LcTprxII Overexpression Enhances Physiological and Biochemical Effects in Maize Under Alkaline (Na2CO3) Stress

Abstract

Alkaline stress limits crop productivity by causing osmotic and oxidative damage. This study investigated the new gene LcTprxII, a type II peroxiredoxin encoded by Leymus chinensis, and its role in enhancing alkaline stress tolerance in transgenic maize. The gene was cloned, overexpressed, and characterized using RT-PCR, phylogenetic analysis, and motif identification. Transgenic maize lines were generated via Agrobacterium-mediated transformation and subjected to physiological, biochemical, and transcriptomic analyses under alkaline stress. Under alkaline stress, the results revealed that LcTprxII overexpression significantly preserved chlorophyll content, mitigated oxidative damage, and maintained growth compared to wild-type plants, as evidenced by elevated activities of antioxidant enzymes (APX, CAT, SOD, and POD) and reduced malondialdehyde (MDA) content. Transcriptomic profiling identified 3733 differentially expressed genes and the upregulation of ABA and MAPK signaling pathways, highlighting the role of these genes in stress signaling and metabolic adaptation. Hormonal analysis indicated reduced ABA and increased GA levels in the transgenic lines. This study identified WRKY, bHLH, and MYB transcription factors as key regulators activated under alkaline stress, contributing to transcriptional regulation in transgenic maize. Field trials confirmed the agronomic potential of LcTprxII-overexpressing maize, with yield maintained under alkaline conditions. The present study revealed that LcTprxII enhances antioxidant defenses and stress signaling, which trigger tolerance to abiotic stress. Future studies should explore the long-term effects on growth, yield, and molecular interactions under diverse environmental conditions.

Keywords: LcTprxII; alkaline stress; hormone regulation; transcriptome; type II peroxiredoxin.

Figure 1

Phylogenetic relationship and expression pattern of LcTprxII. (A) Phylogenetic tree relationship with plant Prxs sequences retrieved through BLAST (https://www.ncbi.nlm.nih.gov/, accessed on 5 April 2025) in the NCBI database. The accession numbers, types, and species of Prxs from different plants are given. The bar indicates the scale for branch length. (B) The map of the pCAMBIA3300-T vector. (C) qRT-PCR was used to measure LcTprxII expression levels across different transgenic lines. The analysis included three biological duplications, with each biological duplication comprising three technical replicates. (D) Plant heights of WT and different transgenics. Results are expressed as mean values ± standard error (SE). Significant differences between wild-type (WT) plants and transgenic lines (#1, #2, and #4) are indicated by asterisks (t-test, *** p < 0.001).

Figure 2

The response of overexpression of LcTprxII when subjected to alkaline conditions was evaluated. (A) The growth performance of WT and transgenic plants before alkaline stress, (B) the growth performances of WT and transgenic plants after 9 days of alkaline stress, (C) the growth rate of WT and transgenic plants, and (D) chlorophyll SPAD values of the third expanded leaves of WT and transgenic lines. WT and transgenic plants were grown in a greenhouse until the leaf stage and treated with 75 mMol of Na₂CO₃. The values were measured during the 9 days of alkaline treatment. Bar = 10 cm. The data are presented as mean ± SE (n = 3).

Figure 3

Comparison of H₂O₂ content, CAT content, and antioxidant enzyme activity in transgenic maize and WT under alkaline (Na₂CO₃) treatment. WT and LcTprxII transgenic plants were grown in a greenhouse for 9 days. H₂O₂ content (A), CAT content (B), MDA (C), APX (D), SOD (E), and POD (F) were measured in WT and transgenic plants after 9 days of alkaline stress. Results are presented as mean values ± SE (n = 3). Asterisks denote significant differences between the wild-type (WT) and transgenic lines (#1, #2, and #4), as determined through the t-test (*** p < 0.001).

Figure 4

The enrichment of DEGs in the Biological Process GO terms and KEGG pathways analysis. The enrichment of DEGs in the biological GO terms: GO:0005737: cytoplasm; GO:0005622: intracellular; GO:0009507: chloroplast; GO:0005575: cellular_component; GO:0016021: integral component of the membrane; GO:0005777: peroxisome; GO:0030054: cell junction; GO:0044237: cellular metabolic process; GO:0008150: biological_process; GO:0008152: metabolic process; GO:0009987: cellular process; GO:0050896: response to stimulus; GO:0042221: response to chemical; GO:0009651: response to salt stress; GO:0006952: defense response; GO:0043169: cation binding; GO:0008289: lipid binding; GO:0020037: heme binding; GO:0046872: metal ion binding; GO:0005488: binding; GO:0003824: catalytic activity; GO:0004601: peroxidase activity. (A) The enrichment of DEGs were divided into three categories of biological process, cellular components, and molecular function with GO analysis. (B) KEGG pathway enrichment analysis.

Figure 5

Determinations of plant hormone in a three leaf stage overexpression LcTprxII treated with 75 mmol/L alkaline stress. (A) The Abscisic acid (ABA) of WT and overexpression LcTprxII treated with water and 75mmol/L alkaline stress for 9 days, (B) ABA-GE content of WT and overexpression LcTprxII treated with water and 75 mmol/L alkaline stress for 9 days, (C) Gibberellin A19 (GA19) WT and overexpression LcTprxII treated with water and alkaline stress for 9 days, (D) Gibberellin A29 (GA29) WT and overexpression LcTprxII treated with water and alkaline stress for 9 days. The data were presented as mean ± SE. The asterisks indicate the signifcant diferences between overexpression LcTprxII and WT plants (t-test, * p < 0.5, ** p < 0.01, *** p < 0.001).

Figure 6

RNA-seq analysis identified the expression patterns of differentially expressed genes (DEGs) associated with abscisic acid (ABA) and transcription factors (TFs) in transgenic maize line #1. (A) The relative expression of DEGs in the ABA metabolism pathway. Zm00001eb294600 (RCP22), Zm00001eb213190 (cyb5-1), Zm00001eb273440 (GLK1). (B) The relative expression of DEGs in TFs. Zm00001eb239380 (HSFTF11), Zm00001eb121380 (NACTF94), Zm00001eb124740 (EREB148), Zm00001eb419370 (MYB36), Zm00001eb212940 (bZIP6), Zm00001eb419370 (WRKY40). Each reaction was conducted with three biological duplicates, and every biological duplicate included three technical replicates. Data analysis employed the 2^−ΔΔCT method for statistical evaluation. Results are displayed as mean values, with error bars representing the standard error (SE).

Figure 7

Two key signaling pathways activated in response to stress: the MAPK cascade and the ABA-dependent pathway. The MAPK cascade involves sequential phosphorylation of MAPKKK, MAPKK, and MAPK, leading to transcription factor activation and stress-responsive gene expression, while the ABA pathway involves PYR/PYL/PCAR-mediated inhibition of PP2C, activation of SnRK2, and subsequent regulation of downstream targets, including SLAC1 and transcription factors, to modulate stress adaptation.