ZmSPAs Modulate Photomorphogenesis and Promote Plant Height in Arabidopsis thaliana

Abstract

SPAs (suppressors of phyA-105) are key modulators of photomorphogenesis that regulate diverse aspects of plant growth. While the role of SPA proteins in Arabidopsis photomorphogenesis is well-characterized, the functions of their maize (Zea mays L.) homologs (ZmSPAs) remain largely unexplored. Here, we show that ZmSPAs have the typical conserved domains of the SPA family and respond to different light qualities and photoperiodic treatments. Further analysis of the subcellular localization of ZmSPAs showed that ZmSPA1 and ZmSPA2 were localized to the nucleus, while ZmSPA3 and ZmSPA4 were localized to both the nucleus and the plasma membrane. The results of tissue-specific expression showed that ZmSPA1 and ZmSPA2 had the highest relative expression level in silks, while ZmSPA3 and ZmSPA4 were mainly expressed in leaves. Interestingly, overexpression of ZmSPAs in Arabidopsis promoted hypocotyl elongation in seedlings, inhibited cotyledon expansion in seedlings, and increased plant height in mature plants. The Y2H and LCI results indicate that ZmSPAs have physical interactions with ZmCOP1a, ZmCOP1b, and AtCOP1. These findings reveal the roles of ZmSPAs in regulating photomorphogenesis in Arabidopsis seedlings and plant height development in mature plants, laying a foundation for future investigations into their endogenous functions in maize.

1. Introduction

Light plays a crucial role in the growth and development of plants. It not only serves as the energy source for photosynthesis but also acts as an environmental signal to regulate the development process [1]. Throughout the plant life cycle, light regulates critical developmental processes, including seed germination [2], seedling de-etiolation, leaf expansion, hypocotyl elongation [3], stomatal development [4], chloroplast movement, anthocyanin biosynthesis, shade avoidance, and flowering time [5,6]. To achieve precise and efficient responses to varying light qualities, intensities, and photoperiodic conditions, plants have evolved distinct photoreceptors (phytochromes, cryptochromes, phototropins, and UVR8) to perceive diverse light signals [7,8]. Previous research has indicated that photoreceptors converge, using perceived light regulators via diverse mechanisms, onto the downstream COP1 (Constitutive Photomorphogenic 1)/SPA1 (Suppressor of phyA) E3 ubiquitin ligase complex [9]. Under dark conditions, the COP1/SPA1 complex degrades photomorphogenesis-promoting factors such as HY5 (Elongated Hypocotyl 5), CO (Constans), and HFR1 (Long Hypocotyl in Far-Red Light 1) in the nucleus, resulting in skotomorphogenic (phenotype of plants growing in dark environments, including elongated hypocotyls, closed cotyledons, and reduced chlorophyll accumulation) phenotypes in seedlings [10,11,12]. Under light conditions, photoreceptors activated by light suppress the COP1/SPA complex, thereby inhibiting its E3 ubiquitin ligase function. This results in the stabilization of photomorphogenesis-promoting transcription factors [13]. COP1 serves as an integral component of the complex, functioning as a central regulator in photomorphogenesis. Its functional architecture comprises three domains: an N-terminal RING-finger domain for ubiquitin transfer, a central coiled-coil dimerization domain, and C-terminal WD-40 repeats mediating substrate recognition [14,15]. The RING domain provides E3 ubiquitin ligase activity to COP1, whereas the coiled-coil (CC) domain facilitates the homo-dimerization of COP1 and mediates hetero-tetramer assembly with SPA proteins. The WD-40 domain acts as a substrate recognition module for photomorphogenesis suppressors. The synergistic effect of the domain enables COP1 to form a stable hetero-tetramer to enhance its E3 ubiquitin ligase activity, thereby efficiently regulating the protein stability of the light signal response factor and ultimately achieving precise control over the plant photomorphogenesis process [16,17].

SPA1 was first identified through a genetic screen for mutations that suppress the phyA null mutant phenotype in Arabidopsis [18]. Subsequent genetic analyses demonstrated that SPA proteins function redundantly to suppress photomorphogenesis in darkness, with the spa1, spa2, spa3, and spa4 quadruple mutants exhibiting constitutive photomorphogenesis similar to cop1 null mutants. Adult spa1, spa2, spa3, and spa4 quadruple mutants display severe dwarfism, with their phenotypic profiles closely resembling those of cop1 null mutants. Enhanced phenotypic severity in weak cop1 alleles by spa mutations confirms the biological relevance of SPA/COP1 interaction [19]. SPA proteins serve as co-factors for the E3 ubiquitin ligase COP1. Each SPA contains three domains: an N-terminal serine/threonine kinase-like motif, a central coiled-coil domain, and a C-terminal WD40 repeat domain. The coiled-coil domain mediates SPA1/COP1 heterodimerization, while the WD40 domain directly binds substrates HY5 and HFR1 [20,21].

The Arabidopsis genome encodes four SPA homologs (AtSPA1 to 4) with light-dependent functional divergence: AtSPA1, AtSPA3, and AtSPA4 primarily suppress photomorphogenesis in light, whereas AtSPA2 promotes skotomorphogenesis in darkness [19,22]. In soybean, loss of GmSPA3a function causes dwarfism, enabling higher planting density and yield increase [23]. AtSPA expression is photoperiod-responsive, and its mutants exhibit early flowering [24,25]. Similarly, a mutation in the BrSPA1 gene of Brassica accelerates bolting under short days [26], collectively highlighting the conserved yet diversified roles of SPA proteins in regulating plant growth and development.

To date, functional studies of the SPA gene family in Arabidopsis thaliana have achieved significant depth. As one of the most important crops, maize (Zea mays L.) plays an indispensable role in human agriculture [27,28]. However, the functional roles of the maize ZmSPA gene family in photomorphogenesis remain poorly characterized. This study aimed to clone the ZmSPA genes and analyze their tissue-specific expression patterns, transcript abundance, and responses to treatments with different light qualities and photoperiods, evaluating functional conservation through heterologous expression in the Arabidopsis spa124 mutant. Since the research on light signal transduction in the model plant Arabidopsis thaliana is relatively in-depth, and the spa124 mutant provides a clean sensitized background with minimal endogenous SPA activity, the experiments are highly operable. However, we acknowledge that cross-species assays cannot fully recapitulate the specific regulatory contexts of maize. By ectopically overexpressing the ZmSPA gene in Arabidopsis thaliana, we explored its important regulatory functions in photomorphogenesis and plant height. The results tested the hypothesis of functional conservation of ZmSPAs in regulating Arabidopsis thaliana photomorphogenesis in the light signal transduction network, providing a basis for future investigation of SPA-mediated light signaling in maize.

2. Results

2.1. Structural Analysis of ZmSPA Genes and Proteins

Using Arabidopsis SPA protein sequences as queries to search the MaizeGDB database, we identified four ZmSPA genes in the maize (Zea mays L.) inbred line B73 genome: ZmSPA1 (Zm00001eb344140), ZmSPA2 (Zm00001eb296410), ZmSPA3 (Zm00001eb154420), and ZmSPA4 (Zm00001eb361760). A phylogenetic tree was constructed using the Neighbor-Joining method in MEGA 10 with 1000 bootstrap replicates. Phylogenetic analysis revealed that ZmSPAs cluster closely with SPA homologs from other Poaceae species, particularly those from MiSPAs and SbSPAs (Figure 1A). Gene structure analysis of four ZmSPA genes in maize revealed that they all contain seven exons and six introns, exhibiting a highly conserved gene structure (Figure 1B). Domain alignments showed that the ZmSPA proteins had highly conserved domains, including an N-terminal serine/threonine kinase-like motif, a central coiled-coil domain, and a C-terminal WD40 repeat domain (Figure 1C). Among them, ZmSPA1 and ZmSPA2 exhibit a relatively high sequence similarity (83.03%), while ZmSPA3 and ZmSPA4 exhibit even higher identity (93.01%). To further evaluate the evolutionary conservation of SPA proteins, multiple sequence alignment of SPA amino acid sequences from maize, Sorghum bicolor, Miscanthus lutarioriparius, and Arabidopsis thaliana was performed using DNAMAN Version 9. The analysis revealed that all SPAs from these species contain three conserved domains: N-terminal kinase-like domain, central coiled-coil domain, and C-terminal WD40-repeat domain. This indicates that the protein structure of SPA is highly conserved across different species (Figure S1).

2.2. Transcription Pattern of ZmSPAs in Maize

To elucidate the tissue-specific expression patterns of ZmSPAs in maize, we collected various tissues from the inbred line B73 grown under field conditions. The results demonstrated that all four ZmSPAs were predominantly expressed in aboveground tissues, with minimal expression detected in roots. Specifically, ZmSPA1 and ZmSPA2 exhibited high expression levels in silks, whereas ZmSPA3 and ZmSPA4 were primarily expressed in leaves (Figure 2A). To further investigate the responsiveness of ZmSPAs to specific light wavelengths, we performed treatments with continuous spectra of distinct light qualities. Under continuous light treatments, ZmSPA1 and ZmSPA2 transcripts were more strongly induced by far-red (FR) and white (W) light, while ZmSPA3 and ZmSPA4 responded preferentially to red (R), blue (B), and white (W) light (Figure 2B). Light-quality transition experiments further uncovered rapid, member-specific transcriptional dynamics (Figure S2). When etiolated seedlings were shifted to far-red (FR), red (R), blue (B), or white (W) light, ZmSPA3 and ZmSPA4 transcripts rose sharply within the first 2 hours, often achieving peak induction shortly thereafter. In comparison, ZmSPA1 and ZmSPA2 showed delayed and less intense responses. Although all ZmSPAs were upregulated upon light exposure, ZmSPA3 and ZmSPA4 consistently exhibited greater fold changes—typically 7.2- to 38.6-fold within 2 hours—far exceeding those observed for ZmSPA1 and ZmSPA2 (Figure S2). Overall, ZmSPA3 and ZmSPA4 demonstrated heightened sensitivity and larger induction amplitudes across all tested light transitions.

To examine photoperiodic regulation of ZmSPA expression, we subjected seedlings to long-day (LD: 16 h light/8 h dark) and short-day (SD: 8 h light/16 h dark) regimes (Figure 2C–F). Under LD, ZmSPA1 and ZmSPA2 transcripts gradually declined during the light phase and reached peak abundance near the end of the dark phase, with the highest value being 4.3 times the lowest value. In contrast, ZmSPA3 and ZmSPA4 showed sharper reductions during the light period, with the highest value being 24 times the lowest value. Under SD conditions, ZmSPA1 exhibited an opposing pattern, accumulating to higher levels throughout the extended dark period, whereas ZmSPA2 peaked early in the light phase, with an overall expression range of 0.1–1.7. ZmSPA3 and ZmSPA4 maintained gradually rising expression during the dark phase, followed by an overall decline under light, with an overall expression range of 0.2–17. Remarkably, ZmSPA3 and ZmSPA4 displayed more pronounced dynamic changes in expression levels than ZmSPA1 and ZmSPA2 under every light regime examined. This pattern suggests that ZmSPA3 and ZmSPA4 may possess enhanced photosensitivity.

2.3. Subcellular Localization of ZmSPAs

To investigate the subcellular distribution of ZmSPAs, we performed transient expression assays in tobacco (Nicotiana benthamiana) leaves, fusing each ZmSPA to GFP and co-expressing a nuclear RFP marker. Transient expression assays in tobacco epidermal cells revealed distinct subcellular localization patterns for the four maize proteins. ZmSPA1 and ZmSA2 exhibited exclusive nuclear localization, with fluorescent signals concentrated specifically within the nucleus. In contrast, ZmSPA3 and ZmSPA4 displayed dual localization, distributing predominantly in both the nucleoplasm and cytoplasm (Figure 3).

2.4. Overexpression of ZmSPAs in Arabidopsis Thaliana Leads to an Etiolated Phenotype

To investigate the roles of four ZmSPA genes in seedling photomorphogenesis, we generated transgenic Arabidopsis thaliana lines by heterologously expressing ZmSPAs in the spa124 triple mutant background (Figure S3A,C). After genomic DNA PCR with specific primers verified positive transgenic lines (Figure S3B), quantitative real-time PCR (Figure S3D) analysis was subsequently employed to select lines ZmSPA1/spa124 (#11, #21), ZmSPA2/spa124 (#1, #5), ZmSPA3/spa124 (#35, #36), and ZmSPA4/spa124 (#100, #110), exhibiting higher expression levels for subsequent experiments.

Four-day-old seedlings grown under different light conditions were analyzed. In the darkness, the spa124 mutant displayed partial photomorphogenic phenotypes, characterized by a 21.4% reduction in hypocotyl length compared to Col-0 and incomplete cotyledon closure. Overexpression of ZmSPAs rescued these constitutive photomorphogenic defects in darkness, partially reversing hypocotyl shortening (Figure 4A,B). Under continuous far-red light (FR) irradiation, the ZmSPA1 and ZmSPA2 strains exhibited phenotypes similar to those of the spa124 mutant, while the ZmSPA3 and ZmSPA4 strains showed significantly elongated hypocotyls, with lengths 1.64-fold and 2.55-fold that of the spa124 mutant, respectively. Following continuous red light (R) treatment, all ZmSPA lines exhibited elongated hypocotyls compared to the spa124 mutant, with the hypocotyl length being 5.12- to 6.96-fold that of the spa124 mutant (Figure 4A,B). After continuous blue light (BL) treatment, no significant difference in hypocotyl length was observed between the ZmSPA1 lines and the spa124 mutant, while ZmSPA2, ZmSPA3, and ZmSPA4 lines exhibited slightly longer hypocotyls compared to the spa124 mutant. Under white light (WL) conditions, all ZmSPA lines showed significantly elongated hypocotyls compared to spa124 (Figure 4A,B). These findings demonstrate that ZmSPAs differentially modulate etiolation phenotypes in Arabidopsis seedlings. ZmSPA1 functions primarily under R and WL conditions, with limited function in darkness, far-red, or blue light. ZmSPA2 shows moderate activity under R, B, and WL (strongest in R, weakest in B), with minimal effects in darkness or FR. ZmSPA3 and ZmSPA4 consistently promote etiolation across all light conditions, with the strongest effects under R, followed by WL, and weakest effects under FR and B. Notably, ZmSPA3 and ZmSPA4 exhibit stronger phenotypic effects than ZmSPA1 and ZmSPA2 under all tested light regimes.

CHS (chalcone synthase) is a key structural gene encoding the enzyme catalyzing the first committed step in flavonoid/anthocyanin biosynthesis. Its light-induced expression is a well-established molecular marker for photomorphogenic signaling. Meanwhile, we measured leaf area and photomorphogenesis-related parameters, including anthocyanin content and chalcone synthase (CHS) expression levels, in seedlings. The results revealed that: Following heterologous expression of ZmSPAs, Arabidopsis thaliana seedlings exhibited reduced leaf area (Figure 5A,B), lower anthocyanin accumulation (Figure 5C), and a significant decrease in CHS relative expression (Figure 5D).

2.5. ZmSPAs Are Involved in Regulating Plant Height in Mature Arabidopsis Thaliana

To investigate the effects of ZmSPA genes on mature plant architecture, we grew transgenic lines alongside spa124 and Col-0 controls under long-day conditions (16 h light/8 h dark) for 50 days. Plant height, petiole length, and total leaf length were measured across all lines. Notably, ZmSPA overexpression partially or fully complemented the reduced height of spa124 mutants. ZmSPA3 nearly fully rescued the reduced plant height phenotype of the spa124 mutant, while ZmSPA1 and ZmSPA4 provided partial phenotypic complementation, and ZmSPA2 exhibited the weakest influence (Figure 6A,B). Petiole length followed a similar trend, with ZmSPA3 lines producing the longest petioles at approximately 5.23 times the length observed in spa124. The ZmSPA1, ZmSPA2, and ZmSPA4 lines developed shorter petioles, reaching 3.91, 3.82, and 4.0 times the spa124 length, respectively (Figure 6C). Similarly, total leaf length analysis demonstrated that ZmSPA3 lines were 1.99-fold longer than spa124, whereas ZmSPA1, ZmSPA2, and ZmSPA4 lines showed relatively smaller increases, reaching 1.43, 1.22, and 1.68 times the spa124 length, respectively (Figure 6D). Collectively, these results establish a hierarchical regulatory pattern among the four ZmSPA genes in plant height control, in which ZmSPA3 exhibits the strongest functional impact, ZmSPA2 displays the weakest, and ZmSPA1 and ZmSPA4 display intermediate regulatory capacities.

2.6. ZmSPAs Interact with AtCOP1 and ZmCOP1s Proteins

Previous studies have demonstrated that in Arabidopsis thaliana, SPA interacts with COP1 to form the COP1-SPA complex, an E3 ubiquitin ligase that targets positive regulators of photomorphogenesis for degradation, thereby promoting skotomorphogenesis [29]. To investigate whether a similar interaction exists between ZmSPAs in maize and their potential role in modulating the etiolation response, we first examined the interactions between ZmSPAs and AtCOP1, ZmCOP1a, and ZmCOP1b using a yeast two-hybrid system. All four ZmSPAs interacted with AtCOP1 as well as the two maize COP1 homologs, ZmCOP1a and ZmCOP1b, as evidenced by yeast growth and blue coloration on selective media supplemented with X-α-Gal (Figure 7A). We further validated these interactions in planta using firefly luciferase complementation imaging (LCI) assays in Nicotiana benthamiana leaves. Co-expression of ZmSPA-nLUC with AtCOP1-cLUC, ZmCOP1a-cLUC, or ZmCOP1b-cLUC produced strong luminescence signals, confirming physical associations in vivo (Figure 7B,C). Additional Y2H assays revealed self- and inter-paralog interactions among ZmSPAs. In yeast two-hybrid assays, co-expression of ZmSPA1 with the other three ZmSPA proteins resulted in robust reporter activation, as evidenced by blue colony coloration on selective medium supplemented with X-α-Gal and Aureobasidin A (Figure S4A). Notably, the N-terminal coiled-coil domain of ZmSPA1 showed particularly strong interactions with ZmSPA3 and ZmSPA4, producing more intense blue coloration compared to interactions with ZmSPA1 or ZmSPA2 (Figure S4B).

3. Discussion

In the model plant Arabidopsis thaliana, the SPA gene family consists of four members (AtSPA1–AtSPA4), which encode SPA proteins characterized by an N-terminal serine/threonine kinase-like domain, a central coiled-coil region, and a C-terminal WD40 repeat domain [18,20]. In this study, four SPA homologs were identified in maize, indicating that the SPA family has remained numerically conserved during monocot evolution. Structural analyses revealed that all ZmSPA proteins retain the canonical kinase-like, coiled-coil, and WD40 domains, highlighting strong conservation at the protein level. However, sequence identity analysis showed that ZmSPA proteins form two closely related paralogous pairs (ZmSPA1/2 and ZmSPA3/4), suggesting lineage-specific duplication events after the divergence of monocots and dicots (Figure 1; Figure S1).

During plant evolution, SPA genes first emerged in pteridophytes and expanded into a multi-member family in angiosperms, typically containing two or more copies [30,31]. Consistent with this hypothesis, our phylogenetic analysis grouped maize SPAs into two major clades corresponding to those reported in other flowering plants. Notably, monocot SPA proteins clustered separately from dicot SPAs, reflecting evolutionary divergence that may underlie functional specialization (Figure 1A). Together, these results indicate that while the overall architecture of SPA proteins is highly conserved, lineage-specific duplication and divergence have shaped the SPA family in maize, potentially contributing to species-specific regulation of light responses.

In Arabidopsis, transcriptional regulation of SPA genes is tightly linked to light quality and photoperiod. AtSPA1, AtSPA3, and AtSPA4 are transcriptionally induced by red, far-red, and blue light, whereas AtSPA2 displays relatively weak light responsiveness and is considered to function predominantly in darkness [32]. Our data show that ZmSPA genes are predominantly expressed in aerial tissues and are generally induced by light, with white light eliciting the strongest transcriptional response (Figure 2A,B). Notably, ZmSPA2 displayed clear light inducibility, in contrast to its Arabidopsis ortholog, suggesting divergence in transcriptional regulation between monocots and dicots. Differential expression patterns among ZmSPA genes under varying light qualities and photoperiods further indicate partial subfunctionalization. ZmSPA3 and ZmSPA4 exhibited highly similar expression profiles, whereas ZmSPA1 and ZmSPA2 showed antagonistic diurnal trends, implying distinct roles in photoperiodic regulation (Figure 2C–F; Figure S2). These findings suggest that transcriptional diversification of ZmSPAs may contribute to fine-tuning light responses in maize.

In Arabidopsis, SPA proteins act as key repressors of photomorphogenesis during seedling development, with distinct SPAs contributing differently depending on light conditions [33,34]. AtSPA1 primarily suppresses photomorphogenesis under light, AtSPA2 functions mainly in darkness, and AtSPA3 and AtSPA4 play prominent roles under illuminated conditions [19,22]. Cotyledon area and anthocyanin content/accumulation are widely recognized as standard, quantitative indicators of photomorphogenesis in Arabidopsis seedlings, particularly during the transition from skotomorphogenesis to de-etiolation under light [35]. By heterologously expressing ZmSPA genes in Arabidopsis, we demonstrated that maize SPAs retain the ability to repress photomorphogenesis, as reflected by altered hypocotyl elongation and cotyledon expansion under various light conditions (Figure 4 and Figure 5). However, individual ZmSPA proteins exhibited distinct functional strengths depending on light quality, with ZmSPA3 and ZmSPA4 showing stronger inhibition of photomorphogenesis, suggesting functional divergence within the maize SPA family. Unlike ZmSPA1 and ZmSPA2, which localize exclusively to the nucleus, ZmSPA3 and ZmSPA4 exhibit dual localization in both the nucleus and the plasma membrane. The plasma membrane association of ZmSPA3/4 may allow additional regulatory inputs, potentially accounting for their enhanced capacity to suppress photomorphogenesis compared to ZmSPA1/2. Importantly, ZmSPAs also influenced adult plant height in Arabidopsis, indicating that their regulatory roles extend beyond early seedling development (Figure 6). While the increased height is consistent with suppression of photomorphogenesis-promoting pathways, we cannot exclude indirect effects via altered gibberellin, brassinosteroid, or autonomous pathway activity. Future hormone measurements and genetic interaction studies in maize are required. These results support the idea that SPA-mediated light signaling regulates plant architecture through conserved mechanisms, while quantitative differences among family members may underlie species-specific growth strategies.

In Arabidopsis, SPA proteins function primarily through forming complexes with COP1, acting as co-factors that enhance COP1 E3 ubiquitin ligase activity [21,29]. Our interaction analyses revealed that all ZmSPA proteins physically interact with ZmCOP1s, indicating strong conservation of the SPA/COP1 interaction module in maize (Figure 7). Moreover, ZmSPA1 was able to form both homo- and heterodimers with other ZmSPAs, and truncation analyses confirmed that the N-terminal regions are essential for these interactions (Figure S4). These findings demonstrate that the molecular mechanism by which SPAs regulate COP1 activity is largely conserved between maize and Arabidopsis, despite evolutionary divergence at the sequence level.

Light signaling pathways play a central role in shaping plant architecture and yield-related traits. In soybean, loss-of-function of GmSPA3a results in dwarfism and improved yield potential under high-density planting [23]. Similarly, manipulation of light receptors such as cryptochromes in maize has been shown to alter plant height [36,37]. Among key agronomic traits in maize, plant height significantly influences planting density, photosynthetic efficiency, lodging resistance, and harvest index, all of which are closely associated with grain yield [38,39,40]. Our findings that ZmSPAs influence plant height in Arabidopsis suggest that SPA-mediated light signaling pathways may serve as potential targets for crop improvement. However, these implications are based on heterologous expression studies in Arabidopsis, and further research using maize mutants and stable transgenic lines will be essential to validate the precise roles of individual ZmSPA genes throughout the maize life cycle. The hypothesis that SPA-mediated pathways can be utilized to optimize maize plant height will be a focus of future research.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

Arabidopsis seeds were sterilized and cold-treated as previously described [41,42]. Subsequently, they were spread on the MS solid medium (1% sucrose, 0.9% agar, 0.44% MS medium salts, and 0.05% MES, pH 5.7). All plant lines were cultivated synchronously under identical environmental conditions except for differences in light quality. Seedlings were grown under far-red light (FR, 1.1 μmol m⁻² s⁻¹), red light (R, 50 μmol m⁻² s⁻¹), blue light (B, 11 μmol m⁻² s⁻¹), or white light (W, 100 μmol m⁻² s⁻¹) conditions at 22 °C for 4 days. Hypocotyl lengths and cotyledon area were measured using ImageJ 11.0.0 1.54g software for at least 30 randomly selected plants per line.

For tissue-specific expression profiling, B73 maize seeds were sown in experimental fields in Zhengzhou, Henan Province, China, and grown under natural conditions for 55 days. Roots, stems, leaves, silks, and stamen were harvested and immediately frozen in liquid nitrogen, followed by storage at −80 °C for subsequent analysis.

Continuous light experiments involved seeds imbibed in water for 20 h, then soil-germinated at 28 °C for 13 days under distinct light conditions: FR (far-red light, 2.5 μmol m⁻² s⁻¹), R (red light, 26 μmol m⁻² s⁻¹), B (blue light, 26 μmol m⁻² s⁻¹), and W (white light, 70 μmol m⁻² s⁻¹). The experimental procedure was performed according to the method described by Chen et al. [42]. Leaves were harvested and frozen at −80 °C. Dark-to-light shifts used seedlings etiolated at 28 °C for 13 days before transfer to FR, R, B, or W light as above. Leaves were sampled at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h post-exposure and stored at −80 °C. For photoperiod experiments, B73 seeds were sown and grown in controlled-environment chambers at 28 °C under two photoperiodic regimes: Long-day (LD): 16 h light/8 h dark or short-day (SD): 8 h light/16 h dark. Leaf samples were collected every 2 hours from 13-day-old seedlings and stored at −80 °C for subsequent analysis.

4.2. Total RNA Extraction and RT-qPCR Assays

Total RNA was isolated using TransZol (Transgen biotech, Beijing, China). The reverse transcription reaction was performed using the EasyScript^® One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen biotech, Beijing, China). The RT-qPCR reaction solutions were prepared according to the instructions of the THUNDERBIRD^® Next SYBR qPCR Mix (TOYOBO, Shanghai, China), and RT-qPCR reactions were performed using the LightCycler 480II real-time PCR detection system (Roche, Basel, Switzerland). Transcript levels of Tubulin 5 (GRMZM2G152466) or Actin 2 (ACT2, AT3G18780) were used as internal controls for RT-qPCR analyses in maize or Arabidopsis, respectively. The primers for RT-qPCR assays are shown in Supplemental Table S2.

4.3. Plasmid Construction and Generation of Transgenic Plants

The full-length coding sequences of ZmSPA genes were amplified by PCR using gene-specific primers (ZmSPAs-F and ZmSPAs-R). Then, the PCR fragments of ZmSPAs, digested with XhoI and SpeI, were inserted into the pJIM19 vector to generate the pJIM19—ZmSPAs binary construct. After sequencing, the proper pJIM19—ZmSPAs plasmid was electroporated into Agrobacterium tumefaciens strain GV3101 and then introduced into the Arabidopsis spa124 mutant using the floral dip method [43]. Transgenic plants were selected as previously described [42]. For each ZmSPAs construct, 12–18 independent T1 transformants were generated; four independent lines per construct were advanced to T3/T4 and confirmed by RT-qPCR. Two lines exhibiting relatively higher transgene expression per genotype were selected for subsequent experiments (Figure S3).

4.4. Subcellular Localization

For subcellular localization analysis, ZmSPA coding sequences were cloned into the pSuper1300 vector downstream of the CaMV35S promoter, generating the recombinant plasmid pSuper1300-35S-ZmSPAs-GFP. The recombinant plasmid and empty vector control (pSuper1300-35S-GFP) were independently transformed into Agrobacterium tumefaciens strain GV3101. Subsequently, bacterial suspensions harboring the target plasmid or empty vector were co-infiltrated with P19 (silencing suppressor) and a nuclear marker (AtWRKY29:AT4G23550) into the abaxial side of 4-week-old Nicotiana benthamiana leaves. After 24 h of cultivation under dark-adapted conditions, confocal laser scanning microscopy (Leica TCS SP8 STED ONE) was employed to visualize fluorescence signals. The primers for subcellular localization assays are shown in Supplemental Table S2.

4.5. Yeast Two-Hybrid (Y2H) Assay

Bait vector pGBKT7 (harboring ZmCOP1s or AtCOP1) and prey vector pGADT7 (harbouring ZmSPAs) were co-transformed into yeast strain Y2HGold. After incubation at 30 °C for 2 d, positive yeast colonies containing bait and prey constructs were selected on SD/-Trp/-Leu plates. The selected yeast colonies were incubated in liquid medium overnight at 30 °C, and then the yeast cells were diluted to an optical density OD600 of 0.5 and spotted on QDO/X/A medium. The primers for Y2H assays are shown in Supplemental Table S2.

4.6. Luciferase Complementation Imaging Assays (LCI)

Nicotiana benthamiana plants were grown at 25 °C under a 12 h light/12 h dark photoperiod for 4 weeks. ZmSPAs were fused to the N-terminal luciferase fragment (nLUC), while ZmCOP1a, ZmCOP1b, and AtCOP1 were fused to the C-terminal fragment (cLUC). Corresponding constructs were introduced into A. tumefaciens GV3101 and co-infiltrated into tobacco leaves. After infiltration, plants were incubated in darkness for 24 h, followed by white light exposure for 24 h. Leaves were sprayed with 20 mg mL⁻¹ luciferin potassium salt and incubated in darkness for 10 min before imaging. Luminescence signals were captured using a NightSHADE LB985 imaging system (Berthold Technologies, Germany) [44,45]. Experiments were repeated three times independently. The primers for LCI assays are shown in Supplemental Table S2.

4.7. Anthocyanin Measurement

For each genotype, 100 Arabidopsis seedlings were incubated overnight at 4 °C in 300 μL methanol containing 1% (v/v) HCl under gentle agitation in darkness. Samples were then mixed with 250 μL distilled water and 500 μL chloroform, vortexed for 30 s, and centrifuged at 12,000 rpm for 5 min. The aqueous phase was collected, and 350 μL was combined with 650 μL 1 M HCl. Anthocyanin content was determined by measuring absorbance at 530 nm and 657 nm. Relative anthocyanin levels were calculated as (A₅₃₀ − 0.25 × A₆₅₇) per 100 seedlings, following a previously described method [41].

4.8. Morphological Measurements and Statistical Analysis

Arabidopsis thaliana seedlings were laid flat on the MS medium. Images were captured and analyzed under a stereomicroscope, and then the hypocotyl length was measured using ImageJ software. Each genotype included at least 30 seedlings. Statistical analyses were performed using Prism software (v.9.5.1, GraphPad). Data are presented as mean ± SD, and significance was assessed using Student’s t-test or one-way ANOVA, with p < 0.05 considered statistically significant.

5. Conclusions

This study characterizes the maize ZmSPA gene family and clarifies its roles in light-regulated development. Four ZmSPA genes were identified, all encoding proteins with conserved kinase-like, coiled-coil, and WD40 domains, supporting structural conservation of SPA proteins across plant lineages. Functional analyses in Arabidopsis thaliana showed that ZmSPAs suppress photomorphogenesis and modulate plant height, with individual members displaying distinct regulatory strengths. Moreover, ZmSPAs interact with COP1 and form homo- and heteromeric complexes through their N-terminal regions, indicating that the COP1–SPA regulatory module is conserved between monocots and dicots. Together, these findings provide molecular insight into SPA-mediated light signaling in maize and offer a basis for future functional studies aimed at improving plant architecture.