Starch depletion in the xylem and phloem ray parenchyma of grapevine stems under drought.

While nonstructural carbohydrate (NSC) storage can support long-lived woody plants during abiotic stress, the timing and extent of their use are less understood, as are the thresholds for cell mortality as NSCs and water supplies are consumed. Here, we combine physiological and imaging tools to study the response of Vitis riparia to a 6-week experimental drought. We focused on the spatial and temporal dynamics of starch consumption and cell viability in the xylem and phloem of the stem. Starch dynamics were further corroborated with enzymatic starch digestion and X-ray microcomputed tomography imaging. Starch depletion in the stems of droughted plants was detected after 2 weeks and continued over time. We observed distinct differences in starch content and cell viability in the xylem and phloem. By the end of the drought, nearly all the starch was consumed in the phloem ray parenchyma (98 % decrease), and there were almost no metabolically active cells in the phloem. In contrast, less starch was consumed in the xylem ray parenchyma (30 % decrease), and metabolically active cells remained in the ray and vessel-associated parenchyma in the xylem. Our data suggest that the higher proportion of living cells in the phloem and cambium, combined with smaller potential NSC storage area, rapidly depleted starch, which led to cell death. In contrast, the larger cross-sectional area of the xylem ray parenchyma with higher NSC storage and lower metabolically active cell populations depleted starch at a slower pace. Why NSC source-sink relationships between xylem and phloem do not allow for a more uniform depletion of starch in ray parenchyma over time is unclear. Our data help to pinpoint the proximate and ultimate causes of plant death during prolonged drought exposure and highlight the need to consider the influence of within-organ starch dynamics and cell mortality on abiotic stress response.

Pathogen-induced hydraulic decline limits photosynthesis and starch storage in grapevines (Vitis sp.).

Xylella fastidiosa (Xf) is the bacterial pathogen responsible for Pierce's Disease (PD) in grapevine (Vitis vinifera L.) and numerous diseases in agriculturally and ecologically important species. Current theory suggests that localized inoculations via insect feeding lead to bacterial spread through the xylem, reducing water transport capacity, leading to declines in productivity, and ultimately death. Yet, the underlying mechanisms of Xf-induced mortality are not fully understood. In this study, we documented the development of PD symptoms over 12-13 weeks postinoculation. Subsequently assessed photosynthetic capacity, starch storage, and stem hydraulics in four grapevine genotypes (two PD-resistant and two PD-susceptible), comparing those physiological changes to control plants. PD-susceptible genotypes showed a coordinated decline in photosynthesis, starch storage, and stem hydraulics, whereas Xf-inoculation led to no change in starch and stem hydraulics in the PD-resistant genotypes. Together these data support the idea of a link between loss of hydraulic conductivity due to tylosis production with a downstream photosynthetic decline and starch depletion in the PD-susceptible genotypes. Our data support the theory that hydraulic failure and carbon starvation underlie plant mortality resulting from PD.

Ecologically driven selection of nonstructural carbohydrate storage in oak trees.

Leaf habit is a major axis of plant diversity that has consequences for carbon balance since the leaf is the primary site of photosynthesis. Nonstructural carbohydrates (NSCs) produced by photosynthesis can be allocated to storage and serve as a resiliency mechanism to future abiotic and biotic stress. However, how leaf habit affects NSC storage in an evolutionary context has not been shown. Using a comparative physiological framework and an analysis of evolutionary model fitting, we examined if variation in NSC storage is explained by leaf habit. We measured sugar and starch concentrations in 51 oak species (Quercus spp.) growing in a common garden and representing multiple evolutions of three different leaf habits (deciduous, brevideciduous and evergreen). The best fitting evolutionary models indicated that deciduous oak species are evolving towards higher NSC concentrations than their brevideciduous and evergreen relatives. Notably, this was observed for starch (the primary storage molecule) in the stem (a long-term C storage organ). Overall, our work provides insight into the evolutionary drivers of NSC storage and suggests that a deciduous strategy may confer an advantage against stress associated with a changing world. Future work should examine additional clades to further corroborate this idea.

Seasonal fluctuation of nonstructural carbohydrates reveals the metabolic availability of stemwood reserves in temperate trees with contrasting wood anatomy.

Nonstructural carbohydrates (NSCs) play a critical role in plant physiology and metabolism, yet we know little about their distribution within individual organs such as the stem. This leaves many open questions about whether reserves deep in the stem are metabolically active and available to support functional processes. To gain insight into the availability of reserves, we measured radial patterns of NSCs over the course of a year in the stemwood of temperate trees with contrasting wood anatomy (ring porous vs diffuse porous). In a subset of trees, we estimated the mean age of soluble sugars within and between different organs using the radiocarbon (14C) bomb spike approach. First, we found that NSC concentrations were the highest and most seasonally dynamic in the outermost stemwood segments for both ring-porous and diffuse-porous trees. However, while the seasonal fluctuation of NSCs was dampened in deeper stemwood segments for ring-porous trees, it remained high for diffuse-porous trees. These NSC dynamics align with differences in the proportion of functional sapwood and the arrangement of vessels between ring-porous and diffuse-porous trees. Second, radial patterns of 14C in the stemwood showed that sugars became older when moving toward the pith. The same pattern was found in the coarse roots. Finally, when taken together, our results highlight how the radial distribution and age of NSCs relate to wood anatomy and suggest that while deeper, and likely older, reserves in the stemwood fluctuated across the seasons, the deepest reserves at the center of the stem were not used to support tree metabolism under usual environmental conditions.

Carbon isotopic tracing of sugars throughout whole-trees exposed to climate warming.

Trees allocate C from sources to sinks by way of a series of processes involving carbohydrate transport and utilization. Yet these dynamics are not well characterized in trees, and it is unclear how these dynamics will respond to a warmer world. Here, we conducted a warming and pulse-chase experiment on Eucalyptus parramattensis growing in a whole-tree chamber system to test whether warming impacts carbon allocation by increasing the speed of carbohydrate dynamics. We pulse-labelled large (6-m tall) trees with 13 C-CO2 to follow recently fixed C through different organs by using compound-specific isotope analysis of sugars. We then compared concentrations and mean residence times of individual sugars between ambient and warmed (+3°C) treatments. Trees dynamically allocated 13 C-labelled sugars throughout the aboveground-belowground continuum. We did not, however, find a significant treatment effect on C dynamics, as sugar concentrations and mean residence times were not altered by warming. From the canopy to the root system, 13 C enrichment of sugars decreased, and mean residence times increased, reflecting dilution and mixing of recent photoassimilates with older reserves along the transport pathway. Our results suggest that a locally endemic eucalypt was seemingly able to adjust its physiology to warming representative of future temperature predictions for Australia.

Climate warming and tree carbon use efficiency in a whole-tree 13 CO2 tracer study.

Autotrophic respiration is a major driver of the global C cycle and may contribute a positive climate warming feedback through increased atmospheric concentrations of CO2 . The extent of this feedback depends on plants' ability to acclimate respiration to maintain a constant carbon use efficiency (CUE). We quantified respiratory partitioning of gross primary production (GPP) and CUE of field-grown trees in a long-term warming experiment (+3°C). We delivered a 13 C-CO2 pulse to whole tree crowns and chased that pulse in the respiration of leaves, whole crowns, roots, and soil. We also measured the isotopic composition of soil microbial biomass and the respiration rates of leaves and whole crowns. We documented homeostatic respiratory acclimation of foliar and whole-crown respiration rates; the trees adjusted to experimental warming such that leaf-level respiration rates were not increased. Experimental warming had no detectable impact on respiratory partitioning or mean residence times. Of the 13 C label acquired by the trees, aboveground respiration consumed 10%, belowground respiration consumed 40%, and the remaining 50% was retained. Experimental warming of +3°C did not alter respiratory partitioning at the scale of entire trees, suggesting that complete acclimation of respiration to warming is likely to dampen a positive climate warming feedback.

Whole-tree nonstructural carbohydrate storage and seasonal dynamics in five temperate species.

Despite the importance of nonstructural carbohydrates (NSC) for growth and survival in woody plants, we know little about whole-tree NSC storage. The conventional theory suggests that NSC reserves will increase over the growing season and decrease over the dormant season. Here, we compare storage in five temperate tree species to determine the size and seasonal fluctuation of whole-tree total NSC pools as well as the contribution of individual organs. NSC concentrations in the branches, stemwood, and roots of 24 trees were measured across 12 months. We then scaled up concentrations to the whole-tree and ecosystem levels using allometric equations and forest stand inventory data. While whole-tree total NSC pools followed the conventional theory, sugar pools peaked in the dormant season and starch pools in the growing season. Seasonal depletion of total NSCs was minimal at the whole-tree level, but substantial at the organ level, particularly in branches. Surprisingly, roots were not the major storage organ as branches stored comparable amounts of starch throughout the year, and root reserves were not used to support springtime growth. Scaling up NSC concentrations to the ecosystem level, we find that commonly used, process-based ecosystem and land surface models all overpredict NSC storage.

Standardized protocols and procedures can precisely and accurately quantify non-structural carbohydrates.

Non-structural carbohydrates (NSCs), the stored products of photosynthesis, building blocks for growth and fuel for respiration, are central to plant metabolism, but their measurement is challenging. Differences in methods and procedures among laboratories can cause results to vary widely, limiting our ability to integrate and generalize patterns in plant carbon balance among studies. A recent assessment found that NSC concentrations measured for a common set of samples can vary by an order of magnitude, but sources for this variability were unclear. We measured a common set of nine plant material types, and two synthetic samples with known NSC concentrations, using a common protocol for sugar extraction and starch digestion, and three different sugar quantification methods (ion chromatography, enzyme, acid) in six laboratories. We also tested how sample handling, extraction solvent and centralizing parts of the procedure in one laboratory affected results. Non-structural carbohydrate concentrations measured for synthetic samples were within about 11.5% of known values for all three methods. However, differences among quantification methods were the largest source of variation in NSC measurements for natural plant samples because the three methods quantify different NSCs. The enzyme method quantified only glucose, fructose and sucrose, with ion chromatography we additionally quantified galactose, while the acid method quantified a large range of mono- and oligosaccharides. For some natural samples, sugars quantified with the acid method were two to five times higher than with other methods, demonstrating that trees allocate carbon to a range of sugar molecules. Sample handling had little effect on measurements, while ethanol sugar extraction improved accuracy over water extraction. Our results demonstrate that reasonable accuracy of NSC measurements can be achieved when different methods are used, as long as protocols are robust and standardized. Thus, we provide detailed protocols for the extraction, digestion and quantification of NSCs in plant samples, which should improve the comparability of NSC measurements among laboratories.

Ecosystem warming extends vegetation activity but heightens vulnerability to cold temperatures.

Shifts in vegetation phenology are a key example of the biological effects of climate change1-3. However, there is substantial uncertainty about whether these temperature-driven trends will continue, or whether other factors-for example, photoperiod-will become more important as warming exceeds the bounds of historical variability4,5. Here we use phenological transition dates derived from digital repeat photography6 to show that experimental whole-ecosystem warming treatments7 of up to +9 °C linearly correlate with a delayed autumn green-down and advanced spring green-up of the dominant woody species in a boreal Picea-Sphagnum bog. Results were confirmed by direct observation of both vegetative and reproductive phenology of these and other bog plant species, and by multiple years of observations. There was little evidence that the observed responses were constrained by photoperiod. Our results indicate a likely extension of the period of vegetation activity by 1-2 weeks under a 'CO2 stabilization' climate scenario (+2.6 ± 0.7 °C), and 3-6 weeks under a 'high-CO2 emission' scenario (+5.9 ± 1.1 °C), by the end of the twenty-first century. We also observed severe tissue mortality in the warmest enclosures after a severe spring frost event. Failure to cue to photoperiod resulted in precocious green-up and a premature loss of frost hardiness8, which suggests that vulnerability to spring frost damage will increase in a warmer world9,10. Vegetation strategies that have evolved to balance tradeoffs associated with phenological temperature tracking may be optimal under historical climates, but these strategies may not be optimized for future climate regimes. These in situ experimental results are of particular importance because boreal forests have both a circumpolar distribution and a key role in the global carbon cycle11.

Detours on the phloem sugar highway: stem carbon storage and remobilization.

For trees to survive, they must allocate resources between sources and sinks to maintain proper function. The vertical transport pathway in tree stems is essential for carbohydrates and other solutes to move between the canopy and the root system. To date, research and models emphasize the role of tree stems as 'express' sugar highways. However, recent investigations using isotopic markers suggest that there is considerable storage and exchange of phloem-transported sugars with older carbon (C) reserves within the stem. Thus, we suggest that stems play an important role not only in long-distance transport, but also in the regulation of the tree's overall C balance. A quantitative partitioning of stem C inputs among storage and sinks, including tissue growth, respiration, and export to roots, is still lacking. Combining methods to better quantify the dynamics and controls of C storage and remobilization in the stem will help to resolve central questions of allocation and C balance in trees.

Energy conserving thermoregulatory patterns and lower disease severity in a bat resistant to the impacts of white-nose syndrome.

The devastating bat fungal disease, white-nose syndrome (WNS), does not appear to affect all species equally. To experimentally determine susceptibility differences between species, we exposed hibernating naïve little brown myotis (Myotis lucifugus) and big brown bats (Eptesicus fuscus) to the fungus that causes WNS, Pseudogymnoascus destructans (Pd). After hibernating under identical conditions, Pd lesions were significantly more prevalent and more severe in little brown myotis. This species difference in pathology correlates with susceptibility to WNS in the wild and suggests that survival is related to different host physiological responses. We observed another fungal infection, associated with neutrophilic inflammation, that was equally present in all bats. This suggests that both species are capable of generating a response to cold tolerant fungi and that Pd may have evolved mechanisms for evading host responses that are effective in at least some bat species. These host-pathogen interactions are likely mediated not just by host physiological responses, but also by host behavior. Pd-exposed big brown bats, the less affected species, spent more time in torpor than did control animals, while little brown myotis did not exhibit this change. This differential thermoregulatory response to Pd infection by big brown bat hosts may allow for a more effective (or less pathological) immune response to tissue invasion.

Distribution and mixing of old and new nonstructural carbon in two temperate trees.

We know surprisingly little about whole-tree nonstructural carbon (NSC; primarily sugars and starch) budgets. Even less well understood is the mixing between recent photosynthetic assimilates (new NSC) and previously stored reserves. And, NSC turnover times are poorly constrained. We characterized the distribution of NSC in the stemwood, branches, and roots of two temperate trees, and we used the continuous label offered by the radiocarbon (carbon-14, (14) C) bomb spike to estimate the mean age of NSC in different tissues. NSC in branches and the outermost stemwood growth rings had the (14) C signature of the current growing season. However, NSC in older aboveground and belowground tissues was enriched in (14) C, indicating that it was produced from older assimilates. Radial patterns of (14) C in stemwood NSC showed strong mixing of NSC across the youngest growth rings, with limited 'mixing in' of younger NSC to older rings. Sugars in the outermost five growth rings, accounting for two-thirds of the stemwood pool, had a mean age < 1 yr, whereas sugars in older growth rings had a mean age > 5 yr. Our results are thus consistent with a previously-hypothesized two-pool ('fast' and 'slow' cycling NSC) model structure. These pools appear to be physically distinct.