Promoter analysis revealed multifunctional role of ALDH superfamily in soybean

Introduction

Environmental fluctuations, including drought, extreme temperatures, salinity, and pathogen attacks, disrupt cellular homeostasis and induce the overproduction of reactive oxygen species (ROS) and toxic lipid-derived aldehydes in plants (Kirch et al., 2005) [1] . If left unchecked, these aldehydes cause severe damage to cellular membranes and macromolecules (Kotchoni et al., 2003) [2] . The aldehyde dehydrogenase (ALDH) superfamily acts as a primary enzymatic detoxification mechanism, irreversibly oxidizing various endogenous and exogenous aldehydes into corresponding, less toxic carboxylic acids. Specific members of this family have multifunctional role. While the structural characteristics and enzymatic functions of ALDHs have been extensively documented, their precise transcriptional regulation in response to multi-stress environments remains a complex challenge in crop biotechnology. Gene expression is largely controlled by the promoter region, which contains specific cis-acting regulatory elements that act as binding sites for transcription factors (Wang et al., 2016) [3] . Identifying these molecular switches in the promoter regions of soybean (Glycine max) ALDH genes is critical for uncovering the complex cross-talk between environmental stress signalling and developmental pathways. Therefore, this study aims to systematically analyse the cis-acting elements within the GmALDH promoters to elucidate their multifunctional roles and provide a theoretical basis for future genetic engineering efforts.

Materials and methods

Sequence retrieval and promoter extraction

To investigate the potential regulatory mechanisms governing the GmALDH family members, the upstream promoter sequences were retrieved for analysis. Specifically, the 1500 bp genomic sequences upstream from the translation start codon (ATG) of all identified GmALDH genes were obtained from the Phytozome v12.1 database (https://phytozome.jgi.doe.gov/pz/portal.html) (Goodstein et al., 2012) [4] . This sequence length was selected as it typically encompasses the core promoter and the most critical proximal regulatory elements required for transcription factor binding.

Cis-acting element analysis

The putative stress or hormone-responsive cis-acting regulatory elements within these 1500 bp promoter sequences were predicted using the PlantCARE online database (Lescot et al., 2002) [5] . The raw data output was systematically filtered to exclude redundant structural elements (such as generic TATA and CAAT boxes), allowing the functional categorization to focus exclusively on motifs associated with biotic stress response, abiotic stress response and phytohormone responsiveness.

Results and discussion

Distribution and mapping of cis-acting elements

To understand the regulatory dynamics of the GmALDH genes, the 1500 bp promoter regions were mapped, revealing a dense and varied distribution of regulatory motifs dispersed across the sequences (Figure 1). This visual mapping demonstrates that multiple responsive elements frequently cluster together within the promoter regions of individual GmALDH genes. Similar to findings in other stress-responsive gene families, such clustering indicates a sophisticated, multifactor transcriptional control system, allowing a single gene to respond to diverse environmental stimuli simultaneously (Ali Q-u et al., 2021) [6] . The presence of these varied cis-regulatory elements within the transcription factor binding sites reveals that the GmALDH genes likely have multiple intersecting roles in growth, metabolism, and stress-related pathways.

Functional categorization

The identified elements were systematically categorized into three primary functional groups: abiotic stress-responsive, biotic stress-responsive, and phytohormone-responsive. The identified cis acting elements were shown in figure 2. While core promoter elements such as the TATA-box and CAAT-box were excluded from functional categorization to focus on stimuli-specific responses, the remaining motifs provided significant insight into the environmental and hormonal regulation of the ALDH superfamily.

Abiotic and environmental stress-responsive cis-acting elements

This category exhibited the highest diversity of motifs, emphasizing the crucial role of the ALDH family in managing environmental fluctuations. Notable abiotic stress-responsive elements included the ARE (essential for anaerobic induction), LTR (low-temperature responsiveness), MBS (MYB binding site involved in drought inducibility), and TC-rich repeats (defense and stress responsiveness). Elements like MYB and ARE were particularly abundant across multiple GmALDH members. The MYB transcription factor binding sites are essential for the expression of drought-inducible genes, highlighting the sensitivity of these genes to dehydration and osmotic stress (Li et al., 2015) [7] . The ARE motif serves as a critical regulatory element for anaerobic induction and is often associated with low oxygen and dehydration responses (Dolferus et al., 2001) [8] .

Furthermore, this category included several motifs known for their environmental and light-responsive roles, such as Box 4, G-Box, GATA-motif, I-box, and MRE. Motifs such as the G-box and Box 4 act as highly conserved molecular switches that are frequently regulated by environmental stresses, UV light, and injuries, suggesting their involvement in complex photoperiod and stress-signaling crosstalk (Shariatipour et al., 2020) [9]

Biotic stress-responsive cis-acting elements

The defensive capabilities of the GmALDH genes against biological threats were highlighted by the presence of specific biotic stress-responsive elements. This response was primarily represented by the widespread presence of the WUN-motif. The WUN-motif is a woundresponsive element known to be rapidly activated upon the perception of mechanical wounding and pathogen invasion (Ali Q-u et al., 2021) [6] . The existence of these elements in the promoter regions suggests that GmALDH genes are actively engaged in biotic stress response cascades and may play a vital role in defense signaling.

Figure 2. Graphical and tabular representation of number of different cis-acting elements in GmALDHs

Phytohormone-responsive cis-acting elements

Phytohormones mediate several crucial biological processes in plant development and stress response cascades (Shu et al., 2018) [10] . A wide array of hormone-responsive elements was identified, directly linking GmALDH gene expression to various plant growth regulators. These included the ABRE (abscisic acid responsiveness), AuxRRcore (auxin), CGTCA-motif and TGACG-motif (methyl jasmonate), ERE (ethylene), GARE-motif, P-box, TATC-box (gibberellin), TCAelement (salicylic acid), and TGA-element. Elements regulating abscisic acid (ABRE) and methyl jasmonate responsiveness showed the highest frequency and widest distribution across the promoters. The abundance of the ABRE motif implies a significant role in abiotic stress signaling, particularly in dehydration and salinity stress, which helps maintain cellular water balance and drives long-term growth responses (Verma et al., 2016) [11] . The methyl jasmonate-responsive elements (CGTCA and TGACG motifs) indicate an association with essential cellular regulators involved in stress tolerance and defense mechanisms. Additionally, the presence of ethylene-responsive (ERE) and salicylic acid-responsive (TCA-element) motifs suggests that these genes participate in anti-oxidative protection mechanisms to mitigate oxidative stress and recover photosynthetic efficiency (Verma et al., 2016) [11] . Overall, the abundance of these regulatory elements proposes that GmALDHs interact with multiple hormones to directly or indirectly induce various adaptive responses

Discussion

The comprehensive mapping of cis-acting elements within the 1500 bp upstream regions of GmALDH promoters provides compelling evidence for the multifunctional role of the ALDH superfamily in soybean. The prominent frequency of abiotic stress elements particularly MYB binding sites and anaerobic responsive elements (ARE) alongside abscisic acid-responsive elements (ABRE), points to a robust regulatory network dedicated to managing abiotic stress factors like drought and waterlogging (Li et al., 2015) [7] . Because ABA is a primary systemic signal for osmotic stress, the co-occurrence of ABRE and MYB motifs suggests that GmALDH expression is heavily reliant on ABA-dependent signaling pathways to maintain cellular integrity under adverse conditions (Verma et al., 2016) [11] .

Furthermore, the widespread presence of methyl jasmonate-responsive elements (CGTCA and TGACG motifs) and salicylic acid elements (TCA), alongside the biotic stress-associated WUN-motif, suggests that GmALDH genes also actively participate in defense signaling pathways (Ali Q-u et al., 2021) [6] . This cross-talk indicates that ALDHs serve as critical hubs integrating developmental signals with both biotic defense and abiotic stress mitigation. These findings are in strong agreement with recent genome-wide characterizations of the ALDH gene family in other vital crops, including maize (Zhang et al., 2024) [12] , common bean (Wang et al., 2024) [13] , and sorghum (Islam et al., 2022). The evolutionary conservation of these stress-and hormone-responsive elements highlights the fundamental necessity of ALDHs in plant survival. More importantly, understanding this complex promoter architecture provides a valuable roadmap for advanced crop improvement. Specific stress-responsive cis-elements identified in this study could serve as precise targets for CRISPR/Cas9-mediated promoter editing, enabling the fine-tuning of GmALDH expression to boost stress resilience without causing pleiotropic yield penalties.

Conclusion

This targeted in silico promoter analysis reveals the highly responsive and dynamic nature of the GmALDH gene family. The diverse array of cis-acting elements indicates that these genes are active participants in complex regulatory networks governing soybean's response to environmental stimuli and hormonal shifts. These insights provide a critical theoretical basis for future functional validation, such as inplanta transformation or targeted promoter editing, paving the way for the development of next-generation, climate-resilient soybean varieties.

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