ZmbZIP27 regulates nitrogen-mediated leaf angle by modulating lignin deposition in maize

Abstract

In grain crops such as maize (Zea mays), leaf angle (LA) is a key agronomic trait affecting light interception and thus planting density and yield. Nitrogen (N) affects LA in plants, but we lack a good understanding of how N regulates LA. Here, we report that N deficiency enhanced lignin deposition in the ligular region of maize seedlings. In situ hybridization showed that the bZIP transcription factor gene ZmbZIP27 is mainly expressed in the phloem of maize vascular bundles. Under N-sufficient conditions, transgenic maize overexpressing ZmbZIP27 showed significantly smaller LA compared with wild type (WT). By contrast, zmbzip27ems mutant showed larger LA under both N-deficient and N-sufficient conditions compared with WT. Overexpression of ZmbZIP27 enhanced lignin deposition in the ligular region of maize in the field. We further demonstrated that ZmbZIP27 could directly bind the promoters of the microRNA genes ZmMIR528a and ZmMIR528b and negatively regulate the expression levels of ZmmiR528. ZmmiR528 knockdown transgenic maize displayed erect architecture in the field by increasing lignin content in the ligular region of maize. Taken together, these results indicate that ZmbZIP27 regulates N-mediated LA size by regulating the expression of ZmmiR528 and modulating lignin deposition in maize.

Headnote

ABSTRACT

In grain crops such as maize (Zea mays), leaf angle (LA) is a key agronomic trait affecting light interception and thus planting density and yield. Nitrogen (N) affects LA in plants, but we lack a good understanding of how N regulates LA. Here, we report that N deficiency enhanced lignin deposition in the ligular region of maize seedlings. In situ hybridization showed that the bZIP transcription factor gene ZmbZIP27 is mainly expressed in the phloem of maize vascular bundles. Under N-sufficient conditions, transgenic maize overexpressing ZmbZIP27 showed significantly smaller LA compared with wild type (WT). By contrast, zmbzip27ems mutant showed larger LA under both N-deficient and N-sufficient conditions compared with WT. Overexpression of ZmbZIP27 enhanced lignin deposition in the ligular region of maize in the field. We further demonstrated that ZmbZIP27 could directly bind the promoters of the microRNA genes ZmMIR528a and ZmMIR528b and negatively regulate the expression levels of ZmmiR528. ZmmiR528 knockdown transgenic maize displayed erect architecture in the field by increasing lignin content in the ligular region of maize. Taken together, these results indicate that ZmbZIP27 regulates N-mediated LA size by regulating the expression of ZmmiR528 and modulating lignin deposition in maize.

Keywords:

Maize

Nitrogen

Leaf angle

Ligular region

Lignin deposition

(ProQuest: ... denotes formulae omitted.)

1. Introduction

Nitrogen (N), as a key component of protein, nucleic acid, and phospholipid, is essential for plant normal growth and development. The concentrations of N in soil range from 100 lmol L-1 to 10 mmol L-1 [1]. In the face of variations in N availability in the soil, plants can change their root architecture to increase N uptake [2]. When maize was subjected to N deficiency for 6 days, the length of axile roots increased, the number of crown roots decreased and the density of lateral roots decreased [3]. Longterm N deficiency also led to discoloration of old leaves. Leaf chlorosis and even senescence are hallmark symptoms of N deficiency in plants. However, we lack a visible indicator of early N deficiency in plants.

Leaf angle (LA), defined as the angle between the vertical stem and the midrib of a leaf blade, is a key agronomic trait affecting planting density and yield in maize [4,5]. In maize, the LA depends on the ligular region, which links the distal blade to the basal sheath [6]. LA is regulated by genes influencing the development of auricles and ligules, genes related to hormone pathways, and genes associated with mechanical strength in the midrib region of leaves [7,8]. Among the known LA-related genes, LIGULELESS1 (LG1) and LG2 are the central regulators of ligular region formation in maize, and liguleless mutants show erect leaves [9,10]. LA is also affected by environmental factors, including N fertilizer, row spacing, and plant density [11,12]. In particular, high N application increases LA and make the leaves more horizontal in maize under field conditions [13]. However, the mechanisms by which N regulates LA remain unclear.

The LA in maize is established by a two-step regulatory process involving initial cell elongation followed by subsequent lignification in the adaxial ligular region [5]. As the second most abundant biological polymer, lignin is important for the structural integrity of the cell wall and stiffness and strength of the stem [14,15]. Peroxidases and/or laccases (LACs) functions in the oxidative polymerization of lignin monomers [16]. Our previous results showed that N deficiency down-regulates, and N luxury up-regulates the abundance of ZmmiR528 in maize [17]. Moreover, the ZmmiR528-ZmLAC3 module affects lodging resistance of maize under N luxury conditions by regulating lignin biosynthesis in the stem and ion homeostasis by modifying root casparian strip formation in the root [17,18]. In rice (Oryza sativa), OsmiR397 and its target OsLAC are expressed mainly in the young panicles and grains, and overexpression of OsmiR397 and OsLAC under the control of the cauliflower mosaic virus 35S promoter led to flat or erect leaves, respectively [19]. Whether and how ZmmiR528 affects nitrogen-mediated LA requires further investigation.

TGACG-BINDING FACTOR (TGA) belongs to subgroup D of the basic region/leucine zipper motif (bZIP) transcription factors [20]. TGA transcription factors participate in plant development and defenses against biotic or abiotic stresses. In Arabidopsis thaliana, NONEXPRESSOR OF PR GENES1 (NPR1) is a receptor for the plant defense hormone salicylic acid. The BTB/POZ domain of NPR1 interacts with and negates the TGA2 repression domain by excluding TGA2 oligomers from their target DNA [21,22]. AtTGA1 and AtTGA4 regulate the expression of the nitrate transporter (NRT) genes AtNRT2.1 and AtNRT2.2 and affect N-regulated lateral root development [23]. Temporal regulatory network analysis further demonstrated that AtTGA1 and AtTGA4 function as important regulatory components of the nitrate response of Arabidopsis thaliana roots [24].

In the present research, we showed that LA was significantly affected by N supply and could be used as a visible indicator to evaluate early symptoms of N deficiency in maize seedlings. We further demonstrated that the maize TGA transcription factor ZmbZIP27 is involved in setting LA size in response to N by regulating lignin deposition in the ligular region of maize.

2. Materials and methods

2.1. Plant materials and growth conditions

Maize seed surface was pre-sterilized for hydroponics as previously described [17]. After the seeds were germinated in paper rolls for 7 d, the endosperms were removed for cultivation. First, the maize seedlings were transferred to 3-L pots containing modified half-strength cultivation Hoagland's nutrient solution for 2 d. Then, the seedlings were supplied with 0.04 mmol L-1 , 4 mmol L-1 , or 8 mmol L-1 NO3 * in full-strength cultivation Hoagland's nutrient solution to simulate N-deficient (ND), N-sufficient (NS), or N-luxury (NL) conditions, respectively. The maize seedlings were grown in a growth chamber with 14 h light/10 h dark and a 28/22 C day/night temperature. The nutrient solution was replaced with fresh solution every 2 d to ensure pH stability.

2.2. Constructs and generation of transgenic maize

To generate ZmbZIP27-overexpressing transgenic maize, the full-length coding sequence of ZmbZIP27 was amplified and cloned into the pCUB vector under the control of the constitutive ubiquitin promoter using the BstEII restriction site by In-Fusion reaction. The pCUB-ZmbZIP27 verified plasmid was electroporated into Agrobacterium tumefaciens EHA105 and transformed into immature embryos of maize inbred line B104 at Bomei Xing'ao Co., Ltd. (Beijing, China). Transformants were selected with bialaphos as previously described [25]. For ZmbZIP27-overexpressing transgenic maize, T3 homozygous lines were used for all experiments. The specific primers are listed in Table S1.

2.3. RNA-Seq analysis

Total RNA was extracted from ligular region of the second leaf from the bottom of maize inbred line B73 that had been subjected to ND or NS treatments for 2 d using the Purelink RNA Micro Scale Kit (Thermo Fisher Scientific, USA), and 3 lg total RNA was used for construction of the RNA libraries. Three biological replicates were performed for each treatment. RNA libraries were constructed and sequenced by Berry Genomics Co., Ltd. (Beijing, China). The clean reads were obtained from the raw reads after exclusion of rRNA sequences, low quality reads, and 50 and 30 adaptor contaminants. The clean reads were then mapped to the maize B73_RefGen_v4 genome (ftp://ftp.ensemblgenomes.org/pub/ plants/release-41/fasta/zea_mays/dna/) using Bowtie2 v2.4.5 with default parameters [26]. Only perfectly matching sequences were used for further analysis. The read counts for each gene were calculated using HTSeq v0.8.0 with default parameters [27]. The DESeq2 package was then used to identify the differentially expressed genes (DEGs) according to the criteria of foldchange 2 and false discovery rate (FDR)* 0.05 [28]. Gene Ontology (GO) enrichment was performed using AgriGO online tools version 2.0 (https://systemsbiology.cau.edu.cn/agriGOv2/) with default parameters, and terms with an FDR * 0.05 were considered significantly enriched [29].

2.4. RNA analysis

Total RNA extraction, first-strand cDNA synthesis, RT-qPCR, and stem-loop RT-qPCR were performed as described previously [17]. RT-qPCR was carried out in an ABI 7500 system (Applied Biosystems, USA) using the SYBR Select Master Mix (Applied Biosystems, USA). Each analysis was based on three replications. The comparative Ct method was used to detect the gene relative expression levels. The expression levels were normalized to ZmActin1. The sequences of the specific primers are listed in Table S1.

2.5. Subcellular localization of ZmbZIP27

For subcellular location of ZmbZIP27 in Nicotiana benthamiana, the full-length coding sequence of ZmbZIP27 was amplified and cloned into the pCAMBIA1305 vector, which was fused to a GFP protein in the N terminus using the BglII restriction site and the C terminus using the BamHI restriction site via an In-Fusion reaction, respectively. The verified plasmids were electroporated into Agrobacterium tumefaciens GV3101 and were transiently expressed in N. benthamiana epidermal cells as described previously [17]. Tobacco epidermal cells were imaged 2 d after transformation by a Zeiss LSM980 confocal microscope. The sequences of the specific primers are listed in Table S1. 2.6. Analysis of lignin content and

The second leaf from the bottom of 12-day-old hydroponically grown maize seedlings and the ligular region of the first leaf above the ear in field-grown maize were sampled for measuring lignin contents and histochemical analysis of vascular structure. The acetyl bromide (AcBr) method was used to determine the total lignin content as described previously [30]. For staining, the samples were embedded in 3% agarose and then sliced into 50-lm sections with a Leica VT1000S vibratome. The vascular structure of the ligular region was visualized by Wiesner staining as previously described [31], and images were collected with a Leica DM4B microscope. ImageJ software was used to calculate the staining signal intensity of the sclerenchymatous hypodermis layer and vascular sclerenchymatous hypodermis layer based on the gray scale pixel values of the images.

2.7. In situ hybridization

The specific fragment of ZmbZIP27 (348 bp) was amplified and cloned into the pGEM-T-easy vector (Promega, USA). Sense and antisense probes were generated by in vitro transcription using T7 or SP6 RNA polymerases with DIG RNA Labeling Kit (Roche, Basel, Switzerland). Ligular region tissue fixation and RNA in situ hybridization for ZmbZIP27 were performed as described previously [17]. Images were collected with a Leica DM4B microscope. The sequences of the specific primers are listed in Table S1.

2.8. Electrophoretic mobility shift assays (EMSAs)

GST-ZmbZIP27 recombinant protein was used for EMSAs. The full-length coding sequence of ZmbZIP27 was amplified and cloned into the pGEX-4T-1 vector using the BamHI restriction site by InFusion reaction to generate GST-ZmbZIP27 expression plasmid. The construct was expressed in Escherichia coli BL21 and further purified using glutathione-Sepharose 4B beads (GE Healthcare, Stockholm, Sweden). DNA probes from the promoters of ZmMIR528a and ZmMIR528b were amplified and then labeled with DIG-ddUTP by DIG Gel Shift Kit (Roche, Basel, Switzerland). The same unlabeled fragments were used as competitor probes and GST protein was used as the negative control. EMSAs procedures were performed as previously described [25]. The sequences of the specific primers are listed in Table S1.

2.9. Statistical analysis

In this study, data are presented as means ± standard deviation (SD). The number of replicates for each experiment can be found in the figure legends. Student's t-test was used to determine statistical significance between two groups by Microsoft Excel; asterisks as followed * or ** indicate significant difference at P < 0.05 or P < 0.01, respectively. For multiple group comparisons, the data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test using SAS (v.8; SAS Institute Inc., Cary, NC, USA); means with the same letter are not significantly different at P < 0.05.

3. Results

3.1. N supply affects LA in maize seedlings

n the field, N supply affects LA in maize hybrids [13]. To test how N affected maize LA, we performed a time-course experiment with maize inbred line B73 under N-deficient (ND), N-sufficient (NS), or N-luxury (NL) conditions. The LA of the first leaf from the bottom of maize did not show obvious difference among various N conditions (Fig. 1A). Compared with NS or NL conditions, short-term (3 days) ND treatment significantly decreased the LA of the second leaf from the bottom of maize though the leaf color was similar among various N conditions (Fig. 1B). When maize seedlings were subjected to ND treatment for 7 d, chlorosis, the typical symptom of N deficiency in plants, appeared in the first leaf from the bottom (Fig. 1C). Compared with NS or NL conditions, seedlings treated with ND showed decreased LA of both the second and the third leaf from bottom (Fig. 1B, D). These results indicated that LA could be used as a visible indicator to evaluate early N deficiency in maize.

3.2. Lignin deposition is important for N-mediated LA in maize

To gain insight into the molecular events involved in the N-mediated LA in maize, we compared the transcriptome profiles of maize seedlings subjected to ND and NS treatments for 2 d, one day earlier than the appearance of difference in LA (Fig. 1B). To avoid the interference of other tissues, we only sampled the ligular region of the second leaf from the bottom of maize inbred line B73 by stereo microscopy (Fig. S1A). Three biological replicates were collected for each treatment. The six transcriptome libraries yielded more than 170 million raw reads, over 90% of the raw reads could be mapped to the B73_RefGen_v4.41 reference genome. Based on the criteria of fold-change 2 and FDR * 0.05, 3,481 DEGs were identified. Among the DEGs, 2104 genes were up-regulated and 1377 genes were down-regulated by ND treatment (Fig. S1B). GO analysis revealed that the top 10 terms of the upregulated DEGs were involved in phenylpropanoid biosynthetic process (GO:0009699, P = 1.6e-9 ), lignin biosynthetic process (GO:0009809, P = 2.7e-8 ), phenylpropanoid catabolic process (GO:0009698, P = 2.2e-8 ), nitrogen compound transport (GO:0071705, P = 1.7e-7 ), and transmembrane transport (GO:0055085, P = 2.6e-9 ) (Fig. 2A). Lignin is depositing in the secondary cell wall and is responsible for the mechanical strength of vascular bundles [14,15]. We thus hypothesized that lignin deposition might be involved in N-mediated LA in maize.

To test this hypothesis, we tried to measure the lignin contents in the ligular region of maize seedlings subjected to ND and NS treatments for 2 d. Due to the small sample size, we used phloroglucinol staining to quantify the effects of N supply on lignin content of maize seedlings. A two-day ND treatment significantly increased the lignin content in both the cortex and the vascular tissue near the cortex of the ligular region (Fig. 2B–D). These results supported the idea that the N-mediated LA might depend on lignin deposition.

3.3. Identification of candidate genes involved in N-mediated LA in maize

To explore early potential regulators involved in the effects of N-mediated LA in maize, we reanalyzed our transcriptome data together with publicly available vascular bundle transcriptome data [32,33]. This analysis identified 42 genes that were both induced by ND treatment and whose expression was enriched in vascular tissue (Fig. S2A; Table S2). Among the identified genes, the bZIP transcription factor gene ZmbZIP27 attracted our attention. ZmbZIP27 is homologous to the Arabidopsis TGA transcription factor genes AtTGA1 (AT5G65210) and AtTGA4 (AT5G10030) (Fig. S2B). AtTGA1 and AtTGA4 are highly induced in response to nitrate treatment and these transcription factors regulate nitrate responses in Arabidopsis roots [23,33]. In contrast to AtTGA1 and AtTGA4, when maize seedlings were subjected to ND treatment for 3 d, the expression levels of ZmbZIP27 significantly increased in the root and shoot, especially in the shoot (Fig. S3A, B).

The RT-qPCR showed that ZmbZIP27 is preferentially expressed in the ligular region and auricles in leaves (Fig. S3C, D). We further examined the expression patterns of ZmbZIP27 by in situ hybridization analysis. A strong signal was detected in the phloem of vascular bundles, and signals were also detected in the bundle sheath and the metaxylem (Fig. 3A). These results suggested that ZmbZIP27 is a potential candidate gene for regulation of LA in response to N in maize.

3.4. ZmbZIP27 regulates N-mediated LA in maize

To characterize the function of ZmbZIP27, we first detect its subcellular localization. Several transcripts were annotated in the maizeGDB database (https://www.maizegdb.org/). These transcripts shared the same 3ʹ terminal. We thus cloned the full length of ZmbZIP27 by 5ʹ-RACE and transiently expressed N-terminal and Cterminal ZmbZIP27GFP fusions in N. benthamiana. ZmbZIP27GFP and GFP-ZmbZIP27 signals were detected only in the nucleus of N. benthamiana (Fig. 3B). Transgenic maize overexpressing ZmbZIP27 were generated under the control of the constitutive ubiquitin promoter. Two transgenic lines (ZmbZIP27-OE #1 and #2) were chosen for further analysis based on their high levels of ZmbZIP27 expression (Fig. 3C). Overexpression of ZmbZIP27 significantly reduced LA in maize under NS condition (Fig. 3D, E). However, the ZmbZIP27OE transgenic maize and wild type (WT) maize had similar LA under ND condition (Fig. 3D, E). Because ND strongly induced the expression of ZmbZIP27 in shoots (Fig. S3B), we deduced that the effects of endogenous ZmbZIP27 would mask the effects of ZmbZIP27 overexpression under ND condition. This hypothesis was further supported by the reduced LA of the flag leaf and the first leaf above ear of ZmbZIP27-overexpressing transgenic maize in the field (Fig. S4A, B).

We obtained an ethyl methanesulfonate (EMS) mutant of ZmbZIP27 in the B73 background from the Maize EMS-induced Mutant Database (mutant ID: EMS3-022b3c). The EMS3-022b3c mutant contains a C/T substitution in the fifth exon of ZmbZIP27, which leads to a premature stop codon in the gene (Fig. 4A). We designated the mutant as zmbzip27ems. Compared with WT maize, the zmbzip27ems mutant had significantly larger LA under both ND and NS conditions (Fig. 4B, C). Consistent with the observations in hydroponics solutions, zmbzip27ems mutant showed large LA of the flag leaf in the field (Fig. S4C, D). These results indicated that ZmbZIP27 regulates N-mediated LA in maize.

3.5. ZmbZIP27 affects lignin deposition in maize

The genes regulating maize LA can be categorized into two classes: (1) genes involved in auricle development; (2) genes associated with mechanical strength in vascular bundles [7]. To determine the pathway by which ZmbZIP27 affects LA in maize, we first measured the auricle margin width and the auricle size of ZmbZIP27OE transgenic maize grown in the field. The auricle margin width and the auricle area of the first leaf above the maize ear did not differ between ZmbZIP27OE transgenic maize and WT plants (Fig. S5). These results suggested that ZmbZIP27 does not affect auricle development.

We therefore investigated whether ZmbZIP27 regulated maize LA by affecting mechanical strength in the vascular bundle. To this end, we used AcBr analysis to measure the lignin content of ligular regions of ZmbZIP27OE transgenic maize grown in the field. Overexpression of ZmbZIP27 significantly increased the lignin content in the ligular region of the first leaf above the maize ear (Fig. 5A). In agreement with AcBr analysis, phloroglucinol staining also showed that lignin accumulated in the sclerenchymatous hypodermis layer and vascular sclerenchymatous cell layer of ZmbZIP27OE transgenic maize (Fig. 5B–D). These results suggested that ZmbZIP27 affects LA by regulating lignin deposition in maize.

To test the potential application of ZmbZIP27 in maize production, we grew ZmbZIP27-overexpressing transgenic maize at three densities in Sanya, Hainan Province. The grain yield showed no obvious difference between ZmbZIP27-overexpressing transgenic maize and WT under densities at 45,000 plants per hectare (Fig. S6). When the planting densities increased to 75,000 and 105,000 plants per hectare, the grain yield of mbZIP27-overexpressing transgenic maize was higher than that of WT (Fig. S6). These results suggested that ZmbZIP27 can improve yield potentials of maize under dense planting conditions.

3.6. ZmmiR528 is involved in ZmbZIP27-regulated lignin deposition in maize

Our previous results showed that the accumulation of ZmmiR528 is regulated by N supply and ZmmiR528 regulates Nmediated lignin deposition in maize [17]. We deduced that ZmmiR528 might contribute to ZmbZIP27-regulated lignin deposition in maize. Analysis of the promoter region (1000-bp region upstream of the transcription start site) of ZmMIR528a and ZmMIR528b detected a TGACG binding motif in the promoter regions of ZmMIR528a and ZmMIR528b (Fig. S7). To test the direct binding of ZmbZIP27 to the ZmMIR528a and ZmMIR528b promoters, we performed electrophoretic mobility shift assays (EMSAs). The EMSAs showed that ZmbZIP27 could bind the ZmMIR528a and ZmMIR528b promoter (Fig. 6A). When unlabeled ZmMIR528a or ZmMIR528b probe was added to the system as a competitor, the signal was suppressed (Fig. 6A), further demonstrating the direct binding of the ZmMIR528a and ZmMIR528b promoter by ZmbZIP27.

We then detected the expression levels of ZmmiR528 precursors in the ligular region of the first leaf above the ear in ZmbZIP27OE transgenic maize by RT-qPCR. Overexpression of ZmbZIP27 significantly decreased the expression levels of ZmMIR528a and ZmMIR528b (Fig. 6B). Stem-loop RT-qPCR showed that the abundance of mature ZmmiR528 was reduced in ZmbZIP27OE transgenic maize (Fig. 6B). These results indicated that ZmbZIP27 negatively regulated the expression levels of ZmmiR528.

To investigate whether ZmmiR528 affects LA in maize, we grew ZmmiR528 knockdown transgenic maize generated by short tandem target mimic technology in the field [17]. Compared with WT plants, ZmmiR528 knockdown transgenic maize showed a more compact plant architecture (Fig. 6C). Knockdown of ZmmiR528 significantly decreased LA of the flag leaf and the first leaf above the ear (Fig. 6D, E). In agreement with the observations in ZmbZIP27OE transgenic maize, the lignin content in the ligular region of the first leaf above maize ear was higher in ZmmiR528 knockdown transgenic maize compared with WT (Fig. 6F). Consistent with the observations in field, knockdown of ZmmiR528 reduced LA under both NS and ND conditions in hydroponic solutions (Fig. S8). These results suggested that ZmmiR528 is involved in ZmbZIP27-regulated lignin deposition in maize.

4. Discussion

N is one of the macro-elements for plant growth and development. Long-term ND treatment induces leaf chlorosis, a typical symptom of N-deficiency in plants [35,36]. However, no visible symptoms were observed in maize subjected to ND treatment for 3 d, even though the N concentration decreased by about 30% in the shoot [3]. Thus, a visible indicator is required to evaluate early N deficiency in maize. In the field, N supply affects LA in maize and rice [13,37]. Here, we observed that short-term (3 d) N deficiency caused a significantly smaller angle in the second leaf from the bottom of maize seedlings compared with those grown with sufficient N. Notably, this phenotype appeared well before chlorosis appeared. Similar N-responsiveness of the LA was observed in maize inbred lines B73 and B104. Thus, this response appears to be independent of genotype. We hypothesize that LA of maize seedlings could be used to evaluate early N deficiency responses.

LA is largely determined by the ligular region at the junction between leaf blade and leaf sheath [6]. In our study, RNAsequencing of tissues from the ligular region revealed that lignindeposition might contribute to N- mediated LA in maize. Lignin is a principal component of plant secondary cell walls and is essential for maintaining mechanical strength [14–16]. Maize LA is influenced by initial cell elongation and subsequent lignification in the ligular adaxial sclerenchyma cells [5]. In rice, lignin also confers vascular bundle strength and thus affects LA [19]. Here, we determined that the lignin contents in both the cortex and the vascular tissue of the ligular region were significantly increased after ND treatment for 2 d. These results support the conclusion that Ninduced changes in LA of maize are dependent on lignin deposition.

In maize, many genes have been identified as regulating LA size, including DE-ETIOLATED2 (ZmDET2/NA1), DWARF1 (ZmDWF1/ NA2), RELATED TO ABI3/VP1-LIKE1 (ZmRAVL1), BRASSINOSTEROID C-6 OXIDASE1 (ZmBRD1), and DROOPING LEAF1 (ZmDRL1). These genes regulate maize LA by affecting biosynthesis of brassinosteroid phytohormones [38–41]. Here, we provide evidence that ZmbZIP27 could regulate maize LA in response to N: (1) ZmbZIP27 was induced by ND; (2) ZmbZIP27 was preferentially expressed in the ligular region and auricle in maize leaves; (3) ZmbZIP27 was expressed in the phloem of vascular bundles; (4) overexpression and loss-of-function of ZmbZIP27 affected LA in maize under different N conditions.

ZmbZIP27 is homologous with AtTGA1 and AtTGA4 in Arabidopsis. In Arabidopsis, AtTGA1 and AtTGA4 are mainly expressed in the root (https://www.ebi.ac.uk/gxa/home), and play important roles in the root developmental responses to nitrate [34]. In contrast, ZmbZIP27 is preferentially expressed in the ligular region and auricles of maize leaves. These results indicate that the TGA transcription factor has functional specificity in regulating the responses of monocotyledonous and dicotyledonous plants to nitrate.

Our previous research indicated that ZmmiR528 is specifically expressed in the phloem of maize leaves and stems and posttranscriptionally regulates the expression levels of ZmLACCASE3 (ZmLAC3) and ZmLAC5 [17]. Laccase is necessary for lignin polymerization during vascular development [42]. Knockdown of ZmMIR528 or overexpression of ZmLAC3 increased maize lodging resistance by regulating lignin biosynthesis under N-luxury conditions [17]. ZmmiR528 also contributes to N-mediated Casparian strip formation in maize roots [18]. There are two members of the miR528 family in maize, and they share the same mature sequences. ZmbZIP27 binding sites were found in the promoters of ZmMIR528a/b, and EMSAs verified that ZmbZIP27 could directly bind the ZmMIR528a and ZmMIR528b promoters. We further demonstrated that ZmbZIP27 negatively regulated the levels of ZmmiR528. Knockdown of ZmmiR528 significantly increased the lignin content in the ligular region of the first leaf above the maize ear and decreased LA. Overall, our results demonstrate that the ZmbZIP27-ZmmiR528 module is an important link between N stress responses and N-mediated LA in maize (Fig. 6G).

CRediT authorship contribution statement

Huan Chen: Data curation, Resources, Validation, Writing – original draft. Xiaoping Gong: Data curation, Resources, Validation. Yu Guo: Data curation, Resources, Validation. Jingjuan Yu: Writing – review & editing. Wen-Xue Li: Conceptualization, Funding acquisition, Writing – review & editing. Qingguo Du: Conceptualization, Resources, Software, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the Biological Breeding-National Science and Technology Major Project (2023ZD04072), the Innovation Program of Chinese Academy of Agricultural Sciences, and the Hainan Yazhou Bay Seed Lab (B23YQ1507).

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ARTICLE INFO

Article history:

Received 12 June 2024

Revised 31 August 2024

Accepted 8 September 2024

Available online 3 October 2024

Footnote

* Corresponding authors.

E-mail addresses: [email protected] (W.-X. Li), [email protected] (Q. Du).

1 These authors contributed equally to this work.

References

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Appendix A. Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2024.09.004.