Research progress on the prediction of drought death point and the mechanism of drought- induced tree mortality

doi:10.17521/cjpe.2023.0319

Abstract

Abstract:

With the global increase in tree mortality events caused by drought, there have been numerous reports on the mechanism of drought-induced tree mortality both domestically and internationally in recent years. However, the exact mechanism that causes tree mortality remains unclear, which increases the uncertainty of predicting the survival probability of forests under future climate changes. This review systematically analyzed the research progress related to tree death caused by extreme drought events, focusing the prediction of death point and physiological mechanism of drought-induced tree mortality. It highlighted that tree death was the result of multiple physiological processes. Furthermore, previous reports have shown that the death judged by visual symptoms may occur after the tree has already been dead for a period, leading to a lack of early warning signals and making the death inevitable. The review analyzed the main characteristics and possible sequence of physiological variables such as the degree of xylem embolism, radial flow, cell membrane permeability, and cambium activity in the process of drought-induced tree mortality. It suggests that the loss of cambium activity ultimately led to irreversible tree death. Therefore, when discussing the mechanism of drought-induced tree mortality, quantifying the loss rate of cambium activity is crucial for accurately determining the time of tree death, which is worth further studying. This paper also proposed relevant issues and research directions in the field of drought-induced tree mortality, providing reference ideas for accurately predicting tree death and formulating efficient and appropriate solutions to future climate change.

Key words: extreme drought, cell damage, cambium activity, radial flow, tree mortality

SHAO Chang-Chang, DUAN Hong-Lang, ZHAO Xi-Zhou, DING Gui-Jie. Research progress on the prediction of drought death point and the mechanism of drought- induced tree mortality[J]. Chin J Plant Ecol, 2025, 49(2): 221-231.

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URL: https://www.plant-ecology.com/EN/10.17521/cjpe.2023.0319

https://www.plant-ecology.com/EN/Y2025/V49/I2/221

Figures/Tables 2

Fig. 1 Dagram representing xylem embolism and repair (the type of pits as an example of conifer species). A, Normal water supply to xylem tracheids. B, Embolization of xylem tracheids. C, Embolized tracheids are unable to restore hydraulic function. D, Re-filling of embolized tracheids. E, Formation of new tracheids.

Fig. 2 A series of physiological changes of trees under extreme drought events. Red diagonals indicate xylem embolism and the cessation of water transport, while blue diagonals represent the cessation of CO2 assimilation and the unavailability for organic matter or failure of organic matter transport. gs, stomatal conductance; RWC, relative water content; SWC, soil water content.

References 79

[1]	Abdalla M, Carminati A, Cai G, Javaux M, Ahmed MA (2021). Stomatal closure of tomato under drought is driven by an increase in soil-root hydraulic resistance. Plant, Cell & Environment, 44, 425-431.
[2]	Adams HD, Zeppel MJB, Anderegg WRL, Hartmann H, Landhäusser SM, Tissue DT, Huxman TE, Hudson PJ, Franz TE, Allen CD, Anderegg LDL, Barron-Gafford GA, Beerling DJ, Breshears DD, Brodribb TJ, et al. (2017). A multi-species synthesis of physiological mechanisms in drought-induced tree mortality. Nature Ecology & Evolution, 1, 1285-1291.
[3]	Andriantelomanana T, Améglio T, Delzon S, Cochard H, Herbette S (2024). Unpacking the point of no return under drought in poplar: insight from stem diameter variation. New Phytologist, 242, 466-478.
[4]	Avila RT, Guan X, Kane CN, Cardoso AA, Batz TA, DaMatta FM, Jansen S, McAdam SAM (2022). Xylem embolism spread is largely prevented by interconduit pit membranes until the majority of conduits are gas-filled. Plant, Cell & Environment, 45, 1204-1215.
[5]	Bai Y, Liu Y, Kueppers LM, Feng X, Yu K, Yang X, Li X, Huang J (2021). The coupled effect of soil and atmospheric constraints on the vulnerability and water use of two desert riparian ecosystems. Agricultural and Forest Meteorology, 311, 108701. DOI: 10.1016/j.agrformet.2021.108701.
[6]	Brodersen CR, McElrone AJ (2013). Maintenance of xylem network transport capacity: a review of embolism repair in vascular plants. Frontiers in Plant Science, 4, 108. DOI: 10.3389/fpls.2013.00108.
[7]	Brodersen CR, McElrone AJ, Choat B, Matthews MA, Shackel KA (2010). The dynamics of embolism repair in xylem: in vivo visualizations using high-resolution computed tomography. Plant Physiology, 154, 1088-1095. DOI PMID
[8]	Chen ZC, Zhu SD, Zhang YT, Luan JW, Li S, Sun PS, Wan XC, Liu SR (2020). Tradeoff between storage capacity and embolism resistance in the xylem of temperate broadleaf tree species. Tree Physiology, 40, 1029-1042. DOI PMID
[9]	Cheng L, Li YL, Ning ZY, Yang HL, Zhan J, Yao B (2024). Response mechanisms of woody plants to drought stress: a review based on plant hydraulic traits. Acta Ecologica Sinica, 44, 2688-2705.
	[程莉, 李玉霖, 宁志英, 杨红玲, 詹瑾, 姚博 (2024). 木本植物应对干旱胁迫的响应机制: 基于水力学性状视角. 生态学报, 44, 2688-2705.]
[10]	Choat B, Brodribb TJ, Brodersen CR, Duursma RA, López R, Medlyn BE (2018). Triggers of tree mortality under drought. Nature, 558, 531-539.
[11]	Choat B, Nolf M, Lopez R, Peters JMR, Carins-Murphy MR, Creek D, Brodribb TJ (2019). Non-invasive imaging shows no evidence of embolism repair after drought in tree species of two genera. Tree Physiology, 39, 113-121. DOI PMID
[12]	Dai YX, Wang L, Wan XC (2015). Progress on researches of drought-induced tree mortality mechanisms. Chinese Journal of Ecology, 34, 3228-3236.
	[代永欣, 王林, 万贤崇 (2015). 干旱导致树木死亡机制研究进展. 生态学杂志, 34, 3228-3236.]
[13]	Dai YX, Wang L, Wan XC (2018). Relative contributions of hydraulic dysfunction and carbohydrate depletion during tree mortality caused by drought. AoB Plants, 10, plx069. DOI: 10.1093/aobpla/plx069.
[14]	Duan H, Resco de Dios V, Wang D, Zhao N, Huang G, Liu W, Wu J, Zhou S, Choat B, Tissue DT (2022). Testing the limits of plant drought stress and subsequent recovery in four provenances of a widely distributed subtropical tree species. Plant, Cell & Environment, 45, 1187-1203.
[15]	Duan H, Shao C, Luo X, Resco de Dios V, Tissue DT, Ding G (2023). Root relative water content is a potential signal for impending mortality of a subtropical conifer during extreme drought stress. Plant, Cell & Environment, 46, 2763-2777.
[16]	Duan HL, Wu JP, Liu WF, Liao YC, Zhang HN, Fan HB (2015). Water relations and carbon dynamics under drought stress and the mechanisms of drought-induced tree mortality. Scientia Silvae Sinicae, 51(11), 113-120.
	[段洪浪, 吴建平, 刘文飞, 廖迎春, 张海娜, 樊后保 (2015). 干旱胁迫下树木的碳水过程以及干旱死亡机理. 林业科学, 51(11), 113-120.]
[17]	Garcia-Forner N, Biel C, Savé R, Martínez-Vilalta J (2017). Isohydric species are not necessarily more carbon limited than anisohydric species during drought. Tree Physiology, 37, 441-455. DOI PMID
[18]	Gauthey A, Peters JMR, Lòpez R, Carins-Murphy MR, Rodriguez-Dominguez CM, Tissue DT, Medlyn BE, Brodribb TJ, Choat B (2022). Mechanisms of xylem hydraulic recovery after drought in Eucalyptus saligna. Plant, Cell & Environment, 45, 1216-1228.
[19]	Gomez-Gallego M, Galiano L, Martínez-Vilalta J, Stenlid J, Capador-Barreto HD, Elfstrand M, Camarero JJ, Oliva J (2022). Interaction of drought- and pathogen-induced mortality in Norway spruce and Scots pine. Plant, Cell & Environment, 45, 2292-2305.
[20]	Guan X, Werner J, Cao K, Pereira L, Kaack L, McAdam SAM, Jansen S (2022). Stem and leaf xylem of angiosperm trees experiences minimal embolism in temperate forests during two consecutive summers with moderate drought. Plant Biology, 24, 1208-1223. DOI PMID
[21]	Güney A, Zweifel R, Türkan S, Zimmermann R, Wachendorf M, Güney CO (2020). Drought responses and their effects on radial stem growth of two co-occurring conifer species in the Mediterranean mountain range. Annals of Forest Science, 77, 105. DOI: 10.1007/s13595-020-01007-2.
[22]	Hammond WM, Johnson DM, Meinzer FC (2021). A thin line between life and death: radial sap flux failure signals trajectory to tree mortality. Plant, Cell & Environment, 44, 1311-1314.
[23]	Hammond WM, Williams AP, Abatzoglou JT, Adams HD, Klein T, López R, Sáenz-Romero C, Hartmann H, Breshears DD, Allen CD (2022). Global field observations of tree die-off reveal hotter-drought fingerprint for Earth’s forests. Nature Communications, 13, 1761. DOI: 10.1038/s41467-022-29289-2.
[24]	Hammond WM, Yu K, Wilson LA, Will RE, Anderegg WRL, Adams HD (2019). Dead or dying? Quantifying the point of no return from hydraulic failure in drought-induced tree mortality. New Phytologist, 223, 1834-1843. DOI PMID
[25]	Han YG, Deng JJ, Zhou WM, Wang QW, Yu DP (2022). Seasonal responses of hydraulic function and carbon dynamics in spruce seedlings to continuous drought. Frontiers in Plant Science, 13, 868108. DOI: 10.3389/fpls.2022.868108.
[26]	Hartmann H, Trumbore S (2016). Understanding the roles of nonstructural carbohydrates in forest trees—From what we can measure to what we want to know. New Phytologist, 211, 386-403. DOI PMID
[27]	Hartmann H, Ziegler W, Trumbore S (2013). Lethal drought leads to reduction in nonstructural carbohydrates in Norway spruce tree roots but not in the canopy. Functional Ecology, 27, 413-427.
[28]	He WQ, Liu HY, Qi Y, Liu F, Zhu XR (2020). Patterns in nonstructural carbohydrate contents at the tree organ level in response to drought duration. Global Change Biology, 26, 3627-3638. DOI PMID
[29]	Igbinosa EO, Beshiru A, Igbinosa IH (2020). Mechanism of action of secondary metabolites from marine-derived Streptoymces on bacterial isolates by membrane permeability. Microbial Pathogenesis, 149, 104532. DOI: 10.1016/j.micpath.2020.104532.
[30]	Jin Y, Li J, Liu C, Liu Y, Zhang Y, Sha L, Wang Z, Song Q, Lin Y, Zhou R, Chen A, Li P, Fei X, Grace J (2018). Carbohydrate dynamics of three dominant species in a Chinese savanna under precipitation exclusion. Tree Physiology, 38, 1371-1383. DOI PMID
[31]	Kaack L, Weber M, Isasa E, Karimi Z, Li S, Pereira L, Trabi CL, Zhang Y, Schenk HJ, Schuldt B, Schmidt V, Jansen S (2021). Pore constrictions in intervessel pit membranes provide a mechanistic explanation for xylem embolism resistance in angiosperms. New Phytologist, 230, 1829-1843. DOI PMID
[32]	Kikuta SB, Hietz P, Richter H (2003). Vulnerability curves from conifer sapwood sections exposed over solutions with known water potentials. Journal of Experimental Botany, 54, 2149-2155. PMID
[33]	Kiorapostolou N, Galiano-Pérez L, von Arx G, Gessler A, Petit G (2018). Structural and anatomical responses of Pinus sylvestris and Tilia platyphyllos seedlings exposed to water shortage. Trees, 32, 1211-1218.
[34]	Knipfer T, Barrios-Masias FH, Cuneo IF, Bouda M, Albuquerque CP, Brodersen CR, Kluepfel DA, McElrone AJ (2018). Variations in xylem embolism susceptibility under drought between intact saplings of three walnut species. Tree Physiology, 38, 1180-1192. DOI PMID
[35]	Körner C (2019). No need for pipes when the well is dry—A comment on hydraulic failure in trees. Tree Physiology, 39, 695-700.
[36]	Lamacque L, Charrier G, Farnese FDS, Lemaire B, Améglio T, Herbette S (2020). Drought-induced mortality: branch diameter variation reveals a point of no recovery in lavender species. Plant Physiology, 183, 1638-1649. DOI PMID
[37]	Levionnois S, Jansen S, Wandji RT, Beauchêne J, Ziegler C, Coste S, Stahl C, Delzon S, Authier L, Heuret P (2021). Linking drought-induced xylem embolism resistance to wood anatomical traits in neotropical trees. New Phytologist, 229, 1453-1466.
[38]	Li S, Feifel M, Karimi Z, Schuldt B, Choat B, Jansen S (2016). Leaf gas exchange performance and the lethal water potential of five European species during drought. Tree Physiology, 36, 179-192. DOI PMID
[39]	Li S, Jansen S (2017). The root cambium ultrastructure during drought stress in Corylus avellana. IAWA Journal, 38, 67-80.
[40]	Li X, Xi B, Wu X, Choat B, Feng J, Jiang M, Tissue D (2022). Unlocking drought-induced tree mortality: physiological mechanisms to modeling. Frontiers in Plant Science, 13, 835921. DOI: 10.3389/fpls.2022.1126049.
[41]	Liang X, Ye Q, Liu H, Brodribb TJ (2021). Wood density predicts mortality threshold for diverse trees. New Phytologist, 229, 3053-3057. DOI PMID
[42]	Lintunen A, Lindfors L, Nikinmaa E, Hölttä T (2017). Xylem diameter changes during osmotic stress, desiccation and freezing in Pinus sylvestris and Populus tremula. Tree Physiology, 37, 491-500. DOI PMID
[43]	Lu RL, Du Y, Yan LM, Xia JY (2019). A methodological review on identification of tree mortality and their applications. Chinese Science Bulletin, 64, 2395-2409.
	[鲁芮伶, 杜莹, 晏黎明, 夏建阳 (2019). 森林树木死亡的判定方法及其应用综述. 科学通报, 64, 2395-2409.]
[44]	Lu Y, Equiza MA, Deng X, Tyree MT (2010). Recovery of Populus tremuloides seedlings following severe drought causing total leaf mortality and extreme stem embolism. Physiologia Plantarum, 140, 246-257.
[45]	Luo DD, Wang CK, Jin Y (2021). Response mechanisms of hydraulic systems of woody plants to drought stress. Chinese Journal of Plant Ecology, 45, 925-941.
	[罗丹丹, 王传宽, 金鹰 (2021). 木本植物水力系统对干旱胁迫的响应机制. 植物生态学报, 45, 925-941.] DOI
[46]	Mantova M, Herbette S, Cochard H, Torres-Ruiz JM (2022). Hydraulic failure and tree mortality: from correlation to causation. Trends in Plant Science, 27, 335-345.
[47]	Mantova M, Menezes-Silva PE, Badel E, Cochard H, Torres-Ruiz JM (2021). The interplay of hydraulic failure and cell vitality explains tree capacity to recover from drought. Physiologia Plantarum, 172, 247-257.
[48]	Martinez-Vilalta J, Anderegg WRL, Sapes G, Sala AN (2019). Greater focus on water pools may improve our ability to understand and anticipate drought-induced mortality in plants. New Phytologist, 223, 22-32. DOI PMID
[49]	McDowell N, Pockman WT, Allen CD, Breshears DD, Cobb N, Kolb T, Plaut J, Sperry J, West A, Williams DG, Yepez EA (2008). Mechanisms of plant survival and mortality during drought: Why do some plants survive while others succumb to drought? New Phytologist, 178, 719-739. DOI PMID
[50]	McDowell NG, Fisher RA, Xu CG, Domec JC, Hölttä T, MacKay DS, Sperry JS, Boutz A, Dickman L, Gehres N, Limousin JM, Macalady A, Martínez-Vilalta J, Mencuccini M, Plaut JA, et al. (2013). Evaluating theories of drought-induced vegetation mortality using a multimodel- experiment framework. New Phytologist, 200, 304-321. DOI PMID
[51]	McDowell NG, Sapes G, Pivovaroff A, Adams HD, Allen CD, Anderegg WRL, Arend M, Breshears DD, Brodribb T, Choat B, Cochard H, de Cáceres M, de Kauwe MG, Grossiord C, Hammond WM, et al. (2022). Mechanisms of woody-plant mortality under rising drought, CO₂ and vapour pressure deficit. Nature Reviews Earth & Environment, 3, 294-308.
[52]	Meinzer FC, Johnson DM, Lachenbruch B, McCulloh KA, Woodruff DR (2009). Xylem hydraulic safety margins in woody plants: coordination of stomatal control of xylem tension with hydraulic capacitance. Functional Ecology, 23, 922-930.
[53]	Morcillo L, Muñoz-Rengifo JC, Torres-Ruiz JM, Delzon S, Moutahir H, Vilagrosa A (2022). Post-drought conditions and hydraulic dysfunction determine tree resilience and mortality across Mediterranean Aleppo pine (Pinus halepensis) populations after an extreme drought event. Tree Physiology, 42, 1364-1376. DOI PMID
[54]	Morris H, Plavcová L, Cvecko P, Fichtler E, Gillingham MAF, Martínez-Cabrera HI, McGlinn DJ, Wheeler E, Zheng J, Ziemińska K, Jansen S (2016). A global analysis of parenchyma tissue fractions in secondary xylem of seed plants. New Phytologist, 209, 1553-1565. DOI PMID
[55]	Nolan RH, Gauthey A, Losso A, Medlyn BE, Smith R, Chhajed SS, Fuller K, Song M, Li X, Beaumont LJ, Boer MM, Wright IJ, Choat B (2021). Hydraulic failure and tree size linked with canopy die-back in eucalypt forest during extreme drought. New Phytologist, 230, 1354-1365. DOI PMID
[56]	Petrucco L, Nardini A, von Arx G, Saurer M, Cherubini P (2017). Isotope signals and anatomical features in tree rings suggest a role for hydraulic strategies in diffuse drought-induced die-back of Pinus nigra. Tree Physiology, 37, 523-535. DOI PMID
[57]	Preisler Y, Hölttä T, Grünzweig JM, Oz I, Tatarinov F, Ruehr NK, Rotenberg E, Yakir D (2022). The importance of tree internal water storage under drought conditions. Tree Physiology, 42, 771-783.
[58]	Preisler Y, Tatarinov F, Grünzweig JM, Yakir D (2021). Seeking the “point of no return” in the sequence of events leading to mortality of mature trees. Plant, Cell & Environment, 44, 1315-1328.
[59]	Rabert C, Inostroza K, Bravo S, Sepúlveda N, Bravo LA (2020). Exploratory study of fatty acid profile in two filmy ferns with contrasting desiccation tolerance reveal the production of very long chain polyunsaturated omega-3 fatty acids. Plants, 9, 1431. DOI: 10.3390/plants9111431.
[60]	Rawat N, Singla-Pareek SL, Pareek A (2021). Membrane dynamics during individual and combined abiotic stresses in plants and tools to study the same. Physiologia Plantarum, 171, 653-676.
[61]	Rowland L, da Costa ACL, Galbraith DR, Oliveira RS, Binks OJ, Oliveira AAR, Pullen AM, Doughty CE, Metcalfe DB, Vasconcelos SS, Ferreira LV, Malhi Y, Grace J, Mencuccini M, Meir P (2015). Death from drought in tropical forests is triggered by hydraulics not carbon starvation. Nature, 528, 119-122.
[62]	Sala AN (2009). Lack of direct evidence for the carbon- starvation hypothesis to explain drought-induced mortality in trees. Proceedings of the National Academy of Sciences of the United States of America, 106, E68. DOI: 10.1073/pnas.0904580106.
[63]	Salmon Y, Dietrich L, Sevanto S, Hölttä T, Dannoura M, Epron D (2019). Drought impacts on tree phloem: from cell-level responses to ecological significance. Tree Physiology, 39, 173-191. DOI PMID
[64]	Salomón RL, Peters RL, Zweifel R, Sass-Klaassen UGW, Stegehuis AI, Smiljanic M, Poyatos R, Babst F, Cienciala E, Fonti P, Lerink BJW, Lindner M, Martinez-Vilalta J, Mencuccini M, Nabuurs GJ, et al. (2022). The 2018 European heatwave led to stem dehydration but not to consistent growth reductions in forests. Nature Communications, 13, 28. DOI: 10.1038/s41467-021-27579-9.
[65]	Sapes G, Roskilly B, Dobrowski S, Maneta M, Anderegg WRL, Martinez-Vilalta J, Sala AN (2019). Plant water content integrates hydraulics and carbon depletion to predict drought-induced seedling mortality. Tree Physiology, 39, 1300-1312. DOI PMID
[66]	Sapes G, Sala A (2021). Relative water content consistently predicts drought mortality risk in seedling populations with different morphology, physiology and times to death. Plant, Cell & Environment, 44, 3322-3335.
[67]	Sevanto S, Hölttä T, Holbrook NM (2011). Effects of the hydraulic coupling between xylem and phloem on diurnal phloem diameter variation. Plant, Cell & Environment, 34, 690-703.
[68]	Sevanto S, McDowell NG, Dickman LT, Pangle R, Pockman WT (2014). How do trees die? A test of the hydraulic failure and carbon starvation hypotheses. Plant, Cell & Environment, 37, 153-161.
[69]	Shao CC, Luo XY, Ding GJ, Duan HL, Zhao XZ, Lou Q (2022). Effects of drought on hydraulic and anatomical characteristics of stem and leaf in Pinus massoniana. Plant Physiology Journal, 58, 937-945.
	[邵畅畅, 罗仙英, 丁贵杰, 段洪浪, 赵熙州, 娄清 (2022). 干旱对马尾松茎叶水力特征及解剖特性的影响. 植物生理学报, 58, 937-945.]
[70]	Sun MS, Hu Y, Chen X, Luo QF, Yang ZQ (2020). Effects of exogenous regulating substances on physiological characteristics of Erythrophleum fordii seedlings under drought stress. Scientia Silvae Sinicae, 56(10), 165-172.
	[孙明升, 胡颖, 陈旋, 罗群凤, 杨章旗 (2020). 外源调节物质对干旱胁迫下格木幼苗生理特性的影响. 林业科学, 56(10), 165-172.]
[71]	Tian Y, Zhang QL, Liu X, Meng M, Wang B (2019). The relationship between stem diameter shrinkage and tree bole moisture loss due to transpiration. Forests, 10, 290. DOI: 10.3390/f10030290.
[72]	Tomasella M, Häberle KH, Nardini A, Hesse B, Machlet A, Matyssek R (2017). Post-drought hydraulic recovery is accompanied by non-structural carbohydrate depletion in the stem wood of Norway spruce saplings. Scientific Reports, 7, 14308. DOI: 10.1038/s41598-017-14645-w.
[73]	Trifilò P, Kiorapostolou N, Petruzzellis F, Vitti S, Petit G, Lo Gullo MA, Nardini A, Casolo V (2019). Hydraulic recovery from xylem embolism in excised branches of twelve woody species: relationships with parenchyma cells and non-structural carbohydrates. Plant Physiology and Biochemistry, 139, 513-520. DOI PMID
[74]	van de Wal BAE, Leroux O, Steppe K (2018). Post-veraison irreversible stem shrinkage in grapevine (Vitis vinifera) is caused by periderm formation. Tree Physiology, 38, 745-754. DOI PMID
[75]	Venturas MD, Sperry JS, Hacke UG (2017). Plant xylem hydraulics: What we understand, current research, and future challenges. Journal of Integrative Plant Biology, 59, 356-389. DOI
[76]	Wagner Y, Volkov M, Nadal-Sala D, Ruehr NK, Hochberg U, Klein T (2023). Relationships between xylem embolism and tree functioning during drought, recovery, and recurring drought in Aleppo pine. Physiologia Plantarum, 175, e13995. DOI: 10.1111/ppl.13995.
[77]	Wang W, English NB, Grossiord C, Gessler A, Das AJ, Stephenson NL, Baisan CH, Allen CD, McDowell NG (2021). Mortality predispositions of conifers across western USA. New Phytologist, 229, 831-844.
[78]	Yuan X, Wang L, Wu P, Ji P, Sheffield J, Zhang M (2019). Anthropogenic shift towards higher risk of flash drought over China. Nature Communications, 10, 4661. DOI: 10.1038/s41467-019-12692-7.
[79]	Zweifel R, Häsler R (2000). Frost-induced reversible shrinkage of bark of mature subalpine conifers. Agricultural and Forest Meteorology, 102, 213-222.