1. Developmental- and tissue-specific expression of NbCMT3-2 encoding a chromomethylase in Nicotiana benthamiana Yu-Ting Lin 1 ( 林郁婷 ), Huei-Mei Wei 1, Syue-Yu Lyu 1, Yung-I Lee 2, and Shih-Feng Fu 1* ( 傅士峰 ) 1 Department of Biology, National Chunghua University of Education ( 彰化師範大學生物系 ) 2 Botany Department, National Museum of Natural Science ( 自然科學博物館植物園 ) Abstract Results DNA methylation is a heritable epigenetic process controlling gene expression and developmental programs in various organisms. The chromomethylase (CMT) protein family is unique to plants and controls non-CpG methylation. Here, we investigated the developmental expression of CMT3-2 in Nicotiana benthamiana (NbCMT3-2) and its significance by analyzing plants with silencing of NbCMT3-2 and analyzing leaf tissues transiently expressing the N-terminal polypeptide. Alignment of the NbCMT3-2 amino acid sequence with other plant CMT3s showed a specific N-terminal extension required for nuclear localization. Transient expression of the N-terminal polypeptide in N. benthamiana resulted in the formation of chlorotic lesions, which indicates its vital role in leaf development. NbCMT3-2 was expressed mainly in proliferating tissues such as the shoot apex and developing leaves. We generated transgenic N. benthamiana harboring a fusion reporter construct linking the NbCMT3-2 promoter region and  -glucuronidase (GUS) reporter (pNbCMT3-2::GUS) to analyze the tissue-specific expression of NbCMT3-2. NbCMT3-2 was expressed in shoot and root apical meristem and leaf primordia in young seedlings and highly expressed in developing leaves and ovary as well as lateral buds in mature plants. Virus- induced gene silencing used to knock down NbCMT3-2 expression led to partial loss of genomic CHG DNA methylation. Silencing NbCMT3-2 interfered with leaf development and the expression of genes involved in jasmonate homeostasis. The differential roles between NbCMT3 and NbCMT3-2 were investigated and compared. We reveal the expression patterns of NbCMT3-2 in proliferating tissues. NbCMT3-2 may play an essential role in leaf development by modulating jasmonate pathways. 1. Nuclear localization of NbCMT3-2 protein Fig. 1 Subcellular localization of NbCMT3-2 proteins in N. benthamiana. (A)Schematic diagram of the constructs used for transient expression of proteins in leaf epidermal cells. The unique N-terminal extension (N) is in yellow, the Bromo- adjacent homology (BAH) domain is in green, and the chromo domain (CD) is in red. The blue boxes indicate the conserved methyltransferase catalytic motifs I, IV, VI, VIII, IX and X. (B) Agrobacterium carrying the fusion constructs under the control of Cauliflower mosaic virus 35S promoter was infiltrated into N. benthamiana epidermal cells. At 4 days after agroinfiltration, fluorescence of NbCMT3-derived proteins was observed by confocal laser scanning microscopy (C) Western blot analysis of NbCMT3-derived protein fusions expressed from pSITEII- 4C1 vectors in agroinfiltrated leaves 2. Expression of NbCMT3 and NbCMT3-2 in tissues of N. benthamiana 2. Expression of NbCMT3 and NbCMT3-2 in tissues of N. benthamiana 3. Shoot apex- and root tip-specific expression of NbCMT3 and NbCMT3-2 promoter 3. Shoot apex- and root tip-specific expression of NbCMT3 and NbCMT3-2 promoter A B Fig. 2 Expression of NbCMT3 and NbCMT3-2 in different tissues. (A) Diagrams of gene-specific primers corresponding to NbCMT3 and NbCMT3-2 cDNA sequences. Apical shoots (A), young leaves (Y), mature leaves (M), flowers (F) and roots (R).The tissue culture (T) of mature leaf explants during shoot regeneration for 2 weeks were collected for gene expression analysis. NbEF1 was a reference gene. Fig. 3 Histochemical localization of GUS activity in transgenic N. benthamiana plants expressing GUS gene driven by the NbCMT3 or NbCMT3-2 promoter region. Cotyledons (Cy), Center zone (CZ), Peripheral zone (PZ), RZ (Ribzone), PM (Primary meristem), AM (Apical meristem), RC (Root cap), Co (Cortex), E (Epidermis). 4. Expression of NbCMT3 or NbCMT3-2 promoter in stigma 4. Expression of NbCMT3 or NbCMT3-2 promoter in stigma 5. Accumulation of NbCMT3– or NbCMT3-2–GFP-GUS fusion protein during organogenesis 5. Accumulation of NbCMT3– or NbCMT3-2–GFP-GUS fusion protein during organogenesis 6. Suppression of endogenous NbCMT3 or NbCMT3-2 expression by VIGS 6. Suppression of endogenous NbCMT3 or NbCMT3-2 expression by VIGS Fig. 4 Distribution of pNbCMT3-2::GUS activity in various organs of mature transgenic N. benthamiana plants. (A) lateral bud, (B) developing leaves, (C) ovary and (D) stigma. Fig. 5 Promoter NbCMT3- or NbCMT3-2-driven GFP and GUS expression during callus formation and organogenesis in vitro. Callus- inducing medium (CIM). Shoot inducing medium (SIM) (A-F). (G and H) Western blot analysis of GFP expressed from transgenic pNbCMT3::GFP- GUS or pNbCMT3- 2::GFP-GUS leaf explants. Wild-type N. benthamiana plants were a negative control. The star (*) denotes the non-specific cross-reaction of antibodies. Fig. 6 Molecular characterization of NbCMT3-, NbCMT3-2- or NbCMT3/3-2- silenced N. benthamiana plants.(A) NbCMT3 or NbCMT3- 2 cDNA structures and the VIGS construct containing the cDNA fragments. (B) Suppression of NbCMT3 or NbCMT3- 2 transcripts by virus- induced gene suppression (VIGS). (C) Quantification of global DNA methylation in NbCMT3-silenced plants. 7. Regulation of leaf development by NbCMT3 and NbCMT3-2 7. Regulation of leaf development by NbCMT3 and NbCMT3-2 Fig. 7 Alternation in N. benthamiana NbCMT3-2 expression led to abnormality in plant growth and development. (A) Phenotypic characterization of NbCMT3-, NbCMT3-2- and NbCMT3/3-2- silenced N.benthamiana plants. (B)Evaluation of growth and development using transient gene expression from a PVX-based vector. Conclusions and future work NbCMT3-2 and its promoter activity were expressed in proliferating tissues such as shoot apex and root tips. The unique N- terminal extension of NbCMT3-2 has a role in nuclear localization and leaf development. Further investigation into the roles of the unique N-terminal extension will help elucidate the molecular actions of NbCMT3-2 in plant epigenetic regulation. C