ZmCYP90D1 regulates maize internode development by modulating brassinosteroid-mediated cell division and growth

Abstract

Plant height (PH) is associated with lodging resistance and planting density, which is regulated by a complicated gene network. In this study, we identified a spontaneous dwarfing mutation in maize, m30, with decreased internode number and length but increased internode diameter. A candidate gene, ZmCYP90D1, which encodes a member of the cytochrome P450 family, was isolated by map-based cloning. ZmCYP90D1 was constitutively expressed and showed highest expression in basal internodes, and its protein was targeted to the nucleus. A G-to-A substitution was identified to be the causal mutation, which resulted in a truncated protein in m30. Loss of function of ZmCYP90D1 changed expression of hormoneresponsive genes, in particular brassinosteroid (BR)-responsive genes which is mainly involved in cell cycle regulation and cell wall extension and modification in plants. The concentration of typhasterol (TY), a downstream intermediate of ZmCYP90D1 in the BR pathway, was reduced. A haplotype conferring dwarfing without reducing yield was identified. ZmCYP90D1 was inferred to influence plant height and stalk diameter via hormone-mediated cell division and cell growth via the BR pathway.

Headnote

ABSTRACT

Plant height (PH) is associated with lodging resistance and planting density, which is regulated by a complicated gene network. In this study, we identified a spontaneous dwarfing mutation in maize, m30, with decreased internode number and length but increased internode diameter. A candidate gene, ZmCYP90D1, which encodes a member of the cytochrome P450 family, was isolated by map-based cloning. ZmCYP90D1 was constitutively expressed and showed highest expression in basal internodes, and its protein was targeted to the nucleus. A G-to-A substitution was identified to be the causal mutation, which resulted in a truncated protein in m30. Loss of function of ZmCYP90D1 changed expression of hormoneresponsive genes, in particular brassinosteroid (BR)-responsive genes which is mainly involved in cell cycle regulation and cell wall extension and modification in plants. The concentration of typhasterol (TY), a downstream intermediate of ZmCYP90D1 in the BR pathway, was reduced. A haplotype conferring dwarfing without reducing yield was identified. ZmCYP90D1 was inferred to influence plant height and stalk diameter via hormone-mediated cell division and cell growth via the BR pathway.

Keywords:

Maize

ZmCYP90D1

BR biosynthesis

Dwarf plant

1. Introduction

PH is an agronomic trait strongly associated with lodging resistance and planting density, which in turn increasing crop yield [1,2]. The Green Revolution genes, such as Ossd1 and TaRht, involved in gibberellin (GA) biosynthesis or signaling, have been widely used in rice and wheat breeding by adopting semi-dwarf varieties that were resistant to lodging [3,4]. But their maize orthologs usually confer unfavorable pleiotropic phenotypes, including reduced crop yields [5–8].

The MaizeGDB database (https://www.maizegdb.org) describes more than 50 PH-associated loci, most of them involved in plant hormone biosynthesis, transport, and signaling. Mutations in GA-biosynthesis genes result in short internodes and dwarf plants. Examples are DWARF1, encoding a GA 3-oxidase that catalyzes the final step in GA biosynthesis, DWARF3, encoding an ent-kaurenoic acid oxidase that catalyzes three steps in GA biosynthesis, and DWARF5, encoding an ent-kaurenoic acid oxidase that catalyzes ent-kaurenoic acid to GA12 [7–10]. Mutations in DWARF8 and DWARF9, two negative regulators of GA signaling, also produce dwarf plants [6]. BR is another important plant hormone that influences PH [11,12]. Several cytochrome P450-catalyzed oxidative reactions play an important role in BR biosynthesis and their disruption reduced PH. Many mutants of CYP724B1, which involve in the generation of 6-deoxoTY and TY, such as dwarf11, clustered primary branch1, grain number and size on chromosome4 in rice, and zmd11 in maize, exhibit reduced PH [13–16]. Mutation of CYP90B1, encoding a 22 alpha-hydroxylase which converts campestanol to 6-deoxocathasterone in Arabidopsis, inhibits cell elongation and produces a dwarf phenotype [17]. Mutants of Arabidopsis CYP90A1, CYP90C1 and CYP90D1 all exhibited reduced stature, although they are involved in different steps of BR biosynthesis [18–21]. In maize and rice, mutations of CYP90D1 ortholog, including d1, EBISU DWARF (d2), CHROMOSOME SEGMENT DELETED DWARF 1 (csdd1), SMALL GRAIN 11 (smg11), and qLTG1, have also been characterized with shorter PH [22–26]. In addition, genes involved in BR signal transduction cascade also influence plant growth and development, such as BR-INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 (BAK1). Loss of function of BAK1 in rice and Arabidopsis both reduced sensitivity to BR and PH [27,28]. Thus, BR biosynthesis and signaling are both required for plant development.

Here, we identified a dwarf mutant, m30, with shorter and fewer internodes in a maize breeding population. To isolate the candidate gene and elucidate its molecular mechanism on PH, we characterized the phenotype and cytological features of m30, positionally cloned the causal gene ZmCYP90D1 of m30, and explored the expression level changes influenced by loss function of ZmCYP90D1. We further performed candidate-based association analysis for ZmCYP90D1 to identify favorable haplotype in natural population for its exploit in practices. Our results show that ZmCYP90D1 influences PH through mediating BR-related genes which involved in cell division, cell wall biosynthesis, and vascular bundle formation. Importantly, there is a favorable haplotype in ZmCYP90D1 for a shorter PH without reducing yield.

2. Materials and methods

2.1. Plant materials

The spontaneous PH mutant m30 from a maize breeding population was crossed with the inbred line YU87-1 to generate an F1, which was backcrossed to m30 to produce a BC1 mapping population. All plant materials were planted at Henan Agricultural University experimental sites in Henan and Hainan, China. The PH phenotypes of BC1 plants were recorded from 10 to 60 days after sowing. The number, length, and diameter of internodes and ear leaves, as well as yield traits, were recorded at maturity. For RNA-sequencing (RNA-seq), the first to third internodes of the wild type (WT, YU87-1) and m30 mutant were sampled at the R1 stage with two replications.

2.2. Cytological analysis

The third internodes above the ground of five plants were sampled and sectioned using a vibratome (VT1000 S, Leica, Germany) [29]. Internode tissues were fixed in formalin:acetic acid:alcohol fixative and evacuated three times for 5 min each time, followed by washing with 1 phosphate buffered saline for 10 min at room temperature. Tissues were then embedded in 3% agarose and processed into 30 lm-thick sections, which were stained with a Saffron-O and Fast Green Stain kit (for plants) (G1375, Solarbio, Beijing, China) and observed under an emicroscope (Axio Scope A1, Zeiss, Germany).

2.3. Map-based cloning of m30 locus

The m30 locus was assayed using 276 InDel markers distributed evenly across the 10 maize chromosomes by bulk-segregant analysis. New InDel or single-nucleotide polymorphism (SNP) markers were developed, and a BC1 mapping population of 1152 plants was used to narrow the map interval containing the m30 locus. All primers used are listed in Table S3. Candidate genes were annotated according to the B73 reference genome (RefGen_V5).

2.4. Subcellular localization of ZmCYP90D1

The coding sequence of ZmCYP90D1 was fused with the enhanced GFP (eGFP) reporter gene and cloned into the pRTL vector (35SPro:ZmCYP90D1-eGFP). The resulting construct and the control nuclear marker Ghd7-RFP were then co-transformed into maize protoplasts. The transfected protoplasts were cultured overnight in the dark at 28 C and observed under a confocal microscope (LSM710, Zeiss, Germany). All primers used are listed in Table S3.

2.5. RNA-seq and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from internodes, roots, ears, tassels, leaves, and husks of the WT and m30 at the R1 stage using the TransZol Plant Kit (ET121, Transgen, Beijing, China).

For RNA-seq, an RNA library for RNA-seq was prepared, and the concentration of the library was measured with a Qubit fluorometer (ThermoFisher). Samples with two biological replicates were subjected to Illumina sequencing (Illumina NovaSeq 6000), and clean reads were mapped to the B73 reference genome(RefGen_V5). For each transcript model, gene expression levels were converted to fragments per kilobase million. Differentially expressed genes (DEGs) were defined by the following criteria: fold change > 2 and adjusted P value < 0.05. Gene Ontology (GO) enrichment analysis of the DEGs was performed with agriGO (https://systemsbiology.cau.edu.cn/agriGOv2/) [30]. Functional annotation of the DEGs was performed using BLAST analyses against the UniProt database (https://www.uniprot.org/blast/) and the NCBI database (https://www.ncbi.nlm.nih.gov/datasets/taxonomy/4577/). Biological processes of the DEGs were identified by the Kyoto Encyclopedia of Genes and Genomes (KEGG) (KOBAS, https://kobas.cbi.pku. edu.cn/) [31].

For reverse transcription, cDNA was synthesized from 1 lg of total RNA using an All-in-One First-Strand Synthesis Master Mix (with dsDNase) (KR0501, Kemix, Zhengzhou, Henan, China), and quantitative polymerase chain reaction (qPCR) was performed on a Bio-Rad CFX96 system using a 2 SYBR Green qPCR Premix (Universal) (KS0601, Kemix, Zhengzhou, Henan, China). Relative expression levels were calculated using the 2DDCT (threshold cycle) method with actin as the internal standard gene [32]. All primers used are listed in Table S3.

2.6. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of endogenous brassinosteroid

Metabolites of BR biosynthesis were measured in internodes of the ear, above the ear, and below the ear of m30 and WT at the R1 stage using MetWare (https://www.metware.cn/) with an AB Sciex QTRAP 6500 liquid chromatography tandem mass spectrometry (LC-MS/MS) instrument. Each sample had three biological replicates, and each replicate represented more than three plants. Student's t-test was used to compare metabolite abundances between WT and m30.

2.7. Candidate gene-based association analysis and haplotype identification

An association mapping population containing 509 maize inbred lines from temperate, tropical/subtropical, and mixed groups (https://www.maizego.org/Resources.html/) was used for candidate gene-based association analysis. The population was planted in three environments (Yongcheng in Henan province, Yuanyang in Henan province, and Xingtai in Heibei province in 2022, China), and kernel weight per ear was measured using five ears for each genotype in each environment. Data for other agronomic traits, and a set of 299 SNPs surrounding or within ZmCYP90D1 were retrieved from MaizeGo (https://www.maizego.org/Resources. html/). SNPs were filtered to ensure a minor allele frequency > 0.05 and a missing rate < 20%. Analysis was performed with TASSEL v.5.0 (https://www.maizegenetics.net/copy-of-tassel/) with a mixed linear model considering kinship and population structure [33]. Linkage disequilibrium and Manhattan plots were generated with LDBlockShow (https://github.com/BGI-shenzhen/LDBlockShow/). The expression data of ZmCYP90D1 in 368 inbred lines were retrieved from the reported transcriptome [34]. Phenotypic values, expression levels, and haplotypes were compared by one-way ANOVA or Student's t-test.

3. Results

3.1. Phenotypic characterization of m30

Compared with the WT, the m30 mutant showed increasingly delayed growth following plant development (Fig. 1A, B) and displayed shorter PH and ear height (EH) with 57.6% and 57.7% reductions at the pollen shedding stage, respectively (Fig. 1C–E). PH measurement for whole developmental stages indicated that the difference in PH and EH between m30 and WT lasted from 10 days after seedling (DAS) to 60 DAS (Fig. 1F). At the mature stage, both the number and the length of internodes, especially the fourth to the eighth, decreased in m30 compared with the WT (Fig. 1G–J). In contrast, the width of internodes, especially the first to the third, was significantly increased in m30 (Fig. 1K). Additionally, the earleaf showed a significant reduction in length (by 35.7%) (Fig. 1L, M) and an increase in leaf angle (by 59.9%) (Fig. 1N) but not in width (Fig. 1O) in m30 compared with the WT. The reduction in PH and EH consequently resulted in a decrease in kernel length (18.8%), kernel width (21.5%), and hundred-kernel weight (26.8%) (Fig. 1P–S). The results indicate that a shorter PH of m30 is derived from a reduction in both the number and length of the internode, which in turn affects kernel yield.

3.2. Cell division and growth are altered in m30

Transverse sections showed light safranin staining in the stem epidermis and vasclar bundles in m30 compared to WT (Fig. 2A, B), indicating reduced lignin in m30. m30 showed an increase in stalk cell area and a decrease in cell number per mm2 (Fig. 2C, D). By contrast, the length of epidermal and parenchyma cells of the internode was significantly decreased (Fig. 2E, F), especially in some extreme m30 individuals with twisted vascular bundles (Fig. 2G), but no significant changes in width were observed between WT and m30 in longitudinal sections (Fig. 2H). These results indicate that cell division and growth are affected in m30.

3.3. ZmCYP90D1 is the causal gene of m30

The PH of F1 was similar to that of YU87-1, and the BC1 population was segregated for PH with a ratio of 1:1 between WT (m30/+) and dwarf plants (m30/m30) (Table S1), indicating that the m30 phenotype was controlled by a single recessive nuclear gene. The candidate gene was mapped on chromosome 3 between two markers, S05065 and S05297. The region was further narrowed to a 271-kb interval between newly developed InDel markers S05141 and S05150 using a population of 518 WT (m30/+) and 634 dwarf mutants (m30/m30) (Fig. 3A). Nine genes were in this interval (Fig. 3A), and cDNA sequencing of these genes revealed a G-to-A mutation in the eighth exon of Zm00001eb120890, which caused a premature termination of the predicted protein translation (Fig. 3B, C). The dCAPS marker developed based on the G-to-A conversion was found to co-segregate with the PH phenotype. As shown in Fig. 3D, all dwarf plants with homozygous m30 genotype were represented by a large uncleaved fragment, whereas all normal individuals with homozygous wild-type or heterozygous genotype showed a small fragment or two fragments. Thus, the G-to-A conversion co-segregated with the dwarf mutation. (Fig 3D).

The full-length cDNA of Zm00001eb120890 comprises an open reading frame of 1512 bp, a 50-untranslated region (UTR) of 248 bp, and a 30-UTR of 490 bp (Fig. 3B). Zm00001eb120890 is the orthologue of Arabidopsis CYP90D1 and rice OsD2, which share several conserved domains, including a proline-rich domain, dioxygen binding domain, steroid binding domain, heme binding domain, and CYP90-like active site (Fig. 3E). The truncated protein in m30 lost 28 amino acids in the C-terminus, resulting in a shortened CYP90-like active site and a conformational change in the CYP90D1 three-dimensional structure (Fig. 3E, F). Thus, the disruption of Zm00001eb120890 was the cause of the reduction in PH and EH of m30. This gene was named ZmCYP90D1.

3.4. ZmCYP90D1 is constitutively expressed and its protein is targeted to the nucleus

To characterize the temporal and spatial expression patterns of ZmCYP90D1, the transcript abundance of ZmCYP90D1 in a wide range of tissues and organs was measured by qRT-PCR. ZmCYP90D1 was constitutively expressed in all examined tissues, with high expression in roots and internodes, especially the first to the third internodes (Fig. 4A), in agreement with online RNA-seq data (https://www.maizegdb.org). A 35SPro:ZmCYP90D1-eGFP vector was transiently expressed in maize leaf protoplasts to ascertain the subcellular location of ZmCYP90D1. As shown in Fig. 4B, eGFP fluorescence signals were concentrated in the nucleus and overlapped with RFP fluorescence signals of the nuclear marker GHD7 (Fig. 4B). These results indicate that ZmCYP90D1 constitutively expresses and its protein localizes to the nucleus.

3.5. BR pathway was changed in m30

To investigate changes at gene expression levels in response to the ZmCYP90D1 mutation, the transcriptome profiles of the first to three internodes above ground of WT and m30 at the rapid growth stage were characterized by RNA-seq. Of 3686 differentially expressed genes (DEGs), 1631 were up-regulated and 2055 were down-regulated in m30 (Fig. 5A; Table S2). Of the DEGs, 2977 were functionally annotated and were enriched in GO: 0,009,408 (response to heat; P = 4.8E-15), GO: 0,009,266 (response to temperature stimulus; P = 4.4E-08), and GO: 0,006,355 (regulation of transcription, DNA-templated; P = 4.8E-10) (Fig. 5B). Among the 290 DEGs in GO: 0006355, 29 were transcription factors (TFs) involved in plant hormone signaling as determined by KEGG, and could be grouped into BR, auxin, ethylene, and abscisic acid biosynthesis pathways (Fig. 5C; Table S2). Other non-TF DEGs involved in BR, GA, and cytokinin biosynthesis pathways were also identified, most of them up-regulated in m30 (Table S2). In the BR biosynthesis pathway, the four up-regulated DEGs encoded cytochrome P450 family proteins: one CYP90D1 (Zm00001eb284090); two CYP90A1, two orthologs of Arabidopsis CPD (Zm00001eb196530, Zm00001eb093140); and one CYP734A1 (Zm00001eb122680) (Fig. 5D). Many DEGs responded to BR levels. ZmBZR1 (Zm00001eb120750), the direct target of the CYP90 gene family, which is a BRresponsive TF negatively regulating cell and fruit size [35], was down-regulated in m30 (Fig. 5C). The expression trend of ZmBZR1 was complementary to that of ZmCYP90D1 in the basal three internodes, suggesting that the BR pathway modulates internode development in maize. A set of 905 DEGs were classified as downstream target genes of BZR1, including 343 up-regulated and 562 down-regulated (Fig. 5E; Table S2) [36]. These DEGs were functionally annotated as involved in the cell cycle and cell wall extension and modification (Fig. 5F, G; Table S2). Surprisingly, only TY among the 11 detected metabolites showed significant a decrease in m30 (Table 1).

3.6. Favorable haplotype of ZmCYP90D1 conferring dwarf PH without reducing yield

Among the 299 SNPs surrounding or within ZmCYP90D1, 15 and 13 SNPs were significantly associated with PH and EH, respectively (Fig. 6A, B). Among them, eight were associated with both PH and EH. SNPs chr3.s_4747984, chr3.s_4748007, and chr3.s_4748739 were found in introns, whereas chr3.s_4752835, chr3.s_4752992, chr3.s_4753166, chr3.s_4753349, and chr3.s_4753446 were found in the promoter region (Fig. 6A, B). Two of the most strongly associated SNPs, chr3.s_4752992 (8.11E-05) and chr3.s_4753166 (4.23E-05), were used to define haplotypes. The 507 maize inbred lines were classified into three haplotypes based on these two SNPs (Fig. 6C). Hap 1 was the largest group and was present in 335 lines, Hap 2 was present in 126 lines and Hap 3 in only 31 lines. In comparison with Hap 1 and Hap 3, inbred lines that carried Hap 2 showed reduced PH and EL (ear length) (Fig. 6D, E), but without a significant difference in ear weight or kernel weight per ear (Fig. 6F, G). Hap 2 should be a favorable haplotype for shorter PH without a significant reduction in yield in natural population. Because Hap 3 was carried by only a few lines and the PH of these lines was similar to that of Hap 2, they were combined into one group. Hap 1 inbred lines showed significantly lower ZmCYP90D1 transcriptions than Hap 3 and Hap 2 lines (Fig. 6H).

4. Discussion

TY has been reported as the downstream intermediate of CYP90D1 in Arabidopsis [20,21]. In this study, we found that the TY content was significantly reduced in m30, which showed down-regulation of many BR-response genes, indicating involvement of ZmCYP90D1 in the hydroxylation of C-23 (Fig. 5C–G; Table 1). CYP90D1 is a direct effector of BZR1 in loquat [35], however, we found that BZR1 gene was significantly down-regulated but ZmCYP90D1 was significantly up-regulated in m30, a complementary expression trend between ZmCYP90D1 and BZR1. A similar complementary expression trend was also identified in previous research in maize [26]. However, the reason for this phenomenon was unknown.

Both stalk length and width are regulated by stem elongation, which is driven by cell division and cell expansion of the internode [37–39]. In Arabidopsis, BR has been found in several studies to be associated with cell division. Balanced BR signaling is required for a normal cell cycle in the root meristem, and mitotic activity decreased in a BR-sensitive mutant bri1-116, which showed a defective meristem [40]. BR-deficient dwarf mutant cpd exhibited a prolonged cell division phase and delayed differentiation in leaves [41]. BIN2 influences cell division by interacting with tubulin protein [42]. BRs also function at the cell wall to influence cell expansion or elongation. In Arabidopsis, BES1 regulated expression levels of cellulose synthase genes, which in turn modulate cellulose synthesis. Cellulose was reduced in BR-deficient mutant det2-1 or BR-perceptional mutant bri1-301 compared with the wild-type, which restricted cell elongation [43]. In Utricularia gibba, defects of BR biosynthesis reduced wall relaxation properties in the epidermis, resulting in twisted internal tissue and shorter and smaller epidermal cells [44]. In contrast, BR stimulated hypocotyl elongation by expanding cell wall in soybean (Glycine max L.) [45]. In the maize BR biosynthesis pathway, several genes have been identified as associated with PH development through the regulation of cell wall biogenesis and cell division. A mutation in ZmDWF1, which encodes 24-sterol reductase, affects the transcript levels of cell wall biogenesis genes. A mutation in Brassinosteroiddeficient dwarf1, which encodes C-6 oxidase, increased cell density per unit area, and reduced PH [46]. In the BR signaling pathway, BRI1 encodes a member of leucine-rich-repeat receptor-like kinases, and RNA interference of BRI1 results in decreased cell division and cell elongation, producing shorter internodes and dwarf plants [47]. In the present study, the mutation in ZmCYP90D1 caused changes in the transcript levels of cell wall biosynthesis (41) and cell division-associated (44) genes (Table S2), similar to those of the cell size-associated genes in ZmD1 mutant [26], a finding consistent with that of other BR-responsive genes, resulting in increased cell area (Fig. 2C), decreased cell length (Fig. 2F), and reduced cell number (Fig. 2D). By contrast, very few common cell size DEGs were identified in these two studies (Table S2), possibly due to differences in sampling stages and internodes. We infer that ZmCYP90D1 likely modulates maize PH via BR-mediated cell wall biogenesis and cell division.

Short and thick stature is crucial for increasing planting density and lodging resistance in maize [48]. Dwarf and semi-dwarf mutants caused by deficiency of GA biosynthesis and signaling genes in rice (e.g., Sd1) and wheat (e.g., Rht) have been widely used in breeding. However, these mutations in maize reduce yield [4,6,8]. PH and 100-kernel weight were reduced in the zmxyl mutant [49], PH was increased and kernel weight per ear was reduced with ZmLBD5 overexpression, and the opposite changes were observed in the loss-of-function mutant [50], PH, EL, and seed weight were reduced in the terminal ear 1 mutant [51], and PH and yield were reduced in the sucrose transporter2 mutant [52]. Although mutation of ZmCYP90D1 reduced PH and yield, we identified a favorable naturally occurring haplotype that reduced PH without affecting yields (Fig. 6D, F, G). Thus, the ZmCYP90D1 mutant allele identified in this study has great potential to be utilized in the breeding of new dwarf varieties suitable for highdensity planting without yield loss.

5. Conclusions

The candidate gene ZmCYP90D1 of the maize dwarf mutant m30 may regulate BR biosynthesis in internodes, influencing internode development. This regulatory function may involve cell division and cell wall synthesis. Natural variation in ZmCYP90D1 was associated with PH but not yield. ZmCYP90D1 may be useful in breeding lodging-resistant maize cultivars for high-density planting.

CRediT authorship contribution statement

Canran Sun: Conceptualization, Investigation, Data curation, Methodology, Writing – original draft, Writing – review & editing. Yang Liu: Conceptualization, Investigation, Data curation. Guofang Li: Investigation, Data curation, Methodology. Yanle Chen: Conceptualization, Investigation. Mengyuan Li: Conceptualization, Investigation. Ruihua Yang: Investigation. Yongtian Qin: Data curation, Methodology. Yongqiang Chen: Investigation, Data curation. Jinpeng Cheng: Investigation, Data curation. Jihua Tang: Conceptualization, Visualization, Supervision, Funding acquisition, Writing – review & editing. Zhiyuan Fu: Conceptualization, Data curation, Writing – original draft, Writing – review & editing, Supervision, Funding acquisition, Project administration.

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 National Natural Science Foundation of China (U2004144, 31971893, 32101743), the Key Technologies R & D Program of Henan Province (232102111080), and Yunnan Academician Expert Workstation (202305AF150082).

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ARTICLE INFO

Article history:

Received 10 August 2023

Revised 17 October 2023

Accepted 3 November 2023

Available online 28 November 2023

Footnote

* Corresponding authors

E-mail addresses: [email protected] (J. Tang), [email protected](Z. Fu).

1 These authors contributed equally to this work.

References

References

[1] A.M. Modarres, R.I. Hamilton, M. Dijak, L.M. Dwyer, D.W. Stewart, D.E. Mather, D.L. Smith, Plant population density effects on maize inbred lines grown in short-season environments, Crop Sci. 38 (1998) 104–108.

[2] Y. Xiao, H. Liu, L. Wu, M. Warburton, J. Yan, Genome-wide association studies in maize: praise and stargaze, Mol. Plant 10 (2017) 359–374.

[3] J. Peng, D.E. Richards, N.M. Hartley, G.P. Murphy, K.M. Devos, J.E. Flintham, J. Beales, L.J. Fish, A.J. Worland, F. Pelica, D. Sudhakar, P. Christou, J.W. Snape, M. D. Gale, N.P. Harberd, 'Green revolution' genes encode mutant gibberellin response modulators, Nature 400 (1999) 256–261.

[4] A. Sasaki, M. Ashikari, M. Ueguchi-Tanaka, H. Itoh, A. Nishimura, D. Swapan, K. Ishiyama, T. Saito, M. Kobayashi, G.S. Khush, H. Kitano, M. Matsuoka, Green revolution: a mutant gibberellin-synthesis gene in rice, Nature 416 (2002) 701–702.

[5] S. Fujioka, H. Yamane, C.R. Spray, M. Katsumi, B.O. Phinney, P. Gaskin, J. Macmillan, N. Takahashi, The dominant non-gibberellin-responding dwarf mutant (D8) of maize accumulates native gibberellins, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 9031–9035.

[6] S.J. Lawit, H.M. Wych, D. Xu, S. Kundu, D.T. Tomes, Maize DELLA proteins dwarf plant8 and dwarf plant9 as modulators of plant development, Plant Cell Physiol. 51 (2010) 1854–1868.

[7] R.G. Winkler, T. Helentjaris, The maize Dwarf3 gene encodes a cytochrome P450-mediated early step in gibberellin biosynthesis, Plant Cell 7 (1995) 1307–1317.

[8] Y. Chen, M. Hou, L. Liu, S. Wu, Y. Shen, K. Ishiyama, M. Kobayashi, D.R. McCarty, B.C. Tan, The maize DWARF1 encodes a gibberellin 3-oxidase and is dual localized to the nucleus and cytosol, Plant Physiol. 166 (2014) 2028–2039.

[9] C.A. Helliwell, P.M. Chandler, A. Poole, E.S. Dennis, W.J. Peacock, The CYP88A cytochrome P450, ent-kaurenoic acid oxidase, catalyzes three steps of the gibberellin biosynthesis pathway, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 2065– 2070.

[10] Z.L. Li, J.L. Zhang, H.L. Wang, M. Wang, S.Y. Guo, P.T. Wang, Z. Li, D.W. Galbraith, D. Li, C.P. Song, GA Associated Dwarf 5 encodes an ent-kaurenoic acid oxidase required for maize gibberellin biosynthesis and morphogenesis, Crop J. 11 (2023) 1742–1751.

[11] U.K. Divi, P. Krishna, Brassinosteroid: a biotechnological target for enhancing crop yield and stress tolerance, Nat. Biotechnol. 26 (2009) 131–136.

[12] Y. Belkhadir, Y. Jaillais, The molecular circuitry of brassinosteroid signaling, New Phytol. 206 (2015) 522–540.

[13] H. Sun, H. Xu, B. Li, Y. Shang, M. Wei, S. Zhang, C. Zhao, R. Qin, F. Cui, Y. Wu, The brassinosteroid biosynthesis gene, ZmD11, increases seed size and quality in rice and maize, Plant Physiol. Biochem. 160 (2021) 281–293.

[14] S. Tanabe, M. Ashikari, S. Fujioka, S. Takatsuto, S. Yoshida, M. Yano, A. Yoshimura, H. Kitano, M. Matsuoka, Y. Fujisawa, H. Kato, Y. Iwasaki, A novel cytochrome P450 is implicated in brassinosteroid biosynthesis via the characterization of a rice dwarf mutant, dwarf11, with reduced seed length, Plant Cell 17 (2005) 776–790.

[15] Y. Zhou, Y. Tao, J. Zhu, J. Miao, J. Liu, Y. Liu, C. Yi, Z. Yang, Z. Gong, G. Liang, GNS4, a novel allele of DWARF11, regulates grain number and grain size in a high-yield rice variety, Rice 10 (2017) 34.

[16] Y. Wu, Y. Fu, S. Zhao, P. Gu, Z. Zhu, C. Sun, L. Tan, CLUSTERED PRIMARY BRANCH 1, a new allele of DWARF11, controls panicle architecture and seed size in rice, Plant Biotechnol. J. 14 (2016) 377–386.

[17] S. Choe, B.P. Dilkes, S. Fujioka, S. Takatsuto, A. Sakurai, K.A. Feldmann, The DWF4 gene of Arabidopsis encodes a cytochrome P450 that mediates multiple 22alpha-hydroxylation steps in brassinosteroid biosynthesis, Plant Cell 10 (1998) 231–243.

[18] M. Szekeres, K. Nemeth, Z. Koncz-Kalman, J. Mathur, A. Kauschmann, T. Altmann, G.P. Redei, F. Nagy, J. Schell, C. Koncz, Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and deetiolation in Arabidopsis, Cell 85 (1996) 171–182.

[19] J. Mathur, G. Molnar, S. Fujioka, S. Takatsuto, A. Sakurai, T. Yokota, G. Adam, B. Voigt, F. Nagy, C. Maas, J. Schell, C. Koncz, M. Szekeres, Transcription of the Arabidopsis CPD gene, encoding a steroidogenic cytochrome P450, is negatively controlled by brassinosteroids, Plant J. 14 (1998) 593–602.

[20] G.T. Kim, S. Fujioka, T. Kozuka, F.E. Tax, S. Takatsuto, S. Yoshida, H. Tsukaya, CYP90C1 and CYP90D1 are involved in different steps in the brassinosteroid biosynthesis pathway in Arabidopsis thaliana, Plant J. 41 (2005) 710–721.

[21] T. Ohnishi, A.M. Szatmari, B. Watanabe, S. Fujita, S. Bancos, C. Koncz, M. Lafos, K. Shibata, T. Yokota, K. Sakata, M. Szekeres, M. Mizutani, C-23 hydroxylation by Arabidopsis CYP90C1 and CYP90D1 reveals a novel shortcut in brassinosteroid biosynthesis, Plant Cell 18 (2006) 3275–3288.

[22] Z. Hong, M. Ueguchi-Tanaka, K. Umemura, S. Uozu, S. Fujioka, S. Takatsuto, S. Yoshida, M. Ashikari, H. Kitano, M. Matsuoka, A rice brassinosteroid-deficient mutant, ebisu dwarf (d2), is caused by a loss of function of a new member of cytochrome P450, Plant Cell 15 (2003) 2900–2910.

[23] H. Li, L. Jiang, J.H. Youn, W. Sun, Z. Cheng, T. Jin, X. Ma, X. Guo, J. Wang, X. Zhang, F. Wu, C. Wu, S.K. Kim, J. Wan, A comprehensive genetic study reveals a crucial role of CYP90D2/D2 in regulating plant architecture in rice (Oryza sativa), New Phytol. 200 (2013) 1076–1088.

[24] N. Fang, R. Xu, L. Huang, B. Zhang, P. Duan, N. Li, Y. Luo, Y. Li, SMALL GRAIN 11 controls grain size, grain number and grain yield in rice, Rice 9 (2016) 64.

[25] S.H. Kim, K.C. Shim, H.S. Lee, Y.A. Jeon, C. Adeva, N.H. Luong, S.N. Ahn, Brassinosteroid biosynthesis gene OsD2 is associated with low-temperature germinability in rice, Front. Plant Sci. 13 (2022) 985559.

[26] L. Le, W. Guo, D. Du, X. Zhang, W. Wang, J. Yu, H. Wang, H. Qiao, C. Zhang, L. Pu, A spatiotemporal transcriptomic network dynamically modulates stalk development in maize, Plant Biotechnol. J. 20 (2022) 2313–2331.

[27] J. Li, J. Wen, K.A. Lease, J.T. Doke, F.E. Tax, J.C. Walker, BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling, Cell 110 (2002) 213–222.

[28] D. Li, L. Wang, M. Wang, Y.Y. Xu, W. Luo, Y.J. Liu, Z.H. Xu, J. Li, K. Chong, Engineering OsBAK1 gene as a molecular tool to improve rice architecture for high yield, Plant Biotechnol. J. 7 (2009) 791–806.

[29] P. Prieto, G. Moore, P. Shaw, Fluorescence in situ hybridization on vibratome sections of plant tissues, Nat. Protoc. 2 (2007) 1831–1838.

[30] T. Tian, Y. Liu, H. Yan, Q. You, X. Yi, Z. Du, W. Xu, Z. Su, agriGO v2.0: a GO analysis toolkit for the agricultural community, update, Nucleic Acids Res. 45 (2017) W122–W129.

[31] D. Bu, H. Luo, P. Huo, Z. Wang, S. Zhang, Z. He, Y. Wu, L. Zhao, J. Liu, J. Guo, S. Fang, W. Cao, L. Yi, Y. Zhao, L. Kong, KOBAS-i: intelligent prioritization and exploratory visualization of biological functions for gene enrichment analysis, Nucleic Acids Res. 49 (2021) W317–W325.

[32] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) method, Methods 25 (2001) 402–408.

[33] P.J. Bradbury, Z. Zhang, D.E. Kroon, T.M. Casstevens, Y. Ramdoss, E.S. Buckler, TASSEL: software for association mapping of complex traits in diverse samples, Bioinformatics 23 (2007) 2633–2635.

[34] J. Fu, Y. Cheng, J. Linghu, X. Yang, L. Kang, Z. Zhang, J. Zhang, C. He, X. Du, Z. Peng, B. Wang, L. Zhai, C. Dai, J. Xu, W. Wang, X. Li, J. Zheng, L. Chen, L. Luo, J. Liu, X. Qian, J. Yan, J. Wang, G. Wang, RNA sequencing reveals the complex regulatory network in the maize kernel, Nat. Commun. 4 (2013) 2832.

[35] W. Su, Z. Shao, M. Wang, X. Gan, X. Yang, S. Lin, EjBZR1 represses fruit enlargement by binding to the EjCYP90 promoter in loquat, Hortic. Res. 8 (2021) 152.

[36] T. Hartwig, M. Banf, G.P. Prietsch, J.Y. Zhu, I. Mora-Ramirez, J.H.M. Schippers, S. J. Snodgrass, A.S. Seetharam, B. Huettel, J.M. Kolkman, J. Yang, J. Engelhorn, Z.Y. Wang, Hybrid allele-specific ChIP-seq analysis identifies variation in brassinosteroid-responsive transcription factor binding linked to traits in maize, Genome Biol. 24 (2023) 108.

[37] S. Muller, Plant cell division - defining and finding the sweet spot for cell plate insertion, Curr. Opin. Cell Biol. 60 (2019) 9–18.

[38] H. Ren, M.Y. Park, A.K. Spartz, J.H. Wong, W.M. Gray, A subset of plasma membrane-localized PP2C.D phosphatases negatively regulate SAURmediated cell expansion in Arabidopsis, PLoS Genet. 14 (2018) e1007455.

[39] W. Yang, S. Cortijo, N. Korsbo, P. Roszak, K. Schiessl, A. Gurzadyan, R. Wightman, H. Jonsson, E. Meyerowitz, Molecular mechanism of cytokininactivated cell division in Arabidopsis, Science 371 (2021) 1350–1355.

[40] M.P. Gonzalez-Garcia, J. Vilarrasa-Blasi, M. Zhiponova, F. Divol, S. Mora-Garcia, E. Russinova, A.I. Cano-Delgado, Brassinosteroids control meristem size by promoting cell cycle progression in Arabidopsis roots, Development 138 (2011) 849–859.

[41] M.K. Zhiponova, I. Vanhoutte, V. Boudolf, C. Betti, S. Dhondt, F. Coppens, E. Mylle, S. Maes, M.P. Gonzalez-Garcia, A.I. Cano-Delgado, D. Inze, G.T.S. Beemster, L. de Veylder, E. Russinova, Brassinosteroid production and signaling differentially control cell division and expansion in the leaf, New Phytol. 197 (2013) 490–502.

[42] X. Liu, Q. Yang, Y. Wang, L. Wang, Y. Fu, X. Wang, Brassinosteroids regulate pavement cell growth by mediating BIN2-induced microtubule stabilization, J. Exp. Bot. 69 (2018) 1037–1049.

[43] L. Xie, C. Yang, X. Wang, Brassinosteroids can regulate cellulose biosynthesis by controlling the expression of CESA genes in Arabidopsis, J. Exp. Bot. 62 (2011) 4495–4506.

[44] R. Kelly-Bellow, K. Lee, R. Kennaway, J.E. Barclay, A. Whibley, C. Bushell, J. Spooner, M. Yu, P. Brett, B. Kular, S. Cheng, J. Chu, T. Xu, B. Lane, J. Fitzsimons, Y. Xue, R.S. Smith, C.D. Whitewoods, E. Coen, Brassinosteroid coordinates cell layer interactions in plants via cell wall and tissue mechanics, Science 380 (2023) 1275–1281.

[45] D.M. Zurek, S.D. Clouse, Molecular cloning and characterization of a brassinosteroid-regulated gene from elongating soybean (Glycine max L.) epicotyls, Plant Physiol. 104 (1994) 161–170.

[46] G. Castorina, M. Persico, M. Zilio, S. Sangiorgio, L. Carabelli, G. Consonni, The maize lilliputian1 (lil1) gene, encoding a brassinosteroid cytochrome P450 C-6 oxidase, is involved in plant growth and drought response, Ann. Bot. 122 (2018) 227–238.

[47] G. Kir, H. Ye, H. Nelissen, A.K. Neelakandan, A.S. Kusnandar, A. Luo, D. Inze, A. W. Sylvester, Y. Yin, P.W. Becraft, RNA interference knockdown of BRASSINOSTEROID INSENSITIVE1 in maize reveals novel functions for brassinosteroid signaling in controlling plant architecture, Plant Physiol. 169 (2015) 826–839.

[48] C. Peng, X. Wang, T. Feng, R. He, M. Zhang, Z. Li, Y. Zhou, L. Duan, System analysis of mirnas in maize internode elongation, Biomolecules 9 (2019) 417.

[49] H. Li, H. Tao, Y. Xiao, L. Qin, C. Lan, B. Cheng, J.A. Roberts, X. Zhang, X. Lu, ZmXYL modulates auxin-induced maize growth, Plant J. 115 (2023) 1699– 1715.

[50] X. Feng, J. Xiong, W. Zhang, H. Guan, D. Zheng, H. Xiong, L. Jia, Y. Hu, H. Zhou, Y. Wen, X. Zhang, F. Wu, Q. Wang, J. Xu, Y. Lu, ZmLBD5, a class-II LBD gene, negatively regulates drought tolerance by impairing abscisic acid synthesis, Plant J. 112 (2022) 1364–1376.

[51] F. Wang, Z. Yu, M. Zhang, M. Wang, X. Lu, X. Liu, Y. Li, X. Zhang, B.C. Tan, C. Li, Z. Ding, ZmTE1 promotes plant height by regulating intercalary meristem formation and internode cell elongation in maize, Plant Biotechnol. J. 20 (2022) 526–537.

[52] K.A. Leach, T.M. Tran, T.L. Slewinski, R.B. Meeley, D.M. Braun, Sucrose transporter2 contributes to maize growth, development, and crop yield, J.Integr. Plant Biol. 59 (2017) 390–408.

Appendix A. Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2023.11.002.