Impact of freeze-thaw-induced pit aspiration on stem water transport in the subalpine conifer Abies veitchii.

During winter, subalpine conifers experience frequent freeze-thaw cycles in stem xylem that may cause embolism and pit aspiration due to increased water volume during the sap to ice transition. This study examined the occurrence and ecological impacts of a combination of freeze-thaw-induced pit aspiration and embolism triggered by natural and artificial stem freezing. In subalpine Veitch's fir (Abies veitchii) trees, the fraction of closed pits and embolized tracheids as well as conductivity losses were measured to examine pit aspiration and its effects. When trees incurred mild drought stress in February and early March, 70%-80% of stem conductivity was lost. Cryo-scanning electron microscopy indicated <20% embolized tracheids but ∼90% closed pits. Severe drought stress in late March caused 96% ± 1.2% (mean ± standard error) loss of stem conductivity, while the fraction of embolized tracheids increased to 64% ± 6.6%, and aspirated pit fraction decreased to 23% ± 5.6%. Experimental freeze-thaw cycles also increased pit aspiration from 7.1% ± 0.89% to 49% ± 10%, and the fraction of closed pits was positively correlated to the percent loss of stem hydraulic conductivity. The results indicated that freezing-induced pit aspiration is an important factor for stem xylem dysfunction under mild drought, and upon severe drought in winter; stem water transport is predominantly inhibited by xylem embolism.

Imperforate tracheary elements and vessels alleviate xylem tension under severe dehydration: insights from water release curves for excised twigs of three tree species.

PREMISE: Water stored in the xylem of woody plants is important for supporting the transpiration stream under prolonged drought, yet the source of stored water within the xylem during drought remains unclear. Insights into xylem water utilization during drought will uncover the adaptation strategies of the test species to stress. METHODS: To fill the existing knowledge gap, we excised twigs of Abies firma (Japanese fir, conifer), Cercidiphyllum japonicum (katsura tree, diffuse-porous) and Quercus serrata (konara oak, ring-porous) to quantify interspecific variation of water transfer in xylem corresponding with increasing cumulative water release (CWR) using micro x-ray computed tomography and cryo-SEM. RESULTS: For all species studied, the main components of water storage within the operating range of water potential were not living cells but cavitation release and capillaries. Abies firma maintained water in the earlywood-like cells, for possible maintenance of the transpiration stream. Cercidiphyllum japonicum maintained water in its vessels over 200 kg m-3 of CWR, while Q. serrata lost most of its water in vessels with increasing CWR up to 100 kg m-3 . Cercidiphyllum japonicum exhibited a higher water storage capacity than Q. serrata. Under high CWR, narrow conduits stored xylem water in C. japonicum and imperforate tracheary elements in Q. serrata. CONCLUSIONS: Among the species examined, increasing CWR appears to indicate differential utilization of stored water in relation to variation of xylem structure, thereby providing insight into the interspecific responses of tree species to drought.

Cutting stems before relaxing xylem tension induces artefacts in Vitis coignetiae, as evidenced by magnetic resonance imaging.

It was recently reported that cutting artefacts occur in some species when branches under tension are cut, even under water. We used non-destructive magnetic resonance imaging (MRI) to investigate the change in xylem water distribution at the cellular level in Vitis coignetiae standing stems before and after relaxing tension. Less than 3% of vessels were cavitated when stems under tension were cut under water at a position shorter than the maximum vessel length (MVL) from the MRI point, in three of four plants. The vessel contents remained at their original status, and cutting artefact vessel cavitation declined to <1% when stems were cut at a position farther than the MVL from the MRI point. Water infiltration into the originally cavitated vessels after cutting the stem, i.e. vessel refilling, was found in <1% of vessels independent of cutting position on three of nine plants. The results indicate that both vessel cavitation and refilling occur in xylem tissue under tension following stem cutting, but its frequency is quite small, and artefacts can be minimized altogether if the distance between the monitoring position and the cutting point is longer than the MVL.

Vulnerability to cavitation differs between current-year and older xylem: non-destructive observation with a compact magnetic resonance imaging system of two deciduous diffuse-porous species.

Development of xylem embolism during water stress in two diffuse-porous hardwoods, Katsura (Cercidiphyllum japonicum) and Japanese white birch (Betula platyphylla var. japonica), was observed non-destructively under a compact magnetic resonance imaging (MRI) system in addition to conventional quantitation of hydraulic vulnerability to cavitation from excised stem segments. Distribution of white and dark areas in MR images corresponded well to the distribution of water-filled/embolized vessels observed by cryo-scanning electron microscopy in both species. Water-filled vessels were observed in MR images as white areas in Katsura and as white dots in Japanese white birch, respectively, and embolisms could be detected as a change to dark areas. The increase in the relative embolized area (REA: %) in the cross-sectional area of total xylem during water stress, which was estimated from the binarized MR images, was consistent with the hydraulic vulnerability curves of these species. From the non-destructive MRI observations, cavitation induced by water stress was shown to develop earlier in 1- or 2-year-old xylem than in the current-year xylem in both species; that is, the vulnerability to cavitation differs between vessels in the current-year xylem and those in older annual rings.

Pit aspiration causes an apparent loss of xylem hydraulic conductivity in a subalpine fir (Abies mariesii Mast.) overwintering at the alpine timberline.

Conifers growing at the alpine timberline are exposed to combinatorial stresses that induce embolism in xylem during winter. We collected branches of Abies mariesii Mast. at the timberline on Mt Norikura of central Japan to evaluate the seasonal changes in the loss of xylem hydraulic conductivity (percent loss of hydraulic conductivity; PLC). Concurrently, we evaluated the distribution of embolized tracheids in native samples via cryo-scanning electron microscopic (cryo-SEM) observation in comparison with the drought-induced embolism samples used for the vulnerability curve. The twigs collected in late winter showed 100 PLC at a water potential of ~-3 MPa, and air-filled tracheids were observed sporadically in the cryo-SEM images. The cryo-SEM images also showed that nearly all pits of the samples from the timberline were aspirated in the xylem with 100 PLC. Conversely, in drought-induced samples used for vulnerability analysis, lower frequency of aspirated pits was observed at similar water potentials and all tracheids in the earlywood of xylem with 100 PLC were filled with air. We hypothesized that pit aspiration is the primary cause of the pronounced winter xylem conductivity loss at the timberline when water potential is near, but still above, the drought-induced vulnerability threshold. Pit aspiration before water loss may be an adaptation to severe winter conditions at timberlines to prevent widespread xylem embolism. The possible causes of pit aspiration are discussed in relation to complex stresses under harsh winter conditions at timberlines.

Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope.

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water distribution in plant tissues in combination with freeze fixation techniques using liquid nitrogen (LN2). For woody species, however, preparations for observing the xylem transverse-cut surface involve some difficulties due to the orientation of wood fibers. Additionally, higher tension in the water column in xylem conduits can occasionally cause artifactual changes in water distribution, especially during sample fixation and collection. In this study, we demonstrate an efficient procedure to observe the water distribution within the xylem of woody plants in situ by using a cryostat and cryo-SEM. At first, during sample collection, measuring the xylem water potential should determine whether high tension is present in the xylem conduits. When the xylem water potential is low (< ca. -0.5 MPa), a tension relaxation procedure is needed to facilitate better preservation of the water status in xylem conduits during sample freeze fixation. Next, a watertight collar is attached around the tree stem and filled with LN2 for freeze fixation of the water status of xylem. After harvesting, care should be taken to ensure that the sample is preserved frozen while completing the procedures of sample preparation for observation. A cryostat is employed to clearly expose the xylem transverse-cut surface. In cryo-SEM observations, time adjustment for freeze-etching is required to remove frost dust and accentuate the edge of the cell walls on the viewing surface. Our results demonstrate the applicability of cryo-SEM techniques for the observation of water distribution within xylem at cellular and subcellular levels. The combination of cryo-SEM with non-destructive in situ observation techniques will profoundly improve the exploration of woody plant water flow dynamics.

Short-time xylem tension relaxation prevents vessel refilling and alleviates cryo-fixation artifacts in diffuse-porous Carpinus tschonoskii and Cercidiphyllum japonicum.

Xylem tension relaxation is an important procedure that closely resembles the in vivo xylem water distribution when measuring conductivity or observing water distribution of plant tissue samples by cryo-scanning electron microscopy (cryo-SEM). Recent studies have shown that partial xylem embolism occurs when samples under tension are cut under water and that gas-filled vessels are refilled during tension relaxation. Furthermore, the frequency of gas-filled vessels has been reported to increase in samples without tension relaxation before cryo-fixation by liquid nitrogen, particularly in samples with significant tension. Here, we examined the effect of tension relaxation on these artifacts in Carpinus tschonoskii and Cercidiphyllum japonicum using magnetic resonance imaging. We observed that xylem embolism rarely occurs in bench-dried samples cut under water. In both species, a small portion of the xylem was refilled within ~1 h after tension relaxation. Cryo-SEM observations revealed that short-time (<1 h) xylem tension relaxation decreases the frequency of gas-filled vessels in samples frozen after xylem tension relaxation regardless of the water potential compared with that in samples frozen without rehydration in both species. Therefore, short-time tension relaxation is necessary to retain xylem water distribution during sample preparation against artifacts.

Repair of severe winter xylem embolism supports summer water transport and carbon gain in flagged crowns of the subalpine conifer Abies veitchii.

Xylem embolism induced by winter drought is a serious dysfunction in evergreen conifers growing at wind-exposed sites in the mountains. Some coniferous species can recover from winter embolism. The aim of this study was to determine whether wind direction influences embolism formation and/or repair dynamics on short windward and long leeward branches of asymmetrical `flagged' crowns. We analyzed the effect of branch orientation on percentage loss of xylem conductive area (PLC), leaf functional traits and the xylem:leaf area ratio for subalpine, wind-exposed flagged-crown Abies veitchii trees in the northern Yatsugatake Mountains of central Japan. In late winter, the shoot water potential was below -2.5 MPa, and the PLC exceeded 80% in 2-year-old branches, independent of branch orientation within a flagged crown. Both of these parameters almost fully recovered by summer. At branch internodes 4 years of age and older, seasonal changes in PLC were not found in either windward or leeward branches, but the PLC was higher in less leafy windward branches. The leaf nitrogen content and water-use efficiency of mature leaves were comparable between windward branches and leafy leeward branches. The ratio of xylem conductive area to total leaf area was the same for windward and leeward branches. These results indicate that the repair of winter xylem embolism allows leaf physiological functions to be maintained under sufficient leaf water supply, even on winter-wind-exposed branches. This permits substantial photosynthetic carbon gain during the following growing season on both windward and leeward branches. Thus, xylem recovery from winter embolism is a key trait for the survival of harsh winters and to support productivity on the individual level in flagged-crown A. veitchii trees.

The dynamics of carbon stored in xylem sapwood to drought-induced hydraulic stress in mature trees.

Climate-induced forest die-off is widespread in multiple biomes, strongly affecting the species composition, function and primary production in forest ecosystems. Hydraulic failure and carbon starvation in xylem sapwood are major hypotheses to explain drought-induced tree mortality. Because it is difficult to obtain enough field observations on drought-induced mortality in adult trees, the current understanding of the physiological mechanisms for tree die-offs is still controversial. However, the simultaneous examination of water and carbon uses throughout dehydration and rehydration processes in adult trees will contribute to clarify the roles of hydraulic failure and carbon starvation in tree wilting. Here we show the processes of the percent loss of hydraulic conductivity (PLC) and the content of nonstructural carbohydrates (NSCs) of distal branches in woody plants with contrasting water use strategy. Starch was converted to soluble sugar during PLC progression under drought, and the hydraulic conductivity recovered following water supply. The conversion of NSCs is strongly associated with PLC variations during dehydration and rehydration processes, indicating that stored carbon contributes to tree survival under drought; further carbon starvation can advance hydraulic failure. We predict that even slow-progressing drought degrades forest ecosystems via carbon starvation, causing more frequent catastrophic forest die-offs than the present projection.

Recovery of Physiological Traits in Saplings of Invasive Bischofia Tree Compared with Three Species Native to the Bonin Islands under Successive Drought and Irrigation Cycles.

Partial leaf shedding induced by hydraulic failure under prolonged drought can prevent excess water consumption, resulting in delayed recovery of carbon productivity following rainfall. To understand the manner of water use of invasive species in oceanic island forests under a fluctuating water regime, leaf shedding, multiple physiological traits, and the progress of embolism in the stem xylem under repeated drought-irrigation cycles were examined in the potted saplings of an invasive species, Bischofia javanica Blume, and three endemic native species, Schima mertensiana (Sieb. Et Zucc,) Koitz., Hibiscus glaber Matsum, and Distylium lepidotum Nakai, from the Bonin Islands, Japan. The progress of xylem embolism was observed by cryo-scanning electron microscopy. The samples exhibited different processes of water saving and drought tolerance based on the different combinations of partial leaf shedding involved in embolized conduits following repeated de-rehydration. Predawn leaf water potential largely decreased with each successive drought-irrigation cycle for all tree species, except for B. javanica. B. javanica shed leaves conspicuously under drought and showed responsive stomatal conductance to VPD, which contributed to recover leaf gas exchange in the remaining leaves, following a restored water supply. In contrast, native tree species did not completely recover photosynthetic rates during the repeated drought-irrigation cycles. H. glaber and D. lepidotum preserved water in vessels and adjusted leaf osmotic rates but did not actively shed leaves. S. mertensiana exhibited partial leaf shedding during the first cycle with an osmotic adjustment, but they showed less responsive stomatal conductance to VPD. Our data indicate that invasive B. javanica saplings can effectively use water supplied suddenly under drought conditions. We predict that fluctuating precipitation in the future may change tree distributions even in mesic or moist sites in the Bonin Islands.