Intraspecific plasticity and co-variation of leaf traits facilitate Ficus tinctoria to acclimate hemiepiphytic and terrestrial habitats.

Despite intensive studies on plant functional traits, the intraspecific variation and their co-variation at the multi-scale remains poorly studied, which holds the potential to unveil plant responses to changing environmental conditions. In this study, intraspecific variations of 16 leaf functional traits of a common fig species, Ficus tinctoria G. Frost., were investigated in relation to different scales: habitat types (hemiepiphytic and terrestrial), growth stages (small, medium and large) and tree crown positions (upper, middle and lower) in Xishuangbanna, Southwest China. Remarkable intraspecific variation was observed in leaf functional traits, which was mainly influenced by tree crown position, growth stage and their interaction. Stable nitrogen isotope (δ15N) and leaf area (LA) showed large variations, while stable carbon isotope (δ13C), stomata width and leaf water content showed relatively small variations, suggesting that light- and nitrogen-use strategies of F. tinctoria were plastic, while the water-use strategies have relatively low plasticity. The crown layers are formed with the growth of figs, and leaves in the lower crown increase their chlorophyll concentration and LA to improve the light energy conversion efficiency and the ability to capture weak light. Meanwhile, leaves in the upper crown increase the water-use efficiency to maintain their carbon assimilation. Moreover, hemiepiphytic medium (transitional stage) and large (free-standing stage) figs exhibited more significant trait differentiation (chlorophyll concentration, δ13C, stomata density, etc.) within the crown positions, and stronger trait co-variation compared with their terrestrial counterparts. This pattern demonstrates their acclimation to the changing microhabitats formed by their hemiepiphytic life history. Our study emphasizes the importance of multi-scaled intraspecific variation and co-variation in trait-based strategies of hemiepiphyte and terrestrial F. tinctoria, which facilitate them to cope with different environmental conditions.

An exploratory steady-state redox model of photosynthetic linear electron transport for use in complete modelling of photosynthesis for broad applications.

A photochemical model of photosynthetic electron transport (PET) is needed to integrate photophysics, photochemistry, and biochemistry to determine redox conditions of electron carriers and enzymes for plant stress assessment and mechanistically link sun-induced chlorophyll fluorescence to carbon assimilation for remotely sensing photosynthesis. Towards this goal, we derived photochemical equations governing the states and redox reactions of complexes and electron carriers along the PET chain. These equations allow the redox conditions of the mobile plastoquinone pool and the cytochrome b6 f complex (Cyt) to be inferred with typical fluorometry. The equations agreed well with fluorometry measurements from diverse C3 /C4 species across environments in the relationship between the PET rate and fraction of open photosystem II reaction centres. We found the oxidation of plastoquinol by Cyt is the bottleneck of PET, and genetically improving the oxidation of plastoquinol by Cyt may enhance the efficiency of PET and photosynthesis across species. Redox reactions and photochemical and biochemical interactions are highly redundant in their complex controls of PET. Although individual reaction rate constants cannot be resolved, they appear in parameter groups which can be collectively inferred with fluorometry measurements for broad applications. The new photochemical model developed enables advances in different fronts of photosynthesis research.

You are what you eat: nutrient and water relations between mistletoes and hosts.

Mistletoes play important roles in biogeochemical cycles. Although many studies have compared nutrient concentrations between mistletoes and their hosts, no general patterns have been found and the nutrient uptake mechanisms in mistletoes have not been fully resolved. To address the water and nutrient relations in mistletoes compared with their hosts, we measured 11 nutrient elements, two isotope ratios and two leaf morphological traits for 11 mistletoe and 104 host species from four sites across a large environmental gradient in southwest China. Mistletoes had significantly higher phosphorus, potassium, and boron concentrations, nitrogen isotope ratio, and lower carbon isotope ratio (δ13 C) indicative of lower water-use efficiency than hosts, but other elements were similar to those in hosts. Sites explained most of the variation in the multidimensional trait space. With increasing host nitrogen concentration, both mistletoe δ13 C and the difference between mistletoe and host δ13 C increased, providing evidence to support the 'nitrogen parasitism hypothesis'. Host nutrient concentrations were the best predictors for that of the mistletoe nutrient elements in most cases. Our results highlight the important roles of environmental conditions and host nutrient status in determining mistletoe nutrient pools, which together explain their trophic interactions with hosts in subtropical and tropical ecosystems.

Physiological response and photosynthetic recovery to an extreme drought: Evidence from plants in a dry-hot valley savanna of Southwest China.

The frequency of extreme drought events has been rising worldwide, but due to its unpredictability, how plants will respond remains poorly understood. Here, we aimed to characterize how the hydraulics and photosynthesis of savanna plants respond to extreme drought, and tested whether they can subsequently recover photosynthesis after drought. There was an extreme drought in 2019 in Southwest (SW) China. We investigated photosynthetic gas exchange, leaf-, stem-, and whole-shoot hydraulic conductance of 18 plant species with diverse leaf habits (deciduous, semi-deciduous and evergreen) and growth forms (tree and shrub) from a dry-hot valley savanna in SW China for three rainy seasons from 2019 to 2021. We also compared photosynthetic gas exchange to those of a regular year (2014). We found that leaf stomatal and hydraulic conductance and maximum photosynthetic rate were significantly lower during the drought in 2019 than in the wetter years. In 2019, all studied plants maintained stomatal conductance at their minimum level observed, which could be related to high vapor pressure deficits (VPD, >2 kPa). However, no significant difference in stem and shoot hydraulic conductance was detected across years. The reductions in leaf hydraulic conductance and stomatal regulation under extreme drought might help keep the stem hydraulic function. Stomatal conductance and photosynthesis after drought (2020 and 2021) showed comparable or even higher values compared to that of 2014, suggesting high recovery of photosynthetic gas exchange. In addition, the response of hydraulic and photosynthetic traits to extreme drought was convergent across leaf habits and growth forms. Our results will help better understand the physiological mechanism underlying the response of savanna ecosystems to climate change.

Leaves as bottlenecks: The contribution of tree leaves to hydraulic resistance within the soil-plant-atmosphere continuum.

Within vascular plants, the partitioning of hydraulic resistance along the soil-to-leaf continuum affects transpiration and its response to environmental conditions. In trees, the fractional contribution of leaf hydraulic resistance (Rleaf ) to total soil-to-leaf hydraulic resistance (Rtotal ), or fRleaf (=Rleaf /Rtotal ), is thought to be large, but this has not been tested comprehensively. We compiled a multibiome data set of fRleaf using new and previously published measurements of pressure differences within trees in situ. Across 80 samples, fRleaf averaged 0.51 (95% confidence interval [CI] = 0.46-0.57) and it declined with tree height. We also used the allometric relationship between field-based measurements of soil-to-leaf hydraulic conductance and laboratory-based measurements of leaf hydraulic conductance to compute the average fRleaf for 19 tree samples, which was 0.40 (95% CI = 0.29-0.56). The in situ technique produces a more accurate descriptor of fRleaf because it accounts for dynamic leaf hydraulic conductance. Both approaches demonstrate the outsized role of leaves in controlling tree hydrodynamics. A larger fRleaf may help stems from loss of hydraulic conductance. Thus, the decline in fRleaf with tree height would contribute to greater drought vulnerability in taller trees and potentially to their observed disproportionate drought mortality.

Xylem conduit deformation across vascular plants: an evolutionary spandrel or protective valve?

The hydraulic system of vascular plants and its integrity is essential for plant survival. To transport water under tension, the walls of xylem conduits must approximate rigid pipes. Against this expectation, conduit deformation has been reported in the leaves of a few species and hypothesized to function as a 'circuit breaker' against embolism. Experimental evidence is lacking, and its generality is unknown. We demonstrated the role of conduit deformation in protecting the upstream xylem from embolism through experiments on three species and surveyed a diverse selection of vascular plants for conduit deformation in leaves. Conduit deformation in minor veins occurred before embolism during slow dehydration. When leaves were exposed to transient increases in transpiration, conduit deformation was accompanied by large water potential differences from leaf to stem and minimal embolism in the upstream xylem. In the three species tested, collapsible vein endings provided clear protection of upstream xylem from embolism during transient increases in transpiration. We found conduit deformation in diverse vascular plants, including 11 eudicots, ginkgo, a cycad, a fern, a bamboo, and a grass species, but not in two bamboo and a palm species, demonstrating that the potential for 'circuit breaker' functionality may be widespread across vascular plants.

Patterns of compound-leaf form and deciduous-leaf habit across forests in China: Their association and key climatic factors.

Leaf form (compound vs. simple) and habit (evergreen vs. deciduous) are key functional traits of trees to adapt to various climates and are vital in determining plant response to climate change. However, their association and climatic determinants remain uncertain, especially in East Asian forests in the largest monsoon region on earth. To fill these knowledge gaps, we compiled a dataset comprising 42 intact forests and over 2200 angiosperm tree species across China (spanning 30 latitudes and 47 longitudes). The geographical and climatic patterns of leaf form and habit were analyzed. The association between compound leaf and deciduousness was tested for tropical, subtropical and temperate climatic zones. We found that both the percentage of compound leaf (CT%) and deciduous tree species (DT%) increased with latitude and decreased with mean annual precipitation (MAP). For all forests, DT% was negatively related to mean annual temperature (MAT), whereas CT% was not. Nevertheless, both DT% and CT% increased with increasing MAT in the tropics, possibly owing to the high vapor pressure deficits (VPD) and canopy water deficits associated with high temperatures. A positive linear relationship between CT% and DT% was found across all forests and within different climatic zones except for temperate, and the intercept of the regression line was significantly higher in the tropics than in the subtropics. Overall, as supported by principal component analysis, deciduousness was negatively associated with both temperature and precipitation, while CT negatively with precipitation only across zones and positively with temperature in the tropics. Different relationships in different climatic zones suggest potentially different selective forces. Our findings provide novel insights into the linkage between leaf form and habit, as well as how climate shapes the landscape of broadleaf forests, which has important implications regarding the response of forest composition to climate change.

Leaf gas exchange and water relations of the woody desiccation-tolerant Paraboea rufescens during dehydration and rehydration.

Desiccation-tolerant (DT) plants can withstand dehydration to less than 0.1 g H2O g-1 dry weight. The mechanism for whole-plant recovery from severe dehydration is still not clear, especially for woody DT plants. In the present study, we evaluated the desiccation tolerance and mechanism of recovery for a potentially new woody resurrection plant Paraboea rufescens (Gesneriaceae). We monitored the leaf water status, leaf gas exchange, chlorophyll fluorescence and root pressure of potted P. rufescens during dehydration and rehydration, and we investigated the water content and chlorophyll fluorescence of P. rufescens leaves in the field during the dry season. After re-watering from a severely dehydrated state, leaf maximum quantum yield of photosystem II of P. rufescens quickly recovered to well-watered levels. Leaf water status and leaf hydraulic conductance quickly recovered to well-watered levels after re-watering, while leaf gas exchange traits also trended to recovery, but at a slower rate. The maximum root pressure in rehydrated P. rufescens was more than twice in well-watered plants. Our study identified P. rufescens as a new DT woody plant. The whole-plant recovery of P. rufescens from extreme dehydration is potentially associated with an increase of root pressure after rehydration. These findings provide insights into the mechanisms of recovery of DT plants from dehydration.

Granal thylakoid structure and function: explaining an enduring mystery of higher plants.

In higher plants, photosystems II and I are found in grana stacks and unstacked stroma lamellae, respectively. To connect them, electron carriers negotiate tortuous multi-media paths and are subject to macromolecular blocking. Why does evolution select an apparently unnecessary, inefficient bipartition? Here we systematically explain this perplexing phenomenon. We propose that grana stacks, acting like bellows in accordions, increase the degree of ultrastructural control on photosynthesis through thylakoid swelling/shrinking induced by osmotic water fluxes. This control coordinates with variations in stomatal conductance and the turgor of guard cells, which act like an accordion's air button. Thylakoid ultrastructural dynamics regulate macromolecular blocking/collision probability, direct diffusional pathlengths, division of function of Cytochrome b6 f complex between linear and cyclic electron transport, luminal pH via osmotic water fluxes, and the separation of pH dynamics between granal and lamellar lumens in response to environmental variations. With the two functionally asymmetrical photosystems located distantly from each other, the ultrastructural control, nonphotochemical quenching, and carbon-reaction feedbacks maximally cooperate to balance electron transport with gas exchange, provide homeostasis in fluctuating light environments, and protect photosystems in drought. Grana stacks represent a dry/high irradiance adaptation of photosynthetic machinery to improve fitness in challenging land environments. Our theory unifies many well-known but seemingly unconnected phenomena of thylakoid structure and function in higher plants.

Linking tree water use efficiency with calcium and precipitation.

Water use efficiency (WUE) is a key physiological trait in studying plant carbon and water relations. However, the determinants of WUE across a large geographical scale are not always clear, limiting our capacity to predict WUE in response to future global climate change. We propose that tree WUE is influenced by calcium (Ca) availability and precipitation. In addition, although it is well-known that transpiration is the major driving force for passive nutrient uptake, the linkage between these two processes has not been well-established. Because Ca uptake is an apoplastic and passive process that purely relies on transpiration, and there is no translocation once assimilated, we further developed a theoretical model to quantify the relationship between tree Ca accumulation and WUE using soil-to-plant calcium ratio (SCa/BCa) and tree WUE derived from δ13C. We tested our theoretical model and predicted relationships using three common tree species across their native habitats in Northern China, spanning 2300 km and a controlled greenhouse experiment with soil Ca concentrations manipulated. We found that tree WUE was negatively related to precipitation of the growing season (GSP) and positively with soil Ca. A multiple regression model and a path analysis suggested a higher contribution of soil Ca to WUE than GSP. As predicted by our theoretical model, we found a positive relationship between WUE and SCa/BCa across their distribution ranges in all three tree species and in the controlled experiment for one selected species. This relationship suggests a tight coupling between water and Ca uptake and the potential use of SCa/BCa to indicate WUE. A negative relationship between SCa/BCa and GSP also suggests a possible decrease in tree Ca accumulation efficiency in a drier future in Northern China.

Drought timing and species growth phenology determine intra-annual recovery of tree height and diameter growth.

Droughts interact with tree phenology to drive declines in growth. As climate change makes drought more likely in the Northeastern USA, it is important to understand how droughts at different times of year will lead to reduced height and diameter growth of trees. To determine how seasonal drought may reduce intra-annual growth, we implemented spring, summer or fall droughts on 288 containerized saplings of six tree species (Acer rubrum, Betula papyrifera, Prunus serotina, Juniperus virginiana, Pinus strobus and Thuja occidentalis). We tracked weekly soil moisture, leaf water potential, height, diameter and survival of all trees before, during and after each 6-week drought. We found that the tree species that conducted the majority of their height or diameter growth in the spring were most sensitive to spring droughts (B. papyrifera and Pi. strobus). Thuja occidentalis also experienced significantly reduced growth from the spring drought but increased growth after the drought ended and achieved total height and diameter growth similar to controls. In contrast, summer droughts halted growth in most species for the remainder of the growing season even after the drought had ended. Fall droughts never impacted growth in the current year. These fine temporal-scale measurements of height and diameter growth suggest that tree response varies among species and is dynamic at intra-annual scales. These relatively rare data on intra-annual height growth sensitivity are important for canopy recruitment of saplings in forest ecosystems. Species-specific sensitivities of intra-annual growth to drought can inform models of forest competition in a changing climate.

Leaf Venation Architecture in Relation to Leaf Size Across Leaf Habits and Vein Types in Subtropical Woody Plants.

Leaves are enormously diverse in their size and venation architecture, both of which are core determinants of plant adaptation to environments. Leaf size is an important determinant of leaf function and ecological strategy, while leaf venation, the main structure for support and transport, determines the growth, development, and performance of a leaf. The scaling relationship between venation architecture and leaf size has been explored, but the relationship within a community and its potential variations among species with different vein types and leaf habits have not been investigated. Here, we measured vein traits and leaf size across 39 broad-leaved woody species within a subtropical forest community in China and analyzed the scaling relationship using ordinary least squares and standard major axis method. Then, we compared our results with the global dataset. The major vein density, and the ratio of major (1° and 2°) to minor (3° and higher) vein density both geometrically declined with leaf size across different vein types and leaf habits. Further, palmate-veined species have higher major vein density and a higher ratio of major to minor vein density at the given leaf size than pinnate-veined species, while evergreen and deciduous species showed no difference. These robust trends were confirmed by reanalyzing the global dataset using the same major vein classification as ours. We also found a tradeoff between the cell wall mass per vein length of the major vein and the major vein density. These vein scaling relationships have important implications on the optimization of leaf size, niche differentiation of coexisting species, plant drought tolerance, and species distribution.

Electrochemiluminescence enhanced by isolating ACQphores in pyrene-based porous organic polymer: A novel ECL emitter for the construction of biosensing platform.

In this work, a pyrene-based porous organic polymer (Py-POP) with strong electrochemiluminescence (ECL) emission was synthesized and used to fabricate an ECL sensor for the extra-sensitive detection of microRNA-155. The ECL intensity of the Py-POP prepared by tetra(p-aminophenyl)methane (TAPM) and 1,3,6,8-tetrakis(4-formylphenyl)pyrene (TFPPy) was about 3.1 times that of TFPPy aggregates, which was primarily ascribed to the elimination of the effect of aggregation-caused quenching (ACQ) by increasing the distance between ACQ luminophores (pyrene cores) in Py-POP. Meanwhile, the strong covalent connections between 1,3,6,8-tetraphenylpyrene (TPPy) and tetraphenylmethane (TPM) units in the rigid framework of Py-POP could partly block the intramolecular motion of TPPy and TPM, which reduced the non-radiative decay and thus further improved the ECL emission. Furthermore, the hydrophobic porous structure of Py-POP was beneficial to the enrichment of lipophilic tripropylamine (TPrA) coreactants in pores of Py-POP, which greatly shortened the electron migration distance between TPrA coreactants and pyrene luminophores on the pore walls of Py-POP, thereby also enhancing the ECL intensity. By using the Py-POP as a new ECL tag and with the help of the strand displacement processes and target recycling, the fabricated ECL biosensor had a sensitive response for microRNA-155 from 1 fM to 1 nM and a detection limit of 0.326 fM. Overall, this work provided a new and feasible strategy to surmount the ACQ effect for enhancing ECL emission, which not only paved a new way to exploit high-performance ECL materials for fabricating extra-sensitive sensors but also broadened the application of POPs in bioanalysis and ECL fields.

Response of four evergreen savanna shrubs to an incidence of extreme drought: high embolism resistance, branch shedding and maintenance of nonstructural carbohydrates.

Extreme drought events are becoming frequent globally, resulting in widespread plant mortality and forest dieback. Although savanna vegetation cover ~20% of the earth's land area, their responses to extreme drought have been less studied than that of forests. Herein, we quantified branch dieback, individual mortality and the associated physiological responses of four evergreen shrubs (Tarenna depauperate Hutch., Maytenus esquirolii (H. Lév.) C.Y. Cheng, Murraya exotica L., Jasminum nudiflorum Lindl.) in a savanna ecosystem in Southwest China to an incidence of extreme drought during 2019 and 2020. We found that 80-100% of the individuals of these species exhibited branch dieback, whereas individual mortality was only found in T. depauperate (4.5%). All species showed high resistance to stem embolism (P50, water potential at 50% loss of hydraulic conductivity ranged from -5.62 to -8.6 MPa), whereas the stem minimum water potentials reached -7.6 to ca -10.0 MPa during the drought. The low water potential caused high native embolism levels (percentage loss of hydraulic conductivity (PLC) 23-65%) in terminal branches, and the remaining stems maintained 15-35% PLC at the end of the drought. Large within-individual variations in stem vulnerability to embolism were detected, and shedding of vulnerable branches could be a mechanism for shrubs to reduce water and carbon consumption. Overall, the content of total nonstructural carbohydrates (NSC) and their components in the stem were generally comparable to or higher than those in the rainy season in three of the four species. Because the leaves were turgor-less for most time during the drought, high NSC levels during the drought could be due to recycling of NSC from dead branches or translocation from roots. Our results suggest high tolerance of savanna shrub species to extreme drought, which could be facilitated by high embolism resistance in some stems and shedding of vulnerable branches to maintain individual water and carbon balance.

Higher water and nutrient use efficiencies in savanna than in rainforest lianas result in no difference in photosynthesis.

Differences in traits between lianas and trees in tropical forests have been studied extensively; however, few have compared the ecological strategies of lianas from different habitats. Here, we measured 25 leaf and stem traits concerning leaf anatomy, morphology, physiology and stem hydraulics for 17 liana species from a tropical seasonal rainforest and for 19 liana species from a valley savanna in south-west China. We found that savanna lianas had higher vessel density, wood density and lower hydraulically weighted vessel diameter and theoretical hydraulic conductivity than tropical seasonal rainforest lianas. Compared with tropical seasonal rainforest lianas, savanna lianas also showed higher leaf dry matter content, carbon isotope composition (δ13C), photosynthetic water use efficiency, ratio of nitrogen to phosphorus, photosynthetic phosphorus use efficiency and lower leaf size, stomatal conductance and nitrogen, phosphorus and potassium concentrations. Interestingly, no differences in light-saturated photosynthetic rate were found between savanna and tropical seasonal rainforest lianas either on mass or area basis. This is probably due to the higher water and nutrient use efficiencies of savanna lianas. A principal component analysis revealed that savanna and tropical seasonal rainforest lianas were significantly separated along the first axis, which was strongly associated with acquisitive or conservative resource use strategy. Leaf and stem functional traits were coordinated across lianas, but the coordination or trade-off was stronger in the savanna than in the tropical seasonal rainforest. In conclusion, a relatively conservative (slow) strategy concerning water and nutrient use may benefit the savanna lianas, while higher nutrient and water use efficiencies allow them to maintain similar photosynthesis as tropical seasonal rainforest species. Our results clearly showed divergences in functional traits between lianas from savanna and tropical seasonal rainforest, suggesting that enhanced water and nutrient use efficiencies might contribute to the distribution of lianas in savanna ecosystems.

Hydraulic prediction of drought-induced plant dieback and top-kill depends on leaf habit and growth form.

Hydraulic failure caused by severe drought contributes to aboveground dieback and whole-plant death. The extent to which dieback or whole-plant death can be predicted by plant hydraulic traits has rarely been tested among species with different leaf habits and/or growth forms. We investigated 19 hydraulic traits in 40 woody species in a tropical savanna and their potential correlations with drought response during an extreme drought event during the El Niño-Southern Oscillation in 2015. Plant hydraulic trait variation was partitioned substantially by leaf habit but not growth form along a trade-off axis between traits that support drought tolerance versus avoidance. Semi-deciduous species and shrubs had the highest branch dieback and top-kill (complete aboveground death) among the leaf habits or growth forms. Dieback and top-kill were well explained by combining hydraulic traits with leaf habit and growth form, suggesting integrating life history traits with hydraulic traits will yield better predictions.

Highly efficient electrochemiluminescence resonance energy transfer material constructed from an AIEgen-based 2D ultrathin metal-organic layer for thrombin detection.

A facile strategy to design a highly efficient electrochemiluminescence resonance energy transfer (ECL-RET) system was proposed by using an AIEgen-based 2D ultrathin metal-organic layer (MOL) to coordinatively immobilize energy donors and acceptors simultaneously, in which the distance between adjacent donor-acceptor pairs was precise and short for obtaining high ECL-RET efficiency.

The hydraulic architecture of an arborescent monocot: ontogeny-related adjustments in vessel size and leaf area compensate for increased resistance.

Bamboos are arborescent monocotyledons that have no secondary growth, but can continually produce conduits with diameters appropriate to the current size of the plant. Here, we studied bamboo hydraulic architecture to address the mechanisms involved in compensating for the increase in hydraulic resistance during ontogeny. We measured the hydraulic weighted vessel diameters (Dh ) at different distances from the apex along the stem of Bambusa textilis. The hydraulic resistance of different components and individuals of different heights were quantified using the high-pressure flowmeter method. The Dh showed tip-to-base widening with a scaling exponent in the range of those reported for trees. Although theoretical hydraulic conductivity decreased from base-to-tip, leaf-specific conductivity did not change. Leaves contributed the most to the whole-shoot hydraulic resistance, followed by the leaf-bearing branches. Roots contributed c. 13% to whole-plant resistance. Interestingly, taller individuals showed lower whole-shoot resistance owing to an increased number of resistances in parallel (side-branches), while leaf-specific resistance was independent of plant size. Tip-to-base vessel widening and height-independent constant leaf-specific conductance could be mechanisms for hydraulic optimization in B. textilis. Similar patterns have also been found in woody plants with secondary growth, but this bamboo exhibits them without secondary growth.

Quantifying vulnerability to embolism in tropical trees and lianas using five methods: can discrepancies be explained by xylem structural traits?

Vulnerability curves (VCs) describe the loss of hydraulic conductance against increasing xylem tension, providing valuable insights about the response of plant water transport to water stress. Techniques to construct VCs have been developed and modified continuously, but controversies continue. We compared VCs constructed using the bench-top dehydration (BD), air-injection-flow (AI), pneumatic-air-discharge (PAD), optical (OP) and X-ray-computed microtomography (MicroCT) methods for tropical trees and lianas with contrasting vessel lengths. The PAD method generated highly vulnerable VCs, the AI method intermediate VCs, whereas the BD, OP and MicroCT methods produced comparable and more resistant VCs. Vessel-length and diameter accounted for the overestimation ratio of vulnerability estimated using the AI but not the PAD method. Compared with directly measured midday embolism levels, the PAD and AI methods substantially overestimated embolism, whereas the BD, MicroCT and OP methods provided more reasonable estimations. Cut-open vessels, uncertainties in maximum air volume estimations, sample-length effects, tissue cracks and shrinkage together may impede the reliability of the PAD method. In conclusion, we validate the BD, OP and MicroCT methods for tropical plants, whereas the PAD and AI need further mechanistic testing. Therefore, applications of VCs in estimating plant responses to drought need to be cautious.