ROS production during abscisic acid (ABA) and methyl jasmonate (MJ) stimuli in Arabidopsis GC chloroplasts were more than tripled

Figure 1.Induction of ROS production in Arabidopsis guard cells’ chloroplasts by ABA and MJ. (A) Leaf disks were prepared from wild-type plants and treated with ABA and MJ. After incubation of leaf discs in solution with the phytohormones, cells were loaded with H2DCFDA as described in methods. Shown are representative images of single section confocal microscopy (1 μm thick) of guard cells from the different treatments. Scale bar = 10 µm. (B) Quantification of the H2DCFDA-dependent fluorescence of individual chloroplasts shown in (A). Quantification was done using ImagePRO-Plus program from projected images of 2–3 confocal Z stack sections of individual chloroplasts, as described in methods. Shown is a representative one of three replicate experiments. n = 30 chloroplasts from 10 guard cells sampled from three different young rosette leaves (± SE). R.U. relative unites.

Zooming into sub-organellar localization of reactive oxygen species in guard cell chloroplasts during abscisic acid and methyl jasmonate treatments

Leshem Y., Levine A. (2013)

Yehoram Leshem, Alex Levine,

Plant Signal Behav. 8(10): – doi: 10.4161/psb.25689 – PMID: 23887496 – PMCID: PMC4091116 –

https://pubmed.ncbi.nlm.nih.gov/23887496/

Abstract

Regulation of stomata movements is crucial for plants ability to cope with their changing environment. Guard cells’ (GC) water potential directs water flux inside/outside this cell, which eventually is causing the stoma to open or close, respectively. Some of the osmolytes which accumulates in the GC cytoplasm and are known to play a role in stomata opening are sugars, arising from chloroplast starch degradation. During stomata closure, the accumulated osmolytes are removed from the GC cytoplasm. Surprisingly little is known about prevention of starch degradation and forming additional sugars which may interfere with osmotic changes that are necessary for correct closure of stomata.

One of the early events leading to stomata closure is production of reactive oxygen species (ROS) in various subcellular sites and organelles of the stoma. Here we report that ROS production during abscisic acid (ABA) and methyl jasmonate (MJ) stimuli in Arabidopsis GC chloroplasts were more than tripled. Moreover, ROS were detected on the suborganelle level in compartments that are typically occupied by starch grains. This observation leads us to suspect that ROS function in that particular location is necessary for stomata closure. We therefore hypothesize that these ROS are involved in redox control that lead to the inactivation of starch degradation that takes place in these compartments, thus contributing to the stoma closure in an additional way.

ABA elevates H2O2 accumulation in guard cell mitochondria to promote stomatal closure

Abscisic Acid Increases Hydrogen Peroxide in mitochondria to facilitate stomatal closure

Postiglione A. E., Muday G. K. (2022)

Anthony E. Postiglione, Gloria K. Muday,

===

Plant Physiology: kiac601 – https://doi.org/10.1093/plphys/kiac601 –

https://academic.oup.com/plphys/advance-article/doi/10.1093/plphys/kiac601/6961288

Abstract

Abscisic acid (ABA) drives stomatal closure to minimize water loss due to transpiration in response to drought. We examined the subcellular location of ABA-increased accumulation of reactive oxygen species (ROS) in guard cells, which drive stomatal closure, in Arabidopsis (Arabidopsis thaliana). ABA-dependent increases in fluorescence of the generic ROS sensor, dichlorofluorescein (DCF), were observed in mitochondria, chloroplasts, cytosol, and nuclei. The ABA response in all these locations was lost in an ABA-insensitive quintuple receptor mutant. The ABA-increased fluorescence in mitochondria of both DCF- and an H2O2-selective probe, Peroxy Orange 1, colocalized with Mitotracker Red. ABA treatment of guard cells transformed with the genetically encoded H2O2 reporter targeted to the cytoplasm (roGFP2-Orp1), or mitochondria (mt-roGFP2-Orp1), revealed H2O2 increases. Consistent with mitochondrial ROS changes functioning in stomatal closure, we found that guard cells of a mutant with mitochondrial defects, ABA overly sensitive 6 (abo6), have elevated ABA-induced ROS in mitochondria and enhanced stomatal closure. These effects were phenocopied with rotenone, which increased mitochondrial ROS. In contrast, the mitochondrially targeted antioxidant, MitoQ, dampened ABA effects on mitochondrial ROS accumulation and stomatal closure in Col-0 and reversed the guard cell closure phenotype of the abo6 mutant. ABA-induced ROS accumulation in guard cell mitochondria was lost in mutants in genes encoding respiratory burst oxidase homolog (RBOH) enzymes and reduced by treatment with the RBOH inhibitor, VAS2870, consistent with RBOH machinery acting in ABA-increased ROS in guard cell mitochondria. These results demonstrate that ABA elevates H2O2 accumulation in guard cell mitochondria to promote stomatal closure.

An Initial Step of Phototropin Signaling in Stomatal Guard Cells

Reconstitution of an Initial Step of Phototropin Signaling in Stomatal Guard Cells

Takemiya A., Doi A., Yoshida S., Okajima K., Tokutomi S., Shimazaki K. (2016)

Atsushi Takemiya 1, Ayaka Doi 1, Sayumi Yoshida 1, Koji Okajima 2, Satoru Tokutomi 3, Ken-Ichiro Shimazaki 1,

  • 1 Department of Biology, Faculty of Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395 Japan
  • 2 Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka, 599-851 Japan Present address: Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Kanagawa, 223-8522 Japan.
  • 3 Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka, 599-851 Japan

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Plant Cell Physiol. 57(1):152-159 – doi: 10.1093/pcp/pcv180 – Epub 2015 Dec 26 – PMID: 26707730 –

https://pubmed.ncbi.nlm.nih.gov/26707730/

Abstract

Phototropins are light-activated receptor kinases that mediate a wide range of blue light responses responsible for the optimization of photosynthesis. Despite the physiological importance of phototropins, it is still unclear how they transduce light signals into physiological responses. Here, we succeeded in reproducing a primary step of phototropin signaling in vitro using a physiological substrate of phototropin, the BLUS1 (BLUE LIGHT SIGNALING1) kinase of guard cells. When PHOT1 and BLUS1 were expressed in Escherichia coli and the resulting recombinant proteins were incubated with ATP, white and blue light induced phosphorylation of BLUS1 but red light and darkness did not. Site-directed mutagenesis of PHOT1 and BLUS1 revealed that the phosphorylation was catalyzed by phot1 kinase. Similar to stomatal blue light responses, the BLUS1 phosphorylation depended on the fluence rate of blue light and was inhibited by protein kinase inhibitors, K-252a and staurosporine. In contrast to the result in vivo, BLUS1 was not dephosphorylated in vitro, suggesting the involvement of a protein phosphatase in the response in vivo. phot1 with a C-terminal kinase domain but devoid of the N-terminal domain, constitutively phosphorylated BLUS1 without blue light, indicating that the N-terminal domain has an autoinhibitory action and prevents substrate phosphorylation. The results provide the first reconstitution of a primary step of phototropin signaling and a clue for understanding the molecular nature of this process.

phot1 and phot2 phosphorylate BLUE LIGHT SIGNALING1 (BLUS1) kinase as a common substrate in stomatal opening

Arabidopsis phot1 and phot2 phosphorylate BLUS1 kinase with different efficiencies in stomatal opening

Takemiya A, Shimazaki K. (2016)

Atsushi Takemiya 1, Ken-ichiro Shimazaki 2,

  • 1 Department of Biology, Faculty of Science, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan.
  • 2 Department of Biology, Faculty of Science, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan.

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J Plant Res. 129(2): 167-174 – doi: 10.1007/s10265-015-0780-1 – Epub 2016 Jan 16 – PMID: 26780063 –

https://pubmed.ncbi.nlm.nih.gov/26780063/

Abstract

In Arabidopsis thaliana, phototropins (phot1 and phot2), light-activated receptor kinases, redundantly regulate various photoresponses such as phototropism, chloroplast photorelocation movement, stomatal opening, and leaf flattening. However, it is still unclear how phot1 and phot2 signals are integrated into a common target and regulate physiological responses. In the present study, we provide evidence that phot1 and phot2 phosphorylate BLUE LIGHT SIGNALING1 (BLUS1) kinase as a common substrate in stomatal opening. Biochemical analysis revealed that the recombinant phot2 protein directly phosphorylated BLUS1 in vitro in a blue light-dependent manner, as reported for phot1. BLUS1 phosphorylation was observed in both phot1 and phot2 mutants, and phot2 mutant exhibited higher phosphorylation of BLUS1 than did phot1 mutant. Transgenic plants expressing phot1-GFP (P1G) and phot2-GFP (P2G) at a similar level under the PHOT2 promoter demonstrated that P1G initiated higher phosphorylation of BLUS1 than P2G, suggesting that phot1 phosphorylates BLUS1 more efficiently. Similarly, P1G mediated a higher activation of the plasma membrane H(+)-ATPase and stomatal opening than P2G, indicating that the phosphorylation status of BLUS1 is a key determinant of physiological response. Together, these findings provide insights into the signal integration and different properties of phot1 and phot2 signaling.

The role of CIPK23 as a newly identified component of phototropin signaling in stomatal guard cells is discussed

CIPK23 regulates blue light-dependent stomatal opening in Arabidopsis thaliana

Inoue S.-I., Kaiserli E., Zhao X., Waksman T., Takemiya A., Okumura M., Takahashi H., Seki M., Shinozaki K., Endo Y., Sawasaki T., Kinoshita T., Zhang X., Christie J. M., Shimazaki K. I. (2020)

Shin-Ichiro Inoue 1, Eirini Kaiserli 2, Xiang Zhao 3, Thomas Waksman 2, Atsushi Takemiya 4, Masaki Okumura 1, Hirotaka Takahashi 5, Motoaki Seki 6 7, Kazuo Shinozaki 8, Yaeta Endo 9, Tatsuya Sawasaki 5, Toshinori Kinoshita 10, Xiao Zhang 3, John M Christie 2, Ken-Ichiro Shimazaki 4,

  • 1 Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan.
  • 2 Institute of Molecular Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK.
  • 3 Institute of Plant Stress Biology, State Key Laboratory of Cotton Biology, School of Life Sciences, Henan University, Kaifeng, 475004, People’s Republic of China.
  • 4 Department of Biology, Faculty of Science, Kyushu University, 744 Motooka, Fukuoka, 819-0395, Japan.
  • 5 Proteo-Science Center, Ehime University, Matsuyama, 790-8577, Japan.
  • 6 RIKEN Cluster for Pioneering Research, 2-1 Hirosawa, Wako, 351-0198, Japan.
  • 7 RIKEN Center for Sustainable Resource Science, 1-7-22, Suehiro, Tsurumi-ku, Yokohama, 230-0045, Japan.
  • 8 Gene Discovery Research Group, RIKEN Center for Sustainable Resource Science, 3-1-1 Koyadai, Tsukuba, 305-0074, Japan.
  • 9 Institute for the Promotion of Science and Technology, Ehime University, Matsuyama, 790-8577, Japan.
  • 10 Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Chikusa, Nagoya, 464-8602, Japan.

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Plant J. 104(3): 679-692 – doi: 10.1111/tpj.14955 – Epub 2020 Sep 1 – PMID: 32780529 – PMCID: PMC7693358 –

https://pubmed.ncbi.nlm.nih.gov/32780529/

Abstract

Phototropins (phot1 and phot2) are plant blue light receptor kinases that function to mediate phototropism, chloroplast movement, leaf flattening, and stomatal opening in Arabidopsis. Considerable progress has been made in understanding the mechanisms associated with phototropin receptor activation by light. However, the identities of phototropin signaling components are less well understood by comparison. In this study, we specifically searched for protein kinases that interact with phototropins by using an in vitro screening method (AlphaScreen) to profile interactions against an Arabidopsis protein kinase library. We found that CBL-interacting protein kinase 23 (CIPK23) interacts with both phot1 and phot2. Although these interactions were verified by in vitro pull-down and in vivo bimolecular fluorescence complementation assays, CIPK23 was not phosphorylated by phot1, as least in vitro. Mutants lacking CIPK23 were found to exhibit impaired stomatal opening in response to blue light but no deficits in other phototropin-mediated responses. We further found that blue light activation of inward-rectifying K+ (K+ in ) channels was impaired in the guard cells of cipk23 mutants, whereas activation of the plasma membrane H+ -ATPase was not. The blue light activation of K+ in channels was also impaired in the mutant of BLUS1, which is one of the phototropin substrates in guard cells. We therefore conclude that CIPK23 promotes stomatal opening through activation of K+ in channels most likely in concert with BLUS1, but through a mechanism other than activation of the H+ -ATPase. The role of CIPK23 as a newly identified component of phototropin signaling in stomatal guard cells is discussed.

A new role for chloroplast membrane oxylipin metabolism in stomatal regulation

Light-induced stomatal opening in Arabidopsis is negatively regulated by chloroplast-originated OPDA signaling

Chang Y., Shi M., Sun Y., Cheng H., Ou X., Zhao Y., Zhang X., Day B., Miao C., Jiang K. (2023)

Yuankai Chang 1, Mianmian Shi 2, Yanfeng Sun 1, Hui Cheng 1, Xiaobin Ou 2, Yi Zhao 3, Xuebin Zhang 1, Brad Day 3, Chen Miao 4, Kun Jiang 5,

  • 1 State Key Laboratory of Cotton Biology, School of Life Sciences, Henan University, Kaifeng 475004, Henan Province, China.
  • 2 College of Life Sciences, Zhejiang University, Hangzhou 310058, Zhejiang Province, China.
  • 3 Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI 48824, USA.
  • 4 State Key Laboratory of Cotton Biology, School of Life Sciences, Henan University, Kaifeng 475004, Henan Province, China.
  • 5 College of Life Sciences, Zhejiang University, Hangzhou 310058, Zhejiang Province, China.

===

Curr. Biol. 33: 1071-1081. e5 – doi: 10.1016/j.cub.2023.02.012 – Epub 2023 Feb 24 –

https://pubmed.ncbi.nlm.nih.gov/36841238/

Abstract

Stomatal movement is orchestrated by diverse signaling cascades and metabolic activities in guard cells. Light triggers the opening of the pores through the phototropin-mediated pathway, which leads to the activation of plasma membrane H+-ATPase and thereby facilitates potassium accumulation through Kin+ channels. However, it remains poorly understood how phototropin signaling is fine-tuned to prevent excessive stomatal opening and consequent water loss. Here, we show that the stomatal response to light is negatively regulated by 12-oxo-phytodienoic acid (OPDA), an oxylipin metabolite produced through enzymatic oxygenation of polyunsaturated fatty acids (PUFAs). We identify a set of phospholipase-encoding genes, phospholipase (PLIP)1/2/3, which are transactivated rapidly in guard cells upon illumination in a phototropin-dependent manner. These phospholipases release PUFAs from the chloroplast membrane, which is oxidized by guard-cell lipoxygenases and further metabolized to OPDA. The OPDA-deficient mutants had wider stomatal pores, whereas mutants containing elevated levels of OPDA showed the opposite effect on stomatal aperture. Transmembrane solute fluxes that drive stomatal aperture were enhanced in lox6-1 guard cells, indicating that OPDA signaling ultimately impacts on activities of proton pumps and Kin+ channels. Interestingly, the accelerated stomatal kinetics in lox6-1 leads to increased plant growth without cost in water or macronutrient use. Together, our results reveal a new role for chloroplast membrane oxylipin metabolism in stomatal regulation. Moreover, the accelerated stomatal opening kinetics in OPDA-deficient mutants benefits plant growth and water use efficiency.

Maintaining water transport integrity rather than fulfilling requirements for carbon uptake is the major factor impacting tree-level stomatal regulation

Tree-level stomatal regulation is more closely related to xylem hydraulic traits than to leaf photosynthetic traits across diverse tree species

Hu Y., Sun Z., Zeng Y., Ouyang S., Chen L., Lei P., Deng X., Zhao Z., Fang X., Xiang W.(2023)

Yanting Hu, Zhihui Sun, Yelin Zeng, Shuai Ouyang, Liang Chen, Pifeng Lei, Xiangwen Deng, Zhonghui Zhao, Xi Fang, Wenhua Xiang,

===

Agricultural and Forest Meteorology 329: 109291 – ISSN 0168-1923 –
https://doi.org/10.1016/j.agrformet.2022.109291 –

https://www.sciencedirect.com/science/article/pii/S0168192322004774

Highlights

• Xylem hydraulic traits were highly correlated to tree-level stomatal regulation.

• Leaf photosynthetic traits showed no correlations with Lcond.

• Tree species with higher hydraulic efficiency took isohydric stomatal regulation.

• Species inhabiting humid region adopt avoidance strategy in stomatal regulation.

• Lcond displayed a phylogenetic signal with a low Blomberg’s K.

Abstract

Understanding stomatal regulatory mechanisms across various woody species is helpful for explaining their adaptations to diverse environmental conditions. Stomatal opening and closing are driven by the requirements for maintaining water transport integrity and carbon uptake; however, distinguishing which factor plays a dominant role in the regulation of tree-level stomatal behavior has seldom been explored. To address this knowledge gap, we investigated differences in tree-level iso/anisohydric stomatal regulation among various tree species (61 and 59 species, at daily and hourly timescales, respectively) across diverse biomes, and analyzed the association of tree-level stomatal regulation with xylem hydraulic and leaf photosynthetic traits. Xylem hydraulic traits were closely related to tree-level stomatal regulation, whereas leaf photosynthetic traits showed non-significant correlations. Reduction in tree-level stomatal conductance with the same vapor pressure deficit increment (ranging from 0.6 to 2 kPa; Lcond), representing the degree of iso/anisohydry, was positively correlated to xylem pressure inducing 50% loss of hydraulic conductivity and vessel diameter, but negatively correlated to the hydraulic safety margin. The associations between xylem hydraulic traits and Lcond revealed that tree species with greater xylem hydraulic efficiency were more likely to adopt an avoidance strategy for tree-level stomatal regulation, whereas tolerance strategy occurred in species with a stronger hydraulic safety system. Furthermore, Lcond was positively correlated to mean annual precipitation and temperature, suggesting that species inhabiting humid and warm regions rely upon isohydric stomatal regulation. Moreover, Lcond displayed a phylogenetic signal, suggesting that variation in Lcond has been influenced by evolutionary history. Overall, tree-level stomatal regulation is more closely related to xylem hydraulic traits than to leaf photosynthetic traits, and maintaining water transport integrity rather than fulfilling requirements for carbon uptake is the major factor impacting tree-level stomatal regulation.

Midday measurements of stomatal conductance and Ψl were significantly correlated with one another

Midday measurements of leaf water potential and stomatal conductance are highly correlated with daily water use of Thompson Seedless grapevines

Williams L. E., Baeza P., Vaughn P. (2012)

Irrig Sci 30: 201–212 – https://doi.org/10.1007/s00271-011-0276-2 –

https://link.springer.com/article/10.1007/s00271-011-0276-2

Abstract

A study was conducted to determine the relationship between midday measurements of vine water status and daily water use of grapevines measured with a weighing lysimeter. Water applications to the vines were terminated on August 24th for 9 days and again on September 14th for 22 days. Daily water use of the vines in the lysimeter (ETLYS) was approximately 40 L vine−1 (5.3 mm) prior to turning the pump off, and it decreased to 22.3 L vine−1 by September 2nd. Pre-dawn leaf water potential (ΨPD) and midday Ψl on August 24th were −0.075 and −0.76 MPa, respectively, with midday Ψl decreasing to −1.28 MPa on September 2nd. Leaf g s decreased from ~500 to ~200 mmol m−2 s−1 during the two dry-down periods. Midday measurements of g s and Ψl were significantly correlated with one another (r = 0.96) and both with ETLYS/ETo (r = ~0.9). The decreases in Ψl, g s, and ETLYS/ETo in this study were also a linear function of the decrease in volumetric soil water content. The results indicate that even modest water stress can greatly reduce grapevine water use and that short-term measures of vine water status taken at midday are a reflection of daily grapevine water use.

Comparing stomatal responses in Grenache and Shiraz grape cultivars to contrasting vapour pressure deficit (VPD)

Grape vine varieties shiraz and grenache differ in their stomatal response to VPD: apparent links with ABA physiology and gene expression in leaf tissue

Soar C. J., Speirs J., Maffei S. M., Penrose A. B., McCarthy M. G., Loveys B. R. (2006)

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Australian Journal of Grape and Wine Research 12(1): 2-12 – https://doi.org/10.1111/j.1755-0238.2006.tb00038.x –

https://onlinelibrary.wiley.com/doi/10.1111/j.1755-0238.2006.tb00038.x

Abstract

Two cultivars of Vitis vinifera L., namely Grenache and Shiraz, have been described as having near-isohydric and near-anisohydric responses respectively to soil water stress (Schultz, Plant Cell and Environment, 26, 1393–1405, 2003). Given that contrast in sensitivity to soil water, a question arises as to whether atmospheric moisture stress will elicit similar differences. The present study was undertaken to investigate this issue by comparing stomatal responses in these same two cultivars to contrasting vapour pressure deficit (VPD). Test material included field grape vines in the Barossa Valley and pot-grown vines under partial shade in Adelaide. Our experiments showed that the same isohydric/anisohydric distinction as described by Schultz (2003) is apparent in leaf responses to atmospheric moisture stress. In the more isohydric cultivar, Grenache, stomatal conductance is more responsive to changes in VPD. This heightened sensitivity (compared with Shiraz) appears to be associated with higher levels of abscisic acid (ABA) in Grenache xylem sap. Expression studies on the key genes in the ABA biosynthetic pathway indicate that regulation of the V.v.nced1 gene expression in leaf tissue, but not in the root tissues, is associated with the changes in the xylem sap ABA. Moreover, the two cultivars (Grenache and Shiraz) differed with respect to both scale and time course of those responses. We conclude that these two Vitis vinifera cultivars do indeed differ significantly in the way that they respond to potentially stressful atmospheric conditions, and that ABA physiology is a key process in these contrasting responses. An understanding of such mechanisms, including the relative importance of roots and shoots in determining vine response to abiotic stress, is highly relevant to irrigation scheduling, and to management of associated variation in vineyard productivity across diverse environments.

Plasticity in stomatal regulation in an isohydric (Eucalyptus camaldulensis) and an anisohydric (Acacia aptaneura) angiosperm species subject to repeated drying cycles

Differences in osmotic adjustment, foliar abscisic acid dynamics, and stomatal regulation between an isohydric and anisohydric woody angiosperm during drought

Nolan R. H., Tarin T., Santini N. S., McAdam S. A. M., Ruman R., Eamus D. (2017)

Rachael H. Nolan, Tonantzin Tarin, Nadia S. Santini, Scott A.M. McAdam, Rizwana Ruman, Derek Eamus,

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Plant, Cell & Environment 40(12): 3122-3134 – https://doi.org/10.1111/pce.13077 –

https://onlinelibrary.wiley.com/doi/10.1111/pce.13077

Abstract

Species are often classified along a continuum from isohydric to anisohydric, with isohydric species exhibiting tighter regulation of leaf water potential through stomatal closure in response to drought. We investigated plasticity in stomatal regulation in an isohydric (Eucalyptus camaldulensis) and an anisohydric (Acacia aptaneura) angiosperm species subject to repeated drying cycles. We also assessed foliar abscisic acid (ABA) content dynamics, aboveground/belowground biomass allocation and nonstructural carbohydrates. The anisohydric species exhibited large plasticity in the turgor loss point (ΨTLP), with plants subject to repeated drying exhibiting lower ΨTLP and correspondingly larger stomatal conductance at low water potential, compared to plants not previously exposed to drought. The anisohydric species exhibited a switch from ABA to water potential-driven stomatal closure during drought, a response previously only reported for anisohydric gymnosperms. The isohydric species showed little osmotic adjustment, with no evidence of switching to water potential-driven stomatal closure, but did exhibit increased root:shoot ratios. There were no differences in carbohydrate depletion between species. We conclude that a large range in ΨTLP and biphasic ABA dynamics are indicative of anisohydric species, and these traits are associated with exposure to low minimum foliar water potential, dense sapwood and large resistance to xylem embolism.