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Article

The Sugarcane ScPetC Gene Improves Water-Deficit and Oxidative Stress Tolerance in Transgenic Tobacco Plants

by
Carolina Ribeiro Liberato Silva
1,†,
César Bueno de Souza
1,†,
Claudiana Moura dos Santos
2,
Bruno Spinassé Floreste
1,
Nicholas Camargo Zani
1,
Andrea Akemi Hoshino-Bezerra
1,
Giane Carolina Bueno
1,
Eder Bedani Ruiz Chagas
1 and
Marcelo Menossi
1,*
1
Department of Genetics, Evolution, Microbiology and Imunology, Institute of Biology, State University of Campinas, Campinas 13083-862, SP, Brazil
2
Reference Center for the Restoration of Degraded Areas of the Lower São Francisco (CRAD), Federal University of Alagoas, Arapiraca Campus, Arapiraca 57309-005, AL, Brazil
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2024, 14(7), 1371; https://doi.org/10.3390/agronomy14071371
Submission received: 17 May 2024 / Revised: 13 June 2024 / Accepted: 20 June 2024 / Published: 26 June 2024
(This article belongs to the Special Issue Sugarcane Challenges: From Germplasm to Biotechnology)
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Versions Notes

Abstract

:
Water deficit is the main limiting factor constraining sugarcane productivity, and its impact is expected to increase due to climate changes. During prolonged drought periods, most plants become extremely vulnerable to ROS accumulation, which can severely damage their photosynthetic apparatus. The PetC gene, encoding a Rieske FeS protein (ISP), has been shown to regulate the electron transport chain and protect photosystems (PSs) under drought conditions in some plant species. In sugarcane, transcriptome analysis revealed that ScPetC is repressed during drought stress in the field. In this study, we have overexpressed ScPetC in tobacco plants and evaluated its role in water-deficit tolerance. Our results indicate that the ScPetC protein structure is conserved when compared to other species. ScPetC overexpression reduced the negative impact of water deficit on plant development. This effect was associated with a positive impact on ScPetC quantum efficiency and the electron transport rate of PSII, the photosynthetic rate, and water use efficiency. The total chlorophyll content under water deficit was higher in plants overexpressing ScPetC, and this was correlated with less chlorophyll degradation from oxidative damage. Together, these results demonstrate that ScPetC confers tolerance to water deficit and oxidative stresses, making it a candidate gene for crop improvement.

1. Introduction

Sugarcane (Saccharum spp.) is a monocot plant that has the capacity to store significant amounts of sugar in its culm, reaching up to 27% of its fresh weight [1]. This singular trait makes sugarcane an ideal source for renewable bioenergy and biofuel production, qualities that hold immense value in the current scenario of increasing demands for lower greenhouse gas emissions [2,3].
Water deficit is the main limiting factor that affects sugarcane productivity [4,5]. As the effects of drought are expected to increase due to ongoing climate changes, many studies aim to select more drought tolerant sugarcane genotypes to improve productivity under water-deficit conditions, promoting more sustainable agriculture [6,7,8].
Under water-deficit conditions, plants suffer a decrease in their photosynthetic rate, as the stomata close to maintain water levels. This limits the assimilation of CO2, which can result in unbalanced biochemical control of photosynthesis, such as the inhibition of ribulose-1,5-bisphosphate carboxylase (Rubisco) synthesis and photosystem damage [9,10,11].
The photosystems PSI and PSII are part of the molecular complex that underlies the photosynthesis process and, alongside the cytochrome b6f (cyt b6f) and ATP synthase complexes, they carry out the light-dependent reactions. The final receptors of PSI reduce NADP+ to NADPH, which is used in the Calvin cycle to reduce CO2 to produce sugars [12]. At the same time, PSI and PSII play a role in the generation of the proton gradient used in ATP synthesis [13].
Water deficit can cause changes in the functional state of the thylakoid membranes, leading to the photoinhibition of the electron transfer from PSII to PSI [14,15], which increases the accumulation of reactive oxygen species (ROS) in the chloroplasts, provoking oxidative stress damage that can cause chlorophyll degradation [16].
Cytochrome b6f is a protein complex that is found in the membrane of thylakoids from plants, green algae, and cyanobacteria [17]. It plays a key role in the electron transfer between PSII and PSI [18] and acts as a limiting factor in the electron transport rate [19]. Cytochrome b6f is composed of two monomers, each consisting of four major subunits—cytochrome f (PetA), cytochrome b6 (PetB), the Rieske FeS protein (PetC), and subunit IV (PetD)—and four minor subunits—PetG, PetL, PetM, and PetN18 [20]. Interestingly, the PetC subunit abundance is directly related to cyt b6t levels [19], and several studies have investigated its influence on the electron transport rate. In A. thaliana, the overexpression of the tobacco (Nicotiana tabacum) PetC protein improved electron transport, the quantum efficiency of both photosystems, the biomass, and the seed yield [21]. Moreover, in Setaria viridis, a C4 plant, the overexpression of the Brachypodium distachyon PetC gene resulted in higher contents of cytochrome b6f and increased overall photosynthesis rates, providing evidence that improved electron transport can increase C4 photosynthesis [22].
In addition to its role in electron transfer, evidence suggests that the Rieske FeS protein (ISP) is also associated with the protection of the electron transport system during drought stress in different plant species. In watermelon, ISP modulated electron transport during drought stress, most likely acting either by depressing the electron transport chain to protect PSI from photoinhibition and/or as a stress-sensitive signaling factor [23]. Furthermore, in triticale, PetC has been shown to be involved in plant recovery from drought stress [24]. Lower PetC levels due to stress damage in this species were associated with irreversible changes in the photosynthetic apparatus activity, indicating that stable levels of PetC during drought stress and rehydration are essential for full plant recovery [24].
In sugarcane, transcriptome analyses have uncovered several genes that are differentially expressed in water-deficit conditions and that may be related to the drought response [25,26]. However, the function of most of these genes remains unknown. One of these genes whose expression is negatively associated with the response to drought corresponds to the sugarcane assembled sequence SCQSLR1018H02.g, which encodes a protein that is similar to ISP.
In this study we named this gene ScPetC and evaluated its role in transgenic tobacco lines. Our results provide evidence that the overexpression of ScPetC increases water-deficit and oxidative stress tolerance by reducing chlorophyll oxidation while improving the photochemical efficiency of photosystems I and II, the quantum efficiency and electron transport rate of photosystem II, the photosynthetic rate, and the water use efficiency.

2. Materials and Methods

2.1. Gene Expression in Field-Grown Sugarcane Plants

Sugarcane plants from two cultivars differing in their tolerance to drought stress were used to quantify ScPetC expression: RB86-7515 (higher tolerance) and RB85-5536 (lower tolerance). Plants were cultivated in the field under irrigation or rainfed (drought treatment) as in a previous work [27]. Two plants grown under the same environmental conditions (biological replicates) were used per treatment. RNA was extracted from the leaves, and RT-qPCRs to quantify ScPetC expression were performed in triplicate for each biological replicate with gene-specific primers (Table S1), using the ubiquitin gene (sugarcane assembled sequence SCCCST2001G02.g; NCBI accession number CA179923.1) described by Papini-Terzi as an internal control [28]. RT-qPCR was performed as described by Rocha et al. [25] and analyzed as described by Tournayre et al. [29].

2.2. ScPetC Protein Sequence Analysis

The ScPetC amino acid sequence was deduced from the coding region of the sugarcane SCQSLR1018H02.g assembled sequence from the SUCEST database [30] (NCBI accession number PP884103; Figure S1) and used for the identification of homologs in other monocot and eudicot plant species in the NCBI database. Homologous amino acid sequences from Sorghum bicolor (XP_002441121.1), Zea mays (NP_001141265.1), Oryza sativa japonica (NP_001409132.1), Brachypodium distachyon (XP_003562932.1), Glycine max (NP_001237648.2), Nicotiana tabacum (P30361.2), and Arabidopsis thaliana (NP_192237.1) were obtained and aligned using the MUSCLE algorithm from the EBI Search [31], and the alignment was used to generate a neighbor-joining phylogenetic tree with the MEGA 11 software [32].

2.3. Construction of the ScPetC Overexpression Vector

SCQSLR1018H02.g is a sugarcane assembled sequence, deduced from the overlap of several expressed sequence tags [30]. One of them, SCVPAM2067H05.g, was the template used to amplify the ScPetC coding region by PCR using gene-specific primers (detailed in Table S1). SCVPAM2067H05.g was obtained from the “Brazilian Clone Collection Center” (BCCCENTER, Jaboticabal, Brazil). The coding sequence was cloned into a pGEM®-T Easy vector (Promega, Madison, WI, USA). An EcoRI insert containing the coding sequence was then cloned under the control of the CaMV 35S promoter and CaMV 35S terminator into the pRT104 vector that had been digested with EcoRI. Subsequently, the p35S::ScPetC::t35S cassette was cloned into the pCAMBIA2301 vector (Cambia, Canberra, Australia) via HindIII sites. The obtained pCAMBIA2301::ScPetC vector, which contains the nptII gene for plant kanamycin selection (Figure S4), was then transformed into the Agrobacterium tumefaciens LBA4404 strain.

2.4. Plant Material and Growth Conditions

Seeds of wild-type Nicotiana tabacum (var. SR1) were sowed in plastic pots filled with 300 g of culture soil and grown in a growth chamber under the following conditions: 25 ± 2 °C, a photoperiod of 16 h light/8 h dark, relative humidity of 75–80%, and a photosynthetic photon flux density of 120 µmol m−2 s−1. Tolerance of transgenic plants to water deficit was examined by germinating T2 transgenic plants. After two months of growth, wild-type control lines and three homozygous transgenic lines named TlScPetC-A, TlScPetC-B, and TlScPetC-C were subjected to 20 days of water withhold followed by 5 days of rewatering. A pot experiment was arranged in a randomized complete block design with four plant lines, two treatments (well-watered and water-deficient) and three replications. The third leaflet of wild-type and transgenic plants, completely expanded, was used for the physiological evaluations. Shoot and root dry mass were determined after drying the plant tissues in an oven at 65 °C.

2.5. Transformation of Tobacco Plants

Foliar disks from tobacco (Nicotiana tabacum, var. SR1) were incubated for 5–10 min in Agrobacterium tumefaciens (LBA4404 strain) harboring the pCAMBIA2301::ScPetC construct (see below). The infected disks were first transferred to MS medium (supplemented with 1 mg/L benzylaminopurine, 0.1 mg/L naphthaleneacetic acid, 0.0059 g/L acetosyringone, and 0.9% w/v agar) for 3 days and then to selective medium (MS salts, 1 mg/L benzylaminopurine, 100 mg/L kanamycin, and 0.9% w/v agar). The developing plants were then transferred to MS medium containing 200 mg/L kanamycin and 0.9% w/v agar. The rooted plants (T0 generation) were transferred to a substrate (Flores e Folhagens, BIOMIX, Guaxupé, Brazil) and kept under greenhouse conditions until flowering and self-fertilization. T1 seeds were germinated on MS media with 200 mg/L kanamycin and resistant plants were self-fertilized. T2 seeds were germinated on MS medium with 200 mg/L kanamycin selection, and the survival of all T2 seeds in selective medium was indicative of homozygous progenitor plants. Six independent kanamycin-resistant tobacco homozygous lines were selected for further characterization. The presence of the transgenes was detected by PCR amplification using gene-specific primers using genomic DNA from T2 homozygous plants as a template.

2.6. Measurement of Gas Exchange, Chlorophyll Fluorescence, and P700 Parameters

Expanded leaflets were used to calculate the transpiration rate (E) and net photosynthetic rate (A) as described by Guo et al. [33]. Measurements were taken with an infrared gas analyzer (IRGA) at a CO2 concentration of 360 µL L−1 and a saturating light intensity of 600 µmol m−2 s−1. The gas exchange measurements were conducted between 8 and 10 h. With the values of A and E, the instantaneous water use efficiency (A/E) was calculated.
The chlorophyll fluorescence and P700 redox state measurement were determined in vivo with detached leaves using a Dual-PAM-100 (Heinz Walz, Effeltrich, Germany). All the PSI and PSII photosynthetic measurements in the light were determined at 25 °C and a PFD of 200 µmol m−2 s−1 after 5 min of light adaptation. WT and transgenic plants were evaluated for their maximum photo-oxidizable P700 (Pm), maximum quantum yield of PSII (Fv/Fm), effective quantum yield of PSII (YII) and PSI (YI), and electron transfer efficiency of PSI (ETR I) and PSII (ETR II) under normal watering, water-deficit, and rewatering conditions.

2.7. Quantification of Chlorophyll Degradation under Oxidative Stress and Water Deficit

The oxidative stress was evaluated with foliar discs of WT and transgenic plants exposed to 0.2 M and 0.8 M of hydrogen peroxide for 24 h [34]. Foliar discs were also treated with 0 µM, 10 µM, 25 µM, and 50 µM Paraquat solutions as described by Dos Santos and De Almeida [8]. For the water-deficit assay, plants were submitted to 20 days of water withholding, followed by 5 days of rewatering. The total chlorophyll content for each experiment was measured at a wavelength of 664 nm using foliar discs incubated with 2 mL of dimethylformamide [35].

2.8. Statistical Analysis

Mean values, standard deviations, and t-tests of the collected experimental data were analyzed with the R software (version 4.3.1) [36]. Shared control estimation analyses were performed with estimation statistics (version 2023.9.12) [37]. Plots were designed with Microsoft Excel (version 2405).

3. Results

3.1. The Sugarcane ScPetC Gene

The productivity and gas exchange parameters of six sugarcane varieties showed marked differences when cultivated under irrigation or when rainfed (i.e., drought stress) in field condition [38]. Later, we evaluated the changes in the transcriptome of these plants using a large-scale DNA chip experiment and found that the ScPetC gene was repressed by drought stress [26]. To confirm this data, we designed gene-specific primers (Table S1) and performed an RT-qPCR using cDNA from the sugarcane leaves. We used two cultivars: RB86-7515, which has higher tolerance to drought, and RB85-5536, which has lower tolerance, as described by Endres et al. [38]. In the RB85-5536 cultivar, there were no changes in ScPetC expression in response to drought, while in the RB86-7515 cultivar, we observed a drastic reduction (Figure 1). Interestingly, the expression levels in the cultivar with lower tolerance were lower than in the high-tolerance cultivar. Moreover, upon drought stress, the ScPetC expression in the high-tolerance cultivar dropped to the levels observed in the lower-tolerance cultivar. This data raises the hypothesis that one of the negative effects of drought is the reduction of ScPetC levels, as pointed out by Hura et al. [24]. It is worth mentioning that ScPetC is conserved between monocots and eudicots, and this conservation is also reflected in the ScPetC 3-D structure (Figure S2).

3.2. The Development of ScPetC-Overexpressing Plants Is Less Affected by Water Deficit

To assess the effects of ScPetC on plant responses to water deficit, transgenic tobacco plants overexpressing ScPetC (named TlScPetC plants) were produced (Figure S3) and cultivated alongside WT plants for 2 months under well-watered conditions and then subjected to water withholding for 20 days. The stress condition caused evident damage in both the wild-type and TlScPetC transgenic plants. However, after 20 days of water deficit, the wild-type plants suffered more damage compared with the transgenic lines (Figure 2).

3.3. ScPetC Protects PSII and PSI Activities in Response to Water Deficit

The maximum photochemical efficiency of photosystem PSII of WT and transgenic plants, represented by the mean Fv/Fm ratio, decreased during the 20 days of water deficit in both the TlScPetC and the WT plants (Figure 3A). However, the mean Fv/Fm ratio of the WT plants was lower when compared with that of transgenic plants, indicating that ScPetC reduced the photoinhibition effects due to water (Figure 3A). After rehydration, all the plants recovered their Fm/Fv ratio (Figure 3A). The maximum photochemical efficiency of photosystem PSI (Pm) showed a similar trend to PSII, as wild-type plants suffered larger reductions over the 20 days of water deficit when compared with the TlScPetC plants (Figure 3B).
Under stress conditions, TlScPetC and WT plants showed diminished values of quantum efficiency for both photosystems PSII (YII) (Figure 3C) and PSI (YI) (Figure 3D). After 20 days of stress, the YII reduction of TlScPetC plants fluctuated, while the mean YII of wild-type plants suffered a 100% reduction (Figure 3C), thus indicating that when under water deficit, PSII of TlScPetC plants can use light in a more efficient way than that of the WT. Regarding the quantum efficiency of PSI (YI), TlScPetC and WT plants were similarly impacted by water deficit (Figure 3D). For both PSII and PSI electron transport rates (ETRII and ETRI, respectively), TlScPetC and WT plants were similarly impacted by water deficit (Figure 3E,F).

3.4. Effect of Water Deficit on Photosynthesis and Water Use Efficiency

The photosynthetic rate (A) was negatively affected in wild-type plants and TlScPetC plants in response to water scarcity (Figure 4A). However, ScPetC overexpression had a small but significant effect, resulting in higher levels of A in comparison with WT plants. We also evaluated the water use efficiency (A/E) (Figure 4B). TlScPetC plants performed significantly better compared with the WT, suggesting that TlScPetC plants have a higher capacity for saving water during water deficit, which may lead to improved drought tolerance.

3.5. ScPetC Overexpression Protects Chlorophyll and Reduces Oxidative Stress Damage

Total chlorophyll degradation was evaluated in plants prior to stress, after 20 days of water deficit, and after 5 days of rehydration (Figure 5A). After 20 days of water suspension, we observed a smaller reduction of total chlorophyll in TlScPetC plants in comparison with the WT (Figure 5A). After rehydration, the TlScPetC plants recovered higher total chlorophyll values in comparison with WT plants, although no statistical difference was observed. We noticed that under control conditions, TlScPetC plants had higher total chlorophyll content, although only in the case of the TlScPetC-B event was p < 0.05, while in the other two events, the p-value was lower than 0.1 (Figure 5A).
To check if the protective role of ScPetC was associated with oxidative stress tolerance, we incubated foliar discs of WT and TlScPetC plants in paraquat and H2O2 solutions (Figure 5B). After the paraquat-induced oxidative stress (25 µM for 18 h), we observed that the total chlorophyll degradation was less pronounced in the transgenic plants. Upon oxidative stress due to the presence of H2O2, two out of three transgenic plants had higher levels of chlorophyll. The reduced chlorophyll degradation observed in the transgenic plants treated with H2O2 or paraquat indicates that the ScPetC gene might act to induce antioxidative mechanisms involved in the dehydration tolerance.

4. Discussion

4.1. Effect of ScPetC on Plant Development under Water Deficit

Water deficit is an important environmental restriction factor that influences a wide array of physiological processes involved in plant growth and development. Here we evaluated the functional role of the sugarcane ScPetC gene overexpressed in tobacco plants under water-deficit stress. Hura et al. observed that the levels of the PetC protein severely decline during water-deficit stress in triticale as a consequence of the elevated ROS concentration, which affects the plant’s capability of coping with the stress [24]. Under field conditions, ScPetC expression was severely reduced in sugarcane plants with a higher tolerance to drought (Figure 1), in line with the data from Hura et al. [24].
Several studies have been made with genes that increase water-deficit tolerance [39,40,41,42,43]. Those studies show that several alterations in the physiological and metabolic pathways can reduce the detrimental effect of stress over the plant. Therefore, genes that have a protective role against one or more of these conditions might improve tolerance to water deficit. For instance, a resistant plant might have better root development and improved water absorption, increased/stable growth during stress, improved protection for water loss, maintenance/increase of photosynthetic rates, and reduced generation of ROS during stress. The results obtained in this work demonstrate that ScPetC is an effective target gene that is capable of influencing several protective mechanisms, thus reducing the effects of water deficit during plant development (Figure 2). Due to its importance in the photosynthetic electron transport chain, the ScPetC amino acid sequence presents a high level of conservation among species (Figure S2A), which is also reflected in the conservation of the 3-D structure of the ScPetC protein (Figure S2C). Phylogenetic analysis of the ScPetC protein sequence and the homologs from monocot and eudicot species grouped monocots and eudicots into two different clades (Figure S2B), indicating that ScPetC most likely appeared before the evolutive separation of both groups. These results are in line with the protective function demonstrated by the ScPetC gene from the monocot sugarcane in effectively improving the overall status of the transgenic tobacco plants after the water-deficit stress.

4.2. Photosystems (PSI and PSII)

Throughout the water stress the maximum photochemical efficiency of PSII and PSI, represented by Fv/Fm and Pm (Figure 3A,B), was reduced in TlScPetC transgenic plants, indicating dynamic photoinhibition due to the over-excitation of PSII in response to drought and an excess of light [18,44]. In this case, the photoinhibition must not be seen as a type of damage, but as a protection mechanism that allows for the dissipation of excess thermal energy through nonphotochemical quenching [44] since there was recovery of the photosystems at the end of the stress. In wild-type plants, the reduction in Fv/Fm and Pm was more severe, indicating that the photoinhibition was more intense in both photosystems. The stress might have been harmful to the functional integrity of the chloroplasts, therefore causing instability of the reaction center from photosystems PSII and PSI, which decreases the photosynthetic capacity and induces oxidative damage in the chloroplasts [45]. Under drought conditions, the wild-type plants showed higher reductions in quantum efficiency. These results indicated that wild-type plants had a lower efficiency of absorption by the chlorophyll associated with photosystem PSII. According to Huang et al., plants subjected to severe drought stress demonstrated a strong reduction in their stomatal conductance, which led the plants to diminish their capacity for using NADPH, consequently causing an inhibition in the quantum efficiency of photosystem PSII (YII) [10].
For the quantum efficiency of photosystem PSI (YI) (Figure 3D) and ETRI (Figure 3F), both TlScPetC and wild-type plants showed similar ratios between the energy absorbed and the electrons transferred under stress, which indicated that photosystem PSI was less affected by the stress.
PSI possesses very effective photoprotection mechanisms, since its damage requires a long time for the cells to repair. These photoprotection mechanisms include the reduction of electron flow into PSI [46] and chemical modifications to P700 that reduce the access to O2, therefore preventing the formation of ROS in PSI [47]. The protective capabilities of those mechanisms could explain the reason for the lack of significant differences between WT and TlScPetC plants despite the stress.
On the contrary, the PSII system does not possess effective protection mechanisms, being dependent on its repair mechanisms. However, the damage caused by ROS can surpass the repair mechanisms’ efficiency, which themselves can also be inhibited by ROS [45]. The TlScPetC plants seem capable of an increased protection of PSII, maintaining an efficiency of PSII that is superior to the WT during the water-deficit stress.
The role of the Rieske ISP protein in photosystems has been studied recently. In Arabidopsis, Simkin et al. demonstrated that overexpression of the Rieske ISP protein increases the electron flow during photosynthesis [21]. Hura et al. found that elevated levels of Rieske ISP are essential for plant recovery after water deficit, preventing saturation of the photosynthetic apparatus, especially through ROS production, which inhibits the system [24]. In wild watermelon, a species considered drought tolerant, Sanda et al. observed that the Rieske ISP protein participated in the suppression of the activity of electron transport from the cytochrome b6f complex, reducing the excessive electron flux transfer to PSI and protecting PSI from damage caused by ROS and photoinhibition [23].
These results suggest that the constitutive overexpression of the ScPetC gene caused a higher amount of Rieske ISP protein to be synthesized in the cytochrome b6f complex in the transgenic plants in response to the excess of electron flux produced during stress on PSII. The overexpression of ScPetC softened the electron transfer from photosystem II to photosystem I, causing less photoinhibition in both systems in the TlScPetC plants. However, it seems that the ScPetC gene did not influence the electron transfer from the PSI side, because the activity of the cytochrome b6f complex had a similar profile in TlScPetC and wild-type plants in protecting PSI against damage and allowing the continuous flux of electrons under stress.

4.3. Gas Exchange

Photosynthesis in the TlScPetC plants was less affected compared with that in the wild-type plants during the water-deficit stress (Figure 4A), which is a consequence of the improvements in the photosystems as detailed above. The TlScPetC plants also demonstrated an increased instantaneous efficiency of water use (A/E) under water deficiency and rehydration conditions (Figure 4B). It has been documented that improved water use efficiency is an important trait that is closely related to a plant’s capacity for resisting water-deficit stress, since it correlates with the plant’s ability to maintain a higher level of carbon assimilation while reducing the water loss through transpiration [48].
During water-deficit stress, the plant closes the stomata to avoid excessive water loss through transpiration; however, this also reduces the CO2 transport rate, decreasing the photosynthetic rate, which is the main factor responsible for reduced growth during stress [49]. Therefore, an increase in water use efficiency is a mechanism of water-deficit tolerance, since it demonstrates the capability of maintaining active photosynthesis despite the activation of water-loss prevention mechanisms. Some works have shown that targeting stomata guard cell genes to decrease the stomatal conductance allows for reduced water loss by transpiration while maintaining similar photosynthetic levels as WT plants, resulting in higher water use efficiency in water-deficit stress [50,51]. In this work, we demonstrated that genes involved in the photosynthesis pathway are also possible candidates for generating higher water use efficiency in plants due to their capabilities in maintaining photosynthesis during stress. Similarly, a recent study in sugarcane compared drought-sensitive and drought-tolerant varieties, with the latter presenting a superior water use efficiency and significant differences in the expression of genes related to photosynthesis, reinforcing the hypothesis that photosynthesis-related genes are good candidates for water-deficit tolerance [52].

4.4. Water and Oxidative Stress

Our results indicated that the overexpression of ScPetC gene in tobacco transgenic plants induced high levels of chlorophyll at the peak of the water-deficit stress as well as after the rehydration period (Figure 5A). Also, ScPetC imparted a better capacity for maintaining chlorophyll integrity during the oxidative stress induced by H2O2 and paraquat (Figure 5B). According to Agathokleous et al., the amount of chlorophyll in leaves is essential for stress tolerance, and in several species, low stress stimulates an increase in chlorophyll synthesis [53]. In several studies with tobacco plants transformed with drought-tolerant candidate genes, there was a higher synthesis of chlorophyll [40] or an improved maintenance of chlorophyll levels compared with wild-type plants during stress [39,54], which contributed to the increase in tolerance to oxidative stress.
In addition, the low degradation of chlorophyll in the TlScPetC plants throughout the drought stress supports a role for the ScPetC gene in the mechanism that protects plants against damage to the photochemistry apparatus from the photosystems, reducing photoinhibition. According to Muhammad et al., the degradation of chlorophyll is one of the consequences of the stress, which, in turn, may be the result of photoinhibition and excessive ROS formation, decreasing photosynthetic efficiency [55].

5. Conclusions

Drought stress is one of the main limiting factors of sugarcane yield; therefore, the development of resistant cultivars is essential for better productivity. In this study, it was possible to show that the ScPetC gene participates in the response to water deficit by improving the water use efficiency and protecting photosystems I and II against photoinhibition and ROS formation. These results indicate that the ScPetC gene is a good candidate for promoting tolerance to drought and oxidative stresses in sugarcane, and evidence that the modulation of other genes from the photosynthetic apparatus is a promising venue for future investigations, as pointed out by López-Calcagno et al. [56], which might help to mitigate the challenges imposed by climate changes.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14071371/s1, Figure S1: The ScPetC nucleotide sequence and deduced protein from the SCQSLR1018H02.g sugarcane assembled sequence (NCBI Acc. No. PP884103). Figure S2: ScPetC protein sequence analysis. Figure S3: PCR identification of recombinant DNA insertion in the genome of TlScPetC plants. Figure S4: Scheme of the pCambia2301::ScPetC vector. Table S1: List of primers used in the study.

Author Contributions

Conceptualization: C.B.d.S. and M.M.; methodology, validation, and formal analysis: C.R.L.S., C.B.d.S., C.M.d.S., B.S.F., N.C.Z., A.A.H.-B., G.C.B. and E.B.R.C.; writing—original draft preparation: C.M.d.S., B.S.F. and N.C.Z.; writing—review and editing: B.S.F., N.C.Z. and M.M.; supervision: M.M.; funding acquisition: M.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Instituto Nacional de Ciência e Tecnologia do Bioetanol—INCT do Bioetanol, Brazil (São Paulo Research Foundation, FAPESP, grant 2014/50884-5) and the National Council for Scientific and Technological Development, CNPq (grants 465319/2014-9 and 404125/2013-1), and FAPESP (grant 2011/23264-8).

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

We express our gratitude to Jose Sergio Soares (in memoriam) for his invaluable contributions to the design of certain experiments in this work. His assistance has left a lasting impact, and we honor his memory for the crucial role he played in shaping our research.

Conflicts of Interest

The authors declare that they have filed a patent application protecting the use of the ScPetC gene in crops. The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. ScPetC expression in field-grown sugarcane. Leaf tissue from RB85536 and RB867515 plants were cultivated in the field under irrigation (orange bars) and under rainfed conditions, i.e., drought stress (purple bars). RT-qPCR was performed using gene-specific primers and normalized to the ubiquitin gene.
Figure 1. ScPetC expression in field-grown sugarcane. Leaf tissue from RB85536 and RB867515 plants were cultivated in the field under irrigation (orange bars) and under rainfed conditions, i.e., drought stress (purple bars). RT-qPCR was performed using gene-specific primers and normalized to the ubiquitin gene.
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Figure 2. Effects of water deficit in TlScPetC and WT plants. Two-month-old plants were grown with full irrigation (Day 0) and kept for 20 days without watering (Day 20), when irrigation was restored for 5 days (Day 25). The experiment was repeated twice with similar results. The pots used for plant cultivation have a diameter of 11 cm.
Figure 2. Effects of water deficit in TlScPetC and WT plants. Two-month-old plants were grown with full irrigation (Day 0) and kept for 20 days without watering (Day 20), when irrigation was restored for 5 days (Day 25). The experiment was repeated twice with similar results. The pots used for plant cultivation have a diameter of 11 cm.
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Figure 3. Chlorophyll fluorescence and P700 parameters of WT and ScPetC transgenic plants. Data were collected prior to stress (0), at 10, 15, and 20 days of water withholding, when they were fully hydrated, and after 5 days of rehydration. (A) Maximum quantum yield of PSII; (B) Maximum photo oxidizable P700; (C) Efficient quantum yield of PSII electron transfer; (D) Efficient quantum yield of PSI; (E) Electron transfer efficiency of PSII; (F) Electron transfer efficiency of PSI. Asterisks (* and **) indicate statistically significant values compared with WT plants (p < 0.05 and p < 0.01, respectively).
Figure 3. Chlorophyll fluorescence and P700 parameters of WT and ScPetC transgenic plants. Data were collected prior to stress (0), at 10, 15, and 20 days of water withholding, when they were fully hydrated, and after 5 days of rehydration. (A) Maximum quantum yield of PSII; (B) Maximum photo oxidizable P700; (C) Efficient quantum yield of PSII electron transfer; (D) Efficient quantum yield of PSI; (E) Electron transfer efficiency of PSII; (F) Electron transfer efficiency of PSI. Asterisks (* and **) indicate statistically significant values compared with WT plants (p < 0.05 and p < 0.01, respectively).
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Figure 4. Gas-exchange parameters in WT and TlScPetC transgenic plants. Measurements were made in the same plants as those described in Figure 3. (A) Photosynthesis rate; (B) Instantaneous water use efficiency. Asterisks (* and **) indicate statistically significant values compared with WT plants (p < 0.05 and p < 0.01, respectively).
Figure 4. Gas-exchange parameters in WT and TlScPetC transgenic plants. Measurements were made in the same plants as those described in Figure 3. (A) Photosynthesis rate; (B) Instantaneous water use efficiency. Asterisks (* and **) indicate statistically significant values compared with WT plants (p < 0.05 and p < 0.01, respectively).
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Figure 5. Spectrophotometric quantification of the chlorophyll content in WT and TlScPetC plants exposed to drought stress or oxidative stress. (A) Drought stress assay. Total chlorophyll content of plants prior to stress, after 20 days of drought stress, and after 5 days of rehydration; (B) Total chlorophyll content of foliar discs incubated with 25 µM of paraquat for 18 h or 0.8 M H2O2 for 48 h. Asterisks (* and **) indicate statistically significant values compared with WT plants (p < 0.05 and p < 0.01, respectively).
Figure 5. Spectrophotometric quantification of the chlorophyll content in WT and TlScPetC plants exposed to drought stress or oxidative stress. (A) Drought stress assay. Total chlorophyll content of plants prior to stress, after 20 days of drought stress, and after 5 days of rehydration; (B) Total chlorophyll content of foliar discs incubated with 25 µM of paraquat for 18 h or 0.8 M H2O2 for 48 h. Asterisks (* and **) indicate statistically significant values compared with WT plants (p < 0.05 and p < 0.01, respectively).
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Silva, C.R.L.; de Souza, C.B.; dos Santos, C.M.; Floreste, B.S.; Zani, N.C.; Hoshino-Bezerra, A.A.; Bueno, G.C.; Chagas, E.B.R.; Menossi, M. The Sugarcane ScPetC Gene Improves Water-Deficit and Oxidative Stress Tolerance in Transgenic Tobacco Plants. Agronomy 2024, 14, 1371. https://doi.org/10.3390/agronomy14071371

AMA Style

Silva CRL, de Souza CB, dos Santos CM, Floreste BS, Zani NC, Hoshino-Bezerra AA, Bueno GC, Chagas EBR, Menossi M. The Sugarcane ScPetC Gene Improves Water-Deficit and Oxidative Stress Tolerance in Transgenic Tobacco Plants. Agronomy. 2024; 14(7):1371. https://doi.org/10.3390/agronomy14071371

Chicago/Turabian Style

Silva, Carolina Ribeiro Liberato, César Bueno de Souza, Claudiana Moura dos Santos, Bruno Spinassé Floreste, Nicholas Camargo Zani, Andrea Akemi Hoshino-Bezerra, Giane Carolina Bueno, Eder Bedani Ruiz Chagas, and Marcelo Menossi. 2024. "The Sugarcane ScPetC Gene Improves Water-Deficit and Oxidative Stress Tolerance in Transgenic Tobacco Plants" Agronomy 14, no. 7: 1371. https://doi.org/10.3390/agronomy14071371

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