Effects of Triacontanol and Light on

Stomatal and Photochemical Responses in

Solanum lycopersicum L.

Emilia Ramos-Zambrano, Tomás

Ernesto Juárez-Yáñez, Daniel Tapia-
Maruri, Brenda Hildeliza Camacho-
Díaz, Antonio Ruperto Jiménez-
Aparicio, et al.
Journal of Plant Growth Regulation

ISSN 0721-7595

J Plant Growth Regul

DOI 10.1007/s00344-020-10262-6

1 23
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 Author's personal copy
Journal of Plant Growth Regulation
https://doi.org/10.1007/s00344-020-10262-6

Effects of Triacontanol and Light on Stomatal and Photochemical

Responses in Solanum lycopersicum L.
Emilia Ramos‑Zambrano1   · Tomás Ernesto Juárez‑Yáñez1 · Daniel Tapia‑Maruri1   ·
Brenda Hildeliza Camacho‑Díaz1   · Antonio Ruperto Jiménez‑Aparicio1   · Alma Leticia Martínez‑Ayala1 

Received: 30 May 2019 / Accepted: 3 November 2020

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract
Triacontanol is a long-chain alcohol that is considered to be a plant growth promoter. Exogenous application of triacontanol
increases dry and fresh weight, plant height, branching, stem and root length, yield, and other biochemical parameters; it also
reduces the effect of different stressors. In this work, the effects of triacontanol on stomatal regulation and photochemical
parameters in tomato plants exposed to three irradiance levels (100, 200, and 600 μmol ­m−2 ­s−1) were examined. Low and
high irradiance levels caused a stress response in plants; photosystem II efficiency (ΦPSII) and photosynthetic-irradiance
parameters were diminished. Triacontanol only reversed the negative effects of high irradiance, increasing photochemical
response and photosynthetic-irradiance parameters. Moreover, a large population of low chloroplast number, chlorophyll
content, and stomatal conductance were stimulated by triacontanol application. In relation to stomatal function, triacontanol
had no effect on stomatal density; however, it affected stomatal size and morphology at the three irradiance levels evaluated.
Bioassays in epidermal peels showed a direct effect on the stomatal aperture and an increase in aperture size in both light
and dark conditions in triacontanol treatments. This is the first report of the role of triacontanol in the regulation of stomata
and photochemical responses in relation to irradiance stress.

Keywords  Growth promoter · Irradiance · Stomatal regulation · Photochemical reaction · Chloroplast

Introduction
Triacontanol (TRIA) is a long-chain alcohol (­ C30H61OH)
found either in free or esterified form in the cuticular wax
of plants and fruits and within waxes that make up the germ,
kernel, seed coat, shell, and skin of various nuts, as well as
animal wax secretions. Due to its broad properties, ranging
from pharmacological activities to its effects as a promoter
of plant growth, TRIA has been obtained from numerous
sources, including sugarcane cuticle, alfalfa plants, agave,
rice seed, sorghum, peanut, Perilla sp. (Méndez et al. 2003;
Irmak and Dunford 2005; Adhikari et al. 2006; Cherif et al.
Handling Editor: Parvaiz Ahmad.
* Alma Leticia Martínez‑Ayala
alayala@ipn.mx
1
Centro de Desarrollo de Productos Bióticos, Instituto
Politécnico Nacional, Carretera Yautepec-Jojutla,
Col. San Isidro, Km. 6, calle CEPROBI No. 8,
62731 Yautepec, Morelos, Mexico
2010; Ertani et al. 2013), and animal sources, such as bees-
wax, wax of Ericerus pela (Jia and Zhao 2004) and, recently,
cochineal wax (Ramos et al. 2019).
Regarding its activity as a growth promoter, this com-
ponent has been used to increase the growth and yield of
vegetable crops, such as rice, tomato, wheat, and maize
(Naeem 2012), metabolite content, and to improve in vitro
tissue culture (Yaseen and Tajuddin 1998; Tantos et al. 1999;
Tantos et al. 2001; Reddy et al. 2002; Giridhar et al. 2004;
Giridhar et al. 2005; Malabadi et al. 2005; Grzegorczyk et al.
2006). Studies of TRIA effects on physiological parameters
have shown enhancements to chlorophyll content in leaves,
photosynthetic rates, stomatal conductance, nitrogen fixa-
tion, enzymatic activity, nutrient uptake, and nitrogen, phos-
phorus, and potassium concentrations, as well as sugar and
protein levels (Misra and Srivastava 1991; Ries et al. 1991;
Kumaralevu et al. 2000; Chen et al. 2002; Verma et al. 2011;
Naeem et al. 2012; Moneruzzaman et al. 2013). Other stud-
ies have focused on the effect of TRIA on plants subjected
to stress as a result of salinity (Krishnan and Kumari 2008);
Aziz et al. 2013; Perveen et al. 2013; Karam and Keramat

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2017), water deficiency (Muthuchelian et al. 2001), heat
(Waqas et al. 2016), and even contaminants, such as cad-
mium (Muthuchelian et al. 1997) and acidic mist (Muth-
uchelian et al. 2003). In these studies, modulation of the lev-
els of defence hormones and antioxidant enzyme activities,
as well as reduction of the effects of stress and augmentation
of plant growth and other biochemical parameters by exog-
enous TRIA application have been demonstrated.
Light intensity is another abiotic stress factor that poten-
tially triggers photoinhibition and photoprotection mecha-
nisms (Raven 2011). Both high and low irradiance cause
plant stress, a high irradiance levels causing stomatal clo-
sure, and therefore, a decrease in the activity of the photo-
synthetic apparatus. Studies by Chen et al. (2002) demon-
strated that TRIA increases the light saturation point and
photosynthesis activity in rice plants; however, more stud-
ies are needed to know how TRIA affects photoinhibition
mechanisms in relation to physiological processes, such as
photochemical response and stomatal opening, among oth-
ers, and how TRIA can alleviate the effect of low light stress.
Solanum lycopersicum L., is one of the main vegetable
crops worldwide, whose annual production is around 170
million tons each year (FAO 2014). This product is highly
appreciated for its antioxidant and vitamin content, but it can
be affected by the conditions in which it is grown (Fanasca
et al. 2006), as well as light intensity aspects (Zushi et al.
2014). It has also been observed that an irradiance level
above 500 µmol ­m−2s−1 over a longer period of time (> 4
weeks), affects net stomatal size, photosynthetic rate, sto-
matal conductance, and transpiration rate (O’Carrigan et al.
2014).
The objective of this work was to determine the effects of
TRIA on photochemical and stomatal responses in Solanum
lycopersicum L. plants subjected to stress by high and low
irradiance; and as a second objective, to know the role of
TRIA in the regulation of stomatal opening and closing.
Materials and Methods
TRIA and Light Effects
Plant Material and Light Treatments
Solanum lycopersicum ‘Sun 7705’ (Bayer Company, Mexico
D.F.) seeds were germinated in polystyrene seed trays (66 ×
34 × 4.8cm) with peat moss as a substrate in a greenhouse,
reaching 600 μmol ­m−2 ­s−1 of maximum irradiance, tem-
peratures of 25–35 °C during the day and 11–22 °C at night,
and a relative humidity of 60%. At 30 days after germination
(DAG), seedlings were transplanted to pots of 5L capacity
with agrolite and peat moss as a substrate (50:50). The plants
were exposed to experimental light conditions in irradiation
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Journal of Plant Growth Regulation
chambers at the same conditions of temperature and humid-
ity for a period of 8 weeks under automatic fertigation and
a 16 h/8 h light/dark cycle. The irradiation chambers were
built with medium-density fibreboard (MDF) (120 × 80 ×
150cm) coated with white paint inside and designed with a
movable ceiling to maintain the same distance between the
plants and the lamps (approx. 20cm) throughout the course
of the experiment. Three irradiance levels were applied
and monitored at the top of the plants in irradiance cham-
­ −2 ­s−1, 200 (202) μmol m
bers, i.e. 100 (113) μmol m ­ −2 ­s−1,
−2 −1
and 600 (580) μmol ­m ­s , which were reached by LED
floodlights (SL Pro Lighting, Mexico), 1 × 30W, 3 × 30W,
and 2 × 100W, respectively, at a distance of approx. 20 cm,
which was measured with a DT-1308 light meter (CEM,
West Bengal, India). Spectral characteristics of the LED
floodlights, including blue (460 nm), green (550 nm), and
red (660 nm), with a high peak intensity in the blue light
range and low emission intensity between blue and green.
Triacontanol (Purity > 98%; Sigma-Aldrich, Toluca, Mex-
ico) was dissolved in 0.1% Tween 20 to 1 mg/L (w/v) and
the control treatment consisted of only the dispersant agent
(0.1% Tween 20). Both treatments were applied via foliar
spraying to the plants at 30 and 45 DAG. After 8 weeks
in light treatments, measurements were conducted in three
plants by treatment.
Photosynthetic Light‑Response Curve
Photosynthetic activity was measured in plants for each irra-
diance treatment after adaptation to darkness for about 20
min, using a FluorPen FP 100 portable fluorimeter (Photon
Systems Instruments, Drasov, Czech Republic); based on
the data, rapid light curves (RLC) were obtained. The RLC
consisted of the fluorescence responses to five different and
increasing actinic irradiances at a duration of 30 s (100, 200,
300, 500, and 1,000 μmol m ­ −2 ­s−1). The light curve graphs
were constructed from the mean response values of each
treatment in triplicate.
The values of the relative electron transport rate (rETR)
at a given actinic irradiance were calculated with the fol-
lowing equation:
rETR = QY × FF × 0.5 × 0.84 (1)
where rETR was the relative electron transport rate in
μmol ­m−2 ­s−1, QY was the energy yield at the given light
conditions, and FF was the flow of photons incident on the
plant during the measurement of QY. The factor 0.84 inte-
grated the fraction of energy captured by the photosynthetic
system, which depends on the species, multiplied by 0.5,
since the energy of two photons is necessary for the transport
of one electron (White and Critchley 1999).
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Journal of Plant Growth Regulation
For analysis of rETR curves as a function of FF, data were
fitted to the Platt equation (Platt et al. 1980):
)] ( 𝛽xEd )
𝛼*Ed
[ (
rETR = Ps 1 − exp − exp − (2)
Ps Ps
where the light gradient (Ed) was FF in μmol m ­ −2 ­s−1, α
was the initial slope of the curve, β was the slope of photoin-
hibition, and Ps was the maximum photosynthetic capacity
in the absence of photoinhibition.
To calculate the photosynthetic capacity (Pm) or the flow
of photons at which the maximum rETR was reached, the
following equation was used:
[ ][ ]𝛽∕𝛼
𝛼 𝛽
Pm = Ps (3)
𝛼+𝛽 𝛼+𝛽
Measurement of Chlorophyll Content
Chlorophyll content was estimated with a Minolta SPAD
502 Plus Chlorophyll Meter (Spectrum Technologies Inc.,
Illinois, USA) on the last expanded leaf of tomato plants.
The chlorophyll readings (SPAD) were converted to µg/cm2,
using the following equation (Monje and Bugbee 1992):
[Chl] = 1.034 + 0.308 × [SPAD] + 0.11 × SPAD2 (4)
[ ]
Chloroplast Number, Size and Volume
Tomato leaf segments of 3 × 3 mm were fixed with 3.5%
glutaraldehyde (v/v) for 1 hour in the dark and subse-
quently transferred to 0.1 M N ­ a2 EDTA solution with a pH
of 9. Samples were stored at 4 °C until micrographs were
obtained.
Samples were observed with a Zeiss LSM800 confocal
laser scanning microscope (Carl Zeiss, Oberkochen, Ger-
many), using a 640 nm laser with an intensity peak char-
acteristic of an autofluorescence emission signal being
captured in the range of 645–700 nm (red fluorescence).
Micrographs with a resolution of 1024 × 1024 pixels, stored
in TIFF format, were obtained using the "z-stack" mode on
different focal planes. Each 0.58 μm slice was imaged to a
depth of 16.5 μm from the beginning of the parenchymal
palisading tissue of the leaf through the epidermal layer.
Image analysis was carried out with sample micrographs
by Fiji ImageJ software version 1.51 (NIH, USA) (Schinde-
lin et al. 2012), using the ImageJ 3D suite for the manipu-
lation and analysis of 3D images (Ollion et al. 2013). The
images were imported for stack rearrangement, adjusting
the scale and depth of each layer (slice). Pre-processing was
performed by applying contrast improvement in a normal-
ised way, with a saturation value of 0.3% and a Gaussian blur
with a sigma value equal to 2. The necessary layers were
eliminated for a depth in z of 16.24 μm, either to eliminate
epidermal tissue or for micrographs with little sharpness
regarding field depth. For segmentation, the "3D Simple
Segmentation" tool with a threshold value of 85 was used,
limiting the particle size to > 500 voxels. The volume of the
segmented zones (chloroplasts) was measured with the "3D
Geometrical Measure" tool.
The percentage of volume occupied by the chloroplasts
(%VolClor) was calculated with the following equation:
%VolClor = (Chloroplast Vol.)∕(Vol.Total) × 100 (5)
To obtain the average volume, chloroplasts were consid-
ered to be ellipsoidal. Thirty measurements of three axes
were taken for each of the samples, with the volume being
calculated with the following equation (Weisstein 2018):
Chloroplast volume = 4∕3𝜋abc (6)
All samples were measured regarding three axes, where
“a” was designated length, “b” was width, and “c” was thick-
ness. The chloroplast number was calculated by dividing the
volume occupied with chloroplasts by the average volume
of a chloroplast corresponding to each sample. The three-
dimensional representations of each treatment were obtained
using the "Z Project" tool, with the "Max intensity" projec-
tion type in the Fiji software v. 1.51 (NIH, USA).
Measurement of Stomatal Conductance, Stomatal Density,
and Morphology
To evaluate stomatal density and size, abaxial epidermal
peels of approximately 0.5 × 0.5 mm were obtained from
the centre of tomato plant leaves exposed to light treatments.
Stomatal density was defined as the number of total stomata
found per 0.6 m­ m2 and extrapolated to 1.0 m
­ m2 from images
of 2,048 × 2,048 pixels obtained by confocal microscopy.
To estimate stomatal size, the length and width of the guard
cells of the total stomata were measured. Stomatal area was
calculated by assuming an oval pore shape and stomatal
conductance was evaluated using a SC-1 Leaf Porometer
(Decagon Devices, Inc., Washington, USA) in triplicate on
the first well-developed leaf of the plant.
Epidermal Bioassays
After observing the effects of TRIA and light on stomata
density, morphology, and size, bioassays were carried out to
evaluate the effect of TRIA concentration on the regulation
of stomatal opening and closing in epidermal peels. For this,
abaxial epidermal peels of approximately 0.5 × 0.5 mm were
obtained from leaves of 8-week-old plants of S. lycopersicum
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and continued to be subjected to light and dark conditions
as detailed below.
To evaluate the effect of TRIA on stomatal closing, the
peels were floated in buffer solution (30 mM KCl, 10 mM
2-(N-morpholino)-ethanesulfonic acid (MES), pH 6.15)
and exposed to light (photosynthetic photon flux density,
PPFD, 200 mol m ­ −2 ­s−1) for 2 hours to open the stomata.
Once the stomata were open, the peels were transferred to
the same buffer solution containing TRIA concentrations
of 0.02, 0.05, 0.1, 0.15, 0.3, or 0.5 mM in a solution of
Tween 20 (0.1%) and control treatment, which consisted in
the buffer solution and Tween 20 (0.1%). Epidermal peels
of both treatments were kept in the dark and stomatal open-
ing was measured. Another experiment was performed to
evaluate the effect of TRIA on stomatal opening. For this
experiment, epidermal peels were floated in a buffer solution
(30 mM KCl, 10 mM MES, 0.5 mM C ­ a2+, pH 6.15) in the
dark to close the stomata. These epidermal peels were then
transferred to the same buffer solution containing TRIA at
concentrations of 0.02, 0.05, 0.1, 0.15, 0.3, or 0.5 mM in a
solution of Tween 20 (0.1%) and control treatment, which
consisted of the buffer solution and Tween 20 (0.1%). Epi-
dermal peels of both treatments were kept in the light for
another 2 hours (Xia et al. 2014) and stomatal opening was
measured.
A Zeiss LSM800 confocal laser scanning microscope
(Carl Zeiss, Oberkochen, Germany) was used to examine
the epidermal peels. The autofluorescence emission signal
was captured in the range of 410–595 nm (green fluores-
cence), using a 20x/0.5 EC lens. Images of 2,048 × 2,048
pixels stored in TIFF format were obtained and total stomata
were analysed in triplicate (n = 3). Stomatal aperture size
was determined on the basis of pore width/stomatal width to
avoid stomata size effects on the stomatal aperture.
Statistical Analyses
The experiment about TRIA and light effects were con-
ducted in a randomised complete block design (RCBD) and
was assessed using two-way analysis of variance (ANOVA).
The statistical significance between TRIA and control treat-
ments in chlorophyll content, stomatal conductance, photo-
chemical response, stomatal density, and size were examined
using the Student’s t test performed with the SigmaPlot soft-
ware (V. 12.0). In the case of rETR, data were fitted to the
Platt equation with R software (2016) version 3.3.1, using
the Levenberg–Marquardt nonlinear least squares regres-
sion algorithm that belongs to the package Minpack.lm ver-
sion 1.2–1 (­ r2 > 0.89) and a T test was performed between
control and TRIA treatments for each actinic irradiance and
photosynthetic-irradiance parameter. All the data are means
± standard deviation (SD), calculated from three biological
replicates (n = 3), except for the analysis of chloroplasts,
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Journal of Plant Growth Regulation
where one individual was taken per treatment and 24 images
were processed for measurement.
For epidermal biossays, a randomised complete
block design (RCBD) was used, which was analysed using
Two-Way ANOVA and a Fisher Test was used to evaluate
the statistical significance between TRIA concentrations and
control (SigmaPlot V. 12.0). All data represent the mean of
three biological replicates (n = 3), with error bars indicat-
ing (SD).
Results
Three irradiance levels were used to evaluate the effect of
TRIA, a low level of 100 µmol m ­ −2s−1, an intermediate level
of 200 µmol ­m s , and a high level of 600 µmol ­m−2s−1.
−2 −1
The low and high levels resulted in decreased photosynthetic
activity; under these conditions, the response efficiency
of photosystem II (ΦPSII) declined as the photosynthetic
photon flux density (PPFD) was increased compared with
intermediate irradiance (Fig. 1). This was consistent with
the response of the relative electron transport rate (rETR),
as seen in Fig. 2.
TRIA increased the rETR response of plants only when
they were grown at 600 μmol m ­ −2 ­s−1, when they were
exposed to 100–1000 PPFD (p < 0.05), as well as some
photosynthetic-irradiance parameters: maximum effective
photosynthetic capacity (Pm) (p < 0.05), the flow of pho-
tons at which the maximum rETR was reached (PPFDmax)
(p < 0.001), and the potential maximum of photosynthetic
capacity in the absence of photoinhibition (Ps) (p < 0.05);
however, TRIA did not enhance the α value (initial slope of
the curve) and irradiance that represents the inflection from
α value to Pm (β) (Fig. 2).
Regarding the other photosynthetic variables, non-photo-
chemical quenching (NPQ) declined in TRIA-treated plants
compared with the control only at irradiances of 600 µmol
­cm−2 ­s−1 (Fig. 1). By contrast, the coefficient of photochemi-
cal quenching (qP) was increased by TRIA only at an irradi-
ance of 600 µmol ­cm−2 ­s−1 (Fig. 1).
The chlorophyll content (µg ­cm−2) was not affected by
irradiance in the control treatment. Nevertheless, plants
treated with TRIA showed an increase at 200 (p < 0.001)
and 600 µmol c­ m−2 ­s−1 (p < 0.01) (Fig. 3). The volume
of chloroplasts was enhanced by TRIA only at irradiances
of 100 and 200 µmol c­ m−2 ­s−1. TRIA treatment increased
the percentage of total volume occupied by chloroplasts by
15.9% at an irradiance of 100 µmol ­cm−2 ­s−1 and by 7.3% at
600 µmol c­ m−2 ­s−1 with respect to the control, likewise chlo-
roplast number was only augmented at irradiance levels of
100 and 600 µmol c­ m−2 ­s−1 (Fig. 4). At high irradiance, the
chloroplast number in plants treated with TRIA was higher
(TRIA = 38195; Control = 27052) and size was lower
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Journal of Plant Growth Regulation
Fig. 1  Effect of TRIA on Photosystem II efficiency (ΦPSII) (a, b, c), photochemical quenching (qP), and non-photochemical quenching (NPQ)
(d, e, f) at different irradiance levels. Control (-○ - -), TRIA-treated (--●--). Values represent mean ± SD of at least three replicates
(Average chloroplast volume TRIA = 10.24 µm3; Control =
12.032 µm3) and therefore, the total volume of chloroplasts
was higher (TRIA = 446.43mm3; Control = 325.49mm3).
Stomatal conductance was negatively affected at low (100
μmol ­m−2 ­s−1) (p < 0.001) and high (600 μmol ­m−2 ­s−1)
(p < 0.05) irradiance levels in comparison with intermedi-
ate levels. Nonetheless, plants treated with TRIA showed
an increase in stomatal conductance for all three irradiance
levels evaluated (Fig. 5). Based on the morphological and
size parameters, stomatal density was not affected by TRIA
treatment; however, TRIA had an effect on the length and
width (only at 200 and 600 μmol ­m−2 ­s−1) of guard cells
(Fig. 5), as well as on the relationship between length and
width and the area of the stoma (Fig.5), which indicates a
major stomatal aperture (Figs. 5 and 6).
Our results show a clear effect on stomatal size and mor-
phology, but in order to understand the role of TRIA in the
regulation of stomatal opening, we performed stomatal
bioassays with epidermal peels isolated from tomato leaves
exposed to light and dark conditions and to different levels
of TRIA. In these bioassays, TRIA increased stomatal aper-
tures in light conditions with respect to the control, reaching
a significantly greater opening above a concentration of 0.05
mM (p < 0.001) (Fig. 7a).
In dark conditions, the addition of TRIA to epidermal
peels inhibited stomatal closing and increasing stomatal
opening to all concentrations tested. At 0.5 mM, stomatal
opening was decreased; however, the apertures were still
larger than those of the control (p < 0.001) (Fig. 7b).
Discussion
We applied three levels of irradiance to tomato plant leaves
to evaluate the effect of TRIA on stomatal and photochemi-
cal responses. The intermediate light level was 200 µmol
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Fig. 2  Effect of TRIA (--●--) in comparison to the control (-○ - -)
on the relative electron transport rate (rETR) at different irradiance
levels: 100 (a), 200 (b), and 600 (c) μmol m­ −2 ­s−1. Curve was fitted
to the Platt model and photosynthetic-irradiance parameters were
Fig. 3  Effect of TRIA (grey bar) in comparison to the control (black
bar) on total chlorophyll content at the different irradiance levels. Val-
ues represent mean ± SD of at least three replicates. T test method p
< 0.05 (*), p < 0.01 (**), p < 0.001 (***) indicates significant differ-
ence between treatments
­ −2s−1, which corresponds to 5–10% of the maximum daily
m responses of plants treated with TRIA in other studies. For
PPFD (Hoshika et al. 2017), while the low and high levels
were 100 and 600 µmol m ­ −2s−1, respectively. At low and
high irradiance levels, photosynthetic efficiency decreased,
based on ΦPSII and rETR responses. These results are in
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Journal of Plant Growth Regulation
obtained. Values represent mean ± SD of at least three replicates. T
test method p < 0.05 (*), p < 0.001 (***) indicates significant differ-
ence between treatments
agreement with the findings of O’Carrigan et al. (2014),
who demonstrated that a light irradiance level above 500
µmol ­m−2s−1 induces stress in tomato plants and decreases
the photosynthetic efficiency. In another study, light intensity
affected growth, photosynthetic rate (Pn), NPQ, ΦPSII, and
rETR in S. lycopersicum seedlings (Wu et al. 2014). Based
on our results, TRIA only reverses the negative effects of
high irradiance levels.
With respect to the curves obtained by the fitted Platt
equation, parameters that characterised the photosynthetic
behaviour of the plants were improved by TRIA treatment,
such as the flow of photons at which maximum rETR was
reached (PPFDmax), maximum effective photosynthetic
capacity (Pm), potential maximum of photosynthetic capac-
ity in the absence of photoinhibition (Ps), except irradiance
that represents the inflection from α value to Pm (β) and
the initial slope of the curve (α value) at 600 µmol c­ m−2
­s−1, indicating that TRIA does not increase the quantum
efficiency at low irradiance levels and confirming the null
effect to in plants grown at low irradiance levels (100 and
200 µmol m ­ −2s−1). Our results are in accordance with the
example, Borowsky et al. (2000) determined that TRIA
increased the maximum efficiency of PSII photochemistry
(Fv/Fm) and the efficiency of excitation capture by open PSII
reaction centres (Fv’/F m’), which led to the plants treated
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Journal of Plant Growth Regulation
Fig. 4  Images of chloroplasts obtained by confocal microscopy from leaves of tomato plants treated with TRIA (a, b, c) in comparison with
untreated controls (d, e, f) at three irradiance levels (100, 200 and 600 μmol ­m−2 ­s−1). Bar scale = 20 µm
with TRIA using the PPFD energy in PSII centres over 22%
more effectively than control plants. Chen et al. (2012) dem-
onstrated that TRIA applied to rice plants increased their
photosynthetic rate to that of the light saturation point, indi-
cating that plants treated with TRIA intensively absorb light.
Furthermore, in this work, TRIA application resulted in an
increase of qP and a decrease of NQP at irradiances of 600
µmol ­cm−2 ­s−1, suggesting less thermal dissipation and a
larger number of open PSII reaction centres. These results
are in agreement with the findings of Chen et al. (2003),
who demonstrated that TRIA increases the minimum fluo-
rescence (F0), maximum fluorescence (Fm) as light yield of
the PSII electron transport (PSII) and qP, and the subsequent
decrease of non-photochemical quenching (NQP).
Similarly, TRIA application resulted in an increase in the
total chlorophyll content (µg ­cm−2) at 200 and 600 µmol
­cm−2 ­s−1. This is consistent with reports for other species,
including rice, wheat, maize, bean, and Lablab purpureus
L. (hyacinth bean; Ries et al. 1985; Chen et al. 2002; Naeem
et al. 2009). An increased chlorophyll content was one of
the main results of TRIA application, even under stress
conditions, such as salinity (Perveen et al. 2012; Azis et al.
2013; Ertani et al. 2013), chilling (Borowsky and Blamowski
2009), and water stress (Muthuchelian et al. 1997). With
respect to the chloroplasts, the current study showed that
chloroplasts of plants exposed to irradiances of 600 µmol
­cm−2 ­s−1 and treated with TRIA had the largest number and
smallest size, which can be associated with the photosyn-
thetic efficiency and the increased parameters of chloro-
phyll fluorescence at 600 μmol ­m−2 ­s−1. Xiong et al. (2017)
determined that a large population of small chloroplasts
was more efficient in terms of photosynthesis because of
changes in ­CO2 diffusion conductance. In the same way,
Moorthy and Kathiresan (1993) found that TRIA increased
the chlorophyll content; the authors attributed this increase
to the inhibition of senescence or iron uptake, regulating
the biosynthesis of chlorophyll. Kathuria et al. (2012) dem-
onstrated that TRIA suppresses chlorophyllase activity, but
does not increase chlorophyll biosynthesis. Muthuchelian
et al. (1990) attributed the increase in chlorophyll to the
number and size of the chloroplasts and a better development
of thylakoid grana. In addition, Ivanov and Angelov (1997)
determined that TRIA modifies the membrane fluidity of
chloroplasts, inducing alterations in the dynamic properties
of protoplast and chloroplast membranes. This indicates that
TRIA application leads to an increased chlorophyll content
and furthermore, to modifications of the chloroplast mem-
brane fluidity. It also affects chloroplast number and size and
13
 Author's personal copy
therefore, the total volume of chloroplasts, as demonstrated
in this work, although this was only observed for high irra-
diance levels.
Furthermore, our results suggest that TRIA inhibits some
effects of high irradiance in relation to stomatal regulation.
TRIA increased stomatal conductance in tomato plants
grown at three different irradiance levels, which is consistent
with the reports for other plants treated with TRIA (Aftab
et al. 2010; Azis et al. 2013; Moneruzzaman et al. 2013).
An increase in stomatal conductance, as a consequence of
a higher density, size, or opening degree of the stomata,
indicates that photosynthetic and transpiration rates are also
potentially higher (Pietragalla and Pask, 2012). The increase
in the stomatal aperture in plants exposed to the different
Fig. 5  Effect of TRIA (grey bar) in comparison to the control (black
bar) on stomatal conductance (a), guard cell width (b), guard cell
length (c), relation length/width (d), stomatal density (e), and stoma-
tal area (f) at the different irradiance levels. Values represent mean ±
13
Journal of Plant Growth Regulation
levels of irradiance indicates that this is not a limiting fac-
tor; however, low irradiance levels decreased the effect
of TRIA. High irradiance levels, above 500 µmol m ­ −2s−1,
induced increases in shoot biomass, leaf number, leaf tem-
perature, vapour pressure deficit, stomatal index, aperture
length, and guard cell length in tomato plants, although
stomatal aperture width was reduced by 31.7 and 46.3%
by short- and long-term exposure to high light intensities,
respectively, with a likely concomitant decrease in photosyn-
thesis (O’Carrigan et al. 2014). In this study, TRIA did not
affect stomatal density, but had an impact on the length and
width of guard cells, as well as on the relationship between
length/width and on stomatal area. A larger stomatal size is
related to a slower stomatal response, as large stomata are
SD of at least three replicates. T test method p < 0.05 (*), p < 0.01
(**), p < 0.001 (***) indicates significant difference between treat-
ments
 Author's personal copy
Journal of Plant Growth Regulation

Fig. 6  Images of epidermal peels obtained by confocal microscopy ­m−2 ­s−1 (a, b), 200 μmol ­m−2 ­s−1 (c, d), and 600 μmol ­m−2 ­s−1 (e, f).
from leaves of tomato plants treated with TRIA (b, d, f) in compari- Bar scale = 50 µm
son to untreated controls (a, c, d) at three irradiance levels 100 μmol

13
 Author's personal copy
Fig. 7  Effect of TRIA (TRIA) on stomatal opening (a) and stomatal closing (b). Values represent mean ± SD of at least three replicates. Differ-
ent letters indicate a significant difference (Fisher p < 0.05). Control (-○ - -), TRIA-treated (--●--)
less able to prevent hydraulic dysfunction in dry habitats;
this lag in response may be advantageous in cool, moist, or
shaded environments (Drake et al. 2013).
However, TRIA did not only affect stomatal size and
morphology, as demonstrated in the epidermal biossays,
where two conclusions were reached. The first was that
TRIA had a greater stomatal opening when compared to the
control group in light conditions; and the second was that
TRIA increases stomatal opening even in dark conditions. A
greater openness is related to greater stomatal conductance
and thus, with greater photosynthetic activity during the
day; however, it is unclear how plants benefit from greater
stomatal conductance at night. Nocturnal stomatal conduct-
ance may be 5–40% of the magnitude of day-time stomatal
conductance (Caird et al. 2007). Even though this causes
a significant percentage (10–25%) of the total daily water
loss (Zeppel et al. 2014), nocturnal stomatal conductance
has advantages, such as embolism removal under conditions
when there is less evaporative demand, transport of oxygen
and nutrients, and refilling of capacitance of the trunk and
stem (Daley and Phillips 2006; Caird et al. 2007). Likewise,
higher night-time stomatal conductance and transpiration
have been associated with higher day-time values for both
photosynthetic pathways, C ­ 3 and C
­ 4 (Snyder et al. 2003).
Oren et al. (2001) suggested stomatal opening at night might
13
Journal of Plant Growth Regulation
improve the ratio of photosynthesis to transpiration and
avoid delays between assimilation and increased stomatal
conductance, perhaps allowing photosynthesis to continue
until sundown and recommence at higher rates earlier in
the morning. Works carried out by Costa et al. (2015) have
shown that mutants of Arabidopsis thaliana that maintain
stomatal opening throughout the night had higher stomatal
conductance values in light conditions. In Eucalyptus cama-
ldulensis, predawn stomatal conductance was positively cor-
related with high conductance and carbon assimilation in
the early morning and midday with increased leaf area and
total plant biomass (Resco de Dios et al. 2016). Likewise,
in potato plants, night transpiration, as well as predawn and
early night conductance, were correlated with increased
yields (Ramírez et al. 2018). This indicates that the increase
in photosynthetic activity given by the application of TRIA
could be due to a greater stomatal opening both during the
day and at night and as a consequence, higher biomass and
yields; however, the amount of light is decisive for the action
of TRIA.
In conclusion, TRIA reverses the negative effects of high
irradiance (600 μmol ­m−2 ­s−1) through improvement of pho-
tochemical responses, like the photosynthetic-irradiance
response, chlorophyll content of leaves, and chloroplast
number, which was smaller in plants exposed to long-term
 Author's personal copy
Journal of Plant Growth Regulation
treatment. Stomatal conductance was negatively affected at
low (100 μmol ­m−2 ­s−1) and high (600 μmol ­m−2 ­s−1) irradi-
ances; however, TRIA application alleviated this response
and it was greater than that of the control, even at 200 μmol
­m−2 ­s−1. TRIA showed an active role in the regulation of
stomatal apertures and inhibition of stomatal closure, as well
as in the size of guard cells in the three irradiance treat-
ments. Stomatal opening in the dark by TRIA application is
in agreement with other reports; this could improve photo-
synthesis during the day. Such results suggest that the limi-
tations to plants exposed to low irradiance did not concern
stomatal aperture, which could be improved by TRIA. As
such, TRIA only alleviated stress at high irradiance levels
at light conditions necessary to carry out photosynthesis (≥
250 μmol ­m−2 ­s−1).
Acknowledgements  We acknowledge the Consejo Nacional de Cien-
cia y Tecnología (CONACYT) and the Instituto Politécnico Nacional
(IPN) for scholarships given to the students Emilia Ramos Zambrano
and Tomás E. Juárez Yáñez during the course of this investigation
and the IPN for financial support. Alma L. Martínez-Ayala, Antonio
R. Jimenez-Aparicio, and Brenda H. Camacho Díaz are fellows of
COFAA and EDI-IPN.
Author Contributions  ERZ: Conceptualization, Methodology, Valida-
tion, Formal analysis, Investigation, Writing - Original Draft; TEJY:
Methodology, Validation, Investigation; ARJA: Resources, Writing -
Review & Editing, Supervision; BHCD: Validation, Resources, Writ-
ing - Review & Editing; DTM: Validation, Writing - Review & Editing;
ALMA: Conceptualization, Resources, Writing - Review & Editing,
Supervision, Project administration, Funding acquisition.
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