We combined 13C pulse-labelling of 8-year-old beech trees (Fagus sylvatica L.) growing outdoors in a nursery with an anatomical study of the phloem tissue in their stems to examine how drought alters carbon transport and phloem transport capacity. For the six trees under drought, predawn leaf water potential ranged from -0.7 to -2.4 MPa, compared with an average of -0.2 MPa in five control trees with no water stress. We also observed a longer residence time of excess 13C in the foliage and the phloem sap in trees under drought compared with controls. Compared with controls, excess 13C in trunk respiration peaked later in trees under moderate drought conditions and showed no decline even after 4 days under more severe drought conditions. We estimated higher phloem sap viscosity in trees under drought. We also observed much smaller sieve-tube radii in all drought-stressed trees, which led to lower sieve-tube conductivity and lower phloem conductance in the tree stem. We concluded that prolonged drought affected phloem transport capacity through a change in anatomy and that the slowdown of phloem transport under drought likely resulted from a reduced driving force due to lower hydrostatic pressure between the source and sink organs.

Estimation of phloem carbon translocation belowground at stand level in a hinoki cypress stand.

At stand level, carbon translocation in tree stems has to match canopy photosynthesis and carbohydrate requirements to sustain growth and the physiological activities of belowground sinks. This study applied the Hagen-Poiseuille equation to the pressure-flow hypothesis to estimate phloem carbon translocation and evaluate what percentage of canopy photosynthate can be transported belowground in a hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.) stand. An anatomical study revealed that, in contrast to sieve cell density, conductive phloem thickness and sieve cell hydraulic diameter at 1.3 m in height increased with increasing tree diameter, as did the concentration of soluble sugars in the phloem sap. At tree level, hydraulic conductivity increased by two orders of magnitude from the smallest to the largest trees in the stand, resulting in a stand-level hydraulic conductance of 1.7 × 10-15 m Pa-1 s-1. The osmotic potential of the sap extracted from the inner bark was -0.75 MPa. Assuming that phloem water potential equalled foliage water potential at predawn, the turgor pressure in the phloem at 1.3 m in height was estimated at 0.22 MPa, 0.59 MPa lower than values estimated in the foliage. With this maximal turgor pressure gradient, which would be lower during day-time when foliage water potential drops, the estimated stand-level rate of carbon translocation was 2.0 gC m-2 day-1 (30% of daily gross canopy photosynthesis), at a time of the year when aboveground growth and related respiration is thought to consume a large fraction of photosynthate, at the expense of belowground activity. Despite relying on some assumptions and approximations, this approach, when coupled with measurements of canopy photosynthesis, may further be used to provide qualitative insight into the seasonal dynamics of belowground carbon allocation.

Reconstruction of root systems in Cryptomeria japonica using root point coordinates and diameters.

MAIN CONCLUSION: We developed simple algorithms for reconstructing tree root system architecture using only the root point coordinate and diameter, which can be systematically obtained without digging up the root systems. Root system architecture (RSA) is strongly related to various root functions of the tree. The aim of this study was to develop a three-dimensional (3D) RSA model using systematically obtained information on root locations and root diameters at the locations. We excavated root systems of Cryptomeria japonica and systematically obtained XYZ coordinates and root diameters using a 10-cm grid. We clarified the patterns of the root point connections and developed a reconstructed root system model. We found that the root diameters farther from the stump centre are smaller. Additionally, we found that the root lengths of the segments running between the base and the connected root point were smaller than those of other root segments, and the inner angle between the base and the stump and between the base and the connected root point was narrower than for the other pairs. The new RSA model developed according to these results had average accuracies of 0.64 and 0.80 for estimates of total volume and length, respectively. The developed model can estimate 3D RSA using only root point data, which can be obtained without digging up root systems. This suggests a wide applicability of this model in root function evaluation.

Seasonal variations drive short-term dynamics and partitioning of recently assimilated carbon in the foliage of adult beech and pine.

13 CO2 pulse-labelling experiments were performed in situ on adult beeches (Fagus sylvatica) and pines (Pinus pinaster) at different phenological stages to study seasonal and interspecific short-term dynamics and partitioning of recently assimilated carbon (C) in leaves. Polar fraction (PF, including soluble sugars, amino acids and organic acids) and starch were purified from foliage sampled during a 10-d chase period. C contents, isotopic compositions and 13 C dynamics parameters were determined in bulk foliage, PF and starch. Decrease in 13 C amount in bulk foliage followed a two-pool exponential model highlighting 13 C partitioning between 'mobile' and 'stable' pools, the relative proportion of the latter being maximal in beech leaves in May. Early in the growing season, new foliage acted as a strong C sink in both species, but although young leaves and needles were already photosynthesizing, the latter were still supplied with previous-year needle photosynthates 2 months after budburst. Mean 13 C residence times (MRT) were minimal in summer, indicating fast photosynthate export to supply perennial organ growth in both species. In late summer, MRT differed between senescing beech leaves and overwintering pine needles. Seasonal variations of 13 C partitioning and dynamics in field-grown tree foliage are closely linked to phenological differences between deciduous and evergreen trees.

In situ 13CO2 pulse labelling of field-grown eucalypt trees revealed the effects of potassium nutrition and throughfall exclusion on phloem transport of photosynthetic carbon.

Potassium (K) is an important limiting factor of tree growth, but little is known of the effects of K supply on the long-distance transport of photosynthetic carbon (C) in the phloem and of the interaction between K fertilization and drought. We pulse-labelled 2-year-old Eucalyptus grandis L. trees grown in a field trial combining K fertilization (+K and -K) and throughfall exclusion (+W and -W), and we estimated the velocity of C transfer by comparing time lags between the uptake of (13)CO2 and its recovery in trunk CO2 efflux recorded at different heights. We also analysed the dynamics of the labelled photosynthates recovered in the foliage and in the phloem sap (inner bark extract). The mean residence time of labelled C in the foliage was short (21-31 h). The time series of (13)C in excess in the foliage was affected by the level of fertilization, whereas the effect of throughfall exclusion was not significant. The velocity of C transfer in the trunk (0.20-0.82 m h(-1)) was twice as high in +K trees than in -K trees, with no significant effect of throughfall exclusion except for one +K -W tree labelled in the middle of the drought season that was exposed to a more pronounced water stress (midday leaf water potential of -2.2 MPa). Our results suggest that besides reductions in photosynthetic C supply and in C demand by sink organs, the lower velocity under K deficiency is due to a lower cross-sectional area of the sieve tubes, whereas an increase in phloem sap viscosity is more likely limiting phloem transport under drought. In all treatments, 10 times less (13)C was recovered in inner bark extracts at the bottom of the trunk when compared with the base of the crown, suggesting that a large part of the labelled assimilates has been exported out of the phloem and replaced by unlabelled C. This supports the 'leakage-retrieval mechanism' that may play a role in maintaining the pressure gradient between source and sink organs required to sustain high velocity of phloem transport in tall trees.

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.

Patterns of root respiration rates and morphological traits in 13 tree species in a tropical forest.

The root systems of forest trees are composed of different diameters and heterogeneous physiological traits. However, the pattern of root respiration rates from finer and coarser roots across various tropical species remains unknown. To clarify how respiration is related to the morphological traits of roots, we evaluated specific root respiration and its relationships to mean root diameter (D) of various diameter and root tissue density (RTD; root mass per unit root volume; gcm(-3)) and specific root length (SRL; root length per unit root mass; mg(-1)) of the fine roots among and within 14 trees of 13 species from a primary tropical rainforest in the Pasoh Forest Reserve in Peninsular Malaysia. Coarse root (2-269mm) respiration rates increased with decreasing D, resulting in significant relationships between root respiration and diameter across species. A model based on a radial gradient of respiration rates of coarse roots simulated the exponential decrease in respiration with diameter. The respiration rate of fine roots (<2mm) was much higher and more variable than those of larger diameter roots. For fine roots, the mean respiration rates for each species increased with decreasing D. The respiration rates of fine roots declined markedly with increasing RTD and increased with increasing SRL, which explained a significant portion of the variation in the respiration among the 14 trees from 13 species examined. Our results indicate that coarse root respiration in tree species follows a basic relationship with D across species and that most of the variation in fine root respiration among species is explained by D, RTD and SRL. We found that the relationship between root respiration and morphological traits provides a quantitative basis for separating fine roots from coarse roots and that the pattern holds across different species.

In situ assessment of the velocity of carbon transfer by tracing 13 C in trunk CO2 efflux after pulse labelling: variations among tree species and seasons.

Phloem is the main pathway for transferring photosynthates belowground. In situ(13) C pulse labelling of trees 8-10 m tall was conducted in the field on 10 beech (Fagus sylvatica) trees, six sessile oak (Quercus petraea) trees and 10 maritime pine (Pinus pinaster) trees throughout the growing season. Respired (13) CO2 from trunks was tracked at different heights using tunable diode laser absorption spectrometry to determine time lags and the velocity of carbon transfer (V). The isotope composition of phloem extracts was measured on several occasions after labelling and used to estimate the rate constant of phloem sap outflux (kP ). Pulse labelling together with high-frequency measurement of the isotope composition of trunk CO2 efflux is a promising tool for studying phloem transport in the field. Seasonal variability in V was predicted in pine and oak by bivariate linear regressions with air temperature and soil water content. V differed among the three species consistently with known differences in phloem anatomy between broadleaf and coniferous trees. V increased with tree diameter in oak and beech, reflecting a nonlinear increase in volumetric flow with increasing bark cross-sectional area, which suggests changes in allocation pattern with tree diameter in broadleaf species. Discrepancies between V and kP indicate vertical changes in functional phloem properties.

Tracing of recently assimilated carbon in respiration at high temporal resolution in the field with a tuneable diode laser absorption spectrometer after in situ 13CO2 pulse labelling of 20-year-old beech trees.

The study of the fate of assimilated carbon in respiratory fluxes in the field is needed to resolve the residence and transfer times of carbon in the atmosphere-plant-soil system in forest ecosystems, but it requires high frequency measurements of the isotopic composition of evolved CO2. We developed a closed transparent chamber to label the whole crown of a tree and a labelling system capable of delivering a 3-h pulse of 99% 13CO2 in the field. The isotopic compositions of trunk and soil CO2 effluxes were recorded continuously on two labelled and one control trees by a tuneable diode laser absorption spectrometer during a 2-month chase period following the late summer labelling. The lag times for trunk CO2 effluxes are consistent with a phloem sap velocity of about 1 m h(-1). The isotopic composition (delta13C) of CO2 efflux from the trunk was maximal 2-3 days after labelling and declined thereafter following two exponential decays with a half-life of 2-8 days for the first and a half-life of 15-16 days for the second. The isotopic composition of the soil CO2 efflux was maximal 3-4 days after labelling and the decline was also well fitted with a sum of two exponential functions with a half-life of 3-5 days for the first exponential and a half-life of 16-18 days for the second. The amount of label recovered in CO2 efflux was around 10-15% of the assimilated 13CO2 for soil and 5-13% for trunks. As labelling occurred late in the growing season, substantial allocation to storage is expected.

Fine root morphological traits determine variation in root respiration of Quercus serrata.

Fine root respiration is a significant component of carbon cycling in forest ecosystems. Although fine roots differ functionally from coarse roots, these root types have been distinguished based on arbitrary diameter cut-offs (e.g., 2 or 5 mm). Fine root morphology is directly related to physiological function, but few attempts have been made to understand the relationships between morphology and respiration of fine roots. To examine relationships between respiration rates and morphological traits of fine roots (0.15-1.4 mm in diameter) of mature Quercus serrata Murr., we measured respiration of small fine root segments in the field with a portable closed static chamber system. We found a significant power relationship between mean root diameter and respiration rate. Respiration rates of roots<0.4 mm in mean diameter were high and variable, ranging from 3.8 to 11.3 nmol CO2 g(-1) s(-1), compared with those of larger diameter roots (0.4-1.4 mm), which ranged from 1.8 to 3.0 nmol CO2 g(-1) s(-1). Fine root respiration rate was positively correlated with specific root length (SRL) as well as with root nitrogen (N) concentration. For roots<0.4 mm in diameter, SRL had a wider range (11.3-80.4 m g(-1)) and was more strongly correlated with respiration rate than diameter. Our results indicate that a more detailed classification of fine roots<2.0 mm is needed to represent the heterogeneity of root respiration and to evaluate root biomass and root morphological traits.