Background: In mammals, nucleostemin (NS), a nucleolar GTPase, is involved in stem cell proliferation, embryogenesis and ribosome biogenesis. Arabidopsis NUCLEOSTEMIN-LIKE 1 (NSN1) has previously been shown to be essential for plant growth and development. However, the role of NSN1 in cell proliferation is largely unknown. Results: Using nsn1, a loss-of-function mutant of Arabidopsis NSN1, we investigated the function of NSN1 in plant cell proliferation and cell cycle regulation. Morphologically, nsn1 exhibited developmental defects in both leaves and roots, producing severely reduced vegetative organs with a much smaller number of cells than those in the wild type. Dynamic analysis of leaf and root growth revealed a lower cell proliferation rate and slower cell division in nsn1. Consistently, the transcriptional levels of key cell cycle genes, including those regulating the transition of G1-S and G2- M, were reduced drastically in nsn1. The introduction of CYCLIN B1::GUS into nsn1 resulted in confined expression of GUS in both the leaf primordia and root meristem, indicating that cell proliferation was hampered by the mutation of NSN1. Upon subjection to treatment with bleomycin and methyl methanesulfonate (MMS), nsn1 plants exhibited hypersensitivity to the genotoxic agents. In the nucleus, NSN1 interacted with nucleosome assembly protein1 (AtNAP1;1), a highly conserved histone chaperone functioning in cell proliferation. Notably, the N-terminal conserved domains of ArabidopsisNSN1werecriticalfor thephysicalinteraction. Conclusions: As a conserved homolog of mammalian nucleostemin, Arabidopsis NSN1 plays pivotal roles in embryogenesis and ribosome biogenesis. In this study, NSN1 was found to function as a positive regulator in cell cycle progression. The interaction between NSN1 and histone chaperone AtNAP1;1, and the high resemblance in sensitivity to genotoxics between nsn1 and atnap1;1 imply the indispensability of the two nuclear proteins for cell cycle regulation. This work provides an insight into the delicate control of cell proliferation throughthe cooperationofaGTP-binding protein with a nucleosome assembly/disassembly proteininArabidopsis. Keywords: Cell cycle, Cell proliferation, Nucleostemin-like1, Nucleosome assembly protein1 Background implicated in stem cell proliferation, cell cycle mainten- In multicellular organisms, organogenesis requires tight ance, ribosome biogenesis and embryogenesis [1–4]. For control of cell proliferation. Nucleostemin (NS), a nucle- example, depletion or overexpression of NS in mouse olar GTP-binding protein, was first identified in stem caused arrest of G1-S and G2-M transition by inhibiting cells and some cancer cell lines in mouse . It has been the activity of Murine Double Minute 2 (MDM2), an E3 ubiquitin ligase targeting p53 for proteasome-mediated degradation, from activating p53 [1, 3, 5, 6]. NS was * Correspondence: firstname.lastname@example.org; email@example.com Zhen Wang and Xiaomin Wang contributed equally to this work. found in a large protein complex containing pre-rRNA Department of Plant Sciences, University of Idaho, Moscow, Idaho 83844, processing related nucleolar proteins, such as Pescadillo USA (Pes1), DDX21 (also known as RHII/Guα) and EBNA1 Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wang et al. BMC Plant Biology (2018) 18:99 Page 2 of 12 binding protein 2 (EBP2) , suggesting a key role type plants under normal growth conditions, suggesting in ribosome biogenesis. the functional redundance of the paralogs [18, 24]. Two As a member of the conserved GTPases present in alleles (m123-1 and m123-2) of the triple mutant Atnap1; prokaryotes and eukaryotes, NS belongs to the YlqF/ 1 Atnap1;2 Atnap1;3 exhibit hypersensitivity to DNA YawG GTPase family [8, 9]. The human NS family con- damage caused by UV-C irradiation , revealing a role sists of nucleostemin, Guanine Nucleotide binding of AtNAP1 genes in nucleotide excision repair of DNA. protein-like 3 (GNL3L) and Ngp-1, and all the three Research on AtNAP1;1 has revealed that like its orthologs members have been shown experimentally to bind GTP in rice and tobacco, some AtNAP1;1-GFP fusion proteins in vivo, which is consistent with the presence of the GTP are targeted to the nucleus at an early stage of leaf devel- binding domain in these proteins [3, 10, 11]. In opment whereas the abundance of the fusion pro- addition, NS also contains an acidic amino acid (AAA) teins are retained in the cytoplasm . Its function in domain at the C-terminus, and a basic amino acid promoting cell division or expansion and the farnesyla- (BAA) domain and a coiled-coil (CC) domain at the N- tion status of the protein has been found to be coupled terminus . The latter two domains contribute pre- with its subcellular localization . In a bimolecular dominantly to its biological functions as a nucleolar pro- fluorescence complementation (BiFC) assay, as well as a tein in cell cycle progression of both animals and plants pull-down experiment, AtNAP1;1 has been demon- [1, 7, 12–14]. strated to interact with ribosomal protein S6 (RPS6), sup- Unlike in mammals, only two subfamilies of NS genes porting its positive regulatory role in plant rDNA (nucleostemin and Ngp-1-like) have been identified in transcription . Analysis of a truncated protein of Arabidopsis with the absence of GNL3L-like family genes AtNAP1;3, which lacks 34 amino acids at the C-terminus, . Nucleostemin-like1 (NSN1) of Arabidopsis has reveals altered Arabidopsis responses to abscisic acid been found to posess the common domains of mama- (ABA) treatment and salt stress by functioning as a dom- lian NS and GFP-fused NSN1 has been mainly localized inant negative factor in ABA responses . to the nucleolus in tobacco BY-2 cells . The nsn1 In addition to NAP1, Arabidopsis has two NAP1- mutant exhibits defects in embryogenesis and in the de- related proteins, AtNRP1 and AtNRP2. Both are primar- velopment of both leaf and flower organs. Consistently, ily localized in the nucleus, and the double mutant NSN1 is highly expressed in the developing embryos, Atnrp1-1Atnrp2-1 exhibits short roots and increased floral and shoot apical meristems, and organ primordia sensitivity to the genotoxic treatment . A recent study [15, 16]. The observation of reduced expression of meri- using reporter constructs of homologous recombination stem genes including WUS, CLV3, STM and ANT in (HR) has demonstrated reduced levels of HR in nsn1 suggests that NSN1 plays critical roles in embryo- the double mutant Atnrp1-1Atnrp2-1  and the genesis . Recently, a role of NSN1 in plant growth AtNAP1 triple mutant m123-1, suggesting that NRP and and senescence by modulating ribosome biogenesis has AtNAP1 act in parallel pathways to synergistically pro- been reported based on the observations that silencing mote somatic HR. NSN1 leads to growth retardation and premature senes- In this study, we focused on the function of NSN1 in cence in Arabidopsis and tobacco . Several ribosomal cell cycle regulation. Molecular, histochemical and gen- proteins including Pescadillo and EBP2 have been found etic analysis demonstrated that the growth retardation of to interact with NSN1 and depletion of NSN1 has been dwarf mutant nsn1 is attributed to its arrested cell cycle. shown to repress global translation probably by delaying The interplay between NSN1 and AtNAP1;1, their in- the biogenesis of the 60S ribosome subunit . Taken volvement in cell cycling progression and the hypersen- together, Arabidopsis NSN1 plays important roles in sitivity of nsn1 and atnap1;1 to the genotoxic agents plant growth and development although the exact mech- imply that in Arabidopsis NSN1 regulates cell cycling anism is not clear. and DNA damage repair by cooperating with histone NUCLEOSOME ASSEMBLY PROTEINS1 (NAP1), a chaperone protein AtNAP1;1. histone chaperone in nucleosome assembly/disassembly, is highly conserved from yeast to human [17–20]. NAP1 Results deficiency is known to cause perturbed expression of Growth defects of nsn1 mutant around 10% of nuclear genes in yeast [17, 20], and embry- In previous studies, Nucleostemin-like1 (NSN1) in Arabi- onic lethality in mammals and fruit fly [20, 21]. In plants, dopsis has been demonstrated to be essential for the NAP1 has been identified in several species including rice, growth of both vegetative and reproductive organs by soybean, tobacco and Arabidopsis [18, 19, 22, 23]. In Ara- participation in regulation of embryogenesis and meri- bidopsis, NAP1 is a multi-gene family containing 4 mem- stem development [14–16]. Here, we investigated the bers: AtNAP1;1, AtNAP1;2, AtNAP1;3 and AtNAP1;4. role of NSN1 in cell proliferation and division in both Phenotypically the individual mutant resembles wild leaf and root organs. Consistent with the findings that Wang et al. BMC Plant Biology (2018) 18:99 Page 3 of 12 Fig. 1 Retarded growth of leaf and root organs in nsn1. a Eight-day-old seedlings of wild type and nsn1, showing reduced size of cotyledons and late initiation of the first leaf in nsn1. Scale bar: 1 mm. b Statistical analysis of cotyledon area of 8-day-old seedlings. c Twenty-one-day-old wild type and nsn1 plants. d Leaf area analysis of the fifth leaf from 21-day-old plants. e Ten-day-old seedlings grown on MS medium showing retarded root growth in nsn1 as compared with the wild type. f Statistical analysis of the primary root length of 10-day-old seedlings. For the statistical analysis, three biological replicates were conducted with 15 plants measured for each experiment cotyledons of nsn1, a loss-of-function mutant of NSN1, are initiated late at the embryo stage [15, 16], delayed emergence of the first leaf in nsn1 dwarf plant was ob- Table 1 Comparison of leaf development between wild type served (Fig. 1a) with the first leaf emerging at 9th day and nsn1 after germination (DAG), compared with the 5th DAG in Emergence of 1st. leaf Full expansion of 5th. leaf the wild type plants (Table 1). For the duration of leaf (DAG) (DAI) expansion, at the 9th day after initiation (DAI), the fifth Wild type 5.1 ± 1.2 9.3 ± 1.8 leaf of wild type reached maximum leaf area, while the nsn1 9.5 ± 1.9 21.2 ± 2.4 counterpart leaf of nsn1 fully expended and achieved the Note: DAG days after germination; DAI days after initiation. Value represents maximum area at the 21st DAI (Table 1). Therefore, mean ± SD. Three biological replicates were performed with at least 15 plants measured for each experiment nsn1 displayed severe growth retardation during the life Wang et al. BMC Plant Biology (2018) 18:99 Page 4 of 12 cycle. The fifth leaf pair was used in this study unless examined and compared with the wild type plants. Kin- otherwise indicated. Analysis of the leaf area revealed etic analysis of leave growth showed that the leaf area of that the size of cotyledons and the fifth leaf of nsn1 wild type increased dramatically after initiation and were about 50 and 40% of their counterparts in wild reached the maximum at 7th DAI, while the progressive type, respectively (Fig. 1b, d). As a result, nsn1 with leaf growth of nsn1 was relatively slow and the max- smaller size of leaves exhibited dwarfism (Fig. 1c). Simi- imum value was achieved at 17th DAI (Fig. 2g). The lar developmental retardation was observed in roots of long duration of cell proliferation in nsn1 indicated a nsn1 plants, whose primary root length was about 40% slower cell proliferation rate in the dwarf mutant. Syn- of that of wild type (Fig. 1e, f). Therefore, both aerial chronically, dynamics of cell number/leaf demonstrated and underground organs of nsn1 plants displayed growth a significant difference of the maximum cell number be- defects, suggesting the indispensability of NSN1 for plant tween nsn1 and wild type, with about 3.4 × 10 at 17th growth. DAI for nsn1 and 4.9 × 10 cells at 7th DAI for wild type (Fig. 2h), respectively. Taken together, the hampered leaf Deficient cell proliferation in nsn1 leaf growth in nsn1 was resulted from the malfunction of Plant organ size is genetically determined by both the NSN1 in cell proliferation. Additionally, in wild type the number and the size of its constituent cells. To investi- palisade cell layer and spongy cell layer are easily gate the cause of dwarfism in nsn1, the number and size differentiated and the longitudinal axial of elongated of palisade cells of the fifth leaf pair were measured and palisade cell is vertical to the leaf epidermis (Fig. 2b). analyzed. In agreement with the dwarf statue of nsn1, In nsn1, however, abnormal palisade cells with the amount of the palisade cells was reduced to 56% of irregular patterning and highly variable size and shape that of the wild type (Fig. 2a). In terms of cell size, al- were observed (Fig. 2c), suggesting that besides though cells in the palisade cell layer from the cross- regulating cell proliferation and division, NSN1 may section of nsn1 leaves were slightly larger than those of function in leaf development as well. the wild type (Fig. 2b-e), statistically the diameter differ- ence of the palisade cells between the two genotypes was not significant (P > 0.05) (Fig. 2f), suggesting that the cell Inefficient cell proliferation in nsn1 roots size is not the major source for the dwarfism in nsn1. In agreement with the retardation of root growth in nsn1 Hence, the reduction of the overall leaf size of nsn1 mu- (Fig. 1e, f), 10-day-old mutant exhibited shorter primary tant is attributed to the decrease of cell numbers rather roots and a smaller number of meristematic cells were than cell size alteration. present in the root meristem region of nsn1 than in wild To search for the cause of cell reduction in nsn1, dy- type (Fig. 3a, b). In terms of root growth rate, root length namics of leaf area and cell proliferation rate were was monitored during the first week after germination. Fig. 2 Inefficient cell proliferation in nsn1 leaves. The fifth leaf pair was used for measurement and analysis. a Statistical analysis of the number of palisade cells from fully expanded leaves. Scanning electron microscope (SEM) images of fully expanded leaves of wild type (b) and nsn1 (c). Arrow heads indicate the palisade cells with similar size from the two genotypes. The palisade cells of fully expanded leaves of wild type (d) and nsn1 (e). f Statistical analysis of the diameter of palisade cells from fully expanded leaves. g Dynamics of leaf expansion of wild type and nsn1. h Dynamic analysis of cell number/leaf of wild type and nsn1. The difference between the two datasets was analyzed by Student’s t test (P < 0.01). Scale bars: 50 μm for (b) and (c); 10 μm for (d) and (e) Wang et al. BMC Plant Biology (2018) 18:99 Page 5 of 12 Fig. 3 Inefficient cell proliferation in nsn1 root. a Longitudinal sections of the primary root from 10-day-old wild type and nsn1. Illustration of the tissue organization of the Arabidopsis primary root apex (left in color) was adapted from . Arrowhead indicated the cortex cells in elongation zone. Scale bar: 100 μm. b Statistical analysis of cell numbers in the root meristem region of 10-day-old plants. c Comparison of the dynamics of the primary root length in wild type and nsn1 within the first week after germination. d Analysis of the daily cell production rate in the primary root of wild type and nsn1. The number of cortex cells at the root tip including elongation and cell division zones was counted under microscope daily from the 4th day to the 10th day after germination. Cortex cells on one side as indicated by arrowheads in (a) were calculated The length of primary root of nsn1 was about 25–35% of [29, 30]. Quantitative analysis revealed that in nsn1, the wild type within the first 7 days (Fig. 3c), and on day 10, expression level of the cyclin genes was about 30–40% nsn1 root reached 40% of wild type (Fig. 1e, f). Consistent of that in wild type, and the transcriptional level of HIS- with the measurement of meristem cells, the number of TONE H4 and RNR, a key enzyme in the DNA synthesis cells produced by nsn1 roots per day was about one third pathway , decreased to approximately 60 and 50% of of that of the wild type roots (Fig. 3d). Therefore, our ana- those in wild type, respectively (Fig. 4a). The notable lysis indicates that slower root elongation and less daily down-regulation of these cell cycle regulating genes in root cell production of nsn1 account for the observed nsn1 implied the disruption of both G1-S and G2-M growth defects in the mutant. transition. To further dissect the involvement of NSN1 in cell cycle Impaired cell cycling progression in nsn1 regulation, flow cytometry analysis was performed. Leaves Given that the dwarfism of nsn1 was resulted from de- from two developmental stages (4-day-old and mature) fective cell proliferation and division, we investigated were subjected to nuclei isolation. Our results showed a whether the nsn1 mutation affected the transcriptional lower percentage of 2C nuclei in nsn1 than wild type at levels of five cell cycle marker genes, including three both the young and mature phases: 79 vs. 95% and 18 vs. cyclin family genes CYCA2;3, CYCB1;1, and CYCD3;1, 60%, respectively (Fig. 4b). Consistently, compared with and two S-phase specific genes HISTONE H4 and Ribo- the wild type, in nsn1, 16% more of 4C nuclei were detected nucleotide reductase (RNR) . For the three cyclin in young leaves, and 42% more of polyploidy including 4C, genes, CYCD3;1 has been reported as a key gene in G1- 8C, 16C and 32C nuclei were observed in mature leaves S transition by controlling cell division rate , (Fig. 4b). The detection of higher percentage of polyploidy while the remaining two genes, CYCB1;1 and CYCA2;3, indicated a condensed DNA content in nsn1 due to the have been found to affect G2/M transition in cell cycle malfunction of nsn1 mutation in cell division. Wang et al. BMC Plant Biology (2018) 18:99 Page 6 of 12 Fig. 4 Analysis of cell cycle defects in nsn1. a Relative transcriptional levels of cell cycle genes in nsn1 as measured by q-RT-PCR. Ten-day-old seedlings shown in Fig. 1e were used for total RNA extraction after the root length measurement. Three biological replicates were conducted with 15 plants for each experiment. b Flow cytometry assay of nuclei isolated from leaves of 4-days after initiation (upper panel) and of fully-expanded fifth leaves (lower panel). c-f Histochemical analysis of GUS signal in ten-day-old seedlings of indicated genotypes. GUS signal in the shoot apical meristem (c)and root meristem (e)of CYCB1;1::GUS expressing wild type plants stained with X-glux solution. GUS signal in the shoot apical meristem (d) and root meristem (f)of CYCB1;1::GUS expressing nsn1 plants stained with X-glux solution. Scale bars: 0.5 mm for (c)and (d); 100 μmfor (e)and (f) To visualize the effect of NSN1 on cell cycle progres- data revealed that the cell cycle progression in both sion, transgenic plants expressing CYCLIN B1;1::GUS, leaves and roots of nsn1 was impaired by the nsn1 which expresses the reporter β-glucuronidase (GUS) mutation. fused to the mitotic destruction sequence (D-box) under the promoter of the cyclin CYCB1;1 , were Hypersensitivity of nsn1 to genotoxic agents crossed with nsn1 mutant to obtain CYCLIN B1;1::GUS- Given that NSN1 is essential for proper cell cycle pro- containing nsn1 lines. The GUS activity marked cells in gression, plant sensitivity to DNA damaging agents me- G2 and early M phase  because the fusion gene thyl methanesulfonate (MMS) and bleomycin was tested. was expressed upon entry into G2 (via the CYCB1:1 pro- MMS is known to result in alkylated DNA, which is moter) and its protein product was degraded upon exit poorly replicated by DNA polymerases , while treat- from metaphase (via D-box) . Histochemical analysis ment with bleomycin, a radiomimetic drug, causes mul- showed that in the wild type background, GUS-positive tiple types of molecular damages including double cells were predominantly detected in the regions of leaf strand breaks (DSBs) . Four-day-old Arabidopsis primordia (Fig. 4c). When CYCLIN B1;1::GUS was intro- plantlets were subjected to treatment with MMS and duced into nsn1, however, the expression of GUS was bleomycin. When nsn1 seedlings were exposed to 25 confined to a much smaller area containing meristem- ppm MMS, rosette leaves turned yellow and root growth atic cells of the shoot apical region (Fig. 4d). In roots, was severely inhibited (Fig. 5a). MMS of 50 ppm inhib- a clearly lower number of GUS-positive cells were ob- ited the growth of leaf and root organs, resulting in leaf served in nsn1 plants than those in the control plant etiolation and plant death. The damages caused by (Fig. 4e, f). The restricted CYCLIN B1;1::GUS signal MMS was dosage-dependent (Fig. 5a). In contrast, the in both the root and shoot apical meristems of nsn1 growth of wild type was not obviously affected by MMS plants provided genetic evidence of arrested cell cyc- at 25 ppm, and for treatment of 100 ppm MMS, no ling in nsn1. Therefore, our molecular and genetic plant death was observed although rosette leaves were Wang et al. BMC Plant Biology (2018) 18:99 Page 7 of 12 Fig. 5 Comparison of sensitivity to genotoxic agent treatment. a Four-day-old seedlings were treated with MMS at the indicated concentrations for 1 week. b Four-day-old seedlings were treated with bleomycin at the different concentrations for 3 weeks. Three biological replicates were performed with at least 15 plants tested per experiment yellowish and root growth was badly inhibited (Fig. 5a). indicating that the presence of both NSN1 and AtNAP1; These results indicated that nsn1 is hypersensitive to 1 is required for the observed interaction. Interestingly, MMS treatment in comparison with the wild type. similar to NSN1, AtNAP1;1 has also been demonstrated The survival rate of nsn1 plants was dramatically de- to regulate cell proliferation and leaf development . creased by bleomycin treatment, especially at dosages NSN1 contains the common conserved domains of higher than 6 mg/L (Fig. 5b). At the dosage of 12 mg/L, nucleostemin, including the basic amino acid domain all nsn1 plantlets died after being treated for 1 week, (BAA), coiled-coil domain (CC), GTP-binding domain while about 60% of wild type plants survived the treat- (GBD), RNA binding domain (RBD) and acidic amino ment (Fig. 5b). Hence, nsn1 displayed more pronounced acid domain (AAA) (Fig. 6a). To investigate which do- sensitivity to the two genotoxic agents than the wild mains of NSN1 were indispensable for the in vitro inter- type plants, implying the involvement of NSN1 in cell action with AtNAP1;1, two adjacent combined domains, cycle progression in plants. including BC standing for the N-terminal BAA and CC domains, and GR for the GBD and RBD domains, were Interaction of NSN1 with AtNAP1;1 in vitro and in vivo tested (Fig. 6a). Domain deletion analysis demonstrated Like mammalian NS, Arabidopsis NSN1 interacts with that with the BC domains, yeast growth and strong blue several nucleolar proteins involved in ribosomal biogen- signal were observed, while the controls comprising ei- esis . To search for potential plant-specific protein(s) ther of the tested peptides with an empty vector were physically interacting with NSN1, a yeast two- negative (Fig. 6b), suggesting that the presence of the hybrid screen was carried out. We screened a cDNA li- two N-terminal domains, BAA and CC, of NSN1 is es- brary of Arabidopsis flower from ABRC (stock CD4-30) sential for the interaction (Fig. 6b). No physical inter- constructed in pAD-GAL4-2.1. NSN1 cDNA was cloned action was detected between AtNAP1;1 and the two into pBD-GAL4 (Strategy, USA) and served as bait. middle domains of NSN1, e.g. GBD and RBD (data not Empty vectors of pBD-GAL4 and pAD-GAL4-2.1 were shown). These results indicated that the N-terminal two used as negative controls for verification of candidate conserved domains (BAA and CC), but not the middle clones. two domains (RBD and AAA), were essential for the Around a million yeast colonies were screened and physical interaction between NSN1 and AtNAP1;1. candidate colonies were verified to eliminate false posi- To test whether NSN1 and AtNAP1;1 interact in vivo, tives. One colony showed consistent growth on the se- the BiFC assay, a widely used tool for visualization of lective media, and the colony was identified as NAP1; protein-protein interaction in living cells, was per- 1 (At4g26110.2), a member of the NUCLEOSOME AS- formed. The coding sequences of NSN1 and AtNAP1;1 SEMBLY PROTEINS1 (NAP1) . Further analysis were fused in frame with the N-terminal and C-terminal demonstrated that the yeast colony expressing the full fragments of enhanced GFP (eGFP), respectively . N C length coding sequence (CDS) of AtNAP1;1 grew health- The fusion proteins (YFP -NSN1 and YFP -NAP1;1) ily on the selective media and turned blue on X-gal indi- were co-expressed in tobacco leaves by Agrobacterium- cator plates (Fig. 6b), suggesting an interaction between infiltration. Leaf epidermal cells of infiltrated tobacco the encoded protein and NSN1. For the control vectors, were checked with confocal laser scanning microscopy as expected, yeast hardly grew on the selective media after 48 h. GFP fluorescence was observed in the nuclei with extremely faint blue in X-gal overlay assay (Fig. 6b of tobacco leaves transfected with YFP -NSN1 and upper panel). Quantification of the interaction strength YFP -NAP1;1 as indicated by DAPI-stained nuclei revealed a significantly strong signal in the cells express- (Fig. 6c). This result is consistent with the previous stud- ing both NSN1 and AtNAP1;1 (Fig. 6b upper panel), ies that NSN1 was predominantly localized in nucleoli Wang et al. BMC Plant Biology (2018) 18:99 Page 8 of 12 Fig. 6 Interaction between NSN1 and NAP1;1 in yeast and tobacco. a Illustration of NSN1 fragments used for yeast two-hybrid assays. NSN1 protein consists of 548 amino acids. Two fragments tested in the yeast two-hybrid assay are the N-terminal fragment (amino acids 1–110) including the basic amino acid (BAA) domain and coiled-coil (CC) domain, and the fragment of amino acids 111–470 representing the GTP binding domain (GBD) and RNA binding domain (RBD). b Analysis of in vitro interaction between NAP1;1 and NSN1 with or without a domain deletion using the yeast two-hybrid system. The full length NSN1 was cloned into pBD vector as bait to screen a library of flower from ABRC (upper panel). A positive interaction colony was confirmed to contain AtNAP1;1 (At4g26110.2) by DNA sequencing. pAD-NAP1;1 represents the full length CDS of AtNAP1;1 cloned into pAD vector. The upper panel displays the interaction between AtNAP1;1 and NSN1; The lower panel shows the interactions between AtNAP1;1 and the N-terminal fragment of NSN1 including the basic amino acid (BAA) domain and coiled-coil (CC) domain, which was abbreviated as BC. Empty vectors pBD and pAD were used as negative controls. X-gal overlay assays and the quantification of ß-galactosidase activity were shown (right panels). Significant difference (p < 0.001) was evaluated by Student’st test. c Analysis of in vivo interaction between NSN1 and NAP1;1 using biomolecule fluorescence complementation (BiFC) in tobacco leaves. An enhanced GFP (eGFP) was used  to test the interaction by fusing the coding sequence of NSN1 and N C AtNAP1;1 with the N-terminal (YFP -NSN1) and the C-terminal fragment of GFP (YFP -NAP1;1), respectively. The two constructs were co-expressed in tobacco leaves by Agrobacterium-infiltration. Infiltrated leaf epidermal cells were checked with confocal laser scanning. The co-expression of YFP -NSN1 and no-fusion pSPYCE (M) 155 (YC) was used as a negative control. Nuclei were stained with DAPI. Scale bars: 10 μm [14, 16]. In contrast, no YFP signal was detected when Arabidopsis NSN1 is involved in plant growth and de- YFP -NSN1 was co-expressed with no-fusion pSPYCE velopment by maintaining proper cell cycle progression. (M) 155 (YC), as the control, in tobacco cells (Fig. 6c). It has been documented that in animals when NS is de- These results demonstrated that NSN1 interacted in vivo leted or overexpressed, cell cycling is arrested and with AtNAP1;1, one of AtNAP1 proteins functioning as a the transition of G1-S and G2-M is stopped [3, 5, 6]. histone chaperone in nucleosome assembly/disassembly. Given that the Arabidopsis loss-of-function mutant nsn1 exhibits dwarfism and NSN1 is localized primarily in the nucleolus where the key features of cell growth Discussion occur, NSN1 may affect plant cell cycle progression. Plant Nucleostemin homologs have been identified in re- This view is supported by the molecular evidence of a cent years from Arabidopsis and tobacco [14, 15]. It has uniform down-regulation of core cell cycling genes, been reported that Arabidopsis NSN1 plays critical roles such as CYCA2;3, CYCB1;1, CYCD3;1, HISTONE H4 in floral meristem development, floral organ identity, em- and RNR,in nsn1 (Fig. 4a). In addition to controlling bryogenesis, leaf development and senescence [14–16]. cell-cycle transition, the core cell cycling components The aim of this work was to functionally characterize have been documented to function in coordinating NSN1 in cell cycle regulation in Arabidopsis. Based on cell division with differentiation and development in the molecular and genetic analysis of the cell cycling plants [36, 37]. The observations of higher percent- components and the dynamics of cell proliferation in ages of polyploidy, severely reduced numbers of CYC- nsn1 mutant, we concluded that NSN1 is indispensable LIN B1;1::GUS-expressing cells and slower cell for correct cell proliferation control. The identification proliferation in nsn1 suggest the deficiency in cell div- of AtNAP1;1, a histone chaperone, as an interacting ision. Consequently, growth retardation was exhibited partner of NSN1 implies that the two proteins might in both leaves and roots of nsn1, and has also function in a nucleolar complex to regulate cell cycle been observed in NbNSN1 silencing tobacco plants as progression. well . These results provided genetic and Wang et al. BMC Plant Biology (2018) 18:99 Page 9 of 12 molecular evidence that like its homolog in tobacco, or simultaneously barely cause visible phenotypes under Arabidopsis NSN1 regulates cell cycling. Therefore, standard laboratory conditions [23, 26], indicating the besides its function in embryogenesis and the devel- dispensability of these genes for plant growth and devel- opment of leaf and flower organs [15, 16], the nuclear opment under normal conditions. When exposed to protein NSN1 is indispensable for maintaining proper bleomysin or UV treatment, nsn1 and Atnap1 mutants cell proliferation in Arabidopsis. including single, double, triple and quadruple ones, to- It appears that nucleosome assembly protein AtNAP1; gether with Atnrp1 and Atnrp2, exhibit hypersensitivity 1 and NSN1 may function together as subunits of a to the genotoxic agents [18, 26, 38]. The similar re- functional protein entity. This view is supported by the sponse of NSN1 and AtNAP1s to genotoxic treatment facts that the two proteins share several common fea- suggests their involvement in the DNA-damage repair tures, including involvement in cell cycle progression, process although the functional mechanism is unclear. nucleus-localization and the resemblance of their mu- It has been reported that in mammals NSN1 forms a tants in response to genotoxics. As conserved histone large protein complex with DDX21, EBNA1 binding pro- chaperones, plant NAP1s and NRPs have been docu- tein 2 (EBP2), Pescadillo (PES), and a subset of riboso- mented in both monocot and dicot species [18, 19, 22, mal proteins . In Arabidopsis, AtEBP2 and AtPES, the 23, 26, 38]. In Arabidopsis, for example, overexpression orthologs of EBP2 and PES, respectively, have also of AtNAP1;1 increases the expression of CYCB1;1 and been found to interact with NSN1, especially its N- shortenes G2 phase, thereby promoting cell division terminal domains (1–174 amino acids) , suggesting . The double mutant nrp1-1 nrp2-1 exhibits arrested that NSN1 and its interacting partners regulate plant cell cycle progression at G2/M . In tobacco, NAP1 growth by modulating ribosome biogenesis. In search for genes have been observed with high expression levels at potential NSN1-interacting partners involved in cell the G1/S transition . Together with our findings that cycling regulation, AtNAP1;1 (At4g26110.2) was identi- mutation of NSN1 caused defect of G1-S and G2-M fied by the yeast two-hybrid system (Fig. 6). In Arabi- transition, we concluded that both AtNAP1;1 and NSN1 dopsis, there are four AtNAP1 family members play roles in the regulation of cell cycle progression. (AtNAP1;1, AtNAP1;2, AtNAP1;3 and AtNAP1;4) and Supportively, intracellular localization analysis of two NRP proteins (AtNRP1 and AtNRP2) [18, 38], clus- NSN1 and AtNAP1;1 has shown that both proteins res- tering in two clades in the phylogenetic tree (Add- ide in the nucleus [14, 15, 18]. The result that the N- itional file 1: Figure S1a). In AtNAP1 subgroup, terminal domain of NSN1 primarily determined its AtNAP1;1 shares a sequence identity of 80.3, 72.4 and localization [14, 15] is in agreement with the findings of 46.4% with AtNAP1;3, AtNAP1;2 and AtNAP1;4, re- mammalian NS, whose nucleolar localization is specified spectively (Additional file 2: Table S1). The interaction by the N-terminal basic domain . Based on our find- of AtNAP1;1 with NSN1 might somehow attribute to ings that the N-terminal domain of NSN1 was essential the divergent C-terminus of AtNAP1;1 (Additional file for its interplay with AtNAP1;1, we postulate that the 1: Figure S1b). Another possible reason why AtNAP1;1 two proteins interact in the nuclei. This postulation was was singled out from the screening might be that the supported by the BiFC experiment showing in vivo inter- coverage of the flower library used as pray in this experi- action between NSN1 and AtNAP1;1 in the nuclei of to- ment was not sufficient to contain low expression genes bacco leaves. Interestingly, like NS in mammals [11, 13], such as AtNAP1;3, whose expression level in flower is AtNAP1;1 is localized in both the nucleus and the cyto- about half of AtNAP1;1 (Additional file 1: Figure S1c) plasm, and the protein appears to shuttle between the according to the data from Arabidopsis eFP Browser two organelles . Studies of gain- or loss-of-function (http://bar.utoronto.ca/efp_arabidopsis). Further in- mutants have revealed that when localized to the nu- vestigation by deleting the domain(s) of AtNAP1;1 will cleus, AtNAP1;1 promotes cell division during the early provide information on the domain(s) of AtNAP1;1 that stage of leaf development . The co-localization of is required for interaction with NSN1. Future study will NSN1 and AtNAP1;1 in the nucleus (Fig. 6) provides focus on the nsn1 Atnap1;1 double mutant to unveil the new evidence that both proteins may function as sub- molecular functions of NSN1 and AtNAP1;1 in regulat- units of a complex in cell cycling regulation. Further in- ing plant growth and development as interacting vestigation of the shuttling mechanism of the two partners. proteins will provide valuable information for the func- tional characterization. Additionally, the phenotypic features of Atnap1;1 are Conclusions highly reminiscent of those of nsn1 upon subjected to The biological functions of Arabidopsis NSN1 in main- UV or other DNA damage agents. AtNAP1;1 belongs to taining proper cell cycle progression were characterized a multi-gene family. Mutations of AtNAP1s individually by molecular and genetic approaches. Our results Wang et al. BMC Plant Biology (2018) 18:99 Page 10 of 12 provide direct evidence that the dwarfism of nsn1 is re- TAG TTC TGA AAG G-3′. The PCR product was sulted from the improper cell proliferation in the meri- cloned into vector pCR2.1, and then sub-cloned into stems of both shoot and roots of the mutant plant. As a pSPYCE (M) 155. positive cell cycle regulator, Arabidopsis NSN1 is co-lo- For infiltration of tobacco leaf, the two constructs were calized and interacts with AtNAP1;1, a nucleosome as- transformed into Agrobacterium tumefaciens GV3101 sembly protein, in the nucleus. The two proteins are and infiltration was carried out as described . involved in regulation of cell cycle progression and their mutants, nsn1 and Atnap1;1, are hypersensitive to treat- Scanning electronic microscopy (SEM) and microscopy ments of genotoxic agents. We propose that NSN1 and SEM was carried out as described . Briefly, plant tis- AtNAP1;1 act together as subunits of a functional protein sues were fixed overnight in 50 mM phosphate buffer complex in regulation of cell cycling progression. Hence, containing 2% glutaraldehyde and 2% paraformaldehyde. like its homologs in mammals, NSN1 in Arabidopsis Fixed tissues were rinsed three times with 50 mM phos- functions conservatively in regulating plant cell cycling. phate buffer and kept in osmium tetroxide (1%) at 4 °C This study sheds new light on the crosstalk between overnight. After being rinsed three times with 50 mM NSN1 and AtNAP1;1 in cell cycle regulation in plants. phosphate buffer, the tissues were dehydrated in a 30, 50, 70, 95 and 100% alcohol gradient. Dehydrated tissues Methods were coated with gold and observed under SEM. Plant materials and growth conditions Leaf initiation was defined as described . For meas- The nsn1-1 T-DNA insertion line (SALK_029201) urement of leaf area, the dissected fifth leaf was pictured was obtained from the Arabidopsis Biological Resource daily and leaf area was measured using ImageJ. Cell Center (ABRC) , and the CYCLIN B1::GUS express- number per leaf was calculated as leaf area divided by ing plant was a gift from Prof. Peter Doerner (University cell area. All dissected leaves were treated with chloral of Edinburg, UK). Plants were grown in soil under nor- hydrate for 3 days. After clearing, photos of palisade mal conditions with 16 h light/8 h darkness at 21 °C. cells were taken using Zeiss microscope with DIC lens. For individual leaf, area of 10 palisade cells was mea- Plant treatment sured using ImageJ and an average area per palisade cell Sterilized Arabidopsis (Col-0 and nsn1) seeds were was calculated accordingly. For each time point, the fifth treated at 4 °C for 2 days before moving to normal con- leaf from 5 plants was measured and three biological ditions for germination on half strength MS plates. replicates were conducted. Plates were placed vertically during seed germination For root length, 70 sterilized Arabidopsis seeds of wild and 4 days after germination, seedlings were transferred type and nsn1 were germinated on 1/2 MS medium with to half strength MS plates (control) or half strengh 0.8% agar. Root tip was marked for daily image capture MS plates supplemented with methyl methanesulfonate and root length was measured by ImageJ. For analysing (MMS) at concentrations of 25, 50, 75 and 100 ppm, or the cell production per day, 4 days after germination, with bleomycin at 3, 6, 9, and 12 mg/L. Images were ten primary root tips grown during the last 24 h were captured and survival rates were recorded after geno- cut and mounted daily on slide using buffer (chloral hy- toxic treatment with MMS and bleomycin for one and drate: glycerol: water = 8:3:1). DIC optics of a Zeiss con- 3 weeks, respectively. focal microscope (Zeiss Axioskop, Germany) was used for image capture. The number of cortex cells on one Constructs and Agrobacterium-mediated transient side of a root tip was counted. expression in N. benthamiana For the biomolecule fluoresence complementation Quantitative RT-PCR (BiFC) experiment, the coding region of NSN1 cDNA Total RNA was isolated from 10-day-old seedlings of was amplified by RT-PCR using primers pBD-NSN F: wild type and nsn1 using the RNeasy plant kit followed 5′-GTC GAC AGA TGG TGA AAC GGA GTA AAA by treatment with RNase-free DNase I according to the AGA G-3′ and pBD-NSN R: 5′-GTC GAC TTT TTC manufacturer instructions (Qiagen, Germantown, MD). TTC GGC AAA AGT CCA G-3′, and cloned into vec- RNA of 2 μg was used as template for first strand cDNA tor pCR2.1 (Invitrogen). To express YFP -NSN1, the synthesis using SuperScrit III (Invitrogen). cDNA was di- NSN1 cDNA fragment was ligated into vector pSPYNE luted 100 times and 5 μl of cDNA was used as PCR tem- (R) 173  after digestion by Sal I. For construction of plate. Real-time PCR was performed using a mix NAP1;1-cYFP, the NAP1;1 (At4g26110.2) coding region containing 10 μl of 2× SYBR Premix Ex Taq (Takara Bio was amplified by RT-PCR using primers pNAP1;1 F: 5′- Inc., Otsu, Shiga, Japan), 0.8 μl of forward and reverse GAA TTC ATG AGC AAC GAC AAG GAT AGC -3′ primer mix (0.2 μM final concentration), 0.4 μlof 50 and pNAP1;1 R: 5′- GTC GAC ACA AAT AAA CTT ROX Reference Dye II, and 3.8 μl of deionized water. Wang et al. BMC Plant Biology (2018) 18:99 Page 11 of 12 PCR was run on an ABI 7500 fast real time PCR system LacZ). The full-length CDS of AtNAP1;1 (At4g26110.2) (Applied Biosystems) using a 2-min initial denaturation cloned into pAD-Gal4-2.1 was amplified with primers at 95 °C, followed by 40 cycles of 95 °C (15 s) and 60 °C (pNAP1;1 F: 5′-GAA TTC ATG AGC AAC GAC AAG (40 s). Primers used for qRT-PCR were as follows: for GAT AGC-3′; pAD-NAP1;1 R: 5′-CTC GAG AAT AAA CYCA2;3 (F: 5′-GGC TAA GAA GCG ACC TGA TG- CTT TAG TTC TGA AAG -3′). Cells of the two yeast 3′ and R: 5′-TAC AAG CCA CAC CAA GCA AC-3′); strains were mated and selected on a plate supplemented for CYCB1;1 (F: 5′-AAG CTT CCA TTG CAG ACG A- with Glu-Trp-Leu-Ade. Yeast colonies were further se- 3′ and R: 5′-AGC AGA TTC AGT TCC GGT C-3′); for lected for growth on an Ade Selection Plate (Glc-Ade- CYCD3;1 (F: 5′-ACA ACT CTC GTG CAT TAA CAG Trp-Leu + His). For X-gal overlay assay, agarose solution GAA-3′ and R: 5′-GAA GAT TGG ATT TGG ATC containing 1 mg/ml 5-bromo-4-chloro-3-indoly-galacto- TGT AAA C-3′); for H4 (F: 5′-TTA GGC AAA GGA pyranoside (X-Gal) in Z buffer was brought to 55 C and GGA GCA AA-3′ and R: 5′-CTC CTC GCA TGC TCA poured to the cooled yeast colonies grown on the Ade GTG TA-3′); and for RNR (F: 5′- CAA GTG GCT CAG selection plates. GAC TGT CA-3′ and R: 5′-TCC ATC AGG TCA ACA GCT TG-3′). Actin2 (At3g18780) was used as an in- Quantification of Y2H ß-Galactosidase assay ternal control (F: 5′-TGG TGT CAT GGT TGG GAT ß-Galactosidase activity was measured using Y2H ß- G-3′ and R: 5′-CAC CAC TGA GCA CAA TGT TAC-3′). Galactosidase Kit (Molecular Biotechnology). Briefly, The relative expression level was calculated based on several yeast colonies picked from selection plate the value of ΔΔCt. were inoculated into 1 ml of the appropriate selective medium andculturedto the late exponentialphase. Flow cytometry Yeast cells were collected and resuspended in lysis Flow cytometry was carried out as described  mixture containing Dye solution. Color development with minor modifications. Briefly, leaves (4-days was monitored by spectrophotometer at 615 nm. after initiation or fully-expended leaves) were Three independent biological replicates were per- chopped with razor blade into fine strips in cold formed and difference was evaluated by Student’st nuclei isolation buffer (Partec, Müster, Germany). test. After filtration, the extracts were kept on ice until measurement. The DNA content of nuclei was mea- Additional files sured using FACS Caliber flow cytometry (BD Bio- sciences, USA). Cell nuclei were stained with Additional file 1: Figure S1. Sequence analysis and expression profiles − 1 2 μgmL DAPI. Each sample was prepared three of NAP1 family members. a. Homology analysis of AtNAP1 and AtNRP times and subjected to FACS Caliber cytometry proteins using DNAMAN version 7. Sequence accession number: AtNAP1;1 (AT4G26110.2); AtNAP1;2 (AT2G19480); AtNAP1;3 (AT5G56950); AtNAP1;4 independently. A total of around 10,000 nuclei were (AT3G13782); AtNRP1 (AT1G74560) and AtNRP2 (AT1G18800). b.Sequence measured per analysis. alignment of AtNAP1s and AtNRPs. Black represents conserved amino acids (consensus), pink for 75% identity, blue for 50% and yellow for 33% identity. c. Comparison of the transcriptional expression pattern of AtNAP1 paralog Yeast two-hybrid assay genes in flower from Arabidopsis eFP Browser (http://bar.utoronto.ca/ Yeast two-hybrid experiments were carried out as de- efp_arabidopsis). (JPG 3822 kb) scribed (Stratagene, USA). ABRC stock CD4-30, an Ara- Additional file 2: Table S1. Analysis of protein identity among AtNRP1s bidopsis cDNA library of inflorescence meristem, floral and AtNRPs. (PDF 98 kb) meristem and floral buds up to stage 8, was used as pray. The cDNA library was cloned into the EcoR I – Xho I Abbreviations site of pAD-Gal4-2.1 (Stratagene), and transformed into DAG: Days after germination; DAI: Days after initiation; GUS: β-glucuronidase; MDM2: Murine Double Minute 2; NAP1: NUCLEOSOME ASSEMBLY yeast strain PJ69-4a (his, leu, ura; Gal1-HIS3, Gal2- PROTEINS1; NRPs: NAP1-RELATED PROTEINs; NS: Nucleostemin; ADE2, Gal7-LacZ). The full length NSN1 coding region NSN: Nucleostemin-like and the fragment corresponding to the BC domain were amplified using primers pBD-NSN (F: 5′-GTC GAC Acknowledgements We thank Prof. Peter Doerner (University of Edinburg, UK) for providing the AGA TGG TGA AAC GGA GTA AAA AGA G-3′;R: seeds of CYCLIN B1::GUS expressing plant and Prof. Changbin Chen (University 5′-GTC GAC TTT TTC TTC GGC AAA AGT CCA G of Minnesota, Saint Paul, USA) for critically reading the manuscript. -3′) and pBD-BC (F: 5′-GTC GAC AGA TGG TGA AAC GGA GTA AAA AGA G-3′;R: 5′-GTC GAC CTC Funding TTC ATG CTT ATT GGG ACC GGC-3′), respectively. This work was financially supported by NSF grants (MCB 0548525 to ZH) and by the Agricultural Science and Technology Innovation Program from China The two fragments were cloned into pBD-GAL4 (Cam) (ASTIP-IAS14 to QY). The funding bodies did not play a role in the design of and used as bait after transformation into yeast strain the study and collection, analysis, or interpretation of data and in writing the PJ69-4α (his, leu, ura; Gal1-HIS3, Gal2-ADE2, Gal7- manuscript. Wang et al. BMC Plant Biology (2018) 18:99 Page 12 of 12 Availability of data and materials nucleosome assembly protein 1 (NAP-1) from soybean. Mol Gen Genet. The datasets used and/or analyzed during the current study are available 1995;249(5):465–73. from the corresponding author on request. 20. Park YJ, Luger K. The structure of nucleosome assembly protein 1. Proc Natl Acad Sci U S A. 2006;103(5):1248–53. Authors’ contributions 21. Lankenau S, Barnickel T, Marhold J, Lyko F, Mechler BM, Lankenau DH. ZH, XW, ZW and QCY designed the research; XW, ZW and BX performed Knockout targeting of the Drosophila nap1 gene and examination of DNA experiments, and XW and ZW wrote the article. All authors read and repair tracts in the recombination products. Genetics. 2003;163(2):611–23. approved the final manuscript. 22. Dong A, Zhu Y, Yu Y, Cao K, Sun C, Shen WH. Regulation of biosynthesis and intracellular localization of rice and tobacco homologues of Ethics approval and consent to participate nucleosome assembly protein 1. Planta. 2003;216(4):561–70. Not applicable 23. Liu ZQ, Gao J, Dong AW, Shen WH. A truncated Arabidopsis NUCLEOSOME ASSEMBLY PROTEIN 1, AtNAP1;3T, alters plant growth responses to abscisic Competing interests acid and salt in the Atnap1;3-2 mutant. Mol Plant. 2009;2(4):688–99. The authors declare that they have no competing interests. 24. Galichet A, Gruissem W. Developmentally controlled farnesylation modulates AtNAP1;1 function in cell proliferation and cell expansion during Arabidopsis leaf development. Plant Physiol. 2006;142(4):1412–26. Publisher’sNote 25. Son O, Kim S, Shin YJ, Kim WY, Koh HJ, Cheon CI. 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