Cloning and functional analysis of ZmMADS42 gene in maize

ABSTRACT

Maize (Zea mays L.) is the most important cereal crop in the world. Flowering period and photoperiod play important roles in the reproductive development of maize. This study, investigated ZmMADS42, a gene that is highly expressed in the shoot apical meristem. Agrobacterium infection was used to successfully obtain overexpressed ZmMADS42 plants. Fluorescence quantitative PCR revealed that the expression of the ZmMADS42 gene in the shoot apical meristem of transgenic plants was 2.8 times higher than that of the wild-type(WT). In addition, the expression of the ZmMADS42 gene in the endosperm was 2.4 times higher than that in the wild-type. The seed width of the T2 generation increased by 5.35%, whereas the seed length decreased by 7.78% compared with that of the wild-type. Dissection of the shoot tips of transgenic and wild-type plants from the 7-leaf stage to the 9-leaf stage revealed that the transgenic plants entered the differentiation stage earlier and exhibited more tassel meristems during their vegetative growth period. The mature transgenic plants were approximately 20 cm shorter in height and had a lower panicle position than the wild-type plants. Comparing the flowering period, the tasseling, powdering, and silking stages of the transgenic plants occurred 10 days earlier than those of the wild-type plants. The results showed that the ZmMADS42 gene played a significant role in regulating the flowering period and plant height of maize.

KEYWORDS:

• Flowering period
• MADS-box
• maize
• plant height
• ZmMADS42

Introduction

Maize (Zea mays L.) is an annual grass that belongs to the genus Zea of the Poaceae family. It is an important source of feed for livestock and aquaculture and serves as a vital raw material for the food, medical, and healthcare industries as well as for the chemical industry.¹ Flowering is a critical stage in plant growth and signifies a transition from reproductive to nutritional growth. Flowering is influenced by various factors, including endogenous hormones and environmental factors, which are regulated by a series of signal transduction pathways.² Through the study of plant flowering, various pathways have been discovered, including cytokinin, brassinosteroid, temperature-sensitive, and sugar metabolic pathways.^3–6

Flowering is a critical stage of plant growth and changes in flowering time can lead to changes in the developmental process of maize. If flowering time is too late, maize growth may be affected by climatic factors, potentially leading to poor growth, crop failure, and other related issues. If the flowering time is too early, it may lead to inadequate fertilization of maize, resulting in reduced seed-setting rate and yield. The MADS-box is a large gene family with a domain composed of 56–58 amino acids that recognizes similar target DNA sequences. All the proteins containing this conserved sequence are collectively referred to as the MADS-box gene family.^7–9 Based on the domain structure, evolutionary rate, and developmental function, the MADS-box transcription factor family can be categorized into two types: Type I (SRF superfamily) and Type II (MEF2 superfamily). Type I genes in plants are clustered with SRF-like genes in animals and their gene structure contains only one to two exons.¹⁰ Ectopic expression of PeMADS5 in Arabidopsis promotes early flowering.¹¹ The Type I gene TaMADS-GS, cloned from wheat, regulates the cell division signaling pathway during the early stages of seed development to regulate wheat seed size and weight.¹² The recently reported LAX1 protein in rice physically interacts with OsMADS1, OsMADS6, and OsMADS7, thereby increasing its influence on axillary meristem initiation, heading days, plant height, panicle length, and spikelet fertility.¹³ FLOWERING LOCUS C (FLC) is a MADS-box gene that is highly expressed at the apex of Arabidopsis and serves as a key repressor of flower transition. In Arabidopsis, it inhibits flowering by directly suppressing the activity of the central flowering promoters SOC1, FLOWERING LOCUS D (FD), and FLOWERING LOCUS T (FT).¹⁴ Recently, TaSOC1, a member of the MADS-box family, has been confirmed to be a flowering inhibitory factor in the vernalization and photoperiodic pathways.¹⁵ The absence of the C-terminal domain of OsMADS57 results in mutant rice plants with more tillers, an increased flower number, and a higher seed yield.¹⁶ To gain a better understanding of rice plants and facilitate plant breeding, the transgenic rice SEP1-like gene OsMADS1, which is regulated by the nopaline synthase (nos) promoter, induces dwarfing and early heading in transgenic plants.¹⁷ In summary, the MADS-box family plays an important role in plant flower development and flowering time.

In this study, we isolated the ZmMADS42 (ID: LOC100381450) gene from maize and demonstrated that ZmMADS42 is a Type I SRF transcription factor through structural domain analysis and sequence alignment. We also constructed a ZmMADS42 overexpression vector and transferred it into maize GSH99 calli via Agrobacterium-mediated transformation to obtain transgenic plants. Overexpressed plants promoted the growth of tassels, enabling them to transition to reproductive growth earlier and flower significantly earlier than the wild-type plants. This study lays the foundation for elucidating gene regulation of tassel development and the tassel regulation network.

Materials and Methods

Plant Materials and Growth Conditions

The maize inbred line GSH99 used in this study was obtained from the Biotechnology Center of Jilin Agricultural University. The seeds were planted in plastic pots filled with sterile soil and regularly supplied with the Hoagland nutrient solution (Table S1). Maize plants were cultivated in a greenhouse under controlled conditions, with a day-night temperature difference of 18–28°C and a 16-hour light/8-hour dark cycle. Relative humidity in the greenhouse was maintained at 65%. The maize plants were allowed to grow until they reached the three-leaf stage before gene extraction was performed.

Generation of Constructs and Transgenic Plants

Total RNA was extracted from maize GSH99 using the TRIzol method and and then the first strand of cDNA was generated by using the M-MLV reverse transcription kit (Takara). Primers were designed using Primer 5.0. Primer pairs containing the BstE II (5’-ACTCTTGACCATGGTAGATCTGACGGATCGTA TCGTAGTACTAGTA-3’) and Bgl II (5’-GGGGAAATTCGAGCTGGTCACCAATAGTAAACCTCAGTTACTTG-3’) restriction enzyme sites were used. Subsequently, PCR was used to amplify the CDS amino acid sequence encoded by ZmMADS42. It was then inserted into the pCAMBIA3301 vector containing cauliflower mosaic virus (CaMV) 35S, and the constructed vector was introduced into Agrobacterium GV3101. The Agrobacterium-mediated technique was used to infect maize GSH99 callus tissue with an infection duration of 20 minutes, followed by a three-day dark incubation. Glyphosate was used for selection and culture, based on the properties of the pCAMBIA3301 vector. We proceeded with differentiation culture, rooting culture, acclimatization, and ultimately transplanting the seedlings.

Sequence Analysis of ZmMads42 Gene

To identify the open reading frame of the ZmMADS42 gene (Gene ID: LOC100381450), we used the NCBI ORF Finder tool (https://www.ncbi.nlm.nih.gov/orffi nder/) for analysis. Subsequently, we used an online tool (https://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb.cgi) to predict the conserved domain of the encoded protein. ZmMADS42 was searched for genes in the same family using PlantTFDB (http://planttfdb.gao-lab.org/). We entered the BLAST results into Molecular Evolutionary Genetics Analysis software (MEGA 7) for multiple sequence alignment and used it to construct the phylogenetic tree using MEGA 7.0. We used the neighbor-joining method with 1000 bootstrap replicates for tree construction.

ZmMads42 Gene Expression Analysis

To analyze the expression of the ZmMADS42 gene, we extracted RNA from the roots (V3), stems (V3), leaves (V3), pollen (R1), endosperm (R6), and shoot apical meristems (V7) of T2 generation plants. All plants were planted at the genetically modified crop experimental base of Jilin Agricultural University in Changchun City, Jilin Province (longitude: 125.410385, latitude: 43.810433). The samples were wrapped in aluminum foil, cooled in liquid nitrogen, and stored at −80°C. TRIzol reagent was used for RNA extraction and M-MLV reverse transcriptase (Takara) was used to generate first-strand cDNA from total RNA, following the manufacturer’s instructions. SYBR Premix Ex Taq (Takara) was used for the qRT-PCR. The PCR program consisted of initial denaturation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds, annealing at 60°C for 20 seconds, and extension at 72°C for 15 seconds, followed by melt curve analysis. The melting point curve and data analyses were performed using the 2^−ΔΔCT method. ZmActin1 was used as an internal reference gene to calculate the Ct values. Three biological replicates were used for each sample.

The primers used in this study are shown in Supplementary Materials Table S2.

Sub‑Cellular Localization

The full-length coding sequence (CDS) of ZmMADS42 was integrated into the pCAMBIA1302 vector (modified with pCAMBIA1302-GFP) to construct the pCAMBIA1302-ZmMADS42-GFP vector. The constructed pCAMBIA1302-ZmMADS42-GFP vector was introduced into tobacco leaves via Agrobacterium-mediated infiltration. Leaves and control leaves were imaged 60 hours after infection with Agrobacterium (GV3101) using a laser confocal fluorescence microscope (Leica sp8, Germany). The excitation wavelength was 488 nm and the fluorescence location was observed.

Agronomic Trait Detection

Transgenic ZmMADS42 maize and wild-type maize plants were cultivated together in a transgenic field at Jilin Agricultural University (longitude: 125.410385, latitude: 43.810433). These plants were planted in rows 4.5 meters long, with a spacing of 0.8 meters between rows and 25 cm between individual plants in 2020, all growing under natural conditions in the wild. Morphological changes in the meristematic tissues of transgenic plants were observed, the stem tips of wild-type and transgenic plants were dissected from the 7-leaf stage to the 9-leaf stage, and images were captured using a microscope. To compare the plant and ear heights of the wild-type and transgenic plants, measurements were taken at the 8-leaf stage using a tape measure. The early and late flowering periods, pollen dispersal periods, and silking periods of transgenic and wild-type plants were recorded. The length and width of the seeds were measured using a ruler and the average value was calculated. Once the maize matured, the 100-seed weight was recorded and the color of the maize kernel was observed. All samples were analyzed in three independent replicates.

Yeast Two-Hybrid

The interaction protein of ZmMADS42 was predicted using the STRING website and was successfully screened for ZmFHA2. Using seamless cloning, the ZmMADS42 and ZmFHA2 genes were cloned into the pGBKT7 bait and pGADT7 prey vectors, respectively. The pGBKT7 empty vector plasmid pGBKT7-ZmMADS42 recombinant vector plasmid was transformed into AH109 yeast receptive state and applied to SD/-Trp-Leu-deficient solid culture medium for toxicity validation. The pGBKT7-ZmMADS42 vector plasmid and pGADT7 empty vector plasmid were co-transformed into AH109 yeast receptive state and coated on SD/- Trp-Leu two-deficient solid culture media. Single colonies were selected and cultured in a YPDA liquid culture medium at a constant temperature of 29°C to approximately OD₆₀₀ (0.8). The bacterial solution was then inoculated onto SD/- Trp-Leu-Ade-His and SD/-Trp-Leu-Ade-His-X-aGal solid culture media, and the growth of colonies in both solid culture media was observed, further determine whether pGBKT7-ZmMADS42 has self-activating activity. PGADT7-T and pGBKT7–53 were used as positive controls and pGADT7-T and pGBKT7-lam were used as negative controls.

Data Statistical Analysis

The data were analyzed using SPSS software (SPSS USA) for variance analysis. All experiments were conducted in three independent replicates. The graphs were created using GraphPad Prism 9.5.

Results

Evolutionary Tree Analysis of the Protein Encoded by ZmMads42

The protein sequences of the same family were then input into MEGA7.0 mapping software for tree construction (Figure 1). The results showed that it belonged to a relatively ancient MADS-box family. The ZmMADS1 and ZmMADS3 genes influence the growth of maize ears, whereas the ZmMADS47 gene affects the storage activity of the endosperm by interacting with Opaque2.¹⁸ AGL61 primarily functions in female gamete differentiation, whereas ZmMADS2 and ZmMADS4 control pollen development and formation, respectively.^19–21 Therefore, it is speculated that ZmMADS42 may be involved in reproductive development in maize and may be closely related to male tassel and seed development.

Figure 1. ZmMADS42 protein homologous family evolutionary tree.

Analysis of the ZmMads42 Coding Sequence and Generation of Transgenic Plants

The protein sequences downloaded from NCBI were analyzed by plotting them (Fig. 2). The results indicate that ZmMADS42 encodes a polypeptide comprising 468 amino acids with a calculated molecular weight of 50.23 kDa. It contains only the MADS-box domain (63–221 amino acids) at the N-terminus. Sequence comparison revealed that ZmMADS42 encodes a Type I SRF transcription factor. We introduced Agrobacterium-mediated infection of maize GSH99 callus tissue, selected glyphosate as the screening medium, and subsequently used differentiation medium, rooting medium, and seedling hardening medium before transplanting the seedlings. The transgenic plants were confirmed using glyphosate test strips and PCR. The results showed that a total of 14 transgenic maize plants overexpressing ZmMADS42 were obtained, and 3 of them were selected for further experiments (Figure 3(b,c)).

Figure 2. Analysis of the structure of ZmMADS42 gene.

Figure 3. Identification of transgenic maizes overexpressing ZmSAMDC by PCR analysis.(a): detection of positive plants by bar test strips; (c):M: 2000bp MARKER; P: pCAMBIA3301-ZmMADS42 plasmid; CK: control plants; WT: wild-type plants; 1–14: overexpressing ZmMADS42 positive plants.

Subcellular Localization of ZmMads42

As a member of the MADS-box transcription factor family, the ZmMADS42 gene is predicted to be located in the nucleus based on its subcellular localization. After 60 hours of infecting tobacco leaves, expression of the ZmMADS42 gene was observed in the nucleus of mesophyll cells using confocal microscopy (Figure 4). No green fluorescence was detected in the cytoplasm or cell membrane, indicating that the ZmMADS42 gene is expressed in tobacco and localized in the nucleus under normal conditions.

Figure 4. The subcellular localization map of pCAMBIA1302-ZmMADS42, with the first row showing 35S:ZmMADS42:GFP and the second row showing 35S∷GFP.35S∷GFP.The results indicate that the ZmMADS42 gene is clearly localized in the nucleus.

ZmMads42 Expression Analysis

To investigate the expression of ZmMADS42 in maize plants, quantitative fluorescence detection was performed on various parts of transgenic T2 generation plants. The ZmMADS42 gene was found to be actively expressed in the shoot apical meristem and endosperm of maize and was also expressed in pollen. The overall trend of gene expression was the highest in the shoot apical meristem, followed by the endosperm, pollen, leaf, stem, embryo, and root (Figure 5). Compared with the WT, the expression level of the shoot apical meristem increased by approximately 2.8-fold, and the expression level of the endosperm increased by approximately 2.4-fold. The ZmMADS42 gene is believed to actively influence the reproductive growth of plants, particularly in the shoot apical meristem. The specific regulatory mechanism of ZmMADS42 gene expression in the shoot apical meristem requires further study.

Figure 5. Relative expression levels of the ZmMADS42 gene in various tissues of T2 recombinant plants.

Anatomy of Stem Apex of the ZmMads42 Transgenic Plant

To observe morphological changes in the shoot apical meristem, we dissected and imaged the shoot apices of wild-type and transgenic plants from the 7-leaf stage to the 9-leaf stage. Our findings indicate that the overexpressing plants exhibited a shortened vegetative growth period and entered the differentiation stage earlier than the wild-type plants. The overexpressing plants exhibited an increased number of male ear meristems, suggesting that the overexpression of ZmMADS42 in maize plants may enhance the reproductive growth of the male ear (Figure 6).

Figure 6. Representative image of dissected maize stem tips from non-transgenic and transgenic ZmMADS42 transgenic plants. (a-e)stem tips of non-transgenic plants; (f-j)Shoot tips of the ZmMADS42 transgenic plants; (a,f)shoot apical meristem; (b, g)inflorescence meristem.; (c,h)branch meristem; (d,i)developing spikes; (e,j)immature spikes.Scale = 2 mm.

Agronomic Traits of Transgenic Plants

The agronomic traits of the transgenic and wild-type plants were evaluated in the field. During the eight-leaf stage, transgenic plants exhibited significantly lower growth rates than wild-type plants, indicating a critical transition period between vegetative growth and reproductive development. Transgenic plants expressing ZmMADS42 exhibited earlier reproductive growth (Figure 7(a)). The mature transgenic plants were approximately 20 cm shorter in height and had a lower spike position than the wild-type plants (Figure 7(b)). By comparing the seed germination rates of wild-type and transgenic plants, it was found that the germination rate of wild-type plants was approximately 17.5% higher than that of the transgenic plants. Through the comparison of flowering periods, the tasseling, pollen dispersal, and silk emergence stages of transgenic plants occurred approximately 10 days earlier than those of wild-type plants (Figure (8a–c)).

Figure 7. Agronomic traits of transgenic plants (a) comparison of phenotypes between wild-type and overexpressed maize at eight leaf stage; (b) comparison between wild-type and overexpression plants of mature maize tassel.

Figure 8. (a)seeding emergence rate; (b)Tasseling stage; silking period; pollen dispersal stage; (c)Plant height; ear height.

Phenotype of Transgenic Plant Seeds

The ZmMADS42 gene was found to have high expression levels in the endosperm, as determined by fluorescence quantitative analysis, and its phenotype was consequently observed in the transgenic plants. By counting the number of seeds on the central axis and measuring the length and width of the seeds, the results showed that the seed width of the T2 generation transgenic plants increased by 5.35% compared with the WT (Figure 9(a)), whereas the seed length decreased by 7.78% compared with the WT (Figure 9(b)). Furthermore, by measuring the 100-seed weight of transgenic and wild-type plants, it was found that the 100-seed weight of transgenic plants was greater than that of wild-type plants, and there was no difference in color (Figure 10).

Figure 9. Phenotype of transgenic plant seeds.(a)Seed width;(b)Seed length.

Figure 10. 100-seed weight.

Yeast Two-Hybrid Verification

To investigate the protein interaction of ZmMADS42, we used ZmMADS42 as the “bait” to fuse the ZmFHA2 gene with the pGADT7 vector, resulting in the construction of the recombinant vector pGADT7-ZmFHA2. We also connected the ZmMADS42 gene to the pDBKT7 vector to create the recombinant vector pGBKT7-ZmMADS42. Subsequently, we co-transformed AH109 competent cells with pGADT7-ZmFHA2 and the constructed vector, pGBKT7-ZmMADS42, to validate their interaction. In this experiment, pGADT7-T and pGBKT7–53 were used as positive controls, and pGADT7-T and pGBKT7-lam were used as negative controls. To confirm the success of the co-transformation, we first validated two of them in SD/-Trp-Leu culture medium (Figure 11(a)). Subsequently, we transformed pGADT7-ZmFHA2 and pGBKT7-ZmMADS42 into SD/-Trp-Leu-His-Ade culture media to verify the results (Figure 11(b)). The results indicated that the yeast strain grew well and exhibited a blue color in X-α-Gal culture media, suggesting an interaction between ZmFHA2 and ZmMADS42 in yeast (Figure 11(c)).

Figure 11. Interaction of ZmMADS42 and ZmFH2. (a) shows the production of colonies on SD/-Trp-Leu culture media by the negative control, positive control, and ZmMADS42-BK+ZmFHA2-AD; (b) demonstrates the production of colonies on SD/-Leu/-Trp/-his/-ade culture media by the positive control and ZmMADS42-BK+ZmFHA2-AD, while the negative control produces no colonies; (c) displays the production of blue colonies on X-α-gal-coated SD/-Leu/-Trp/-his/-ade culture media by the positive control and ZmMADS42-BK+ZmFHA2-AD, while the negative control produces no colonies.

Discussion

Most transcription factors regulate the growth and development of various plant organs. More than 100 MADS-box genes have been identified in Arabidopsis, making it a versatile transcription factor family that is involved in nearly all aspects of plant organ formation and the entire life cycle.^22,23 These roles include hormone response, seed germination, flower development, and overall plant morphogenesis.^24,25 Research indicates that the ZmMADS1 gene functions as a regulatory factor for flowering time in plants. When ZmMADS1 is downregulated by RNA interference, maize exhibits delayed flowering, whereas its overexpression results in earlier flowering, establishing it as a flowering activator.²⁶

The OsMADS1 and OsMADS3 genes identified in rice also play critical roles in the development of flower meristems through physical and genetic interactions. OsMADS1 and OsMADS58 physically and genetically interact to regulate the determination of flower meristems and inhibit the reversion of spikelet meristems.²⁷ Suppressor of Overexpression of Constans 1 (SOC1) is a key pathway integrator and activator of flower development.^28,29 As a typical regulator of flowering, the suppressor of Constans 1 overexpression belongs to the MADS-box family. Moreover, many homologous genes have been identified and shown to activate flowering.^30,31 It has been reported that BoMADS50, a homolog of the soc1 gene identified in bamboo, interacts with APETALA1/FRUITFULL (AP1/FUL)-like proteins BoMADS14–1/2 and BoMADS15–1/2 in vivo. BoMADS14–1 significantly enhanced the expression of BoMADS50, demonstrating the synergistic effect of BoMADS50 and BoAP1/FUL on the flowering of Bambusa oldhamii in green bamboo.³² Overexpression of the Zea mays SOC gene promotes flowering, reduces plant height, and does not reduce grain production per plant, suggesting an enhanced yield potential, at least in part, by increasing planting density.³³

The ABCDE model was used to classify flower development within the MADS-box transcription factor family. In this model, homologous genes of the flower were divided into five functional classes (A-E). Class A genes include APETALA1 (AP1) and APETALA2 (AP2), class B includes APETALA3 (AP3) and PISTILLATA, class C includes AGAMOUS (AG), class D includes FLORAL-BINDING PROTEIN 7 and 11, and class E encompasses SEPALLATA1–4.^34–36 The AGAMOUS gene is an ancient MADS-box gene. In wheat, a lack of AGL6 function results in low floret fertility and sterility. However, manipulating AGL6 expression appropriately can increase spikelet and grain numbers per ear.³⁷ OsMADS6 and SlMBP11 in rice, and ZAG3 in maize, are homologs of AGL6 gene. In maize mutant zag3, the upper flower forms additional floral organs, whereas the lower flower initiates additional florets. In the rice osmads6 mutant, additional carpels are produced in the florete center.^38–40 In addition, AGL15 and AGL18 play a role in regulating the flowering process in plants.⁴¹

Maize is one of the most important food and industrial crops in China, and its yield is an important measure for evaluating the quality of maize varieties. The function of the MADS-box gene family is also closely associated with crop yield. One study reported that LeMADS-RIN is essential for fruit ripening at the tomato ripening inhibitor (rin) locus.⁴² In addition, FUL proteins from the MADS-box family can regulate tomato fruit ripening by finely adjusting the expression of genes related to ethylene biosynthesis and ripening.⁴³ In soybean, the expression of the ZmSOC1 gene can result in early flowering and reduced plant height in transgenic plants. Additionally, the grain weight per plant of transgenic plants is 13.5–23.2% higher than that of non-transgenic plants.⁴⁴ Overexpression of the GmAP3 gene, found in soybeans, accelerates the flowering time of plants and leads to changes in flower organ morphology as well as increased yield.⁴⁵

Conclusions

This study analyzed ZmMADS42 transgenic plants, and the results showed that overexpression of this gene promoted meristem tissue growth and enabled plants to enter reproductive growth earlier.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21645698.2024.2328384

Additional information

Funding

This research is supported by the Science and Technology Research Project ofFunding: This work was supported by Jilin Province Science and Technology Development Plan Project [20230202003NC, 20230508005RC].