Enhanced expression of the ScTpx2 gene confers tolerance to drought stress in transgenic sugarcane

ABSTRACT

Drought events can have a devastating impact on agriculture, and due to climate change, such extreme events are expected to become more frequent. Sugarcane plays a critical role in the Brazilian economy by producing sugar and bioethanol, contributing positively to the reduction of CO₂ emissions. Although sugarcane is considered resilient to drought, this stress remains the primary abiotic factor reducing sugar and biomass yields. Here, we describe the role of a sugarcane gene, ScTpx2, which is induced by drought in sugarcane leaves under field conditions. When overexpressed in Arabidopsis, ScTpx2 enhanced plant survival under extreme water deficit and improved performance under mild stress conditions, which better represent field scenarios. We subsequently overexpressed the ScTpx2 gene in sugarcane plants. After 10 days of water deficit at 30% field capacity in a greenhouse, net photosynthesis in ScTpx2-overexpressing lines (ScTpx2OE) was 12–23% higher than in wild-type plants. While malondialdehyde (MDA) content, a marker of oxidative stress, increased by 129% in wild-type plants under water deficit, in ScTpx2OE plants, the increase ranged from 20% to 107%. Additionally, the vascular bundles and xylem areas were larger in ScTpx2OE compared to WT. These findings suggest that the ScTpx2 protein influences the development of the vascular system, thereby improving water transport efficiency. Our results demonstrate that overexpression of the ScTpx2 gene mitigates the effects of water deficit in sugarcane, offering promising opportunities for biotechnological applications in developing drought-tolerant commercial cultivars.

Introduction

Sugarcane is a crop of considerable economic significance both in Brazil and globally, with Brazil being the world’s leading producer. This versatile crop holds immense potential for renewable energy production, as it can be converted into bioelectricity and second-generation bioethanol.^1–3 As concerns about fossil fuel depletion intensify, the transportation sector, a major contributor to greenhouse gas emissions, is projected to see a sixfold increase in biofuel demand by 2050.⁴ This shift highlights the urgent need for sustainable energy alternatives.

Climate change poses a critical threat to agricultural productivity, impacting all crop species, including sugarcane. In Brazil, sugarcane cultivation is primarily concentrated in regions with favorable rainfall patterns.^1,5 In areas where natural precipitation is inadequate, sugarcane production relies on supplementary or full irrigation.⁶ However, climate models predict an increase in the frequency, duration, and intensity of water deficits, presenting a significant challenge for plant breeding programs aimed at developing drought-tolerant varieties.⁷

Advancements in stress biology, encompassing genetic, agronomic, and molecular biology research, have driven the development of biotechnological strategies to enhance water stress tolerance in sugarcane.^1,2,8–11 Drought stress triggers a wide range of physiological and molecular responses in sugarcane, modulating the expression of hundreds of genes.^12–14 These drought-responsive genes serve diverse functions, such as enhancing oxidative stress protection, promoting physiological plasticity, increasing sugar production, and improving agronomic traits under water-limited conditions.^12,15–18 Identifying key genes involved in stress tolerance is crucial for understanding and optimizing plant responses to drought.

In this study, we focus on ScTpx2, a sugarcane gene encoding a TPX2-like protein that was found to be upregulated under drought stress in field-grown sugarcane.¹² TPX2 family proteins are characterized by a conserved TPX2 domain (Pfam: PF06886), initially identified in Xenopus laevis within a kinesin-like protein called Targeting Protein for Xklp2 (TPX2). According to Evrard et al.,¹⁹ proteins containing the PF06886 domain, along with additional functional motifs, are classified as TPX2-related proteins. Some members of this family are microtubule-associated and play a critical role in the cell cycle, particularly in the assembly of the mitotic spindle.^19–23 Notably, studies in Xenopus cells have shown that TPX2 accumulates at DNA damage sites following ionizing radiation exposure and interacts with damage mediators such as MDC1 and ATM, indicating a potential role in the DNA damage response.²³ Additionally, TPX2 has been implicated in the activation of Aurora A kinase²⁴ and in the interaction with GTPase Ran during spindle assembly.²⁵

The TPX2 protein family in plants has been associated with the regulation of abiotic stress responses, hypocotyl elongation, and vascular development.²⁶ It is hypothesized that TPX2 proteins, through their interaction with microtubules, may mediate responses to abiotic stress by protecting DNA from cellular damage. In Arabidopsis thaliana, TPX2 has also been shown to activate Aurora A kinase,²⁷ while in Eucalyptus grandis, TPX2-like proteins localize to the cytoskeleton, associating with microtubules.²¹ Intriguingly, Smertenko et al.²⁶ provided evidence that MAP20, a TPX2-like protein, plays a role in xylem development, and that reduced levels of MAP20 were linked to decreased water deficit tolerance in Brachypodium distachyon.

Here, we demonstrate that the overexpression of ScTpx2 in Arabidopsis thaliana enhances survival under extreme water deficit and promotes growth under milder stress conditions, which more closely resemble field drought scenarios. Furthermore, transgenic sugarcane lines overexpressing ScTpx2 exhibited higher photosynthetic rates and reduced oxidative stress. Notably, increased levels of ScTpx2 led to the formation of larger xylem vessels. These findings provide new insights into the role of TPX2-like proteins in plant drought responses and suggest that ScTpx2 could be a promising candidate for developing crops with enhanced drought resilience.

Materials and Methods

Plant Material

Arabidopsis thaliana (Columbia genotype) and sugarcane plants (cultivar SP803280) were used for the production of transgenic lines. Arabidopsis seeds were obtained from The Arabidopsis Information Resource (TAIR), while sugarcane plants were sourced from the germplasm bank of the Functional Genomics Laboratory, Department of Genetics, Evolution, Microbiology, and Immunology, Institute of Biology, Campinas State University (Campinas, Brazil).

Gene expression Cassettes

The complete coding sequence (CDS) of the ScTpx2 gene, derived from SAS SCJFRT2059C10.g, was amplified via PCR using the primers: 5‘-CACCATGGCCAGAGAAATGGAGAT-3‘ and 5’-GATGGCCTTATAGTTGG-3‘. The amplified fragment was cloned into the pENTR-D Topo vector (Invitrogen), resulting in the construct ScTpx2-pENTR-D Topo, which was subsequently recombined into the pGWB608 vector²⁸ using the Gateway system (Thermo Fisher, USA). The resulting construct, pScTpx2.GWB608, was used for Arabidopsis transformation.

For sugarcane transformation, the pGVG vector,²⁹ also compatible with the Gateway system, was used. Given that both pGVG and pENTR-D Topo vectors possess the same selective marker (kanamycin resistance), the ScTpx2 insert flanked by attL1 and attL2 sites from ScTpx2-pENTR-D was amplified using primers 5’-GTAAAACGACGGCCAG-3‘ and 5’-CAGGAAACAGCTATGAC-3’ and cloned into the pGEM-T Easy vector (Promega), which is ampicillin-resistant. The resulting construct, ScTpx2-pGEM-T Easy, was then recombined with the pGVG vector, generating the ScTpx2-pGVG construct.

Production of Transgenic Arabidopsis Plants

Arabidopsis seeds were germinated either directly in a soil and vermiculite mix (3:1 ratio) or on plates containing Murashige-Skoog (MS) medium. Seeds underwent vernalization at 4°C in the dark for 3–4 days before being transferred to growth conditions (22–24°C, 16-hour photoperiod, ~120 μE m^{− 2} s^{− 1}). For transformation 30 plants (10 plants per pot) were grown for 5 weeks, after which the initial inflorescences were pruned to encourage multiple inflorescences.

Transformation was performed using the floral dip method.³⁰ A single colony of Agrobacterium tumefaciens containing the gene construct was cultured in 5 mL YEB medium with 100 mg/L spectinomycin, 50 mg/L gentamicin, and 30 mg/L rifampicin at 28°C with shaking. This pre-culture was expanded in 200 mL YEB medium under the same conditions. After 16 hours, the culture was diluted with a fresh 5% sucrose solution containing 0.03% Silwet L-77 (in a 4:1 ratio) before dipping the plants. Post-transformation, plants were covered with plastic wrap for 24 hours to enhance efficiency, then returned to standard growth conditions until seed collection.

To confirm successful transformation, leaves from the resulting transgenic plants were collected for DNA extraction, followed by a PCR assay using primers specific 5‘-CTATCCTTCGCAAGACCCTTCCT-3‘ and 5’-AACGATCGGGGAAATTCGAGCTC-3‘ to the pGWB608 vector to verify the presence of the gene of interest. Plants that tested negative for transgene integration were discarded, while confirmed transformants were allowed to continue growing under standard conditions. When the first inflorescences appeared, plants were isolated using the Arasystem (Arasystem, Belgium). After approximately 6 weeks, T2 seeds were collected.

The T2 seeds were germinated on MS medium containing 50 μM ammonium glufosinate to select for transgenic plants. Successful events displayed a 3:1 ratio of normal seedlings to seedlings with only cotyledons, indicating a single copy insertion of the transgene. The plates were kept under a 16-hour light period with a light intensity of 100 μmol m⁻² s⁻¹. Segregation patterns were evaluated after two weeks.

From each T2 line with confirmed single-copy integration, 10 individual plants were selected and grown to maturity. Seeds from these T2 plants were collected, and T3 seeds were subsequently sown on selective MS medium to screen for homozygous lines. RNA was extracted from Arabidopsis approximately 3 weeks after germination. Homozygous T3 leaves were frozen with liquid nitrogen and ground using two 2 mm metal balls in a Retsch machine (Retsch, Germany). The Trizol protocol (Life Technologies, USA) described by the manufacturer was used. For RT-PCR, the forward primer specific for the sugarcane ScTpx2 gene (5‘-CCCAGGAAATCATTCGCAGA-3‘) and the reverse primer, within the NOS terminator region (5’-CCGGCAACAGGATTCAATCT-3’), were used. Plants confirmed as homozygous for the gene of interest were selected for further experiments.

Water Deficit experiment in Arabidopsis

For severe stress in soil, seedlings were sown in separated pots (55 mm) filled with jiffy-7 (Jiffypot, Netherlands). Three different T3 homozygous events for each gene and two events transformed with pGWB608 were randomized in the same tray (35 pots), grown under normal conditions (16 h light, at 22°C). After 10 days of well-watered conditions, the weight of all pots was normalized until the maximum water capacity, and the watering was withheld for approximately two weeks. The positions of the pots were randomly changed every day to avoid differential water loss among the pots. When the majority of the plants showed clear symptoms of wilting, the plants were re-watered and one day after survivors was counted.

For moderate stress in soil, we used the automated phenotyping platform WIWAM³¹ to apply moderate drought stress treatment. Seeds from homozygous transgenic plants were sown directly in the pots. Initially, 32 pots per line were treated under control (well-watered) conditions of soil water content (2.2 g H₂O/g dry soil). After 10 days, 16 pots for each line received the moderate stress treatment (1.2 g H₂O/g dry soil) and 16 were maintained under well-watered conditions until day 21. After the treatments, the rosette and leaf area were measured using ImageJ (http://imagej.nih.gov/ij/).

Production of Transgenic Sugarcane Plants

Transgenic sugarcane plants were generated by PangeiaBiotech (Campinas, Brazil) using A. tumefaciens strain EHA105 containing the ScTpx2-pGVG construct, following the method described by Guidelli et al..²⁹ After 4 months of in vitro culture, transformed seedlings were delivered. Integration of the transgene was confirmed via PCR using leaf-derived genomic DNA and the CTAB method.³² The specific primers used were 5’-ATGGCCAGAGAAATGGAGAT-3‘ (coding region) and 5’-AGGTCACTGGATTTTGGTTTTAGGA-3’ (35S terminator).

Analysis of Transgene expression in Transgenic Sugarcane Plants

RNA from leaf tissue (leaf +1, fully expanded) was extracted using the Trizol method (Invitrogen, USA) followed by DNase I treatment. cDNA synthesis was carried out with the iScript Reverse Transcription Kit (Bio-Rad, USA). The expression of ScTpx2 was quantified by RT-qPCR using the primers 5”-GAAGCTTGCACAGTTGATGGA-3‘ and 5’-GCTGGCATAAGTTGGGCCTT-3,‘ with Ubiquitin as the internal control (primers 5’-CCGGTCCTTTAAACCAACTCAGT-3’ and 5’-CCCCTCTGGTGTACCTCCATTTG-3’ NCBI access CA179923.1,³³). Reactions were performed using the SYBR® Green PCR Master Mix kit (Applied Biosystems, USA), in the 7500 Real-Time PCR System (Applied Biosystems, USA) and data were analyzed using the 2^{^−ΔΔCT} method.³⁴

Clonal Multiplication of Transgenic Sugarcane Events

For clonal multiplication, a single plant from each of the five independent transgenic sugarcane events with the highest ScTpx2 expression levels was selected. These plants were grown in a greenhouse for 8 months. Culm cuttings containing lateral buds were used for vegetative propagation. After 30 days of bud germination, five replicates of each transgenic event, along with wild-type (WT) controls, were transplanted into 20 L pots. The pots were filled with a substrate composed of one-third vermiculite, one-third sand, and one-third soil.

Water Deficit Stress experiment in Sugarcane

Four-month-old transgenic ScTpx2 and WT plants were arranged in a completely randomized block design in the greenhouse to minimize positional effects. On day zero, all pots were weighed, and the plants were placed on an automated weighing scale system. Irrigation was stopped on day one, and daily water loss was monitored until the soil moisture reached a field capacity of 30%. From that point, water was added daily to maintain 30% field capacity until day 18. Afterward, all plants were re-irrigated to 100% pot capacity, and their recovery was assessed.

Gas Exchange and Photochemical Activity Analysis

Gas exchange and photochemical parameters were measured before, during, and after water stress, as well as after rehydration, using an infrared gas analyzer (LCpro-SD, ADC BioScientific Ltd., UK). Measurements were taken between 9:00 AM and 1:00 PM under a photosynthetically active radiation (PAR) of 2,000 μmol m⁻² s⁻¹, with a leaf chamber temperature of 25°C and a CO₂ concentration of 380 μmol mol⁻¹. The parameters measured included photosynthetic rate (A), stomatal conductance (gs), intercellular CO₂ concentration (Ci), and transpiration rate (E).

Proline Concentration and Lipid Peroxidation Analysis

To determine proline concentration, 50 mg of leaf tissue was ground to a fine powder in liquid nitrogen and extracted with 3 mL of 3% (w/v) sulfosalicylic acid, following the method of Bates et al.,³⁵ with modifications. The extract (1 mL) was mixed with 1 mL of acid ninhydrin reagent (0.63 g of ninhydrin, 15 mL of glacial acetic acid, 10 mL of 6 M phosphoric acid, and 1 mL glacial acetic acid) and incubated at 100°C for 1 hour. After cooling on ice, the samples were analyzed spectrophotometrically at 520 nm (Lambda 40, Perkin Elmer, USA). The results were expressed as μg proline per gram of fresh weight.

Lipid peroxidation was assessed by measuring thiobarbituric acid reactive substances (TBARS) following the method of Heath and Packer,³⁶ with modifications. Leaf tissue (200 mg) was pulverized in liquid nitrogen and homogenized in 80:20 (v/v) ethanol-water to a final volume of 3 mL. After centrifugation at 3000 rpm for 10 minutes, 1 mL of the supernatant was mixed with 1 mL of 20% (w/v) trichloroacetic acid (TCA) containing 0.65% (w/v) thiobarbituric acid (TBA). The mixture was shaken vigorously and incubated at 100°C for 25 minutes, then cooled on ice, and centrifuged again for 10 min. Absorbance was measured at 532 nm and 600 nm (Lambda 40 spectrophotometer, Perkin Elmer, USA). The malondialdehyde (MDA) content was calculated using an extinction coefficient of 155 mM⁻¹ cm⁻¹ and expressed as nmol MDA per gram of fresh weight.

Microscopy and Histochemistry Analyzes

For microscopy analysis, samples were taken from the median region of the 3^rd and 6^th internodes of both WT and transgenic ScTpx2 sugarcane plants. Samples were fixed in FAA 50 (formalin-acetic acid-alcohol) for 48 hours³⁷ and then stored in 70% ethanol. Sections, 18–20 µm thick, were prepared using a Leica SM 2010 R microtome. Quantitative analysis involved measuring the total area of 25 vascular bundles per sample, as well as the xylem area, using the ImageJ software. Histochemical tests were performed on additional sections using Mäule’s reagent to detect syringyl (S) and guaiacyl (G) lignin and Phloroglucinol-HCl³⁷ for total lignin content. The results were documented using an Olympus DP71 camera attached to an Olympus B× 51 microscope.

Statistical Analysis

Statistical analysis was done according to Ho et al.,³⁸ using a shared control (WT) and as effect size the mean difference (https://www.estimationstats.com/).

Results

The ScTpx2 Gene is Induced by Drought Stress and Encodes a Protein from the TPX2 Family

Drought-induced changes in the transcriptome of field-grown sugarcane plants were previously analyzed using Agilent oligoarrays, which uncovered a wide range of cellular functions modulated by this critical abiotic stress.¹² Among the genes identified, SCCCST2001G02.g was notably upregulated in the leaves of sugarcane plants cultivated for seven months under drought field conditions. To validate the expression pattern observed in the oligoarray analysis, RT-qPCR assays were conducted using leaf cDNA from two sugarcane cultivars: RB867515 and RB855536. The RB867515 cultivar exhibited higher rates of photosynthesis and transpiration, along with lower proline accumulation and less reduction in water potential under drought conditions, compared to the drought-sensitive cultivar RB855536.^12,39 A significant induction of SCCCST2001G02.g expression was observed in RB867515 plants under water-deficit conditions, whereas the less tolerant RB855536 showed only a modest increase (Figure 1(A)).

Figure 1.

The ScTpx2 gene from sugarcane. A) gene expression in response to drought stress. RT-qPCR assay in 7 months-old plants from two cultivars, grown in the field with irrigation or drought-stressed (without irrigation, rainfed). B). Phylogenic analysis of ScTpx2 compared to the Arabidopsis TPX2 family members. The 19 Arabidopsis proteins belonging to the TPX2 family, described by Smertenko et al.,²⁰ were aligned with TPX2 (NCBI accession SCJFRT2059C10.G) using clustal Omega.^40,41 C). Alignment of TPX2 proteins from nine plant species. The blue color scale indicates amino acid similarity of the sequences aligned with clustal Omega. Sequences were obtained from accession numbers B8BE75 (Oryza sativa), A0A3B6LJH1 (Triticum aestivum), I1IPA2 (Brachypodium distachyon), A0A1D6ENV3 (Zea mays), K3ZX18 (Setaria italica), SCJFRT2059C10.G (TPX2), C5X918 (Sorghum bicolor), C6SWV7 (Glycine max) and F4K773 (Arabidopsis thaliana).

Smertenko et al.²⁰ identified 19 members of the TPX2 family in the Arabidopsis genome. Phylogenetic analysis of the protein encoded by SCCCST2001G02.g indicated that it is closely related to AtTPXL6/MAP20 (Figure 1(B)), and the gene was therefore designated ScTpx2. Multiple sequence alignment further demonstrated that TPX2 shares strong homology with proteins from other crop species and model plants (Figure 1(C)).

Expression of ScTpx2 Enhances Arabidopsis Responses to Severe and Mild Water Deficit

The complete coding sequence of the ScTpx2 gene was cloned into the pGWB608 vector²⁸ for overexpression in Arabidopsis, resulting in the pScTpx2.GWB608 construct. The gene was also cloned into the pGVG vector,²⁹ and the resulting pScTpx2-pGVG construct was used to generate transgenic sugarcane plants.

Ten-day-old Arabidopsis plants grown in soil were subjected to water withdrawal for two weeks until severe wilting symptoms were observed. Water was then supplied, and plant survival was assessed one day after rehydration. The ScTpx2 overexpressing lines (ScTpx2-OE) exhibited an average survival rate of 76%, with independent lines showing survival rates of 86%, 71%, and 71%, compared to the 29% survival rate observed in control plants (Figure 2).

Figure 2.

Assessment of tolerance to severe water deficit. A) DNA constructs used to overexpress ScTpx2 in Arabidopsis. (B) survival rates after exposure to drought stress. Ten-day-old Arabidopsis plants were subjected to two weeks of water withdrawal, followed by rehydration. Photos were taken at the end of the withdrawal period (before) one day after rehydration (after), and the percentage of recovered plants was determined. Plants transformed with an empty vector were used as controls. Ten plants from each of the three independent events were assessed, and the experiment was repeated with consistent results (supplementary Figure S1). ScTpx2-OE1, -OE2, and -OE3 indicate independent transgenic lines overexpressing ScTpx2; EV represents plants transformed with pGWB608 lacking the ScTpx2 gene.

Most genes that confer improved survival rates under severe water deficit when overexpressed have minimal effects on plant growth under mild stress conditions, which more closely resemble drought stress encountered in field environments.³¹ Consequently, WT and ScTpx2-OE Arabidopsis plants were also evaluated under mild water stress. This treatment, conducted with ten-day-old plants using the WIWAM system,³¹ reduced leaf area by approximately 30%. Leaf areas of individual plants were assessed after 11 days of moderate stress, with ScTpx2-OE plants consistently exhibiting larger leaf areas compared to WT plants (Figure 3).

Figure 3.

Effect of moderate water deficit on leaf area. the graphs represent the total rosette area (A) and the area of each leaf (B-D) of transgenic lines overexpressing ScTpx2 and plants transformed with an empty vector (EV) under moderate water deficit conditions. The X-axis indicates the cotyledons (cot) and numbered leaves (1 to 10). Plants transformed with the empty vector (EV) were used as controls. Data represent rosette and leaf area from two independent experiments (n = 32).

Overexpression of ScTpx2 Confers Water Deficit Tolerance in Sugarcane

The pScTpx2-pGVG construct (Figure 4(A)) was transferred via Agrobacterium into sugarcane calli and fourteen putative transgenic plants, along with wild-type plants regenerated in vitro, were subjected to PCR using oligonucleotides specific to the ScTpx2 coding region and the 35S terminator. Amplification of a 916 bp fragment confirmed the presence of the transgene in nine events, whereas no amplification was observed in wild-type plants, as expected (Figure 4(B)).

Figure 4.

Overexpression of ScTpx2 in transgenic sugarcane plants. (A) construct used to overexpress ScTpx2. The line indicates the region amplified to confirm the transgenic events. (B) confirmation of transgenic events. Nine transgenic events were confirmed (1, 2, 3, 4, 7, 9, 10, 12, and 14). MM: molecular weight marker; WT: wild-type plants. (C) relative quantification (RQ, 2^−ΔΔϹt) values of ScTpx2 gene expression determined by real-time PCR. On the X-axis, WT represents wild-type plants, and the numerical identifiers represent transgenic events overexpressing ScTpx2. Events marked with an asterisk were selected for water deficit tests.

The nine confirmed transgenic events were further analyzed by real-time RT-qPCR to verify ScTpx2 overexpression. Five events with the highest expression levels of ScTpx2 were selected for water deficit tolerance tests, as indicated by asterisks in Figure 4(C).

To evaluate the impact of water deficit on gas exchange parameters, five transgenic events (7, 9, 10, 12, and 14) and wild-type (WT) plants were analyzed, with five replicates for each group obtained through clonal propagation. Plants were grown for four months in the greenhouse under full irrigation. At the beginning of the experiment (day 0), photosynthesis measurements were initiated. On day 1, irrigation was suspended until pots reached 30% of their water-holding capacity. Daily weighing of pots was performed to monitor water loss, achieving 30% capacity on day 7, and this level of water was kept for another 10 days. After this period, plants were rehydrated to 100% capacity, and their recovery was assessed three- and six-days post-rehydration.

Both WT and ScTpx2-OE plants exhibited reduced photosynthesis during the water deficit period (Figure 5(A)). After 10 days of water deficit, WT plants displayed a 96% reduction in photosynthetic rate, while the reduction in ScTpx2 transgenic plants ranged from 73% to 84% across different events. Transgenic events 9, 10, 12, and 14 showed the highest photosynthetic rates on day 16, and events 7, 9, and 12 stood out on day 18. WT plants demonstrated a more pronounced reduction in photosynthesis and a slower recovery after rehydration compared to ScTpx2-OE plants.

Figure 5.

Gas exchange parameters in transgenic and wild sugarcane plants under water deficit conditions. Black bars represent wild plants and white bars represent transgenic events (7, 9, 10, 12, 14, from left to right). (A) photosynthetic rate (A). (B) stomatal conductance (gs). (C) transpiration rate (E). Values are presented as mean ± SEM. Asterisks within the bars indicate the statistical significance of the p-values: p < .05 (*), p < .01 (**), and p < .001 (***). The p-values < 0.05, p < .01, and p < .001 indicate different levels of statistical significance for the transgenic events. Data are available in supplementary tables S1, S2, and S3.

Stomatal conductance during water deficit is shown in Figure 5(B). After 10 days at 30% pot capacity, WT plants exhibited an 85% reduction in stomatal conductance, whereas reductions in ScTpx2-OE plants ranged from 42% to 85% across the five transgenic events. Similar to the photosynthetic rates, stomatal conductance in transgenic events recovered more rapidly to pre-stress levels compared to WT plants.

Transpiration rates were also analyzed during the water deficit period (Figure 5(C)). Reductions in transpiration were observed in both WT and ScTpx2-OE plants on days 9, 16, and 18. After 10 days, WT plants exhibited an 84% decrease in transpiration rates, while reductions in transgenic events ranged from 46% to 83%. However, differences between WT and transgenic plants were less pronounced compared to photosynthesis and stomatal conductance.

ScTpx2 Overexpression Reduces Lipid Peroxidation without Affecting Proline Levels

Lipid peroxidation, a biochemical marker of various stress conditions in sugarcane, including high temperature, water deficit, and salinity,⁴² was evaluated by measuring malondialdehyde (MDA) content. All plants subjected to water deficit exhibited an increase in MDA levels after 10 days at 30% pot capacity (Figure 6(A)). WT plants showed a 129% increase in MDA content, while increases in ScTPX2OEplants ranged from 20% to 107% across different events.

Figure 6.

Lipid peroxidation and proline content in sugarcane under water deficit. (A) Lipid peroxidation, measured as MDA content (nmol/g fresh mass): (B) proline content (µg/g fresh mass). Samples were taken from transgenic and WT plants on day 0 (control) and after 10 days of water stress at 30% pot capacity. Values represent means ± SEM (n = 5). Statistical analysis was performed using ANOVA and Tukey’s test at a 5% significance level. Different letters on each sampling day indicate significant differences between means (p < .05). The asterisks on the bars indicate that there is a significant difference in the events compared to the other events and WT. Data supporting these results are available in supplementary tables S4 and S5.

Proline accumulation, an indicator of water deficiency,⁴³ has been linked to the protection of sugarcane against oxidative stress during water deficit.¹⁷ All plants subjected to water deficit exhibited increased proline levels after 10 days. While ScTPX2OE plants displayed varying proline levels, only event 14 showed a statistically significant increase compared to WT plants (Figure 6(B); see supplementary table S5). For the remaining transgenic events, differences were small and not statistically significant.

ScTpx2 Is Involved in Vascular Bundle Development

In Brachypodium distachyon, the knockout of the TPX2 homolog MAP20 resulted in reduced vascular bundle area.²⁶ This observation led to the hypothesis that overexpression of ScTpx2 in sugarcane would lead to enlarged vascular bundles. To test this hypothesis, anatomical analyses of sugarcane internodes were conducted.

Distinct lignin deposition patterns were observed between WT and ScTpx2-OE plants in the 3^rd (immature) and 6^th (mature) internodes. In the 3^rd internode, the Mäule tests revealed that WT plants predominantly displayed G-lignin (yellowish coloration) in vascular sheath fibers and epidermal cells (Figure 7(a)), whereas ScTpx2-OE plants exhibited S-lignin (reddish coloration) in the fibers of the vascular sheaths near xylem vessels (Figure 7(e)). In both WT and ScTpx2-OE, the more peripheral fibers of the vascular bundle are still differentiating and not lignified (Figure 7(a,e)). In the medullary region, vascular fibers and cell wall of xylem conductor cells contained primarily S-lignin in ScTpx2-OE (Figure 7(f)) and G-lignin in WT (Figure 7(b)).

Figure 7.

Anatomical analysis of sugarcane internodes. Transverse sections from different regions of the 3^rd and 6^th internode, from rind and pith of WT and ScTpx2 plants. A-H. Mäule region for the detection of lignins S and G. I-P. Phloroglucinol for total lignin detection. Fp = filling parenchyma; vb = vascular bundle. Bar: 200 µm.

In the 6^th internode, WT plants showed G-lignin in the epidermis and in the layers beneath the epidermis (Figure 7(c)), while ScTpx2-OE plants displayed significant S-lignin deposition in these cells, vascular sheath fibers, in the cell wall of the fundamental parenchyma of the peripheral region, and xylem vessel walls (Figure 7(g)). Parenchyma lignification was complete in ScTpx2-OE (Figure 7(g)) but incomplete in WT (Figure 7(c)). In the medullary region, G-lignin was exclusively observed in WT plants (Figure 7(d)), whereas S-lignin predominated in ScTpx2-OE plants (Figure 7(h)), along with signs of early lignification in ScTpx2-OE parenchyma cells (Figure 7(h)).

Staining with acidified phloroglucinol revealed total lignin deposition patterns in both internodes. In the 3^rd internode, more peripheral fibers of the vascular bundle are still differentiating and not lignified in both WT and ScTpx2-OE plants (Figure 7(i,m)). The pith region shows only the fibers of the vascular bundles with a lignified wall in both (7j, 7n)

In the 6^th internode, ScTpx2-OE plants exhibited intense lignification in vascular fibers, cell wall of xylem conductor cells and fundamental parenchyma cells wall evidenced by the pink color (Figure 7(o)). Whereas WT plants showed less intense lignification in outer fibers and fundamental cells (Figure 7(k)). No differences in lignification were observed in the medullary region, both of which show lignification in the cell walls of the vascular bundle cells and the fundamental parenchyma (Figure 7(l,p)).

Quantitative measurements of the vascular bundles and xylem areas in the 6^th internode revealed significant differences between WT and ScTpx2-OE plants. The vascular bundle area was larger in ScTpx2-OE plants, with a mean of 64 mm² compared to 46 mm² in WT (Figure 8(A)). Similarly, xylem area was greater in ScTpx2 plants, averaging 19 μm², compared to 15 μm² in WT (Figure 8(B)).

Figure 8.

Quantitative analysis of vascular bundles and xylem areas. (A) vascular bundle area and (B) xylem area of WT and ScTpx2-OE plants in the 6^th internode. Blue dots represent WT plants (n = 4), and orange bars represent ScTpx2-OE plants (n=4, with one plant of each of the following events: 7, 10, 12 and 14). Bootstrap sampling distributions show mean differences, with 95% confidence intervals indicated by error bars.³⁸.

Discussion

Water deficit induces changes in physiological processes that significantly affect plant development. Silva et al.¹⁸ observed that TCP02-4587, a drought-tolerant sugarcane genotype, exhibited a greater photosynthetic capacity under water deficit conditions compared to the drought-susceptible genotype HoCP93-776. Similarly, Dos Santos et al.⁴⁴ studied six sugarcane varieties (SP79-1011, RB855113, RB92579, RB867515, RB72454, and RB855536) under water deficit and found that the stress effects were more pronounced during the phase of intense growth. Interestingly, the RB867515 and RB92579 varieties were less impacted, demonstrating a greater ability to adapt to water deficit. Consistent with these findings, our study showed that the ScTpx2 gene is highly induced by drought in the RB867515 cultivar, with induction levels significantly higher than those observed in the less drought-tolerant RB855536 cultivar.

Drought stress is closely associated with reductions in plant biomass, as reported in several studies.^45,46 Jangpromma et al.⁴⁵ found that drought stress reduced stalk diameter, biomass, root length, root surface area, root volume, and root dry weight in sugarcane, although it did not significantly affect the root-to-shoot ratio. Marchiori et al.¹⁶ evaluated the initial growth phase of two sugarcane genotypes, IACSP95-5000 and IACSP94-2094, and demonstrated that their ability to adapt to abiotic stress and recover physiological status is crucial for productivity In our study using Arabidopsis as a model, we observed that independent events overexpressing the ScTpx2 gene exhibited a greater leaf area compared to control plants.

Drought stress also directly affects gas exchange parameters in plants.⁴⁷ Graça et al.⁴⁸ compared the photosynthetic rate, stomatal conductance, and transpiration in two drought-tolerant sugarcane cultivars (SP83-2847 and CTC15) and a drought-susceptible cultivar (SP86-155). They reported reductions in stomatal conductance and photosynthetic rates across all cultivars but noted higher photosynthetic rates in the drought-tolerant genotypes. In sugarcane, both wild-type (WT) and ScTpx2-OE plants experienced reductions in photosynthesis under stress. However, transgenic events overexpressing ScTpx2 demonstrated greater physiological plasticity, maintaining higher rates of photosynthesis, stomatal conductance, and transpiration under water deficit and recovering more effectively after rehydration.

Recovery of photosynthetic activity following rehydration varies among genotypes and is influenced by the physiological and molecular mechanisms specific to each plant species.⁴⁹ For instance, C4 plants exhibit greater efficiency in photorespiration compared to C3 plants, providing a physiological advantage under stress conditions.⁵⁰ In our study, plants overexpressing ScTpx2 exhibited a significantly enhanced recovery of photosynthesis compared to WT plants, reaching higher levels of photosynthetic activity three and seven days after rehydration. These findings highlight the role of ScTpx2 in improving drought tolerance and facilitating recovery under water deficit conditions.

Several species exhibit increased proline accumulation in response to the intensity and duration of water stress, as observed in maize⁵¹ and wheat.⁵² In sugarcane, Queiroz et al.⁴³ reported elevated proline levels in two genotypes, IAC91-5155 and IAC91-2195, under water deficit. Similarly, in our study, both wild-type (WT) and ScTpx2 overexpressing plants (ScTpx2-OE) showed increased proline accumulation after 10 days of water stress. Notably, one event, ScTpx2-OE-Ev.14, exhibited a significant increase in proline content (see Table S5), while the other four ScTpx2 events demonstrated a trend toward increased proline levels, though not statistically significant.

Sawahel and Hassan⁵³ observed increased proline accumulation in transgenic wheat plants overexpressing the P5CS gene, which encodes an enzyme involved in proline biosynthesis, in response to salt stress. Molinari et al.¹⁷ also found increased proline levels in sugarcane plants overexpressing the P5CS gene, which enhanced their ability to withstand water deficit. However, the relatively small effect of ScTpx2 overexpression on proline accumulation in our study suggests that the protective role of this gene may not be directly related to proline levels.

Lipid peroxidation, a marker of oxidative stress, is a key response to environmental stressors.⁵⁴ Molinari et al.¹⁷ observed an increase in MDA content in sugarcane under abiotic stress. Morais et al.⁴² reported that MDA levels rose in response to water deficit, high temperatures, and salt stress in several sugarcane genotypes, with the increase varying by genotype and stress type. Similarly, Zeng et al.⁵⁵ observed increased MDA content in two sugarcane genotypes subjected to cadmium (Cd) stress. In our study, after 10 days of water deficit, both WT and transgenic plants showed elevated MDA levels. However, WT plants exhibited a higher MDA content compared to those overexpressing the ScTpx2 gene. These results suggest that ScTpx2-OE plants experience less oxidative stress, which may contribute to improved gas exchange parameters and better overall plant performance under drought conditions.

Specialized tissues such as vascular bundles, xylem, and phloem perform essential functions in plants.^56–59 The lignification patterns we observed in the stems of the SP8032-80 sugarcane cultivar are similar to those described in the H5-7052 cultivar.⁶⁰ Sugarcane varieties can differ in vascular bundle attributes, such as their distribution, as observed in six cultivars from Pakistan.⁶¹ Salleo et al.⁶² found that larger xylem vessels are associated with higher water conduction but are also more prone to cavitation, which is one of the primary causes of plant death due to drought. Meinzer et al.⁶³ evaluated two sugarcane cultivars (H65-7052 and H69-8235) alongside the ancestors S. officinarum and S. spontaneum and found that maximum water conductivity per unit of leaf width was linked to larger metaxylem vessel diameters. Furthermore, H65-7052, which had larger metaxylem vessels, was more efficient in maintaining water conductivity at lower leaf water potential. In our study, we observed that overexpression of the ScTpx2 gene led to an increase in the size of vascular bundles and xylem, which was associated with enhanced tolerance to water deficit.

The study of promoters or genes that are activated by drought is relevant in genetic engineering in sugarcane.^64,65 Sun et al.⁶⁵ cloned six different promoters from sugarcane bacilliform virus (SCBV) genotypes. Their results show that expression levels increased under ABA treatment and water stress. Gao et al.⁶⁴ studied the plant viral promoter SCBV21, and they observed that the promoter has activity in the vascular bundle of the culm and in the storage parenchyma. In our study to determine the expression of the ScTpx2 gene in plants, RT-qPCR was performed using RNA extracted from leaf +1. However, to better understand the role of ScTPX2 during water deficit, it would be interesting to perform expression assays in the vascular bundle of the culm and storage parenchyma.

Conclusion

Analysis of Arabidopsis plants overexpressing the ScTpx2 gene demonstrated that this gene confers tolerance to both mild and severe water deficit. In sugarcane, overexpression of ScTpx2 clearly improved the regulation of photosynthesis and stomatal conductance during water deficit, as well as during the rehydration phase. Although proline accumulation showed a tendency to be higher in ScTpx2-OE plants, it does not appear to be the primary factor underlying the protective role of this gene. The malondialdehyde (MDA) content was higher in wild-type plants than in those overexpressing ScTpx2, indicating that the ScTPX2 protein helps reduce oxidative stress. Additionally, the area of vascular bundles and xylem was greater in ScTpx2-OE plants compared to wild-type plants. Taken together, these data suggest that manipulating the anatomy of vascular bundles by modulating ScTpx2 levels can increase water deficit tolerance in crops, providing a potential strategy to cope with the challenges posed by climate change.

Funding Statement

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 National Council for Scientific and Technological Development, CNPq, grant 465319/2014-9 and grant 2022/04006-2 (FAPESP)). Fellowships: NT (Coordination for the Improvement of Higher Education Personnel, CAPES), WSB (São Paulo Research Foundation, FAPESP).

Disclosure Statement

No potential conflict of interest was reported by the author(s).

Data Availability Statement

The DNA constructs used in this study can be made accessible upon completion of a Material Transfer Agreement.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21645698.2025.2612426