Maize Plant Architecture Is Regulated by the Ethylene Biosynthetic Gene ZmACS7

Abstract

Plant height and leaf angle are two crucial determinants of plant architecture in maize (Zea mays) and are closely related to lodging resistance and canopy photosynthesis at high planting density. These two traits are primarily regulated by several phytohormones. However, the mechanism of ethylene in regulating plant architecture in maize, especially plant height and leaf angle, is unclear. Here, we characterized a maize mutant, Semidwarf3 (Sdw3), which exhibits shorter stature and larger leaf angle than the wild type. Histological analysis showed that inhibition of longitudinal cell elongation in the internode and promotion in the auricle were mainly responsible for reduced plant height and enlarged leaf angle in the Sdw3 mutant. Through positional cloning, we identified a transposon insertion in the candidate gene ZmACS7, encoding 1-aminocyclopropane-1-carboxylic acid (ACC) Synthase 7 in ethylene biosynthesis of maize. The transposon alters the C terminus of ZmACS7. Transgenic analysis confirmed that the mutant ZmACS7 gene confers the phenotypes of the Sdw3 mutant. Enzyme activity and protein degradation assays indicated that the altered C terminus of ZmACS7 in the Sdw3 mutant increases this protein's stability but does not affect its catalytic activity. The ACC and ethylene contents are dramatically elevated in the Sdw3 mutant, leading to reduced plant height and increased leaf angle. In addition, we demonstrated that ZmACS7 plays crucial roles in root development, flowering time, and leaf number, indicating that ZmACS7 is an important gene with pleiotropic effects during maize growth and development.

Figure 1.

Phenotypic characterization of the Sdw3 mutant. A, Overall morphology of three NILs of Sdw3 at anthesis. The wild type (N18), heterozygous mutant (Sdw3/−), and homozygous mutant (Sdw3) are shown. Scale bars = 20 cm. B, Quantitative measurements of plant height of N18, Sdw3/−, and Sdw3 at anthesis. Error bars represent ± sd (n = 24). C, Quantitative measurements of the length of tassels and aboveground internodes at anthesis. Internodes above the primary ear are labeled 1, 2, 3, 4, and 5, and internodes below the ear are labeled −1, −2, −3, −4, −5, −6, and −7. All internodes were measured except the internodes below −7 due to obstruction from strong brace roots. Error bars represent ± sd (n = 20). T, Tassels. D, sem observation of longitudinal sections of parenchyma tissue in the middle region of the second internode above the primary ear of N18, Sdw3/−, and Sdw3 at anthesis. Scale bars = 100 μm. E, Quantitative measurements of the angle of the first leaf above the primary ear of N18, Sdw3/−, and Sdw3 at anthesis. Error bars represent ± sd (n = 24). F, Adaxial auricles of the first leaf above the primary ear of N18, Sdw3/−, and Sdw3 at anthesis. The red dotted lines indicate the outer edge of the auricles. Scale bars = 1 cm. G, sem observation of adaxial epidermal cells in auricles in the middle region near the outer edge (framed by a white rectangle in Fig. 1F) of the first leaf above the primary ear of N18, Sdw3/−, and Sdw3 at anthesis. Scale bars = 50 μm. Different letters above the error bars in B, C and E indicate significant differences at P < 0.05 using Tukey’s multiple comparison test. Images in A and F were digitally extracted from their original photos and placed on a black background.

Figure 2.

Map-based cloning of the Sdw3 mutant and mRNA expression pattern of ZmACS7. A, Fine mapping of the Sdw3 mutant. M1 to M10 indicate molecular markers. Numbers below the markers indicate the number of recombinants between the locus and the markers shown. Double hyphens below the markers indicate that the markers shown displayed no molecular polymorphism between the Sdw3 mutant and the corresponding inbred line. The final mapping interval (M8 to M9) overlaps with a partial sequence of only one candidate gene, GRMZM5G89619 (ZmACS7). B, Gene structure and mutated sequences of ZmACS7. ZmACS7 has four exons and three introns. The boxes indicate exons, and the lines between boxes indicate introns. The black boxes represent the coding sequence. The empty boxes represent the 5′ untranslated region (left) and 3′ untranslated region (right). The red triangle indicates the 205-bp transposon. The DNA sequence and the deduced amino acid sequence of mutated ZmACS7 in the Sdw3 mutant are in red. The transposon deleted the last four amino acids VHAS of ZmACS7, but extended the C terminus, containing two additional SP motifs (encircled by red rectangles). C, RT-qPCR analysis of ZmACS7 in multiple N18 and Sdw3 tissues. The tissues and stages used for RNA extraction are described in the legend of Supplemental Fig. S14B. ZmActin1 (GRMZM2G126010) served as the reference gene for normalization of RT-qPCR data. Error bars represent ± sd (n = 3). Asterisks indicate significant differences between N18 and Sdw3 using Student’s t test (*P < 0.05 and **P < 0.01; n.s. = no significant difference).

Figure 3.

Excessive ACC and ethylene confer reduced plant height and enlarged leaf angle in the Sdw3 mutant. A, Quantitative measurements of endogenous ACC content in three tissues between N18 and Sdw3 seedlings at the V7 stage. Error bars represent ± sd (n = 3). FW, fresh weight. B, Quantitative measurements of endogenous ethylene production in the leaf blade between N18 and Sdw3 seedlings at the V7 stage. Error bars represent ± sd (n = 9). DW, dry weight. C, Overall morphology of N18 and Sdw3 seedlings after hydroponic cultivation. The distance between two yellow arrowheads refers to the length of the fifth leaf sheath. Scale bars = 10 cm. D and E, Quantitative measurements of the fifth leaf sheath length (D) and the fifth leaf angle (E) of N18 and Sdw3 plants after hydroponic cultivation. Error bars represent ± sd (n = 12). F, Overall morphology of various concentrations of ethephon (ETH)-treated N18 plants at anthesis. Scale bars = 30 cm. G and H, Quantitative measurements of plant height (G) and the first leaf angle above the primary ear (H) of ETH-treated N18 plants at anthesis. Error bars represent ± sd (n = 20). Asterisks in A and B indicate significant differences in the corresponding tissue between N18 and Sdw3 (*P < 0.05 and **P < 0.01; Student’s t test). Different letters above the error bars in D, E, G, and H indicate significant differences at P < 0.05 using Tukey’s multiple comparison test. Images in C and F were digitally extracted from their original photos and placed on a black background.

Figure 4.

ZmACS7 is responsible for reduced plant height and enlarged leaf angle in the Sdw3 mutant. A, Schematic diagram of the pZmACS7^L329:ZmACS7^Sdw3 construct including the coding sequence of ZmACS7 from the Sdw3 mutant under the native promoter of ZmACS7 from L329. RB, right border; Tnos, terminator of nopaline synthase gene; p35S, cauliflower mosaic virus 35S promoter; Bar, (bialaphos resistance gene) serves as the selective marker; LB, left border. B, Overall morphology of seven positive pZmACS7^L329:ZmACS7^Sdw3 T₀ plants (C#1 to C#7) grown in the greenhouse from four batches of transformations. NT#1 (nontransgenic plant 1) is the control for C#1 and C#2, whereas NT#2 is the control for C#3; all these lines share the same genetic background of germplasm L329. NT#3 is the control of C#4, and NT#4 is the control of C#5, C#6, and C#7 with genetic background of B104. Scale bars = 15 cm. C, Gene structure of ZmACS7 with the target (marked in red) for CRISPR/Cas9-specific binding to perturb the C terminus of ZmACS7. D, Alignment of C-terminal domains of ZmACS7 in L329, PC#1, and PC#2. Background colors indicate the similarity level of amino acid sequences (dark blue = 100%; baby blue <75% and ≥50%). Red rectangles were used to mark the SP and TP motifs in the CTDs of ZmACS7. The homologous sequence of the last 16 amino acids of AtACS6 in ZmACS7 was marked by a black line. E, Schematic diagram of the construct used for transgenic overexpression of ZmACS7. The maize ubiquitin promoter was applied to dominate the expression of the ZmACS7 coding sequence from L329. The Bar gene serves as the selective marker. pUbi, the maize ubiquitin-1 promoter. F, Overall morphology of PC and ZmACS7 OX lines at anthesis. Scale bars = 30 cm. The Cas9 constructs were screened out from PC lines. The wild-type parent L329 served as the control of both PC and ZmACS7 OX lines, because L329 displayed similar phenotypes with L329’, a transgenic line introduced by an empty vector, similar to pBCXUN (Supplemental Fig. S6). G, Quantitative measurements of endogenous ACC content of the shoot at V1 stage in PC and ZmACS7 OX seedlings. Error bars represent ± sd (n = 3). FW, fresh weight. H, Quantitative measurements of endogenous ethylene production in leaf 8 and leaf 9 at V7 stage in PC and ZmACS7 OX seedlings. Error bars represent ± sd (n = 5). DW, dry weight. I and J, Quantitative measurements of plant height (I) and angle of the first leaf above the primary ear (J) in PC and ZmACS7 OX lines at anthesis. Error bars represent ± sd (n = 20). Asterisks above the error bars in G and H indicate significant differences between the corresponding line and L329 (**P < 0.01; Student’s t test). Different letters above the error bars in I and J indicate significant differences at P < 0.05 using Tukey’s multiple comparison test. Images in B and F were digitally extracted from their original photos and placed on a black background.

Figure 5.

Specific activity and stability of ZmACS7 in N18 and Sdw3 plants. A, Specific activity of ZmACS7^N18 and ZmACS7^Sdw3 measured based on the ethylene synthesis rate in vitro. CK refers to HIS-tagged protein from the empty vector, which served as the negative control. Error bars represent ± sd (n = 9); n.s. indicates no significant difference between ZmACS7^N18 and ZmACS7^Sdw3 (Student’s t test). B, Immunoblot analysis of HIS-tagged CTD^N18 and CTD^Sdw3 proteins after incubation in a cell-free degradation system at different time points. NC indicates the negative control (sample without adding the target protein to the cell-free extract). RbcL (Rubisco large subunit) was used as a nondegraded control to indicate the same loading quantity. All the samples were equally diluted to test protein quantity of RbcL by Western blotting. Numbers above the Western blotting bands refer to relative protein content measured by ImageJ software. C, Quantitative measurements of HIS-tagged CTD^N18 and CTD^Sdw3 proteins obtained by immunoblot analysis using ImageJ software. The mean values from three biological replicates are shown. Error bars represent ± sd (n = 3). DMSO, dimethyl sulfoxide.