Tree Physiology

A long-established ecological paradigm predicts a functional relationship determining vulnerability to cavitation: vulnerability increases with vessel hydraulic efficiency and vessel diameter. Even within a species, big vessels cavitate before small ones. Some centrifuge methods for measuring vulnerability are prone to artifacts due to nano-particles seeding early embolism, as the particles are drawn into vessels during measurements. Both the Sperry and Cochard rotors are prone to early cavitation due to nano-particles drawn into long and wide vessels in Robinia pseudoacacia and Quercus acutissima, whereas extraction centrifuge methods produce vulnerability curves more resistant to cavitation. Sufficient nano-particles pass through the stems to seed early embolism in all rotor designs. For several years, people have thought that early embolism is induced by nano-particles present in laboratory water. One new hypothesis is that the origin of nano-particles is from cut-open living cells but a much bigger study including many species is required to confirm this idea. This paper confirms the hypothesis in comparisons between short-vesselled Acer, and long-vesselled Robinia, and Quercus. Our new results and a review of old results justifies bigger study. Hypothetical nano-particles might explain why different methods for measuring vulnerability curves cause different T50 = tensions causing 50% loss of hydraulic conductivity. Hence the hypothesis for future research should be that the open-vessel artifact is consistent with long vessels surrounded by cut open living cells. One sentence SummaryNano-particles induced early cavitation in species with vessel lengths about [1/4] the stem length used in all centrifuge rotors, and the origin of nano-particles might be from living cells nearby vessels

O_LIIsohydric species reduce water potential fluctuations through more stringent stomatal regulation. It is unclear whether isohydric behavior leads to smaller reversible diurnal stem shrinkage (i.e. greater nocturnal stem rehydration) during drought and a lower drought sensitivity of radial growth compared to anisohydric behavior. C_LIO_LIWith synchronous high-resolution sap flow and dendrometer measurements in mature anisohydric European beech and isohydric Douglas fir trees in pure and mixed stands, we quantified declines in stem water content (SWC), stem rehydration, sap flow and radial growth during soil dry-down and determined the critical soil moisture levels (expressed as Relative Extractable Water, REW) and elapsed desiccation time until 30-90% reductions in these traits. C_LIO_LISap flow and growth started to decline in both species at REW [~]0.6, preceding declines in stem rehydration. Water-spending Douglas fir approached 50% drops in SWC, sap flow, stem rehydration and growth during soil dry-down faster than beech, indicating higher drought sensitivity. In mixture, both species reached these reduction levels later than in monoculture, suggesting positive mixing effects on the species drought resistance. C_LIO_LIOur findings demonstrate that SWC, sap flow and radial growth decrease earlier than nocturnal stem rehydration, with isohydric and anisohydric species exhibiting different timelines of physiological downregulation during soil dry-down. C_LI

Understanding xylem embolism formation is challenging due to dynamic changes and multiphase interactions in conduits. If embolism spread involves gas movement in xylem, we hypothesise that it is affected by time. We measured hydraulic conductivity (Kh) in flow-centrifuge experiments over one hour at a given pressure and temperature for stem samples of three angiosperm species. Temporal changes in Kh at 5, 22, and 35{degrees}C, and at various pressures were compared to modelled gas pressure changes in a recently embolised vessel in the centre of a centrifuge sample. Temporal changes in Kh at 22{degrees}C showed maximum relative increases between 6% and 40%, and maximum decreases between 41% and 61% at low and high centrifugal speed, respectively. Logarithmic changes in Kh were species-specific, and most pronounced during the first 15 minutes. Embolism formation started near the edges of centrifuge samples and gradually increased at the centre. Moreover, measured decreases in Kh strongly correlated with modelled increases in gas concentration in a recently embolised vessel. Although embolism is mostly pressure-driven, our experimental and modelled data indicate that time, conduit characteristics, and temperature are involved due to their role in gas diffusion. Gas diffusion, however, does not cover the entire process of embolism spread.

The ability of trees to survive and naturally regenerate in increasing drought conditions will depend on their capacity to vary key hydraulic and morphological traits that increase drought tolerance. Despite many studies investigating variability in these drought-tolerant traits, there has been limited investigation into this variability under recurrent severe drought conditions since the establishment phase. We investigated the long-term hydraulic and leaf trait adjustments of Scots pine trees settled in an abandoned slag quarry by comparing them across three different topographic positions inducing contrasted effects on growth and development. We measured xylem and foliar traits to compare the water status of trees according to tree location and to evaluate the respective risk for xylem hydraulic failure using the soil-plant hydraulic model SurEau. Compared to upslope and downslope trees, slope trees exhibited lower growth, vulnerability to embolism, specific hydraulic conductivity and photosynthetic pigment contents, as well as higher water potential at turgor loss point and midday water potentials. The hydraulic adjustments of trees settled on slag slopes reduced the risk for hydraulic failure and thus prevented an increase in embolism compared to downslope and upslope trees. These results suggest a prioritization of hydraulic safety over growth in Scots pine developed in a harsh environment, resulting in a dwarf phenotype.

Residual water losses after stomatal closure have recently been identified as key determinants of drought-induced hydraulic failure, particularly under heatwave conditions. However, little is known about the intraspecific variability of residual conductance (gres) and its thermal sensitivity. Here, we investigated the genetic and environmental sources of variation in gres and its associated thermal parameters (phase transition temperature T_, and temperature sensitivities Q10a and Q10b) in Abies alba Mill., together with vulnerability to xylem embolism (P50). Measurements were performed using the Drought-Box on seven French provenances grown in a common garden to assess genetic variability, and on trees growing across contrasting forest sites to quantify phenotypic plasticity. Seasonal dynamics and within-canopy microclimatic effects were also examined, and linked to needle biochemical traits. Residual conductance exhibited a marked seasonal decline, with high values in newly formed needles followed by a stabilization from late summer to the following spring, closely tracking the accumulation of cuticular waxes. In contrast, Klason lignin content showed little seasonal variation. Difference between provenances was weak for all investigated parameters, suggesting strong constraints on these safety-related traits. By contrast, gres showed significant environmental plasticity, with lower values at more climatically constrained sites, while thermal parameters and P50 remained relatively conserved. Our results identify gres as a developmentally dynamic and environmentally plastic trait in silver fir, potentially representing a key lever of acclimation to drought. Incorporating such variability into mechanistic models should improve predictions of tree vulnerability under future climates combining intensified droughts and heatwaves. Key message.Residual conductance in Abies alba is developmentally dynamic and environmentally plastic but genetically constrained, highlighting its key role in acclimation to drought and heatwave-driven hydraulic failure.

Wood formation is a crucial process for carbon sequestration, yet how variations in carbon supply affect wood formation and carbon dynamics in trees more generally remains poorly understood. To better understand the role of carbon supply in wood formation, we restricted phloem transport using girdling and compression around the stem of mature white pines and monitored the effects on local wood formation and stem CO2 efflux, as well as nonstructural carbon concentrations in needles, stems, and roots. Growth and stem CO2 efflux varied with location relative to treatment (i.e., above or below on the stem). We observed up to a two-fold difference in the number of tracheids formed above versus below the manipulations over the remaining growing season. In contrast, the treatments did not affect mean cell size noticeably and mean cell-wall area decreased only slightly below them. Surprisingly, nonstructural carbon pools and concentrations in the xylem, needles, and roots remained largely unchanged, although starch reserves declined and increased marginally below and above the girdle, respectively. Our results suggest that phloem transport strongly affects cell proliferation and respiration in the cambial zone of mature white pine, but has little impact on nonstructural carbon concentrations. These findings contribute to our understanding of how wood formation is controlled. HighlightRestrictions in phloem transport designed to affect carbon supply, lead to changes in wood formation and stem respiration of mature white pines without substantially changing local nonstructural carbon concentrations.

Xylem embolism is a major threat to tree function and survival under drought, in natural and agricultural settings alike, with its impact increasing in light of global climate change. Conversely, potassium (K+) has been shown to increase xylem conductivity (Ks) in trees, and carbohydrates were reported to impact leaf gas exchange. In this study we examined the effects of K+ and carbohydrates on Ks in two divergent evergreen tree species that are regularly exposed to drought: pine (Pinus brutia) and lemon (Citrus x limon). Five-year-old trees were pretreated with zero, moderate, and high K+, and with ambient or elevated CO2, to experimentally increase their xylem K+ or carbohydrates levels, respectively. Trees were then monitored for Ks and embolism (using a microCT), along with leaf gas exchange and water potential, before and after a 1.5-2.5 month drought period. Potassium fertigation had a positive effect on Ks, in both species when irrigated, which was eliminated following drought. Drought decreased Ks about 10-fold in lemon, with little effect in pine. CO2-treated trees had the same Ks as control trees before and after drought. Our results indicate a positive effect of K+ on tree hydraulics, which was more pronounced in lemon than in pine, supporting the hypothesis of interaction with the angiosperm pit membrane, and not with the gymnosperm bordered pit. Yet, the elimination of this benefit following drought, and the lack of benefit from elevated carbohydrates following a short-term CO2 treatment, question the relevance of these components to tree drought resistance mechanisms. Key massagePotassium fertigation increases hydraulic conductivity and reduces xylem embolism in the gymnosperm pine, and more so in the angiosperm lemon tree, benefits which were eliminated following drought.

Drought-induced embolism formation in conifers is associated with several tracheid and pit traits, which vary in parallel from stem apex to base. We tested whether this axial anatomical variability is associated with a progressive variation in embolism vulnerability along the stem from apex to base. We assessed the xylem pressure at 50% loss of conductivity (P50), the tracheid hydraulic diameter (Dh) and mean pit membrane area (PMA) on longitudinal stem segments extracted at different distances from the stem apex (DFA) in a Picea abies and an Abies alba tree. In both trees, Dh and PMA scaled with DFA0.2. P50 varied for more than 3 MPa from the treetop to the stem base, according to a scaling of -P50 with DFA-0.2. The largest Dh, PMA and P50 variation occurred for DFA<1.5 m. PMA and Dh scaled isometrically (exponent b=1). Pit traits vary proportionally with tracheid lumen diameter. Apex-to-base trends in tracheid and pit traits determine a large DFA-dependent P50 variability. Such a DFA effect on P50 did not receive sufficient attention so far, although analysing the relationships P50 vs. DFA is fundamental for the assessment of embolism vulnerability at the individual level. HighlightsO_LIConifer embolism vulnerability depends on pit properties, in agreement with published data. C_LIO_LIPit dimensions increase with tracheid lumen diameter, in agreement with published data C_LIO_LITracheid lumen diameter and pit dimensions increase progressively from the stem apex to base, in agreement with published data. C_LIO_LIXylem vulnerability to embolism formation (P50) varies for > 3 MPa from the stem apex to base, with the largest variation occurring within 1.5 m from the stem apex. C_LIO_LIAxial anatomical patterns should be accounted for when analyzing hydraulic properties at individual, intra- and inter-specific scales. C_LI

The dye uptake and heat transfer methods are used to understand sap movement in mature tree stems, but correlation of results between both methods is weak. The circumferential variation of sap movement is detected generally in the heated transfer method. On the other hand, the tangential movement such as helical ascent is tracked in dye uptake method. It is still unclear whether the circumferential and the tangential movements in both methods mean same and/or related phenomenon. In this study, we monitored sap flux density (Fd) on the east and west sides in intact stems of Betula ermanii Cham. using the thermal-dissipation method to identify Fd response to the creation of artificial flows through severed vessels by drill treatments. We then tracked sap flow by injecting dye solution. The Fd values in east side tended to be higher than that in west side before drilling treatment. Similar Fd values in both sides were detected after drilling treatments, and dye heights were similar to Fd values after drilling in any sides. Simple comparison of water ascent using both methods can lead to errors, because water ascent from severed vessels may differ from water ascent via roots.

Non-structural carbon compounds (NSCs) serve to buffer short-term imbalances between carbon supply and demand in trees; however, their seasonal dynamics throughout the entire tree remain inadequately understood. We quantified year-round non-structural carbohydrate storage and fluxes in a temperate pine forest by integrating monthly measurements of soluble sugars, starch, and lipids across five tissues with biometric scaling to ecosystem stocks. Soluble sugars were consistently highest in canopy tissues and maintained a relatively stable concentration, even as sugar fluxes exhibited pronounced seasonal variations and reversals. In contrast, starch showed clear seasonality, increasing during the mid-growing season and decreasing later, whereas lipid pools remained relatively stable and contributed minimally to short-term fluctuations. Ecosystem-scale analyses indicated that sugars predominantly contributed to NSC turnover, accounting for approximately 80% of the total annual flux, while stored pools exhibited slower changes. The net annual NSC flux, approximately 65 g C m-2 yr-1, was relatively modest in comparison to biomass production, which totaled around 522g C m-2 yr -1. These findings indicate that seasonal changes in carbon balance are primarily driven by rapid redistribution of soluble carbon rather than by significant changes in overall NSC storage.

O_LIStemflow of forest trees can contribute a significant fraction of water to forests water fluxes; however, it is still unclear if and to what extent trees use stemflow for water supply and how much stemflow is lost by percolating below the root zone. C_LIO_LIWe applied deuterium-enriched stemflow equivalent to a throughfall depth of 23 mm to adult Fagus sylvatica trees to trace stemflow through the soil and trees. We continuously measured in-situ water stable isotope compositions in soil and xylem water and destructively sampled xylem water in the crowns of adult labelled (n=18), unlabelled F. sylvatica (n=15) and unlabeled Picea abies (n=9), complemented by destructive xylem water sampling of neighboring juvenile F. sylvatica (n=45). C_LIO_LIStemflow water supported 3.9 - 14.0% of daily sap flux of adult labelled F. sylvatica trees. In the soil, deuterium-enriched stemflow was detectable at a max. of 0 - 0.40 m to the labelled tree. However, unlabeled juvenile trees within a distance of [~]2 m showed label water uptake, indicating rooting into soil compartments affected by stemflow label. C_LIO_LIWe demonstrate the importance of stemflow as a water source for both adult and neighboring juvenile F. sylvatica, strongly profiting from stemflow infiltration. C_LI

The shoot system of all loblolly pine (Pinus taeda L.) contains abundant resin ducts, and the oleoresins contained within them have demonstrated roles in constitutive defenses. This study is providing a quantitative assessment of oleoresin biosynthesis and accumulation in resin ducts. Morphometric analyses of representative tissue sections indicate that the fractional volume of resin ducts is particularly high in the cortex of young stems and their needles, representing a major portion of total pine resins from primary growth of the canopy. We demonstrate that it is possible to extrapolate oleoresin formation from the microscopic scale (tissues sections) to the macroscale (entire trees), which has implications for assessing resins as renewable feedstocks for bioproducts.

Mangifera indica is the fifth most consumed fruit worldwide, and the most important in tropical regions, but its anatomy is quite unexplored. Previous studies examined the effect of chemicals on the xylem structure in the stems of mango, but the anatomy of the phloem has remained elusive, leaving the long distance transport of photo assimilates understudied. In this work, we used a combination of fluorescence and electron microscopy to evaluate in detail the structure of the sieve tube elements composing the phloem tissue in the tapering branches of mango trees. We then used this information to better understand the hydraulic conductivity of the sieve tubes following current models of fluid transport in trees. Our results revealed that the anatomy of the phloem in the stems changes from current year branches, where it was protected by pericyclic fibers, to older ones, where the lack of fibers was concomitant with laticiferous canals embedded in the phloem tissue. Callose was present in the sieve plates, but also in the walls of the phloem conduits, making them discernible from other phloem cells in fresh sections. A scaling geometry of the sieve tube elements, including the number of sieve areas and the pore size across tapering branches resulted in an exponential conductivity from current year branches to the base of the tree. Our measurements of the phloem in mango fit with measurements of the phloem architecture in the stems of forest woody species, and imply that, despite agronomic pruning practices, the sieve conduits of the phloem scale with the tapering branches. As a result, the pipe model theory applied to the continuous tubing system of the phloem appears as a good approach to understand the "long distance" hydraulic transport of photoassimilates in fruit trees.

Compound droughts, i.e. the co-occurrences of heat and drought, represent a serious challenge for temperate forest trees leading to significant losses in forest biomass. We studied the physiological response of Norway spruce (Picea abies) saplings to heat and drought individually, and in combination. Continuous measurements of leaf gas exchange and VOC emission allowed us to identify fast-response reactions, while discrete VOC and root exudate samplings added qualitative information on compositional changes. Additionally, we used 13CO2 and 2H2O label pulses to investigate C-allocation and root water uptake in response to stress. Heat as well as drought reduced assimilation rates in the saplings, whereas transpiration, leaf VOC emission and root exudation rates increased in response to heat. Drought alone increased VOC emission but decreased exudation rates. Combined heat and drought triggered an amplified response in both processes despite negative net CO2 assimilation rates. Label incorporation showed compromised water uptake capacity of drought-stressed plants and illustrated de novo C-allocation to VOC emission and root exudates. The results point at the high susceptibility of Norway spruce saplings to drought and heat. Combined stress resulted in synergistic responses in VOC emissions and root exudates, showing the detrimental effect of compound droughts on Norway spruce. HighlightIn this study, we found synergistic effects of heat and drought on carbon losses from leaf VOC emission and root exudates despite negative assimilation rates in Norway spruce saplings.

Centrifuges provide a fast and standard approach to quantify embolism resistance of xylem in vulnerability curves (VCs). Traditionally, embolism formation in centrifuge experiments is assumingly driven by centrifuge speed, and thus pressure, but unaffected by spin time. Here, we explore to what extent embolism resistance is not only pressure but also spin time dependent, and hypothesise that time-stable hydraulic conductivity (Kh) values could shift VCs. We quantified time-based shifts in flow- centrifuge VCs and their parameter estimations for six angiosperm species by measuring Kh at regular intervals over 15 minutes of spinning at a particular speed before a higher speed was applied to the same sample. We compared various VCs per sample based on cumulative spin time, and modelled the relationship between Kh, xylem water potential ({Psi}), and spin time. Time-based changes of Kh showed considerable increases and decreases at low and high centrifuge speeds, respectively, which generally shifted VCs towards more positive {Psi} values. Values corresponding to 50% loss of hydraulic conductivity (P50) increased up to 0.72 MPa in Acer pseudoplatanus, and on average by 8.5% for all six species compared to VCs that did not consider spin time. By employing an asymptotic exponential model, we estimated time-stable Kh, which improved the statistical significance of VCs in 5 of the 6 species studied. This model also revealed the instability of VCs at short spin times, and showed that embolism formation in flow-centrifuges followed a saturating exponential growth curve. Although pressure remains the major determinant of embolism formation, spin time should be considered in flow- centrifuge VCs to avoid overestimation of embolism resistance. This spin-time artefact is species- specific, and likely based on relatively slow gas diffusion associated with embolism spreading. It can be minimized by determining time-stable Kh values for each centrifuge speed, without considerably extending the experimental time to construct VCs.

Fast-growing perennial trees such as Populus nigra L. are important species for wood, plywood, pulp, and bioenergy feedstock production, yet tree vigor in a changing climate is poorly understood. This research aimed to identify breeding targets for yield in water-limited environments, alongside unraveling the relationship between drought, yield, and glucose release in P. nigra. A diversity panel of 20 P. nigra genotypes, selected from a wide natural association population, was grown at three divergent European sites. Through extensive phenotyping of physiological and morphological productivity and water-use traits, under irrigated conditions and when exposed to a progressive drought, we elucidated the adaptive and plastic drivers underlying tree productivity. We have identified the underpinning traits for drought tolerance, whereby high yields can be maintained under water deficit, in this key species. This highlighted the importance of examining the yield stress index (YSI) over the drought resistance index (DRI) to assess genotypes for performance under moderate drought. In this way, we found genotypes with high hydraulic capacity, and large leaves made up of many cells to be best suited to multiple European environments, with contrasting water availability. Moreover, we identified genotypes that combine yield and water use efficiency, with good glucose release potential, which will be important traits for the future of poplar as a bioenergy crop. Vigorous poplar genotypes, which are adapted to wet climates showed high environmental plasticity. However, in European drought scenarios, these trees outperform drought resistant genotypes, and some exhibit good glucose release. These trees are a valuable resource for the future.

Spring freezing is an unforgiving stress for young leaves, often leading to death, with consequences for tree productivity and survival. While both the plant water transport system and living tissues are vulnerable to freezing, we do not know whether damage to one or both of these systems causes death in leaves exposed to freezing. Whole saplings of Liriodendron tulipifera were exposed to freezing and thawing trajectories designed to mimic natural spring freezes. We monitored the formation of freeze-thaw xylem embolism and damage to photosynthetic tissues to reveal a predictable progression of ice formation across the leaf surface that is strongly influenced by leaf vein architecture, notably the presence or absence of bundle sheath extensions. Our data also show that freeze-thaw embolism occurs only in the largest vein orders where mean vessel diameter exceeds 30{micro}m. With evidence of both freeze-thaw embolism and damage to photosynthetic tissue, we conclude that this dual-mode lethality may be common among other wide-vesseled angiosperm-leaves, potentially playing a role in limiting geographic distributions, and show that bundle sheath extensions may stall or even prevent freezing spread. HighlightBoth ice and air lead likely lead to death in young L.tulipfera leaves exposed to freezing, with the spread of both governed by physical characteristics of these leaves.

Despite a long research history, we do not fully understand why plants are able to transport xylem sap under negative pressure without constant failure. Microbubble formation via direct gas entry is assumed to cause hydraulic failure, while the concentration of gas dissolved in xylem sap is traditionally supposed to be constant, following Henrys law. Here, the concentration of soluble gas in xylem sap was estimated in vivo using well-watered Citrus plants under varying levels of air temperature and photoperiodic exposure, and compared to modelled data. The gas concentration in xylem sap showed non-equilibrium curves, with a minimum over- or undersaturation of 5% compared to gas solubility based on Henrys law. A similar diurnal pattern was obtained from the gas concentration in the cut-open conduits and discharge tube, and oversolubility was strongly associated with decreasing xylem water potentials during transpiration. Although our model did not explain the daily changes in gas solubility for an anisobaric situation, oversolubility characterises nanoconfined liquids, such as sap inside cell walls. Thus, plants are able to transport sap under negative pressure with relatively high amounts of dissolved gas, providing them with a buffering capacity to prevent hydraulic failure, despite diurnal changes in pressure and temperature.

Woody stems conduct both photosynthetic assimilation and respiration. The two processes work in concert, as stem photosynthesis helps refix CO2 released by stem respiration, thereby increasing carbon-use efficiency and generating a local pool of non-structural carbohydrates supporting cambial growth and stem hydraulic function. Despite its importance, little is known about seasonal variation in stem photosynthesis and the factors underlying its activity throughout the season. To fill this gap, we measured stem gas exchange together with growth activity, water status and photosynthetic pigment contents in two temperate species, Acer platanoides L. and Prunus avium L., over the season. In both species, gross photosynthetic rates (Pg) and dark respiration (Rd) changed significantly over the season in a similar pattern, indicating strong coordination between the two processes. Both Pg and Rd reached the highest values in May, during the period of rapid leaf expansion and secondary growth, and declined later in the growing season. At each measurement date, Rd exceeded Pg, resulting in a net CO2 efflux from the stems. The seasonal changes in Pg and Rd translated into seasonal variability in relative refixation of CO2, ranging from 3 to 59% and gradually decreasing towards the end of the season. Additionally, the Pg corresponded with the tissue hydration and increased significantly with increasing stem water potential. In contrast, total chlorophyll content showed less pronounced seasonal variation and thus explained substantially lower seasonal variability in Pg, except for the chlorophyll a/b ratio, which changed dynamically over the season and reached a minimum during the peak of the growing season. Overall, our results reveal that stem photosynthesis varies seasonally in accord with stem growth and water status, while the chlorophyll content has a lower impact on the seasonal changes. These findings are important for our understanding of the carbon relations of trees.

O_LIWe investigated the potential of wood anatomical traits to improve the reconstruction of masting events--variable and synchronized patterns of seed production --which are key to understanding tree species responses to current and predicted climate variability. Traditional reliance on tree-ring width as a proxy for reproduction is limited, as growth reductions can also result from drought and other stressors. C_LIO_LIWe analyzed 12 beech cores from North-East Germany, building a 52-year dataset. A wide range of wood anatomical traits was assessed to disentangle the effects of masting and drought. We used multivariate regression and developed a random forest model to evaluate the predictive power of these traits compared to tree ring width alone. C_LIO_LIResults suggested a complex mechanism of carbon reallocation towards reproduction, while reflecting a compensatory strategy to maintain hydraulic function and mechanical stability under resource-constrained conditions. Number of parenchyma cells, vessel density, and lignin content estimates emerged as key predictors for masting, outperforming tree-ring width in capturing the reproductive signal. C_LIO_LIOur findings establish a novel link between wood anatomy and masting events, demonstrating that quantitative wood anatomical traits offer a more accurate and ecologically relevant approach for reconstructing past masting dynamics. C_LI