Enhanced Photosynthesis and Water-Use Efficiency in Transgenic Sugarcane Expressing Arabidopsis DREB1A Under Drought Stress

Summary

This study evaluates Arabidopsis DREB1A-mediated drought tolerance in transgenic sugarcane. Under stress, enhanced photosynthesis, water-use efficiency, and biomass accumulation were observed. DR-21 exhibited the highest DREB1A expression, improving drought resilience. The findings provide insights into molecular mechanisms and offer a potential strategy for developing climate-resilient sugarcane varieties in water-limited regions.

Abstract

Drought stress imposes a critical limitation on sugarcane productivity, thereby necessitating the development of stress-resilient cultivars for sustainable agriculture. The present study aimed to enhance drought tolerance in sugarcane by introducing the Arabidopsis DREB1A gene under the control of the stress-inducible rd29A promoter. Transgenic sugarcane lines were generated via particle bombardment-mediated transformation and subsequently evaluated through molecular, physiological, and agronomic assessments under controlled and drought-stressed conditions.

Molecular analysis confirmed stable transgene integration, with transgenic lines exhibiting up to a 10-fold increase in DREB1A expression relative to wild-type plants. Physiological assessments demonstrated that, under drought stress at 60% field capacity (FC), transgenic lines maintained photosynthetic rates (P_N) that were 224-270% higher, stomatal conductance (g_s) increased by 84-167%, and relative water content (RWC) was enhanced by 25-31% compared to non-transgenic controls. Moreover, the leaves of transgenic sugarcane displayed improved osmotic regulation and water-use efficiency. Agronomic evaluations further revealed significant improvements in plant growth and productivity. Under drought stress (60% FC), transgenic lines exhibited 76-109% greater cane height, with cane diameter 71-86% larger. Shoot biomass increased by 39-87%, and root biomass was enhanced by 65-103%.

Additionally, the Brix percentage, indicative of sucrose accumulation, increased by 36-55% in the transgenic plants at 60% FC. These findings establish a robust correlation between DREB1A expression, enhanced physiological resilience, and improved agronomic performance under drought conditions. The capacity of DREB1A-expressing transgenic sugarcane to sustain higher photosynthetic activity, superior water-use efficiency, and increased biomass accumulation underscores its potential as a genetic strategy for developing drought-resilient sugarcane varieties. This study offers novel insights into the molecular mechanisms underlying drought tolerance and provides a promising approach for ensuring sustainable sugarcane cultivation in water-scarce regions.

Introduction

Sugarcane (Saccharum officinarum L.) is Pakistan's second major cash crop, primarily cultivated for its high sucrose content. However, optimal production is rarely achieved due to periodic water shortages^1,2. It is a member of the Poaceae grass family and is grouped in the PACCAD clade of the subfamily Panicoideae³. Members of this clade are often referred to as warm-season grasses and exhibit a C₄ photosynthetic system⁴. The primary mode of CO₂ fixation in sugarcane is C₄ photosynthesis with minimum photorespiration⁵. C₄ grasses, such as sugarcane, can fix more CO₂ than C₃ grasses, producing higher sucrose levels and exhibiting better nitrogen use efficiency (NUE) and water use efficiency (WUE)^6,7,8. However, the soil moisture requirements for sugarcane are quite high^9,10,11.

Due to the changing climate and limited water availability, producing high-yielding water-stress-tolerant sugarcane varieties is crucial^12,13,14. Plant tolerance to water stress is a multigenic trait influenced by the intensity, time, and duration of stress. Due to the geographical location of Pakistan (30.3753° N, 69.3451° E), conventional breeding practices are not feasible because of low flowering and pollen viability¹⁵. Therefore, varietal improvement through traditional breeding depends on genetic variation and the subsequent selection of desired agronomic traits¹⁶. In this context, transforming stress-related genes through genetic engineering is one of the most promising alternative approaches to improve plant stress tolerance^17,18,19.

Abiotic stress responses in plants are regulated by multiple transcription factor (TF) families and cis-acting elements, including WRKY, MYB, bHLH, ERF, and NAC. These TFs regulate networks in osmotic adjustment, reactive oxygen species (ROS) scavenging, stomatal regulation, and stress signaling pathways^{20,21,22,23,24,25,26}. For instance, WRKY TFs modulate stress signaling via ABA-dependent pathways, while MYB proteins regulate flavonoid biosynthesis and stomatal function under drought stress²⁷. Similarly, bHLH and ERF factors interact with ethylene signaling, thereby enhancing drought resilience²⁸. Given the complexity of stress responses, targeting key regulators such as DREB1A is a promising strategy for enhancing drought tolerance.

In this study, we investigated the role of ArabidopsisDREB1A in transgenic sugarcane, focusing on its impact on photosynthesis, water relations, and yield traits under water stress conditions. The role of DRE-binding (DREB) proteins in modulating water stress tolerance has been reported previously^27,29,30. For instance, Kasuga et al. observed that DREB positively regulates DRE sequence expression in Arabidopsis thaliana³¹. Moreover, many researchers have suggested that transgenic sugarcane overexpressing DREB1A exhibits constitutive expression of stress-inducible genes and higher sugar levels than wild-type sugarcane^17,28,32,33.

Protocol

Generation of rd29A: DREB1A expression vector (Figure 1)
The full-length DREB1A open reading frame (ORF, 651 bp) was amplified from Arabidopsis thaliana L. cDNA using gene-specific primers incorporating BamHI and EcoRI restriction sites: forward 5^/-GGCGGATCCATGAACTCATTTTCTGCT-3^/ and reverse 5^/-GGCGAATTCTTAATAACTCCATAACGA-3^/. Similarly, the rd29A promoter (499 bp) was amplified from Arabidopsis genomic DNA using primers containing KpnI and BamHI sites: forward 5^/-GCGGGTACCCCTATTAGAACGATTAAGGAG-3^/ and reverse 5^/-GGCGGATCCGGTGGTTCCTCTGTTTGATCC-3^/. PCR reactions were carried out in 25 µL volume containing 2.5 µL of 10x PCR buffer, 1.5 mM MgCl₂, 0.2 mM of each dNTP, 0.4 µM of each primer, 1 U of Taq DNA polymerase, and 1 µL of DNA template (approximately 100 ng for genomic DNA or 50 ng for cDNA), with nuclease-free water added to the final volume. Amplification conditions for both DREB1A and rd29A promoter fragments were as follows: initial denaturation at 95 °C for 3 min; 35 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 1 min; followed by a final extension at 72 °C for 7 min. Amplified products were resolved on a 0.7% agarose gel and gel-purified using a Gel Extraction Kit according to the manufacturer's protocol.

The amplified DREB1A and rd29A promoter PCR products were cloned into the cloning vector, pTZ57R/T, using TA cloning, and the products were subjected to Sanger Sequencing for confirmation. The DREB1A sequence (Accession No. AM992886) was submitted to GenBank (rd29A promoter was inserted into the KpnI and BamHI sites, and the DREB1A coding sequence was inserted downstream into the BamHI and EcoRI sites, thereby replacing the original 2x35S promoter and positioning DREB1A under the control of rd29A. The final cassette (rd29A::DREB1A::CamV) was excised using KpnIand EcoRV and cloned into the T-DNA region of the binary vector pGA482 to generate the construct used for plant transformation (Figure 1). After the initial Sanger sequencing to validate the cloned products, no further sequencing verification was carried out.

Gene sequence analysis
Using the referenced bioinformatics tool, a phylogenetic analysis of the protein sequences of AtDREB1A and DREB1 from 13 different dicot and monocot species was performed and compared via the neighbor-joining method. The amino acid sequences were retrieved from Phytozome (http://phytozome.jgi.doe.gov/pz/portal.html) and GenBank (http://www.ncbi.nlm.nih.gov/genbank/). Multiple sequence alignment analysis was performed using the Clustal Omega online program. Once the essential sequences have been acquired from Phytozome and GenBank, no more sequence retrieval is necessary.

Preparation of explants and callus induction gun^20,34
The sugarcane cultivar was grown in the experimental field at NIBGE, Faisalabad. After 6 months, the top portion of the healthy cane was excised to retrieve the apical region, which was trimmed to the terminal bud. Under sterile conditions, the leaf rolls were peeled into ~5 mm cylindrical segments and surface sterilized with 70% ethanol. From the region immediately above the apical meristem, approximately 10-15 apical discs (~3 mm in size) were prepared per cane. The discs were cultured on the Callus Induction Medium (CIM) as previously described^1,20,34. After 4-6 weeks, the resulting embryogenic Calli were subcultured on fresh CIM for further proliferation. NOTE: The use of 70% ethanol for surface disinfection was found sufficient, and no additional sterilization steps were required.

Genetic transformation of rd29A: DREB1A in Sugarcane through Gene Gun^20,34
The proliferated embryogenic callus was arranged in the center of the Petri plate containing CIM. After 3 days, the calli were bombarded with a gold-coated plant-expressible binary vector, pGA482, containing rd29A: DREB1A, using a PDS-1000/He Biolistic gun as described in detail³⁴. After bombardment, small portions of calli were cultured on CIM for 3 days and placed in the dark at 26 ± 2 ^oC. Then, the calli were cultured on callus selection medium (CSM) containing 60 mg/L geneticin, subcultured on CSM every 10 days, and placed in the dark at 26 ± 2 ^oC.

After 30 days, healthy calli were cultured on a regeneration selection medium (RSM) containing 60 mg/L antibiotic to get shoots. The plates were placed in a growth room for 4 weeks at a controlled temperature (26 ± 2 °C), 16 h/8 h light/dark photoperiod, 500 µmol m^-2 s^-1 light intensity, and controlled humidity³⁵. The regenerated health shoots were cultured on rooting selection medium (RtSM) containing 60 mg/L antibiotic in glass jars and placed in the growth room under the abovementioned conditions. After 4-6 weeks, putative transformants were transferred into small plastic pots containing sterilized sandy soil and placed in the growth room under the abovementioned conditions. No more sub-culturing on callus selection medium (CSM) is required beyond 10 days.

Molecular confirmation of transgenic sugarcane plants
As previously described, genomic DNA was isolated from the young leaves of putative transgenic sugarcane plantlets using the CTAB extraction method³⁶. DNA quality and concentration were assessed using a NanoDrop spectrophotometer and agarose gel electrophoresis. PCR amplification of the selectable marker gene nptII (750 bp) and transgene DREB1A (651 bp) was performed using gene-specific primers listed in Table 1. Each 25 µL PCR reaction contained 1x PCR buffer, 2.5 mM MgCl₂, 0.2 mM dNTPs each, 0.4 µM of each primer, 1 U of Taq DNA polymerase, ~100 ng of genomic DNA, and deionized water to volume. Reactions were run in a thermocycler under the following conditions: initial denaturation at 95 °C for 3 min; 35 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 45 s; followed by a final extension at 72 °C for 5 min. PCR products were resolved on a 1% agarose gel and visualized under UV light to confirm amplification. PCR-positive (T₀) transgenic plantlets were then transferred to large earthen pots and hardened in a controlled glasshouse environment until maturity. Upon maturation, cane segments from five randomly selected PCR-confirmed T₁ events were planted and propagated in microplots for 12 months to generate sufficient clonal sets for the drought stress experiment. NOTE: Once nptII and DREB1A genes have been confirmed, no additional PCR amplifications were performed.

DREB1A expression study
The relative expression of DREB1A in stressed leaves of transgenic and non-transgenic sugarcane plants was quantified using real-time PCR (qPCR). Total RNA was isolated from ~100 mg of leaf tissue using the extraction reagent according to the manufacturer's protocol. RNA quality was verified by agarose gel electrophoresis and quantified using a spectrophotometer. For each sample, 2 µg of RNA was reverse-transcribed using the cDNA Synthesis Kit following the supplier's instructions, with oligo(dT) primers.

The resulting cDNA was used as a template to amplify a 188 bp DREB1A using gene-specific primers: forward 5^'-ACAGAGGAGTTCGTCGGAGA-3^' and reverse 5^'-GAGTCTCCAAGCCGAGTCAG-3^'. Each 25 µL qPCR reaction contained 12.5 µL of 2x SYBR Green qPCR Master Mix, 0.4 µM of each primer, 2 µL of diluted cDNA (~100 ng template), and nuclease-free water to bring the volume to 25 µL. Reactions were performed in triplicate on a thermal cycler using the following program: initial denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s, with a final extension at 72 °C for 10 min.

To confirm the efficiency and linearity of the qPCR reactions, a standard curve was initially generated using 10-fold serial dilutions of plasmid DNA containing the cloned DREB1A fragment. However, for expression analysis, relative quantification was performed using the 2^−ΔΔCt method³⁷. The Actin gene served as the endogenous reference, and the expression level of DREB1A in each transgenic line was normalized to that of the wild-type control. All C_t values were collected using automatic baseline and threshold settings recommended by the instrument software. Only melt curves with single, sharp peaks were accepted, confirming specificity. High-quality, non-degraded RNA was essential for accurate quantification. Impure or degraded RNA samples were excluded to avoid compromising the reliability of cDNA synthesis and downstream gene expression analysis.

Study of DREB1A -mediated changes in sugarcane: water stress experiment^20,34
After 1 year, three replicates of five cane segments (two nodes/segment) of transgenic and non-transgenic (wild-type) plants were sown at 5 cm depth in earthen pots containing 18 kg of soil (EC_e 1.34; SAR 2.72; pH 7.8). Three water stress regimes:100, 80, and 60% FC were applied ( Table 2). Sugarcane photosynthetic activity (P_N, E, and g_s) and plant water relations (Ψ_w, Ψ_s, and RWC) were studied after 12 months. Do not change the established water regimes, as it will affect the consistency of the treatments.

Determination of photosynthetic activity
After 180 days of water stress, gas exchange parameters -- photosynthetic rate (P_N), transpiration rate (E), and stomatal conductance (g_s) -- were measured in both transgenic and wild-type sugarcane plants using a portable handheld photosynthesis system. Measurements were taken between 09:00 and 11:00 AM using the third fully expanded leaf from the top of each plant. The selected leaf was gently inserted into the built-in transparent leaf chamber (3 cm² clamp area), ensuring complete contact without creasing or obstruction. Ambient conditions were recorded in real-time by the system, while light intensity was maintained at approximately 1,000 µmol m^-2 s^-1 under natural sunlight. The device was allowed to equilibrate for 30-60 s until steady-state readings were obtained for CO₂ assimilation and water vapor exchange. The instrument's internal sensors automatically logged the leaf temperature, relative humidity, and ambient CO₂ concentration. For each genotype and treatment, measurements were replicated on at least three biologically independent plants, and data were stored internally and later exported for analysis. Care was taken to maintain consistent environmental conditions across replicates to ensure the reliability of the data.

Determination of leaf water potential and osmotic potential
The water potential (Ψ_w) of the third fully expanded leaf from the top of the plants was determined using the Scholander-type pressure chamber. The leaves were then frozen at -20 ^oC in 1.5 mL tubes, and the cell sap obtained from the frozen leaves was used to analyze osmotic potential (Ψ_s) using the Osmometer.

Determination of leaf relative water content
The water content of the transgenic and wild-type plant leaves was examined in detail. The fresh weights of the leaves were measured, and the leaves were immersed in distilled water overnight. Subsequently, the turgid weight of the leaves was recorded, whereas the dry weight was determined by incubating the leaves at 70 °C for 72 h. Leaf RWC was calculated using the following formula:

NOTE: To accurately analyze the osmotic potential using an osmometer, leaves must be frozen at -20 °C once the water potential has been determined.

Yield attributes
Sugarcane yield traits were measured at the harvest stage, such as cane height (cm), diameter, number of millable canes, brix (%), and shoot and root biomass.

Statistical analyses
Field experiments (controlled using a fence) were performed using a Randomized Complete Block Design layout with three replicates per treatment (five plants per replicate; n = 15). A two-way analysis of variance was performed to analyze the changes in different agronomic and physiological traits at a significance level of P ≤ 0.05.

Results

Molecular characterization of sugarcane transgenic lines
The rd29A: DREB1A expression cassette was constructed and cloned in the plant-expressible binary vector pGA482 (Figure 1), which was then transformed into the sugarcane genome using the biolistic-mediated transformation technique and successfully developed transgenic sugarcane plantlets as described (Figure 2). The presence of transgenes in sugarcane lines was confirmed by amp...

Discussion

Developing water stress tolerance in plants is crucial due to the ongoing expansion of drought-prone regions worldwide. As a potentially feasible biotechnological approach, we studied the role of DREB1A in water stress tolerance in transgenic sugarcane lines. Previous studies have reported the role of the DREB1A transcription factor in inducing the expression of multiple genes that confer tolerance to abiotic stress^28,38,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cloning vector, pTZ57R/T	Fermentas, Vilnius, Lithuania
infrared gas-exchange analyzer	Bio-Rad, California, USA
Osmometer	Bio-Rad, California, USA
PCR machine	Bio-Rad, California, USA
PDS-1000/He Biolistic gun	ThermoFisher Sceintific Inc, USA
Real-time PCR	ThermoFisher Sceintific Inc, USA
RevertAid cDNA Synthesis Kit	CI-340, CID Bio-Science, Inc., USA
Scholander-type pressure chamber	Skye instruments, Llandrindod Wells, Birmingham, UK
TRIzol reagent	Wescor-5500, Logan, Utah, USA