Root exudation of carbon and nitrogen compounds varies over the day-night cycle in pea: The role of diurnal changes in internal pools.

Rhizodeposition is the export of organic compounds from plant roots to the soil. Carbon allocation towards rhizodeposition has to be balanced with allocation for other physiological functions, which depend on both newly assimilated and stored nonstructural carbohydrate (NSC). To test whether the exudation of primary metabolites scales with plant NSC status, we studied diurnal dynamics of NSC and amino acid (AA) pools and fluxes within the plant and the rhizosphere. These diurnal dynamics were measured in the field and under hydroponic-controlled conditions. Further, C-limiting treatments offered further insight into the regulation of rhizodeposition. The exudation of primary metabolites fluctuated diurnally. The diurnal dynamics of soluble sugars (SS) and AA concentrations in tissues coincided with exudate pool fluctuations in the rhizosphere. SS and AA pools in the rhizosphere increased with NSC and AA pools in the roots. C starvation treatments offset the balance of exudates: AA exudate content in the rhizosphere significantly decreased while SS exudate content remained stable. Our results suggest that rhizodeposition is to some extent controlled by plant C:N status. We propose that SS exudation is less controlled than AA exudation because N assimilation depends on controlled C supply while SS exudation relies to a greater extent on passive diffusion mechanisms.

Comparison of phenological traits, growth patterns, and seasonal dynamics of non-structural carbohydrate in Mediterranean tree crop species.

Despite non-structural carbohydrate (NSC) importance for tree productivity and resilience, little is known about their seasonal regulations and trade-off with growth and reproduction. We characterize the seasonal dynamics of NSC in relation to the aboveground phenology and temporal growth patterns of three deciduous Mediterranean species: almond (Prunus dulcis (Mill.) D. A. Webb), walnut (Juglans regia L.) and pistachio (Pistacia vera L.). Seasonal dynamics of NSC were synchronous between wood tissues from trunk, branches and twigs. Almond had almost identical levels and patterns of NSC variation in twigs, branches and trunks whereas pistachio and walnut exhibited clear concentration differences among plant parts whereby twigs had the highest and most variable NSC concentration, followed by branches and then trunk. While phenology had a significant influence on NSC seasonal trends, there was no clear trade-off between NSC storage and growth suggesting that both were similarly strong sinks for NSC. A temporal trade-off observed at the seasonal scale was influenced by the phenology of the species. We propose that late senescing species experience C allocation trade-off at the end of the growing season because of C-limiting thermal conditions and priority allocation to storage in order to survive winter.

Sodium interception by xylem parenchyma and chloride recirculation in phloem may augment exclusion in the salt tolerant Pistacia genus: context for salinity studies on tree crops.

Working in tandem with root exclusion, stems may provide salt-tolerant woody perennials with some additional capacity to restrict sodium (Na) and chloride (Cl) accumulation in leaves. The Pistacia genus, falling at the nexus of salt tolerance and human intervention, provided an ideal set of organisms for studying the influences of both variable root exclusion and potentially variable discontinuities at the bud union on stem processes. In three experiments covering a wide range of salt concentrations (0 to 150 mM NaCl) and tree ages (1, 2 and 10 years) as well as nine rootstock-scion combinations we show that proportional exclusion of both Na and Cl reached up to ~85% efficacy, but efficacy varied by both rootstock and budding treatment. Effective Na exclusion was augmented by significant retrieval of Na from the xylem sap, as evidenced by declines in the Na concentrations of both sap and wood tissue along the transpiration stream. However, while we observed little to no differences between the concentrations of the two ions in leaves, analogous declines in sap concentrations of Cl were not observed. We conclude that some parallel but separate mechanism must be acting on Cl to provide leaf protection from toxicity specific to this ion and suggest that this mechanism is recirculation of Cl in the phloem. The presented findings underline the importance of holistic assessments of salt tolerance in woody perennials. In particular, greater emphasis might be placed on the dynamics of salt sequestration in the significant storage volumes offered by the stems of woody perennials and on the potential for phloem discontinuity introduced with a bud/graft union.

Diurnal Variation in Nonstructural Carbohydrate Storage in Trees: Remobilization and Vertical Mixing.

Nonstructural carbohydrate (NSC) storage plays a critical role in tree function and survival, but understanding and predicting local NSC storage dynamics is challenging because NSC storage pools are dispersed throughout the complex architecture of trees and continuously exchange carbon between source and sink organs at different time scales. To address these knowledge gaps, characterization and understanding of NSC diel variation are necessary. Here, we analyzed diurnal NSC dynamics in the overall architecture of almond (Prunus dulcis) trees. We also analyzed the allocation of newly assimilated carbon using isotopic labeling. We show that both components of NSC (i.e. soluble carbohydrates and starch) are highly dynamic at the diurnal time scale and that these trends are influenced by tissue type, age, and/or position within the canopy. In leaves, starch reserves can be depleted completely during the night, while woody tissue starch levels may vary by more than 50% over a daily cycle. Recently assimilated carbon showed a dispersed downward allocation across the entire tree. NSC diurnal fluctuations within the tree's structure in combination with dispersed carbon allocation patterns provide evidence for the presence of vertical mixing and suggest that the xylem acts as a secondary NSC redistribution pathway.

In vivo quantification of plant starch reserves at micrometer resolution using X-ray microCT imaging and machine learning.

Starch is the primary energy storage molecule used by most terrestrial plants to fuel respiration and growth during periods of limited to no photosynthesis, and its depletion can drive plant mortality. Destructive techniques at coarse spatial scales exist to quantify starch, but these techniques face methodological challenges that can lead to uncertainty about the lability of tissue-specific starch pools and their role in plant survival. Here, we demonstrate how X-ray microcomputed tomography (microCT) and a machine learning algorithm can be coupled to quantify plant starch content in vivo, repeatedly and nondestructively over time in grapevine stems (Vitis spp.). Starch content estimated for xylem axial and ray parenchyma cells from microCT images was correlated strongly with enzymatically measured bulk-tissue starch concentration on the same stems. After validating our machine learning algorithm, we then characterized the spatial distribution of starch concentration in living stems at micrometer resolution, and identified starch depletion in live plants under experimental conditions designed to halt photosynthesis and starch production, initiating the drawdown of stored starch pools. Using X-ray microCT technology for in vivo starch monitoring should enable novel research directed at resolving the spatial and temporal patterns of starch accumulation and depletion in woody plant species.

PtxtPME1 and homogalacturonans influence xylem hydraulic properties in poplar.

While the xylem hydraulic properties, such as vulnerability to cavitation (VC), are of paramount importance in drought resistance, their genetic determinants remain unexplored. There is evidence that pectins and their methylation pattern are involved, but the detail of their involvement and the corresponding genes need to be clarified. We analyzed the hydraulic properties of the 35S::PME1 transgenic aspen that ectopically under- or over-express a xylem-abundant pectin methyl esterase, PtxtPME1. We also produced and analyzed 4CL1::PGII transgenic poplars expressing a fungal polygalacturonase, AnPGII, under the control of the Ptxa4CL1 promoter that is active in the developing xylem after xylem cell expansion. Both the 35S::PME1 under- and over-expressing aspen lines developed xylem with lower-specific hydraulic conductivity and lower VC, while the 4CL1::PGII plants developed xylem with a higher VC. These xylem hydraulic changes were associated with modifications in xylem structure or in intervessel pit structure that can result in changes in mechanical behavior of the pit membrane. This study shows that homogalacturonans and their methylation pattern influence xylem hydraulic properties, through its effect on xylem cell expansion and on intervessel pit properties and it show a role for PtxtPME1 in the xylem hydraulic properties.

Temperature gradients assist carbohydrate allocation within trees.

Trees experience two distinct environments: thermally-variable air and thermally-buffered soil. This generates intra-tree temperature gradients, which can affect carbon metabolism and water transport. In this study, we investigated whether carbohydrate allocation within trees is assisted by temperature gradients. We studied pistachio (Pistacia integerrima) to determine: (1) temperature-induced variation in xylem sugar concentration in excised branches; (2) changes in carbon allocation in young trees under simulated spring and fall conditions; and (3) seasonal variability of starch levels in mature orchard trees under field conditions. We found that warm branches had less sugar in perfused sap than cold branches due to increasing parenchyma storage. Simulated spring conditions promoted allocation of carbohydrates from cold roots to warm canopy and explained why starch levels surged in canopies of orchard trees during early spring. This driving force of sugar transport is interrupted in fall when canopies are colder than roots and carbohydrate redistribution is compartmentalized. On the basis of these findings, we propose a new mechanistic model of temperature-assisted carbohydrate allocation that links environmental cues and tree phenology. This data-enabled model provides insights into thermal "fine-tuning" of carbohydrate metabolism and a warning that the physiological performance of trees might be impaired by climatic changes.

Spring bud growth depends on sugar delivery by xylem and water recirculation by phloem Münch flow in Juglans regia.

MAIN CONCLUSION: During spring, bud growth relies on long-distance transport of remotely stored carbohydrates. A new hypothesis suggests this transport is achieved by the interplay of xylem and phloem. During the spring, carbohydrate demand of developing buds often exceeds locally available storage, thus requiring the translocation of sugars from distant locations like limbs, stems and roots. Both the phloem and xylem have the capacity for such long-distance transport, but their functional contribution is unclear. To address this ambiguity, the spatial and temporal dynamics of carbohydrate availability in extension shoots of Juglans regia L. were analyzed. A significant loss of extension shoot carbohydrates in remote locations was observed while carbohydrate availability near the buds remained unaffected. This pattern of depletion of carbohydrate reserves supports the notion of long-distance translocation. Girdling and dye perfusion experiments were performed to assess the role of phloem and xylem in the transport of carbohydrate and water towards the buds. Girdling caused a decrease in non-structural carbohydrate concentration above the point of girdling and an unexpected concurrent increase in water content associated with impeded xylem transport. Based on experimental observations and modeling, we propose a novel mechanism for maintenance of spring carbohydrate translocation in trees where xylem transports carbohydrates and this transport is maintained with the recirculation of water by phloem Münch flow. Phloem Münch flow acts as a pump for generating water flux in xylem and allows for transport and mobilization of sugars from distal locations prior to leaves photosynthetic independence and in the absence of transpiration.

Modelling the mechanical behaviour of pit membranes in bordered pits with respect to cavitation resistance in angiosperms.

BACKGROUND AND AIMS: Various correlations have been identified between anatomical features of bordered pits in angiosperm xylem and vulnerability to cavitation, suggesting that the mechanical behaviour of the pits may play a role. Theoretical modelling of the membrane behaviour has been undertaken, but it requires input of parameters at the nanoscale level. However, to date, no experimental data have indicated clearly that pit membranes experience strain at high levels during cavitation events. METHODS: Transmission electron microscopy (TEM) was used in order to quantify the pit micromorphology of four tree species that show contrasting differences in vulnerability to cavitation, namely Sorbus aria, Carpinus betulus, Fagus sylvatica and Populus tremula. This allowed anatomical characters to be included in a mechanical model that was based on the Kirchhoff-Love thin plate theory. A mechanistic model was developed that included the geometric features of the pits that could be measured, with the purpose of evaluating the pit membrane strain that results from a pressure difference being applied across the membrane. This approach allowed an assessment to be made of the impact of the geometry of a pit on its mechanical behaviour, and provided an estimate of the impact on air-seeding resistance. KEY RESULTS: The TEM observations showed evidence of residual strains on the pit membranes, thus demonstrating that this membrane may experience a large degree of strain during cavitation. The mechanical modelling revealed the interspecific variability of the strains experienced by the pit membrane, which varied according to the pit geometry and the pressure experienced. The modelling output combined with the TEM observations suggests that cavitation occurs after the pit membrane has been deflected against the pit border. Interspecific variability of the strains experienced was correlated with vulnerability to cavitation. Assuming that air-seeding occurs at a given pit membrane strain, the pressure predicted by the model to achieve this mechanical state corresponds to experimental values of cavitation sensitivity (P50). CONCLUSIONS: The results provide a functional understanding of the importance of pit geometry and pit membrane structure in air-seeding, and thus in vulnerability to cavitation.

Hydrolase treatments help unravel the function of intervessel pits in xylem hydraulics.

Intervessel pits are structures that play a key role in the efficiency and safety functions of xylem hydraulics. However, little is known about the components of the pit membrane (PM) and their role in hydraulic functions, especially in resistance to cavitation. We tested the effect of commercial chemicals including a cellulase, a hemicellulase, a pectolyase, a proteinase and DTT on xylem hydraulic properties: vulnerability to cavitation (VC) and conductance. The effects were tested on branch segments from Fagus sylvatica (where the effects on pit structure were analyzed using TEM) and Populus tremula. Cellulose hydrolysis resulted in a sharp increase in VC and a significant increase in conductance, related to complete breakdown of the PM. Pectin hydrolysis also induced a sharp increase in VC but with no effect on conductance or pit structure observable by TEM. The other treatments with hemicellulase, proteinase or DTT showed no effect. This study brings evidence that cellulose and pectins are critical components underpinning VC, and that PM components may play distinct roles in the xylem hydraulic safety and efficiency.

Growth and molecular responses to long-distance stimuli in poplars: bending vs flame wounding.

Inter-organ communication is essential for plants to coordinate development and acclimate to mechanical environmental fluctuations. The aim of this study was to investigate long-distance signaling in trees. We compared on young poplars the short-term effects of local flame wounding and of local stem bending for two distal responses: (1) stem primary growth and (2) the expression of mechanoresponsive genes in stem apices. We developed a non-contact measurement method based on the analysis of apex images in order to measure the primary growth of poplars. The results showed a phased stem elongation with alternating nocturnal circumnutation phases and diurnal growth arrest phases in Populus tremula × alba clone INRA 717-1B4. We applied real-time polymerase chain reaction (RT-PCR) amplifications in order to evaluate the PtaZFP2, PtaTCH2, PtaTCH4, PtaACS6 and PtaJAZ5 expressions. The flame wounding inhibited primary growth and triggered remote molecular responses. Flame wounding induced significant changes in stem elongation phases, coupled with inhibition of circumnutation. However, the circadian rhythm of phases remained unaltered and the treated plants were always phased with control plants during the days following the stress. For bent plants, the stimulated region of the stem showed an increased PtaJAZ5 expression, suggesting the jasmonates may be involved in local responses to bending. No significant remote responses to bending were observed.

Arabidopsis thaliana as a model species for xylem hydraulics: does size matter?

While Arabidopsis thaliana has been proposed as a model species for wood development, the potential of this tiny herb for studying xylem hydraulics remains unexplored and anticipated by scepticism. Inflorescence stems of A. thaliana were used to measure hydraulic conductivity and cavitation resistance, whereas light and electron microscopy allowed observations of vessels. In wild-type plants, measured and theoretical conductivity showed a significant correlation (R (2) = 0.80, P < 0.01). Moreover, scaling of vessel dimensions and intervessel pit structure of A. thaliana were consistent with structure-function relationships of woody plants. The reliability and resolution of the hydraulic methods applied to measure vulnerability to cavitation were addressed by comparing plants grown under different photoperiods or different mutant lines. Sigmoid vulnerability curves of A. thaliana indicated a pressure corresponding to 50% loss of hydraulic conductance (P 50) between -3 and -2.5MPa for short-day and long-day plants, respectively. Polygalacturonase mutants showed a higher P 50 value (-2.25MPa), suggesting a role for pectins in vulnerability to cavitation. The application of A. thaliana as a model species for xylem hydraulics provides exciting possibilities for (1) exploring the molecular basis of xylem anatomical features and (2) understanding genetic mechanisms behind xylem functional traits such as cavitation resistance. Compared to perennial woody species, however, the lesser amount of xylem in A. thaliana has its limitations.

Embolism resistance as a key mechanism to understand adaptive plant strategies.

One adaptation of plants to cope with drought or frost stress is to develop wood that is able to withstand the formation and distribution of air bubbles (emboli) in its water conducting xylem cells under negative pressure. The ultrastructure of interconduit pits strongly affects drought-induced embolism resistance, but also mechanical properties of the xylem are involved. The first experimental evidence for a lower embolism resistance in stems of herbaceous plants compared to stems of their secondarily woody descendants further supports this mechanical-functional trade-off. An integrative approach combining (ultra)structural observations of the xylem, safety-efficiency aspects of the hydraulic pipeline, and xylem-phloem interactions will shed more light on the multiple adaptive strategies of embolism resistance in plants.