Agricultural Water Management 212 (2019) 12–22

Contents lists available at ScienceDirect

Agricultural Water Management

journal homepage: www.elsevier.com/locate/agwat

Morpho-anatomical, physiological and biochemical adaptive responses to T

saline water of Bougainvillea spectabilis Willd. trained to different canopy
shapes
Petronia Carilloa, Chiara Cirillob, Veronica De Miccob, Carmen Arenac,d, Stefania De Pascaleb,
⁎
Youssef Rouphaelb,
a
Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania “Luigi Vanvitelli”, Caserta, Italy
b
Department of Agricultural Sciences, University of Naples Federico II, Portici, Italy
c
Department of Biology, University of Naples Federico II, Naples, Italy
d
CNR—National Research Council, Institute for Sustainable Plant Protection, Florence, Italy

A R T I C LE I N FO A B S T R A C T

Keywords: Bougainvillea is widely used as potential ornamental shrub in revegetation and landscaping projects as well as in
Adaptive mechanisms urban landscape design in Mediterranean areas characterized by saline and sodic soils. The current research
Gas exchange aimed at elucidating the morphological, physiological and biochemical changes occurring in Bougainvillea
Ion content spectabilis ‘Alexandra’ grown under salt stress conditions (1 mM [S1, electrical conductivity EC = 2.0 dS m−1]
Leaf anatomy and eco-physiology
non-salt control, 40 mM [S40, EC = 6.5 dS m−1] and 80 mM [S80, EC = 11.0 dS m−1] NaCl). The response of
Ornamental potted plant
plants trained to two canopy shapes (globe [GSh] and pyramid [PSh]) has been compared in order to unravel
Salinity
Water relations possible adaptive mechanisms, combining morpho-functional traits at different levels, to cope with saline water.
PSh plants under moderate saline water (40 mM NaCl) showed higher leaf dry weight, leaf number and area as
well as higher number of flowers per plant than those observed in GSh Bougainvillea plants. Saline water-trig-
gered leaf anatomical changes were also harmonized to physiological adjustments in both canopy shapes in such
a way to counteract the reduction in transpiration due to stomatal limitations by adopting traits improving CO2
diffusion in the mesophyll and investing more resources in the assimilatory tissue (e.g. increase in intercellular
spaces and incidence of palisade parenchyma over the total mesophyll thickness). Bougainvillea plants enact
many-sided strategies to acclimate to S80 salinity by harmonizing several mechanisms including sodium and
chloride inclusion in leaves and their compartmentalization in vacuoles, retention of high levels of potassium in
the cytosol to osmotically balance the cytoplasm and other organelles within the vacuole, modulation of leaf
functional anatomical traits, in addition to a C2 Kranz mechanism of photosynthesis which allow plants to
successfully cope with stress.

1. Introduction
In Mediterranean basin more than 40% of soils are affected by
salinity and sodicity (Nedjimi, 2014). In this area, daily exposure to
hyperosmotic stress due to seawater intrusion into groundwater and
irrigation with saline water, as well as to seasonal accumulation of salts
in the roots due to scarce winter rainfalls increase salinity and reduce
crop productivity (Rana and Katerji, 2000; Maggio et al., 2011). Sali-
nity decreases plant capacity to extract water from soil and determines
accumulation of ions like Na+ and Cl− at toxic concentrations in cell
tissues. This dual osmotic and ionic stress reduces cell and tissues ex-
pansion, and causes nutritional imbalance and oxidative stress affecting
plant growth, development and survival (Hasegawa et al., 2000; Munns
and Tester, 2008; Gorham et al., 2010). The extent of damage to plants
depends not only on salt concentration and toxicity level, but also on
the specific sensitivity of the plant species that may vary with the
phenological stage (Lutts et al., 1995; Hasegawa et al., 2000) and may
be eventually influenced by crop management practices (Colla et al.,
2010; Rouphael et al., 2012).
Sodium and chloride are both toxic if accumulated at high con-
centration in cytosol and organelles (Tavakkoli et al., 2010). Sodium
reduces potassium and calcium uptake, affecting stomatal conductance;
moreover, it is able to replace potassium in key enzymatic reactions.
Therefore, it is crucial for salt tolerance not only limiting the absolute

⁎
Corresponding author.
E-mail address: youssef.rouphael@unina.it (Y. Rouphael).

https://doi.org/10.1016/j.agwat.2018.08.037
Received 23 December 2017; Received in revised form 5 August 2018; Accepted 25 August 2018
0378-3774/ © 2018 Elsevier B.V. All rights reserved.
P. Carillo et al.
amount of sodium in the cytosol, but also increasing the potassium to
sodium ratio present in the cell (Shabala and Cuin, 2008; Annunziata
et al., 2017). Instead, chloride toxic effects concern chlorophyll de-
gradation, decline of PSII quantum yield and photosynthetic electron
transport rate (Tavakkoli et al., 2010, 2011). However, plants irrigated
with saline water take specific measures to minimize stress damage. In
particular, they compartmentalize toxic ions in the vacuole as cheap
osmotica and synthesize compatible metabolites to osmotically balance
the cytosol, controlling ion and water homeostasis to maintain normal
cellular turgor, reducing oxidative stress and reactivating growth
(Hasegawa et al., 2000; Gao et al., 2007; Carillo et al., 2008; Puniran-
Hartley et al., 2014; Rouphael et al., 2017a, d). Moreover, they modify
suites of leaf anatomical traits, such as leaf thickness and density, the
ratio between palisade and spongy mesophyll as well as stomata fea-
tures in order to improve gas diffusion inside leaf lamina tissues to
reach carboxylation sites (Acosta-Motos et al., 2015; Tattini et al., 2006;
Rouphael et al., 2017d).
Crop plants irrigated with saline water have been extensively stu-
died under single or combined salt stress (Annunziata et al., 2017;
Woodrow et al., 2017 and references therein). However, few authors
have focused their attention on the effect of salinity on ornamental
shrub and bush species, completely neglecting them in spite of their
wide range of tolerance levels since many of them are naturally oc-
curring in coastal areas and have evolved peculiar combinations of
structural and physiological traits to cope with drought, salinity and
high solar irradiation stresses (Tattini et al., 2002, 2006). Therefore,
they provide interesting models to be studied because they would not
only provide relevant additional information on general stress tolerance
mechanisms in plants, but also furnish know-how on their adaptability
for use in landscaping and urban greening (Acosta-Motos et al., 2016,
2017; Cassaniti et al., 2009, 2012; Cirillo et al., 2014; Kumar et al.,
2017).
In Mediterranean arid and semiarid areas, as well as in marginal
soils, brackish water could be used in landscaping projects, using or-
namental species showing relative degree of salt tolerance (Acosta-
Motos et al., 2015). Bougainvillea spectabilis Willd., also known as great
bougainvillea or ‘paper flower’ for its thin and papery bracts, is an
important ornamental flowering species of high aesthetic value, native
of Brazil and present nowadays all over the world in areas with tropical
or warm climates (Lack, 2012; Ghogar and Jiraungkoorskul, 2017). In a
previous study including several landscape species, Cassaniti et al.,
(2009) classified Bougainvillea glabra Choisy in DC. as moderately salt
tolerant based on the reduction of shoot dry weight under long-term
salinity stress (Cassaniti et al., 2009). Also B. spectabilis was classified as
high tolerant to salt stress for its ability to cope with soil salinity until
10 dS m−1 and accumulated high contents of sodium and chloride in
the leaves (Francois, 1980). Moreover, taking into account the visual
appearance is the most important criterion to be considered for orna-
mental plants (Acosta-Motos et al., 2016), the ability of B. spectabilis to
maintain a high visual quality without showing any symptoms of ne-
crosis under sprinkle irrigation with reused water with a salinity level
around 2.0 dS m−1 (Devitt et al., 2005; Cassaniti et al., 2012), could
make this salt-tolerant ornamental shrub a potential candidate in re-
vegetation projects in arid and degraded land, landscaping projects and
public areas (Navarro et al., 2007, 2008). The degree of adaptation of a
species to a major abiotic stress such as drought could also be affected
by the canopy shape as demonstrated earlier by Cirillo et al. (2014,
2017), which proved that plants can modify the growth behavior and
anatomical hydraulic properties depending on the canopy-shape
training and irrigation level.
Based on these considerations, the aim of this study was to in-
vestigate morphological, physiological, and biochemical changes in B.
spectabilis ‘Alexandra’ irrigated with saline water and trained at two
canopy shapes (globe and pyramid) in order to unravel possible adap-
tive strategies, combining morpho-functional traits at different levels,
to cope with salinity. The salinity-tolerance of globe and pyramid
13
Agricultural Water Management 212 (2019) 12–22
trained Bougainvillea were compared in terms of growth, ornamental
quality, ions content, gas exchange parameters, photosystem efficiency,
water relations and leaf functional anatomical traits. The knowledge
acquired from this study can be directly translated into specific man-
agement measures for revegetation and landscaping programs.
2. Materials and methods
2.1. Plant material, growth conditions and experimental design
The experimental trial was conducted in the 2014 growing season in
a glasshouse at the experimental station of the University of Naples
Federico II, located in Bellizzi (Salerno), southern Italy (43° 31ʹ N, 14°
58ʹ E; 60 m a.s.l.). On April 2, one year-old rooted cuttings of B.
spectabilis Willd. ‘Alexandra’ were purchased from a specialized nursery
(Vivaio Floramici, Catania, Italy) and transplanted into plastic pots
containing 3 L of peat moss. The pots were placed on 1.8 × 7.0 m
troughs, at a plant density of 2.5 plants m−2. Plants were grown under a
50% black shading net. Inside the glasshouse, the daily air temperature
varied between 18 and 33 °C and the relative humidity was 55%/75%
during day/night, respectively.
The experiment was designed as a factorial combination of three
water salinity treatments (S) (1 mM a non-salt [S1] and two salinized
solutions 40 mM [S40; moderate salt stress] or 80 mM [S80; severe salt
stress] NaCl) and two canopy shape treatments (Sh) [pyramid (PSh) and
a globe (GSh) shape]. The globe shape was obtained by regular pruning
based on the new shoot thinning and cut back, whereas the pyramid
Bougainvillea plants were grown as a vine on a tutor and pruned by
trimming exceeding shoots as described previously in Cirillo et al.
(2014). The treatments were arranged in a randomized complete-block
design with four replicates, amounting to a total of 24 experimental
units with four plants each (n = 96 plants).
2.2. Nutrient solution management and salinity application
The non-salt basic nutrient solution had the following composition:
13.6 mM N-NO3, 2.0 mM S, 1.4 mM P, 6.0 mM K, 4.5 mM Ca, 2.0 mM
Mg, 1 mM Na, 1 mM Cl, 20 μM Fe, 9 μM Mn, 1.5 μM Cu, 3 μM Zn, 20 μM
B and 0.3 μM Mo with an electrical conductivity (EC) of 2.0 dS m−1.
The two saline water treatments had the same basic composition plus
an additional 39 and 79 mM NaCl, giving an EC values of 6.5 and
11.0 dS m−1, respectively. The pH in the three nutrient solutions was
6.2 ± 0.5. All nutrient solutions have been prepared using irrigation
water typical of the area, which is characterized by a high bicarbonate
concentration (5.5 mM HCO3), 0.7 mM Ca, 0.7 mM Mg, 0.1 mM K,
0.4 mM Na and 0.3 mM Cl. Values of the pH and EC of the irrigation
water were 7.3 and 0.5 dS m−1, respectively. Saline treatments were
initialized on April 14 (12 days after transplanting, 12 DAT).
The nutrient solution was pumped from independent tanks and
delivered through a drip irrigation system with one emitter per plant at
a flow rate of 2 L h−1. Irrigation scheduling was performed by low-
tension tensiometers (LT-Irrometer, California, USA) which control ir-
rigation based on substrate matric potential as described by Norrie et al.
(1994). Tensiometers were installed at about the midpoint of the plastic
pots. In each replication two tensiometers were installed randomly and
were connected to an electronic programmer that managed the begin-
ning and the end of irrigation cycle in response to maximum (−10 kPa)
and minimum (−1 kPa) tension set points, respectively for the major
part of the growing media (Kiehl et al., 1992). The timing of irrigation
varied from 2 to 7 fertigations per day of 1–3 min. The watering was
performed with a volume of water of 20% (leaching fraction) higher
than the amount of water required to restore field capacity. Leaching
fractions are needed to maintain the EC in the substrate at re-
commended level (Colla et al., 2012, 2013).
P. Carillo et al.
2.3. Plant growth and ornamental quality measurements
Shoot trimming and pruning, were performed at three weeks in-
terval during the growing cycle starting 43 days after transplanting
(DAT), where the Bougainvillea pruned biomass was recorded and the
cumulative pruning dry weight per plant was calculated. At the end of
the experiment (July 25, 114 DAT), all plants were separated into
leaves, stems and roots. All plant tissues were dried at 80 °C for 72 h
until they reached a constant weight and weighed again to determine
the corresponding dry biomasses. The total above ground dry weight
per plant was calculated as the sum of shoot dry weight and the cu-
mulative pruning dry weight per plant. Shoot dry weight was equal to
the sum of the aerial vegetative parts (leaves + stems), and the root-to-
shoot ratio (root dry weight/shoot dry weight) was also calculated.
Dried plant tissues were then sampled for ion analyses. The total leaf
area per plant was measured using an electronic area meter (Li-
Cor3000, Li-Cor, Lincoln, NE, USA). Lastly, the number of leaves and
flowers per plant were also counted.
2.4. SPAD index and flower colorimetry
On the same date of plant growth measurements, the leaf Soil Plant
Analysis Development index (i.e., greenness readings) and flower col-
orimetry were carried out. SPAD index was measured on fully expanded
leaves by means of a portable chlorophyll meter SPAD-502 (Konica
Minolta, Japan). Fifteen healthy and fully expanded leaves were ran-
domly measured and averaged to a single SPAD value for each ex-
perimental plot. Moreover, six representative leaves per replicate were
analyzed for flower colorimetry. The Commission Internationale de
L’Eclairage (CIE) color space parameters: L* (lightness), a* (−a*
greenness), and b* (+b* yellowness) were measured at the centre of
the upper part of the flower surface using a Minolta CR-300 Chroma
Meter (Minolta Camera Co. Ltd., Japan). Minolta Chroma Meter was
calibrated with the Minolta standard white plate before sampling the
Bougainvillea flowers.
2.5. Leaf gas exchange and fluorescence parameters
At 65 DAT, the net CO2 assimilation rate (Pn), stomatal conductance
(gs) and transpiration rate (E) were measured with a portable gas ex-
change analyzer (LCA-4; ADC BioScientific Ltd., Hoddesdon, UK)
equipped with a broadleaf chamber (cuvette window area, 6.25 cm2).
This measurement was carried out within 2 h across solar noon (i.e.
between 11.00 and 13.00) on six fully expanded leaves per treatment.
Photosynthetically Active Radiation (PAR), Relative humidity (RH) and
CO2 concentration (682 ± 13 μmol m−2 s−1, RH 50 ± 0.6%, and
364 ± 0.5 ppm, respectively) were set at ambient value and the flow
rate of air was 400 ml s−1.The intrinsic Water Use Efficiency (WUEint)
was calculated as Pn/gs.
At 95 DAT, fluorescence measurements were performed out on six
replicates for each treatment. The chlorophyll a fluorescence emission
measurements were conducted using a portable FluorPen FP100max
fluorometer, equipped with a light sensor (Photon System Instruments,
Brno, Czech Republic). The ground fluorescence signal, Fo, was induced
on 30′ dark adapted leaves, by a blue LED internal light of about
1–2 μmol m−2 s−1. The maximal fluorescence level in the dark, Fm, was
induced by a 1 s saturating light pulse of 3000 μmol m−2 s−1. The
maximum quantum efficiency of PSII photochemistry, Fv/Fm, was cal-
culated as (Fm − Fo)/Fm, according to Butler and Kitajima (1975). For
the fluorescence measurements in the light, the fluorometer FluorPen
FP100max was equipped with an open leaf-clip suitable for measure-
ments under ambient light. The quantum yield of PSII electron trans-
port (QY) was determined according to Genty et al. (1989). The linear
electron transport rate (ETR) was expressed following Krall and
Edwards (1992), whereas the photochemical (qP) and non-photo-
chemical quenching (NPQ) were calculated as described by Quick and
14
Agricultural Water Management 212 (2019) 12–22
Horton (1984) and Bilger and Björkman (1990), respectively.
2.6. Leaf water relation measurements
Leaf water potential measurements were performed at the same
time of the gas exchange measurement (65 DAT) on four replicates for
each treatment, using a dew-point psychrometer (WP4; Decagon
Devices, Pullman, WA). Leaf total water potential (Ψl) was measured at
midday. The osmotic potential (Ψπ) was recorded on frozen/thawed
leaf samples, whereas the pressure potential (Ψp) was estimated as the
difference between Ψl and Ψπ, assuming a matrix potential equal to
zero.
2.7. Ion analyses
Dried plant tissues (leaf, stem and root) were ground separately in a
Wiley mill (IKA, MF10.1, Staufen, Germany) to pass through an 841 μm
screen, and then portions of the dried tissues were used for ion analyses.
Total nitrogen (N) concentration in all plant tissues was determined by
Kjeldahl method following mineralization with sulphuric acid (96%,
Carlo Erba Reagents, Milan, Italy) in the presence of potassium sulfate
(K2SO4) and low concentration of copper (Cu) catalyst (Bremner,
1965).
For the cations (K+, Ca2+, Mg2+ and Na+) and anions (PO43− and
−
Cl ) analysis, 250 mg of dried material was extracted in 50 mL of ul-
trapure water (Milli-Q, Merck Millipore, Darmstadt, Germany) using a
shaking water bath (ShakeTemp SW22, Julabo, Seelbach, Germany) at
80 °C for 10 min as described previously by Rouphael et al. (2017 a,b).
The mixture was centrifuged at 6000 rpm for 10 min (R-10 M, Remi
Elektrotechnik Limited, India), then filtered through a 0.20 μm filter
paper (Whatman International Ltd., Maidstone, U.K.). The four cations
(K+, Ca2+, Mg2+ and Na+) were separated by ion chromatography
(ICS-3000, Dionex, Sunnyvale, CA, USA) and quantified through an
electrical conductivity detector. A conductivity detector with IonPac
CG12 A (4 × 250 mm, Dionex, Corporation) guard column and IonPac
CS12 A (4 × 250 mm, Dionex, Corporation) analytical column were
used for the analysis of the four cations whereas for the two anions
(PO43− and Cl-), an IonPac AG11-HC guard (4 × 50 mm) column and
IonPac AS11-HC analytical column (4 × 250 mm) were used.
2.8. Microscopy analysis
Sampling of leaves for microscopy analyses was also performed at
the end of the experiment (114 DAT). Three fully expanded leaves were
sampled from three plants per combination of treatments and im-
mediately fixed in FAA (5 mL 40% formaldehyde, 5 mL glacial acetic
acid, 90 mL 50% ethanol). Each leaf was dissected to remove the apical
and basal portions, while keeping the median region of the lamina
which was divided into two sub-samples: one devoted to stomata
characterization, the other to the preparation for thin sectioning.
For stomata characterization, the abaxial epidermis of the leaf la-
mina was carefully peeled off with a pointed tweezer, in a region of the
lamina not including the main vein. The epidermis strip was flattened
on a glass slide and mounted with distilled water. Three film strips from
each leaf were observed under a transmitted light microscope (BX60,
Olympus, Hamburg, Germany). Digital images of the epidermis were
collected by means of a camera (CAMEDIA C4040, Olympus), avoiding
veins. The following parameters were measured through the software
program AnalySIS 12.0 (Olympus, Germany): stomata frequency (SF,
calculated by counting the number of stomata in five regions of the
epidermis and expressed as the number of stomata per mm2), and guard
cell length and guard cell width (GCL, GCW, quantified by measuring
the length, pole to pole, and width, in the median position, of 20 sto-
mata).
The second group of subsamples was dissected under a dissection
microscope (SZX16, Olympus, Germany): 5 × 5 mm portions of the leaf
P. Carillo et al.
lamina were dehydrated in an ethanol series (up to 95%), infiltrated
and embedded in the JB4 acrylic resin (Polysciences, USA). Thin cross
sections (5 μm thick) were cut by means of a rotary microtome, stained
with 0.025% toluidine blue in 0.1 M citrate buffer at pH 4 (Reale et al.,
2012), and permanently mounted with mineral oil for microscopy.
Sections were analyzed under the BX60 light microscope (Olympus,
Germany) and digital images were collected as reported above. Un-
stained sections were observed through polarized light microscopy
(BX61, Olympus, Germany), to highlight the presence of crystals, and
digital images were captured as well with a XC50 camera (Olympus,
Germany). Images were analyzed through the Olympus AnalySIS soft-
ware to quantify some functional anatomical traits. More specifically,
the following parameters were analyzed: thickness of palisade and
spongy parenchyma tissues (TPP, TSP - measured in five positions of the
leaf lamina avoiding veins); quantity of intercellular spaces in the
spongy parenchyma (ISS - expressed as the percentage of tissue occu-
pied by intercellular spaces over a given surface, in five positions of the
leaf lamina) (De Micco et al., 2011); volume of the cell occupied by the
vacuole/cytoplasm in the spongy parenchyma (VCS, expressed as the
percentage of cell surface occupied by vacuole over the whole cell area
occupied also by other organelles in the two-dimension space, in 10
cells); number of chloroplasts per unit surface of palisade and spongy
parenchyma tissues (ChPP, ChSP, calculated by counting the number of
chloroplasts per unit area in five regions of the palisade and spongy
parenchyma respectively, and expressed as the number of chloroplasts
per mm2); number of crystals per unit surface of mesophyll (NCM,
calculated by counting the number of crystals per unit area in five re-
gions of the mesophyll, and expressed as the number of crystals per
mm2) as well as the area of crystals (CA, measured as surface of each
single crystal).
2.9. Statistical analysis of data
All experimental data were statistically analyzed by two-way ana-
lysis of variance (ANOVA) using the SPSS 13 software package (www.
ibm.com/software/analytics/spss). To separate treatment means within
each measured parameter, Duncan’s multiple range test was performed
at a significance level of P ≤ 0.05. Shapiro-Wilk and Kolmogorov-
Smirnov tests were performed to check for normality. Percent data were
transformed through the arcsine function before statistical analysis.
Principal component analysis (PCA) method aimed to extract trends
when several variables were used by formulating new variables corre-
lated to the original ones (Lawless and Heymann, 2010; Rouphael et al.,
2017c). The PCA outputs include variable loading to each selected
component and treatment component scores using Minitab 16.2.1 sta-
tistical software (Ciarmiello et al., 2015; Rouphael et al., 2017b).
3. Results
3.1. Morphological and physiological parameters
The shoot biomass in particular leaf and stem dry weight as well as
the number of leaves per plant decreased only under severe (S80) salt
stress conditions (Table 1). For instance, irrespective of canopy shape
treatments leaf and stem dry weight as well as the number of leaves
were significantly reduced by 37.2%, 37.4% and 30.1% in S80 com-
pared to non-saline control treatment (S1). Moreover, the root dry
weight was significantly lowered by 21.3% and 31.1% in S40 and S80
treatments, respectively compared to the control treatment with no
significant difference between the two saline treatments (Table 1).
Averaged over saline water treatments, dry weight of both stem and
roots resulted lower in the GSh plants (Table 1). Furthermore, there was
also a decrease of leaf dry weight (37.2%) and total leaf area (33.9%) in
S40 GSh compared to the pyramid-shaped (PSh) plants (Tables 1 and
2). The total above ground dry weight (shoot biomass + cumulative
pruning dry weight) produced by the plants during the growing cycle
15
Agricultural Water Management 212 (2019) 12–22
was influenced by salinity and canopy shape, but not by their interac-
tion (Table 1). The increased salinity induced a significant and linear
decrease of the total above ground dry weight, whereas the GSh plants
showed a significantly lower amount (14.9%) of produced dry biomass
during the entire growing cycle (Table 1).
Under moderate salt stress conditions (S40) PSh Bougainvillea plants
exhibited more leaves and flowers (35.8% and 82.8%, respectively)
compared to GSh plants, whereas no significant effects between the two
shapes were observed at S80 (Table 1). Both saline water and canopy
shape significantly affected the flower brightness (L*). Only S80 treat-
ment yielded darker flower, expressed by decreasing L* values, with no
significant difference between globe- and pyramide-trained plants
(Table 1).
Except for transpiration rate which was affected by water sali-
nity × canopy shape interaction with the highest values recorded in
PSh plants under control treatment, the physiological measurements in
particular the net photosynthetic rate (Pn), stomatal conductance (gs)
and intrinsic water use efficiency (WUEi) were only influenced by water
salinity. When averaged over canopy shape treatments, increasing the
NaCl concentration in the irrigation water reduced Pn (by 20.2% and
27.8% for S40 and S80, respectively) and gs (by 33.7% and 45.5% for
S40 and S80, respectively) in comparison to the control treatment with
no significant differences between the two water saline treatments (S40
and S80). Our results also showed that S80 elicited significant increase
(30.8%) of WUEi compared to non-saline treatment, whereas S40 ex-
hibited intermediate values (Table 2).
The SPAD index incurred significant water salinity × canopy shape
interaction, whereas Ψl and Ψπ, were influenced by water salinity and
canopy shape with no significant water salinity × shape interaction
(Table 2). Increasing the NaCl concentration in the irrigation water
from 1 to 80 mM decreased the greenness readings (i.e., SPAD index)
with the lowest values recorded in PSh plants supplied with 80 mM
NaCl (Table 2). When averaged over canopy shape, the Ψl decreased as
the water salinity level increased (by 50.5% and 165.9% in S40 and
S80, respectively), whereas the Ψπ was lowered only under severe salt
stress conditions (Table 2). When averaged over water salinity treat-
ments, the ψl and ψp in pyramid-trained plants were respectively lower
than those in globe-trained plants (Table 2).
Among the biochemical parameters measured, the photochemical
quenching (qP), maximum quantum efficiency of PSII photochemistry
(Fv/Fm) as well as linear electron transport rate (ETR) were significantly
affected by water salinity × canopy shape interaction, whereas the
quantum yield of PSII electron transport (QY) and non-photochemical
quenching (NPQ) were mainly influenced by canopy shape and to a
lesser degree (only NPQ) by water salinity (Table 3). For instance, the
highest values of (Fv/Fm) and ETR were recorded in PSh Bougainvillea
plants grown under non-saline conditions (Table 3). S80 elicited sig-
nificant increase (57.2%) of NPQ compared to non-saline treatment,
whereas S40 exhibited intermediate values (Table 3). Finally, when
averaged over water salinity, the highest QY was recorded in GSh plants
whereas an opposite trend was observed for NPQ (Table 3).
3.2. Leaf functional anatomical traits
In Bougainvillea leaves, stomatal frequency was significantly influ-
enced by the water salinity × canopy shape interaction with the highest
value recorded in globe-trained plants supplied with non-salt control
treatment and the lowest value measured in globe-shape Bougainvillea
supplied with 80 mM of NaCl (Table 4). Similarly to stomatal fre-
quency, stomata size (guard cell length, GCL and guard cell width,
GCW) were both affected by the water salinity × shape interaction with
the highest and lowest values recorded in pyramid-trained plants with
1 mM and 80 mM NaCl, respectively (Table 4).
Although irrigating with saline water as well as canopy shape did
not affect the tissue organization of Bougainvillea leaves, from a quan-
titative viewpoint there were significant differences in most analyzed
P. Carillo et al. Agricultural Water Management 212 (2019) 12–22

Table 1
Analysis of variance and means comparison for dry weight of leaves, stems and root, total above ground dry weight (leaf + stem + pruning), root-to-shoot (R/S)
ratio, number of leaves and flowers, Hunter colour parameters L* (brightness), a* (+a*= red; -a* = green) and b* (+b* = yellow;-b*= blue) of potted Bougainvillea
plants irrigated with three saline water concentrations and trained to two shapes.
Treatments Leaf dry weight Stem dry weight Root dry weight Total above ground R/S Leaves Flower Flower colorimetry
(g plant−1) (g plant−1) (g plant−1) dry weight (n. plant−1) (n. plant−1)
(g plant−1) L a* b*

Saline water (S; mM NaCl)

S1 32.8 a 51.6 a 6.1 a 112.7 a 0.07 1048.1 a 40.9 b 51.9 a 17.2 b −3.8
S40 28.2 a 46.6 a 4.8 b 94.0 b 0.07 916.6 a 58.4 a 50.9 a 20.9 a −4.5
S80 20.6 b 32.3 b 4.2 b 67.0 c 0.09 732.2 b 33.9 b 47.5 b 16.2 b −4.7

Shape (Sh)
Pyramid 29.6 a 56.6 a 5.9 a 98.6 a 0.07 928.6 53.2 a 50.2 19.0 −4.0
Globe 24.8 b 30.4 b 4.2 b 83.9 b 0.08 869.3 35.6 b 50.1 17.1 −4.6

S × Sh
S1 Pyramid 33.9 a 64.0 7.5 114.9 0.08 1084.2 a 52.5 b 53.0 a 19.7 −4.2
Globe 31.6 a 39.2 4.7 110.5 0.07 1011.9 ab 29.3 b 50.7 b 14.6 −3.4
S40 Pyramid 34.6 a 64.7 5.4 112.2 0.06 1055.9 a 75.5 a 50.8 b 20.8 −3.9
Globe 21.7 b 28.5 4.2 75.8 0.09 777.3 c 41.3 b 51.0 ab 21.0 −5.1
S80 Pyramid 20.2 b 41.1 4.8 68.5 0.09 645.6 c 31.6 b 46.7 c 16.6 −4.1
Globe 21.0 b 23.5 3.6 65.4 0.08 818.8 bc 36.2 b 48.4 c 15.8 −5.4

Significance
Salinity (S) *** *** * *** NS *** ** *** * NS
Shape (Sh) * *** *** * NS NS ** NS NS NS
S × Sh * NS NS NS NS * * * NS NS

*; **, ***Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to
NS,
Duncan’s multiple-range test (P ≤ 0.05).

parameters (Table 5). For instance, the thickness of palisade and spongy
parenchyma (TPP and TSP) tissues were influenced by both factors:
water salinity and shape (Table 5). In PSh plants, palisade and spongy
parenchyma tissues respectively increased (by 24.1% at S40 and by
13% at S80) and decreased according to increasing salinity already in
S40 plants (by 13.5%). In GSh plants, the increase of palisade par-
enchyma was significant only at the highest level of salinity (+37.1%),
while an increase in spongy tissue thickness was evident only in S40
plants (+19.4%). Increasing salinity in irrigation water from 1 mM to
80 mM determined an increase in the percentage of intercellular spaces
which was also higher in GSh than PSh plants (Table 5). Moreover, the
volume of the cell occupied by the vacuole in the spongy parenchyma
(VCS) decreased according to increasing water salinity (by 11.8% and
18.3% in S40 and S80, respectively) and was also higher in PSh than
GSh Bougainvillea (Table 5). Salinity significantly decreased the number
of chloroplasts in the palisade parenchyma already in similar extent in
both S40 and S80 GSP (by 21%). In both tissues, GSh plants were
characterized by higher chloroplasts frequency than PSh plants
(Table 5).
Crystals of calcium oxalate were evidenced by polarized light

Table 2
Analysis of variance and means comparison for total leaf area (LA), net photosynthetic rate (Pn), stomatal conductance (gs) transpiration rate (E), intrinsic water use
efficiency (WUEi), SPAD index, leaf water potential (Ψl), leaf osmotic potential (Ψπ) and leaf pressure potential (Ψp) of potted Bougainvillea plants irrigated with
three saline water concentrations and trained to two shapes.
Treatments LA Pn gs E WUEi SPAD ψl ψπ ψp
(cm2plant−1) (μmol CO2 m−2 (mmol m−2 (mol H2O m−2 (μmol CO2 mmol−1 Index (MPa) (MPa) (MPa)
s−1) s−1) s−1) H2O)

Saline water (S; mM

NaCl)
S1 7447.9 a 7.9 a 78.6 a 1.7 a 112.8 b 51.2 a −0.91 a −2.20 a 1.29
S40 6646.9 a 6.3 b 52.1 b 1.4 b 129.9 ab 49.4 b −1.37 b −2.67 a 1.30
S80 4902.1 b 5.7 b 42.8 b 1.1 b 147.6 a 46.7 c −2.42 c −3.34 b 0.92

Shape (Sh)
Pyramid 6682.1 a 7.2 69.3 1.6 a 123.2 48.3 b −1.70 b −3.06 b 1.36
Globe 5982.5 a 6.1 46.4 1.2 b 137.0 49.9 a −1.43 a −2.41 a 0.98

S × Sh
S1 Pyramid 7665.3 a 9.3 106.2 2.1 a 93.5 51.3 a −0.96 −2.43 1.47
Globe 7230.5 a 6.5 51.0 1.3 b 132.1 51.2 a −0.85 −1.96 1.11
S40 Pyramid 8006.5 a 6.9 59.4 1.5 b 130.7 48.9 b −1.47 −3.02 1.55
Globe 5287.4 b 5.7 44.8 1.2 b 129.0 49.9 ab −1.27 −2.31 1.04
S80 Pyramid 4374.6 b 5.4 42.1 1.1 b 145.3 44.7 c −2.67 −3.72 1.05
Globe 5429.7 b 6.0 43.4 1.1 b 149.9 48.6 b −2.17 −2.96 0.78

Significance
Salinity (S) *** ** *** *** ** *** *** ** NS
Shape (Sh) NS NS NS ** NS ** * ** NS
S × Sh * NS NS * NS * NS NS NS

*; **, ***Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to
NS,
Duncan’s multiple-range test (P ≤ 0.05).

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P. Carillo et al. Agricultural Water Management 212 (2019) 12–22

Table 3 Bougainvillea was trained to a pyramid shape (Table 5).

Analysis of variance and means comparison for photochemical quenching (qP),
quantum yield of PSII electron transport (QY), maximum quantum efficiency of 3.3. Ions content and partitioning
PSII photochemistry (Fv/Fm), non-photochemical quenching (NPQ) and linear
electron transport rate (ETR) of potted Bougainvillea plants irrigated with three
Sodium (Na+) and chloride (Cl−) contents in leaves, stem and roots
saline water concentrations and trained to two shapes.
occurred in increasing pattern with increasing external water salinity;
Treatments qP QY Fv/Fm NPQ ETR the highest concentrations of toxic ions being found in leaves, and in
particular in those from S80 treated plants (Table 6). Under S40 and
Saline water (S;
mM NaCl)
S80, Na+ and Cl− concentrations in leaves increased by 16.8/34 fold
S1 0.866 0.521 0.759 1.17 b 298.9 a and 6.3/17.2 fold, respectively, compared to non-salt control, showing
S40 0.890 0.488 0.753 1.60 ab 255.4 b the maximum relative increase in ion levels and the maximum con-
S80 0.917 0.475 0.748 1.84 a 250.6 b centration in PSh plants at S80. The GSh and PSh plants accumulated in
Shape (Sh) similar manner Na+ and Cl-, with the only exception in S80 PSh plants
Pyramid 0.875 0.458 b 0.745 b 1.75 a 249.9 b that accumulated a concentration of Cl- that was 1.55 fold higher than
Globe 0.907 0.531 a 0.762 a 1.32 b 286.7 a
that of Na+, and almost double compared to the Cl- in GSh Bougainvillea
S × Sh plants. These latter were able, in general, to accumulate less Na+ and
S1 Pyramid 0.807 b 0.456 0.738 b 1.25 242.4 bc Cl− in roots than PSh plants under S80 treatment; while they did not
Globe 0.924 a 0.586 0.781 a 1.08 355.5 a
S40 Pyramid 0.895 a 0.469 0.753 b 1.88 223.2 c
differ significantly under moderate stress condition (S40).
Globe 0.884 a 0.506 0.753 b 1.33 287.6 b The total N in leaves was negatively affected by salt stress treatment
S80 Pyramid 0.922 a 0.450 0.744 b 2.14 284.0 b especially with S80, whereas an opposite trend was observed in roots
Globe 0.913 a 0.500 0.751 b 1.55 217.1 c (Table 7). The GSh treatment averaged over water salinity levels, af-
Significance fected the N concentration in leaf tissue which was higher than in PSh-
Salinity (S) NS NS NS * * trained Bougainvillea (Table 7). Phosphate (PO43−) was significantly
Shape (Sh) NS ** ** * *
higher in all tissues of GSh plants than in PSh ones, but it did not de-
S × Sh ** NS ** NS ***
crease under salinity (Table 7).
NS, *; **, ***Non significant or significant at P ≤ 0.05, 0.01, and 0.001, re- Potassium (K+) concentration was higher in stems and roots of GSh
spectively. Different letters within each column indicate significant differences than in PSh plants. It decreased in stems and roots as the external
according to Duncan’s multiple-range test (P ≤ 0.05). salinity increased getting a 30% maximum decrease at S80 (Table 7).
Leaves showed a milder decrease of K+ by around 7% and 6% in S40
Table 4 and S80, respectively; but when considering the interaction between
Analysis of variance and means comparison for stomatal frequency (SF), guard the main factors (water salinity × canopy shape), K+ did not decrease
cell length (GCL), guard cell width (GCW) of potted Bougainvillea plants irri- in PSh, and only slightly in GSh plants. K+/Na+ ratio was higher in
gated with three saline water concentrations and trained to two shapes. stem and roots of GSh than in PSh plants. The decrease of K+/Na+ ratio
Treatments SF GCL GCW in relation to the non-stressed control was higher in leaves than in stem
(n. mm−2) (μm) (μm) and roots, according to the lower levels of Na+ retained in these plant
tissue and notwithstanding the stronger K+ decrease. Furthermore, leaf
Saline water (S; mM NaCl)
S1 314.8 a 27.9 11.6 b
K+/Na+ ratio decreased to a final value corresponding to 5.4% and
S40 300.7 a 27.5 11.6 b 2.7% of the initial value, in S40 and S80 treatments, respectively
S80 248.6 b 27.5 12.3 a (Table 7). The concentration of calcium (Ca2+) was significantly af-
Shape (Sh) fected only by salinity and it also decreased in leaves and roots (on
Pyramid 280.7 28.0 12.0 a average -37% and -23%, respectively; Table 7). Similarly to the
Globe 295.4 27.2 11.6 b monovalent cation, magnesium (Mg2+) concentration was higher in
S × Sh roots of GSh than in PSh plants, mainly at S80. It decreased by 33% on
S1 Pyramid 274.9 b 29.1 a 11.9 b average in all tissues of plants supplied with 40 and 80 mM NaCl
Globe 354.7 a 26.6 b 11.3 c (Table 7). The statistical analysis of the Na+ and Cl− concentrations
S40 Pyramid 300.9 b 27.4 b 11.5 bc
Globe 300.5 b 27.5 b 11.7 bc
among the different parts of Bougainvillea plants indicated that both
S80 Pyramid 266.1 b 27.4 b 12.6 a toxic ions reached the highest concentration in leaves (13.0 and 15.5 g
Globe 230.9 c 27.6 b 11.9 b kg−1 dw for Na+ and Cl−, respectively) compared to stem (5.7 and
Significance 3.6 g kg-1 dw for Na+ and Cl−, respectively) and roots (6.3 and 3.3 g
Salinity (S) *** NS *** kg−1 dw for Na+ and Cl−, respectively) (data not shown).
Shape (Sh) NS NS ***
S × Sh *** *** ** 3.4. Leaf osmolality
NS, *; **, ***Non significant or significant at P ≤ 0.05, 0.01, and 0.001, re-
spectively. Different letters within each column indicate significant differences Bougainvillea sap osmolality was 993 and 801 mOsmol kg−1 in
according to Duncan’s multiple-range test (P ≤ 0.05). leaves of pyramid- and globe-trained plants, respectively. However, the
sap osmolality increased after the addition of NaCl in irrigation water,
microscopy in leaf lamina of plants in all treatments. For instance, the reaching a value of 21% and 52% higher than the control in both
number of crystals per surface unit was significantly influenced by the pyramid and globe under 40 and 80 mM NaCl, respectively (Table 8).
water salinity × shape interaction, whereas the crystal size was only The relative contribution of the main inorganic ions to osmolality was
affected by the salinity treatment (Table 5). In fact, increasing salinity on average 25%, 35% and 45% for non-salt control, S40 and S80
in irrigation water from 1 to 80 mM reduced both the number and the treatments, respectively. In particular, in PSh plants the relative ion
size of crystals, with no significant differences between S40 and S80. contribution towards osmolality increased from 1% to 17% for both Cl-
Finally, a sharp reduction in the number of crystals was observed in and Na+; while in GSh Bougainvillea it increased from 2% to 11% and
both Bougainvillea canopy shapes at S80 and in GSh plants supplied with from 1% to 16% for Cl−and Na+, respectively. On the contrary, the K+
S40, whereas the crystals number was not affected by S40 when contribution towards osmolality decreased under salinity from 21% to
13% in PSh plants and from 25 to 14% in the GSh ones (Table 8).

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P. Carillo et al. Agricultural Water Management 212 (2019) 12–22

Table 5
Analysis of variance and means comparison for thickness of palisade parenchyma (TPP), thickness of spongy parenchyma (TSP), quantity of intercellular spaces in the
spongy parenchyma (ISS) volume of the cell occupied by the vacuole in the spongy parenchyma (VCS). number of chloroplasts per unit surface of palisade and spongy
parenchymas (ChPP and ChSP), number of crystals per unit surface of mesophyll (NCM) and area of crystals (CA) in leaves of potted Bougainvillea plants irrigated
with three saline water concentrations and trained to two shapes.
Treatments TPP TSP (μm) ISS VCS ChPP ChSP NCM CA
(μm) (%) (%) (n. mm−2) (n. mm−2) (n. mm−2) (μm2)

Saline water (S; mM NaCl) Na)

S1 72.06 c 75.59 a 19.23 b 50.66 a 10.53 a 14.65 8.01 a 741.8 a
S40 79.25 b 76.93 a 21.57 a 44.67 b 8.90 b 14.99 5.41 b 457.3 b
S80 90.42 a 69.59 b 20.26 ab 41.40 c 9.01 b 15.48 4.52 b 430.5 b

Shape (Sh)
Pyramid 81.25 72.5 19.39 b 46.96 a 8.67 b 13.23 b 6.34 544.8
Globe 79.91 75.57 21.34 a 44.07 b 10.29 a 16.85 a 5.62 541.6

S × Sh
S1 Pyramid 72.11d 80.96 a 18.66 52.76 9.02 b 12.75 8.58 a 668.2
Globe 72.01 d 70.22 b 18.56 48.56 12.03 a 16.55 7.44 a 815.3
S40 Pyramid 89.51 b 70.02 b 22.23 47.06 8.15 b 12.93 6.62 ab 497.2
Globe 68.99 d 83.86 a 20.89 42.19 9.64 b 17.05 4.19 c 417.4
S80 Pyramid 82.12 c 66.56 b 19.83 41.11 8.84 b 14.01 3.82 c 468.9
Globe 98.73 a 72.64 b 21.99 41.60 9.18 b 16.94 4.72 bc 392.1

Significance
Salinity (S) *** ** * *** *** NS *** **
Shape (Sh) NS NS ** ** ** *** NS NS
S × Sh *** *** NS NS * NS * NS

*; **, ***Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to
NS,
Duncan’s multiple-range test (P = 0.05).

Table 6
Analysis of variance and means comparison for sodium, chloride and potassium-to-sodium ratio of leaves, stems and roots of potted Bougainvillea plants irrigated with
three saline water concentrations and trained to two shapes.
Treatments Na+ (g kg−1 dw) Cl− (g kg−1 dw) K+/Na+

leaves stem roots leaves stem roots leaves stem roots

Saline water (S; mM NaCl)

S1 0.8 c 1.4 c 2.4 c 2.3 c 0.6 c 0.7 c 54.1 a 17.7 a 7.8 a
S40 13.8 b 6.3 b 6.8 b 14.7 b 3.3 b 3.2 b 2.9 b 3.0 b 2.0 b
S80 27.9 a 9.2 a 9.6 a 36.9 a 6.7 a 6.0 a 1.4 b 1.8 c 1.3 b

Shape (Sh)
Pyramid 14.9 6.4 a 7.2 a 22.1 4.3 a 3.9 a 20.5 5.4 b 2.2 b
Globe 13.3 4.9 b 5.3 b 13.8 2.8 b 2.7 b 18.5 9.7 a 5.2 a

S × Sh
S1 Pyramid 0.8 1.8 3.0 d 2.1 0.7 1.0 c 56.6 12.1 b 4.2 b
Globe 0.8 1.1 1.8 e 2.4 0.5 0.4 c 51.6 23.4 a 11.4 a
S40 Pyramid 12.3 6.8 7.2 bc 14.7 3.6 3.1 b 3.4 2.6 c 1.5 c
Globe 15.2 5.7 6.4 c 14.6 3.1 3.3 b 2.4 3.4 c 2.5 bc
S80 Pyramid 31.8 10.5 11.4 a 49.4 8.5 7.6 a 1.4 1.5 c 0.8 c
Globe 24.0 7.9 7.8 b 24.4 4.9 4.5 b 1.5 2.2 c 1.8 c

Significance
Salinity (S) *** *** *** *** *** *** *** *** ***
Shape (Sh) NS ** *** NS ** * NS *** ***
S × Sh NS NS ** NS NS * NS *** ***

*; **, ***Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to
NS,
Duncan’s multiple-range test (P = 0.05).

According to Puniran-Hartley et al., (2014), it is might be possible that
other ions and metabolites present in the cell might have contribute to
osmolality, and their relative contribution can be calculated as differ-
ence between the total sap osmolality and the contribution of the
measured major inorganic ions as shown in Table 8. The calculated
other osmolytes contribution was therefore on average 75%, 65% and
55% in non-salt control, S40 and S80 treatments, respectively.
3.5. Heat map analysis
integrated view of the effect of saline water on Bougainvillea perfor-
mance (Fig. 1). GSh accumulated lower Na+ and Cl− ions in leaves at
the highest salinity treatment, and in stem and roots independently of
salinity, showing in these tissues higher K+/Na+ ratio, as well as higher
PO43−concentration than the PSh plants. Moreover, GSh plants showed
higher incidence of intercellular spaces, higher frequency of chlor-
oplasts and a better photochemical efficiency (indicated by Fv/Fm, QY
and ETR) as well as lower non-photochemical processes (NPQ) than PSh
plants. However, the dry weights of stem and roots were lower in GSh
than in PSh Bougainvillea plants.

A heat map representing the changes in the analyzed parameters in

dependence on the shape and different salinity treatments provided an

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P. Carillo et al. Agricultural Water Management 212 (2019) 12–22

Table 7
Analysis of variance and means comparison for total nitrogen, phosphate, potassium, calcium and magnesium concentration of leaves, stems and roots of potted
Bougainvillea plants irrigated with three saline water concentrations and trained to two shapes.
Treatments Ntot PO43− K+ Ca2+ Mg2+
(g kg−1 dw) (g kg−1 dw) (g kg−1 dw) (g kg−1 dw) (g kg−1 dw)

leaves stem roots leaves stem roots leaves stem roots leaves stem roots leaves stem roots

Saline water (S; mM NaCl)

S1 51.8 a 20.3 21.3 ab 24.9 34.4 22.2 42.1 a 23.8 a 16.6 a 0.7 a 0.6 0.5 a 3.9 a 1.3 a 1.2 a
S40 47.5 b 20.0 22.4 a 30.9 33.4 20.2 39.2 b 18.8 b 13.7 b 0.4 b 0.6 0.4 b 2.6 b 0.9 b 0.9 b
S80 45.2 c 19.1 19.5 b 27.3 32.4 19.5 38.7 b 16.5 c 11.7 c 0.4 b 0.5 0.4 c 2.6 b 0.8 b 0.7 b

Shape (Sh)
Pyramid 47.2 b 19.9 20.6 22.7 b 30.8 b 15.8 b 41.7 a 18.6 b 11.0 b 0.5 0.6 0.4 3.1 0.9 b 0.7 b
Globe 49.2 a 19.7 21.5 32.7 a 35.9 a 25.5 a 38.2 b 20.8 a 16.9 a 0.5 0.5 0.4 2.9 1.1 a 1.2 a

S × Sh
S1 Pyramid 51.2 20.0 a 20.3 21.2 30.4 13.9 42.8 22.3 12.7 0.7 0.6 0.5 3.6 b 1.1 0.7 ab
Globe 52.4 20.5 a 22.2 28.6 38.3 30.5 41.4 25.3 20.5 0.7 0.6 0.5 4.1 a 1.5 1.7 a
S40 Pyramid 47.2 19.4 ab 21.7 25.2 31.0 16.4 41.1 18.3 11.3 0.4 0.7 0.4 2.6 d 0.8 0.7 ab
Globe 47.8 20.5 a 23.1 36.5 35.8 24.0 37.2 19.2 16.1 0.4 0.5 0.4 2.5 d 0.9 1.1 b
S80 Pyramid 43.1 20.3 a 19.8 21.6 31.1 17.0 41.3 15.2 9.1 0.5 0.5 0.4 3.0 c 0.7 0.6 c
Globe 47.3 17.9 b 19.2 32.9 33.6 22.0 36.1 17.8 14.2 0.4 0.5 0.3 2.1 e 0.8 0.9 b

Significance
Salinity (S) *** NS * NS NS NS ** *** *** *** NS *** *** *** *
Shape (Sh) ** NS NS ** ** ** *** ** *** NS NS NS NS ** ***
S × Sh NS * NS NS NS NS NS NS NS NS NS NS *** NS *

*; **, ***Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to
NS,
Duncan’s multiple-range test (P ≤ 0.05).

Table 8 and the type of salts. These are the main factors to take into account
Relative contribution (%) of main inorganic ions and other osmolytes towards when irrigation with saline/brackish water is considered, because their
the total osmolality. Values are mean ± SD (n = 4). interaction will induce plant physiological, anatomical and molecular
Osmolality Cl− Na+ K+ Main ions Other changes (Álvarez and Sánchez-Blanco, 2015; Acosta-Motos et al.,
(mOsmol contribution osmolytes 2017). In the present study, pyramid (PSh)- and globe (GSh)- shaped B.
kg−1) spectabilis plants supplied with saline water showed similar alterations
only at the highest salt concentration (S80), while at lower con-
Saline S1 897 1 1 23 25 75
water S40 1090 7 10 17 35 65 centration (S40) PSh plants were more tolerant to saline water. The first
(mM S80 1365 14 17 14 45 55 evident salt constrain was the reduction in dry weight of shoot, roots, as
NaCl) well as total above ground dry weight produced during the growing
Shape Pyramid 1250 9 10 16 36 64 cycle, taking in account all the cumulative pruning dry weight, under
Globe 985 8 11 19 38 62
salt stress conditions, which was milder in PSh plants supplied with
Pyramid S1 993 1 1 21 23 77
S40 1235 6 8 16 31 69 40 mM NaCl. Indeed, even though the amount of removed shoots by
S80 1521 17 17 13 48 52 pruning was on average nearly doubled in the GSh plants compared to
Globe S1 801 2 1 25 28 72 the PSh plants (which is due to the different pruning systems adopted to
S40 944 8 13 19 41 59
keep the defined and specific shape), the plant growth parameters (leaf
S80 1210 11 16 14 42 58
dry weight, leaf number and total area) of Bougainvillea plants trained
to pyramid shape were clearly higher than those observed in globe
3.6. Principal component analysis plants. However, in landscape getting the maximum growth is not
crucial for plants like Bougainvillea; quite the contrary, excessive shoot
Multivariate analysis was applied to all studied datasets (see Tables vigor is undesirable and the use of moderate saline water (S40) could be
1–7). The principal component analysis (PCA) scatter-plot clearly se- useful for obtaining more compact plants (Cassaniti et al., 2009;
parated the six diverse treatments. The first two principal components Álvarez and Sánchez-Blanco, 2015). Indeed, a decrease in growth and
(PCs) were associated with Eigen values higher than one and explained development, and therefore in biomass may also affect the visual and
cumulatively 72.8% of the total variance, with PC1 and PC2 accounting therefore the ornamental quality of the plants (Gómez-Bellot et al.,
for 48.1% and 24.7%, respectively (data not shown). Saline water 2013). An important visual characteristic of B. spectabilis is the presence
contributed to the clear separation on PC1, while shape contributed to of the thin and papery bracts/flowers which confer harmony to the
separation on PC2 (Fig. 2). PC1 was highly and positively correlated plant. The number of these bracts was higher in PSh plants under S40
with total N, K+, K+/Na+, leaf water potential and SPAD index, and treatments compared to GSh plants; while similar behavior was ob-
was negatively correlated with Na+, Cl− concentrations as well as with served when plants were supplied with 80 mM NaCl. Under salt stress
NPQ. Additionally, PC2 received the main positive contribution from conditions the control of ion homeostasis (increase of osmolality),
PO43- concentration, photosystem efficiency (i.e. qP, Fv/Fm and QY) regulation of crystal accumulation/utilization, reduction of leaf water
and also from WUEi. potential (Ψl) and stomatal conductance (gs), is useful for reducing
transpiration (E) thus limiting water loss (Acosta-Motos et al., 2017).
Leaf anatomy in B. spectabilis under salinity was also harmonized to
4. Discussion physiological adjustments in both canopy shapes in such a way to
counteract the reduction in transpiration by means of the increase in
Plant responses to saline water are not only dependent on species intercellular spaces and in the incidence of palisade parenchyma over
but also on the duration of salt exposure as well as the concentration the total mesophyll thickness. Indeed, the increase in the ratio between

19
P. Carillo et al.
Fig. 1. Heat map analysis summarizing the plant responses to shape and water salinity treatments. Results were calculated as Logarithm base 2 (Log2) of GSh values/
PSh values under the three different salt treatments (non salt control 1 [S1], 40 [S40] and 80 [S80] mM NaCl) and were visualized using a false colour scale with blue
indicating an increase and red a decrease of GSh plants values compared to PSh ones. No differences were visualized by white squares (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article).
Fig. 2. Principal component loading plot and scores of principal component
analysis (PCA) of morphological, physiological, anatomical traits and ion con-
tents of potted Bougainvillea irrigated with three saline water concentrations (1
[S1], 40 [S40] and 80 [S80] mM NaCl) and trained to two shapes (Globe [GS]
and Pyramide [PS]).
palisade and spongy parenchyma thickness and in the amount of in-
tercellular spaces, have been reported also in other species: they are
considered adaptive traits of plants to cope with salinity in so far as
such modifications improve CO2 diffusion in the mesophyll, thus
compensating for salinity-induced increased stomatal limitations
(Acosta-Motos et al., 2015; Tattini et al., 2006; Rouphael et al., 2017d).
The increase in palisade parenchyma thickness occurring in PSh plants
already at S40 would indicate a better ability to modulate resource
allocation under stressful conditions in order to invest more resources
towards the assimilatory tissue of these plants if compared to GSh
plants (Fernández-García et al., 2014). The two canopy shapes also
seem to induce a different structure-mediated regulation of transpira-
tion in B. spectabilis, but only in non-saline conditions. The different
structure-mediated regulation of transpiration rate could be attributed
to an alteration of mesophyll traits, but the PSh plants seem to have also
a better control at the stomata level because salinity-treated PSh plants
20
Agricultural Water Management 212 (2019) 12–22
are able to maintain the same stomata frequency than the control
plants, although reducing the size of stomata aperture. Such an ad-
justment of stomatal traits would allow a faster control of stomata
aperture/closure that is considered an advantage under stressful con-
ditions (Raven, 2014). Similar morphological and physiological effects
were already reported in three different shrub species belonging to the
genus Bougainvillea grown at globe or pyramid under deficit irrigation
conditions (Cirillo et al., 2014). In addition, another mechanism
adopted by GSh plants, and even more by PSh plants, to minimize water
loss is also that of restricting leaf area. Both halophytes and non–-
halophytes plants show particular anatomical changes under high
salinity (Maas and Niemann, 1978), and most of them are displayed in
leaves and are similar to those present under drought stress (Hameed
et al., 2010).
Generally, photosynthesis is strongly inhibited by salinity, as con-
sequence of both stomatal and non-stomatal limitations, such as the
decrease of maximum quantum efficiency of PSII photochemistry (Fv/
Fm), quantum yield of PSII electron transport (QY) and chlorophyll
content, as well as stomata partial closure (Steduto et al., 2000; Mehta
et al., 2010). Accordingly, there was a reduction in chlorophyll content
and number of chloroplasts accompanied by a decrease in net photo-
synthetic rate. Alteration in the localization of chloroplasts and in their
structure have also been reported as common responses to salinity or
other stresses (Fernández-García et al., 2014). Moreover, the thermal
dissipation processes, namely NPQ, increased as also found in other
mainly tolerant species, under salt stress conditions (Acosta-Motos
et al., 2015). These strategies allow plant to reduce safely the excess of
excitation energy to reaction centers that cannot be utilized in photo-
chemistry, thus protecting photosynthetic apparatus from the over-ex-
citation of the electron chain and Reactive Oxygen Species (ROS) pro-
duction (Demmig-Adams et al., 2014). Otherwise, other photosynthetic
parameters like Fv/Fm and QY remained almost unvaried, in particular
in PSh, partially due to the alteration of mesophyll organization that
enabled a valuable gas diffusion throughout the spongy parenchyma,
notwithstanding the reduced stomatal conductance resulting from
partial stomatal closure and reduction of stomata frequency.
Moreover, the previous results could be explained also by the fact
that ‘Alexandra’ like ‘Mary Palmer’, a cultivar belonging to the same
Bouganvillea species, might use a photorespiratory glycine shuttle to
P. Carillo et al.
concentrate CO2 from the mesophyll tissue into a bundle-sheath-like
compartment surrounding the vascular tissue (Sage et al., 2014). This
mechanism has been described as an intermediate stage between C3
and C4 plants (Sage et al., 2014). Indeed, the areas in which Bou-
gainvillea thrives are often characterized by recurring dryness, scarce
atmospheric humidity and periodic phenomena of drought and salinity.
These constrains, and in particular salinity, can cause high evapo-
transpiration potentials and water stress, maximizing the impact of
photorespiration and being important drivers for C3-C4 transition (Sage
et al., 2013).
Another important feature of Bougainvillea plants was the ability to
retain higher Na+ and Cl− levels in leaves than in stem and roots under
salinity, suggesting the existence of an inclusion mechanism operating
in the plants, which is supposed to be a trait of salt tolerance (Läuchli
and Epstein, 1990; Rodrı́guez et al., 2005). The salinity-related de-
crease in the number and size of crystals in B. spectabilis may also
suggest a possible role of such crystals in immobilizing specific ele-
ments in case of excess (Pérez Cuadra and Cambi, 2017). Furthermore,
under NaCl salinity, the increase of cellular Na+ interferes with K+
translocation from roots to shoots and with its transport at the plasma
membrane, therefore strongly lowering K+ uptake (Gao et al., 2016).
Na+ excess contributes both to depolarize and damage the plasma
membrane favoring K+ leaking, causing a decrease of cytosolic K+
(Wang et al., 2013). Moreover, Na+ tends to replace K+ in key enzy-
matic reactions, damaging cytosol and organelles. For this reasons,
usually plants are more sensitive to K+ to Na+ ratio than to the abso-
lute amount of Na+ (Cuin et al., 2009; Shabala and Cuin, 2008).
However, in Bougainvillea, K+ concentration was maintained in all
tissues at about 87% and 70% of the corresponding value in leaves and
other plant tissues of untreated plants, respectively. Greenway and
Munns (1980) reported that even if Na+ concentration in plant tissues
increased under salinity, not necessarily the K+ to Na+ ratio in the
cytoplasm was drastically reduced. On the contrary, cytosolic K+ con-
centration could be maintained at a constant level or even increased by
using the K+ stored in the vacuole. The capability to satisfy plant me-
tabolic demand for K+ under salinity by compartmentalizing Na+ in
the vacuole and the majority of K+ in the cytosol could be another
essential mechanism for Bougainvillea salt tolerance (Wang et al., 2013)
and may also be used by B. spectabilis where the volume of the cell
occupied by the vacuole seemed to be decreased under salinity if
compared with volume occupied by other organelles such as chlor-
oplasts. The increase in K+ concentration in the cytosol might also
contribute to osmotically balance the toxic ions compartmentalized in
the vacuole. In fact, also a low K+ content alone, without considering
metabolites, could be enough to balance the mesophyll cells, assuming
that, under salinity, K+ is for the most located in the cytosol, and that
the cytosolic compartments (including cytosol and hyaloplasmic orga-
nelles), vacuoles and apoplast occupy about 34.1%, 45.5% and 20.4%
of the cell volume fraction, respectively (Lawlor, 1993; Cuin et al.,
2009).
5. Conclusions
Greenhouse Bougainvillea plant performance and the morpho-ana-
tomical, physiological and biochemical changes were assessed in a
multifactorial approach accounting for the effects of increasing NaCl
concentration in irrigation water and canopy-shape training. The re-
sults of this study confirmed that Bougainvillea is a moderately tolerant
ornamental species, since increasing NaCl concentration up to 40 mM in
irrigation water did not reduce the leaf dry weight, leaf number and
total area, but improved the ornamental quality (higher number of
flowers). Our findings also demonstrated that Bougainvillea plants and
in particular the PSh shaped ones can enact morpho-physiological
changes which enable them to maintain a better growth performance
(higher number of leaves, total leaf area and leaf dry weight) and or-
namental quality (higher number of flowers) under moderate salinity
21
Agricultural Water Management 212 (2019) 12–22
compared to GSh plants. Among the different mechanisms, toxic ions
inclusion in leaves and their compartmentation in vacuoles, retention of
high levels of K+ ions in the cytosol for osmotic adjustment and me-
tabolic function, leaf area restriction, modulation of leaf functional
anatomical traits, in addition to a C2 Kranz mechanism of photo-
synthesis allow plants to successfully cope even with long-term S80
salinity. Therefore, Bougainvillea, due to its ability to maintain leaf
traits, good blooming and also a more compact size even under S80
salinity can be considered for revegetation and landscaping project also
in semiarid and saline areas as well as in urban landscape design where
low quality water is used for irrigation.
Acknowledgements
This work was funded by the Italian Ministry of Education,
University and Research Project No. PON01-01611, ‘Sustainability of
Potted Plant Production in a Mediterranean Environment’.
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