Dissecting the Regulatory Network of Maize Phase Change in ZmEPC1 Mutant by Transcriptome Analysis

The developmental phase changes of maize are closely associated with the life span, environmental adaption, plant height, and disease resistance of the plant and eventually determines the grain yield and quality of maize. A natural mutant, Early Phase Change 1 (ZmEPC1), was selected from the inbred line KN5585. Compared with the wild type plant, the ZmEPC1 mutant exhibits deceased plant stature, accelerated developmental stages, and decreased leaf size. Through the transcriptome sequencing analysis of leaf samples at flowering stage, a total of 4583 differentially expressed genes (DEGs) were screened between the mutant and wild type, including 2914 down-regulated genes and 1669 up-regulated genes. The GO enrichment and KEGG enrichment analysis revealed that the DEGs were mainly involved in hormone response, hormone signal transduction, autophagy, JA response and signal response, photosynthesis, biotic/abiotic stress, and circadian rhythms. The RT-qPCR results revealed that the most tested DEGs display consistent expression alterations between V5 and FT stages. However, several genes showed opposite expression alterations. Strikingly, most of the JA biosynthesis and signaling pathway-related genes displayed diametrically expression alterations between V5 and FT stages. miR156, a key regulator of plant phase transition, exhibited significant down-regulated expression at V5 and FT stages. The expression of two miR156 target genes were both significantly different between mutants and wild type. In conclusion, ZmEPC1 was identified to be mainly involved in the regulation of JA-mediated signaling pathways and hormone response and signaling, which is possible to confer developmental phase change through miR156-SPLs pathway.


Introduction
Maize is one of the most popular grain crops for human food, animal feed, and industrial materials. Plant growth and development largely determine plant height, yield, quality, and disease resistance in maize [1]. The development process of maize includes two developmental phase changes, juvenile to adult vegetative phase and vegetative to reproductive phase, with significant phenotypic alterations. In maize, the juvenile stage is usually from germination to five or six leaves old in most genotypes, and the adult vegetative stage is form the end of juvenile vegetative stage to flowering time [2]. The juvenile and adult leaves are distinguished primarily by features of the epidermis of the leaf blade, the most obvious of which are the presence of epicuticular wax on the juvenile leaf and epidermal hairs on the adult leaf. Flowering represents the onset of reproductive phase that mainly displays ear development, grain development, and leaf senescence. The two developmental phase transition play a crucial regulatory role in maize environmental adaption, development, and yield, which provides breeders the opportunities for selecting different types of maize varieties through manipulating the developmental phase transition timing. fied, such as indeterminate1 (id1) [44], delayed flowering1 (dlf1) [40], ZEA CENTRORADIALIS 8 (ZCN8) [39], ZCN12 [45], ZmMADS1 [46], ZEA MAYS MADS4 (ZMM4) [47], Vegetative to generative transition 1 (Vgt1) [48], ZmCCT9 [49], ZmCCT10 [50], ZmMADS69 [51], High Phosphatidyl Choline 1 (HPC1) [52], ZmNF-YC2 [53], and ZmCOL3 [34]. Additionally, GA and JA play opposite roles in regulating maize flowering [16,18].
In the present work, we isolated a natural mutation, Early Phase Change 1 (ZmEPC1), exhibiting accelerated developmental phase changes. To identify the potential developmental phase transition related genes and construct the corresponding regulatory model, we conducted comparative transcriptome analysis between ZmEPC1 mutant and wild type (WT) NILs.

Plant Materials and Growth Condition
In our previous study, an early developmental phase change mutant ZmEPC1 was screened form the inbred line KN5585. The ZmEPC1 mutant displays serious male and female imbalance, which is difficult for pollination and seed-setting. For mapping the mutant gene, we crossed the mutant with the inbred line KN5585 twice to construct a segregation population. In the constructed BC 1 F 2 population, the dominant homozygous material was WT (almost without any phenotypic difference from KN5585), and the recessive material with early flowering and decreased plant stature was the early phase change type ZmEPC1. In the summer of 2020, the BC 1 F 3 population was planted in the field and photographed during growth and development. Leaf samples (the 5th leaf of 5-leaf (V5) stage and ear leaf of flowering stage (FT)) of ZmEPC1 mutant plants and the corresponding control were collected (3 biological replicates, respectively) and frozen in liquid nitrogen immediately. The treated samples were stored in the −80 • C freezer for further transcriptome sequencing and RT-qPCR analysis.

Total RNA Isolation and Transcriptome Analysis
Total RNA was extracted from the collected leaf samples of ZmEPC1 mutants and the WT at V5 and FT stage using Trizol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. The RNA samples of the ear leaf samples (collected at FT stage) were used to construct 6 sequencing libraries, and the libraries were sequenced using the Illumina HiSeq 4000 platform (Berry Gene, Beijing, China). The entire original sequence data in fastq format have been uploaded to the NCBI Short Read Archive (accession number: PRJNA869324).
In order to identify the changes at the transcriptome level involved in the developmental changes of ZmEPC1 mutant, the obtained sequencing data was analyzed. First, we performed quality control for the obtained raw data using FastQC (http://www. bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 18 August 2022). The Q30 ratios of the 6 libraries were all greater than 92%. The trimmed and low-quality (Q < 30) sequencing data were removed by Trimmomatic Software V0. 39

Construction of Regulatory Network in Flowering Stage
The online tool STRING V11 (https://string-db.org/, accessed on 18 August 2022) was used to build connect network of those GO terms [54].

Real-Time qPCR Is Used for Gene Expression Validation
Total RNA of the leaf samples collected at the V5 and FT phases was extracted with TRIzol reagent (Invitrogen). The expression levels of miR156-SPLs, miR172-gl15, and those selected key DEGs were detected using the PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real Time) and the SYBR ® Premix EX Taq™ II (Tli RNaseH Plus) Kit (TaKaRa, Dalian, China). RT-qPCR primers (http://primer3.ut.ee/, accessed on 18 August 2022) are listed in Table S1. The RT-qPCR was performed using the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The ACTIN gene and U6 small RNA was used as the endogenous control for the tested genes and miRNAs, respectively. The data thus obtained were calculated by the 2 −∆∆ct method [55]. All experiments included 3 biological replicates and 3 technical replicates.

Statistical Analysis
All the collected data from RT-qPCR analysis was subjected to one-way variance analysis (ANOVA) and Student's t-test using software SPSS 22.0 (IBM, Armonk, NY, USA). p < 0.05 indicates the statistical differences to reach the significant different level, p < 0.01 and p < 0.001 for very significant different level.

Phenotypic Alterations of ZmEPC1 Mutant
The phenotypic alterations of ZmEPC1 mutants were identified in the field ( Figure 1). During the vegetative stage, the ZmEPC1 mutant plants displayed significantly smaller plant size and more internodes than that in the WT plants ( Figure 1A,B). Compared with the WT plants, the ZmEPC1 mutant plants exhibited early tasseling ( Figure 1C). The ZmEPC1 mutant plants have significantly reduced plant height and decreased leaf size ( Figure 1C). These phenotypic alterations indicated that ZmEPC1 was involved in the regulation of maize development and the gene mutation could accelerate the developmental phase changes.

Identification of Differentially Expressed Genes
We obtained 23.2GB of raw data by constructing cDNA libraries and RNA-seq for 6 samples (3 replicates each for WT and ZmEPC1 plants). Between ZmEPC1 mutant and the WT, 4583 significantly differentially expressed genes (DEGs) were screened, including 2914 down-regulated genes and 1669 up-regulated genes. Of these DEGs, the up-regulated genes Zm00001d051093 (encodes LRR receptor-like serine/threonineprotein kinase EFR, involved in the regulation of shoot apical meristem development), Zm00001d039437 (encodes dbb3, involved in light signaling pathway), and Zm00001d003811 (involved in controlling photoperiod flowering response) and down-regulated genes Zm00001d004573 (encodes JA-inducible protein), Zm00001d050837 (encodes gibberellin receptor-GID1L2), Zm00001d026271 (encodes AP2/EREBP), and Zm00001d029940 (encodes ethylene-responsive transcription factor ERF105) exhibited the most significant differences (Figures 2 and 3, Table 1). These genes may play important roles in regulating maize vegetative to reproductive stage transition.

Identification of Differentially Expressed Genes
We obtained 23.2GB of raw data by constructing cDNA libraries and RNA-seq for 6 samples (3 replicates each for WT and ZmEPC1 plants). Between ZmEPC1 mutant and the WT, 4583 significantly differentially expressed genes (DEGs) were screened, including 2914 down-regulated genes and 1669 up-regulated genes. Of these DEGs, the up-regulated genes Zm00001d051093 (encodes LRR receptor-like serine/threonine-protein kinase EFR, involved in the regulation of shoot apical meristem development), Zm00001d039437 (encodes dbb3, involved in light signaling pathway), and Zm00001d003811 (involved in controlling photoperiod flowering response) and down-regulated genes Zm00001d004573 (encodes JA-inducible protein), Zm00001d050837 (encodes gibberellin receptor-GID1L2), Zm00001d026271 (encodes AP2/EREBP), and Zm00001d029940 (encodes ethylene-responsive transcription factor ERF105) exhibited the most significant differences (Figures 2 and 3, Table 1). These genes may play important roles in regulating maize vegetative to reproductive stage transition.

GO Enrichment Analysis
Based on the Annotation Hub database, gene ontology (GO) enrichment analysis was performed using the screened DEGs. The results revealed that the DEGs were mainly enriched in biological pathways, such as during photosynthesis, hormone response, cell response to hormone stimulation, cellular response to endogenous stimulation, response to endogenous stimulation, JA response, JA-mediated signal response pathway, and damage response (Figure 4).

KEGG Enrichment Analysis
The KEGG enrichment analysis of those DEGs indicated that the ZmEPC1 mutation gene is mainly associated with plant hormone signal transduction, photosynthesis, linoleic acid metabolism, benzoxazinoid biosynthesis, plant-pathogen interaction, glycerophospholipid metabolism, and photosynthetic organisms. Significant biological processrelated pathways were phytohormone signaling, photosynthesis, linoleic acid metabolism, and benzoxazinoid biosynthesis ( Figure 5).

KEGG Enrichment Analysis
The KEGG enrichment analysis of those DEGs indicated that the ZmEPC1 mutation gene is mainly associated with plant hormone signal transduction, photosynthesis, linoleic acid metabolism, benzoxazinoid biosynthesis, plant-pathogen interaction, glycerophospholipid metabolism, and photosynthetic organisms. Significant biological process-related pathways were phytohormone signaling, photosynthesis, linoleic acid metabolism, and benzoxazinoid biosynthesis ( Figure 5).

Regulatory Network Analysis
The DEGs were further analyzed to construct a biological process regulatory network involving in flowering. Photosynthesis, hormone-mediated signaling, and JA-mediated signaling are at the central places of the regulatory network ( Figure 6). This indicated that the JA signaling pathway and the cellular response to JA stimulation play a crucial role in the control of maize developmental phase transition.

Regulatory Network Analysis
The DEGs were further analyzed to construct a biological process regulatory network involving in flowering. Photosynthesis, hormone-mediated signaling, and JA-mediated signaling are at the central places of the regulatory network ( Figure 6). This indicated that the JA signaling pathway and the cellular response to JA stimulation play a crucial role in the control of maize developmental phase transition. . Connect network of those GO terms in biological processes Where the relations between the GO terms are represented as edges: is a (is a subtype of); part of (part of whole); regulates (the former regulates the latter).

Expression Analysis of Key DEGs
In order to further verify the results of transcriptome analysis and the potential involved regulatory pathways in ZmEPC1 mutant mediated early phase changes, 12 upregulated genes and 18 down-regulated genes were selected for RT-PCR analysis in the samples of V5 and FT stages (Figure 7). The selected genes were mainly associated with Figure 6. Connect network of those GO terms in biological processes. Where the relations between the GO terms are represented as edges: is a (is a subtype of); part of (part of whole); regulates (the former regulates the latter).

Expression Analysis of Key DEGs
In order to further verify the results of transcriptome analysis and the potential involved regulatory pathways in ZmEPC1 mutant mediated early phase changes, 12 upregulated genes and 18 down-regulated genes were selected for RT-PCR analysis in the samples of V5 and FT stages (Figure 7). The selected genes were mainly associated with phytohormone signaling, shoot meristem development, and photoperiod pathways. Most of the selected DEGs exhibited significantly different at V5 and FT stages. The expression trends of most genes in the V5 phase were consistent with the transcriptome results in the FT phase, which confirmed that ZmEPC1 not only has an important regulatory role in flowering but is also involved in the regulation of vegetative phase change. The selected up-regulated DEGs mainly involved in ethylene, GA, IAA, CTK, BR signaling pathway, as well as the photoperiod regulation pathway. Ethylene signaling pathway-related genes Zm00001d043247 (ETHYLENE RESPONSE SENSOR 1) and Zm00001d013338 (ETHYLENE RESPONSE SENSOR 1) were up-regulated in both V5 and FT stages. The GA signaling-related genes Zm00001d018617 (ga2ox12) and Zm00001d002999 (ga2ox2) were up-regulated and showed significant differences between mutants and WT at FT stage. The IAA signaling pathway-related genes Zm00001d001945 (arftf4) and Zm00001d053819 (arftf16) showed a down-regulated expression trend in the V5 phase and a very significant up-regulated expression trend in the FT stage. The CTK The selected up-regulated DEGs mainly involved in ethylene, GA, IAA, CTK, BR signaling pathway, as well as the photoperiod regulation pathway. Ethylene signaling pathway-related genes Zm00001d043247 (ETHYLENE RESPONSE SENSOR 1) and Zm00001d013338 (ETHYLENE RESPONSE SENSOR 1) were up-regulated in both V5 and FT stages. The GA signaling-related genes Zm00001d018617 (ga2ox12) and Zm00001d002999 (ga2ox2) were up-regulated and showed significant differences between mutants and WT at FT stage. The IAA signaling pathway-related genes Zm00001d001945 (arftf4) and Zm00001d053819 (arftf16) showed a down-regulated expression trend in the V5 phase and a very significant up-regulated expression trend in the FT stage. The CTK biosynthesisrelated genes Zm00001d041763 (encodes UDP-glucose) and Zm00001d052209 (encodes glycosyltransferase) showed an up-regulated expression trend in both periods, but the difference reached a significant level only at the FT stage. The BR biosynthesis-related gene Zm00001d033180 (brassinosteroid-deficient dwarf1) showed a significant down-regulation at V5 stage, while it was significantly up-regulated at the FT stage. The photoperiod regulationrelated genes Zm00001d039437 (double B-box zinc finger protein3) and Zm00001d005366 (PSEUDO-RESPONSE REGULATOR 1) were significantly up-regulated at the V5 and FT stages.

Expression Analysis of Flowering Time, JA Synthesis, JA Signaling Related Genes and miR156-SPLs
Based on the results of GO and KEGG analysis, we selected the key flowering-related genes, JA biosynthesis and signaling-related genes, miR156-SPLs and miR172-gl15, for RT-qPCR analysis (Figure 8). Of the detected FT homologues, only ZCN18 showed extremely significant up-regulation ( Figure 8A). ZCN7/8/12, MADS32/56/68, and several AP2/EREBP genes all showed a significant down-regulated expression. Of the JA synthesis and JA signaling related genes ( Figure 8B), only DAD1 showed a very significant up-regulated expression, OSAOS1, LOX1, AOC, Zm00001d004573, Zm00001d028744, Zm00001d048021 and other genes showed a significant down-regulated expression trend at FT stage. This suggests that ZCN18 and DAD1 genes may play important roles in the regulation of the early flowering of ZmEPC1 mutant. Most of those JA biosynthesis and signaling-related genes displayed up-regulated expression at V5 stage, only Zm00001d004573 exhibited significant down-regulated expression. Between V5 and FT stage, OSAOS1, LOX1, AOC, Zm00001d028744, and Zm00001d048021 displayed opposite expression alterations.
The miR156-SPLs regulatory module has been proved to be a key regulator in plant phase transition [56]. In ZmEPC1 mutant, the expression of miR156 was significantly reduced compared with the WT at the two detected stages ( Figure 8C). The expression of the miR156 target gene not1 was significantly increased, while piip2 was consistent with the expression level of miR156, suggesting that piip2 might feedback-regulated the expression of miR156. In turn, miR172 exhibited up-regulated expression in ZmEPC1 mutant at V5 stage but displayed the down-regulated expression at FT stage ( Figure 8D). The target gene of miR172, gl15, displayed opposite expression trends compared with the expression of miR172. The miR156-SPLs regulatory module has been proved to be a key regulator in plant phase transition [56]. In ZmEPC1 mutant, the expression of miR156 was significantly reduced compared with the WT at the two detected stages ( Figure 8C). The expression of the miR156 target gene not1 was significantly increased, while piip2 was consistent with the expression level of miR156, suggesting that piip2 might feedback-regulated the expression of miR156. In turn, miR172 exhibited up-regulated expression in ZmEPC1 mutant at V5 stage but displayed the down-regulated expression at FT stage ( Figure 8D). The target gene of miR172, gl15, displayed opposite expression trends compared with the expression of miR172.

ZmEPC1 Is Involved in the Regulation of Maize Developmental Phase Transition
In plants, the post-embryonic development of the shoot usually occurs in three more or less discrete temporal phases: juvenile vegetative phase, adult vegetative phase, and reproductive phase [15]. The timing of developmental phase transitions is important for plant growth, environmental adaptation, and crop production. In maize, an early phase change mutant displayed reduced juvenile vegetative phase, early flowering, and decreased plant height and leaf size [2]. Several early flowering-related mutants, such as ZmCCT9-KO [49] and ZmMADS69-OE [51], have also been identified to exhibit decreased plant height and leaf size and the late flowering mutants gl15 [3], ZmCOL3-OE [34], dlf1 [40], id1 [44] and ZmCCT10-OE [50] exhibit increased plant height and leaf size. This research indicated that the developmental phase transition is tightly associated with the plant height and leaf size. In this study, ZmEPC1 mutant plants exhibited obviously phenotypic changes, including early developmental phase transition, decreased plant height, and small leaves. These phenotypic changes revealed ZmEPC1 to be an important regulator in maize juvenile to adult vegetative phase transition and vegetative to reproductive phase transition.

ZmEPC1 Acts on Phytohormones Signaling Pathway
Phytohormones, auxin, GA, CTK, ethylene, ABA, and BR, have been proved to act as crucial regulators in plant development and response to various environmental stimulus, including drought, heat, salinity stress, chilling damage, and heavy metal toxicity [17,57]. In control of plant vegetative phase transition and flowering, the regulatory roles of GA, JA, ABA, BR, auxin, ethylene, and CTK have been explored [6,9,12,14,16,17,21,22,[34][35][36][37][38][39][40]. Especially, GA and JA have been defined to affect maize vegetative phase change and flowering [16,18]. In the regulation of flowering, GA signaling pathway acts a crucial determinant not only through its interaction with other endogenous signaling pathways and environmental stimulus but also via its crosstalk with other phytohormones [22]. DELLA proteins has been proved to link the GA signaling pathway with other phytohormone signaling pathways, such as JA, CTK, ABA, auxin, ethylene, and BR. The JA signaling pathway regulates flowering via controlling floral induction and its crosstalk with the GA signaling pathway [37]. In maize, GA promotes vegetative phase transition and flowering, but JA acts the opposite role in vegetative phase transition [16,18]. In the present study, numerous DEGs were screened in the transcriptome analysis of ZmEPC1 mutants. The GO, KEGG and regulatory network analysis of these DEGs revealed that ZmEPC1 is mainly involved in the regulation of biological pathways, including photosynthesis, hormone response, hormone-stimulated cell response, endogenous stimulation cell response, endogenous stimulation response, JA response, and JA-mediated signal response pathway. We analyzed the expression of JA biosynthesis-and signaling-genes in V5 and FT leaf samples, which demonstrated that most genes express significant down-regulation in mutant ZmEPC1 at the FT stage but up-regulated expression at the V5 stage. The down-regulated expression of JA biosynthesis-and signaling-related genes at FT stage may contribute the early flowering, these genes down-regulated expression at the V5 stage are possible to result in narrow and short leaves. GA, ethylene, IAA, CTK, and BR signaling-related DEGs were selected for RT-qPCR verification in V5 and FT samples. Of the detected genes, two GA biosynthesis-related genes displayed up-regulated expression at the FT stage, which may promote flowering and a GA receptor encoding gene exhibited down-regulated expression at the V5 and FT stages, which can possibly result in the early developmental phase transition in ZmEPC1. ZmEPC1 mutation also caused the expression alterations of other phytohormone signaling-related genes, such as IAA biosynthesis-and signaling-related genes, CTK biosynthesis-related genes, BR biosynthesis-and signaling-genes, and ethylene signaling-related genes. These results indicated that ZmEPC1 mutation acts as an essential regulator in phytohormones signaling.

Potential Regulatory Mechanism of ZmEPC1 in Developmental Phase Changes
Regulatory modules, miR156-SPLs and miR172-AP2s, are crucial determinants in juvenile to adult vegetative phase transition [3][4][5][6][7][8]. In addition, embryonic regulators, sugar, meristem regulators, hormones, and epigenetic modifications may affect the juvenile to adult vegetative phase transition [9,15]. In the control of the vegetative to reproductive phase transition, signaling pathways, including photoperiod and circadian clock pathways, vernalization, and autonomous pathways, the GA pathway, ambient temperature pathway, age pathway, and meristem responses, have been identified to play important roles [19,[21][22][23][24][25][26]. In maize, Tp1, Tp2, Tp3, gl15, and Cg1 have been identified to confer vegetative phase change [3,4,18] and id1, dlf1, ZCN8, ZCN12, ZmMADS1, ZMM4, Vgt1, ZmCCT9, ZmCCT10, ZmMADS69, HPC1, ZmNF-YC2, and ZmCol3 have been proven to affect vegetative to productive phase transition [34,39,40,[44][45][46][47][48][49][50][51][52]. In the present work, the expression level of miR156 in ZmEPC1 mutant leaves at the V5 and FT stage showed a significant down-regulation trend. However, miR172 exhibited up-regulated expression in ZmEPC1 mutant at the V5 stage but down-regulated the FT stage. One target gene Zm00001d014794-piip2 showed a similar expression trend, the other target gene Zm00001d049824-not1 showed an opposite expression alteration to that of miR156. The target gene of miR172, gl15, displayed opposite expression trends compared with the expression of miR172. These results indicated that ZmEPC1 may regulate maize developmental stage transitions through the miR156-SPLs and miR172-gl15 regulatory modules. The transcriptome analysis revealed ZmEPC1 to be involved in GA and JA signaling pathways. In ZmEPC1 mutants, the GA and JA signaling-related genes display differentially expression in consistent with the early flowering. Two shoot meristem development related genes, WOX2 (Zm00001d042920 at V5 and FT stages) and Zm00001d051093 (encodes LRR receptor-like serine/threonine-protein kinase EFR at the FT stage) displayed significantly down-or up-regulated expression in ZmEPC1 mutants, which indicated meristem regulators could act as important determinants in developmental phase change. Moreover, the number of flowering time-related genes displayed differential expressions between the mutant and the wild type. These genes may contribute the early developmental phase change in ZmEPC1 mutant. Collectively, the mutation gene can possibly act as a regulator of JA and GA signaling, which mediates the expression alterations of miR156-SPLs, miR172-gl15, to further modulate shoot meristem development and to determine the developmental phase changes in the maize ZmEPC1 mutant.

Conclusions
A natural mutant ZmEPC1 with significantly reduced plant height and early developmental phase was screened from an inbred line. By transcriptome analysis, major early developmental phase change-related genes were identified, and the underlying regulatory pathways in the mutant were analyzed. The present work provides the necessary support for cloning the candidate gene of ZmEPC1 and dissecting the genetic mechanism in the maize developmental phase transition.