Volume 172, Issue 2 pp. 748-761

Special Issue Article

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Unraveling the roles of brassinosteroids in alleviating drought stress in young Eucalyptus urophylla plants: Implications on redox homeostasis and photosynthetic apparatus

Udson O. Barros Junior,

Udson O. Barros Junior

orcid.org/0000-0002-9562-9391

Núcleo de Pesquisa Vegetal Básica e Aplicada, Universidade Federal Rural da Amazônia, Paragominas, Pará, Brazil

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Michael D. R. Lima,

Michael D. R. Lima

orcid.org/0000-0002-4789-1205

Núcleo de Pesquisa Vegetal Básica e Aplicada, Universidade Federal Rural da Amazônia, Paragominas, Pará, Brazil

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Abdulaziz A. Alsahli,

Abdulaziz A. Alsahli

orcid.org/0000-0002-7734-4886

Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia

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Allan K. S. Lobato,

Corresponding Author

Allan K. S. Lobato

[email protected]

orcid.org/0000-0002-2641-6122

Núcleo de Pesquisa Vegetal Básica e Aplicada, Universidade Federal Rural da Amazônia, Paragominas, Pará, Brazil

Correspondence

Allan K. da Silva Lobato, Núcleo de Pesquisa Vegetal Básica e Aplicada, Universidade Federal Rural da Amazônia, Paragominas, Pará, Brazil.

Email: [email protected]

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Udson O. Barros Junior,

Udson O. Barros Junior

orcid.org/0000-0002-9562-9391

Núcleo de Pesquisa Vegetal Básica e Aplicada, Universidade Federal Rural da Amazônia, Paragominas, Pará, Brazil

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Michael D. R. Lima,

Michael D. R. Lima

orcid.org/0000-0002-4789-1205

Núcleo de Pesquisa Vegetal Básica e Aplicada, Universidade Federal Rural da Amazônia, Paragominas, Pará, Brazil

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Abdulaziz A. Alsahli,

Abdulaziz A. Alsahli

orcid.org/0000-0002-7734-4886

Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia

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Allan K. S. Lobato,

Corresponding Author

Allan K. S. Lobato

[email protected]

orcid.org/0000-0002-2641-6122

Núcleo de Pesquisa Vegetal Básica e Aplicada, Universidade Federal Rural da Amazônia, Paragominas, Pará, Brazil

Correspondence

Allan K. da Silva Lobato, Núcleo de Pesquisa Vegetal Básica e Aplicada, Universidade Federal Rural da Amazônia, Paragominas, Pará, Brazil.

Email: [email protected]

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First published: 27 November 2020

https://doi.org/10.1111/ppl.13291

Citations: 35

Edited by: P. Ahmad

Funding information: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/Brazil); Fundação Amazônia de Amparo a Estudos e Pesquisas (FAPESPA/Brazil); Researches Supporting Project Number RSP 2020/236 from King Saud University; Universidade Federal Rural da Amazônia (UFRA/Brazil)

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Abstract

Water deficit is the most limiting abiotic stress to plants because it affects several physiological and biochemical processes. Brassinosteroids, including 24-epibrassinolide (EBR), are steroids that regulate growth and positively act on gas exchange. This research aims to determine whether EBR can attenuate the negative effects of water deficit, revealing possible contributions of this steroid on photosynthetic machinery of young Eucalyptus urophylla plants under water deficit. The experiment had a completely randomized factorial design with two water conditions (control and water deficit) and three levels of EBR (0, 50, and 100 nM EBR). Water deficit caused a decrease in the levels of total chlorophyll and carotenoids, but these photosynthetic pigments increased by 135 and 226%, respectively, in plants sprayed with EBR when compared to the water deficit + 0 nM EBR treatment. Regarding the antioxidant system, 100 nM EBR induced significant increments in superoxide dismutase (42%), catalase (52%), ascorbate peroxidase (147%), and peroxidase (204%). Steroid application in E. urophylla plants exposed to water deficit increased the effective quantum yield of the photosystem II (PSII) photochemistry and electron transport rate. However, interestingly, it decreased the nonphotochemical quenching and relative energy excess at the PSII level, indicating improvements related to PSII efficiency. This research revealed that application of 100 nM EBR attenuated the negative effects caused by water deficit, being explained by the positive repercussions on antioxidant enzyme activities, chloroplastic pigments, PSII efficiency, electron flux, and net photosynthetic rate.

Abbreviations

Φ_PSII: effective quantum yield of PSII photochemistry
APX: ascorbate peroxidase
BR: brassinosteroid
Car: carotenoid
CAT: catalase
Chl a: chlorophyll a
Chl b: chlorophyll b
C_i: intercellular CO₂ concentration
E: transpiration rate
EBR: 24-epibrassinolide
EL: electrolyte leakage
ETR: electron transport rate
EXC: relative energy excess at the PSII level
g_s: stomatal conductance
HBL: 28-homobrassinolide
LDM: leaf dry matter
MDA: malondialdehyde
NPQ: nonphotochemical quenching
P_N: net photosynthetic rate
POX: peroxidase
PQ: plastoquinone
PSI: photosystem I
PSII: photosystem II
q_P: photochemical quenching
RDM: root dry matter
ROS: reactive oxygen species
SDM: stem dry matter
SOD: superoxide dismutase
TDM: total dry matter
WUE: water-use efficiency

1 INTRODUCTION

The genus Eucalyptus has more than 700 species, distributed across more than 20 million hectares of the world's temperate and tropical regions (Mora et al., 2017; Mora & Serra, 2014). Of this total, approximately 5.7 million hectares are cultivated in commercial forests in Brazil (IBA, 2017). The interest in species of this genus is justified by their rapid growth and broad environmental adaptability in addition to the wide variety of products generated by their cultivation, such as wood, cellulose pulp, and essential oils (Mora et al., 2009).

Chloroplasts are fundamental organelles in photosynthetic organisms. They are composed of thylakoids and stroma, in which reside the components intrinsically linked to the photosynthetic machinery (Staehelin, 2003; Zoschke & Bock, 2018). In the granum lamellae, the solar energy that reaches the antenna of the light-harvesting systems (light-harvesting complex II) is captured by the photosynthetic pigments, including chlorophylls a (Chl a) and b (Chl b) and carotenoids (Car), which transfer the electrons to the plastoquinone (PQ), primary acceptor of the reaction center (P680) in the photosystem II (PSII) (Baker, 2008; Kume et al., 2018). The PQ then transfers the excited energy to the reaction center (P700) of photosystem I (PSI) through the electron transport chain in the thylakoid membrane. This energy is used for the synthesis of ATP and nicotinamide adenine dinucleotide phosphate during reactions connected to carbon dioxide (CO₂) fixation (Belyaeva et al., 2016; Cherepanov et al., 2017).

Water deficit is the abiotic stress most limiting to plants (Liu et al., 2019; Lobato et al., 2020; Wyka et al., 2019), causing a reduction in leaf water potential (Rivas et al., 2016), a reduction in stomatal conductance and transpiration (Pereira et al., 2016), and thus a decrease in the photosynthetic rate (Silva et al., 2015). This stress also reduces the synthesis of photosynthetic pigments (Santos et al., 2015) and photochemical efficiency (Singh & Reddy, 2011) and induces the accumulation of reactive oxygen species (ROS), leading to oxidative stress (Sheikh-Mohamadi et al., 2017; Silva et al., 2012), which reduces growth rates (Caser et al., 2017).

The exogenous application of brassinosteroids (BRs) may represent a mitigation option for plants' development under water deficit conditions. BRs are natural polyhydroxy steroid compounds classified according to the number of carbons. They are present in various plant organs, such as leaves, roots, seeds, and fruits, where they are rapidly absorbed and metabolized (Bajguz & Hayat, 2009; Bajguz & Tretyn, 2003). There are approximately 70 types of BRs, but 24-epibrassinolide (EBR), 28-homobrassinolide (HBL), and brassinolide are the most frequently used (Divi & Krishna, 2009; Oklestkova et al., 2015).

EBR is recognized as a steroid with intrinsic effects on growth. They participate in several physiological, biochemical, and molecular processes (Fonseca et al., 2020; Nazir et al., 2021; Planas-Riverola et al., 2019), including osmotic regulation (Shahid et al., 2014), gas exchange (Ma & Guo, 2014), chlorophyll biosynthesis (Yuan et al., 2012), and photochemical efficiency (Wang, Zheng, et al., 2015). This molecule can mitigate oxidative stress (Talaat et al., 2015) by stimulating the activity of antioxidant enzymes (Ali et al., 2008) and increasing growth (Shahbaz et al., 2008). In addition, EBR regulates the tolerance mechanisms to salinity stress (Reyes Guerrero et al., 2015), low and high temperatures (Cui et al., 2016; Wu et al., 2014), heavy metal toxicity (Ramakrishna & Rao, 2015), nutrient uptake (Karlidag et al., 2011), and nutrient deficiency (Wang, Li, & Zhang, 2015).

Researchers have demonstrated the beneficial effects of the application of EBR on plants exposed to water deficit, such as increases in the growth, relative water content, and minor production of H₂O₂ in Lycopersicon esculentum plants (Jangid & Dwivedi, 2017). In Hordeum vulgare plants sprayed with EBR, biomass production and gas exchange were maximized (Gill et al., 2017). In Vigna unguiculata, plants pretreated with EBR had an increase in leaf water potential and carbon fixation (Lima & Lobato, 2017).

The hypothesis of this research considered the negative effects induced by water deficit on absorption and utilization of light (Ju et al., 2018; Lang et al., 2018). Important results on the protective roles exercised by the EBR are available in literature, including benefits on carbon fixation (Talaat, 2020). Therefore, the objective of this research is to determine whether EBR can attenuate the negative effects resulting from water deficit, revealing possible contributions of this steroid on antioxidant system and photosynthetic machinery in Eucalyptus urophylla under water deficit.

2 MATERIALS AND METHODS

2.1 Location and growth conditions

The experiment was performed at the Campus of Paragominas of the Universidade Federal Rural da Amazônia, Paragominas, Brazil (2°55′S, 47°34′W). The study was conducted in a greenhouse with the temperature and humidity semi-controlled. The minimum, maximum, and median temperatures were 20, 31, and 24.5°C, respectively. The relative humidity during the experimental period varied between 60 and 80%. Day length of 12 h. During the measurement period (12:00 p.m.), the photosynthetically active radiation varied between 585 and 1854 μmol photons m⁻² s⁻¹.

2.2 Plants, containers, and acclimation

Thirty-eight-day-old seedlings of E. urophylla, similar in aspects and sizes, were selected and placed in 1.2 L containers filled with a mixed substrate of sand and vermiculite in a 3:1 ratio supplied with 500 ml of nutrient solution (semi-hydroponic conditions) in agreement with Oliveira et al. (2019), with minor modifications. Ionic strength started at 50% (38th day) and was modified to 100% after 5 days (40th day). After this period, the nutritive solution remained at total ionic strength.

2.3 Experimental design

The experiment was a factorial design with the completely randomized factors of two water conditions (control and water deficit) and three levels of EBR (0, 50, and 100 nM EBR). A total of 30 experimental units (five replicates for each of six treatments) was used, with one plant in each unit. The steroid concentrations were chosen according to Amzallag and Vaisman (2006).

2.4 EBR preparation and application

Forty-day-old seedlings were sprayed with EBR or Milli-Q water (containing a proportion of ethanol equal to that used to prepare the EBR solution) at 6-day intervals until Day 76. The 0, 50, and 100 nM EBR (Sigma-Aldrich) solutions were prepared by dissolving the solute in ethanol (100 μl) followed by dilution with Milli-Q water (ethanol: water [v/v] = 1:10,000) (Ahammed et al., 2013). On Day 74, half of the plants were subjected to water restriction, while the others were properly watered.

2.5 Water deficit treatment

Plants received the following macro- and micronutrients in the nutrient solution in agreement with Oliveira et al. (2019), with minor modifications. To simulate water deficit, the nutrient solution was removed completely, and the root system was placed in similar pots without water/solution. The water deficit was applied for 4 days (between Day 74 and Day 78). During the study, the nutrient solutions were changed at 07:00 h at 3-day intervals with the pH adjusted to 6.0 using HCl or NaOH. On Day 78 of the experiment, physiological and morphological parameters were measured for all plants, and leaf tissues were harvested for nutritional and biochemical analyses.

2.6 Measurement of chlorophyll fluorescence and gas exchange

The chlorophyll fluorescence was measured in fully expanded leaves under light using a modulated chlorophyll fluorometer (model OS5p; Opti-Sciences). Preliminary tests determined the location of the leaf, the part of the leaf, and the time required to obtain the highest F_v/F_m ratio; therefore, the acropetal third of leaves located at the middle third of the plant and adapted to the dark for 30 min, was used in the evaluation. The intensity and duration of the saturation light pulse were 7500 μmol m⁻² s⁻¹ and 0.7 s, respectively. The gas exchange was evaluated in all plants under constant CO₂ concentration using an infrared gas analyzer (model LCPro⁺; ADC BioScientific). The photosynthetically active radiation, air-flow rate, and temperature were 360 μmol mol⁻¹ CO₂, 800 μmol photons m⁻² s⁻¹, 300 μmol s⁻¹, and 28°C, respectively, when the measurements occurred (between 10:00 a.m. and 12:00 p.m.).

2.7 Determination of antioxidant enzymes, superoxide, and soluble proteins

Antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT], ascorbate peroxidase [APX], and peroxidase [POX]), superoxide (O₂⁻), and soluble proteins were extracted from totally expanded leaf (0.5 g) according to the method of Badawi et al. (2004), in which this extraction methodology presents as advantages to be rapid, low cost, and efficient. Total soluble proteins were quantified using the methodology described by Bradford (1976). The SOD activity was measured at 560 nm by Unit m⁻¹ proteins (Giannopolitis & Ries, 1977). The CAT activity was detected at 240 nm (Havir & McHale, 1987) by μmol H₂O₂ mg⁻¹ protein min⁻¹. The APX activity was measured at 290 nm (Nakano & Asada, 1981) in μmol AsA mg⁻¹ protein min⁻¹. POX activity was detected at 470 nm (Cakmak & Marschner, 1992), with the activity expressed in μmol tetraguaiacol mg⁻¹ protein min⁻¹. The determination of O₂⁻ was measured at 530 nm (Elstner & Heupel, 1976).

2.8 Quantification of hydrogen peroxide, malondialdehyde, and electrolyte leakage

Stress indicators (hydrogen peroxide [H₂O₂] and malondialdehyde [MDA]) were extracted from leaf tissue using the methodology described by Wu et al. (2006). It has the advantage of applying a single extraction procedure to both compounds. H₂O₂ was measured with the procedures defined by Velikova et al. (2000). MDA was determined by the method of Cakmak and Horst (1991), using the extinction coefficient of 155 mM⁻¹ cm⁻¹. Electrolyte leakage (EL) was measured according to Gong et al. (1998), being calculated by the formula EL (%) = (EC₁/EC₂) × 100.

2.9 Quantification of photosynthetic pigments and biomass

The chlorophyll and Car determinations were performed using a spectrophotometer (model UV-M51; Bel Photonics), according to the methodology of Lichtenthaler and Buschmann (2001). The biomass of roots, stems, and leaves was measured based on constant dry weights (g) after drying in a forced-air ventilation oven at 65°C.

2.10 Data analysis

The normality of data was verified with Shapiro–Wilk test. Data were submitted to two-way ANOVA (water condition and EBR concentration) and applied Scott–Knott test at a probability level of 5% (Steel et al., 2006). All statistical procedures used the Assistat software.

3 RESULTS

3.1 EBR contributions to water status and light capture

Plants exposed to water deficit had a significant reduction in leaf water potential (Ψw) values, but the application of 100 nM of EBR caused an increase of 18% when compared to plants under water deficit without EBR (Figure 1). Water restriction led to significant decreases in maximal quantum yield of PSII photochemistry (F_v/F_m) and maximal fluorescence yield of the dark-adapted state (F_m) values, while plants sprayed with EBR (100 nM) increased those parameters by 40 and 54%, respectively, compared to water deficit + 0 nM EBR. Water deficit significantly increased the minimal fluorescence yield of the dark-adapted state (F₀) values, while the application of 100 nM EBR to plants under water deficit induced a significant reduction of 15% in F₀ values when compared to the equal treatment without EBR.

Details are in the caption following the image — **FIGURE 1**
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Leaf water potential (Ψw), maximal quantum yield of PSII photochemistry (F_v/F_m), minimal (F₀) and maximal (F_m) fluorescence yield of the dark-adapted state in *Eucalyptus urophylla* plants sprayed with 24-epibrassinolide (EBR) and exposed to water deficit. Different uppercase letters between EBR levels (0, 50, and 100 nM EBR under equal water condition) and lowercase letters between water conditions (control and water deficit under equal EBR concentration) indicate significant differences from the Scott–Knott test (P < 0.05). Means ± sd, n = 5

3.2 Steroid increased photosynthetic efficiency and electron transport

The effective quantum yield of PSII photochemistry (Φ_PSII), photochemical quenching (q_P), and electron transport rate (ETR) were significantly reduced under water deficit. In contrast, plants under water deficit + 100 nM of EBR increased those parameters by 40, 124, and 62%, respectively, compared to the water deficit + 0 nM EBR treatment. Significant increases in nonphotochemical quenching (NPQ), relative energy excess at the PSII level (EXC), and the ratio between the ETR and net photosynthetic rate (P_N) were detected after water deficit. The application of 100 nM EBR reduced NPQ, EXC, and ETR/P_N by 30, 6, and 33%, respectively, compared to the equal treatment without EBR (Table 1).

Table 1. Chlorophyll fluorescence in Eucalyptus urophylla plants sprayed with 24-epibrassinolide (EBR) and exposed to water deficit

Water condition	EBR (nM)	Φ_PSII	q_P	NPQ	ETR (μmol m⁻² s⁻¹)	EXC (μmol m⁻² s⁻¹)	ETR/P_N
Control	0	0.79 ± 0.01Ba	0.67 ± 0.01Ca	0.36 ± 0.03Ab	80.9 ± 1.8Ba	0.30 ± 0.01Ab	5.6 ± 0.5Ab
Control	50	0.83 ± 0.01Aa	0.76 ± 0.03Ba	0.35 ± 0.03Ab	89.9 ± 2.7Aa	0.26 ± 0.02Bb	5.7 ± 0.3Ab
Control	100	0.84 ± 0.01Aa	0.88 ± 0.04Aa	0.34 ± 0.02Ab	94.8 ± 2.8Aa	0.23 ± 0.01Bb	5.3 ± 0.1Ab
Water deficit	0	0.53 ± 0.02Cb	0.25 ± 0.02Cb	0.61 ± 0.01Aa	23.2 ± 2.9Bb	0.70 ± 0.02Aa	46.4 ± 1.7Aa
Water deficit	50	0.67 ± 0.02Bb	0.40 ± 0.03Bb	0.55 ± 0.03Ba	27.3 ± 1.9Bb	0.72 ± 0.01Aa	39.0 ± 2.1Ba
Water deficit	100	0.74 ± 0.02Ab	0.56 ± 0.03Ab	0.43 ± 0.01Ca	37.5 ± 3.7Ab	0.66 ± 0.02Ba	31.2 ± 2.4Ca
Interaction effects
W × E (P-value)		0.025	<0.001	0.027	0.018	0.022	0.019

Notes: Columns with different uppercase letters between EBR levels (0, 50, and 100 nM EBR under equal water condition) and lowercase letters between water conditions (control and water deficit under equal EBR concentration) indicate significant differences from the Scott–Knott test (P < 0.05). W = water condition, E = EBR, means ± sd, n = 5.
Abbreviations: Φ_PSII, effective quantum yield of PSII photochemistry; ETR, electron transport rate; EXC, relative energy excess at the PSII level; ETR/P_N, ratio between the electron transport rate and net photosynthetic rate; NPQ, nonphotochemical quenching; q_P, photochemical quenching coefficient.

3.3 Gas exchange was maximized by EBR

Plants under water deficit suffered significant reductions in P_N, transpiration rate (E), and stomatal conductance (g_s), but the application of 100 nM EBR increased them by 140, 33, and 133%, respectively, compared to the water deficit + 0 nM EBR treatment. Water-use efficiency (WUE) and carboxylation instantaneous efficiency (P_N/intercellular CO₂ concentration [C_i]) were significantly reduced during water deficit; however, they increased by 88% and 300%, respectively, upon 100 nM EBR application compared to cultivated plants without EBR (Table 2).

Table 2. Gas exchange in Eucalyptus urophylla plants sprayed with 24-epibrassinolide (EBR) and exposed to water deficit

Water condition	EBR (nM)	P_N (μmol m⁻² s⁻¹)	E (mmol m⁻² s⁻¹)	g_s (mol m⁻² s⁻¹)	C_i (μmol mol⁻¹)	WUE (μmol mmol⁻¹)	P_N/C_i (μmol m⁻² s⁻¹ Pa⁻¹)
Control	0	14.3 ± 0.3Ca	2.5 ± 0.1Ba	0.26 ± 0.01Ca	258 ± 8Ab	5.7 ± 0.3Ba	0.055 ± 0.002Ca
Control	50	15.7 ± 0.4Ba	2.7 ± 0.1Aa	0.29 ± 0.01Ba	254 ± 7Ab	5.8 ± 0.1Ba	0.062 ± 0.003Ba
Control	100	17.7 ± 0.5Aa	2.8 ± 0.1Aa	0.34 ± 0.02Aa	244 ± 9Ab	6.3 ± 0.1Aa	0.072 ± 0.003Aa
Water deficit	0	0.5 ± 0.1Bb	0.6 ± 0.0Bb	0.03 ± 0.01Ab	358 ± 21Aa	0.8 ± 0.1Bb	0.001 ± 0.001Ab
Water deficit	50	0.7 ± 0.2Bb	0.8 ± 0.1Ab	0.04 ± 0.01Ab	344 ± 21Aa	0.9 ± 0.1Bb	0.002 ± 0.002Ab
Water deficit	100	1.2 ± 0.1Ab	0.8 ± 0.1Ab	0.07 ± 0.01Ab	334 ± 22Aa	1.5 ± 0.1Ab	0.004 ± 0.001Ab
Interaction effects
W × E (P-value)		0.034	0.041	0.029	0.047	0.038	0.026

Notes: Columns with different uppercase letters between EBR levels (0, 50, and 100 nM EBR under equal water condition) and lowercase letters between water conditions (control and water deficit under equal EBR concentration) indicate significant differences from the Scott–Knott test (P < 0.05). W = water condition, E = EBR, means ± sd, n = 5.
Abbreviations: C_i, intercellular CO₂ concentration; E, transpiration rate; g_s, stomatal conductance; P_N, net photosynthetic rate; P_N/C_i, carboxylation instantaneous efficiency; WUE, water-use efficiency.

3.4 Antioxidant metabolism improved in plants pretreated with EBR

Water deficit caused an increase in all enzyme activities evaluated (Figure 2). The application of 100 nM EBR intensified this effect, and SOD, CAT, APX, and POX activities significantly increased by 42, 52, 147, and 204%, respectively, when compared to plants cultivated under the water deficit + 0 nM EBR.

3.5 The oxidative stress was mitigated in plants exposed to water deficit

Plants exposed to water deficit had significant increases in O₂⁻, H₂O₂, MDA, and EL levels; however, the application of 100 nM EBR significantly attenuated these effects with a reduction of 33, 33, 20, and 16%, respectively, when compared to plants grown under water deficit without EBR (Figure 3).

3.6 Attenuation of the effects of water deficit on photosynthetic pigments

The water deficit promoted significant reductions in Chl a, Chl b, total chlorophyll (total Chl), and Car (Table 3). However, treatment with 100 nM EBR caused a significant increase in those pigments by 109, 212, 135, and 226%, respectively, when compared to the water deficit + 0 nM EBR treatment. In addition, the Chl a/Chl b and total Chl/Car ratios significantly increased in plants under water deficit compared with control plants under the same concentration of EBR (Table 3).

Table 3. Photosynthetic pigments in Eucalyptus urophylla plants sprayed with 24-epibrassinolide (EBR) and exposed to water deficit

Water condition	EBR (nM)	Chl a (mg g⁻¹ FM)	Chl b (mg g⁻¹ FM)	Total Chl (mg g⁻¹ FM)	Car (mg g⁻¹ FM)	Ratio Chl a/Chl b	Ratio total Chl/Car
Control	0	8.24 ± 0.27Ba	6.27 ± 0.19Ca	14.51 ± 0.79Ba	1.17 ± 0.04Ca	1.31 ± 0.11Ab	12.40 ± 1.22Ab
Control	50	8.43 ± 0.29Ba	7.08 ± 0.19Ba	15.51 ± 0.61Ba	1.31 ± 0.06Ba	1.19 ± 0.09Ab	11.84 ± 0.83Ab
Control	100	9.23 ± 0.37Aa	8.08 ± 0.28Aa	17.31 ± 1.03Aa	1.47 ± 0.05Aa	1.14 ± 0.08Ab	11.78 ± 0.85Ab
Water deficit	0	2.75 ± 0.25Bb	0.98 ± 0.09Cb	3.74 ± 0.32Cb	0.19 ± 0.01Cb	2.80 ± 0.18Aa	19.68 ± 1.02Aa
Water deficit	50	5.58 ± 0.24Ab	2.01 ± 0.20Bb	7.60 ± 0.48Bb	0.46 ± 0.04Bb	2.78 ± 0.23Aa	16.52 ± 1.31Ba
Water deficit	100	5.74 ± 0.31Ab	3.06 ± 0.17Ab	8.80 ± 0.37Ab	0.62 ± 0.03Ab	1.88 ± 0.14Ba	14.20 ± 0.41Ca
Interaction effects
W × E (P-value)		0.024	<0.001	0.010	<0.001	0.041	0.027

Notes: Columns with different uppercase letters between EBR levels (0, 50, and 100 nM EBR under equal water condition) and lowercase letters between water conditions (control and water deficit under equal EBR concentration) indicate significant differences from the Scott–Knott test (P < 0.05). W = water condition, E = EBR, means ± sd, n = 5.
Abbreviations: Car, carotenoids; Chl a, chlorophyll a; Chl b, chlorophyll b; Total chl, total chlorophyll.

3.7 EBR maximized the growth rate in control and water deficit conditions

Water deficit reduced leaf dry matter (LDM), root dry matter (RDM), stem dry matter (SDM), and total dry matter (TDM) of E. urophylla plants, while plants sprayed with both concentrations of EBR (50 and 100 nM) suffered less of the water deficit. The best effect was observed with 50 nM EBR, where LDM, RDM, SDM, and TDM increased by 15, 18, 18, and 16%, respectively, compared to plants under water deficit without EBR treatment (Figure 4).

4 DISCUSSION

Our study unraveled the roles of BRs in alleviating drought stress in young E. urophylla plants with clear benefits on redox homeostasis and photosynthetic apparatus (Figure 5). This research confirms by physiological and biochemical approaches the insights obtained mainly at molecular level by other research groups. In this context, Xia et al. (2011) evaluated the EBR properties inducing stress tolerance in plants, and they confirmed that the overexpression of CPD and DWF4 genes is connected to the biosynthetic pathway of this steroid. Li et al. (2016) found that tomato dwarf mutants modified in EBR biosynthesis presented an increased gas exchange, also ratified by our results.

The pretreatment with EBR promoted the increase in Ψw values in plants exposed to water deficit, suggesting that this steroid improved the osmotic adjustment of E. urophylla plants through the accumulation of organic solutes, such as proline and soluble sugars (Behnamnia, Kalantari, & Ziaie, 2009; Liu et al., 2014). Farooq et al. (2009) reported an increase in Ψw values after foliar treatment of two forms of BRs (HBL and EBR) in Oryza sativa plants grown under water deficit. These results are due to the improvements in leaf water economy and CO₂ assimilation promoted by the steroid. The beneficial contributions of EBR on Ψw and growth were associated with enhanced levels of soluble proteins and free proline in three cultivars of Sorghum vulgare exposed to osmotic stress with application of 2 and 3 μM of this steroid (Vardhini & Rao, 2003).

Higher values of F_v/F_m revealed that EBR increased the photochemical efficiency of the reaction center linked to PSII, attenuating the photoinhibition due to water deficit. The reductions in F₀ and increase in F_m indicated that EBR application reduced the photo-oxidative damage caused by water deficit on the structure and function of the primary PQ acceptor in the reaction center of PSII, increasing the cyclic electron flow in the photosystems (Huang et al., 2012; Lima Neto et al., 2017). Wang, Zheng, et al. (2015) evaluated chlorophyll fluorescence, leaf morphology, and cell ultrastructure and also found increases in the F_v/F_m of Vitis vinifera plants under 12 days of water deficit. Li et al. (2012) reported similar results for F₀ and F_m in Chorispora bungeana plants sprayed with 0.1 μM EBR and exposed to water deficit for 3 days: F₀ reduced by 6% and F_m increased by 7% after application of EBR.

Increases in Φ_PSII and q_P in the plants treated with EBR were associated with higher values of F_v/F_m, indicating that this steroid increased the ratio linked to the reaction center of PSII and maximized the light capture efficiency by the light-harvesting system (Figure 5). The increase in ETR is related to the reduction of PSII overexcitation, represented by the lower EXC values. It revealed that EBR increased the photosynthetic electron transport efficiency of the primary PQ acceptor preventing damage to the electron transport chain in the thylakoid membrane (Fariduddin et al., 2014; Zhang et al., 2013). Jiang et al. (2012) evaluated the CO₂ assimilation and photosynthetic electron transport in Cucumis sativus plants and reported increases in Φ_PSII and q_P after application of 0.1 μM EBR. An increase in ETR was also verified by Thussagunpanit et al. (2015) investigating the photosynthetic efficiency of O. sativa plants sprayed with 1 nM EBR.

The reduction in NPQ and EXC in E. urophylla plants by EBR indicates that this steroid improved the efficiency of light conversion, maximizing the energy proportion available to the photosynthetic electron transport after the light was absorbed by the antenna of PSII, attenuating the photoinhibitory damages imposed to PSII. EXC is intrinsically related to the energy that is neither used for electron transport or dissipated as heat (Kato et al., 2003). Additionally, the reduction in ETR/P_N was related to the increase in P_N and reduction of O₂⁻. The application of EBR induced the plants to use more photochemical energy in the fixation of CO₂ instead of other metabolic processes, such as the photoreduction of O₂ to O₂⁻ (Lima Neto et al., 2017; Silva et al., 2012). Lima and Lobato (2017) evaluated the PSII efficiency, gas exchange, antioxidant enzymes, and growth in V. unguiculata plants exposed to water deficit for 2 days and reported reductions of 30, 19, and 12% in NPQ, EXC, and ETR/P_N, respectively.

EBR spray increased P_N, which was explained by the higher electron flow between the photosystems (ETR) detected in this study, resulting in greater photosynthetic activity (Figure 5). Increases in E and g_s are corroborated by higher water content in leaf tissue (Ψw) induced by EBR, which led to the increased opening of the stomata (Xia et al., 2014; Zhang et al., 2008). Zhao et al. (2017) evaluated the effects of the application of 0.1 mg L⁻¹ of EBR on photosynthesis and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) of Triticum aestivum plants exposed to water deficit for 8 h and verified an increase in P_N. Increases in P_N (29%), E (43%), and g_s (70%) were found by Hayat et al. (2011) studying the comparative effects of two EBR homologs on the growth, carbonic anhydrase activity, and photosynthetic efficiency in L. esculentum plants.

Steroid application attenuated the effect of water deficit on WUE, and this response was related to the combined increases in P_N and E, verified in this study, because WUE is the physiological variable that expresses the amount of carbon fixed (P_N) per plant per unit of lost water (E) (Barros Junior et al., 2017). The increase in P_N/C_i in plants treated with EBR indicated that the steroid increased the assimilation of CO₂ (P_N) with concomitant reduction of the concentrations of C_i due to the increase in the activity of the enzyme RuBisCO (Lima & Lobato, 2017; Yusuf et al., 2011). Fariduddin et al. (2009) evaluated the effects of EBR in Brassica juncea plants exposed to water deficit for 7 days and verified an increase in WUE after the application of 0.01 μM EBR. Anjum et al. (2011) studied the modulation of enzymatic antioxidants and leaf gas exchange and verified an increase in P_N/C_i induced by the application of EBR in Zea mays plants after 6 days of water restriction.

The increase in enzyme activity (SOD, CAT, APX, and POX) in plants sprayed with 100 nM EBR revealed the intrinsic action of this molecule on the antioxidant metabolism. These increases in activity contributed to the reduction of the photoinhibitory damage caused to PSII (detected by lower NPQ), resulting in increases in F_v/F_m and ETR, ratified by the lower oxidative stress (MDA and EL). NPQ is a variable that measures the amount of absorbed light energy dissipated in heat (Ruban, 2016); the excess light often causes deleterious effects on the photosynthetic efficiency due to inefficient electron flux and ROS accumulation into chloroplasts that damages membranes. In other words, ROS removal by the antioxidant enzymes is essential to the adequate performance of the photosynthetic apparatus. Li et al. (2008) evaluated the effects of different concentrations of EBR on Robinia pseudoacacia seedlings exposed to three water regimes (75, 55, and 35% of the field capacity) for 10 days and detected peaks in SOD, CAT, and APX activities upon treatment with 0.2 mg L⁻¹ EBR. Zhang et al. (2008) studied the adverse effects of water deficits on photosynthesis and the antioxidant metabolism of Glycine max after application of 0.1 mg L⁻¹ of EBR in plants exposed to 7 days of water deficit and found increases of 29 and 30% for POX and SOD, respectively.

Decreases in O₂⁻ and H₂O₂ levels in plants sprayed with 100 nM EBR are related to the increases in the activities of SOD, CAT, APX, and POX enzymes promoted by the steroid (Figures 2 and 5). The lower level of O₂⁻ is associated with the increased activity of the SOD enzyme, which is responsible for converting O₂⁻ into H₂O₂ and O₂ (Gill & Tuteja, 2010; Reyes Guerrero et al., 2015), while the reduction in H₂O₂ concentrations is linked to APX activity. This enzyme is responsible for dismutation of H₂O₂ into H₂O and O₂, contributing to cell detoxification and reducing oxidative stress (El-Beltagi & Mohamed, 2013; Gratão et al., 2005). Reduction in O₂⁻ level induced by the application of EBR was observed by Lima and Lobato (2017) with V. unguiculata plants exposed to water deficit for 2 days. Behnamnia, Kalantari, and Rezanejad (2009) evaluated the mitigation effects of exogenous EBR application on drought-induced oxidative stress in L. esculentum plants and reported significant reductions in H₂O₂ levels after the application of two concentrations of EBR (0.01 and 1 μM) in plants under water restriction for 3 and 5 days.

The reductions in MDA and EL levels in E. urophylla plants after treatment with EBR were directly connected to the lower accumulation of O₂⁻ and H₂O₂, influenced by the action of the antioxidant enzymes. O₂⁻ and H₂O₂ are highly reactive and toxic biomolecules that, in high concentrations, cause the structural and functional deterioration of membranes leading to lipid peroxidation in response to water deficit (Siddiqui et al., 2015; Yuan et al., 2010). In addition, EBR improves membrane structure and stability, decreasing lipid peroxidation (Rady, 2011). Li and Feng (2011) verified the effects of four concentrations of EBR (0.1, 0.2, 0.3, and 0.4 mg L⁻¹) on Xanthoceras sorbifolia plants exposed to mild (12–13% soil moisture) and severe (7–8% soil moisture) water deficit and reported reductions in MDA and EL levels promoted by EBR. Zhu et al. (2014) confirmed the protective role triggered by BRs in Salvia miltiorrhiza plants under drought stress due to the synergistic effects of a more performant antioxidant system and membrane stability.

Increases in the Chl a, Chl b, total Chl, and Car levels of E. urophylla plants sprayed with EBR revealed a lower accumulation of ROS (O₂⁻ and H₂O₂), reducing the oxidative damages, benefiting the chloroplast membranes (Figure 5). In parallel, Ali et al. (2020) described that BRs protect plant cells against environmental stresses, including drought. These results were confirmed by the reductions in the MDA and EL levels previously described in this study, combined with increases in the biosynthesis of these pigments (Hayat et al., 2010). The lowest Chl a/Chl b and total Chl/Car ratios found after the application of EBR were related to the beneficial effects on Chl b and Car. Our results are similar to what observed in H. vulgare plants sprayed with three concentrations of EBR (0.01, 0.1, and 1 μM) and exposed to water deficit (PEG 6000 at 8%) for 15 days where Chl a, Chl b, total Chl, and Car increased 30, 59, 18, and 54%, respectively (Gill et al., 2017).

Increments in LDM, RDM, SDM, and TDM after the exogenous application of EBR suggested that this steroid probably accelerated cell division and expansion in leaves, stems, and roots, increasing the biomass. In agreement with Ahmad et al. (2018), this molecule plays a crucial role connected to growth and development in higher plants. In addition, EBR increased the growth based on the higher P_N and enzymatic activities (SOD, CAT, APX, and GPX) previously verified in this study (Khalid & Aftab, 2016; Shahbaz et al., 2008). Talaat et al. (2015) studied the effects of dual application of 0.1 mg L⁻¹ of EBR and 25 mg L⁻¹ of spermine in two Z. mays hybrids under water deficit (50 and 75% of field capacity) for 30 days and found increases in RDM in both treatments (only EBR and EBR + spermine) related to the elevation of antioxidant enzyme activities and the improvement in the redox state of ascorbate and glutathione. Gomes et al. (2013) evaluated the effects of the analogue of BRs (Biobras 16) in two genotypes of Carica papaya plants exposed to 15 days of water restriction. They reported an increase in shoot biomass (leaf + stem) of the UC 01 genotype and suggested that BR improves the flux of photoassimilates and other compounds to the youngest leaves.

Based on our results and the ones from the literature, we propose an action mechanism triggered by the EBR in E. urophylla plants under drought stress (Figure 5). Pretreatment with this molecule clearly induced direct and indirect responses on chloroplasts, modulating positively antioxidant enzymes (SOD, CAT, APX, and POX), and consequently protecting the membranes against the oxidative stress generated by ROS as measured by the reductions in EL, MDA, O₂⁻, and H₂O₂. Indirectly, this protective role alleviated the damages on chloroplastic pigments, improving the performance of the photosynthetic apparatus, more specifically the PSII efficiency (Φ_PSII), electron flux (ETR), and CO₂ fixation (P_N).

5 CONCLUSIONS

This research confirmed the negative effects induced by water deficit on photosynthetic machinery, more specifically on absorption and utilization of light. Our results confirmed that the water deficit tolerance triggered by EBR in E. urophylla plants was associated with the enhancement on photosynthetic pigments, PSII efficiency, electron flux, and CO₂ fixation, being explained by the increments detected in all antioxidant enzymes evaluated, contributing to minor accumulation of oxidative compounds. Our results revealed that 100 nM EBR gives the best results.

ACKNOWLEDGMENTS

This research had financial support from Fundação Amazônia de Amparo a Estudos e Pesquisas (FAPESPA/Brazil), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/Brazil), and Universidade Federal Rural da Amazônia (UFRA/Brazil) to Allan K. da Silva Lobato. Udson de Oliveira Barros Junior and Michael D. R. Lima were supported by scholarships from Universidade Federal Rural da Amazônia (UFRA/Brazil). The authors would like to extend their sincere appreciation to the Researches Supporting Project Number (RSP-2020/236) from King Saud University, Riyadi, Saudi Arabia.

AUTHOR CONTRIBUTIONS

Allan K. da Silva Lobato was the advisor of this project, planning all phases of the research, and critically revised the manuscript. Udson de Oliveira Barros Junior and Michael D. R. Lima conducted the experiment and performed the physiological, biochemical and morphological determinations, as well as wrote and edited the manuscript. Abdulaziz A. Alsahli critically revised and edited the manuscript. All authors read and approved the final version of the manuscript.

Open Research

DATA AVAILABILITY STATEMENT

Data are available upon request from the corresponding author.

REFERENCES

Ahammed, G.J., Choudhary, S.P., Chen, S., Xia, X., Shi, K., Zhou, Y., et al. (2013) Role of brassinosteroids in alleviation of phenanthrene–cadmium co-contamination-induced photosynthetic inhibition and oxidative stress in tomato. Journal of Experimental Botany, 64, 199–213.
10.1093/jxb/ers323
CAS PubMed Web of Science® Google Scholar
Ahmad, F., Singh, A. & Kamal, A. (2018) Crosstalk of brassinosteroids with other phytohormones under various abiotic stresses. Journal of Applied Biology & Biotechnology, 6, 56–62.
CAS Google Scholar
Ali, B., Hayat, S., Fariduddin, Q. & Ahmad, A. (2008) 24-Epibrassinolide protects against the stress generated by salinity and nickel in Brassica juncea. Chemosphere, 72, 1387–1392.
10.1016/j.chemosphere.2008.04.012
CAS PubMed Web of Science® Google Scholar
Ali, Q., Shahid, S., Nazar, N., Hussain, A.I., Ali, S., Chatha, S.A.S., et al. (2020) Use of phytohormones in conferring tolerance to environmental stress. In: Plant ecophysiology and adaptation under climate change: mechanisms and perspectives II. Singapore: Springer Singapore, pp. 245–355.
Google Scholar
Amzallag, G.N. & Vaisman, J. (2006) Influence of brassinosteroids on initiation of the root gravitropic response in Pisum sativum seedlings. Biologia Plantarum, 50, 283–286.
10.1007/s10535-006-0021-5
CAS Web of Science® Google Scholar
Anjum, S.A., Wang, L.C., Farooq, M., Hussain, M., Xue, L.L. & Zou, C.M. (2011) Brassinolide application improves the drought tolerance in maize through modulation of enzymatic antioxidants and leaf gas exchange. Journal of Agronomy and Crop Science, 197, 177–185.
10.1111/j.1439-037X.2010.00459.x
CAS Web of Science® Google Scholar
Badawi, G.H., Yamauchi, Y., Shimada, E., Sasaki, R., Kawano, N., Tanaka, K., et al. (2004) Enhanced tolerance to salt stress and water deficit by overexpressing superoxide dismutase in tobacco (Nicotiana tabacum) chloroplasts. Plant Science, 166, 919–928.
10.1016/j.plantsci.2003.12.007
CAS Web of Science® Google Scholar
Bajguz, A. & Hayat, S. (2009) Effects of brassinosteroids on the plant responses to environmental stresses. Plant Physiology and Biochemistry, 47, 1–8.
10.1016/j.plaphy.2008.10.002
CAS PubMed Web of Science® Google Scholar
Bajguz, A. & Tretyn, A. (2003) The chemical characterisitic and distribution of brassinosteroids in plants. Phytochemistry, 62, 1027–1046.
10.1016/S0031-9422(02)00656-8
CAS PubMed Web of Science® Google Scholar
Baker, N.R. (2008) Chlorophyll fluorescence: a probe of photosynthesis in vivo. Annual Review of Plant Biology, 59, 89–113.
10.1146/annurev.arplant.59.032607.092759
CAS PubMed Web of Science® Google Scholar
Barros Junior, U.O., Barbosa, M.A.M., Lima, M.D.R., Viana, G.D.M. & Lobato, A.K.S. (2017) Short-time of rehydration is not effective to re-establish chlorophyll fluorescence and gas exchange in two cowpea cultivars submitted to water deficit. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 45, 238–244.
10.15835/nbha45110569
Web of Science® Google Scholar
Behnamnia, M., Kalantari, K.M. & Rezanejad, F. (2009) Exogenous application of brassinosteroid alleviates drought-induced oxidative stress in Lycopersicon esculentum L. General and Applied Plant Physiology, 35, 22–34.
CAS Google Scholar
Behnamnia, M., Kalantari, K.M. & Ziaie, J. (2009) The effects of brassinosteroid on the induction of biochemical changes in Lycopersicon esculentum under drought stress. Turkish Journal of Botany, 33, 417–428.
Web of Science® Google Scholar
Belyaeva, N.E., Bulychev, A.A., Riznichenko, G.Y. & Rubin, A.B. (2016) Thylakoid membrane model of the Chl a fluorescence transient and P700 induction kinetics in plant leaves. Photosynthesis Research, 130, 491–515.
10.1007/s11120-016-0289-z
CAS PubMed Web of Science® Google Scholar
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.
10.1016/0003-2697(76)90527-3
CAS PubMed Web of Science® Google Scholar
Cakmak, I. & Horst, W.J. (1991) Effect of aluminium on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (Glycine max). Physiologia Plantarum, 83, 463–468.
10.1111/j.1399-3054.1991.tb00121.x
CAS Web of Science® Google Scholar
Cakmak, I. & Marschner, H. (1992) Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves. Plant Physiology, 98, 1222–1227.
10.1104/pp.98.4.1222
CAS PubMed Web of Science® Google Scholar
Caser, M., Lovisolo, C. & Scariot, V. (2017) The influence of water stress on growth, ecophysiology and ornamental quality of potted Primula vulgaris “Heidy” plants. New insights to increase water use efficiency in plant production. Plant Growth Regulation, 83, 361–373.
10.1007/s10725-017-0301-4
CAS Web of Science® Google Scholar
Cherepanov, D.A., Milanovsky, G.E., Petrova, A.A., Tikhonov, A.N. & Semenov, A.Y. (2017) Electron transfer through the acceptor side of photosystem I: interaction with exogenous acceptors and molecular oxygen. The Biochemist, 82, 1249–1268.
CAS Google Scholar
Cui, L., Zou, Z., Zhang, J., Zhao, Y. & Yan, F. (2016) 24-Epibrassinoslide enhances plant tolerance to stress from low temperatures and poor light intensities in tomato (Lycopersicon esculentum Mill.). Functional & Integrative Genomics, 16, 29–35.
10.1007/s10142-015-0464-x
CAS PubMed Web of Science® Google Scholar
Divi, U.K. & Krishna, P. (2009) Brassinosteroid: a biotechnological target for enhancing crop yield and stress tolerance. New Biotechnology, 26, 131–136.
10.1016/j.nbt.2009.07.006
CAS PubMed Web of Science® Google Scholar
El-Beltagi, H.S. & Mohamed, H.I. (2013) Reactive oxygen species, lipid peroxidation and antioxidative defense mechanism. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 41, 44–57.
10.15835/nbha4118929
CAS Web of Science® Google Scholar
Elstner, E.F. & Heupel, A. (1976) Inhibition of nitrite formation from hydroxylammoniumchloride: a simple assay for superoxide dismutase. Analytical Biochemistry, 70, 616–620.
10.1016/0003-2697(76)90488-7
CAS PubMed Web of Science® Google Scholar
Fariduddin, Q., Khanam, S., Hasan, S.A., Ali, B., Hayat, S. & Ahmad, A. (2009) Effect of 28-homobrassinolide on the drought stress-induced changes in photosynthesis and antioxidant system of Brassica juncea L. Acta Physiologiae Plantarum, 31, 889–897.
10.1007/s11738-009-0302-7
CAS Web of Science® Google Scholar
Fariduddin, Q., Yusuf, M., Ahmad, I. & Ahmad, A. (2014) Brassinosteroids and their role in response of plants to abiotic stresses. Biologia Plantarum, 58, 9–17.
10.1007/s10535-013-0374-5
CAS Web of Science® Google Scholar
Farooq, M., Wahid, A., Basra, S.M.A. & Din, I. (2009) Improving water relations and gas exchange with brassinosteroids in rice under drought stress. Journal of Agronomy and Crop Science, 195, 262–269.
10.1111/j.1439-037X.2009.00368.x
CAS Web of Science® Google Scholar
Fonseca, S.S., Silva, B.R.S. & Lobato, A.K.S. (2020) 24-Epibrassinolide positively modulate leaf structures, antioxidant system and photosynthetic machinery in rice under simulated acid rain. Journal of Plant Growth Regulation, 39(4), 1559–1576. https://doi.org/10.1007/s00344-020-10167-4
10.1007/s00344-020-10167-4
Web of Science® Google Scholar
Giannopolitis, C.N. & Ries, S.K. (1977) Superoxide dismutases: I. Occurrence in higher plants. Plant Physiology, 59, 309–314.
10.1104/pp.59.2.309
CAS PubMed Web of Science® Google Scholar
Gill, S.S. & Tuteja, N. (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry, 48, 909–930.
10.1016/j.plaphy.2010.08.016
CAS PubMed Web of Science® Google Scholar
Gill, M.B., Cai, K., Zhang, G. & Zeng, F. (2017) Brassinolide alleviates the drought-induced adverse effects in barley by modulation of enzymatic antioxidants and ultrastructure. Plant Growth Regulation, 82, 447–455.
10.1007/s10725-017-0271-6
CAS Web of Science® Google Scholar
Gomes, M.M.A., Netto, A.T., Campostrini, E., Bressan-smith, R., Zullo, M.A.T., Ferraz, T.M., et al. (2013) Brassinosteroid analogue affects the senescence in two papaya genotypes submitted to drought stress. Theoretical and Experimental Plant Physiology, 25, 186–195.
Web of Science® Google Scholar
Gong, M., Li, Y.-J. & Chen, S.-Z. (1998) Abscisic acid-induced thermotolerance in maize seedlings is mediated by calcium and associated with antioxidant systems. Journal of Plant Physiology, 153, 488–496.
10.1016/S0176-1617(98)80179-X
CAS Web of Science® Google Scholar
Gratão, P.L., Polle, A., Lea, P.J. & Azevedo, R.A. (2005) Making the life of heavy metal-stressed plants a little easier. Functional Plant Biology, 32, 481–494.
10.1071/FP05016
CAS Web of Science® Google Scholar
Havir, E.A. & McHale, N.A. (1987) Biochemical and developmental characterization of multiple forms of catalase in tobacco leaves. Plant Physiology, 84, 450–455.
10.1104/pp.84.2.450
CAS PubMed Web of Science® Google Scholar
Hayat, S., Hasan, S.A., Hayat, Q. & Ahmad, A. (2010) Brassinosteroids protect Lycopersicon esculentum from cadmium toxicity applied as shotgun approach. Protoplasma, 239, 3–14.
10.1007/s00709-009-0075-2
CAS PubMed Web of Science® Google Scholar
Hayat, S., Yadav, S., Wani, A.S., Irfan, M. & Ahmad, A. (2011) Comparative effect of 28-homobrassinolide and 24-epibrassinolide on the growth, carbonic anhydrase activity and photosynthetic efficiency of Lycopersicon esculentum. Photosynthetica, 49, 397–404.
10.1007/s11099-011-0051-x
CAS Web of Science® Google Scholar
Huang, W., Yang, S.J., Zhang, S.B., Zhang, J.L. & Cao, K.F. (2012) Cyclic electron flow plays an important role in photoprotection for the resurrection plant Paraboea rufescens under drought stress. Planta, 235, 819–828.
10.1007/s00425-011-1544-3
CAS PubMed Web of Science® Google Scholar
IBA. (2017) Indústria Brasileira de Arvores (IBA). Report 2017: Base year 2016. Brasilia. https://iba.org/.
Google Scholar
Jangid, K.K. & Dwivedi, P. (2017) Physiological and biochemical changes by nitric oxide and brassinosteroid in tomato (Lycopersicon esculentum Mill.) under drought stress. Acta Physiologiae Plantarum, 39(3), 73.
10.1007/s11738-017-2373-1
Web of Science® Google Scholar
Jiang, Y.P., Cheng, F., Zhou, Y.H., Xia, X.J., Shi, K. & Yu, J.Q. (2012) Interactive effects of CO2 enrichment and brassinosteroid on CO2 assimilation and photosynthetic electron transport in Cucumis sativus. Environmental and Experimental Botany, 75, 98–106.
10.1016/j.envexpbot.2011.09.002
CAS Web of Science® Google Scholar
Ju, Y., Yue, X., Zhao, X., Zhao, H. & Fang, Y. (2018) Physiological, micro-morphological and metabolomic analysis of grapevine (Vitis vinifera L.) leaf of plants under water stress. Plant Physiology and Biochemistry, 130, 501–510.
10.1016/j.plaphy.2018.07.036
CAS PubMed Web of Science® Google Scholar
Karlidag, H., Yildirim, E. & Turan, M. (2011) Role of 24-epibrassinolide in mitigating the adverse effects of salt stress on stomatal conductance, membrane permeability, and leaf water content, ionic composition in salt stressed strawberry (Fragaria×ananassa). Scientia Horticulturae (Amsterdam), 130, 133–140.
10.1016/j.scienta.2011.06.025
CAS Web of Science® Google Scholar
Kato, M.C., Hikosaka, K., Hirotsu, N., Makino, A. & Hirose, T. (2003) The excess light energy that is neither utilized in photosynthesis nor dissipated by photoprotective mechanisms determines the rate of photoinactivation in photosystem II. Plant & Cell Physiology, 44, 318–325.
10.1093/pcp/pcg045
CAS PubMed Web of Science® Google Scholar
Khalid, A. & Aftab, F. (2016) Effect of exogenous application of 24-epibrassinolide on growth, protein contents, and antioxidant enzyme activities of in vitro-grown Solanum tuberosum L. under salt stress. In Vitro Cellular & Developmental Biology—Plant, 52, 81–91.
10.1007/s11627-015-9745-2
CAS Web of Science® Google Scholar
Kume, A., Akitsu, T. & Nasahara, K.N. (2018) Why is chlorophyll b only used in light-harvesting systems? Journal of Plant Research, 131, 1–12. https://doi.org/10.1007/s10265-018-1052-7.
10.1007/s10265-018-1052-7
Web of Science® Google Scholar
Lang, Y., Wang, M., Xia, J. & Zhao, Q. (2018) Effects of soil drought stress on photosynthetic gas exchange traits and chlorophyll fluorescence in Forsythia suspensa. Journal of Forest Research, 29, 45–53.
10.1007/s11676-017-0420-9
CAS Web of Science® Google Scholar
Li, K.R. & Feng, C.H. (2011) Effects of brassinolide on drought resistance of Xanthoceras sorbifolia seedlings under water stress. Acta Physiologiae Plantarum, 33, 1293–1300. https://doi.org/10.1007/s11738-010-0661-0.
10.1007/s11738-010-0661-0
CAS Web of Science® Google Scholar
Li, K.R., Wang, H.H., Han, G., Wang, Q.J. & Fan, J. (2008) Effects of brassinolide on the survival, growth and drought resistance of Robinia pseudoacacia seedlings under water-stress. New Forest, 35, 255–266.
10.1007/s11056-007-9075-2
Web of Science® Google Scholar
Li, Y.H., Liu, Y.J., Xu, X.L., Jin, M., An, L.Z. & Zhang, H. (2012) Effect of 24-epibrassinolide on drought stress-induced changes in Chorispora bungeana. Biologia Plantarum, 56, 192–196.
10.1007/s10535-012-0041-2
CAS Web of Science® Google Scholar
Li, X., Guo, X., Zhou, Y., Shi, K., Zhou, J., Yu, J., et al. (2016) Overexpression of a brassinosteroid biosynthetic gene dwarf enhances photosynthetic capacity through activation of Calvin cycle enzymes in tomato. BMC Plant Biology, 16(33), 33.
10.1186/s12870-016-0715-6
PubMed Web of Science® Google Scholar
Lichtenthaler, H.K. & Buschmann, C. (2001) Chlorophylls and carotenoids: measurement and characterization by UV-VIS spectroscopy. In: Current protocols in food analytical chemistry. Hoboken, NJ: Wiley, pp. 431–438.
Google Scholar
Lima, J.V. & Lobato, A.K.S. (2017) Brassinosteroids improve photosystem II efficiency, gas exchange, antioxidant enzymes and growth of cowpea plants exposed to water deficit. Physiology and Molecular Biology of Plants, 23, 59–72.
10.1007/s12298-016-0410-y
CAS PubMed Web of Science® Google Scholar
Lima Neto, M.C., Cerqueira, J.V.A., da Cunha, J.R., Ribeiro, R.V. & Silveira, J.A.G. (2017) Cyclic electron flow, NPQ and photorespiration are crucial for the establishment of young plants of Ricinus communis and Jatropha curcas exposed to drought. Plant Biology, 19, 650–659.
10.1111/plb.12573
CAS PubMed Web of Science® Google Scholar
Liu, J., Gao, H., Wang, X., Zheng, Q., Wang, C., Wang, X., et al. (2014) Effects of 24-epibrassinolide on plant growth, osmotic regulation and ion homeostasis of salt-stressed canola. Plant Biology, 16, 440–450.
10.1111/plb.12052
CAS PubMed Web of Science® Google Scholar
Liu, J., Hu, T., Feng, P., Liu, J., Hu, T., Feng, P., et al. (2019) Tomato yield and water use efficiency change with various soil moisture and potassium levels during different growth stages. PLoS One, 14, 1–14.
Web of Science® Google Scholar
Lobato, S.M.S., Santos, L.R., Silva, B.R.S., Paniz, F.P., Batista, B.L. & Lobato, A.K.S. (2020) Root-differential modulation enhances nutritional status and leaf anatomy in pigeonpea plants under water deficit. Flora, 262, 151519.
10.1016/j.flora.2019.151519
Web of Science® Google Scholar
Ma, Y.H. & Guo, S.R. (2014) 24-epibrassinolide improves cucumber photosynthesis under hypoxia by increasing CO2 assimilation and photosystem II efficiency. Photosynthetica, 52, 96–104.
10.1007/s11099-014-0010-4
CAS Web of Science® Google Scholar
Mora, F. & Serra, N. (2014) Bayesian estimation of genetic parameters for growth, stem straightness, and survival in Eucalyptus globulus on an Andean Foothill site. Tree Genetics & Genomes, 10, 711–719.
10.1007/s11295-014-0716-2
Web of Science® Google Scholar
Mora, F., Gleadow, R., Perret, S. & Scapim, C.A. (2009) Genetic variation for early flowering, survival and growth in sugar gum (Eucalyptus cladocalyx F. Muell) in southern Atacama Desert. Euphytica, 169, 335–344.
10.1007/s10681-009-9962-z
Web of Science® Google Scholar
Mora, F., Arriagada, O., Ballesta, P. & Ruiz, E. (2017) Genetic diversity and population structure of a drought-tolerant species of Eucalyptus, using microsatellite markers. Journal of Plant Biochemistry and Biotechnology, 26, 274–281.
10.1007/s13562-016-0389-z
CAS Web of Science® Google Scholar
Nakano, Y. & Asada, K. (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant & Cell Physiology, 22, 867–880.
10.1093/oxfordjournals.pcp.a076232
CAS Web of Science® Google Scholar
Nazir, F., Fariduddin, Q., Hussain, A. & Khan, T.A. (2021) Brassinosteroid and hydrogen peroxide improve photosynthetic machinery, stomatal movement, root morphology and cell viability and reduce Cu-triggered oxidative burst in tomato. Ecotoxicology and Environmental Safety, 207, 111081.
10.1016/j.ecoenv.2020.111081
CAS PubMed Web of Science® Google Scholar
Oklestkova, J., Rárová, L., Kvasnica, M. & Strnad, M. (2015) Brassinosteroids: synthesis and biological activities. Phytochemistry Reviews, 14, 1053–1072.
10.1007/s11101-015-9446-9
CAS Web of Science® Google Scholar
Oliveira, V.P., Lima, M.D.R., Silva, B.R.S., Batista, B.L. & Lobato, A.K.S. (2019) Brassinosteroids confer tolerance to salt stress in Eucalyptus urophylla plants enhancing homeostasis, antioxidant metabolism and leaf anatomy. Journal of Plant Growth Regulation, 38, 557–573.
10.1007/s00344-018-9870-3
Web of Science® Google Scholar
Pereira, T.S., Lima, M.D.R., Paula, L.S. & Lobato, A.K.S. (2016) Tolerance to water deficit in cowpea populations resulting from breeding program: detection by gas exchange and chlorophyll fluorescence. Indian Journal of Plant Physiology, 21, 171–178.
10.1007/s40502-016-0218-3
Google Scholar
Planas-Riverola, A., Gupta, A., Betegón-Putze, I., Bosch, N., Ibañes, M. & Caño-Delgado, A.I. (2019) Brassinosteroid signaling in plant development and adaptation to stress. Development, 146, 1–11.
10.1242/dev.151894
Web of Science® Google Scholar
Rady, M.M. (2011) Effect of 24-epibrassinolide on growth, yield, antioxidant system and cadmium content of bean (Phaseolus vulgaris L.) plants under salinity and cadmium stress. Scientia Horticulturae (Amsterdam), 129, 232–237.
10.1016/j.scienta.2011.03.035
CAS Web of Science® Google Scholar
Ramakrishna, B. & Rao, S.S.R. (2015) Foliar application of brassinosteroids alleviates adverse effects of zinc toxicity in radish (Raphanus sativus L.) plants. Protoplasma, 252, 665–677.
10.1007/s00709-014-0714-0
CAS PubMed Web of Science® Google Scholar
Reyes Guerrero, Y., Martínez González, L., Dell'Amico, J., Núñez, M. & Pieters, A.J. (2015) Reversion of deleterious effects of salt stress by activation of ROS detoxifying enzymes via foliar application of 24-epibrassinolide in rice seedlings. Theoretical and Experimental Plant Physiology, 27, 31–40.
10.1007/s40626-014-0029-8
CAS Web of Science® Google Scholar
Rivas, R., Falcão, H.M., Ribeiro, R.V., Machado, E.C., Pimentel, C. & Santos, M.G. (2016) Drought tolerance in cowpea species is driven by less sensitivity of leaf gas exchange to water deficit and rapid recovery of photosynthesis after rehydration. South African Journal of Botany, 103, 101–107.
10.1016/j.sajb.2015.08.008
CAS Web of Science® Google Scholar
Ruban, A.V. (2016) Nonphotochemical chlorophyll fluorescence quenching: mechanism and effectiveness in protecting plants from photodamage. Plant Physiology, 170, 1903–1916.
10.1104/pp.15.01935
CAS PubMed Web of Science® Google Scholar
Santos, C.M., Silva, M.A., Lima, G.P.P., Bortolheiro, F.P.A.P., Brunelli, M.C., Holanda, L.A., et al. (2015) Physiological changes associated with antioxidant enzymes in response to sugarcane tolerance to water deficit and rehydration. Sugar Tech, 17, 291–304.
10.1007/s12355-014-0325-2
Web of Science® Google Scholar
Shahbaz, M., Ashraf, M. & Athar, H.U.R. (2008) Does exogenous application of 24-epibrassinolide ameliorate salt induced growth inhibition in wheat (Triticum aestivum L.)? Plant Growth Regulation, 55, 51–64.
10.1007/s10725-008-9262-y
CAS Web of Science® Google Scholar
Shahid, M.A., Balal, R.M., Pervez, M.A., Garcia-Sanchez, F., Gimeno, V., Abbas, T., et al. (2014) Treatment with 24-epibrassinolide mitigates NaCl-induced toxicity by enhancing carbohydrate metabolism, osmolyte accumulation, and antioxidant activity in Pisum sativum. Turkish Journal of Botany, 38, 511–525.
10.3906/bot-1304-45
CAS Web of Science® Google Scholar
Sheikh-Mohamadi, M.H., Etemadi, N., Nikbakht, A., Arab, M., Majidi, M.M. & Pessarakli, M. (2017) Antioxidant defence system and physiological responses of Iranian crested wheatgrass (Agropyron cristatum L.) to drought and salinity stress. Acta Physiologiae Plantarum, 39, 1–16.
10.1007/s11738-017-2543-1
CAS Web of Science® Google Scholar
Siddiqui, M.H., Al-Khaishany, M.Y., Al-Qutami, M.A., Al-Whaibi, M.H., Grover, A., Ali, H.M., et al. (2015) Response of different genotypes of faba bean plant to drought stress. International Journal of Molecular Sciences, 16, 10214–10227.
10.3390/ijms160510214
CAS PubMed Web of Science® Google Scholar
Silva, E.N., Ribeiro, R.V., Ferreira-Silva, S.L., Vieira, S.A., Ponte, L.F.A. & Silveira, J.A.G. (2012) Coordinate changes in photosynthesis, sugar accumulation and antioxidative enzymes improve the performance of Jatropha curcas plants under drought stress. Biomass and Bioenergy, 45, 270–279.
10.1016/j.biombioe.2012.06.009
CAS Web of Science® Google Scholar
Silva, E.N., Silveira, J.A.G., Ribeiro, R.V. & Vieira, S.A. (2015) Photoprotective function of energy dissipation by thermal processes and photorespiratory mechanisms in Jatropha curcas plants during different intensities of drought and after recovery. Environmental and Experimental Botany, 110, 36–45.
10.1016/j.envexpbot.2014.09.008
Web of Science® Google Scholar
Singh, S.K. & Reddy, K.R. (2011) Regulation of photosynthesis, fluorescence, stomatal conductance and water-use efficiency of cowpea (Vigna unguiculata [L.] Walp.) under drought. Journal of Photochemistry and Photobiology B: Biology, 105, 40–50.
10.1016/j.jphotobiol.2011.07.001
CAS PubMed Web of Science® Google Scholar
Staehelin, L.A. (2003) Chloroplast structure: from chlorophyll granules to supra-molecular architecture of thylakoid membranes. Photosynthesis Research, 76, 185–196.
10.1023/A:1024994525586
CAS PubMed Web of Science® Google Scholar
Steel, R.G., Torrie, J.H. & Dickey, D.A. (2006) Principles and procedures of statistics: a biometrical approach, 3rd edition. Moorpark: Academic Internet Publishers.
Google Scholar
Talaat, N.B. (2020) 24-Epibrassinolide and spermine combined treatment sustains maize (Zea mays L.) drought tolerance by improving photosynthetic efficiency and altering phytohormones profile. Journal of Soil Science and Plant Nutrition, 20, 516–529.
10.1007/s42729-019-00138-4
CAS Web of Science® Google Scholar
Talaat, N.B., Shawky, B.T. & Ibrahim, A.S. (2015) Alleviation of drought-induced oxidative stress in maize (Zea mays L.) plants by dual application of 24-epibrassinolide and spermine. Environmental and Experimental Botany, 113, 47–58.
10.1016/j.envexpbot.2015.01.006
CAS Web of Science® Google Scholar
Thussagunpanit, J., Jutamanee, K., Sonjaroon, W., Kaveeta, L., Chai-Arree, W., Pankean, P., et al. (2015) Effects of brassinosteroid and brassinosteroid mimic on photosynthetic efficiency and rice yield under heat stress. Photosynthetica, 53, 312–320.
10.1007/s11099-015-0106-5
CAS Web of Science® Google Scholar
Vardhini, B.V. & Rao, S.S.R. (2003) Amelioration of osmotic stress by brassinosteroids on seed germination and seedling growth of three varieties of sorghum. Plant Growth Regulation, 41, 25–31.
10.1023/A:1027303518467
CAS Web of Science® Google Scholar
Velikova, V., Yordanov, I. & Edreva, A. (2000) Oxidative stress and some antioxidant systems in acid rain-treated bean plants protective role of exogenous polyamines. Plant Science, 151, 59–66.
10.1016/S0168-9452(99)00197-1
CAS Web of Science® Google Scholar
Wang, B., Li, G. & Zhang, W.H. (2015) Brassinosteroids are involved in Fe homeostasis in rice (Oryza sativa L.). Journal of Experimental Botany, 66, 2749–2761.
10.1093/jxb/erv079
CAS PubMed Web of Science® Google Scholar
Wang, Z., Zheng, P., Meng, J. & Xi, Z. (2015) Effect of exogenous 24-epibrassinolide on chlorophyll fluorescence, leaf surface morphology and cellular ultrastructure of grape seedlings (Vitis vinifera L.) under water stress. Acta Physiologiae Plantarum, 37, 1–12.
10.1007/s11738-014-1746-y
CAS Web of Science® Google Scholar
Wu, Q.-S., Xia, R.-X. & Zou, Y.-N. (2006) Reactive oxygen metabolism in mycorrhizal and non-mycorrhizal citrus (Poncirus trifoliata) seedlings subjected to water stress. Journal of Plant Physiology, 163, 1101–1110.
10.1016/j.jplph.2005.09.001
CAS PubMed Web of Science® Google Scholar
Wu, X.X., He, J., Zhu, Z.W., Yang, S.J. & Zha, D.S. (2014) Protection of photosynthesis and antioxidative system by 24-epibrassinolide in Solanum melongena under cold stress. Biologia Plantarum, 58, 185–188.
10.1007/s10535-013-0377-2
CAS Web of Science® Google Scholar
Wyka, T.P., Bagniewska-Zadworna, A., Kuczyńska, A., Mikołajczak, K., Ogrodowicz, P., Żytkowiak, M., et al. (2019) Drought-induced anatomical modifications of barley (Hordeum vulgare L.) leaves: an allometric perspective. Environmental and Experimental Botany, 166, 103798.
10.1016/j.envexpbot.2019.103798
Web of Science® Google Scholar
Xia, X.-J., Zhou, Y.-H., Ding, J., Shi, K., Asami, T., Chen, Z., et al. (2011) Induction of systemic stress tolerance by brassinosteroid in Cucumis sativus. The New Phytologist, 191, 706–720.
10.1111/j.1469-8137.2011.03745.x
CAS PubMed Web of Science® Google Scholar
Xia, X.-J., Gao, C.-J., Song, L.-X., Zhou, Y.-H., Shi, K. & Yu, J.-Q. (2014) Role of H2O2 dynamics in brassinosteroid-induced stomatal closure and opening in Solanum lycopersicum. Plant, Cell & Environment, 37, 2036–2050.
10.1111/pce.12275
CAS PubMed Web of Science® Google Scholar
Yuan, G.F., Jia, C.G., Li, Z., Sun, B., Zhang, L.P., Liu, N., et al. (2010) Effect of brassinosteroids on drought resistance and abscisic acid concentration in tomato under water stress. Scientia Horticulturae (Amsterdam), 126, 103–108.
10.1016/j.scienta.2010.06.014
CAS Web of Science® Google Scholar
Yuan, L., Shu, S., Sun, J., Guo, S. & Tezuka, T. (2012) Effects of 24-epibrassinolide on the photosynthetic characteristics, antioxidant system, and chloroplast ultrastructure in Cucumis sativus L. under Ca(NO3)2stress. Photosynthesis Research, 112, 205–214.
10.1007/s11120-012-9774-1
CAS PubMed Web of Science® Google Scholar
Yusuf, M., Fariduddin, Q., Hayat, S., Hasan, S.A. & Ahmad, A. (2011) Protective response of 28-homobrassinolide in cultivars of Triticum aestivum with different levels of nickel. Archives of Environmental Contamination and Toxicology, 60, 68–76.
10.1007/s00244-010-9535-0
CAS PubMed Web of Science® Google Scholar
Zhang, M., Zhai, Z., Tian, X., Duan, L. & Li, Z. (2008) Brassinolide alleviated the adverse effect of water deficits on photosynthesis and the antioxidant of soybean (Glycine max L.). Plant Growth Regulation, 56, 257–264.
10.1007/s10725-008-9305-4
CAS Web of Science® Google Scholar
Zhang, Y.P., Zhu, X.H., Ding, H.D., Yang, S.J. & Chen, Y.Y. (2013) Foliar application of 24-epibrassinolide alleviates high-temperature-induced inhibition of photosynthesis in seedlings of two melon cultivars. Photosynthetica, 51, 341–349.
10.1007/s11099-013-0031-4
CAS Web of Science® Google Scholar
Zhao, G., Xu, H., Zhang, P., Su, X. & Zhao, H. (2017) Effects of 2,4-epibrassinolide on photosynthesis and Rubisco activase gene expression in Triticum aestivum L. seedlings under a combination of drought and heat stress. Plant Growth Regulation, 81, 377–384.
10.1007/s10725-016-0214-7
CAS Web of Science® Google Scholar
Zhu, J., Lu, P., Jiang, Y., Wang, M. & Zhang, L. (2014) Effects of brassinosteroid on antioxidant system in Salvia miltiorrhiza under drought stress. Journal of Research Agriculture and Animal Science, 2, 1–6.
Google Scholar
Zoschke, R. & Bock, R. (2018) Chloroplast translation: structural and functional organization, operational control and regulation. Plant Cell, 30, 745–770.
10.1105/tpc.18.00016
CAS PubMed Web of Science® Google Scholar

Citing Literature

Volume172, Issue2

Special Issue:Drought Tolerance

June 2021

Pages 748-761

Unraveling the roles of brassinosteroids in alleviating drought stress in young Eucalyptus urophylla plants: Implications on redox homeostasis and photosynthetic apparatus