Measuring sap flow and stem water content in trees : a critical analysis and development of a new heat pulse method (Sapflow+)

Just as carbon, water is indispensable for plants to develop and grow. A lack of water causes turgor loss in plant cells which prevents further expansion of these cells and the coupled incorporation of carbon sources in the cell wall. This inhibits growth and, if this water scarcity continues, plant dimensions such as the stem diameter will start to decrease. Finally the plant will lose its vital functions and die. Worldwide, sap flow methods are applied to monitor plant water status and validate vegetation models. These methods determine flow direction as well as relative and absolute flow, forming the link between plant water uptake, release and storage. Hence, whether to assess the correct irrigation dose, to monitor forest vitality or to obtain trustworthy modelling results, reliable sap flow measurements are indispensable. The most commonly applied sap flow methods are based on heat dissipation in the sapwood. Within this group, a distinction can be made between those methods determining the total flow per time inside a stem or stem section and those assessing sap flux density, the flow per surface per time. While the former are widely applied in irrigation and other applications necessitating an estimation of total plant water use, the latter are applied to investigate specific hydraulic pathways and processes as they allow to distinguish spatial patterns in sap flow, both axially, radially and azimuthally. In this PhD study, the accuracy and applicability of the most important sap flux density methods were investigated. To this end, the underlying thermodynamic theory was studied, Finite Element Modelling (FEM) conducted and lab experiments on cut tree stem segments were undertaken, complemented with a field study on Avicennia marina (Forssk.) Vierh. and Rhizophora stylosa Griff. By investigating the thermodynamic interpretation of the thermal diffusivity as sapwood property, it became clear that the link between the Heat Field Deformation (HFD) temperature ratio and sap flux density, based on this thermal diffusivity, was incorrect. It was concluded that therefore, the continuous HFD method should be considered merely empirical, similar to the Thermal Dissipation method. Moreover, based on FEM, an improved empirical correlation between the HFD temperature ratio and sap flux density was proposed. Also for the methods based on the application of heat pulses, a flaw in the basic theory was noted. These methods are based on the isotropic heat conduction-convection equation for an ideal heater in an infinite medium. Sapwood, however, is known to be anisotropic. Fortunately, the Compensation Heat Pulse, Tmax as well as the Heat Ratio method are based on derivations of this basic equation in a way that is independent of the assumption of isotropy. Hence, for these methods the results are still theoretically correct. Nevertheless, attention should be paid to apply the correct anisotropic equation in modelling and method development, as recent examples show that by neglecting anisotropy, errors can be induced. Within the heat pulse sap flux density methods, the Heat Ratio method enables measurements of low and reverse flow, unlike the Compensation Heat Pulse and Tmax method. This method, however, is dependent on accurate estimations of axial thermal sapwood diffusivity. In this PhD, it was shown that in the currently applied method of mixtures to determine this diffusivity, no distinction was made between bound and unbound water, resulting in over- or underestimations of axial thermal diffusivity dependent on the dry sapwood density and sapwood water content. A correction to this method was proposed, differentiating between bound and unbound water based on the fibre saturation point. This correction has the disadvantage that fibre saturation point is a sapwood characteristic that is not measurable in-situ and, hence, has to be estimated based on dry sapwood density. In response to the difficulties encountered when studying the different sap flux density methods, a new method was developed: the Sapflow+ method. This method is based on a curve fitting procedure during which the anisotropic heat conduction-convection equation is directly fitted to measured temperature profiles located both axially and tangentially from the heater. As was shown by the conducted identifiability analysis and the lab experiments on stem segments of Fagus sylvatica L., the Sapflow+ method enables simultaneous measurements of heat velocity, across the entire naturally occurring range, and thermal sapwood properties, from which sap flux density and sapwood water content can be derived. The applicability of the method to determine heat velocity was confirmed in a field experiment on Avicennia marina (Forssk.) Vierh. and Rhizophora stylosa Griff. For the determination of sapwood water content, further validation experiments and possible optimization of the method are needed. Next to providing an opportunity to test the Sapflow+ method in harsh field conditions, the experiments conducted on Avicennia and Rhizophora led to the remarkable finding that both species show a completely different pattern in stem diameter variation, despite being influenced by the same environmental conditions. This led to the hypothesis that endogenous control of stem diameter fluctuations and growth might be much more crucial than previously assumed and could play an important role in plant growth strategies. In conclusion, the presented PhD study has exposed some limitations of and inaccuracies in existing sap flux density methods and has provided an alternative based on correct theoretical principles. This Sapflow+ method is sensitive towards the entire naturally occurring sap flow range and holds the promise of accurately determining sapwood water content

Magnetic resonance imaging of plants: plant water status and drought stress response

This Thesis presents an approach for the study of plant water balance during drought stress, using a combination of in vivo NMR experiments and computer simulations. The ultimate aim is the interpretation of the NMR parameters in terms of physiologically relevant characteristics, such as cell dimensions and membrane permeability. Especially the latter has raised a growing interest in plant science, and up to now the measurement of this parameter in vivo was limited to single cells and short experiment time spans.NMR microscopy of plants yields information on various levels of organisation. The NMR images provide clear anatomical details, which have been used to monitor the response of stem growth rates to osmotic stress. On the tissue and cell levels, the NMR parameters T 2 (transverse spin relaxation time) and D app (apparent diffusion coefficient) provide information on the physical and chemical properties. Correct quantitative values for T 2 and D app are crucial for a useful interpretation. Therefore, Chapter 2 evaluates the accuracy of different fitting procedures.The physical and chemical properties can vary considerably between and within different tissues, cells, and intracellular compartments, resulting in distinctly different relaxation and diffusion characteristics for these compartments. The interpretation of these parameters is not straightforward. A numerical model of restricted diffusion and relaxation behaviour was therefore developed, based on Fick's second law of diffusion (Chapter 3). This model expands previous one-dimensional models to a two-dimensional space, consisting of multiple concentric cylindrical compartments, separated by membranes. Numerical simulation experiments using this model demonstrate the importance of modelling two-dimensional diffusion in relation to the effects of spatial restrictions, and spin exchange between the different compartments.This model has been applied to investigate the effects of diffusive exchange on the transverse spin relaxation times, the apparent diffusion coefficients, and the NMR signal amplitudes of water in plant cells (Chapter 4). For different multi-compartment model systems a Pulsed Field Gradient Multiple Spin Echo (PFG-MSE) experiment was simulated, and intrinsic physiological parameters, i.e. the bulk diffusion constant, the cell radius and the membrane permeability were afterwards extracted using common theoretical models. The results justify the use of these models to interpret the in vivo experiments, since meaningful diffusion constants, cell radii and membrane permeabilities can be extracted for a large range of conditions. This is still true if not all conditions of the theory are known or met, e.g . for intact plants.Chapters 5 and 6 study the effect of mild osmotic stress on maize and pearl millet by in vivo1H NMR microscopy, and water uptake measurements. Single NMR parameter images of (i) the water content, (ii) the transverse relaxation time ( T 2 ) and (iii) the apparent diffusion coefficient ( D app ) were used to follow the water status of the stem apical region during osmotic stress. The results are interpreted using the multi-compartment model (Chapter 4), tailored to suit plant cells. For this particular case, an equation was derived to describe the relation between the observed T 2 , the cell dimensions, the bulk T 2 , and the membrane permeability, based on the Brownstein & Tarr theory. Experimentally determined T 2 values of non-stressed stem tissue are indeed correlated to the cell dimensions, which is in agreement with the derived equation. The T 2 of maize cells is higher than the T 2 of equally sized millet cells, implying that the membrane permeability of the latter is higher.The growth rate was strongly inhibited by mild stress in both species, even though the water uptake was only mildly affected. During stress, there are hardly any changes in water content or T 2 of the stem region of maize. In contrast, the apical tissue of pearl millet showed a ~30% decrease of T 2 within 48 hours of stress, whereas the water content and D app hardly changed. This decrease in T 2 can be caused by a decreasing cell radius, a decreasing bulk T 2 , and/or an increasing membrane permeability for water. To distinguish between these contributions, additional scanning electron microscopy was used, showing no apparent changes in cell size. Transverse spin relaxation measurements of a wide range of sugar solutions showed only very small effects of osmotic adjustment on the bulk T 2 . Together, these results point to an increase in membrane permeability during stress. This conclusion is confirmed by numerical simulations of the plant cell model, which showed that only an increasing membrane permeability yields a similar combination of water content, T 2 , and D app values during stress.Under severe osmotic stress, the effects on the plant water balance are naturally larger (Chapter 7). During stress, no significant changes occurred in the maize stem, though the leaves wilted, and the plant died after two days of stress. Pearl millet showed again changes in T 2 , especially in the secondary shoots, which were more pronounced than during mild stress. Furthermore, the stem tissue shrunk, implying that the cell dimensions changed; the secondary shoots showed far less decrease in water content, however. Despite these changes, the plants recovered once stress was relieved. In the framework of the plant cell model, the decreasing T 2 is interpreted as the result of a combination of decreasing cell size and increasing membrane permeability. The latter can result in a higher tissue conductance, thereby facilitating water re-allocation to young, expanding tissues to prevent irreparable damage.The combination of experimental data and simulations as presented in this Thesis has proven to be an effective tool to link NMR information to physiology (Chapter 8). This approach promises to be of great use to plant science, and to NMR microscopy in general

Stable Isotopes in Tree Rings

This Open Access volume highlights how tree ring stable isotopes have been used to address a range of environmental issues from paleoclimatology to forest management, and anthropogenic impacts on forest growth. It will further evaluate weaknesses and strengths of isotope applications in tree rings. In contrast to older tree ring studies, which predominantly applied a pure statistical approach this book will focus on physiological mechanisms that influence isotopic signals and reflect environmental impacts. Focusing on connections between physiological responses and drivers of isotope variation will also clarify why environmental impacts are not linearly reflected in isotope ratios and tree ring widths. This volume will be of interest to any researcher and educator who uses tree rings (and other organic matter proxies) to reconstruct paleoclimate as well as to understand contemporary functional processes and anthropogenic influences on native ecosystems. The use of stable isotopes in biogeochemical studies has expanded greatly in recent years, making this volume a valuable resource to a growing and vibrant community of researchers

Transpiration, tracheids and tree rings : linking stem water flow and wood formation in high-elevation conifers

Conifers show a biogeographical distribution across a wide range of contrasting environmental conditions, stretching from the Arctic Circle to the equator and Southern Hemisphere. In mountainous ecosystems, conifers can dominate at high elevations with low temperatures severely limiting tree growth and survival. Conifers growing at sites with temperature limiting conditions are highly sensitive to ongoing climatic change, where warmer and drier conditions will impact their growth. Understanding how high-elevation conifers will respond to these changes in climate is critical, as they play a role in regulating terrestrial carbon storage (facilitated by the formation of woody tissue) and water balance (by releasing water to the atmosphere via transpiration). The environmental regulation of wood formation (i.e., tracheid development in conifers), which dictates annual ring-width patterns, is commonly associated with the tree’s photosynthetic activity, while other growth-limiting factors might also be relevant. For example, tree growth requires turgidity in the cambium to exert the pressure necessary for cell expansion, assimilates to lengthen and thicken cell walls, warmth to allow the metabolic reactions to take place, and time for these processes to be completed. Yet, an in-depth study on how important tree hydraulics (i.e., transpiration dynamics) are in regulating “turgor-driven” growth in high elevation forests is lacking. As part of the LOTFOR project, the general objective of this work is to develop a better mechanistic understanding on how tree hydraulics and environmental factors interact in regulating wood formation and shaping tree rings in high-elevation conifer trees. More specifically, the coupling between stem hydrological cycles and structural carbon dynamics is investigated in the context of increasing temperature and water scarcity. This thesis combines multi-annual records of both intra-annual wood formation data and high-resolution hydraulic measurements within a mechanistic growth model to explain inter- and intra-annual tree growth patterns. To simulate the impact from recent climate change on these mechanisms, a space-for-time experimental setting is applied within the Lötschental, located in the Swiss Alps, where we collected data of two commonly occurring conifer species (Larix decidua Mill. and Picea abies Karst. L.) along an elevation/thermal gradient and contrasting wet and dry sites. Additionally, evaluations are performed on existing methodologies for measuring sap flow and handling large wood anatomical datasets. Analysing how climate affects tree growth at high elevations requires measurements on inter- and intra-annual growth, frequently obtained from tree rings and wood formation observations, respectively. In CHAPTER 2 of this thesis, more than 150 years of inter-annual growth dynamics along the elevational gradient (derived from tree rings) are assessed in relation to temperature, precipitation and insect activity. An analysis of the recent forest biomass increment increase, derived from the tree-ring width measurements, indicates that the absence of insect outbreaks (since 1981) has caused an equal or even greater impact on carbon sequestration compared to the observed warmer summer temperatures. The presented analysis reveals the relevance of including such biotic drivers and their interactions with climate in models assessing the future productivity and carbon sink capacity of forests. In CHAPTER 4, using the algorithms presented in CHAPTER 3, we monitored intra-annual tracheid development across an 8 °C thermal gradient including two elevational transects (in the Lötschental and Vosges Mountains in France) to investigate cell enlargement and wall thickening dynamics in relation to environmental conditions. Results show that at colder sites, differentiating tracheids compensate for lower rates of cell enlarging and wall thickening by increasing the cell development time, except for the wall-thickening latewood cells. This compensation allows conifer trees to mitigate the influence of temperature on the final tree-ring structure, with important implications for the ring’s size and functioning. The production of carbohydrates and generation of turgidity in the cambium to initiate growth are tightly linked to the way a tree regulates the flow of water through the soil-plant-atmosphere continuum. For high elevation conifers, the regulation of the stomatal conductance in the leaves is important, as transpiration has to be optimised for minimal water losses during winter and maximum photosynthetic yield during the short vegetative season. Interestingly, sap flow (measured with thermal dissipation probes installed into the water-conducting wood) can be used to derive stomatal conductance, although this application requires proper data processing of raw sap flow measurements to reduce uncertainties. CHAPTER 5 presents a quantification of the uncertainties generated by commonly applied data-processing methods for conifer sap flow measurements. The uncertainty analysis reveals the importance of performing species-specific calibrations of the sap flow probes, determining zero sap flow conditions with environmental measurements, and applying a dampening correction for better estimates of both the variability and absolute values of whole-tree water use. The processed sap flow measurements are used in CHAPTER 6 to address the ability of L. decidua and P. abies in the Lötschental to adjust their conductance response to environmental conditions when growing under persistently colder and drier conditions. The results indicate that the pioneer L. decidua is more plastic in optimizing its conductance response to temperature with increasing elevation, compared to P. abies. Surprisingly, drought sensitive P. abies did not show a stronger downregulation of its stomatal conductance during drought episodes compared to L. decidua. The stronger plasticity of stomatal conductance response to environmental conditions and the higher water-conductance efficiency of L. decidua, compared to P. abies, provides a new insight into how trees differ in water-use strategies and indicates that L. decidua may be well equipped to function under changing future climatic conditions, compared to a climax species such as P. abies. While mechanistic models can now simulate turgor-driven growth and potentially improve current growth predictions, they lack validation on annual timescales. CHAPTER 7 uses the processed intra-daily sap flow together with site-specific environmental measurements in a mechanistic whole-tree model. The simulated growth dynamics show good agreement with the observed inter- and intra-annual growth in high-elevation conifers (obtained from tree-ring width and xylogenesis observations, respectively). Four years of high-resolution measurements on sap flow and diameter variations were used to apply the mechanistic model for L. decidua or P. abies trees growing along the elevational gradient and in contrasting dry and wet sites in the Lötschental. Good agreement was found between the simulated and observed radial stem growth. Growth was unlikely to occur at temperatures below 2 °C (which is above the photosynthetic minimum) or soil water potentials lower than -0.6 MPa. These results suggest that turgor and its environmental drivers are important for regulating radial growth and should be considered when assessing forest productivity under changing environmental conditions. If one message becomes clear from this thesis, it is that elevational transect studies provide crucial insights into the effect of persistent changes in growing season temperature (as induced by climate change) on annual tree growth patterns, wood formation dynamics and tree hydraulics. Furthermore, collecting a large variety of tree physiological measurements is vital for testing and validating the mechanisms that regulate tree growth and forest productivity patterns

Effects of CO2 concentration on photosynthesis, transpiration and production of greenhouse fruit vegetable crops

The effect of the C0 2 concentration of the greenhouse air (C) in the range 200 to 1100 μmol mol -1was investigated in tomato ( Lycopersicon esculentum Mill.), cucumber ( Cucumis sativus L.), sweet pepper ( Capsicum annuum L.) and eggplant ( Solanum melongena L.), grown in greenhouses.The effect of C on canopy net photosynthetic C0 2 assimilation rate (or photosynthesis, P) was expressed by a set of regression equations, relating P to PAR, C and LAI. A rule of thumb ('CO 2 -rule') was derived, approximating the relative increase of P caused by additional C0 2 at a certain C. This C0 2 -rule is: X = (1000/C) 2* 1.5 (X in % per 100 μmol mol -1, and C in μmol mol -1). Two models for canopy photosynthesis were examined by comparing them with the experimental photosynthesis data. No 'midday depression' in P was observed.The effects of C on leaf conductance ( g ) and on rate of crop transpiration ( E ) were investigated. An increase of 100 μmol mol -1in C reduced g by about 3-4% in sweet pepper, tomato and cucumber and by about 11 % in eggplant. The effect of C on E was analyzed by combining the regression equation for g with the Penman-Monteith equation for E . C had only a relatively small effect on E , owing to thermal and hydrological feedback effects. The decoupling of g and E was quantified. No timedependent variation or 'midday depression' in E was observed, and no significant effect of C on average leaf temperature was established.In five experiments, the effect of C on growth and production and on specific features were analyzed: light use efficiency was increased by about 10 to 15% per 100 μmol mol -1increase in C; fruit set of sweet pepper was greatly increased by high C; allocation of biomass to fruits was increased by high C in sweet pepper and cucumber; specific leaf area (SLA) was reduced by 15 to 20% at 150 to 250 μmol mol -1increase in C (except in cucumber); dry matter content (DMC) of vegetative organs slightly increased at high C (also not in cucumber); fruit production (dry weight) was most affected by C in sweet pepper; fresh weight fruit production per unit CO 2 was highest in cucumber; fruit quality was not influenced by C. High C promoted the 'short leaves syndrome' in tomato and 'leaf tip chlorosis' in eggplant, probably related to calcium and boron translocation, respectively. The observed effect of C on production was larger than expected on the basis of the CO 2 -rule. Intermittent CO 2 supply (ICS) could under normal ventilation accomplish only a limited increase in average C, and hence a limited increase in production. No physiological advantages of ICS were revealed