AtPRMT5 Regulates Shoot Regeneration through Mediating Histone H4R3 Dimethylation on KRPs and Pre-mRNA Splicing of RKP in Arabidopsis  Hui Liu, Xu Ma, Hua Nan Han, Yu Jin Hao, Xian Sheng Zhang  Molecular Plant  Volume 9, Issue 12, Pages 1634-1646 (December 2016) DOI: 10.1016/j.molp.2016.10.010 Copyright © 2016 The Author Terms and Conditions

Figure 1 AtPRMT5 Regulates Shoot Regeneration. (A) Shoot regeneration frequencies of calli of wild-type (Col), atprmt5-1, atprmt5-4, 35S:KRP1-2#, and 35S:KRP1-3# after culture on shoot-induction medium (SIM) at indicated times. Calli were cultured on callus-induction medium (CIM) for 6 days using roots as explants and then transferred to SIM for shoot induction. The frequencies were calculated using the number of all calli cultured on SIM divided by the number of calli with shoots. (B) Regenerative shoot number per callus of Col, atprmt5-1, atprmt5-4, 35S:KRP1-2#, and 35S:KRP1-3# after culture on SIM at indicated times. Calli were cultured on CIM for 6 days using roots as explants and then transferred to SIM for shoot induction. Only the calli giving rise to at least one shoot were used to count. (C) Morphology of calli shown in Col, atprmt5-1, atprmt5-4, 35S:KRP1-2#, and 35S:KRP1-3# after culture on SIM at indicated times. Calli were cultured on CIM for 6 days using roots as explants and then transferred to SIM for shoot induction. S0 represents the calli cultured on CIM for 6 days using roots as explants before transferring to SIM. S4, S8, S12, S16, S20, S24, S28, and S32 represent the calli cultured on SIM for 4, 8, 12, 16, 20, 24, 28, and 32 days, respectively. Scale bars represent 500 μm. Standard errors were calculated from three sets of biological replicates and more than 100 calli were examined in each replicate. Significant difference (Student's t test, **P < 0.01). Molecular Plant 2016 9, 1634-1646DOI: (10.1016/j.molp.2016.10.010) Copyright © 2016 The Author Terms and Conditions

Figure 2 Different Expression Genes Analyzed by RNA-Seq Analyses. (A) Cluster analysis of transcript levels of genes in the wild-type (Col) and the atprmt5 mutant. Col-A, Col-B, and Col-C are the three repeats of Col. atprmt5-A, atprmt5-B, and atprmt5-C are the three repeats of the atprmt5 mutant. (B) Analysis of different expression genes by edgeR software. A 2-fold change in the gene expression levels between the wild-type Col and the mutant atprmt5 with an adjusted P value (FDR) cutoff of 0.05 was used for analyses. (C) GO analysis of the upregulated genes in the atprmt5 mutant. (D) GO analysis of the downregulated genes in the atprmt5 mutant. Molecular Plant 2016 9, 1634-1646DOI: (10.1016/j.molp.2016.10.010) Copyright © 2016 The Author Terms and Conditions

Figure 3 AtPRMT5 Regulates KRPs Expression through Histone Modifications. (A) Expression level of KRP1 in the wild-type (Col) and the atprmt5-1 mutant calli after culture on SIM for 0, 4, 8, 12, 16, and 20 days. TUBULIN2 (TUB2) was used as an internal control. (B) Histone H4R3sme2 levels in regions (a, b, c, and d) of the KRP1 promoter in Col and atprmt5-1 calli after culture on SIM for 16 days revealed by ChIP analysis. (C) Expression level of KRP2 in the wild-type (Col) and the atprmt5-1 mutant calli after culture on SIM for 0, 4, 8, 12, 16, and 20 days. TUB2 was used as an internal control. (D) Histone H4R3sme2 levels in regions (a, b, c, and d) of the KRP2 promoter in Col and atprmt5-1 calli after culture on SIM for 16 days revealed by ChIP analysis. (E) Expression of KRP1 in Col and atprmt5-1 calli cultured on SIM for 0, 4, 8, 12, 16, and 20 days as indicated by GUS staining. Calli were cultured on CIM for 6 days using roots as explants and then transferred to SIM for shoot induction. (F) Spatial-temporal expression of KRP1 in Col and atprmt5-1 calli cultured on SIM as indicated by paraffin section of (E). S0 represents the calli cultured on CIM for 6 days using roots as explants before transferring to SIM. S4, S8, S12, S16, and S20 represent the calli cultured on SIM for 4, 8, 12, 16, and 20 days, respectively. Scale bars represent 500 μm. Standard errors were calculated from three sets of biological replicates and more than 100 calli were examined in each replicate. Significant difference (Student's t test, **P < 0.01). Molecular Plant 2016 9, 1634-1646DOI: (10.1016/j.molp.2016.10.010) Copyright © 2016 The Author Terms and Conditions

Figure 4 AtPRMT5 Regulates Shoot Regeneration through Repression of KRPs Expression. (A) Shoot regeneration frequencies of calli of wild-type (Col), atprmt5-1, krp1-1, krp2-1, krp1-1krp2-1, atprmt5-1krp1-1, atprmt5-1krp2-1, and atprmt5-1krp1-1krp2-1. Calli were cultured on CIM for 6 days using roots as explants and then transferred to SIM for shoot induction. The frequencies were calculated using the number of all calli cultured on SIM divided by the number of calli with shoots. (B) Regenerative shoot number per callus of wild-type (Col), atprmt5-1, krp1-1, krp2-1, krp1-1krp2-1, atprmt5-1krp1-1, atprmt5-1krp2-1, and atprmt5-1krp1-1krp2-1. Calli were cultured on CIM for 6 days using roots as explants and then transferred to SIM for shoot induction. Only the calli giving rise to at least one shoot were used to count. (C) The morphology of the calli shown in Col, atprmt5-1, krp1-1, krp2-1, krp1-1krp2-1, atprmt5-1krp1-1, atprmt5-1krp2-1, and atprmt5-1krp1-1krp2-1 cultured on SIM at indicated times. Calli were cultured on CIM for 6 days using roots as explants and then transferred to SIM for shoot induction. S0 represents the calli cultured on CIM for 6 days using roots as explants before transferring to SIM. S4, S8, S12, S16, S20, S24, S28, and S32 represent calli cultured on SIM for 4, 8, 12, 16, 20, 24, 28, and 32 days, respectively. Scale bars represent 500 μm. Standard errors were calculated from three sets of biological replicates and more than 100 calli were examined in each replicate. Significant difference (Student's t test, **P < 0.01). Molecular Plant 2016 9, 1634-1646DOI: (10.1016/j.molp.2016.10.010) Copyright © 2016 The Author Terms and Conditions

Figure 5 AtPRMT5 Regulates Shoot Regeneration through Pre-mRNA Splicing of RKP. (A) Schematic illustration of pre-mRNA splicing of RKP in the wild-type (Col) and atprmt5-1. *The intron with pre-mRNA splicing defects. (B) Pre-mRNA splicing of RKP of Col and atprmt5-1 calli after culture on SIM for 12, 16, and 20 days. TUBULIN2 (TUB2) was used as an internal control. *The amplified fragment including the intron with pre-mRNA splicing defects. S12, S16, and S20 represent the calli cultured on SIM for 12, 16, and 20 days, respectively. (C) The relative indensity of the bands in (B). Normal indicates the right spliced mRNA of RKP, and abnormal indicates the unspliced mRNA of RKP. (D) Expression patterns of RKP are shown by GUS staining in Col calli after culture on SIM at indicated times. Calli were cultured on CIM for 6 days using roots as explants and then transferred to SIM for shoot induction. S0 represents the calli cultured on CIM for 6 days using roots as explants before transferring to SIM. S4, S8, S12, S16, and S20 represent the calli cultured on SIM for 4, 8, 12, 16, and 20 days, respectively. (E) The vector pRKP:wtRKP with the wild-type RKP gene and the mutated vector pRKP:mRKP with the mutation in the two sides of sixth intron of RKP. (F) Pre-mRNA splicing of wild-type (Col), rkp-2, pRKP:wtRKP-1#, pRKP:wtRKP-5#, pRKP:wtRKP-8# in rkp-2 and pRKP:mRKP-2#, pRKP:mRKP-10#, pRKP:mRKP-12# in rkp-2 calli after culture on SIM for 16 days. TUB2 was used as an internal control. (G) Frequencies of calli of wild-type (Col), rkp-2, pRKP:wtRKP-1#, pRKP:wtRKP-5#, pRKP:wtRKP-8# in rkp-2 and pRKP:mRKP-2#, pRKP:mRKP-10#, pRKP:mRKP-12# in rkp-2 after culture on SIM for 32 days. The frequencies were calculated using the number of all calli cultured on SIM divided by the number of calli with shoots. (H) The regenerative shoot number per callus of wild-type (Col), rkp-2, pRKP:wtRKP-1#, pRKP:wtRKP-5#, pRKP:wtRKP-8# in rkp-2 and pRKP:mRKP-2#, pRKP:mRKP-10#, pRKP:mRKP-12# in rkp-2 after culture on SIM for 32 days. Only the calli giving rise to at least one shoot were used to count. Scale bars represent 500 μm. Standard errors were calculated from three sets of biological replicates, and more than 100 calli were examined in each replicate. Significant difference (Student's t test, **P < 0.01). ns indicates no significant difference. Molecular Plant 2016 9, 1634-1646DOI: (10.1016/j.molp.2016.10.010) Copyright © 2016 The Author Terms and Conditions

Figure 6 Mutation of RKP Represses Shoot Regeneration. (A) Shoot regeneration frequencies of calli of wild-type (Col), atprmt5-1, rkp-1, and atprmt5-1 rkp-1 after culture on SIM at indicated times. Calli were cultured on CIM for 6 days using roots as explants and then transferred to SIM for shoot induction. The frequencies were calculated using the number of all calli cultured on SIM divided by the number of calli with shoots. (B) Regenerative shoot number per callus of Col, atprmt5-1, rkp-1, and atprmt5-1 rkp-1 after culture on SIM at indicated times. Calli were cultured on CIM for 6 days using roots as explants and then transferred to SIM for shoot induction. Only the calli giving rise to at least one shoot were used to count. (C) Morphology of calli shown in Col, atprmt5-1, rkp-1, and atprmt5-1 rkp-1 cultured on SIM at indicated times. Calli were cultured on CIM for 6 days using roots as explants and then transferred to SIM for shoot induction. S0 represents the calli cultured on CIM for 6 days using roots as explants before transferring to SIM. S4, S8, S12, S16, S20, S24, S28, and S32 represent the calli cultured on SIM for 4, 8, 12, 16, 20, 24, 28, and 32 days, respectively. Scale bars represent 500 μm. Standard errors were calculated from three sets of biological replicates and more than 100 calli were examined in each replicate. Significant difference (Student's t test, **P < 0.01). Molecular Plant 2016 9, 1634-1646DOI: (10.1016/j.molp.2016.10.010) Copyright © 2016 The Author Terms and Conditions

Figure 7 RKP Regulates Shoot Regeneration through KRP1 Function. (A) Shoot regeneration frequencies of calli of wild-type (Col), rkp-1, 35S:KRP1-3#, and rkp-135S:KRP1-3# after culture on SIM at indicated times. Calli were cultured on CIM for 6 days using roots as explants and then transferred to SIM for shoot induction. The frequencies were calculated using the number of all calli cultured on SIM divided by the number of calli with shoots. (B) Regenerative shoot number per callus of Col, rkp-1, 35S:KRP1-3#, rkp-135S:KRP1-3# after culture on SIM at indicated times. Calli were cultured on CIM for 6 days using roots as explants and then transferred to SIM for shoot induction. Only the calli giving rise to at least one shoot were used to count. (C) Morphology of calli shown in Col, rkp-1, 35S:KRP1-3#, rkp-135S:KRP1-3# cultured on SIM at indicated times. Calli were cultured on CIM for 6 days using roots as explants and then transferred to SIM for shoot induction. S0 represents the calli cultured on CIM for 6 days using roots as explants before transferring to SIM. S4, S8, S12, S16, S20, S24, S28, and S32 represent the calli cultured on SIM for 4, 8, 12, 16, 20, 24, 28, and 32 days, respectively. Scale bars represent 500 μm. Standard errors were calculated from three sets of biological replicates and more than 100 calli were examined in each replicate. Significant difference (Student's t test, **P < 0.01). ns indicates no significant difference. Molecular Plant 2016 9, 1634-1646DOI: (10.1016/j.molp.2016.10.010) Copyright © 2016 The Author Terms and Conditions

Figure 8 Co-regulation of Shoot Regeneration by AtPRMT5-Mediated Histone Modifications and RNA Splicing. Molecular Plant 2016 9, 1634-1646DOI: (10.1016/j.molp.2016.10.010) Copyright © 2016 The Author Terms and Conditions