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, type II peroxiredoxin,
   transcriptome, hormone regulation

1. Introduction

   Infections and alkaline salts, as biotic and abiotic stresses, severely
   affect crop growth and agricultural output worldwide. The total area of
   saline–alkaline soils worldwide exceeds 434 million hectares
   [[54]1,[55]2]. Moreover, alkaline stress is recognized as a more
   detrimental factor than salt stress. It significantly impairs plant
   growth and development, making it a critical concern for crop
   productivity [[56]3]. Over 70% of the land areas that have undergone
   varying degrees of alkalinization continue to expand in northeastern
   China [[57]4]. Chinese alkaline soils contain substantial amounts of
   (Na[2]CO[3]) and (NaHCO[3]) [[58]5]. Alkaline and salt affect plant
   development and growth via osmotic variables, ionic toxicity, dietary
   abnormalities, and ROS bursts [[59]6].

   The two-phase model of alkaline damage outlined indicates that plant
   development first decreases because of osmotic stress and subsequently
   because of the accumulation of sodium ions (Na^+) [[60]7].
   Nevertheless, the two stages are similar. However, because it increases
   soil pH, sodium carbonate (Na[2]CO[3]) has a more noticeable adverse
   influence on plants than neutral salts, such as sodium chloride (NaCl).
   This suggests that alkaline stress is typically more destructive to
   plants than neutral salt stress.

   Alkaline stress induces significant amounts of different ROS, such as
   hydrogen peroxide (H[2]O[2]), superoxide (O[2]^−•), hydroperoxide
   (ROOH), hydroxyl peroxide (HO[2]^−•), and hydroxyl radicals (OH•). The
   rise of ROS levels can lead to oxidative damage to plant proteins and
   lipids [[61]8]. This damage significantly restricts plant growth and
   maturity, resulting in wilting and plant mortality [[62]9].

   Plants have evolved complex systems to preserve redox homeostasis when
   challenged by adverse environments [[63]10]. In response to
   environmental stress, plants minimize ROS using enzymes like POD, SOD,
   CAT, AsA, GSH, enzymes of the AsA–GSH cycle, and other antioxidants
   [[64]11]. Recent studies have demonstrated the antioxidant functions of
   mercaptan reductase in plants [[65]12].

   Chinese wildrye (Leymus chinensis), a monocot perennial grass,
   flourishes in alkaline–sodic soil. It has developed methods to adapt to
   unique living conditions through evolution. There are reports that the
   expressed sequence tag (EST) of type II Prx in Chinese wildrye, which
   was treated with Na[2]CO[3], was described to be ascending [[66]13].

   Peroxiredoxins (Prxs) are classified into four types according to their
   sequence homology and catalytic function: (a) 1-Cys Prx, (b) Prx II,
   (c) 2-Cys Prx, and (d) PrxQ [[67]14]. Chinese cabbage was initially
   characterized as a type II Prx [[68]15]. Subsequent genetic and
   biochemical studies revealed several type II Prxs in dicots. Studies
   have indicated that Arabidopsis thaliana has H[2]O[2]-reducing activity
   in type II Prxs [[69]16]. Additionally, poplar type II Prx has been
   shown to lower H[2]O[2], tBOOH, and CM-OOH levels in vitro [[70]17].
   Peroxiredoxins (Prxs) are involved in various cellular functions, such
   as providing antioxidant protection [[71]18], facilitating
   H[2]O[2]-dependent cell signaling [[72]19], and performing molecular
   chaperone roles [[73]20]. These signaling pathways encompass abscisic
   acid (ABA) and jasmonic acid (JA) and influence the alkaline stress
   response by modulating the transcriptional activity of Prx II genes,
   although transcription levels remain unchanged with plant age
   [[74]21,[75]22]. Previous research on type II peroxiredoxin (Prxs)
   physiology, plant hormones, and biochemistry in maize (Zea mays L.),
   Arabidopsis thaliana, and rice (Oryza sativa) has demonstrated this and
   transcription regulation of plants [[76]23,[77]24].

   Chinese-wildrye-growing plants have evolved responses that adapt to
   various environmental threats. Finding functional gene(s) primarily
   responsible for stress response could help improve crop yields via
   biotechnological breeding. In a previous study, a peroxiredoxin-like
   sequence was identified in Leymus chinensis that was Na[2]CO[3]-treated
   under simulated alkaline conditions [[78]13]. We cloned a type II
   peroxiredoxin gene, LcTprxII, which is induced by alkaline stress and
   shows elevated antioxidant activity. In this study, we overexpressed
   the LcTprxII gene in maize to investigate whether it can increase maize
   alkalinity resistance and its regulatory pathways. This study provides
   a novel approach with the potential to improve alkaline stress
   tolerance in maize dicots and monocots.

2. Results

2.1. Phylogenetic Relationship and Expression Pattern of LcTprxII

   To investigate the evolution of LcTpxII, a phylogenetic tree was
   constructed based on the similarities of the deduced amino acid
   sequences of 28 available type II Prx genes from various plant species
   ([79]Figure 1A). Based on Dietz’s classification, the Prx proteins
   formed four major types, and LcTpxII clustered most closely with type
   II Prx members, including those from Zea mays, Oryza sativa, and
   Arabidopsis thaliana. Sequence analysis indicated that LcTpxII shares
   approximately 90% identity with Zea mays type II Prx, 87% with Oryza
   sativa, and 75% with Arabidopsis thaliana. The conserved cysteine
   residue at position 51 was identified as potentially involved in
   intramolecular disulfide bond formation during the catalytic cycle.
   Further sequence characterization of LcTpxII confirmed a 489 bp open
   reading frame encoding a 162 amino acid polypeptide with an estimated
   molecular weight of 17,400 Da and an isoelectric point of 5.12. Motif
   analysis revealed a thioredoxin domain and four predicted motifs: an
   N-myristoylation site, a casein kinase II phosphorylation site, an
   amidation site, and a protein kinase C phosphorylation site
   ([80]Supplementary Figure S1). Overall, the phylogenetic placement and
   high sequence identity with monocot Prx proteins strongly indicate that
   LcTpxII is a member of the type II Prx subfamily ([81]Table S1). A
   binary plasmid was assembled for plant transformation ([82]Figure 1B).
   The LcTpxII coding sequence was placed under the control of the maize
   polyubiquitin (ZmUbi) promoter. At the same time, the bar selection
   marker was governed by the promoter of the Cauliflower mosaic virus 35S
   (CaMV 35S). A Nos terminator was positioned downstream of LcTpxII to
   ensure appropriate transcription termination. Following
   Agrobacterium-mediated transformation, transgenic lines (#1, #2, and
   #4) were verified, and the expression of LcTpxII was quantified
   relative to wild-type (WT) plants through qRT-PCR ([83]Figure 1C). The
   expression levels of LcTpxII in each line were assessed using qRT-PCR.
   It was observed that the levels varied among the three lines, with line
   #1 exhibiting the highest expression, line #4 showing the lowest, and
   line #2 displaying an intermediate level. Notably, no expression of
   LcTpxII was detected in the wild-type (WT) samples. These findings
   align with expectations and confirm the normal expression of LcTpxII in
   the transgenic plants, confirming that the introduced construct
   effectively drove the overexpression of LcTpxII in the transgenic
   plants. Measurements of plant height are presented in [84]Figure 1D.
   The WT plants reached a mean height of approximately 160 cm. In
   contrast, each of the transgenic lines (#1, #2, and #4) displayed
   reduced height, ranging between 120 cm and 130 cm. These decreases were
   statistically significant, with p < 0.001 for all three lines compared
   to WT. Among the transgenic lines, the smallest average height was
   observed in line #1 (approximately 120 cm), although no statistically
   significant differences in height were detected among the three lines
   themselves.

Figure 1.

   [85]Figure 1
   [86]Open in a new tab

   Phylogenetic relationship and expression pattern of LcTprxII. (A)
   Phylogenetic tree relationship with plant Prxs sequences retrieved
   through BLAST ([87]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).

2.2. Physical and Biochemical Interactions of LcTprxII Overexpression with
Alkaline Stress

   To assess the impact of LcTprxII overexpression on alkaline stress
   tolerance, three T[2] transgenic maize lines (#1, #2, and #4) were
   analyzed under alkaline-stressed conditions. The growth rate showed
   slight variations after reaching the three-leaf stage in a greenhouse
   with normal conditions. Still, no other noticeable phenotypic
   differences were observed between transgenic lines (#1, #2, and #4) and
   control plants ([88]Figure 2A). Following nine-day exposure to 75
   mmol/L of alkaline treatment, control plants exhibited visible
   phenotypic changes. In contrast, transgenic lines demonstrated reduced
   damage ([89]Figure 2B). Under alkaline stress conditions, chlorophyll
   content and plant growth rate were monitored over time. Prior to
   alkaline treatment, transgenic maize exhibited higher chlorophyll
   content and shorter plant height compared to the WT. Upon exposure to
   alkaline stress, WT plants displayed a slower growth rate than
   transgenic maize ([90]Figure 2C). The reduction in chlorophyll content
   occurred at a significantly slower rate in transgenic plants compared
   to WT ([91]Figure 2D). These findings suggest that transgenic maize
   experienced less disruption in growth and development under alkaline
   stress than WT plants.

Figure 2.

   [92]Figure 2
   [93]Open in a new tab

   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 Na[2]CO[3]. The values were measured during the
   9 days of alkaline treatment. Bar = 10 cm. The data are presented as
   mean ± SE (n = 3).

2.3. Effects of Alkaline Stress on Antioxidant Enzymes and Oxidative Markers

   Antioxidant enzymes and oxidation markers are widely recognized as key
   indicators of plant response to environmental stress. These factors
   play critical roles in mitigating osmotic and oxidative stress, thereby
   contributing to tolerance to alkaline stress. These specific indicators
   were examined in the current investigation, with results presented in
   [94]Figure 3A. Transgenics exhibited significantly lower levels of
   H[2]O[2] compared to the WT. The levels in the transgenic lines were
   0.10–0.01-fold, 0.05–0.01-fold, and 0.10–0.01-fold those in control
   plants. These results indicate that the overexpression of LcTprxII
   contributes to a reduction in the accumulation of this ROS. The MDA
   content of transgenic maize after 9 days of alkaline stress treatment
   was reduced by 20%, 30%, and 40% when compared to the WT ([95]Figure
   3B), leading to lower levels of oxidative damage to cell membranes.
   Antioxidant reductases, including APX, SOD, CAT, and POD, have been
   reported to be associated with the elimination of ROS. In the present
   study, compared to the WT, in APX activity, transgenic lines exhibit
   significantly lower reduction by 1.29- to 2.49-fold ([96]Figure 3C),
   and the activity levels of SOD, CAT, and POD in transgenic maize
   increased by 2.38–2.51-fold ([97]Figure 3D), 3.30–2.32-fold ([98]Figure
   3E), and 2.78–2.37-fold ([99]Figure 3F), respectively, when under
   alkaline stress conditions. However, the results indicate that LcTprxII
   overexpression enhances antioxidant enzyme activity, reduces the
   accumulation of ROS, and decreases oxidative damage to the cell
   membrane in maize.

Figure 3.

   [100]Figure 3
   [101]Open in a new tab

   Comparison of H[2]O[2] content, CAT content, and antioxidant enzyme
   activity in transgenic maize and WT under alkaline (Na[2]CO[3])
   treatment. WT and LcTprxII transgenic plants were grown in a greenhouse
   for 9 days. H[2]O[2] 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).

2.4. Differential Transcriptome Analysis Between Transgenic Maize and Control
Plants

   Differentially expressed genes from comparisons between transgenic and
   wild-type plants were analyzed using GO functional enrichment
   methodology.

   First, 3733 DEGs were annotated within the Biological Process (BP), the
   Cellular Component (CC), and Molecular Function (MF) categories
   ([102]Table S3). Pathways related to metabolic and cellular processes,
   such as cellular metabolic processes (GO:0044237), are prominently
   enriched, emphasizing active biochemical and physiological functions.
   In the Molecular Function (MF) category, terms like binding
   (GO:0005488) and catalytic activity (GO:0003824) underscore the
   importance of molecular interactions and enzymatic processes. The
   Cellular Component (CC) analysis identifies the cytoplasm (GO:0005737)
   and other intracellular compartments as key loci for these activities,
   reflecting the centrality of intracellular dynamics. Furthermore, a
   KEGG analysis was performed in the present research. It was shown that
   the enrichment of 1793 DEGs in 14 pathways was coupled with alkaline
   stress. The plant–pathogen interaction and plant hormone signal
   transduction pathways are the most enriched, indicating a strong
   emphasis on defense signaling and hormonal regulation in response to
   environmental stimuli. The MAPK signaling pathway further highlights
   its role in transducing stress signals into adaptive cellular responses
   (see [103]Figure 4). Metabolic pathways, such as carbon metabolism,
   starch, sucrose metabolism, and phenylpropanoid biosynthesis, are
   significantly enriched, emphasizing the importance of energy
   production, carbohydrate utilization, and secondary metabolite
   synthesis for structural and defensive functions. Stress-related
   pathways, including glutathione metabolism, peroxisomes, and oxidative
   phosphorylation, reveal mechanisms for maintaining redox balance and
   energy homeostasis. Additionally, enrichment in photosynthesis and
   photosynthesis antenna proteins suggests regulation of light harvesting
   and energy capture ([104]Table S4).

Figure 4.

   [105]Figure 4
   [106]Open in a new tab

   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.

2.5. Differential Regulation of ABA Metabolites in LcTprxII Overexpression

   To investigate the role of LcTprxII in hormone signaling, we quantified
   the levels of key hormones involved in the stress response and growth
   regulation ([107]Figure 5A), including ABA levels in WT and OE lines
   under controlled and treated conditions. Under control conditions, no
   significant difference in ABA content was observed between WT and OE
   lines (p > 0.05). However, following treatment, the OE line exhibited
   significantly lower ABA accumulation than the WT line (p < 0.001).
   Specifically, ABA levels in the WT treated plants remained comparable
   to control levels, whereas the treated OE line showed a marked
   reduction. ABA-GE, a conjugated form of ABA, displayed a distinct
   pattern ([108]Figure 5B). Under control conditions, the ABA-GE content
   did not differ significantly between the WT and OE lines (p > 0.05).
   Similarly, no significant difference was found between WT and OE lines
   after treatment (p > 0.05). However, the ABA-GE content in both the OE
   lines and the WT plants under control and treatment conditions was
   insignificant. Furthermore, the levels of GA19 and GA20, two precursors
   of the gibberellin biosynthesis pathway, were examined. Under control
   conditions, GA19 levels were similar between WT and OE lines (p > 0.05)
   ([109]Figure 5C). Following treatment, the OE line exhibited a notable
   increase in GA19 content compared with the treated WT line (p < 0.05).
   While the treated WT line showed a slight, non-significant decrease in
   GA19, the OE line showed a trend towards increased GA19. In contrast,
   the GA20 response presented a significant difference between groups
   ([110]Figure 5D). Under control conditions, GA20 levels were not
   significantly different between the WT and OE lines. However, after
   treatment, the OE line showed a considerable increase compared to the
   WT group (p < 0.01).

Figure 5.

   [111]Figure 5
   [112]Open in a new tab

   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).

2.6. Difference in Agronomic Traits Due to LcTprxII Overexpression

   To investigate whether the results obtained in the greenhouse were
   reproducible under field conditions, homozygous transgenic and control
   plants were evaluated in a field trial. The field was established in
   GMO fields in Jilin province, China (125° E, 44° N), following three or
   more biological replications in randomized blocks per line (transgenic,
   control), respectively. The results were significantly higher in the
   transgenic maize plants under alkaline conditions with regard to
   parameters like plant height, ear height, grain weight, and cob
   diameter, while #1 and #2 exhibited an increase in plant height of
   23.93%, 20.07%, and 21.16% and an increase in ear height of 31.27%,
   33.10%, and 34.87%, respectively, compared with the wild type
   ([113]Table S5). Moreover, interestingly, we found that the plant
   height improvement was effective in transgenic lines under alkaline
   stress compared with the WT plant height. Furthermore, the agronomic
   trait between transgenic lines and WT was investigated. Transgenic
   plants achieved a significant increase in ear height, grain weight, and
   cob diameter ([114]Table S5). The results of this study indicated that
   the transgenic lines of the LcTprxII gene express resistance to
   alkaline tolerance in maize.

2.7. Differentially Expressed TFs Under Alkaline (Na[2]CO[3]) Stress

   To investigate the potential functions of the LcTprxII-OE gene,
   transcriptome analysis revealed significant differential expression of
   several transcription factor families in response to stress treatment
   in maize. The major families included 25 DEGs encoding vimyb avian
   myeloblastosis viral oncogene homolog (MYB), 28 coding for
   domain-containing protein (WRKY), 13 coding for NAC domain-containing
   proteins (NAC), 7 coding for bZIP basic leucine zipper (bZIP), 2 coding
   for ethylene-responsive factor (ERF), 4 coding for heat stress factor
   (HSF), and 16 coding for basic helix-loop-helix (bHLH). Among these, 95
   differentially expressed genes (DEGs) showed consistent expression
   patterns in transgenic overexpression wild type ([115]Table S6).
   However, DEGs of these families showed different expression patterns;
   there are 5 upregulated and 20 downregulated DEGs for MYB, 4
   upregulated and 24 downregulated DEGs for WRKY, 2 upregulated and 11
   downregulated DEGs for NAC, 3 upregulated and 4 downregulated DEGs for
   bZIP, 0 upregulated and 2 downregulated DEGs for AP2/ERF, 1 upregulated
   and 3 downregulated DEGs for HSF, and 8 upregulated and 9 downregulated
   DEGs for bHLH.

2.8. The Verification of Gene Expression Candidates

   To verify the integrity of the RNA-Seq data, three abscisic acids
   (ABAs) and six DEGs were selected from each TF family, and qRT-PCR also
   verified the expression patterns. Zm00001eb396390 (MYB),
   Zm00001eb419370 (WRKY), Zm00001eb121380 (NAC), Zm00001eb212940 (bZIP),
   Zm00001eb124740 (AP2/ERF), and Zm00001eb239380 (HSF) were upregulated
   in LcTprxII-OE maize, while they were downregulated in LcTprxII-OE
   maize and randomly chosen for qRT-PCR ([116]Table S7). These results
   are consistent with those of the transcriptome data ([117]Figure 6B and
   Table 1). Therefore, this indicates that these TFs play an important
   role in the tolerance of transgenic maize to drought stress.

Figure 6.

   [118]Figure 6
   [119]Open in a new tab

   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).

3. Discussion

   Type II peroxiredoxins (Prxs) play a crucial role in regulating plant
   physiological processes, serving as key modulators of growth and
   development mechanisms. Plants must navigate complex challenges
   involving metabolic adaptations, protective responses, and reproductive
   strategies. Environmental stressors like alkaline conditions can
   significantly impair plant function by triggering ROS accumulation and
   undermining cellular stress resilience [[120]24]. To explore the
   genetic modification of LcTprxII overexpression in transgenic maize, it
   provides a molecular strategy for enhancing alkaline stress tolerance,
   revealing intricate mechanisms of plant adaptive responses.
   Interestingly, protein alignment indicated that LcTprxII and its
   homologs contain an N-terminal secretory signal peptide
   ([121]Supplementary Figure S1). These results supported the idea that
   LcTprxII is secreted and active in type II peroxiredoxin ([122]Figure
   1A). Different types of peroxiredoxin were induced by differential
   stress and have been characterized by alkaline stress [[123]25].

   Phylogenetic analysis showed that LcTprxII is a homolog of several
   reported type II peroxiredoxin (PrxsII) proteins, including OsPrxIIC,
   ZmPrx5, AtPrxIIB, AtPrxIIC, and AtPrxIID [[124]26,[125]27]. These
   PrxsII proteins were proposed to be involved in plant defense
   responses. The physiological and biochemical indicators and
   transcriptome analysis revealed the remarkable role of LcTprxII
   overexpression in transgenic maize for the enhancement of alkaline
   tolerance ([126]Figure 3). This research indicated that overexpression
   of LcTprxII in transgenic maize significantly improves alkaline stress
   tolerance through various mechanisms, including enhanced antioxidant
   enzyme activity, improved stress signaling, and transcriptional
   regulation. Under alkaline stress, the transgenic plants showed
   increased activity of APX, SOD, CAT, and POD, which collectively
   reduced ROS levels and limited membrane lipid peroxidation ([127]Figure
   3C–F). These results are consistent with the established role of type
   II peroxiredoxins in strengthening antioxidant defenses and promoting
   stress resilience [[128]25,[129]26,[130]27]. Importantly, homologous
   proteins, such as ZmPrx5-1 in maize and OsPrxII2 in rice, have also
   been shown to enhance ROS scavenging and maintain cellular metabolism
   under various stress conditions [[131]28], emphasizing the conserved
   function of PrxII proteins across plant species.The transcriptome
   analysis revealed that the DEGs were significantly enriched in the
   plant hormone signal transduction pathways and MAPK signal pathways
   ([132]Figure 4A,B).

   Transcriptomic profiling revealed significant differential expression
   of genes associated with hormone signaling, particularly in the
   abscisic acid (ABA) and mitogen-activated protein kinase (MAPK)
   pathways. ABA signaling, known for regulating ion homeostasis and
   metabolic adjustments under stress [[133]29,[134]30,[135]31], was
   enriched in transgenic lines. This included genes encoding PYR/PYL/RCAR
   receptors, which modulate downstream stress responses. In [136]Figure
   7, ABA signaling involves PYR/PYL receptors that inhibit PP2C
   phosphatases, leading to the activation of SnRK2 kinases. These kinases
   then phosphorylate downstream targets, such as AREB/ABF transcription
   factors and SLAC1, which regulate stomatal closure and
   stress-responsive gene expression. Additionally, MAPK pathway
   activation, critical for amplifying stress signals [[137]32], was
   observed, suggesting a synergistic interaction between ABA and MAPK
   signaling. Alkaline stress likely induces ABA-mediated PYR expression,
   which relieves SnRK2 inhibition and activates MAPK cascades
   (ZmMAPKKK18, homologous to drought-responsive NPK1 in tobacco)
   [[138]25]. This sequential activation enhances stress signal
   transduction, as evidenced by the rapid upregulation of Zm00001d043742
   (50-fold at 6 h), an ortholog of rice OsMAPKKK63 implicated in salt
   tolerance [[139]33,[140]34]. The MAPK pathway, depicted in [141]Figure
   7 as a cascade involving MAPKKK, MAPKK, and MAPK, amplifies stress
   signals and interacts with ABA signaling to enhance stress responses.
   This synergistic interaction is critical for the plant’s ability to
   adapt to alkaline stress. By integrating the roles of ABA and MAPK
   signaling pathways, our findings highlight the complex regulatory
   networks that plants utilize to respond to environmental stresses. The
   interplay between these pathways not only enhances stress signal
   transduction but also ensures a coordinated response that mitigates the
   adverse effects of alkaline stress. This integrated approach provides a
   comprehensive understanding of the molecular mechanisms underlying
   stress tolerance in transgenic maize overexpressing LcTprxII.

Figure 7.

   [142]Figure 7
   [143]Open in a new tab

   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.

   Plants have evolved a range of stress resistance mechanisms in which
   transcription factors play a key regulatory role. Although LcTprxII is
   a peroxiredoxin, it likely regulates TF genes indirectly by altering
   the redox status of the cell. This modulates ROS levels, which are
   known to affect transcriptional responses. TFs linked to stress
   adaptation were differentially expressed in transgenic plants
   ([144]Table 1).

Table 1.

   DEGs of the transcription factors involved in abiotic stress responses.
   Group Gene ID Log[2]FC Swiss_Prot Annotation NR_Annotation Reference
   MYB Zm00001eb396390 1.09 Myb-related protein P OS = Zea mays OX = 4577
   GN = P PE = 2 SV = 1 myb-related protein Myb4 [Zea mays] [[145]35]
   WRKY/40 Zm00001eb419370 2.68 WRKY transcription factor SUSIBA2 OS =
   Hordeum vulgare OX = 4513 GN = WRKY46 PE = 1 SV = 1 WRKY transcription
   factor SUSIBA2-like [Zea mays] [[146]36]
   Zm00001eb149570 1.24 WRKY DNA-binding transcription factor 70 OS =
   Solanum lycopersicum OX = 4081 GN = WRKY70 PE = 2 SV = 1 WRKY DNA
   binding domain-containing protein [Zea mays] [[147]37]
   NAC Zm00001eb121380 1.01 NAC domain-containing protein 75 OS =
   Arabidopsis thaliana OX = 3702 GN = NAC075 PE = 2 SV = 1 ANAC075
   isoform X2 [Zea mays] [[148]38]
   bZIP Zm00001eb212940 1.03 bZIP transcription factor RISBZ2 OS = Oryza
   sativa subsp. japonica OX = 39947 GN = RISBZ2 PE = 1 SV = 1 unknown
   [Zea mays] [[149]39]
   AP2/ERF Zm00001eb124740 −1.42 AP2/ERF and B3 domain-containing protein
   Os01g0141000 OS = Oryza sativa subsp. japonica OX = 39947 GN =
   Os01g0141000 PE = 2 SV = 1 AP2/ERF and B3 domain-containing protein
   Os01g0141000 [Zea mays] [[150]36,[151]40]
   HSF Zm00001eb198620 −1.38 Heat stress transcription factor B-2b OS =
   Oryza sativa subsp. japonica OX = 39947 GN = HSFB2B PE = 2 SV = 1
   uncharacterized protein LOC100283530 isoform X1 [Zea mays] [[152]37]
   Zm00001eb239380 1.47 Heat stress transcription factor C-2a OS = Oryza
   sativa subsp. japonica OX = 39947 GN = HSFC2A PE = 2 SV = 1 Unknown
   [Zea mays] [[153]41,[154]42]
   [155]Open in a new tab

   The first column represents the group name of DEGs involved in abiotic
   stress responses; the second column represents the gene ID; and the
   subsequent columns represent the fold change (log2FC) of the DEGs. The
   last column represents the annotation information of DEGs in Swiss-Prot
   and NR databases.

   Upregulated MYB (Zm00001eb396390) and WRKY TFs (Zm00001eb419370,
   Zm00001eb149570) align with their roles in oxidative stress mitigation
   and defense [[156]34]. Conversely, the downregulation of AP2/ERF
   (Zm00001eb124740) implies a potentially negative regulatory role in
   stress responses [[157]43]. The divergent expression of heat shock
   factors (HSFs) in upregulated Zm00001eb239380 versus downregulated
   Zm00001eb198620 further highlights the functional complexity within TF
   families during stress [[158]44]. These findings mirror observations in
   drought-tolerant maize [[159]45,[160]46], suggesting conserved TF
   networks across stress types. Critically, transgenic maize maintained
   robust growth and yield in natural alkaline soils, in contrast to prior
   studies that were limited to controlled environments [[161]47]. This
   field validation underscores the translational potential of LcTprxII
   overexpression for breeding alkaline-resistant crops. The absence of
   growth penalties under field conditions further supports its
   agricultural applicability. Although our work elucidates key
   mechanisms, several functional characterizations of specific
   differentially expressed genes (DEGs) are needed to dissect their roles
   in alkaline tolerance. Additionally, exploring ABA, MAPK, and other
   hormone pathways (e.g., auxin and ethylene) could unravel broader
   regulatory networks. Extended field trials across diverse alkaline
   environments will validate the universality of these findings and
   assess the stability of LcTprxII-mediated tolerance under various
   conditions. In addition, LcTprxII overexpression is expected to
   modulate ROS levels, thereby impacting redox-sensitive TF activation,
   hormone (ABA) signaling, and MAPK kinase cascades. These interconnected
   pathways collectively shape the differential TF expression observed in
   the transgenic maize. Such a mechanism aligns with reports that
   peroxiredoxins regulate H[2]O[2]-dependent transcription and ABA
   responses.

4. Materials and Methods

4.1. Cloning and Transformation of LcTprxII

   In this study, the LcTprxII gene, which confers alkaline resistance,
   was previously cloned from Leymus chinensis [[162]48]. The LcTprxII
   gene (GenBank accession no [163]GQ397275) was inserted into the monocot
   expression vector pCAM3300, resulting in the construct designated as
   pCAM3300-LcTprxII. The expression of the LcTprxII gene was driven by
   the ubiquitin promoter ([164]Figure 1A). Transgenic maize plants were
   generated through Agrobacterium-mediated transformation, following the
   methodology described by [[165]49]. The LcTprxII gene (T[0]) was
   introduced into the inbred maize through backcrossing. BC[1]F[1] was
   produced, BC[1]F[1] was backcrossed to produce BC[2]F[1], and this
   process was repeated until BC[4]F[1] lines were generated. Transgenic
   plants were selected based on herbicide resistance and PCR confirmation
   of the LcTprxII gene. Four independent transgenic lines were used for
   further analysis.

4.2. Phenotyping and Relative Chlorophyll Content Analysis

   Plant height, measured from the first internode to the tip of the first
   leaf, was recorded starting from the 3-leaf stage in seedlings grown in
   the greenhouse, with three biological replicates. The relative
   chlorophyll content was assessed using a SPAD-502 meter (Konica
   Minolta, Chiyoda City, Japan) on the third fully expanded leaf, also
   with three replicates.

4.3. Physiological and Biochemical Analysis

   Frozen leaf tissues (1 g) were homogenized in 10 mL of 0.1 M potassium
   phosphate buffer (pH 7.0) containing 0.1 mM EDTA-Na[2], 0.5 mM
   ascorbate, and 1% polyvinyl polypyrrolidone (PVPP). After filtration,
   the homogenate underwent centrifugation at 28,710× g for 10 min while
   maintained at 4 °C. Protein content and antioxidant enzyme activities
   were analyzed using the supernatant. Following established protocols,
   nitroblue tetrazolium (NBT) staining was performed on leaves collected
   from both wild-type (‘WT’) and overexpression (‘OE’) seedlings that had
   been exposed to saline–alkaline stress conditions [[166]50]. The
   H[2]O[2] concentration was calculated using a standard curve [[167]51].
   Superoxide dismutase activity was determined according to [[168]52].
   Peroxidase activity was determined according to [[169]53]. Catalase
   activity was assayed using the method described by [[170]54]. Ascorbate
   peroxidase activity and MDA content were determined according to
   [[171]55,[172]56]. The biological data were used for all measurements,
   and statistical differences were determined using Student’s two-tailed
   t-test.

4.4. Determination of Endogenous Plant Hormones (GA, ABA)

   The shoot tips from vegetative stage V3 homozygous mutants and
   wild-type controls grown in the greenhouse were harvested, with ten
   individual plants combined to create a single biological replicate. The
   previously mentioned endogenous hormone levels were analyzed using
   NetWare ([173]http://www.metware.cn, accessed on 17 August 2024) on the
   AB Sciex QTRAP 6500 LC-MS/MS platform. The procedures for the analysis
   are outlined as follows: for the determination of endogenous
   gibberellins (GAs), refer to the outlined method [[174]57]; for
   abscisic acid (ABA) determination, refer to the specified method
   [[175]58].

4.5. Field Trials and Yield Assessment of LcTprxII Overexpression

   Transgenic LcTprxII plants and wild-type controls were grown in
   genetically modified organism (GMO) fields in Jilin province, China
   (125° E, 44° N), following a randomized block design with two
   replications for each genotype. Each block comprised 40 plants arranged
   in two rows of 20, with 25 cm × 30 cm spacing between each plant. In
   general, two transgenic plants of #1 and #2 were selected to be planted
   in the field; the #4 transgenic was not selected due to its poor
   performance at the seedling stage. The plants were subjected to
   watering and alkaline stress treatments. Morphological and yield
   parameters were assessed, with plant height measured from the ground to
   the tassel tip and ear height measured from the ground to the base of
   the first ear. Stem diameter was measured at the first internode above
   ground using a Vernier caliper oriented perpendicular to the direction
   of leaf growth. The harvest index (HI) was calculated as the seed
   weight ratio to total biomass, including dry vegetative tissue and
   panicle weight. Unit yield was obtained by extrapolating seed weight
   from the block area to a unit area. All measurements were conducted on
   at least 24 plants per maize line, excluding those from the two
   outermost rows in each block.

4.6. RNA-Seq Analysis and Expression Patterns of LcTprxII-OE

   RNA sequencing was conducted on LcTprxII-OE (overexpression) seedlings
   and wild-type seedlings at the V3 developmental stage. Leaf tissues
   were harvested, and RNA was extracted from transgenic overexpression
   and wild-type plants and divided into three biological replicates.
   Sequencing libraries were generated using the NEBNext UltraTM RNA
   Library Prep Kit for Illumina (NEB, Ipswich, MA, USA). Clean reads were
   mapped to the maize reference genome
   ([176]http//ftp.ensemblgenomes.org/pub/plants/release-32/fasta/zea_mays
   /, accessed on 17 August 2024) using the Tophat2 algorithm, which was
   employed to process the data. Sequences exhibiting exact matches or
   containing a single mismatch with the reference genome underwent
   additional examination and classification according to two reference
   databases: the KEGG Orthology database
   ([177]https://www.kegg.jp/kegg/ko.html, accessed on 17 August 2024) and
   the Gene Ontology (GO) database ([178]http://www.geneontology.org/,
   accessed on 17 August 2024). Gene expression was measured as fragments
   per kilobase of the transcript per million mapped fragments.
   Statistical significance was assessed using q-values for p-value
   adjustment following the method of Storey and Tibshirani [[179]59].
   Differentially expressed genes were identified using dual criteria: a
   Q-value below 0.005 and a log2 (fold change) equal to or exceeding 1.
   The raw sequencing data have been uploaded to the NCBI public database
   under project number PRJNA1222582.

4.7. Real-Time PCR (qRT-PCR) Analysis

   Total RNA was extracted using the TRIzol reagent from Sangon Biotech,
   Shanghai Co., Ltd. For cDNA synthesis, 500 ng of DNase-treated RNA was
   utilized with an RT reagent kit from TakaRa (Shanghai, China),
   following the manufacturer’s instructions. Quantitative reverse
   transcription PCR (qRT-PCR) was conducted on an ABI7500 qRT-PCR System
   using the SYBR^® RT-PCR Kit from Takara (Dalian, China). The maize
   actin1 gene served as the internal control. Specific primer sequences
   for the LcTprxII gene were designed for the qRT-PCR assays. The thermal
   cycling conditions included an initial denaturation at 95 °C for 5 min,
   followed by 40 cycles of 95 °C for 15 s (denaturation), 54–62 °C for 15
   s (annealing), and 72 °C for 37 s (extension). A mixed solution without
   a template was included as a negative control. Statistical analyses
   were performed using the 2^−∆∆CT [[180]58,[181]60] Method. The qRT-PCR
   experiments were conducted with three biological replicates. Gene
   expression pattern analysis was performed by using qRT-PCR. To verify
   the RNA sequencing, leaves of the WT and transgenic plants at the V3
   stage were harvested for qRT-PCR. The actin gene (GenBank accession no
   [182]AB181991) was used as an internal control. Each experiment had
   three biological replicates.

4.8. Statistical Data Analysis

   Statistical analysis was conducted on phenotyping, relative chlorophyll
   content assessment, alkaline treatment, and physiological/biochemical
   evaluations. The data were statistically processed, and the means of
   each transgenic line (#1, #2, #4) and the wild-type control (WT) were
   compared using a t-test, with calculations performed using GraphPad
   version 10.0 software (San Diego, CA, USA) [[183]61]. Results are
   presented as the mean accompanied by the standard error (SE) of the
   mean. All experiments included three or more biological replicates.

5. Conclusions

   The study shows that LcTprxII overexpression in transgenic maize
   improves alkaline stress tolerance through enhanced antioxidant
   defense, hormonal regulation, and transcriptional reprogramming. The
   transgenic lines showed elevated antioxidant enzyme activities and
   reduced oxidative damage, with lower malondialdehyde levels.
   Transcriptomic profiling identified 3733 differentially expressed genes
   (DEGs) enriching ABA and MAPK signaling pathways. Hormonal analysis
   revealed reduced ABA and increased GA levels under stress, promoting
   growth. Field trials confirmed the agronomic potential of LcTprxII-OE
   maize by maintaining yield and biomass under alkaline conditions. These
   findings suggest LcTprxII as a promising candidate for breeding
   stress-tolerant crops, particularly in alkaline-soil-affected regions.
   Future research should focus on the functional characterization of
   specific DEGs, exploring LcTprxII and other hormone pathways, and
   developing LcTprxII-OE lines in other crops.

Supplementary Materials

   The following supporting information can be downloaded at
   [184]https://www.mdpi.com/article/10.3390/plants14101467/s1.
   [185]plants-14-01467-s001.zip^ (688.7KB, zip)

Author Contributions

   Conceptualization, D.P.J.M.; methodology, Q.L., Y.Y., F.U.R., A.T.,
   H.X. and J.H.; validation, F.U.R., A.T. and J.H.; writing—original
   draft preparation, D.P.J.M.; writing—review and editing, X.W. and J.L.;
   visualization, B.Q.; supervision, X.L., L.J., X.Q., R.S., Z.C. and
   J.Z.; project administration, J.Z.; funding acquisition, J.Z. All
   authors have read and agreed to the published version of the
   manuscript.

Data Availability Statement

   The original contributions presented in this study are included in the
   article/[186]Supplementary Materials. Further inquiries can be directed
   to the corresponding author(s).

Conflicts of Interest

   The authors declare no conflict of interest.

Funding Statement

   This work was supported by the Science and Technology Development Plan
   Project of Jilin Province, China (20240303004NC).

Footnotes

   Disclaimer/Publisher’s Note: The statements, opinions and data
   contained in all publications are solely those of the individual
   author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI
   and/or the editor(s) disclaim responsibility for any injury to people
   or property resulting from any ideas, methods, instructions or products
   referred to in the content.

References