Volume 10, Issue 3, Pages 470-482 (March 2017) The U6 Biogenesis-Like 1 Plays an Important Role in Maize Kernel and Seedling Development by Affecting the 3′ End Processing of U6 snRNA  Jiankun Li, Junjie Fu, Yan Chen, Kaijian Fan, Cheng He, Zhiqiang Zhang, Li Li, Yunjun Liu, Jun Zheng, Dongtao Ren, Guoying Wang  Molecular Plant  Volume 10, Issue 3, Pages 470-482 (March 2017) DOI: 10.1016/j.molp.2016.10.016 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Kernel and Plant Phenotypes. (A) Ear segregation for wild-type and ubl1 mutant kernel (arrow) at 20 days after pollination (DAP). (B) Ear segregation for wild-type and mutant kernel (arrow) at 30 DAP. (C) Isolated wild-type (left) and mutant (right) embryos at 20 DAP. (D) Isolated wild-type (left) and mutant (right) embryos at 30 DAP. (E) Longitudinal section of mature wild-type (left) and mutant (right) kernels. (F) Wild-type (left) and mutant (right) seedlings at 11 days after planting. (G) Wild-type (left) and mutant (right) plants at 55 days after planting. Scale bars represent 1 cm (A and B), 0.25 cm (C–E), 2.5 cm (F), and 8 cm (G). Molecular Plant 2017 10, 470-482DOI: (10.1016/j.molp.2016.10.016) Copyright © 2016 The Authors Terms and Conditions

Figure 2 BETL Were Altered in ubl1 Mutant Kernels. (A) The 10-μm longitudinal wax sections of wild-type kernel at 14 DAP. Red arrows indicate the BETL. (B) The 10-μm longitudinal wax sections of ubl1 mutant kernel at 14 DAP. Red arrows indicate the BETL. (C) Magnified BETL of wild-type kernel at 14 DAP is indicated by red arrows. (D) Magnified BETL of mutant kernel at 14 DAP is indicated by red arrows, and ectopic BETL cells are indicated by red triangles. (E) Cell wall ingrowth of BETL cells in wild-type kernel at 14 DAP is indicated by red arrows. (F) Abnormal cell wall ingrowth of BETL cells in ubl1 kernel at 14 DAP is indicated by red arrows. Black arrows indicate protein bodies. En, endosperm; Em, embryo; BETL, basal endosperm transfer layer. Scale bars represent 200 μm (A and B), 80 μm (C and D), and 10 μm (E and F). Molecular Plant 2017 10, 470-482DOI: (10.1016/j.molp.2016.10.016) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Cloning of UBL1. (A) Modified DLA method is used to the analysis of the ubl1 segregation population. PAGE result of the co-segregation analysis using the adapter primer (NSP-15tgc) is indicated by the arrowhead. (B) The wild-type mRNA of UBL1 is absent in the ubl1 mutant. The RNA used in this experiment was extracted from the wild-type and ubl1 mutant seedlings. (C) Schematic of the UBL1 gene. The Mu insertion location in ubl1, ubl1-3, and ubl1-4 are indicated by a black triangle. The gray portion represents a replacement by a Mu element in the ubl1-2 mutant. (D–F) Seedling phenotypes of ubl1-2/ubl1-2 (D), ubl1/ubl1-3 (E), and ubl1/ubl1-4 (F). Scale bars represent 2.5 cm.WT, wild-type. Molecular Plant 2017 10, 470-482DOI: (10.1016/j.molp.2016.10.016) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Phenotypic Complementation of the ubl1 and At ubl1 Mutant. (A–D) Seedling phenotypes of complementary lines L1–L4 from the construct 35S:UBL1-CDS. L1–L4 represent F2 seedlings with the transgenic locus (35S:UBL1-CDS) and homozygous ubl1/ubl1 genotype derived from the self-pollinated ears of double heterozygous plants. (E) Phenotypic complementation of the At ubl1 mutant with At UBL1. 1-1, 1-2, and 1-3 represent T3 seedlings of three independent complementary lines from the overexpression of At UBL1. (F) Phenotypic complementation of the At ubl1 mutant with UBL1. 2-1, 2-2, and 2-3 represent T3 seedlings of three independent complementary lines from the overexpression of UBL1. WT, wild-type. Scale bars represent 2.5 cm (A–D) and 3 cm (E and F). Molecular Plant 2017 10, 470-482DOI: (10.1016/j.molp.2016.10.016) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Characterization of UBL1 Protein. (A) Analysis of UBL1 activity in vitro. RNA substrate used in this study corresponding in sequence to the 3′ end of maize U6 snRNA (nucleotides G86–U103) carrying five nontemplated uridine residues (indicated in red). RNA substrate was incubated with UBL1 for 10, 30, and 60 min, respectively. UBL1 containing two histidine mutations (H132A and H221A) was incubated with RNA substrate for 60 min. RNA substrate was incubated in buffer alone for 60 min as control. (B) Two invariant histidine mutations in UBL1 destroyed the function of UBL1. At ubl1 is a T-DNA insertion mutant from the Salk Institute. L1–L3 represent three overexpression transgenic lines of 35S:UBL1H132A,H221A. Scale bar represents 3 cm. (C) Expression of UBL1H132A,H221A gene was detected using RT–PCR in transgenic lines. The upper bands represent amplification of UBL1H132A,H221A. The lower bands represent amplification of the Arabidopsis Actin2 gene.WT, wild-type. Molecular Plant 2017 10, 470-482DOI: (10.1016/j.molp.2016.10.016) Copyright © 2016 The Authors Terms and Conditions

Figure 6 UBL1 Is Important for Functional U6 snRNA. (A and B) Relative expression levels of UBL1 in wild-type and ubl1 mutant seedlings (A), and relative expression levels of U6 in wild-type and mutant seedlings (B). The cDNA was generated by wild-type and ubl1 mutant seedlings from three different heterozygous ears (928, 938, and 951). qRT–PCR values for UBL1 and U6 are means of three technical replicates normalized with the maize Actin1 gene. Error bars represent SDs. (C) Increasing 3′-terminal heterogeneity of U6 in the ubl1 mutant. Circularized RNA RT–PCR analysis of U6 snRNA from UBL1 and ubl1 mutant is shown. The RNA was treated by HCl and rSAP to remove the phosphate moiety from the 3′ end. RNase H cleavage was performed to remove the 5′ end structure of U6. After reverse transcription PCR was performed, and the products were cloned and sequenced. The numbers indicate the amount of independent clones sequenced.WT, wild-type. Molecular Plant 2017 10, 470-482DOI: (10.1016/j.molp.2016.10.016) Copyright © 2016 The Authors Terms and Conditions

Figure 7 Loss of Function of UBL1 Leads to Intron Splicing Defect. (A) Density plot of reads mapped to introns expressed as reads per kilobase per million mapped reads (RPKM). Introns with zero read were excluded from the distribution plot. The x axis is represented in log10 scale to improve visualization of the data. (B) RT–PCR validation for 15 intron retention (IR) events. The RNA samples were extracted from three biological replications of ubl1 mutant and wild-type seedlings derived from three independent F7 (successive inbreeding of three F4 siblings) heterozygous ears. The primers used for RT–PCR were designed according the sequences of upstream and downstream exons of the detected IR events and are listed in Supplemental Table 6. The primers for amplification of GAPC1 are across three introns to eliminate DNA contaminant. IR event coordinates are given below each gel in the format: transcript, chromosome, strand, and the start and end positions of the intron in the B73 reference genome (AGPv2). The black pentastars indicate the intron-retained products and the bands at the bottom of the gel indicate completely spliced isoforms. All of the IR products detected by RT–PCR were confirmed by sequencing. The number of amplification cycles was 35 cycles for IR events and 25 cycles for GPAC1.WT, wild-type. Molecular Plant 2017 10, 470-482DOI: (10.1016/j.molp.2016.10.016) Copyright © 2016 The Authors Terms and Conditions