Introduction
⌅Stomatal conductance is considered a major determinant of photosynthesis and a breeding target for improving crop productivity under the expected rising temperatures and more frequent episodes of drought (Faralli et al., 2019Faralli M, Matthews J, Lawson T, 2019. Exploiting natural variation and genetic manipulation of stomatal conductance for crop improvement. Curr Opin Plant Biol 49: 1-7. https://doi.org/10.1016/j.pbi.2019.01.003). Evaluating the stomatal response to water shortage seems also interesting under a scenario of increasing atmospheric CO2, Contrary to what has been frequently accepted, that plants respond with decreasing stomatal conductance to elevated CO2, Purcell et al. (2018)Purcell C, Batke SP, Yiotis C, Caballero R, Soh WK, Murray M, et al., 2018. Increasing stomatal conductance in response to rising atmospheric CO2. Ann Bot 121: 1137-1149. https://doi.org/10.1093/aob/mcx208 found that stomatal conductance may increase in woody species adapted to warm, low-humidity conditions.
Stomata sensitivity to external and internal factors differs between and within species (Klein, 2014Klein T, 2014. The variability of stomatal sensitivity to leaf water potential across tree species indicates a continuum between isohydric and anisohydric behaviours. Funct Ecol 28: 1313-1320. https://doi.org/10.1111/1365-2435.12289; Cañete-Salinas et al., 2019Cañete-Salinas P, Zamudio F, Yáñez M, Gyenge J, Valdés H, Espinosa C et al., 2019. Responses in growth and physiological traits in two Populus × canadensis clones (‘I-214’ and ‘I-488’) submitted to different irrigation frequencies in central Chile. For Ecol Manage 449: 117455. https://doi.org/10.1016/j.foreco.2019.117455), affecting carbon uptake and contributing to explain why plant species differ so much in their rate of biomass production. In this regard, Brodribb & Holbrook (2004)Brodribb TJ, Holbrook NM, 2004. Stomatal protection against hydraulic failure: a comparison of coexisting ferns and angiosperms. New Phytol 162: 663-670. https://doi.org/10.1111/j.1469-8137.2004.01060.x suggested that early stomatal closure could be an explanation for the low evolutionary success of pteridophytes relative to angiosperms in seasonally and perennially dry habitats. Stomatal closure decreases the chance for evaporative cooling, a common mechanism to cope with high temperatures, which seems particularly relevant given the increased frequency of heat waves (López et al., 2022López R, Ramírez-Valiente JA, Pita P, 2022. How plants cope with heatwaves in a drier environment. Flora 295: 152148. https://doi.org/10.1016/j.flora.2022.152148). In a previous study, we measured the lowest stomatal conductance in the Eucalyptus globulus Labill. clone that reached the lowest growth (Hernandez et al., 2016Hernandez MJ, Montes F, Ruiz F, López G, Pita P, 2016. The effect of vapour pressure deficit on stomatal conductance, sap pH and leaf-specific hydraulic conductance in Eucalyptus globulus clones grown under two watering regimes. Ann Bot 117: 1063-1071. https://doi.org/10.1093/aob/mcw031).
Plant growth depends on the uptake of carbon, nitrogen and other essential resources, but also on how those resources are used. Biomass allocation has widely been considered a major trait conditioning plants’ performance, growth and productivity (Poorter & Sack, 2012Poorter H, Sack L, 2012. Pitfalls and possibilities in the analysis of biomass allocation patterns in plants. Front Plant Sci 3. https://doi.org/10.3389/fpls.2012.00259). For fast-growing tree species, a rapid development of leaf area would be most relevant in determining growth (Mira et al., 2023Mira E, Cochard H, Evette A, Dulormne M, 2023. Growth, xylem vulnerability to cavitation and leaf cell response to dehydration in tree seedlings of the Caribbean dry forest. Forests 14: 697. https://doi.org/10.3390/f14040697). Prioritizing leaf area growth would come at the cost of decreasing biomass allocation to other organs and/or producing leaves with a higher specific leaf area (SLA). This parameter, calculated as the ratio of leaf area to leaf dry weight, is considered a key factor in determining slow growth in herbaceous species (Lambers & Oliveira, 2019Lambers H, Oliveira RS, 2019. Plant physiological ecology, 3rd Ed. Springer, Cham, Switzerland, 736 pp. https://doi.org/10.1007/978-3-030-29639-1) and has shown to be a reliable indicator of growth in a number of woody species (Diémé et al., 2019Diémé JS, Armas C, Rusch GM, Pugnaire FI, 2019. Functional responses of four Sahelian tree species to resource availability. Flora 254: 181-187. https://doi.org/10.1016/j.flora.2018.10.009; Lambers & Oliveira, 2019Lambers H, Oliveira RS, 2019. Plant physiological ecology, 3rd Ed. Springer, Cham, Switzerland, 736 pp. https://doi.org/10.1007/978-3-030-29639-1; Rodríguez et al., 2020Rodríguez ME, Lauff D, Cortizo S, Luquez VMC, 2020. Variability in flooding tolerance, growth and leaf traits in a Populus deltoides intraspecific progeny. Tree Physiol 40: 19-29. https://doi.org/10.1093/treephys/tpz128).
Breeding programs established in dry environments must seek a balance between improved growth and improved stress tolerance. This is a complex task, since most of the responses that help tolerate water stress involve a loss of growth for the plant. For instance, species or provenances adapted to drought-prone environments typically show a low vulnerability to water stress-induced cavitation (López et al., 2013López R, López de Heredia U, Collada C, Cano FJ, Emerson BC, Cochard H, et al., 2013. Vulnerability to cavitation, hydraulic efficiency, growth and survival in an insular pine (Pinus canariensis). Ann Bot 111(6): 1167-1179. https://doi.org/10.1093/aob/mct084). Cavitation resistance tends to increase with wood density (Barotto et al., 2018Barotto AJ, Monteoliva S, Gyenge J, Martinez-Meier A, Fernandez ME, 2018. Functional relationships between wood structure and vulnerability to xylem cavitation in races of Eucalyptus globulus differing in wood density. Tree Physiol 38: 243-251. https://doi.org/10.1093/treephys/tpx138), which is costly in terms of biomass allocation to non-photosynthetic tissues. In addition, leaf-specific hydraulic conductance may decrease under water shortage as reported for E. globulus (Hernandez et al., 2016Hernandez MJ, Montes F, Ruiz F, López G, Pita P, 2016. The effect of vapour pressure deficit on stomatal conductance, sap pH and leaf-specific hydraulic conductance in Eucalyptus globulus clones grown under two watering regimes. Ann Bot 117: 1063-1071. https://doi.org/10.1093/aob/mcw031), leading to a reduction in water supply to the leaves, photosynthesis and growth (Zhang & Cao, 2009Zhang JL, Cao KF, 2009. Stem hydraulics mediates leaf water status, carbon gain, nutrient use efficiencies and plant growth rates across dipterocarp species. Funct Ecol 23: 658-667. https://doi.org/10.1111/j.1365-2435.2009.01552.x).
While numerous studies highlight the relevance of tree breeding for improved drought resistance, mainly in the scope of global change (Polle et al., 2019Polle A, Chen SL, Eckert C, Harfouche A, 2019. Engineering drought resistance in forest trees. Front Plant Sci 9: 1875. https://doi.org/10.3389/fpls.2018.01875; Taylor et al., 2019Taylor G, Donnison IS, Murphy-Bokern D, Morgante M, Bogeat-Triboulot MB, Bhalerao R, et al., 2019. Sustainable bioenergy for climate mitigation: developing drought-tolerant trees and grasses. Ann Bot 124: 513-520. https://doi.org/10.1093/aob/mcz146), studies facing the challenge of improving tree growth in water-limited environments are much less abundant and show contrasting results. According to Wikberg & Ögren (2007)Wikberg J, Ögren E, 2007. Variation in drought resistance, drought acclimation and water conservation in four willow cultivars used for biomass production. Tree Physiol 27: 1339-1346. https://doi.org/10.1093/treephys/27.9.1339, increased drought resistance results in decreased growth in Salix. Conversely, selection for both drought resistance and improved growth would be possible in Pinus contorta (Wang et al., 2003Wang T, Aitken SN, Kavanagh KL, 2003. Selection for improved growth and wood quality in lodgepole pine: effects on phenology, hydraulic architecture and growth of seedlings. Trees 17: 269-277. https://doi.org/10.1007/s00468-002-0236-9). Growth was unaffected by mild water stress in poplar (Populus tremula × Populus alba) selected lines showing enhanced tolerance to water deficit (Sow et al., 2021Sow MD, Le Gac AL, Fichot R, Lanciano S, Delaunay A, Le Jan I, et al., 2021. RNAi suppression of DNA methylation affects the drought stress response and genome integrity in transgenic poplar. New Phytol 232(1): 80-97. https://doi.org/10.1111/nph.17555). Moreover, breeding for improved growth had opposite effects on drought tolerance in coastal and interior provenances of Pinus radiata in Chile (Espinoza et al., 2016Espinoza SE, Magni CR, Santelices RE, Ivković M, Cabrera AM, 2016. Changes in drought tolerance of Pinus radiata in Chile associated with provenance and breeding generation. Ann For Sci 73: 267-275. https://doi.org/10.1007/s13595-015-0498-1).
Fast-growing species are prone to water stress in the early stages of growth because of their tendency to allocate a greater proportion of biomass to leaves than to roots (Brodribb & Hill, 2000Brodribb TJ, Hill RS, 2000. Increases in water potential gradient reduce xylem conductivity in whole plants. Evidence from a low-pressure conductivity method. Plant Physiol 123: 1021-1028. https://doi.org/10.1104/pp.123.3.1021). Eucalypt plantations established in southwest Spain often suffer from the combination of drought and high summer temperatures. Breeding programs established for E. globulus have taken advantage of hybrid vigor, also called heterosis, which is widely used in plant and animal production. Heterosis can be defined as the phenomenon by which hybrids (F1 genotypes) are superior to parents (F0) in terms of yield, growth rate, viability, and disease resistance (Wu et al., 2021Wu X, Liu Y, Zhang Y, Gu R, 2021. Advances in research on the mechanism of heterosis in plants. Front Plant Sci 12: 745726. https://doi.org/10.3389/fpls.2021.745726). The mechanisms underlying hybrid vigor remain to be fully elucidated (Wu et al., 2021Wu X, Liu Y, Zhang Y, Gu R, 2021. Advances in research on the mechanism of heterosis in plants. Front Plant Sci 12: 745726. https://doi.org/10.3389/fpls.2021.745726).
We carried out a greenhouse experiment with two F0 and four F1 E. globulus clones differing in survival and growth in dry Mediterranean climate conditions, as shown in two trial plantations established in SW Spain. We aimed at identifying differences in the response to water shortage between clones that could contribute to explain the observed differences in growth and survival in the field. We investigated the following hypotheses: (1) regardless of water availability, clones with lower SLA will grow less; (2) in the event of a water scarcity, the maximum leaf-specific hydraulic conductivity will drop and remain greater in clones that develop the fastest; (3) clones that grow more quickly in dry conditions should be able to achieve larger stomatal conductances.
Material and methods
⌅Plant material, irrigation treatments and growth conditions
⌅ENCE Energía y Celulosa S.A. supplied the plants, which belonged to two F0 and four F1 clones from the company’s breeding program. The parental (F0) clones, C13 and C14, have been used regularly in commercial plantations established in SW Spain. The hybrid (F1) clones (H463, H354, H231) come from crosses of clone C14 with other F0 clones, while clone H491 comes from the self-crossing of C14. Table 1 shows the results obtained by the company three years after planting, in two field trials. Survival and growth were higher in the trial established on better soil, as expected. The worst results in survival and growth were measured for clone H491. Excluding this clone, survival values were similar for F0 and F1 clones, while growth was generally higher in F1 clones.
| C13 | C14 | H231 | H354 | H463 | H491:inbred | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | |
| Survival (%) | 75 | 73 | 75 | 80 | 70 | 95 | 60 | 55 | 55 | 80 | 55 | 50 |
| Height (m) | 6.4 | 12.4 | 5.7 | 12.4 | 6.7 | 14.0 | 7.6 | 13.8 | 7.4 | 13.1 | 4.8 | 8.4 |
| DBHob (cm) | 6.4 | 11.0 | 5.4 | 10.9 | 6.8 | 13.7 | 6.9 | 13.5 | 7.1 | 12.9 | 4.5 | 6.0 |
Data kindly provided by ENCE Energía y Celulosa S.A.
Twenty plants of each clone were transplanted into 3-L pots filled with the same weight of a mixture of peat and sand (3:1 v/v). At this time the plants had an average of ten whorls. The average height ranged 20-25 cm and was highest in clone C14 and lowest in H463. Plants were placed inside a greenhouse where minimum temperature remained above 16ºC and maximum temperature was kept below 32ºC. Maximum photosynthetic photon flux density (PPFD) was 1700 mmol m-2 s-1.
The plants were maintained under optimum irrigation conditions for six weeks and then two irrigation treatments were established, as explained below. Ten plants per clone were randomly assigned to each treatment. From this day (April 29, d0) all the plants were irrigated once to three times a week, when the first symptoms of turgor loss were observed. Irrigation was carried out by placing the plants on a balance and adding water until reaching the values established as target weight in each treatment. The maximum target weight was established for the high-watering (HW) treatment based on previous tests and corresponds to the maximum amount of water that could be added guaranteeing that there would be no loss of water by percolation after irrigation. For the low-watering (LW) treatment, a target weight 10% lower than that of the HW treatment was set. These irrigation treatments were applied for 35 days. From d35 till the end (d55), all plants were watered in the same way, using the target weight of the HW treatment.
In order to better characterize the irrigation treatments, predawn leaf water potential (Y) was measured in the first fully expanded leaf, on days d9 and d14. Plants were moved to a growth chamber in the afternoon of days d8 and d13, kept in the dark until measured in the early morning and then returned to the greenhouse. After measuring the water potential, we recorded the weight of each plant (WT) and measured volumetric soil water content (SWC) with a TDR probe (Trime-FM, IMKO Micromodultechnik Gmbh, Ettlingen, Germany). These measurements were made on four plants per clone and treatment on day d9 and eight plants per clone of the LW treatment on day d14. Soil water content and WT were also measured at the time of harvest. We found a strong linear relationship between both parameters, with no differences between lines fitted to data measured on different dates, so we used the whole data set to obtain the following regression equation:
We used this equation to calculate SWC at the time of gas exchange measurements to minimize damages to the root system from the TDR probe.
Growth and specific leaf area
⌅To estimate leaf area values at the beginning of the experiment, we made tracings of all leaves in a subsample of two plants per clone. We measured leaf area (LA) (LI-3000, Li-Cor Inc), leaf blade length (LL) and maximum leaf blade width (LW) on the tracings. For each clone we fitted these values by linear regression (R2>0.90):
Using Eqn. (2) we calculated the leaf area per plant (LA) in a total of six plants per clone, in which the length and width of the leaf blade of all leaves were measured.
At the time of harvesting (d35-d55), we measured plant leaf area directly from leaves, as well as plants’ height. We separated those leaves attached to branches (auxiliary) from those in the main stem. We also measured separately all leaves distal to the stem portion used to measure the hydraulic conductance of the stem. This value was used to calculate the maximum leaf specific hydraulic conductivity. We calculated growth rates in leaf area for each plant as the difference between the leaf area measured at the time of harvesting and the initial leaf area estimated from leaf blade length and width, divided by the time elapsed between measurements.
After measuring leaf area, we oven-dried the leaves at 70ºC up to constant weight to determine leaf dry weight. We calculated SLA by dividing the surface area of the leaves by their dry weight.
Gas exchange and hydraulic conductivity
⌅In order to answer our third hypothesis, we measured stomatal conductance in plants of the LW treatment on four occasions (d35, d43, d50, d51), on 4-9 plants per clone and day of measurement. Stomatal conductance and other gas exchange parameters were measured on a fully developed leaf of the sixth whorl with a LCA4 IRGA (Analytical Development Co, Hoddesdon, UK). Measurements were made in batches of six plants, with one plant of each clone in each batch, between 9:30 and 11:30 (local solar time), on sunny days, with a PPFD > 1100 µmol m-2 s-1.
We carried out hydraulic measurements between d35 and d55. Measurements were made between 8:00 and 09:30 (local solar time) on sunny days, with a maximum air temperature of 32°C and minimum temperatures between 18 and 20°C. Each day, we measured between one and two plants per clone, from the same irrigation treatment. Measurements of plants from different treatments were taken on alternate days. As explained above, SWC and WT were measured in each plant at this time, as was plant height. Then, water potential was measured on a leaf of the sixth whorl with a pressure chamber (PMS Instrument Co., Albany, OR, USA) and hydraulic conductance was measured following Sperry et al. (1988)Sperry JS, Donnelly JR, Tyree MT, 1988. A method for measuring hydraulic conductivity and embolism in xylem. Plant Cell Environ 11: 35-40. https://doi.org/10.1111/j.1365-3040.1988.tb01774.x.
We used the portion of the stem between the base and the sixth whorl for hydraulic conductivity measurements. The stem was cut under water, and all leaves and branches were also removed under water. The stems were left in trays with water at room temperature for approximately 20 minutes before measuring the initial hydraulic conductance (Ki), at a pressure drop of 0.0064 MPa. A pressure of 0.08 MPa was then applied for 30 min, which was enough to eliminate any embolism in the stem. Maximum hydraulic conductance (Km) was then measured as explained for Ki and the percentage loss of hydraulic conductance (PLC) was calculated from Ki and Km:
A filtered (0.2 µm), degassed and slightly acidified (1‰ HCl) distilled water solution was used throughout the process. We measured the length of the stems used to determine the hydraulic conductance (L). To calculate the maximum leaf-specific hydraulic conductivity (KLmax) we used Eqn. (4), where LAd is the surface area of all leaves fed by the stem.
Statistical analyses
⌅We carried out the analyses of variance by fitting General Linear Models, considering the effect of the watering treatment and clone, together with the possible interactions. Analysis of covariance was used to analyze the effect of clone and watering regime on leaf area and plant height, taking the day of harvest as continuous predictor. We used factorial ANOVA to analyze differences between clones and watering regimes in SLA, PLC and KLmax and one-way ANOVA to analyze the differences between clones in the maximum values of stomatal conductance. The effect of the watering treatment was further analyzed clone by clone by means of ANCOVA or one-way ANOVA. We checked the validity of the basic assumptions regarding the normality, independence and homocedasticity of residuals; and log-transformed the data when necessary. We used simple linear regression to analyze the relationship between WT and SWC and multiple linear regression to analyze the relationship between LA, LW, and LL. Percentage values were arcsin transformed prior to analysis. Mean values were compared using Fisher’s LSD test. Differences were considered significant at p<0.05. All analyses were run using the 6.0 version of STATISTICA (StatSoft, Tulsa, OK, USA).
Results
⌅Soil water content and predawn Ѱ
⌅Figure 1 shows the average SWC values for both treatments, calculated from WT values using Eqn. (1). The mean minimum SWC remained above 5% in the HW treatment and decreased to 4% in plants from the LW treatment (Fig. 1). There was a tight relationship between predawn Y and the values of SWC estimated from Eqn. (1) (Fig. 2), which further supports the validity of the parameter WT to estimate SWC. When SWC was higher than 5%, predawn Y remained above -0.8 MPa (Fig. 2). As SWC decreased below this threshold value of 5%, predawn Y tended to decrease steeply.
Growth and specific leaf area
⌅We found no significant differences in leaf area between clones at d0. At harvest, leaf area differed between clones (F5,92=3.49, p=0.006) and irrigation treatments (F1,92=16.5, p<0.001). The effect of time was also significant (F1,92=63.1, p<0.001). We measured the highest growth in the F1 clones H463 and H354 grown under the most favorable HW treatment and the lowest growth in the F0 clone C14 in both irrigation treatments. The inbred clone showed the highest decrease in growth with water shortage (Table 2).
| C13 | C14 | H231 | H354 | H463 | H491 | ||
|---|---|---|---|---|---|---|---|
| SLA (main) (m2 kg-1) | HW | 24.60±1.00a* | 19.38±0.31c | 20.75±0.65bc | 20.10±1.00bc | 22.08±1.05b | 20.85±0.74bc |
| LW | 21.67±0.44a* | 18.78±0.55b | 20.03±0.54ab | 19.36±0.54b | 21.52±0.79a | 18.96±0.33b | |
| SLA (aux.) (m2 kg-1) | HW | 27.3±1.63a | 25.2±0.58ab | 24.7±1.4ab | 23.7±0.83b | 25.5±0.83ab | 25.7±0.91ab |
| LW | 24.9±0.88a | 23.5±0.96a | 23.5±0.60a | 23.1±0.54a | 23.7±0.83a | 24.2±0.68a | |
| Growth-LA·10-3 (m2 day-1) | HW | 3.50±0.18abc | 3.10±0.14c | 3.38±0.17bc | 3.65±0.16ab | 3.90±0.18a | 3.50±0.17abc* |
| LW | 3.55±0.14a | 2.80±0.19c | 3.09±0.11bc | 3.30±0.15ab | 3.51±0.20ab | 3.06±0.12bc* | |
| Height (cm) | HW | 98.7±3.6bc | 91.7±3.1c | 96.5±3.1c | 108.2±3.2ab* | 111.0±3.3a* | 101.8±3.4bc* |
| LW | 93.5±3.0ab | 81.2±3.3c | 91.0±3.3bc | 99.2±3.1a* | 99.7±3.3a* | 84.9±3.0c* |
There were significant differences in height between clones at the time of transplanting (F5,113=14.45, p<0.001). Average height was largest in clone C14 (25.0±0.5 cm) and lowest in H463 (20.1±0.7 cm). At the time of harvest, plant height was lower in the LW treatment compared with the HW treatment (F1,90=39.7, p<0.001). We found a significant effect of time (F1,90=39.7, p<0.001) and clone (F5.90=9.51, p<0.001) on plant height. The lowest values were measured in clone C14 and the highest in H463, showing no effect of the initial height on growth. The effect of the irrigation treatment was highest in the inbred clone, as found for leaf area (Table 2).
Specific leaf area (SLA) decreased under water shortage in leaves directly attached to the main stem (F1,88=8.1, p<0.01) and also in auxiliary leaves (F1,88=7.6, p<0.01). Auxiliary leaves had a higher SLA than main leaves (Table 2), most probably because the proportion of growing leaves was higher in auxiliary leaves than in main leaves. Differences between clones in SLA were only significant for main leaves (F5,88=8.9, p<0.001), with no clone × treatment interaction. For main leaves, we measured the highest and lowest values of SLA in clones C13 and C14 respectively (Table 2). Interestingly, clone C14 attained the lowest growth under both watering regimes in the present study and under field conditions.
Hydraulic traits
⌅Water shortage significantly decreased KLmax (F1,92=7.75, p=0.0065). Neither the effect of the clone nor the interaction between clone and treatment were significant. We measured the highest values of KLmax in HW plants from clone H463 and the lowest in LW plants from the inbred clone, H491. The highest decrease in KLmax with treatment was measured in clones H463 (40%) and H491 (37%) and the lowest in clone H354 (4.5% decrease, Fig. 3).
We found substantial hysteresis between Y and PLC (Fig. 4). The lower boundary of the set of points showed an almost exact coincidence with the vulnerability curve obtained by Pammenter & Vander Willigen (1998)Pammenter NW, Vander Willigen C, 1998. A mathematical and statistical analysis of the curves illustrating vulnerability to cavitation. Tree Physiol 18: 589-593. https://doi.org/10.1093/treephys/18.8-9.589 for 7-8-year-old E. globulus trees (Fig. 4). There were no significant differences in PLC, either between clones or watering treatments.
Gas exchange
⌅There was a tight relationship between stomatal conductance and the net photosynthetic rate (Fig. 5). The graphical output strongly suggested a diminished ability in the inbred clone (H491) to reach the highest values of stomatal conductance. We only measured a stomatal conductance higher than 0.5 mol m-2 s-1 in one plant from clone H491, while for the other clones 4-5 different plants were able to exceed this value (Fig. 5). We analyzed the differences between clones in the maximum stomatal conductance calculated as the average of the four highest values measured throughout the experiment. Differences between clones were close to be significant (F5,18=2.74, p=0.052). According to the Fisher LSD test, the average value for the inbred clone was significantly lower than the average values measured in clones C13 and H463. Low-watered plants from these two clones reached the highest growth in leaf area and an average height larger than clones H491, C14 and H231 (Table 2).
The highest values of stomatal conductance were measured at a SWC higher than 15% (Fig. 6), far above the critical level of 5% for predawn Y drop. We fitted a second-grade polynomial to the boundary values of stomatal conductance measured in the 5.8-23% range of SWC. The regression line cut the x-axis at SWC=5.3%, showing a tendency of maximum stomatal conductance to decrease as SWC decreased and become zero before reaching the threshold for predawn leaf water potential drop.
Discussion
⌅Breeding programs for E. globulus have mainly focused on improving growth. Given the economic relevance of this species, it seems crucial to gain a better understanding of the effects of climate stress on growth and survival, which could help to design management strategies to reduce climate-associated risks (Tomé et al., 2021Tomé M, Almeida MH, Barreiro S, Branco MR, Deus E, Pinto G, et al., 2021. Opportunities and challenges of Eucalyptus plantations in Europe: The Iberian Peninsula experience. Eur J Forest Res 140: 489-510. https://doi.org/10.1007/s10342-021-01358-z). By analyzing the suitability of some morpho-physiological traits to characterize the response to water shortage in six selected genotypes, our study could contribute to fill this gap.
Growth and specific leaf area
⌅Water shortage decreased growth in height and leaf area, SLA and hydraulic conductance. Our results contrast with those of Maseda & Fernández (2016)Maseda PH, Fernández RJ, 2016. Growth potential limits drought morphological plasticity in seedlings from six Eucalyptus provenances. Tree Physiol 36: 243-251. https://doi.org/10.1093/treephys/tpv137, who found no significant effect of the watering regime on SLA in greenhouse-grown E. globulus and Eucalyptus camaldulensis. This discrepancy could be partly explained by a comparatively low light intensity in the study by Maseda & Fernández (2016)Maseda PH, Fernández RJ, 2016. Growth potential limits drought morphological plasticity in seedlings from six Eucalyptus provenances. Tree Physiol 36: 243-251. https://doi.org/10.1093/treephys/tpv137. Decreasing SLA under water shortage would be more advantageous under high solar radiation, which increases leaf temperature and consequently increases the water vapor gradient between the mesophyll and the outside air, promoting higher rates of transpiration. A comparatively low SLA often results in an increase in net photosynthesis per unit of transpiring leaf area, which increases water use efficiency (Tortosa et al., 2022Tortosa I, Escalona JM, Opazo I, Douthe C, Medrano H, 2022. Genotype variations in water use efficiency correspond with photosynthetic traits in Tempranillo grapevine clones. Agronomy 12: 1874. https://doi.org/10.3390/agronomy12081874), improving plants’ performance under partial stomata closure. On the other hand, a large investment in biomass to grow new leaves typically translates into diminished growth. Accordingly, we measured the lowest SLA in clone C14, which reached the lowest growth among outcrossed genotypes both in the present study and in field trials.
The highest average heights at harvest were reached by F1 clones, independently of the watering regime. The highest growth in leaf area was also measured in F1 clones. A similar result was observed in field trials established in SW Spain for the clones selected for the present study. Under the most favorable irrigation treatment, the inbred clone achieved higher growth in leaf area than both F0 clones and one F1 clone. This result contrasts with the poor development of the inbred clone in field trials established in SW Spain, under a dry Mediterranean climate. In the present study, we measured the highest reduction in growth under water shortage in clone H491. This result points at water stress as a main factor causing depressed growth in the inbred clone. Despite being a generalized trait, the magnitude of inbreeding depression may depend on the trait measured and environmental factors, often increasing in stressful environments (Sandner et al., 2021Sandner TM, Matthies D, Waller DM, 2021. Stresses affect inbreeding depression in complex ways: disentangling stress-specific genetic effects from effects of initial size in plants. Heredity 127: 347-356. https://doi.org/10.1038/s41437-021-00454-5). For example, Johnsen et al. (2003)Johnsen K, Major JE, Maier CA, 2003. Selfing results in inbreeding depression of growth but not of gas exchange of surviving adult black spruce trees. Tree Physiol 23: 1005-1008. https://doi.org/10.1093/treephys/23.14.1005 reported inbreeding depression of growth, but detected no differences in gas exchange or stable carbon isotope discrimination among inbred and out-crossed adult black spruce trees. Similarly, Isakov (2021)Isakov Y, 2021. The effect of a single inbreeding on the growth and development of fast-growing tree species, Betula pendula and Betula pubescens. IOP Conf Ser: Earth Environ Sci 875: 012014. https://doi.org/10.1088/1755-1315/875/1/012014 found both positive and negative effects of inbreeding in seedlings of Betula sp.
Hydraulic traits
⌅Values of KLmax were similar to previously measured values in species from the genera Eucalyptus and Populus ( Vander Willigen & Pammenter, 1998Vander Willigen C, Pammenter NW, 1998. Relationship between growth and xylem hydraulic characteristics of clones of Eucalyptus spp. at contrasting sites. Tree Physiol 18: 595-600. https://doi.org/10.1093/treephys/18.8-9.595; Sparks & Black, 1999Sparks JP, Black RA, 1999. Regulation of water loss in populations of Populus trichocarpa: the role of stomatal control in preventing xylem cavitation. Tree Physiol 19: 453-459. https://doi.org/10.1093/treephys/19.7.453), but higher than values measured in Mediterranean species (Tognetti et al., 1998Tognetti R, Longobucco A, Raschi A, 1998. Vulnerability of xylem to embolism in relation to plant hydraulic resistance in Quercus pubescens and Quercus ilex co-occurring in a Mediterranean coppice stand in central Italy. New Phytol 139: 437-447. https://doi.org/10.1046/j.1469-8137.1998.00207.x; Vilagrosa et al., 2003Vilagrosa A, Bellot J, Vallejo VR, Gil-Pelegrín E, 2003. Cavitation, stomatal conductance, and leaf dieback in seedlings of two co-occurring Mediterranean shrubs during an intense drought. J Exp Bot 54: 2015-2024. https://doi.org/10.1093/jxb/erg221). The effect of losing hydraulic conductance when KLmax is high would be easier to tolerate, since it is not PLC what limits gas exchange and therefore growth, but rather the amount of hydraulic conductance that remains after embolism (Vander Willigen et al., 2000Vander Willigen C, Sherwin HW, Pammenter NW, 2000. Xylem hydraulic characteristics of subtropical trees from contrasting habitats grown under identical environmental conditions. New Phytol 145: 51-59. https://doi.org/10.1046/j.1469-8137.2000.00549.x).
We measured the highest values of KLmax in HW plants from clone H463 and the lowest in LW plants from the inbred clone, H491. Interestingly, the maximum and minimum growth in leaf area were measured in these clones, under the same treatments. It is worth noting that clone H463 has been rejected as a commercial clone; because of its tendency to produce epicormic shoots under harsh conditions (F. Ruiz, ENCE, pers. comm.). Therefore, a high KLmax alone would not be a sufficiently reliable selection criterion.
Since we measured hydraulic conductance in plants that had undergone natural dehydration, PLC values quantify native embolism, which is the result of two different processes, cavitation and refilling. Results from several studies suggest that xylem conduits, in some species at least, undergo frequent cycles of cavitation and embolism repair (refilling) (Knipfer et al., 2016Knipfer T, Cuneo IF, Brodersen CR, McElrone AJ, 2016. In situ visualization of the dynamics in xylem embolism formation and removal in the absence of root pressure: A study on excised grapevine stems. Plant Physiol 171: 1024-1036. https://doi.org/10.1104/pp.16.00136). Though the underlying mechanism still remains unclear, refilling may be concurrent with transpiration (Secchi et al., 2017Secchi F, Pagliarani C, Zwieniecki MA, 2017. The functional role of xylem parenchyma cells and aquaporins during recovery from severe water stress. Plant Cell Environ 40: 858-871. https://doi.org/10.1111/pce.12831), causing hydraulic conductance to vary diurnally as a result of both processes (Knipfer et al., 2016Knipfer T, Cuneo IF, Brodersen CR, McElrone AJ, 2016. In situ visualization of the dynamics in xylem embolism formation and removal in the absence of root pressure: A study on excised grapevine stems. Plant Physiol 171: 1024-1036. https://doi.org/10.1104/pp.16.00136). Interestingly, despite the differences in the methodologies used, we found a good fit between boundary PLC values showing the highest resistance to cavitation in our study and the vulnerability curve obtained from field-grown E. globulus by Pammenter & Vander Willigen (1998)Pammenter NW, Vander Willigen C, 1998. A mathematical and statistical analysis of the curves illustrating vulnerability to cavitation. Tree Physiol 18: 589-593. https://doi.org/10.1093/treephys/18.8-9.589. In their study, these authors measured embolism in branches cut from the stem and allowed to dry for two days. Not surprisingly, their set of data points showed a low degree of scattering, since there was no chance for refilling. Therefore, PLC increased progressively while water potential decreased with tissue dehydration. The high degree of scattering in our study is consistent with the phenomenon of hysteresis in the vulnerability curve (Sperry et al., 2003Sperry JS, Stiller V, Hacke UG, 2003. Xylem hydraulics and the soil-plant-atmosphere continuum: opportunities and unresolved issues. Agron J 95: 1362-1370. https://doi.org/10.2134/agronj2003.1362), which could result from partial (incomplete) refilling of the embolized vessels.
Stomatal conductance
⌅Recent studies highlight the relevance of Y when modelling stomatal conductance (Anderegg et al., 2017Anderegg WRL, Wolf A, Arango-Velez A, Choat B, Chmura DJ, Jansen S, et al., 2017. Plant water potential improves prediction of empirical stomatal models. PLoS ONE 12: e0185481. https://doi.org/10.1371/journal.pone.0185481). The dependence of stomatal conductance on Y has often been interpreted as an attempt to prevent hydraulic impairment from xylem embolism. In this regard, Creek et al. (2020)Creek D, Lamarque LJ, Torres-Ruiz JM, Parise C, Burlett R, Tissue DT, et al., 2020. Xylem embolism in leaves does not occur with open stomata: evidence from direct observations using the optical visualization technique. J Exp Bot 71: 1151-1159. https://doi.org/10.1093/jxb/erz474 found that stomatal closure preceded the onset of leaf hydraulic dysfunction in three tree species. The range of PLC values suggests that this would not be the case in our study. Rather, our results point at SWC as a critical factor to stomata closure.
We measured rather small values of stomatal conductance at any SWC. The well-known lagged effect of ABA-mediated stomata closure (Klein, 2014Klein T, 2014. The variability of stomatal sensitivity to leaf water potential across tree species indicates a continuum between isohydric and anisohydric behaviours. Funct Ecol 28: 1313-1320. https://doi.org/10.1111/1365-2435.12289) could contribute to explain this result. On the other hand, we found that maximum values of stomatal conductance decreased as SWC decreased, pointing at a strict stomata control to delay, as far as possible, reaching a critical threshold in SWC. Interestingly, this threshold corresponded to the SWC below which further dehydration would translate into an increasingly steeper decrease in soil water potential, as estimated from predawn leaf water potentials. In agreement with this, Carminati et al. (2020)Carminati A, Ahmed MA, Zarebanadkouki M, Cai G, Lovric G, Javaux M, 2020, Stomatal closure prevents the drop in soil water potential around roots. New Phytol 226: 1541-1543. https://doi.org/10.1111/nph.16451 recently reviewed the relevance of stomatal closure to prevent the formation of large gradients in water potential around the roots. Such gradients would translate into decreasing xylem water potentials, which, according to Knipfer et al. (2016)Knipfer T, Cuneo IF, Brodersen CR, McElrone AJ, 2016. In situ visualization of the dynamics in xylem embolism formation and removal in the absence of root pressure: A study on excised grapevine stems. Plant Physiol 171: 1024-1036. https://doi.org/10.1104/pp.16.00136 would jeopardize the refilling of embolized vessels.
The maximum values of stomatal conductance were within the range reported by Franks et al. (2009)Franks PJ, Drake PL, Beerling DJ, 2009. Plasticity in maximum stomatal conductance constrained by negative correlation between stomatal size and density: an analysis using Eucalyptus globulus. Plant Cell Environ 32: 1737-1748. https://doi.org/10.1111/j.1365-3040.2009.002031.x and were lowest in the inbred clone (H491). The ability to reach high values of stomatal conductance allows plants to improve leaf cooling through transpiration and is therefore relevant to tolerate high temperatures. In Pima cotton (Gossypium barbadense L.), breeding for enhanced heat resistance and high yield resulted in higher stomatal conductance in the improved cultivars (Lu & Zeiger, 1994Lu ZM, Zeiger E, 1994. Selection for higher yields and heat resistance in Pima cotton has caused genetically determined changes in stomatal conductance. Physiol Plantarum 92: 273-278. https://doi.org/10.1034/j.1399-3054.1994.920212.x), highlighting the importance of stomatal conductance for breeding programs in heat-prone environments (Lu et al., 2000Lu ZM, Quiñónez MA, Zeiger E, 2000. Temperature dependence of guard cell respiration and stomatal conductance co-segregate in an F2 population of Pima cotton. Aust J Plant Physiol 27: 457-462. https://doi.org/10.1071/PP98128).
The diminished ability of the inbred clone to reach high values of stomatal conductance may explain its poor performance in the field, not only because of the decrease in the net photosynthetic rate, but also because of its effect in limiting the cooling capacity of the leaves. The wide range of stomatal conductance values in the other five clones shows greater plasticity, allowing the adjustment of water loss with little effect on photosynthesis. By contrast, for almost all the stomatal conductance values measured in the inbred clone, decreasing water loss by means of partial stomata closure would have a larger negative effect on photosynthesis.
Conclusions
⌅Maximum values of stomatal conductance were the lowest in the inbred clone, showing the lowest plasticity and a diminished ability to tolerate high temperatures, which could explain its poor development under field conditions. This parameter, together with SLA could help to identify genotypes unsuitable for improving growth in drought-prone environments.
Soil water content played a relevant role in stomata control. A critical threshold for stomata closure would be the SWC at which further dehydration leads to a drop in predawn leaf water potential. This should be considered when defining watering treatments to test the response to water shortage.
We found no differences in KLmax between clones that could contribute to explain the differences observed in growth, under controlled and field conditions. Further research is needed to find the traits that allow the early selection of genotypes for improving growth in water-limited environments.