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Effects of reduced irradiance on hydraulic architecture and water relations of two olive clones with different growth potentials
Environmental and Experimental Botany 66 (2009) 249–256
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Effects of reduced irradiance on hydraulic architecture and water relations of two olive clones with different growth potentials F. Raimondo a , P. Trifilò a , M. A. Lo Gullo a,∗ , R. Buffa b , A. Nardini c , S. Salleo c a b c
Dipartimento di Scienze della Vita “M. Malpighi”, Università di Messina, Salita Sperone 31, 98166 Messina S. Agata, Italy Dipartimento di Colture Arboree - Università di Palermo, Via delle Scienze 11, 90128 Palermo, Italy Dipartimento di Scienze della Vita, Università di Trieste, Via L. Giorgieri 10, 34127 Trieste, Italy
a r t i c l e
i n f o
Article history: Received 23 July 2008 Received in revised form 9 March 2009 Accepted 11 March 2009 Keywords: Olive Shading Hydraulic architecture Xylem features Transpiration rate Water potential
a b s t r a c t The hydraulic architecture and water relations of two olive genotypes, ‘Leccino Dwarf’ (LD) and ‘Leccino Minerva’ (LM) growing at two irradiance levels i.e. full sunlight irradiance (HI) and 50% sunlight irradiance (LI) were studied. The two clones showed similar plant hydraulic conductances (Kplant ) and similar conductance of roots and leaves (Kroot and Kleaf ) when growing at equal irradiance levels. However, both Kplant and Kroot were significantly lower in LI plants than in HI ones. On the contrary, Kleaf was unaffected by the light regime. One-year-old twigs of LI plants produced longer xylem conduits but lower average diameter of conduits and less conduits per unit xylem cross-sectional area compared to HI plants. As a consequence total conductive cross-sectional area of twigs was computed to be about 16% smaller in LI individuals than in HI ones. The LM genotype resulted potentially more vulnerable to cavitation than the LD one, although shading did not influence this variable. Shading influenced root biomass negatively with stronger reduction in LM genotype than in LD one. Although transpiration rates were substantially lower in shaded than in HI plants minimum diurnal leaf water potential was about −1.2 MPa for both clones regardless the irradiance regime. Our conclusion is that the hydraulic efficiency of both olive clones was adjusted to meet the evaporative demand imposed by the irradiance regime with consequently similar equal hydraulic sufficiency. © 2009 Elsevier B.V. All rights reserved.
1. Introduction In modern orchard management, grafting techniques are widespread. The rootstock is usually selected for performance in different soil types whereas the scion is selected in the view of obtaining high productivity and/or quality of fruits (Cohen and Naor, 2002). Low-vigour rootstocks are increasingly used in highdensity orchards with the purpose of reducing the scion growth potential with consequent reduction of cultural costs associated with harvesting and pruning (Troncoso et al., 1990; Webster, 1995; Tous et al., 1999). Recent studies suggest that the dwarfing effect on the scion as induced by low-vigour rootstocks is related to constraint on the ‘hydraulic architecture’ of grafted plants (e.g. Atkinson et al., 2003; Basile et al., 2003; Clearwater et al., 2004; Nardini et al., 2006; Cohen et al., 2007). The term ‘hydraulic architecture’ first coined by Zimmermann (1978) refers to the partitioning of hydraulic conductances in a plant and includes changes in xylem efficiency and vulnerability to cavitation (Tyree and Ewers, 1991).
∗ Corresponding author. Tel.: +39 090 6765627; fax: +39 090 392686. E-mail address: [email protected] (M.A.L. Gullo). 0098-8472/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2009.03.022
It is generally agreed that hydraulic architecture influences plant water relations and growth potential (e.g. Hubbard et al., 1999; Sperry, 2000; Meinzer, 2002) in that plants with high hydraulic conductance undergo smaller water potential drops between leaves and roots and run lower risk of xylem cavitation which favours gas exchange and growth (Tyree, 2003). In turn, plant hydraulic properties vary in response to several environmental factors including water and nutrient availability (Cruiziat et al., 2002; Ewers et al., 2000) and irradiance (Cochard et al., 1999; Barigah et al., 2006). Any of these factors may profoundly affect the hydraulic efficiency of plant organs. As an example, water stress has negative effects on radial water transport in the root (Lo Gullo et al., 1998), triggers xylem cavitation (Sperry and Tyree, 1988; Sperry and Ikeda, 1997) and may cause vein collapse and aquaporin inactivation in the leaves (Cochard et al., 2004; Kim and Steudle, 2007). Plasticity in the response to environmental factors involves morpho-anatomical changes e.g. in xylem conduit dimensions and root-to-shoot ratio and is age-dependent (Barigah et al., 2006). Variations of plant hydraulic efficiency have also been measured over the short term as a consequence of cycles of xylem embolism and refilling (Salleo et al., 2004), modifications of xylem sap ionic concentration (Zwieniecki et al., 2004) or as the result of
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expression/regulation of aquaporins (Henzler et al., 1999). Recent studies have shown that light regulates the hydraulic properties of roots and leaves both over the long (Tyree et al., 1998; Sack et al., 2005) and the short term (Lo Gullo et al., 2005; Cochard et al., 2007). Several studies have been conducted on crops grafted on low- and high-vigour rootstocks with contrasting results in terms of effects of changes in plant hydraulics as determined by the rootstock’s vigour. In some species (e.g. Malus domestica L., Atkinson et al., 2003), dwarfing included decrease in whole-plant hydraulic conductance (Kplant ) which may depend on decrease in root hydraulic conductance (Nardini et al., 2006). By contrast, Clearwater et al. (2004) have shown that the scion growth potential of Actinidia was independent on plant hydraulics. In the case of Olea europaea L., the genotype ‘Minerva’ of the cultivar Leccino (LM) known for good oil quality, high and constant productivity, resistance to pathogens and tolerance to a wide range of habitats (Marsilio and Lanza, 1988; Del Río et al., 2000, 2003) has been measured for hydraulic architecture when grafted on ‘Leccino Dwarf’ (LD) which is a genotype characterized by strongly reduced vegetative growth. This scion/rootstock combination showed low hydraulic conductance of the rootstock which effectively depressed the scion growth (Nardini et al., 2006). Nonetheless, although the dwarfing effect reduced the transpiring surface of the grafted plant, no clearly increased resistance to xylem cavitation of the scion was observed in LM/LD combinations (Trifilò et al., 2007). In the Mediterranean Basin area where olive cultivation is widespread, plant growth is not only limited by summer water scarcity but also by high irradiance causing chlorophyll degradation and reduction in photosynthesis, in particular during the juvenile period (e.g. Gussakovsky et al., 1993; Jifon and Syvertsen, 2001, 2003; Blanke, 2002). For this reason, it is a common cultivation practice to protect young individuals in the nursery using shading nets until plants are transferred to the field. Shading has been reported to induce increase of productivity, gas exchange and photosynthetic rate, as well as resource allocation (e.g. Sasaki and Mori, 1981; Popma and Bongers, 1988; Bjorkman and Demmig-Adams, 1994; Cohen et al., 1997, 2003, 2005; Stanhill and Cohen, 2001; Medina et al., 2002; Raveh et al., 2003). By contrast Barigah et al. (2006) have found that saplings of six forest trees grown under severe shading (4–36% of full sunlight) showed increased vulnerability to xylem cavitation, decreased Kroot and Kplant as well as the root-to-shoot ratio with quantitatively different species-specific responses to reduced irradiance. Accordingly, Nardini et al. (2005) have shown that woody and herbaceous species adapted to shade conditions showed lower leaf hydraulic conductance than sungrowing species due to narrower vein conduits in the former. This feature was interpreted as a consequence of low water demand in shade-growing plants. Previous studies by some of us (Nardini et al., 2006; Gascò et al., 2007; Lovisolo et al., 2007; Trifilò et al., 2007) on olive genotypes with different growth potentials (see below) and on their grafting combinations have revealed the need of a better understanding of changes in hydraulic architecture as influenced by environmental factors in the view of correctly interpreting the influence of the rootstock vigour on scion growth potential. On the basis of the above studies, saplings of the olive genotype ‘Leccino Dwarf’ (LD) and the vigorous ‘Leccino Minerva’ (LM) were compared for hydraulic architecture when under the effect of shade conditions commonly used in olive nurseries (50% of full sunlight, see below). In particular, the hydraulic conductance of whole plants (Kplant ) was measured as partitioned between roots (Kroot ) and leaves (Kleaf ) that are thought to be the sites of major hydraulic bottlenecks in a plant (Nardini and Tyree, 1999). Shading conditions were also studied as potentially modifying xylem conduit dimensions and vulnerability to cavitation, leaf water status, gas exchange and growth.
2. Material and methods 2.1. Plant material and growing conditions All experiments were conducted in July 2007 on two genotypes of Olea europaea cv Leccino characterized by different growth potentials (see above) i.e. Leccino ‘Minerva’ and Leccino ‘Dwarf’ (LM and LD, respectively, Nardini et al., 2006; Gascò et al., 2007). One year before experiments (July 2006), 20 plants per genotype two years of age were transferred to an experimental field in Sciacca (Sicily, southern Italy) in 3000-L containers filled with a 3:2 (v/v) mixture of peat and fine pumice stone. Plants of each genotype were divided into two groups one of which was grown in full sunlight (high irradiance, HI) while the second one was grown at 50% solar irradiance (low irradiance, LI). Reduced irradiance was obtained by covering plants with shading nets and was estimated by measuring the photosynthetically active radiation (PAR) with a quantum sensor (LI190S1, LiCor Inc., Lincoln, NE, USA) at 10 cm from the top of the canopy. Shading nets effectively reduced maximum daily irradiance from about 1900 mol m−2 s−1 (full sunlight) to 1000 mol m−2 s−1 (shade). The soil was fertilized with 2 kg m−3 of a commercial slow-release N, P, K fertilizer and 2 kg m−3 of Biotron (Cifo S.p.a., S. Giorgio di Piano, Bologna, Italy). All plants were kept well irrigated throughout the study period by irrigating soil to field capacity twice per week. 2.2. Gas exchange and plant water status Maximum transpiration rate (EL ) and minimum diurnal leaf water potential ( min ) were measured between 12.00 and 14.00 h of two successive sunny days on ten leaves from different plants of each genotype under study at each irradiance level tested using a steady-state porometer (LI-1600, LICor Inc., Lincoln, NE, USA) and a portable pressure chamber (3005 Plant Water Status Console, Soilmoisture Equipment Corp., Goleta, CA, USA), respectively. Air temperature (Tair ) and relative humidity (RH) were recorded at 10 cm from the top of the canopy using the porometer immediately prior to collect samples for hydraulic measurements (see below). Air temperature and relative humidity were 30.2 ± 0.7 ◦ C and 44.2 ± 5.9%, respectively with no statistically significant difference between the two irradiance levels (n = 10) in this regard. 2.3. Hydraulic measurements In order to get an overall picture of the hydraulic map of the two genotypes under study, measurements of hydraulic conductance (K) were performed on five plants of each genotype at each irradiance level tested. The hydraulic conductance of whole plants and roots was measured using the ‘evaporative flux’ method (Nardini et al., 2003). In particular, hydraulic resistances of both whole plant (Rplant = 1/Kplant ) and root (Rroot = 1/Kroot ) were calculated on the basis of the Ohm’s law hydraulic analogue as: (
Rplant =
Rroot =
(
soil
− EL
− EL
soil
min )
x)
where EL is the transpiration rate measured at midday and min is the midday leaf water potential. The midday xylem water potential ( x ) was estimated by measuring of leaves inserted near the base of plants that had been covered with plastic film and aluminium foil the evening preceding experiments. Soil water potential ( soil ) was measured using a Dew Point Hygrometer (WP4, Decagon Devices, Pullman, WA, USA) on soil samples collected at different depths (50 and 120 cm). Kplant and Kroot were then calculated as K = 1/R.
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Leaves were measured for hydraulic conductance (Kleaf ) from five plants per genotype and irradiance level using a high pressure flow meter (HPFM). A detailed description of this instrument can be found in Tyree et al. (1995). Single 1-year-old twigs from different plants were first cut off under distilled water and connected to the HPFM. The hydraulic resistance of leafy twigs (Rshoot = 1/Kshoot ) was first measured in the quasi-steady state mode at a pressure (P) of 0.3 MPa. Leaves were then removed and R was measured again, thus obtaining the resistance of the leafless stem (Rresidual = 1/Kresidual ). Leaf hydraulic resistance (Rleaf = 1/Kleaf ) was calculated as: Rleaf = Rstem − Rresidual To minimize transpiration and facilitate quasi-steady state conditions during measurements, the samples were enclosed in a transparent plastic bag and sprayed with water. Measurements were performed in the open under daylight conditions (the PAR at the sample level was between 500 and 700 mol m−2 s−1 as measured with the quantum sensor) to prevent down-regulation of leaf hydraulic conductance in the dark as observed in some species (Tyree et al., 2005). At the end of measurements, total leaf surface area of the twig (AL ) was determined with a portable leaf area meter (LI-3000A, LiCor Inc., Lincoln, NE, USA), and the hydraulic resistance of leaves was scaled by AL (Tyree et al., 1998). All HPFM measurements were corrected for the calibration temperature of the instrument (Tcal = 22 ◦ C). All hydraulic measurements were completed between 12.00 and 14.00 h so as to minimize the impact of possible short-term changes of hydraulic resistance (Tsuda and Tyree, 2000; Lo Gullo et al., 2005). 2.4. Anatomical measurements and vulnerability to stem xylem cavitation Five 1-year-old twigs from five different plants of both LM and LD genotype growing at HI and LI were collected and immediately fixed in FAA (formalin, acetic acid, ethanol, 1:1:1, v:v:v). Transverse 30m sections were then made from the middle third of twigs using a microtome (mod. Cut 4055, SLEE Technik GmbH, Mainz, Germany). Sections were first double-stained with 0.1% (w:v) safranin and 1% (w:v) fast green (Lo Gullo et al., 2007) and then they were observed under a microscope equipped with a digital camera. Digital images were analyzed with the Sigma Scan Pro software package v. 5.0 (Aspire Software International, Ashburn, VA) to determine xylem conduit numbers and internal diameter distribution (Trifilò et al., 2007). The potential cross-sectional conductive area was estimated as r2 where r is the conduit radius (Lo Gullo et al., 2000). Analogous measurements were performed of five petioles and midribs of leaves from LD and LM plants at the two irradiance levels. Petioles and midribs were trimmed and sectioned with a fresh razor blade and sections were stained and analyzed as described above. Vessel length distribution was estimated using the technique described by Sperry et al. (2005). Five 1-year-old twigs of the two genotypes at the two irradiance levels tested were injected with silicone (Rhodorsil RTV-141, Rhodia, Cranbury, NJ, USA) mixed with a blue pigment (Pentasol, Prochima, Pesaro, Italy) for 3 h at P = 0.5 MPa. Silicone hardening was complete after about 12 h for stems in air. Stems were then cross-sectioned into serial 2-cm-long segments using fresh razor blades and observed immediately under a microscope to count the number of stained conduits and the total number of conduits per section. Vessel length distribution was then calculated using the equations reported by Sperry et al. (2005). To estimate xylem vulnerability to cavitation, curves were constructed of the hydraulic conductance of 1-year-old twigs as a function of the pressure difference applied across the interconduit pit membranes (Salleo et al., 1996). Five twigs from five different LM and LD plants at both irradiance levels were tested. One-year-old
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twigs were cut off under distilled water, immediately connected to a hydraulic apparatus (XYL’EM, Bronkhorst, Montigny les Cormeilles, France) and perfused at P = 8 kPa with 50 mM KCl solution filtered to 0.1 m to measure native stem hydraulic conductivity (Kh ). Stems were then ‘flushed’ at P = 0.2 MPa for 20 min in order to remove eventual emboli, and the newly established hydraulic conductivity (Khmax ) was re-measured at P = 8 kPa. Xylem embolism was experimentally induced in twigs still connected to the hydraulic apparatus using the air-injection technique (Trifilò et al., 2007). Air pressures of 0.5, 1.0, 1.5, 2.0 and 2.5 MPa were sequentially applied in steps of 10 min each. After each pressurization, Kh was re-measured at P = 8 kPa (Khp ) and the percentage loss of hydraulic conductivity (PLC) as referred to each pressure applied was computed as:
PLC = 1 −
Khp Khmax
· 100
2.5. Assessment of root biomass At the end of experiments all plants under experiment were carefully excavated. The soil was first removed from root systems under a gentle jet of water, and the root systems were cut off. Fine roots (<2 mm in diameter) which are thought to be most responsible for water uptake were excised, dried in oven at 70 ◦ C for three days and their dry weight (DW) was determined (Lovisolo et al., 2007). The DW of fine roots from plants used for hydraulic measurements was measured separately. Total dry weight of root systems was measured too. 2.6. Statistics Data were analyzed with the SigmaStat 3.1 (SPSS, Chicago, IL, USA) statistics package. One-way ANOVA in combination with post-hoc pairwise comparisons using Tukey’s test were used. The statistical significance of correlations between parameters was tested by Pearson Product Moment Correlation. 3. Results LD plants developed significantly smaller leaf surface area than LM ones (AL and AL , Table 1). AL was about 3 cm2 in LD and 5 cm2 in LM plants while AL (total leaf surface area of 1-year-old twigs) was about 550 versus 830 cm2 , respectively. In both genotypes, low irradiance (LI) induced an increase in AL compared to HI conditions, by about 15% in LM plants (5.35 for LI versus 4.61 cm2 for HI plants) and by about 30% in LD (3.47 for LI and 2.48 cm2 for HI, Table 1). By contrast, the length of 1-year-old twigs was unaffected by LI conditions and statistically significant differences were only observed between genotypes. LD plants grown in full sunlight had a fine root dry biomass over 50% less than LM plants under the same light regime (Table 1). Shading resulted in a decrease in root dry biomass in both genotypes with the noticeable difference between the two plants. The effect of LI caused root biomass of LD plants to decrease to one third that of LD plants growing in full sunlight whereas the reduction in fine root biomass of LM plants at LI was much smaller (one third less). Similar differences between HI and LI conditions were detected in both genotypes in terms of total root dry biomass (Table 1). Equal transpiration rates (EL ) per unit leaf surface area were measured for the two genotypes when growing under the same PAR conditions (Fig. 1A). Substantial reduction of this variable was measured at LI compared to HI conditions. In fact, EL was about 5 mmol m−2 s−1 for HI plants versus about 3 mmol m−2 s−1 for LI ones. The 50% reduction of irradiance tested did not influence min-
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Table 1 1-year-old twig length, total leaf surface area of 1-year-old twigs (AL ), mean leaf surface area (AL ) and root dry biomass of Leccino Minerva (LM) and Leccino Dwarf (LD) clones growing at two irradiance levels (full sunlight irradiance, HI, and 50% full sunlight irradiance, LI). Root biomass was measured for fine roots (<2 mm in diameter) and as total biomass. Means are reported ± SD (n = 10 for twig length and AL ; n = 65 for AL ; n = 5 for root dry biomass). Different letters indicate significant differences for Tukey’s pairwise comparisons (P < 0.001).
LM, HI LM, LI LD, HI LD, LI
1-year-old twig length (cm)
AL (cm2 )
AL (cm2 )
Fine root dry biomass (g)
Total root dry biomass (g)
65.9 ± 10.4a 63.6 ± 9.9a 44.0 ± 4.1b 49.4 ± 8.4b
790.7 ± 135a 884.1 ± 214a 571.2 ± 98b 546.6 ± 78b
4.61 ± 1.1a 5.35 ± 1.4b 2.48 ± 0.7c 3.47 ± 0.6d
322.2 ± 44.7a 202.7 ± 30.9b 141.9 ± 31.6c 41.3 ± 18.9d
588.5 ± 123.6a 400.5 ± 50.8b 197.2 ± 53.7c 66.7 ± 24.2d
imum diurnal leaf (Fig. 1B) which was about −1.25 MPa for both LM and LD plants growing in full sunlight and about −1.15 MPa for shaded ones with no statistically significant difference between the two genotypes or irradiance levels in this regard. Similar whole-plant hydraulic conductances (Kplant , Fig. 2A) were measured for both genotypes. However, Kplant was significantly lower in LI plants compared to that measured in HI ones (Kplant was about 6.0 e-5 in the former versus 8.0 e5 kg s−1 m−2 MPa−1 in the latter). Analogous depression in K was measured for the roots (Kroot , Fig. 2B) of LM and LD plants when grown under shading nets whereas leaf hydraulic conductance (Kleaf ) was apparently unaffected by lower irradiance (Fig. 2C). The two genotypes showed no difference in Kroot or Kleaf when grown at equal PAR values. Anatomical measurements showed that LD twigs had substantially shorter and narrower xylem conduits than LM ones
Fig. 1. (A) Maximum transpiration rate (EL ) and (B) Minimum leaf water potential ( leaf ) as measured between 12.00 and 14.00 h in Leccino Minerva (LM) and Leccino Dwarf (LD) clones growing in full sunlight (black columns) and at 50% full sunlight irradiance (white columns). Different letters indicate significant differences for Tukey’s pairwise comparisons (P < 0.01). Vertical bars are SD of the mean (n = 5).
Fig. 2. (A) Plant hydraulic conductance scaled by plant leaf surface area (Kplant ), (B) root hydraulic conductance scaled by plant leaf surface area (Kroot ) and (C) leaf hydraulic conductance scaled by leaf surface area (Kleaf ) all measured in Leccino Minerva (LM) and Leccino Dwarf (LD) clones growing in full sunlight (black columns) and at 50% full sunlight irradiance (white columns). Means are reported ± SD (n = 5). Different letters indicate significant differences for Tukey’s pairwise comparisons (P < 0.01).
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Table 2 Xylem cross-sectional area, number of conduits per unit xylem cross-sectional area and cross-sectional conductive area (r2 ) as measured in 1-year-old twigs of Leccino Minerva (LM) and Leccino Dwarf (LD) clones growing at two irradiance levels (full sunlight irradiance, HI, and 50% full sunlight irradiance, LI). Means are given ± SD (n = 5). Different letters indicate significant differences for Tukey’s pairwise comparisons (P < 0.01).
LM, HI LM, LI LD, HI LD, LI
Xylem cross-sectional area (mm2 )
Number of conduits (mm−2 )
r2 (mm2 )
10.11 ± 1.86a 9.71 ± 1.63a 5.78 ± 2.11b 5.73 ± 1.91b
209 ± 24a 154 ± 16b 233 ± 16a 154 ± 20b
0.80 ± 0.08a 0.68 ± 0.06b 0.71 ± 0.07b 0.58 ± 0.07c
(Tables 3 and 4). Shading induced the production of longer xylem conduits in both genotypes and in particular in 1-year-old twigs of shaded plants where longest vessels were 41–50 cm long in LM genotype and of 31–40 cm in LD ones (Table 3). Moreover, reduced irradiance influenced conduit diameter negatively in stems of both genotypes. Conduits less than 20 m in diameter increased to about 40% in LM shaded plants and to about 45% in LD ones. Moreover, the percentage of conduits wider than 30 m decreased in shaded plants. In petiole and midribs, differences in conduit diameter were only recorded between genotypes (LM had wider conduits than LD) but not as a consequence of reduction in irradiance (data not shown). In particular, petioles and midribs of LM plants growing at HI had, respectively, 19.1 ± 5.3 and 35 ± 8% conduits with a diameter of 15–20 m (which was the maximum conduit diameter measured in these leaf regions) versus only 7.67 ± 3.2 (for petiole) and 10 ± 4.1% (for midrib) in LD plants. Effects of LI on xylem conduits also emerged from comparison of the different experimental groups in terms of number of conduits per unit xylem cross-sectional area and total cross-sectional conductive area (r2 , Table 2). The number of conduits mm−2 was by 25 to 33% lower in twigs of shaded plants of both genotypes than in those of plants grown in full sunlight over 210 conduits mm−2 in HI plants versus about 150 conduits mm−2 in LI ones. Accordingly, total conductive cross-sectional area was by about 16% smaller in LI twigs than in HI ones. In summary, in both genotypes low irradiance induced the production of less conduits per unit xylem cross-sectional area (Table 2) with single conduits slightly narrower (Table 4) but noticeably longer (Table 3) than in plants growing in full sunlight. Fig. 3 reports vulnerability to xylem cavitation measured on 1-year-old twigs of LM and LD plants grown under the two irradiance levels tested. At pressures ranging between −1.0 and −1.2 MPa corresponding to the min interval recorded during field experiments (Fig. 1B), the percentage loss of hydraulic conductivity (PLC) ranged between 35 and 45% in LM twigs at both irradiance levels, while it was 22–35% in LD twigs. In other words, the LM genotype was potentially more vulnerable to cavitation than the LD one with no influence of shading on this variable in both genotypes.
Fig. 3. Percent loss of hydraulic conductivity (PLC) of 1-year-old twigs measured in Leccino Minerva (LM) and Leccino Dwarf (LD) clones growing in full sunlight (black circles) and at 50% full sunlight irradiance (white circles). Vertical bars are SD of the mean (n = 5).
4. Discussion The olive genotypes ‘Leccino Minerva’ and ‘Leccino Dwarf’ were apparently different for plant size and growth potential as well as for leaf surface area (Table 1). By contrast, they did not differ from each other for hydraulic sufficiency or water status. Under optimal water availability conditions, the two genotypes showed similar plant hydraulic conductances (Kplant ) as well as similar conductance of roots and leaves (Kroot and Kleaf , Fig. 2A) on a leaf area basis. In other words, the hydraulic sufficiency of plants of LM and LD genotypes was the same both at the whole plant and at the organ level. The above finding helps explain the otherwise surprisingly similar transpiration rates (EL , Fig. 1A) and minimum leaf water potentials ( leaf , Fig. 1B) measured in the two clones. The recorded differences between the two genotypes in terms of xylem conduit dimensions, with LD twigs showing substantially shorter (Table 3) and slightly narrower (Table 4) conduits than LM ones did translate into some decrease in total conductive area in the former (r2 was by about 12% smaller in LD compared to LM). Nonetheless, equal Kplant values as expressed per unit leaf area were recorded in LM and LD plants suggesting that the
Table 3 Vessel length distribution measured on 1-year-old twigs of Leccino Minerva (LM) and Leccino Dwarf (LD) clones growing in full sunlight (HI) and at 50% full sunlight irradiance (LI). Means are ± SD (n = 5) is reported. Different letters indicate significant differences for Tukey’s pairwise comparisons (P < 0.01).
Table 4 Xylem conduit diameter distribution measured on 1-year-old twigs, petioles and midribs of Leccino Minerva (LM) and Leccino Dwarf (LD) clones growing in full sunlight (HI) and at 50% full sunlight irradiance (LI). Means are ± SD (n = 5). Different letters indicate significant differences for Tukey’s pairwise comparisons (P < 0.01).
Conduit length (cm)
LM, HI
LM, LI
LD, HI
LD, LI
Conduit diameter (m)
LM, HI
LM, LI
LD, HI
LD, LI
1–10 11–20 21–30 31–40 41–50
35.5 ± 4.9ab 42.0 ± 4.1bf 16.5 ± 2.2c 5.5 ± 0.9cd
28.5 ± 8.9a 39.5 ± 4.7bf 16.0 ± 3.3c 8.0 ± 2.2cd 2.5 ± 0.5d
60.5 ± 8.7f 32.5 ± 5.5ab 5.0 ± 1.8d
50 ± 6.5f 39 ± 4.8ab 10 ± 3.3cd 1 ± 0.3d
<10 11–20 21–30 31–40 >40
0.5 ± 0.6a 22.4 ± 5.0b 59.3 ± 4.8d 17.8 ± 4.8b 0.3 ± 0.2a
1.3 ± 1.2a 37.6 ± 6.7cd 53.1 ± 5.1d 10.1 ± 3.6ef 0.2 ± 0.4a
1.7 ± 1.9a 31.9 ± 8.0bc 50.4 ± 4.0cd 15.8 ± 3.1e
1.7 ± 0.4a 45.1 ± 5.0d 48.2 ± 3.5cd 7.8 ± 2.5f
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unit photosynthesizing area was similarly supplied with water in both cases. This finding is in accordance with the fact that the major portion (about 66%) of the whole plant hydraulic resistance (Rplant = 1/Kplant ) resided in the root system in both genotypes (Fig. 2). Such a partitioning of hydraulic resistances within the plant implies that any increase in hydraulic resistance of stem and leaves of both LD and LM plants (e.g. due to narrower conduits or to smaller numbers of conduits) would be minimized at the whole plant level (Meinzer, 2002). As an example, an increase in stem and leaf hydraulic resistance of LD stems as high as, say, 50% would lead Rplant of LD to increase by only 17%. In accordance with previous studies (e.g. Sellin, 1993; Tyree et al., 1998; Nardini et al., 2005; Sack et al., 2005; Barigah et al., 2006), shading induced significant reduction in Kplant in both genotypes (Fig. 2A). In principle, such a reduction could be a consequence of modification in the hydraulics of one or more plant organs (leaves, stems or roots). Our data clearly indicate that leaf hydraulic properties were unaffected by the light regime imposed (Fig. 2C). This conclusion is in contrast with previous reports by Nardini et al. (2005) and Sack et al. (2005). In both these studies, leaf hydraulic conductance was found to be higher in sun- than in shade-growing species. Similar light-dependency of Kleaf has been recently described in silver birch by Sellin et al. (2008). The lack of differences in Kleaf between our HI and LI plants might be due to the fact that shaded leaves experienced still high PAR values (about 1000 mol m−2 s−1 ). In other words, it is possible that substantial changes in leaf hydraulic properties of olive plants would only be triggered when leaves are more heavily shaded than in our experimental conditions. Xylem architecture was apparently influenced by the light regime, in that LI plants produced narrower (but longer) conduits than HI ones. In fact, the potential cross-sectional conductive area was reduced by 16 to 18% under shade conditions in both LM and LD clones (r2 , Table 2). In addition, shaded plants of the two clones produced one fourth to one third less conduits than plants growing in full sunlight (Table 2). This feature might contribute to the reduced Kplant recorded for shaded plants of the two clones compared to the same variable measured for plants in full sunlight. Root hydraulic conductance was found to be substantially lower in LI plants with respect to HI ones. The fractional magnitude of this reduction was near the same as that measured for Kplant suggesting that the recorded modification of root hydraulic properties was most responsible for the overall changes in Kplant , in accordance with plant hydraulics being dominated by the root compartment (see above). This conclusion is in accordance with previous studies by Kyllo et al. (2003) and Barigah et al. (2006) showing that in several tropical and temperate woody species, root hydraulic conductance decreased when plants were grown under shade conditions. In most of the above study cases, lower Kplant was due to a general reduction of root biomass, while Kroot expressed on a root biomass basis was often found to be unchanged. In other words, reduction of Kroot under shade conditions was due to reduced allocation to root growth and not to modifications of root hydraulic properties per se as in the case of changes in aquaporin expression (Tyermann et al., 1999). Our data are in accordance with these studies in that low irradiance showed to depress root production with larger effects in the dwarf genotype than in the vigorous one (Table 1). Previous studies by some of us had revealed that: (a) LM and LD rootstocks did not substantially differ for Kroot when expressed on a root biomass basis and (b) LD clone had strongly reduced root biomass compared to LM (Lovisolo et al., 2007). On the basis of our present data, the observed reduction of Kroot measured in plants of both LM and LD when growing in the shade was due to strongly reduced allocation of biomass to the root system. The basically similar Kplant values recorded in both genotypes was apparently the result of the similar allometry of the two genotypes
with the relative root growth proportional to that of the shoot. In turn, the shading effect leading to reduction of root biomass led to reduction in Kplant in both study cases. Interestingly, vulnerability to cavitation of LM and LD stems tended to shift towards a slight decrease under shade conditions (Fig. 3) contrary to expectations suggested by data reported in the literature (Cochard et al., 1999; Lemoine et al., 2002; Barigah et al., 2006). It is possible that some decrease in vulnerability to cavitation in shaded plants was a consequence of narrower xylem conduits (e.g. Lo Gullo et al., 1995). However, the magnitude of the shift in xylem vulnerability was small so that it is unlikely that this physiological trait may play any role in the adaptation of olive clones to LI, at least under the experimental conditions tested in the present study. It can be noted that Olea europea genotypes resulted more vulnerable to xylem embolism when compared with other olive cultivars as “Chemlali” and “Meski”, where PLC of 50% were recorded at −7.5 MPa (Ennajeh et al., 2008). Our data suggest that the hydraulic architecture of LM and LD clones was adjusted to meet the evaporative demand imposed by the irradiance regime at which plants were growing. In fact, HI plants were more hydraulically efficient than LI ones, as clearly indicated by higher Kroot and wider diameters of xylem conduits. The higher Kplant of HI plants compared to LI ones allowed the former to maintain water potential levels similar to those measured in the latter, despite the evaporative demand per unit leaf surface area was about 60% higher under full sunlight conditions than in the shade. It is of interest to note that the minimum leaf water potential recorded in the field (Fig. 1B) was close to the xylem water potential inducing 50% loss of hydraulic conductivity (Fig. 3), possibly indicating a runaway cavitation avoidance strategy based on buffering leaf to prevent catastrophic xylem dysfunction (Tyree and Sperry, 1988; Nardini and Salleo, 2000). The same goal, however, was achieved by LI plants that kept leaf water potential within critical thresholds by investing less energy and biomass in the production of roots and xylem conduits. In fact, production of root biomass and vascular tissue in excess of that required to compensate for evaporation would represent a useless, expensive investment under shade conditions. The shade-induced reduction in hydraulic efficiency of LI plants might have negative consequences for water balance of these plants when they are transplanted to the open at natural irradiance. The increase of transpiration rates under these new environmental conditions together with low hydraulic efficiency of plants may result in severe drops in leaf water potential with consequent stomatal closure, reduction of photosynthesis and growth until plants readapt to higher irradiance conditions. In turn, larger drops of leaf water potential might induce extensive cavitation and higher loss of hydraulic conductivity (larger in LM than in LD plants, Fig. 3) which would limit the necessary cambial activity for producing new roots and more efficient xylem. The scenario outlined is in accordance with the mortality recorded by farmers after transplanting plants grown under shading nets. We also note that even if the ability of plants to modify their hydraulic efficiency in response to changed irradiance conditions has been reported in detail (e.g. Barigah et al., 2006), no studies have been conducted yet on the time course of this adaptation in the case on sun species like olive. A corollary of the present study is that the hydraulic efficiency of plants is shaped to meet the evaporative demand. To this purpose, the composition of xylem, the hydraulic efficiency of conduits as well as the hydraulic conductance of the root system are adjusted to yield water potentials not exceeding levels triggering catastrophic embolism (Tyree and Sperry, 1988; Nardini and Salleo, 2000). Hydraulic sufficiency is addressed at maintaining the photosynthesizing apparatus well hydrated which was evidently the case both in dwarf plants and in vigorous ones (LD versus LM clone) as well as in plants grown under reduced evaporation compared to those with opposite behaviour (LI versus HI plants).
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Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot
Effects of reduced irradiance on hydraulic architecture and water relations of two olive clones with different growth potentials F. Raimondo a , P. Trifilò a , M. A. Lo Gullo a,∗ , R. Buffa b , A. Nardini c , S. Salleo c a b c
Dipartimento di Scienze della Vita “M. Malpighi”, Università di Messina, Salita Sperone 31, 98166 Messina S. Agata, Italy Dipartimento di Colture Arboree - Università di Palermo, Via delle Scienze 11, 90128 Palermo, Italy Dipartimento di Scienze della Vita, Università di Trieste, Via L. Giorgieri 10, 34127 Trieste, Italy
a r t i c l e
i n f o
Article history: Received 23 July 2008 Received in revised form 9 March 2009 Accepted 11 March 2009 Keywords: Olive Shading Hydraulic architecture Xylem features Transpiration rate Water potential
a b s t r a c t The hydraulic architecture and water relations of two olive genotypes, ‘Leccino Dwarf’ (LD) and ‘Leccino Minerva’ (LM) growing at two irradiance levels i.e. full sunlight irradiance (HI) and 50% sunlight irradiance (LI) were studied. The two clones showed similar plant hydraulic conductances (Kplant ) and similar conductance of roots and leaves (Kroot and Kleaf ) when growing at equal irradiance levels. However, both Kplant and Kroot were significantly lower in LI plants than in HI ones. On the contrary, Kleaf was unaffected by the light regime. One-year-old twigs of LI plants produced longer xylem conduits but lower average diameter of conduits and less conduits per unit xylem cross-sectional area compared to HI plants. As a consequence total conductive cross-sectional area of twigs was computed to be about 16% smaller in LI individuals than in HI ones. The LM genotype resulted potentially more vulnerable to cavitation than the LD one, although shading did not influence this variable. Shading influenced root biomass negatively with stronger reduction in LM genotype than in LD one. Although transpiration rates were substantially lower in shaded than in HI plants minimum diurnal leaf water potential was about −1.2 MPa for both clones regardless the irradiance regime. Our conclusion is that the hydraulic efficiency of both olive clones was adjusted to meet the evaporative demand imposed by the irradiance regime with consequently similar equal hydraulic sufficiency. © 2009 Elsevier B.V. All rights reserved.
1. Introduction In modern orchard management, grafting techniques are widespread. The rootstock is usually selected for performance in different soil types whereas the scion is selected in the view of obtaining high productivity and/or quality of fruits (Cohen and Naor, 2002). Low-vigour rootstocks are increasingly used in highdensity orchards with the purpose of reducing the scion growth potential with consequent reduction of cultural costs associated with harvesting and pruning (Troncoso et al., 1990; Webster, 1995; Tous et al., 1999). Recent studies suggest that the dwarfing effect on the scion as induced by low-vigour rootstocks is related to constraint on the ‘hydraulic architecture’ of grafted plants (e.g. Atkinson et al., 2003; Basile et al., 2003; Clearwater et al., 2004; Nardini et al., 2006; Cohen et al., 2007). The term ‘hydraulic architecture’ first coined by Zimmermann (1978) refers to the partitioning of hydraulic conductances in a plant and includes changes in xylem efficiency and vulnerability to cavitation (Tyree and Ewers, 1991).
∗ Corresponding author. Tel.: +39 090 6765627; fax: +39 090 392686. E-mail address: [email protected] (M.A.L. Gullo). 0098-8472/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2009.03.022
It is generally agreed that hydraulic architecture influences plant water relations and growth potential (e.g. Hubbard et al., 1999; Sperry, 2000; Meinzer, 2002) in that plants with high hydraulic conductance undergo smaller water potential drops between leaves and roots and run lower risk of xylem cavitation which favours gas exchange and growth (Tyree, 2003). In turn, plant hydraulic properties vary in response to several environmental factors including water and nutrient availability (Cruiziat et al., 2002; Ewers et al., 2000) and irradiance (Cochard et al., 1999; Barigah et al., 2006). Any of these factors may profoundly affect the hydraulic efficiency of plant organs. As an example, water stress has negative effects on radial water transport in the root (Lo Gullo et al., 1998), triggers xylem cavitation (Sperry and Tyree, 1988; Sperry and Ikeda, 1997) and may cause vein collapse and aquaporin inactivation in the leaves (Cochard et al., 2004; Kim and Steudle, 2007). Plasticity in the response to environmental factors involves morpho-anatomical changes e.g. in xylem conduit dimensions and root-to-shoot ratio and is age-dependent (Barigah et al., 2006). Variations of plant hydraulic efficiency have also been measured over the short term as a consequence of cycles of xylem embolism and refilling (Salleo et al., 2004), modifications of xylem sap ionic concentration (Zwieniecki et al., 2004) or as the result of
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expression/regulation of aquaporins (Henzler et al., 1999). Recent studies have shown that light regulates the hydraulic properties of roots and leaves both over the long (Tyree et al., 1998; Sack et al., 2005) and the short term (Lo Gullo et al., 2005; Cochard et al., 2007). Several studies have been conducted on crops grafted on low- and high-vigour rootstocks with contrasting results in terms of effects of changes in plant hydraulics as determined by the rootstock’s vigour. In some species (e.g. Malus domestica L., Atkinson et al., 2003), dwarfing included decrease in whole-plant hydraulic conductance (Kplant ) which may depend on decrease in root hydraulic conductance (Nardini et al., 2006). By contrast, Clearwater et al. (2004) have shown that the scion growth potential of Actinidia was independent on plant hydraulics. In the case of Olea europaea L., the genotype ‘Minerva’ of the cultivar Leccino (LM) known for good oil quality, high and constant productivity, resistance to pathogens and tolerance to a wide range of habitats (Marsilio and Lanza, 1988; Del Río et al., 2000, 2003) has been measured for hydraulic architecture when grafted on ‘Leccino Dwarf’ (LD) which is a genotype characterized by strongly reduced vegetative growth. This scion/rootstock combination showed low hydraulic conductance of the rootstock which effectively depressed the scion growth (Nardini et al., 2006). Nonetheless, although the dwarfing effect reduced the transpiring surface of the grafted plant, no clearly increased resistance to xylem cavitation of the scion was observed in LM/LD combinations (Trifilò et al., 2007). In the Mediterranean Basin area where olive cultivation is widespread, plant growth is not only limited by summer water scarcity but also by high irradiance causing chlorophyll degradation and reduction in photosynthesis, in particular during the juvenile period (e.g. Gussakovsky et al., 1993; Jifon and Syvertsen, 2001, 2003; Blanke, 2002). For this reason, it is a common cultivation practice to protect young individuals in the nursery using shading nets until plants are transferred to the field. Shading has been reported to induce increase of productivity, gas exchange and photosynthetic rate, as well as resource allocation (e.g. Sasaki and Mori, 1981; Popma and Bongers, 1988; Bjorkman and Demmig-Adams, 1994; Cohen et al., 1997, 2003, 2005; Stanhill and Cohen, 2001; Medina et al., 2002; Raveh et al., 2003). By contrast Barigah et al. (2006) have found that saplings of six forest trees grown under severe shading (4–36% of full sunlight) showed increased vulnerability to xylem cavitation, decreased Kroot and Kplant as well as the root-to-shoot ratio with quantitatively different species-specific responses to reduced irradiance. Accordingly, Nardini et al. (2005) have shown that woody and herbaceous species adapted to shade conditions showed lower leaf hydraulic conductance than sungrowing species due to narrower vein conduits in the former. This feature was interpreted as a consequence of low water demand in shade-growing plants. Previous studies by some of us (Nardini et al., 2006; Gascò et al., 2007; Lovisolo et al., 2007; Trifilò et al., 2007) on olive genotypes with different growth potentials (see below) and on their grafting combinations have revealed the need of a better understanding of changes in hydraulic architecture as influenced by environmental factors in the view of correctly interpreting the influence of the rootstock vigour on scion growth potential. On the basis of the above studies, saplings of the olive genotype ‘Leccino Dwarf’ (LD) and the vigorous ‘Leccino Minerva’ (LM) were compared for hydraulic architecture when under the effect of shade conditions commonly used in olive nurseries (50% of full sunlight, see below). In particular, the hydraulic conductance of whole plants (Kplant ) was measured as partitioned between roots (Kroot ) and leaves (Kleaf ) that are thought to be the sites of major hydraulic bottlenecks in a plant (Nardini and Tyree, 1999). Shading conditions were also studied as potentially modifying xylem conduit dimensions and vulnerability to cavitation, leaf water status, gas exchange and growth.
2. Material and methods 2.1. Plant material and growing conditions All experiments were conducted in July 2007 on two genotypes of Olea europaea cv Leccino characterized by different growth potentials (see above) i.e. Leccino ‘Minerva’ and Leccino ‘Dwarf’ (LM and LD, respectively, Nardini et al., 2006; Gascò et al., 2007). One year before experiments (July 2006), 20 plants per genotype two years of age were transferred to an experimental field in Sciacca (Sicily, southern Italy) in 3000-L containers filled with a 3:2 (v/v) mixture of peat and fine pumice stone. Plants of each genotype were divided into two groups one of which was grown in full sunlight (high irradiance, HI) while the second one was grown at 50% solar irradiance (low irradiance, LI). Reduced irradiance was obtained by covering plants with shading nets and was estimated by measuring the photosynthetically active radiation (PAR) with a quantum sensor (LI190S1, LiCor Inc., Lincoln, NE, USA) at 10 cm from the top of the canopy. Shading nets effectively reduced maximum daily irradiance from about 1900 mol m−2 s−1 (full sunlight) to 1000 mol m−2 s−1 (shade). The soil was fertilized with 2 kg m−3 of a commercial slow-release N, P, K fertilizer and 2 kg m−3 of Biotron (Cifo S.p.a., S. Giorgio di Piano, Bologna, Italy). All plants were kept well irrigated throughout the study period by irrigating soil to field capacity twice per week. 2.2. Gas exchange and plant water status Maximum transpiration rate (EL ) and minimum diurnal leaf water potential ( min ) were measured between 12.00 and 14.00 h of two successive sunny days on ten leaves from different plants of each genotype under study at each irradiance level tested using a steady-state porometer (LI-1600, LICor Inc., Lincoln, NE, USA) and a portable pressure chamber (3005 Plant Water Status Console, Soilmoisture Equipment Corp., Goleta, CA, USA), respectively. Air temperature (Tair ) and relative humidity (RH) were recorded at 10 cm from the top of the canopy using the porometer immediately prior to collect samples for hydraulic measurements (see below). Air temperature and relative humidity were 30.2 ± 0.7 ◦ C and 44.2 ± 5.9%, respectively with no statistically significant difference between the two irradiance levels (n = 10) in this regard. 2.3. Hydraulic measurements In order to get an overall picture of the hydraulic map of the two genotypes under study, measurements of hydraulic conductance (K) were performed on five plants of each genotype at each irradiance level tested. The hydraulic conductance of whole plants and roots was measured using the ‘evaporative flux’ method (Nardini et al., 2003). In particular, hydraulic resistances of both whole plant (Rplant = 1/Kplant ) and root (Rroot = 1/Kroot ) were calculated on the basis of the Ohm’s law hydraulic analogue as: (
Rplant =
Rroot =
(
soil
− EL
− EL
soil
min )
x)
where EL is the transpiration rate measured at midday and min is the midday leaf water potential. The midday xylem water potential ( x ) was estimated by measuring of leaves inserted near the base of plants that had been covered with plastic film and aluminium foil the evening preceding experiments. Soil water potential ( soil ) was measured using a Dew Point Hygrometer (WP4, Decagon Devices, Pullman, WA, USA) on soil samples collected at different depths (50 and 120 cm). Kplant and Kroot were then calculated as K = 1/R.
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Leaves were measured for hydraulic conductance (Kleaf ) from five plants per genotype and irradiance level using a high pressure flow meter (HPFM). A detailed description of this instrument can be found in Tyree et al. (1995). Single 1-year-old twigs from different plants were first cut off under distilled water and connected to the HPFM. The hydraulic resistance of leafy twigs (Rshoot = 1/Kshoot ) was first measured in the quasi-steady state mode at a pressure (P) of 0.3 MPa. Leaves were then removed and R was measured again, thus obtaining the resistance of the leafless stem (Rresidual = 1/Kresidual ). Leaf hydraulic resistance (Rleaf = 1/Kleaf ) was calculated as: Rleaf = Rstem − Rresidual To minimize transpiration and facilitate quasi-steady state conditions during measurements, the samples were enclosed in a transparent plastic bag and sprayed with water. Measurements were performed in the open under daylight conditions (the PAR at the sample level was between 500 and 700 mol m−2 s−1 as measured with the quantum sensor) to prevent down-regulation of leaf hydraulic conductance in the dark as observed in some species (Tyree et al., 2005). At the end of measurements, total leaf surface area of the twig (AL ) was determined with a portable leaf area meter (LI-3000A, LiCor Inc., Lincoln, NE, USA), and the hydraulic resistance of leaves was scaled by AL (Tyree et al., 1998). All HPFM measurements were corrected for the calibration temperature of the instrument (Tcal = 22 ◦ C). All hydraulic measurements were completed between 12.00 and 14.00 h so as to minimize the impact of possible short-term changes of hydraulic resistance (Tsuda and Tyree, 2000; Lo Gullo et al., 2005). 2.4. Anatomical measurements and vulnerability to stem xylem cavitation Five 1-year-old twigs from five different plants of both LM and LD genotype growing at HI and LI were collected and immediately fixed in FAA (formalin, acetic acid, ethanol, 1:1:1, v:v:v). Transverse 30m sections were then made from the middle third of twigs using a microtome (mod. Cut 4055, SLEE Technik GmbH, Mainz, Germany). Sections were first double-stained with 0.1% (w:v) safranin and 1% (w:v) fast green (Lo Gullo et al., 2007) and then they were observed under a microscope equipped with a digital camera. Digital images were analyzed with the Sigma Scan Pro software package v. 5.0 (Aspire Software International, Ashburn, VA) to determine xylem conduit numbers and internal diameter distribution (Trifilò et al., 2007). The potential cross-sectional conductive area was estimated as r2 where r is the conduit radius (Lo Gullo et al., 2000). Analogous measurements were performed of five petioles and midribs of leaves from LD and LM plants at the two irradiance levels. Petioles and midribs were trimmed and sectioned with a fresh razor blade and sections were stained and analyzed as described above. Vessel length distribution was estimated using the technique described by Sperry et al. (2005). Five 1-year-old twigs of the two genotypes at the two irradiance levels tested were injected with silicone (Rhodorsil RTV-141, Rhodia, Cranbury, NJ, USA) mixed with a blue pigment (Pentasol, Prochima, Pesaro, Italy) for 3 h at P = 0.5 MPa. Silicone hardening was complete after about 12 h for stems in air. Stems were then cross-sectioned into serial 2-cm-long segments using fresh razor blades and observed immediately under a microscope to count the number of stained conduits and the total number of conduits per section. Vessel length distribution was then calculated using the equations reported by Sperry et al. (2005). To estimate xylem vulnerability to cavitation, curves were constructed of the hydraulic conductance of 1-year-old twigs as a function of the pressure difference applied across the interconduit pit membranes (Salleo et al., 1996). Five twigs from five different LM and LD plants at both irradiance levels were tested. One-year-old
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twigs were cut off under distilled water, immediately connected to a hydraulic apparatus (XYL’EM, Bronkhorst, Montigny les Cormeilles, France) and perfused at P = 8 kPa with 50 mM KCl solution filtered to 0.1 m to measure native stem hydraulic conductivity (Kh ). Stems were then ‘flushed’ at P = 0.2 MPa for 20 min in order to remove eventual emboli, and the newly established hydraulic conductivity (Khmax ) was re-measured at P = 8 kPa. Xylem embolism was experimentally induced in twigs still connected to the hydraulic apparatus using the air-injection technique (Trifilò et al., 2007). Air pressures of 0.5, 1.0, 1.5, 2.0 and 2.5 MPa were sequentially applied in steps of 10 min each. After each pressurization, Kh was re-measured at P = 8 kPa (Khp ) and the percentage loss of hydraulic conductivity (PLC) as referred to each pressure applied was computed as:
PLC = 1 −
Khp Khmax
· 100
2.5. Assessment of root biomass At the end of experiments all plants under experiment were carefully excavated. The soil was first removed from root systems under a gentle jet of water, and the root systems were cut off. Fine roots (<2 mm in diameter) which are thought to be most responsible for water uptake were excised, dried in oven at 70 ◦ C for three days and their dry weight (DW) was determined (Lovisolo et al., 2007). The DW of fine roots from plants used for hydraulic measurements was measured separately. Total dry weight of root systems was measured too. 2.6. Statistics Data were analyzed with the SigmaStat 3.1 (SPSS, Chicago, IL, USA) statistics package. One-way ANOVA in combination with post-hoc pairwise comparisons using Tukey’s test were used. The statistical significance of correlations between parameters was tested by Pearson Product Moment Correlation. 3. Results LD plants developed significantly smaller leaf surface area than LM ones (AL and AL , Table 1). AL was about 3 cm2 in LD and 5 cm2 in LM plants while AL (total leaf surface area of 1-year-old twigs) was about 550 versus 830 cm2 , respectively. In both genotypes, low irradiance (LI) induced an increase in AL compared to HI conditions, by about 15% in LM plants (5.35 for LI versus 4.61 cm2 for HI plants) and by about 30% in LD (3.47 for LI and 2.48 cm2 for HI, Table 1). By contrast, the length of 1-year-old twigs was unaffected by LI conditions and statistically significant differences were only observed between genotypes. LD plants grown in full sunlight had a fine root dry biomass over 50% less than LM plants under the same light regime (Table 1). Shading resulted in a decrease in root dry biomass in both genotypes with the noticeable difference between the two plants. The effect of LI caused root biomass of LD plants to decrease to one third that of LD plants growing in full sunlight whereas the reduction in fine root biomass of LM plants at LI was much smaller (one third less). Similar differences between HI and LI conditions were detected in both genotypes in terms of total root dry biomass (Table 1). Equal transpiration rates (EL ) per unit leaf surface area were measured for the two genotypes when growing under the same PAR conditions (Fig. 1A). Substantial reduction of this variable was measured at LI compared to HI conditions. In fact, EL was about 5 mmol m−2 s−1 for HI plants versus about 3 mmol m−2 s−1 for LI ones. The 50% reduction of irradiance tested did not influence min-
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Table 1 1-year-old twig length, total leaf surface area of 1-year-old twigs (AL ), mean leaf surface area (AL ) and root dry biomass of Leccino Minerva (LM) and Leccino Dwarf (LD) clones growing at two irradiance levels (full sunlight irradiance, HI, and 50% full sunlight irradiance, LI). Root biomass was measured for fine roots (<2 mm in diameter) and as total biomass. Means are reported ± SD (n = 10 for twig length and AL ; n = 65 for AL ; n = 5 for root dry biomass). Different letters indicate significant differences for Tukey’s pairwise comparisons (P < 0.001).
LM, HI LM, LI LD, HI LD, LI
1-year-old twig length (cm)
AL (cm2 )
AL (cm2 )
Fine root dry biomass (g)
Total root dry biomass (g)
65.9 ± 10.4a 63.6 ± 9.9a 44.0 ± 4.1b 49.4 ± 8.4b
790.7 ± 135a 884.1 ± 214a 571.2 ± 98b 546.6 ± 78b
4.61 ± 1.1a 5.35 ± 1.4b 2.48 ± 0.7c 3.47 ± 0.6d
322.2 ± 44.7a 202.7 ± 30.9b 141.9 ± 31.6c 41.3 ± 18.9d
588.5 ± 123.6a 400.5 ± 50.8b 197.2 ± 53.7c 66.7 ± 24.2d
imum diurnal leaf (Fig. 1B) which was about −1.25 MPa for both LM and LD plants growing in full sunlight and about −1.15 MPa for shaded ones with no statistically significant difference between the two genotypes or irradiance levels in this regard. Similar whole-plant hydraulic conductances (Kplant , Fig. 2A) were measured for both genotypes. However, Kplant was significantly lower in LI plants compared to that measured in HI ones (Kplant was about 6.0 e-5 in the former versus 8.0 e5 kg s−1 m−2 MPa−1 in the latter). Analogous depression in K was measured for the roots (Kroot , Fig. 2B) of LM and LD plants when grown under shading nets whereas leaf hydraulic conductance (Kleaf ) was apparently unaffected by lower irradiance (Fig. 2C). The two genotypes showed no difference in Kroot or Kleaf when grown at equal PAR values. Anatomical measurements showed that LD twigs had substantially shorter and narrower xylem conduits than LM ones
Fig. 1. (A) Maximum transpiration rate (EL ) and (B) Minimum leaf water potential ( leaf ) as measured between 12.00 and 14.00 h in Leccino Minerva (LM) and Leccino Dwarf (LD) clones growing in full sunlight (black columns) and at 50% full sunlight irradiance (white columns). Different letters indicate significant differences for Tukey’s pairwise comparisons (P < 0.01). Vertical bars are SD of the mean (n = 5).
Fig. 2. (A) Plant hydraulic conductance scaled by plant leaf surface area (Kplant ), (B) root hydraulic conductance scaled by plant leaf surface area (Kroot ) and (C) leaf hydraulic conductance scaled by leaf surface area (Kleaf ) all measured in Leccino Minerva (LM) and Leccino Dwarf (LD) clones growing in full sunlight (black columns) and at 50% full sunlight irradiance (white columns). Means are reported ± SD (n = 5). Different letters indicate significant differences for Tukey’s pairwise comparisons (P < 0.01).
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Table 2 Xylem cross-sectional area, number of conduits per unit xylem cross-sectional area and cross-sectional conductive area (r2 ) as measured in 1-year-old twigs of Leccino Minerva (LM) and Leccino Dwarf (LD) clones growing at two irradiance levels (full sunlight irradiance, HI, and 50% full sunlight irradiance, LI). Means are given ± SD (n = 5). Different letters indicate significant differences for Tukey’s pairwise comparisons (P < 0.01).
LM, HI LM, LI LD, HI LD, LI
Xylem cross-sectional area (mm2 )
Number of conduits (mm−2 )
r2 (mm2 )
10.11 ± 1.86a 9.71 ± 1.63a 5.78 ± 2.11b 5.73 ± 1.91b
209 ± 24a 154 ± 16b 233 ± 16a 154 ± 20b
0.80 ± 0.08a 0.68 ± 0.06b 0.71 ± 0.07b 0.58 ± 0.07c
(Tables 3 and 4). Shading induced the production of longer xylem conduits in both genotypes and in particular in 1-year-old twigs of shaded plants where longest vessels were 41–50 cm long in LM genotype and of 31–40 cm in LD ones (Table 3). Moreover, reduced irradiance influenced conduit diameter negatively in stems of both genotypes. Conduits less than 20 m in diameter increased to about 40% in LM shaded plants and to about 45% in LD ones. Moreover, the percentage of conduits wider than 30 m decreased in shaded plants. In petiole and midribs, differences in conduit diameter were only recorded between genotypes (LM had wider conduits than LD) but not as a consequence of reduction in irradiance (data not shown). In particular, petioles and midribs of LM plants growing at HI had, respectively, 19.1 ± 5.3 and 35 ± 8% conduits with a diameter of 15–20 m (which was the maximum conduit diameter measured in these leaf regions) versus only 7.67 ± 3.2 (for petiole) and 10 ± 4.1% (for midrib) in LD plants. Effects of LI on xylem conduits also emerged from comparison of the different experimental groups in terms of number of conduits per unit xylem cross-sectional area and total cross-sectional conductive area (r2 , Table 2). The number of conduits mm−2 was by 25 to 33% lower in twigs of shaded plants of both genotypes than in those of plants grown in full sunlight over 210 conduits mm−2 in HI plants versus about 150 conduits mm−2 in LI ones. Accordingly, total conductive cross-sectional area was by about 16% smaller in LI twigs than in HI ones. In summary, in both genotypes low irradiance induced the production of less conduits per unit xylem cross-sectional area (Table 2) with single conduits slightly narrower (Table 4) but noticeably longer (Table 3) than in plants growing in full sunlight. Fig. 3 reports vulnerability to xylem cavitation measured on 1-year-old twigs of LM and LD plants grown under the two irradiance levels tested. At pressures ranging between −1.0 and −1.2 MPa corresponding to the min interval recorded during field experiments (Fig. 1B), the percentage loss of hydraulic conductivity (PLC) ranged between 35 and 45% in LM twigs at both irradiance levels, while it was 22–35% in LD twigs. In other words, the LM genotype was potentially more vulnerable to cavitation than the LD one with no influence of shading on this variable in both genotypes.
Fig. 3. Percent loss of hydraulic conductivity (PLC) of 1-year-old twigs measured in Leccino Minerva (LM) and Leccino Dwarf (LD) clones growing in full sunlight (black circles) and at 50% full sunlight irradiance (white circles). Vertical bars are SD of the mean (n = 5).
4. Discussion The olive genotypes ‘Leccino Minerva’ and ‘Leccino Dwarf’ were apparently different for plant size and growth potential as well as for leaf surface area (Table 1). By contrast, they did not differ from each other for hydraulic sufficiency or water status. Under optimal water availability conditions, the two genotypes showed similar plant hydraulic conductances (Kplant ) as well as similar conductance of roots and leaves (Kroot and Kleaf , Fig. 2A) on a leaf area basis. In other words, the hydraulic sufficiency of plants of LM and LD genotypes was the same both at the whole plant and at the organ level. The above finding helps explain the otherwise surprisingly similar transpiration rates (EL , Fig. 1A) and minimum leaf water potentials ( leaf , Fig. 1B) measured in the two clones. The recorded differences between the two genotypes in terms of xylem conduit dimensions, with LD twigs showing substantially shorter (Table 3) and slightly narrower (Table 4) conduits than LM ones did translate into some decrease in total conductive area in the former (r2 was by about 12% smaller in LD compared to LM). Nonetheless, equal Kplant values as expressed per unit leaf area were recorded in LM and LD plants suggesting that the
Table 3 Vessel length distribution measured on 1-year-old twigs of Leccino Minerva (LM) and Leccino Dwarf (LD) clones growing in full sunlight (HI) and at 50% full sunlight irradiance (LI). Means are ± SD (n = 5) is reported. Different letters indicate significant differences for Tukey’s pairwise comparisons (P < 0.01).
Table 4 Xylem conduit diameter distribution measured on 1-year-old twigs, petioles and midribs of Leccino Minerva (LM) and Leccino Dwarf (LD) clones growing in full sunlight (HI) and at 50% full sunlight irradiance (LI). Means are ± SD (n = 5). Different letters indicate significant differences for Tukey’s pairwise comparisons (P < 0.01).
Conduit length (cm)
LM, HI
LM, LI
LD, HI
LD, LI
Conduit diameter (m)
LM, HI
LM, LI
LD, HI
LD, LI
1–10 11–20 21–30 31–40 41–50
35.5 ± 4.9ab 42.0 ± 4.1bf 16.5 ± 2.2c 5.5 ± 0.9cd
28.5 ± 8.9a 39.5 ± 4.7bf 16.0 ± 3.3c 8.0 ± 2.2cd 2.5 ± 0.5d
60.5 ± 8.7f 32.5 ± 5.5ab 5.0 ± 1.8d
50 ± 6.5f 39 ± 4.8ab 10 ± 3.3cd 1 ± 0.3d
<10 11–20 21–30 31–40 >40
0.5 ± 0.6a 22.4 ± 5.0b 59.3 ± 4.8d 17.8 ± 4.8b 0.3 ± 0.2a
1.3 ± 1.2a 37.6 ± 6.7cd 53.1 ± 5.1d 10.1 ± 3.6ef 0.2 ± 0.4a
1.7 ± 1.9a 31.9 ± 8.0bc 50.4 ± 4.0cd 15.8 ± 3.1e
1.7 ± 0.4a 45.1 ± 5.0d 48.2 ± 3.5cd 7.8 ± 2.5f
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unit photosynthesizing area was similarly supplied with water in both cases. This finding is in accordance with the fact that the major portion (about 66%) of the whole plant hydraulic resistance (Rplant = 1/Kplant ) resided in the root system in both genotypes (Fig. 2). Such a partitioning of hydraulic resistances within the plant implies that any increase in hydraulic resistance of stem and leaves of both LD and LM plants (e.g. due to narrower conduits or to smaller numbers of conduits) would be minimized at the whole plant level (Meinzer, 2002). As an example, an increase in stem and leaf hydraulic resistance of LD stems as high as, say, 50% would lead Rplant of LD to increase by only 17%. In accordance with previous studies (e.g. Sellin, 1993; Tyree et al., 1998; Nardini et al., 2005; Sack et al., 2005; Barigah et al., 2006), shading induced significant reduction in Kplant in both genotypes (Fig. 2A). In principle, such a reduction could be a consequence of modification in the hydraulics of one or more plant organs (leaves, stems or roots). Our data clearly indicate that leaf hydraulic properties were unaffected by the light regime imposed (Fig. 2C). This conclusion is in contrast with previous reports by Nardini et al. (2005) and Sack et al. (2005). In both these studies, leaf hydraulic conductance was found to be higher in sun- than in shade-growing species. Similar light-dependency of Kleaf has been recently described in silver birch by Sellin et al. (2008). The lack of differences in Kleaf between our HI and LI plants might be due to the fact that shaded leaves experienced still high PAR values (about 1000 mol m−2 s−1 ). In other words, it is possible that substantial changes in leaf hydraulic properties of olive plants would only be triggered when leaves are more heavily shaded than in our experimental conditions. Xylem architecture was apparently influenced by the light regime, in that LI plants produced narrower (but longer) conduits than HI ones. In fact, the potential cross-sectional conductive area was reduced by 16 to 18% under shade conditions in both LM and LD clones (r2 , Table 2). In addition, shaded plants of the two clones produced one fourth to one third less conduits than plants growing in full sunlight (Table 2). This feature might contribute to the reduced Kplant recorded for shaded plants of the two clones compared to the same variable measured for plants in full sunlight. Root hydraulic conductance was found to be substantially lower in LI plants with respect to HI ones. The fractional magnitude of this reduction was near the same as that measured for Kplant suggesting that the recorded modification of root hydraulic properties was most responsible for the overall changes in Kplant , in accordance with plant hydraulics being dominated by the root compartment (see above). This conclusion is in accordance with previous studies by Kyllo et al. (2003) and Barigah et al. (2006) showing that in several tropical and temperate woody species, root hydraulic conductance decreased when plants were grown under shade conditions. In most of the above study cases, lower Kplant was due to a general reduction of root biomass, while Kroot expressed on a root biomass basis was often found to be unchanged. In other words, reduction of Kroot under shade conditions was due to reduced allocation to root growth and not to modifications of root hydraulic properties per se as in the case of changes in aquaporin expression (Tyermann et al., 1999). Our data are in accordance with these studies in that low irradiance showed to depress root production with larger effects in the dwarf genotype than in the vigorous one (Table 1). Previous studies by some of us had revealed that: (a) LM and LD rootstocks did not substantially differ for Kroot when expressed on a root biomass basis and (b) LD clone had strongly reduced root biomass compared to LM (Lovisolo et al., 2007). On the basis of our present data, the observed reduction of Kroot measured in plants of both LM and LD when growing in the shade was due to strongly reduced allocation of biomass to the root system. The basically similar Kplant values recorded in both genotypes was apparently the result of the similar allometry of the two genotypes
with the relative root growth proportional to that of the shoot. In turn, the shading effect leading to reduction of root biomass led to reduction in Kplant in both study cases. Interestingly, vulnerability to cavitation of LM and LD stems tended to shift towards a slight decrease under shade conditions (Fig. 3) contrary to expectations suggested by data reported in the literature (Cochard et al., 1999; Lemoine et al., 2002; Barigah et al., 2006). It is possible that some decrease in vulnerability to cavitation in shaded plants was a consequence of narrower xylem conduits (e.g. Lo Gullo et al., 1995). However, the magnitude of the shift in xylem vulnerability was small so that it is unlikely that this physiological trait may play any role in the adaptation of olive clones to LI, at least under the experimental conditions tested in the present study. It can be noted that Olea europea genotypes resulted more vulnerable to xylem embolism when compared with other olive cultivars as “Chemlali” and “Meski”, where PLC of 50% were recorded at −7.5 MPa (Ennajeh et al., 2008). Our data suggest that the hydraulic architecture of LM and LD clones was adjusted to meet the evaporative demand imposed by the irradiance regime at which plants were growing. In fact, HI plants were more hydraulically efficient than LI ones, as clearly indicated by higher Kroot and wider diameters of xylem conduits. The higher Kplant of HI plants compared to LI ones allowed the former to maintain water potential levels similar to those measured in the latter, despite the evaporative demand per unit leaf surface area was about 60% higher under full sunlight conditions than in the shade. It is of interest to note that the minimum leaf water potential recorded in the field (Fig. 1B) was close to the xylem water potential inducing 50% loss of hydraulic conductivity (Fig. 3), possibly indicating a runaway cavitation avoidance strategy based on buffering leaf to prevent catastrophic xylem dysfunction (Tyree and Sperry, 1988; Nardini and Salleo, 2000). The same goal, however, was achieved by LI plants that kept leaf water potential within critical thresholds by investing less energy and biomass in the production of roots and xylem conduits. In fact, production of root biomass and vascular tissue in excess of that required to compensate for evaporation would represent a useless, expensive investment under shade conditions. The shade-induced reduction in hydraulic efficiency of LI plants might have negative consequences for water balance of these plants when they are transplanted to the open at natural irradiance. The increase of transpiration rates under these new environmental conditions together with low hydraulic efficiency of plants may result in severe drops in leaf water potential with consequent stomatal closure, reduction of photosynthesis and growth until plants readapt to higher irradiance conditions. In turn, larger drops of leaf water potential might induce extensive cavitation and higher loss of hydraulic conductivity (larger in LM than in LD plants, Fig. 3) which would limit the necessary cambial activity for producing new roots and more efficient xylem. The scenario outlined is in accordance with the mortality recorded by farmers after transplanting plants grown under shading nets. We also note that even if the ability of plants to modify their hydraulic efficiency in response to changed irradiance conditions has been reported in detail (e.g. Barigah et al., 2006), no studies have been conducted yet on the time course of this adaptation in the case on sun species like olive. A corollary of the present study is that the hydraulic efficiency of plants is shaped to meet the evaporative demand. To this purpose, the composition of xylem, the hydraulic efficiency of conduits as well as the hydraulic conductance of the root system are adjusted to yield water potentials not exceeding levels triggering catastrophic embolism (Tyree and Sperry, 1988; Nardini and Salleo, 2000). Hydraulic sufficiency is addressed at maintaining the photosynthesizing apparatus well hydrated which was evidently the case both in dwarf plants and in vigorous ones (LD versus LM clone) as well as in plants grown under reduced evaporation compared to those with opposite behaviour (LI versus HI plants).
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