Does acclimation in cavitation resistance due to mechanical perturbation support the pit area or conduit reinforcement hypotheses in Phaseolus vulgaris?

Abstract

Two Phaseolus vulgaris L. cultivars were exposed to reduced water and stem mechanical perturbation treatments (flexing) to determine if acclimation to these treatments induced hydraulic changes, altered cavitation resistance and changed stem mechanical properties. Additionally, this study sought to determine if changes in cavitation resistance would support the pit area or conduit reinforcement hypotheses. Flexing reduced biomass, leaf area, xylem vessel area and hydraulic conductivity. One cultivar had greater measures of stem strength and cavitation resistance. Flexing increased cavitation resistance (P₅₀) but did not increase Young’s modulus, rigidity, or flexural strength on dried stems. Stem rigidity and basal diameter were correlated with leaf mass. The ratio of conduit wall thickness to span ((t/b)_h²) increased under high water and flexing treatments while rigidity decreased for one cultivar exposed to both flexing and lower water suggesting an inability to compensate for two simultaneous stresses. Although P₅₀ was not correlated with measures of mechanical strength, P₅₀ was correlated with vessel diameter, consistent with the pit area hypothesis. This study confirmed that mechanical perturbation can impact xylem structural properties and result in altered plant water flow characteristics and cavitation resistance. Long-term hydraulic acclimation in these herbaceous annuals was constrained by similar tradeoffs that constrain hydraulic properties across species.

Introduction

Phenotypic plasticity is an important mechanism causing traits to vary across environments and understanding the extent of plasticity in hydraulic traits is important for predicting the ability of plants and ecosystems to respond to climate change impacts such as increases in drought and increasing temperatures (Anderegg 2015). Recently there has been greater appreciation for the importance of plasticity in hydraulic traits in response to various environmental factors (Anderegg 2015, Badel et al. 2015, Fichot et al. 2015). For example, cavitation resistance has been shown to shift in response to various environmental conditions including drought (Wortemann et al. 2011, Plavcová and Hacke 2012, López et al. 2016). Much of this work has been done using woody perennial species and there are still relatively few studies that have focused on plasticity in cavitation resistance and its importance in herbaceous annual plants. Understanding the contribution of plasticity to variation in cavitation resistance is critical because cavitation resistance is a key trait, which can limit growth and survival of plants (López et al. 2016, El Aou-ouad et al. 2017) and within species variation in cavitation resistance is substantial and ecologically relevant (Anderegg 2015).

Differences between species in drought induced cavitation resistance are thought to occur as a result of variation in inter-conduit pore size (Tyree et al. 1994) and/or through differences in conduit wall reinforcement (Hacke et al. 2001). Although a number of studies have shown that cavitation resistance can shift, few studies have provided evidence that the same tradeoffs that constrain cavitation resistance across species also work to limit phenotypic plasticity in cavitation resistance within species. In addition, recent work has suggested that these tradeoffs can to a certain extent be decoupled (Plavcová and Hacke 2012, Fichot et al. 2015), but there is little understanding of the extent of this decoupling or the conditions under which it might occur.

According to the pit area hypothesis (Christman et al. 2012), species with larger conduits have a tendency for greater pitted wall area and a greater probability of a pit membrane pore that is more vulnerable to air seeding. These more vulnerable pits could be more susceptible to cavitation because they are defective, because of factors related to size, or due to other specific geometrical and physiochemical characteristics of the pit pore structure (Meyra et al. 2007, Badel et al. 2015). The pit area hypothesis has given rise to the observed “safety from cavitation versus efficiency of hydraulic transport” tradeoff (Tyree et al. 1994). There has been support for this hypothesis. In a study across 335 angiosperm and 89 gymnosperm species a weak correlation between specific conductivity and cavitation resistance was observed (Gleason et al. 2016). Choat et al. (2003) also found a relationship between cavitation resistance and hydraulic efficiency, however they did not observe differences in pit membrane porosity suggesting the relationship is indirect (Badel et al. 2015). Even if the effect is indirect, environmental factors that decrease conduit size, could have the potential to increase cavitation resistance if the smaller conduits also resulted in less pitted wall area and a smaller probability of a more vulnerable pit membrane pore. Plasticity in conduit size has been documented in a number of studies in response to environmental factors such as canopy release (Noyer et al. 2017), low phosphorus concentration (Holste et al. 2006), irradiance (Matzner et al. 2014) or reduced moisture (Holste et al. 2006). In addition, cavitation resistance typically changes in predictable ways in response to various environmental factors (Fichot et al. 2015) but relatively few studies have specifically looked at whether the changes in conduit size resulted in altered cavitation resistance.

Differences in cavitation resistance between species are hypothesized to occur through alterations in conduit reinforcement (conduit reinforcement hypothesis, Hacke et al. 2001), which is thought to enable plants to resist vessel implosion. Across species, greater reinforcement is a function of greater conduit wall thickness at the cellular level and higher wood density at the tissue level resulting in a tradeoff between “cavitation resistance and the cost of construction” (Hacke et al. 2001, Jacobsen et al. 2005, Pratt et al. 2007). Relatively few studies have observed acclimation of conduit reinforcement (mostly in woody perennial species) in response to environmental changes and often measures such as wood density (WD) have been shown to remain unchanged. For example, Pinus pinaster populations growing under different climatic conditions did not show differences in wood density (Lamy et al. 2012). In a study by Christensen-Dalsgaard and Ennos (2012) only one of three species studied exhibited changes in WD due to drought. Wood density did not change dramatically in response to canopy release (Noyer et al. 2017). One study that did observe changes in wood density (WD) was able to show variation in response to a number of environmental factors including drought, nitrogen fertilization, and shade treatments in Poplar (Plavcová and Hacke 2012). While there is strong evidence that species with greater cavitation resistance typically have greater measures of conduit reinforcement (Hacke et al. 2001, Jacobsen et al. 2005, Pratt et al. 2007), it is less clear whether environmental factors that alter stem mechanical strength can drive changes in cavitation resistance

In studying plasticity in xylem conduit size, conduit reinforcement and their impact on cavitation resistance, two environmental factors that might be particularly useful in altering stem structural traits are drought and mechanical perturbation. In response to reduced water, cavitation resistance has been shown to increase (Awad et al. 2010, Lopez et al. 2016) and conduit size decrease (Holste et al. 2006, Plavcova and Hacke 2012) but the effect of drought on measures of reinforcement is less clear (Holste et al. 2006, Plavcova and Hacke 2012, Awad et al. 2010, Lopez et al. 2016). Mechanical perturbation can result in thicker stems and increased stem rigidity (Jaffe et al. 1984) and these changes can be correlated with an increased production of strengthening tissues and a decrease in hydraulic conductivity (Smith and Ennos 2003, Braam 2005). Looking specifically at the effects of mechanical perturbation on P. vulgaris plants, Jaffe et al. (1984) found that stem elongation was retarded and stem thickening increased. They also saw that Young’s modulus decreased (increasing flexibility) and the force required to cause rupture increased, but stem flexural stiffness did not change. Greater mechanical strength of stems has been shown to correlate with greater cavitation resistance across species (Hacke et al. 2001, Jacobsen et al. 2005, Pratt et al. 2007), and since wood density is highly correlated with conduit size and cell wall thickness (Badel et al. 2015), mechanical perturbation may also affect xylem vessel size. In response to one or more stresses such as drought or mechanical perturbation, plants may be subject to tradeoffs between conductive efficiency, resistance to embolism and mechanical strength (Badel et al. 2015). Both water deficit and mechanical perturbation have the potential to alter conduit reinforcement or conduit size, which could theoretically result in a shift in cavitation resistance. It is unclear whether the effect of the combination of two stresses could be additive or result in competition between tradeoffs. Work with Arabidopsis for example; has shown that different stressors such as herbivory and drought can activate different stress pathways (Abscisic Acid and Jasmonic Acid) and they can negatively interact with each other (Fujita et al. 2006, Asselbergh et al. 2008).

The objectives of this study were threefold. The first objective was to determine if mechanical stimulation and reduced water treatments caused acclimation in xylem structure or stem mechanical support in the herbaceous Phaseolus vulgaris. A second objective was to determine if these treatments resulted in shifts in cavitation resistance individually and in combination. Lastly, this study sought to conclude if acclimation of cavitation resistance would be constrained as predicted by either the pit area hypothesis or the conduit reinforcement hypothesis.

Materials and methods

Experimental material

In 2009, 2010, and 2012 Phaseolus vulgaris L. (common bean), of the cultivars Othello (Durango race) and G4523 (Nueva Grenada race) were grown from seed with high or low water treatment (high water treatment only in 2012) and with or without mechanical perturbation (stem flexing). Plants were grown in a greenhouse on the campus of Augustana University (Sioux Falls, South Dakota, USA). Between 36 and 45 bean seeds were planted for each of four blocks in May (Block 1), June of 2009 (Block 2), May/April of 2010 (Block 3), and May of 2012 (Block 4). The plants were grown for 5–6 weeks before harvesting. Temperature and humidity varied with ambient conditions with supplemental heating below 4.4°C and cooling above 23.9°C. For 2009 and 2010, midday temperature and humidity were measured at the same time that maximum stomatal conductance was measured. Midday temperatures averaged 26.6 and 29.2°C on stomatal conductance measurement days for 2009 and 2010 respectively and humidity averaged 46, and 38.5%, for the same time periods. In 2012, humidity and temperature were monitored using a temperature/RH probe (Onset HOBO Pro v2) and daytime temperature averaged 26.0°C and humidity averaged 55.3%. Plants were grown in 4.2-l pots with one plant per pot. Treatments were imposed approximately 1 week after seedling emergence and continued for the duration of the experiment until harvested. The mechanical perturbation (flexing) treatment was similar to that used by Smith and Ennos (2003) and consisted of holding the base of the stem with thumb and forefinger and bending stems at a 45° angle in four cardinal directions, beginning at 10 and scaling up to 50 times per day. Water treatments consisted of saturating the soil media within the pots and allowing them to drain overnight; pots were then weighed to get the field capacity weight (FC). Pots were allowed to dry down and high water plants were kept at ~95% FC and low water plants at ~80% FC. Soil media for all treatments was composed of a black soil, sand, fritted clay mixture (1:1:1, by volume) with supplemental nutrients provided by osmocote enrichment (14:14:14, NPK, Scotts- Sierra, Marysville, OH). All treatments were watered as needed with a 1/10 strength nutrient solution to keep within desired water treatment range.

Maximum vessel lengths

Additional bean plants of both cultivars were grown during the summer of 2018 under greenhouse conditions with similar soil media as described above in order to measure maximum vessel length for both cultivars. Plants were watered daily and fertilized once per week with a nutrient solution (24:8:14 NPK) as well as being supplied with supplemental osmocote enrichment (14:14:14 NPK). Using a method similar to that described by Greenidge (1952) and also by Jacobsen et al. (2012), the maximum vessel length was measured for the two cultivars. Stem sections were cut under water and tubing connected to an air tank was attached to the distal end. A latex grommet filled with water was attached to the proximal end. Air pressures up to 100 kPa were applied and maximum vessel length was determined when bubbles appeared at the proximal end. The Othello cultivar averaged 9.04 cm (SE = 3.52, n = 20) while the G4523 cultivar averaged 9.26 cm (SE = 3.54, n = 32).

Vulnerability curves

Stem segments of P. vulgaris (at least 14.5 cm in length with one or more nodes) were cut under water and measures of conductance and cavitation resistance were determined following similar protocols as Matzner et al. (2014). Segments were flushed with a 0.22 μm filtered water solution for at least fifteen minutes to refill any embolized vessels and ensure the maximum conductance. After flushing, stems were perfused with a 0.22 μm filtered and degassed 10 mM KCl solution, adjusted to pH 7.0. Hydraulic conductance was calculated based on stem exudate mass collected over time. Xylem cavitation was induced using the centrifugal method. The amount of cavitation was quantified by measuring hydraulic conductance after applying tensions of 0.0, −0.1, −0.5, −1.0, −1.5 and −2.0 MPa. Hydraulic conductivity measured at 0.0 MPa was expressed as the maximum conductance, all conductance measurements at the more negative tensions were expressed as a percent loss from this initial conductance (PLC) and used to generate vulnerability curves. The tensions resulting in a 50% loss of hydraulic conductance was estimated using these curves as a measure of cavitation resistance. A Weibull function was used to fit hydraulic vulnerability curves using the program R (Duursma and Choat 2017).

Vessel diameters and conduit reinforcement

During the summer of 2010 and 2011, sections of stem segments frozen from 2009 and 2010 were used for measurements of vessel diameter and conduit reinforcement. The stems were sectioned with a hand-microtome and stained with a Safranin O solution in 95% EtOH. Stained stem sections were viewed under a confocal microscope (Olympus, Fluoview FV300, HeNe green 543 nm laser, TRITC, magnification 20×) and images of xylem conduits were obtained. An image analysis program (Fluoview) was used to calculate the cell areas of the xylem vessels, and diameter was calculated as the diameter of an equivalent circle. Hydraulic mean diameter (HmD) was calculated as the ∑ d^5/ ∑d^4 with d as the vessel diameter (Kolb and Sperry 1999) and used to target desired sizes of vessels (± 5 μm of the target HmD) for further analysis of wall thickness. Wall thickness was measured using the Fluoview analysis program and the ratio of conduit wall thickness (t) to diameter (b), or (t//b)_h² was used as a measure of conduit reinforcement (Hacke et al. 2001) across all treatments.

Water use, conductance, δ¹³C, and xylem water potential measures

Maximum stomatal conductance measurements (max g_s) were made on upper canopy leaves between 1000 and 1400 h (solar time) with a Li-Cor 1600 steady-state porometer (Li-Cor, Lincoln, NE). Midday xylem water potential (Ψ_mid) was measured for bagged plant leaves during the same time period (Soil Moisture Equipment Corp., Santa Barbara, CA). To determine δ¹³C values, leaf samples were dried, ground (< 0.5 μm) and analyzed for ¹³C:¹²C on a VG SIRA series II triple trap isotope ratio mass spectrometer (GV Instruments, Hudson, NH and Manchester, UK). Values were expressed as per ml (‰) ¹³C values. Whole plant water use was measured over a three to five day period just before plants were harvested. Pots were bagged to minimize evaporation from the soil surface or pot drainage and water loss was monitored gravimetrically (Weigh-Tronix, Fairmont, MN). Pot water loss per day was divided by plant leaf area to give the leaf area specific whole plant transpiration (T_LA). Leaf area was estimated based on the relationship between leaf area and leaf dry weight. Stem hydraulic conductance measures were made on the same stems used for the cavitation measures. Hydraulic conductance (k_h) was defined as the mass flow rate through the stem divided by the pressure gradient across the stem segment (kg m s⁻¹ MPa⁻¹). For leaf specific conductivity (k_L), k_h values were divided by plant leaf area.

Three point bending measurements

During the summer of 2012 and 2013, stem segment sections used to measure the percent loss in conductivity curves from 2009, 2010, and 2012 were used for measurements of whole stem stress through a three point bending test using an MTS 858 Materials Testing System (www.sd-metlab.org). Upon completion of the PLC curves, the mostly embolized stems were frozen for future vessel diameter measures and in order to use the same segments for the mechanical strength measures, the stems were dried at 60°C for at least 24 h. Jacobsen et al., 2005 also used dried stem material for their measurements of mechanical strength. While the three point bending measures were used as a measure of mechanical strength, we are not suggesting these measures are equivalent to measures made on fresh stems. Stem bending rigidity, and Young’s modulus were calculated similar to the calculations of Paul-Victor and Rowe (2011).

Stem bending rigidity of elliptical stems (EI; N mm²) was calculated as:

$$\text{EI = }{\text{L}}^{\text{3}}\text{c/4b}{\text{d}}^{\text{3}}$$ (1)

Where L is the distance in mm between the two supports in three-point bending, c is the slope of the force deflection curve (N mm⁻¹), b is the mean horizontal radius in the direction of the applied load and d is the mean vertical radius.

Values of Young’s modulus (E) measured in bending were calculated as:

$$\text{E = EI/I}$$ (2)

Where I (mm⁴) is the axial second moment of area of the stem approximated as an ellipse by the following:

$$\text{I = }{\text{d}}^{\text{3}}\text{bπ/4}$$ (3)

The flexural strength (σ), or ability of the stems to resist deformation under load and the highest stress at moment of rupture was calculated as:

$$\text{σ = PLd/π}{\text{d}}^{\text{3}}\text{b}$$ (4)

where P is the peak applied force, L is the distance in mm between the two supports in three-point bending, b is the width of the stem and d is the depth of the stem.

Statistics

Differences in dependent variables (biomass, root:shoot, basal diameter, etc.) were analyzed using a univariate, general linear model (JMP 13, SAS Inst.). Variables that did not meet normality assumptions were transformed using a Box-Cox transformation. Cultivar, drought, and flexing were fixed factors, and Block (date and year plants were grown) was listed as a random factor. Pearson correlation coefficients and probabilities were also calculated using JMP and correlations between all variables can be seen in Table S1. Replication for each variable for each treatment and cultivar combination varied and ranged between 12–27 for total biomass, root:shoot, basal diameter, leaf area, maximum g_s, k_h, k_L, P₅₀, and T_LA, and between 7–13 for vessel diameter, vessel area, δ¹³C, E, EI, σ, (t/b)_h², and Ψ_mid.

Results

Flexing did result in a significant increase in stem basal diameter (P < 0.003, Table 1, Table 2), although the differences in basal diameter were slight. Flexing also resulted in decreases in total biomass and leaf area (P < 0.015) and an increase in root:shoot (P < 0.05). Flexing resulted in lower measures of hydraulic support including lower vessel diameter (P < 0.005, Table 1, Fig. 1A), lower vessel area, and leaf specific conductance (All P < 0.003, Tables 1 and 2). Plant stems exposed to the flexing treatment maintained a higher percent loss in conductivity with increasing applied tension for both cultivars (Fig. S1A, B). The flexing treatment did result in lower P₅₀ values (greater cavitation resistance, P < 0.0001, Table 1, Fig. 1B); however, it did not increase measures of stem mechanical strength including Young’s modulus (E), flexural strength (σ ), and stem bending rigidity (EI, Tables 1 and 2, Fig. 1C).

Table 1.

Analysis of variance (ANOVA) probability values for various dependent variables for the treatments and interactions included in the statistical model. Interactions included flexing by cultivar (T*C), flexing by water (T*W), cultivar by water (C*W), and flexing by cultivar by water (T*C*W). Abbreviated variables included the maximum stomatal conductance (max g_s), leaf specific conductance (k_L), leaf specific whole plant transpiration (T_LA), carbon isotope ratio (δ¹³C), cavitation resistance (P₅₀), Young’s Modulus (E), flexural strength (σ), Stem rigidity (EI), the ratio of xylem vessel wall thickness to vessel span ((t/b)_h²), and midday water potential (Ψ_mid). The n ranged from 12–27 except for vessel diameter, vessel area, δ¹³C, E, σ, EI, (t/b)_h², and Ψ_mid where n ranged from 7–13.

	Treatment (T)	Cultivar (C)	Water (W)	T*C	T*W	C*W	T*C*W	Block
Biomass	0.001	0.01	NS	NS	NS	NS	NS	NS
Root:Shoot	0.05	0.0001	NS	NS	0.005	NS	NS	NS
Basal Diameter	0.003	0.0001	0.04	NS	NS	NS	0.01	NS
Leaf Area	0.015	0.0001	NS	NS	NS	NS	NS	NS
max gs	NS	0.0001	0.0001	NS	NS	0.05	NS	NS
kL	0.002	0.0001	NS	NS	NS	NS	NS	NS
T(LA)	NS	0.0001	0.004	NS	NS	NS	NS	NS
Vessel Dia.	0.005	0.0001	NS	NS	NS	NS	NS	NS
Vessel Area	0.003	0.0001	NS	NS	NS	NS	NS	NS
δ 13C	NS	0.0001	0.03	NS	NS	NS	NS	NS
P50	0.0001	0.0001	NS	NS	NS	NS	0.03	NS
E	NS	0.05	NS	NS	NS	0.03	NS	NS
σ	NS	0.0001	NS	NS	NS	NS	NS	NS
EI	NS	0.0001	NS	0.03	0.002	NS	0.002	NS
(t/b)h2	NS	0.0001	0.008	NS	0.04	NS	NS	NS
Ψmid	NS	NS	0.0001	NS	NS	0.02	NS	NS

Table 2.

Effect of stem flexing (F) or not flexed (N) and high water treatment (H) or low water treatment (L) on the long-term hydraulic acclimation of various plant growth and hydraulic properties for the Othello and G4523 cultivars of Phaseolus vulgaris. Abbreviated variables included maximum stomatal conductance (max g_s) leaf specific conductance (k_L), leaf specific whole plant transpiration (T_LA), carbon isotope ratio (δ¹³C), Young’s Modulus (E), flexural strength (σ), and midday water potential (Ψ_mid). The numbers in bold represent the mean with the standard error values below the means in parentheses. The n ranged from 12–27 except for vessel area, δ¹³C, E, σ, and Ψ_mid where n ranged from 7–13

	G4523 Cultivar				Othello Cultivar
	FL	FH	NL	NH	FL	FH	NL	NH
Biomass (g)	11.9 (1.8)	11.4 (1.1)	16.1 (1.9)	14.4 (1.0)	10.2 (1.4)	10.0 (0.9)	16.1 (1.4)	11.0 (0.8)
Root:Shoot (g/g)	0.20 (0.01)	0.18 (0.01)	0.16 (0.01)	0.20 (0.02)	0.27 (0.02)	0.29 (0.04)	0.20 (0.03)	0.26 (0.02)
Basal Dia. (mm)	5.6 (0.3)	6.1 (0.2)	5.9 (0.3)	5.5 (0.2)	4.8 (0.2)	4.7 (0.2)	4.6 (0.2)	4.6 (0.2)
Leaf Area (m2)	0.198 (0.030)	0.237 (0.013)	0.266 (0.031)	0.267 (0.011)	0.146 (0.025)	0.168 (0.013)	0.213 (0.028)	0.179 (0.012)
max gs (mmol m−2 s−1)	151.5 (19.4)	254.9 (28.1)	129.4 (23.5)	239.3 (25.2)	185.6 (27.7)	420.4 (29.3)	187.4 (24.8)	365.9 (22.7)
kL (kg m−1 s−1 MPa−1)	4.4E-05 (7.4E-06)	3.6E-05 (7.1E-06)	8.3E-05 (11.5E-06)	5.3E-05 (8.7E-06)	7.2E-05 (6.5E-06)	6.6E-05 (6.1E-06)	7.6E-05 (7.4E-06)	8.8E-05 (8.1E-06)
T(LA) (kg d−1 m−2)	1.37 (0.13)	1.45 (0.12)	1.29 (0.14)	1.42 (0.07)	1.92 (0.16)	2.60 (0.18)	1.91 (0.16)	2.42 (0.18)
Vessel Area (μm2)	1402.8 (115.1)	1350.5 (109.5)	1714.5 (86.8)	1687.9 (162.7)	2147.7 (129.8)	2078.1 (113.5)	2624.5 (226.1)	2296.2 (179.2)
δ 13C (‰)	−26.83 (0.40)	−27.01 (0.20)	−26.87 (0.24)	−27.33 (0.14)	−28.70 (0.47)	−28.93 (0.35)	−28.77 (0.19)	−29.33 (0.45)
E (GPa)	3.47 (0.30)	5.51 (0.49)	4.42 (0.58)	5.94 (0.29)	4.11 (0.60)	4.36 (0.71)	3.71 (0.91)	4.09 (0.62)
σ (N mm−2)	54.03 (4.62)	56.38 (4.90)	58.05 (9.95)	59.66 (4.25)	35.75 (5.30)	48.79 (7.32)	34.26 (7.01)	44.09 (6.99)
Ψmid (MPa)	−0.60 (0.10)	−0.41 (0.03)	−0.45 (0.03)	−0.45 (0.04)	−0.45 (0.04)	−0.45 (0.04)	−0.43 (0.03)	−0.45 (0.03)

Fig. 1.

The average (± SE) effect of flexing (F) or non-flexing (N) treatments for both the G4523 (G) and Othello (O) cultivars of P. vulgaris at either high water level (H) or low water level (L) for (A) vessel diameter (VD), (B) cavitation resistance (P₅₀), (C) stem rigidity (EI), and (D) the ratio of conduit wall thickness to conduit span ((t/b)_h²). The n ranged from 8–13 for except for P₅₀ where n ranged from 12–23.

The two cultivars differed in measures of plant size with the G4523 cultivar having greater total plant biomass, leaf area, and basal stem diameter (All P < 0.01, Tables 1 and 2). The G4523 cultivar also had higher values of δ¹³C (an indirect measure of WUE), P₅₀, and measures of stem strength such as E, EI, σ and (t/b)_h² (All P < 0.05; Tables 1 and 2). In contrast, the Othello cultivar had greater measures of hydraulic support including larger vessel area and average diameter, greater maximum stomatal conductance (max g_s), leaf specific conductance (k_L), leaf area specific transpiration (T_LA) and root:shoot (All P <0.0001, Tables 1 and 2, Fig. 1A). The “low” water treatment used in this study ended up being a rather mild water stress as neither plant biomass or leaf area was significantly affected by the treatment. Plant water potential was decreased (P < 0.0001) under the lower water treatment while water use efficiency (measured as δ¹³C) was increased (P < 0.03). Cavitation resistance curves were similar between high and low water treatments for the two cultivars and flexing treatments (Fig. S2A, B). The lower water treatment resulted in lower max g_s T_LA, and δ¹³C (P < 0.004; Tables 1 and 2) suggesting reduced stomatal opening. Other measures of hydraulic support were not significantly different. The high water treatment also had higher (t/b)_h² (P < 0.008; Table 1, Fig. 1D).

There were a number of cultivar×water or flexing×cultivar×water interactive effects (for basal diameter, max g_s, E and EI, all P < 0.05) reflecting differences in how the two cultivars responded to the water and flexing treatments (Tables 1 and 2, Fig. 1C). There were also several flexing×water treatment interactions (for root:shoot and (t/b)_h², all P < 0.04) that were due to different effects of flexing under the two water treatments (Tables 1 and 2, Fig. 1D).

Measures of stem strength were tightly correlated and a significant positive correlation was observed between E and σ (Fig. 2). There was also a strong positive correlation between EI and basal diameter (Fig. 3A) and a significant negative relationship between EI and the T_LA reflecting a tradeoff between plant hydraulic flow and mechanical strength (Fig. 3B). Both EI and stem basal diameter were also highly correlated with leaf biomass (Fig. 4 A,B). Measures of (t/b)_h² were not significantly correlated with changes in cavitation resistance (Fig. 5A); in contrast, there was a positive correlation between cavitation resistance and vessel diameter (P < 0.01, Fig. 5B).

Fig. 2.

The relationship between Young’s modulus (E) and the flexural strength (σ) for both cultivars of P. vulgaris exposed to flexing and water treatments. The correlation coefficients and probability values for the relationships are also given.

Fig. 3.

(A) The relationship between stem rigidity and the stem basal diameter and (B) the relationship between stem rigidity and the leaf specific transpiration rate (T_LA) for both cultivars of P. vulgaris exposed to flexing and water treatments. The correlation coefficients and probability values for the relationships are also given.

Fig. 4.

(A) The relationship between leaf biomass and stem rigidity and (B) the relationship between leaf biomass and stem basal diameter for both cultivars of P. vulgaris exposed to flexing and water treatments. The correlation coefficients and probability values for the relationships are also given.

Fig. 5.

(A) The relationship between the ratio of conduit wall thickness to conduit span ((t/b)_h²) and cavitation resistance (P₅₀) and (B) the relationship between vessel diameter (VD) and cavitation resistance (P₅₀) for both cultivars of P. vulgaris exposed to flexing and water treatments. Each point represents the average (± SE) for each of the eight treatment and cultivar combinations. The n ranged from 8–12 for vessel diameter and (t/b)_h² and 12–23 for P₅₀. The correlation coefficients and probability values for the relationships are also given. Note that the data for the two cultivars were grouped together as the slopes were not significantly different.

Discussion

Effects of mechanical stimulation and water treatments

Mechanical stimulation of P. vulgaris stems resulted in a number of alterations such as increased stem diameter, reduced growth, a decrease in leaf area, and reduced hydraulic conductivity that are consistent with studies that exposed plants to either wind or mechanical stimulation (Jaffe et al. 1984, Smith and Ennos 2003). In addition, flexing resulted in a decrease in vessel diameter and an increase in cavitation resistance. Smith and Ennos (2003) also observed a decrease in hydraulic capability as a result of mechanical stimulation; however, this is the first study to show that mechanical stimulation can alter cavitation resistance.

Flexing did not result in an increase in stem strength, rigidity, or flexibility of the dried stems in this study. Although cavitation resistance has been shown to be greater for species with greater mechanical support (Hacke et al. 2001, Jacobsen et al. 2005, Pratt et al. 2007), the increased cavitation resistance observed under the flexing treatment did not appear to result from an increase in mechanical support. Because the stems used for cavitation and hydraulic conductance were frozen for later measures of vessel diameter and xylem structure, mechanical strength was measured on dried stem material. It is possible that the use of fresh material would have had different results. While three-point bending was used as a measure of mechanical strength, we are not suggesting these measures are equivalent to measures made on fresh stems. Other studies have also used dried stem material for measures of mechanical strength (Jacobsen et al. 2005). There were clear differences between the mechanical properties of the dried stems between the two cultivars with the G4523 cultivar having greater measures of mechanical support. This is consistent with its more upright growth form and greater leaf area. The fact that mechanical stimulation did not result in increases in most measures of mechanical support in P. vulgaris was surprising and is in contrast with studies by Jaffe et al. (1984) and Smith and Ennos (2003) that did observe changes in mechanical properties in beans and sunflowers respectively. The effects of mechanical stimulation can be quite variable however. Goodman and Ennos (1996) also did not find changes in rigidity, strength and stiffness in shoots of sunflower and maize plants (although they did see changes in root tissue). In general, studies have observed reductions in the Young’s modulus (E) in response to mechanical perturbation (Paul-Victor and Rowe 2011, Anten et al. 2010) and that the maximum stress applied before rupture can increase (Smith and Ennos 2003) or remain unchanged (Goodman and Ennos 1996). Rigidity in response to mechanical perturbation can increase (Smith and Ennos 2003), decrease (Anten et al. 2010, Paul-Victor and Row 2011) or remain unchanged (Goodman and Ennos 1996) depending on the species and tissue (Braam 2005).

Stem flexing did increase (t/b)_h² in this study, but only under the high water treatment. Relatively few studies have looked at acclimation of mechanical properties due to drought (mostly in woody perennials) and the plasticity of these traits appear to vary with species. Studies by Niez et al. (2018) and Roignant et al. (2018) found significant cross-sectional ovalization in response to periodic bending of young popular trees and alteration of anatomical traits (vessel and fiber diameter, microfibril angle between compressed and stretched woody zones. Niez et al. (2018) also observed mechanical acclimation under both high and low water treatments and an increase in bending rigidity regardless of hydric conditions. In general, measures of stem structural support for P. vulgaris in this study tended to be lower under drought or under flexing and drought combined. This is in contrast to the observations by Niez et al. (2018) and to other studies showing that several woody perennial species exhibited greater measures of mechanical support in response to drought. Christensen-Dalsgaard and Ennos (2012) found that drought resulted in both stiffer and stronger stems in several woody species. Plavcová and Hacke (2012) observed acclimation in response to drought (WD increased) in distal stem segments (but not basal segments) in Poplar. Beikircher and Mayr (2009) found changes in ((t/b)_h² in response to drought in Ligustrum vulgare although not in Viburnum lantana. Awad et al. (2012) also did not see acclimation in Young’s modulus for transgenic poplars modified for lignin metabolism. Increases in various measures of mechanical strength in stems can be a result of a number of factors (Badel et al. 2015); however, this type of morphological plasticity typically comes with the cost of additional carbon allocation (Grime & Mackey 2002). The inability of P. vulgaris to increase (t/b)_h² under the lower water treatment in this study may reflect constraints on carbon allocation. The positive relationship between stem rigidity and stem basal diameter in this study may also reflect constraints on carbon allocation. One way to increase stem rigidity is to increase basal diameter; from a carbon cost perspective, it is more efficient to increase the number of large thin walled cells than to have fewer smaller cells with thicker cell walls (Awad et al. 2012). In the study by Matzner et al. (2014), lower WD in P. vulgaris exposed to low light may also have reflected carbon allocation constraints. The carbon cost of increasing measures of stem structural support in response to environmental factors may make plasticity in the ability of annual herbaceous species to increase stem structural support challenging.

The reduced water treatment also did not have a significant effect on vessel diameter, vessel area, or P₅₀ in this study. Given that it was a mild water stress that did not significantly affect biomass or leaf area this may not be surprising. Previous work with the herbaceous P. vulgaris (Othello cultivar) did observe a reduction of vessel diameter in response to drought, but only found a marginally significant effect of drought on cavitation resistance (Holste et al. 2006).

Cultivar effects

Previous work with the G4523 and Othello cultivars found distinct differences in both growth and hydraulic characteristics. The G4523 cultivar, selected for use under rain fed conditions typically has a more upright growth form, greater leaf area, and greater cavitation resistance while the Othello cultivar, selected for use under irrigated conditions has greater measures of hydraulic support (Matzner et al., 2014). The G4523 cultivar in this study also had higher values of leaf area, δ¹³C, basal diameter, stem mechanical strength and cavitation resistance. The Othello cultivar in this study exhibited greater measures of hydraulic support with higher g_s, k_h, k_L, T_LA, and vessel area. With the G4523 cultivar having greater leaf area, it is not surprising that it also had greater stem mechanical support. Overall, both stem rigidity and basal diameter were highly correlated with total leaf mass across both cultivars. Herbaceous plant stems need to support the weight of their leaf material against the forces of gravity and side forces such as wind (Schulgasser and Witztum 1997). The fact that the G4523 cultivar also had greater cavitation resistance compared to the Othello cultivar is also consistent with the higher values of basal diameter, EI, and σ observed for the G4523 cultivar. Positive relationships between cavitation resistances and mechanical reinforcement have been observed across species (Jacobsen et al. 2005, Pratt et al. 2007) as well as across genotypes within a species (Guet et al. 2015).

Plasticity constraints and importance

Most of the studies that have looked for safety vs efficiency or mechanical support vs construction cost tradeoffs have used interspecific comparisons of woody species from a range of native habitats or life history strategies (Tyree et al. 1994, Jacobsen et al. 2005, Pratt et al. 2007, Jacobsen et al. 2016). Within a single species, these relationships may be more difficult to find (Martínez-Vilalta et al. 2009), so it is not entirely clear whether these same tradeoffs also work to constrain plasticity and whether changes in either vessel diameter or conduit reinforcement can drive changes in cavitation resistance. If these tradeoffs occur within individual species then it would be expected that environmental conditions that increase vessel size or decrease mechanical strength (or both) would be most likely to shift cavitation resistance. Support for the idea that changes in conduit reinforcement could drive shifts in cavitation resistance can be found in the study by Plavcová and Hacke (2012) who observed that differences in WD due to various environmental treatments correlated with P₅₀ for Poplar. Matzner et al. (2014) also found that decreasing WD with decreasing light levels was correlated with a decrease in P₅₀ in P. vulgaris plants. The correlation between xylem vessel diameter and cavitation resistance in this study would also be consistent with the possibility that changes in conduit size may also be able to drive changes in cavitation resistance through a mechanism such as the pit area hypothesis. One caveat is that this relationship could be the result of an open vessel artifact. To address this question, maximum vessel lengths were measured for each cultivar in a separate experiment. Maximum vessel lengths averaged between 9–9.3 cm (SE = 3.5), which is well below the 14.5 cm stem lengths used in this study. There was considerable variation however so the possibility of an open vessel artifact contributing to the correlation between vessel diameter and cavitation resistance cannot be completely discounted. For a positive correlation between vessel diameter and cavitation resistance to occur due to an open vessel artefact, vessel length and vessel diameter would need to be correlated. Jacobsen et al. (2012) however, did not find that vessel diameter was strongly predictive of mean vessel length. In this study with the two bean cultivars, vessel diameter was significantly greater in the Othello cultivar, but the maximum vessel length was not different between the two cultivars; this does not appear to support a strong correlation between vessel diameter and maximum vessel length for these two cultivars.

Variation in hydraulic and structural traits and acclimation of those traits were also constrained by genotype. The two cultivars differed in both structural and hydraulic measures, but also differed in the ability to adjust. For example, stem rigidity (EI) varied significantly for the G4523 cultivar but there was little difference within the Othello cultivar. Other studies have also indicated that the ability for acclimation can vary with species or genotype (Rosenthal et al. 2010, Wortemann et al. 2011).

In response to the flexing and reduced water treatments there were significant shifts in hydraulic and structural traits within the two herbaceous P. vulgaris cultivars. Acclimation of cavitation resistance has been shown for a number of woody perennial species (Beikircher and Mayr 2009, Awad et al. 2012, Lopez et al. 2016). While a number of studies have documented acclimation in woody species, relatively few studies have focused on hydraulic acclimation of herbaceous annual plants and its importance. Studies showing that cavitation resistance can limit growth and survival of woody species (López et al. 2016, El Aou-ouad et al. 2017) highlight the importance of cavitation and that the ability for acclimation could be quite important for herbaceous plants as well. The few studies that have focused on plasticity in herbaceous plants have confirmed its importance. For example, differences in the ability to adjust cavitation resistance helped explain the distribution differences between two species of sunflower growing in dune vs off-dune sites in a study by Rosenthal et al. (2010). In a study on Solidago canadensis, Nolf et al. (2013) observed plasticity in hydraulic and anatomical traits and suggested this ability is an important factor in explaining its invasive potential. Although few studies have focused on the importance of cavitation resistance in herbaceous plants, the capacity of P. vulgaris in this study to adjust hydraulically indicates that it has the potential to be quite important.

Conclusions

In response to mechanical perturbation this herbaceous annual plant exhibited significant changes in growth, hydraulic, and structural traits. Mechanical perturbation resulted in an increase in P₅₀ and this is the first study to show that mechanical perturbation can alter cavitation resistance. Despite the fact that mechanical perturbation has been shown to alter stem mechanical properties, the changes in cavitation resistance were not related to changes in conduit reinforcement. Mechanical stimulation did cause a decrease in conduit size, and changes in cavitation resistance were correlated with changes in vessel diameter, which appears to support the pit pore hypothesis. The two cultivars responded similarly to both flexing and water treatments but also exhibited distinct differences. The G4523 cultivar had greater levels of mechanical strength and cavitation resistance, while the Othello cultivar exhibited higher values of hydraulic support. While most measures of mechanical strength were not altered by mechanical perturbation, values of (t/b)_h² were only higher under both flexing and high water treatments suggesting an inability to increase mechanical support under two simultaneous stresses. Measures of mechanical strength were highly correlated with each other, and stem rigidity was positively correlated with leaf biomass and negatively correlated with hydraulic efficiency. This study highlights the potential for acclimation of hydraulic and structural traits within an herbaceous annual and suggests that plasticity may be constrained by tradeoffs similar to tradeoffs observed within interspecific comparisons.

Abbreviations –

δ¹³C

carbon isotope ratio

P₅₀

cavitation resistance

σ

flexural strength

k_h

hydraulic conductivity

k_L

leaf specific conductivity

T_LA

leaf area specific whole plant transpiration

max g_s

maximum stomatal conductance

Ψ_mid

midday water potential

(t/b)h2

ratio of conduit wall thickness to span

EI

stem bending rigidity

E

Young’s modulus

WD

wood density