Improving the Traits of Perilla frutescens (L.) Britt Using Gene Editing Technology.

Plant breeding has evolved significantly over time with the development of transformation and genome editing techniques. These new strategies help to improve desirable traits in plants. Perilla is a native oil crop grown in Korea. The leaves contain many secondary metabolites related to whitening, aging, antioxidants, and immunity, including rosmarinic acid, vitamin E, luteolin, anthocyanins, and beta-carotene. They are used as healthy and functional food ingredients. It is an industrially valuable cosmetics crop. In addition, perilla seeds are rich in polyunsaturated fatty acids, such as α-linolenic acid and linoleic acid. They are known to be effective in improving neutral lipids in the blood, improving blood circulation, and preventing dementia and cardiovascular diseases, making them excellent crops whose value can be increased through improved traits. This research will also benefit perilla seeds, which can increase their stock through various methods, such as the increased production of functional substances and improved productivity. Recently, significant attention has been paid to trait improvement research involving gene-editing technology. Among these strategies, CRISPR/Cas9 is highly adaptable, enabling accurate and efficient genome editing, targeted mutagenesis, gene knockouts, and the regulation of gene transcription. CRISPR/Cas9-based genome editing has enormous potential for improving perilla; however, the regulation of genome editing is still at an early stage. Therefore, this review summarizes the enhancement of perilla traits using genome editing technology and outlines future directions.

Overexpression of R2R3-MYB IbMYB1a induces anthocyanin pigmentation in soybean cotyledon.

KEY MESSAGE: This is the first report showing anthocyanin accumulation in the soybean cotyledon via genetic transformation of a single gene. Soybean [Glycine max (L.) Merrill] contains valuable components, including anthocyanins. To enhance anthocyanin production in Korean soybean Kwangankong, we utilized the R2R3-type MYB gene (IbMYB1a), known for inducing anthocyanin pigmentation in Arabidopsis. This gene was incorporated into constructs using two promoters: the CaMV 35S promoter (P35S) and the ß-conglycinin promoter (Pß-con). Kwangankong was transformed using Agrobacterium, and the presence of IbMYB1a and Bar transgenes in T0 plants was confirmed through polymerase chain reaction (PCR), followed by gene expression validation. Visual inspection revealed that one P35S:IbMYB1a and three Pß-con:IbMYB1a lines displayed seed color change. Pß-con:IbMYB1a T1 seeds accumulated anthocyanins in cotyledon outer layers, whereas P35S:IbMYB1a and non-transgenic black soybean (Cheongja 5 and Seum) accumulated anthocyanins in the seed coat. During the germination and growth phase, T1 seedlings from Pß-con:IbMYB1a lines exhibited anthocyanin pigmentation in cotyledons for up to 1 month without growth aberrations. High-performance liquid chromatography confirmed cyanidin-3-O-glucoside as the major anthocyanin in the Pß-con:IbMYB1a line (#3). We analyzed the expression patterns of anthocyanin biosynthesis genes, chalcone synthase 7,8, chalcone isomerase 1A, flavanone 3-hydroxylase, flavanone 3'-hydroxylase, dihydroflavanol reductase 1, dihydroflavanol reductase 2, anthocyanidin synthase 2, anthocyanidin synthase 3, and UDP glucose flavonoid 3-O-glucosyltransferase in transgenic and control Kwangankong and black soybean (Cheongja 5 and Seum) seeds using quantitative real-time PCR. We conclude that the induction of gene expression in transgenic plants in comparison with Kwangankong was attributable to IbMYB1a transformation. Notably, flavanone 3-hydroxylase, flavanone 3'-hydroxylase, and dihydroflavanol reductase 1 were abundantly expressed in black soybean seed coat, distinguishing them from transgenic cotyledons.

Marine plant-based biorefinery for sustainable 2,5-furandicarboxylic acid production: A review.

Marine plants, including macroalgae and seagrass, show promise as biorenewable feedstocks for sustainable chemical manufacturing. This study explores their potential in producing 2,5-furandicarboxylic acid (FDCA), a versatile platform chemical for commodity polymers. FDCA-based polyethylene 2,5-furandicarboxylate offers a sustainable alternative to petroleum-derived polyethylene terephthalate, commonly used in plastic bottles. Our research pioneers the concept of a marine plant-based FDCA biorefinery, introducing innovative approaches for sustainability and cost-effectiveness. This review outlines the use of ionic liquid-based solvents (ILS) and deep eutectic solvent (DES) systems in FDCA production. Additionally, we propose biomodification strategies involving target enzyme-encoding genes to enhance the depolymerization of non-structural storage glucans in marine plants. Our findings pave the way for eco-friendly biorefineries and biorenewable plastics.

Genome-edited HEADING DATE 3a knockout enhances leaf production in Perilla frutescens.

Environmental cues regulate the transition of many plants from vegetative to flowering development. Day length, or photoperiod, is one cue that synchronizes flowering by changing seasons. Consequently, the molecular mechanism of flowering control is prominent in Arabidopsis and rice, where essential genes like FLOWERING LOCUS T (FT) homolog, HEADING DATE 3a (Hd3a), have been connected to flowering regulation. Perilla is a nutrient-rich leaf vegetable, and the flowering mechanism remains largely elusive. We identified flowering-related genes under short-day conditions using RNA sequencing to develop an enhanced leaf production trait using the flowering mechanism in the perilla. Initially, an Hd3a-like gene was cloned from the perilla and defined as PfHd3a. Furthermore, PfHd3a is highly rhythmically expressed in mature leaves under short-day and long-day conditions. Ectopic expression of PfHd3a in Atft-1 mutant plants has been shown to complement Arabidopsis FT function, resulting in early flowering. In addition, our genetic approaches revealed that overexpression of PfHd3a in perilla caused early flowering. In contrast, the CRISPR/Cas9 generated PfHd3a-mutant perilla showed significantly late flowering, resulting in approximately 50% leaf production enhancement compared to the control. Our results suggest that PfHd3a plays a vital role in regulating flowering in the perilla and is a potential target for molecular breeding in the perilla.

Strategic biomodification for raw plant-based pretreatment biorefining toward sustainable chemistry.

Plant-based pretreatment biorefining is the initial triggering process in biomass-conversion to bio-based chemical products. In view of chemical sustainability, the raw plant-based pretreatment biorefining process is more favorable than the fossil-based one. Its direct use contributes to reducing CO2 emissions and the production cost of the target products by eliminating costly steps, such as the separation and purification of intermediates. Three types of feedstock plant resources have been utilized as raw plant feedstock sources, such as: lignocellulosic, starchy, and inulin-rich feedstock plants. These plant sources can be directly used for bio-based chemical products. To enhance the efficiency of their pretreatment biorefining process, well-designed biomodification schemes are discussed in this review to afford important information on useful biomodification approaches. For lignocellulosic feedstock plants, the enzymes and regulatory elements involved in lignin reduction are discussed using: COMT, GAUT4, CSE, PvMYB4 repressor, etc. For inulin-rich feedstock plants, 1-SST, 1-FFT, 1-FEH, and endoinulinase are illustrated in relation with the reduction of chain length of inulin polymer. For starchy feedstock plants, their biomodification is targeted to enhancing the depolymerization efficiency of starch to glucose monomer units. For this biomodification target, six candidates are discussed. These are SBE I, SBE IIa, SBE IIb, GBSS I, PTSTI, GWD 1, and PTSTI. The biomodification strategies discussed here promise to be conducive to enhancing the efficiency of the plant-based pretreatment biorefining process.

Conversion of inulin-rich raw plant biomass to 2,5-furandicarboxylic acid (FDCA): Progress and challenge towards biorenewable plastics.

The current commercial plastic manufactures have been produced using petroleum-based resource. However, due to concerns over the resource depletion and the environmental sustainability, bioresource-based manufacturing processes have been developed to cope against these concerns. Bioresource-derived 2,5-furandicarboxylic acid (FDCA) can be utilized as a building block material for plastic manufactures. To date, numerous technologies have been developed for the production of FDCA using various types of bio-based feedstocks such as hydroxymethylfurfural (HMF), 6-C sugars, and polysaccharides. The commercial companies produce FDCA using HMF-based production processes due to their high production efficiency, but the high price of HMF is a problem bottleneck. Our review affords important information on breakthrough approaches for the cost-efficient and sustainable production of FDCA using raw plant feedstocks rich in inulin. These approaches include bioprocessing technology based on the direct use of raw plant feedstocks and biomodification of the target plant sources. For the former, an ionic liquid-based processing system is proposed for efficient pretreatment of raw plant feedstocks. For the latter, the genes encoding the key enzymes; sucrose:sucrose 1-fructoyltransferase (1-SST), fructan:fructan 1-fryuctosyltransferase (1-FFT), fructan 1-exohydrolase (1-FEH), and microbe-derived endoinulinase, are introduced for biomodification conducive to facilitating bioprocess and improving inulin content. These approaches would contribute to cost-efficiently and sustainably producing bio-based FDCA.

Seagrass-based platform strategies for sustainable hydroxymethylfurfural (HMF) production: toward bio-based chemical products.

Today, sustainable chemistry is a key trend in the chemical manufacturing industry due mainly to concerns over the global environment and resource security. In sustainable chemical manufacture, the choice of a bio-based feedstock plays a pivotal pillar. In terms of feedstock utilization for producing HMF, which is a multivalent platform intermediate easily convertible to valuable chemical products; biopolymers, biofuels, and other important chemicals, seagrass biomasses can be more favorable feedstocks compared with land plant resources due primarily to easy availability and no systematic farming. Moreover, seagrass feedstocks could contribute cost-effectively and sustainably producing HMF by exploiting the beach-cast seagrasses on seagrass-prairies with no feedstock cost, indicating that seagrass biomasses could be a most promising biofeedstock source for sustainable HMF production. We afford a platform bioprocessing technology that has not been attempted before for sustainable HMF production using raw seagrass biomass. This bioprocess can be operated by simple reaction conditions using inorganic Brønsted acids (mainly HCl) and ionic liquid solvents at relatively low temperatures (120-130 °C). In addition, some bioengineering strategies for improving the growth of seagrass biomass and the quantity/quality of nonstructural carbohydrates (starch, sucrose) that can be used as the feeding substrates for HMF production are also discussed. The main aim of this review is to provide some important information about breakthrough bio/technologies conducive to cost-effective and sustainable HMF production.

Nuclear import of LIKE HETEROCHROMATIN PROTEIN1 is redundantly mediated by importins α-1, α-2 and α-3.

LIKE HETEROCHROMATIN PROTEIN1 (LHP1) encodes the only plant homologue of the metazoan HETEROCHROMATIN PROTEIN1 (HP1) protein family. The LHP1 protein is necessary for proper epigenetic regulation of a range of developmental processes in plants. LHP1 is a transcriptional repressor of flowering-related genes, such as FLOWERING LOCUS T (FT), FLOWERING LOCUS C (FLC), AGAMOUS (AG) and APETALA 3 (AP3). We found that LHP1 interacts with importin α-1 (IMPα-1), importin α-2 (IMPα-2) and importin α-3 (IMPα-3) both in vitro and in vivo. A genetic approach revealed that triple mutation of impα-1, impα-2 and impα-3 resulted in Arabidopsis plants with a rapid flowering phenotype similar to that of plants with mutations in lhp1 due to the upregulation of FT expression. Nuclear targeting of LHP1 was severely impaired in the impα triple mutant, resulting in the de-repression of LHP1 target genes AG, AP3 and SHATTERPROOF 1 as well as FT. Therefore, the importin proteins IMPα-1, -2 and -3 are necessary for the nuclear import of LHP1.

Toward Sustainable Hydroxymethylfurfural Production Using Seaweeds.

Chemical manufacturing involves carbon sources releasing CO2 into the atmosphere. By contrast, seaweeds are carbon sinks that can absorb released CO2 and therefore have great potential for use as feedstocks in sustainable chemical manufacturing. In particular, seaweeds could contribute to mitigating vast amounts of global CO2 emissions. Accordingly, seaweeds could be an excellent candidate biomaterial for sustainable production of hydroxymethylfurfural (HMF), called a 'sleeping giant' platform chemical due to its wide versatility in chemical manufacturing. HMF is produced through sugar dehydration mechanisms, and seaweed storage glucans comprised of glucose can be appropriate feeding substrates for its production. This opinion article introduces a new opportunity for sustainable production of HMF using storage glucan-rich seaweeds.

Strigolactone Signaling Genes Showing Differential Expression Patterns in Arabidopsis max Mutants.

Strigolactone (SL) is a recently discovered class of phytohormone that inhibits shoot branching. The molecular mechanism underlying SL biosynthesis, perception, and signal transduction is vital to the plant branching phenotype. Some aspects of their biosynthesis, perception, and signaling include the role of four MORE AXILLARY GROWTH genes, MAX3, MAX4, MAX1, and MAX2. It is important to identify downstream genes that are involved in SL signaling. To achieve this, we studied the genomic aspects of the strigolactone biosynthesis pathway using microarray analysis of four max mutants. We identified SL signaling candidate genes that showed differential expression patterns in max mutants. More specifically, 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE 4 (ACC4) and PROTEIN KINASE 3 (PKS3) displayed contrasting expression patterns, indicating a regulatory mechanism in SL signaling pathway to control different phenotypes apart from branching phenotype.

Raw plant-based biorefinery: A new paradigm shift towards biotechnological approach to sustainable manufacturing of HMF.

Unlike petrorefinery, biorefinery uses carbon-based biomaterials, such as plant feedstocks, as the major feeding input materials in chemical manufacturing. To date, petroleum-based resources have been used for the production of wide spectrums of chemical products. However, petrorefinery is currently associated with a variety of issues, i.e., concerns over adverse impacts on the environment and human society. As an alternative technology, the sustainable biorefinery is a matter of great importance in industrial chemical manufacturing due primarily to its sustainability. As carbon-based resources, plants are paramount biomaterials for biorefinery process required in sustainable chemical manufacturing. In particular, raw plant-based biorefinery is a breakthrough technology for chemical manufacturing due mainly to its sustainable benefits. Nowadays, numerous biorefinery technologies have been developed for the production of industrially valuable chemicals. HMF, a versatile platform chemical, can be produced by dehydrating hexose sugars using raw plant feedstocks such as inulin-rich, starch-rich, and lignocellulosic plants and now, it is generally recognized as a chemical feedstock for future chemical manufacturing and bioenergy production. In this review article, this emerging hybrid technology is discussed in relation to the production of HMF from raw plant feedstocks mentioned above. In addition, the plant candidates useful for biorefinery processing of raw plant feedstocks are introduced and bioengineering strategy for their genetic modification is together described to provide current knowledge on sustainable biorefinery.

Interplay between ABA and GA Modulates the Timing of Asymmetric Cell Divisions in the Arabidopsis Root Ground Tissue.

In multicellular organisms, controlling the timing and extent of asymmetric cell divisions (ACDs) is crucial for correct patterning. During post-embryonic root development in Arabidopsis thaliana, ground tissue (GT) maturation involves an additional ACD of the endodermis, which generates two different tissues: the endodermis (inner) and the middle cortex (outer). It has been reported that the abscisic acid (ABA) and gibberellin (GA) pathways are involved in middle cortex (MC) formation. However, the molecular mechanisms underlying the interaction between ABA and GA during GT maturation remain largely unknown. Through transcriptome analyses, we identified a previously uncharacterized C2H2-type zinc finger gene, whose expression is regulated by GA and ABA, thus named GAZ (GA- AND ABA-RESPONSIVE ZINC FINGER). Seedlings ectopically overexpressing GAZ (GAZ-OX) were sensitive to ABA and GA during MC formation, whereas GAZ-SRDX and RNAi seedlings displayed opposite phenotypes. In addition, our results indicated that GAZ was involved in the transcriptional regulation of ABA and GA homeostasis. In agreement with previous studies that ABA and GA coordinate to control the timing of MC formation, we also confirmed the unique interplay between ABA and GA and identified factors and regulatory networks bridging the two hormone pathways during GT maturation of the Arabidopsis root.

Rice serine/threonine kinase 1 is required for the stimulation of OsNug2 GTPase activity.

Several GTPases are required for ribosome biogenesis and assembly. We recently identified rice (Oryza sativa) nuclear/nucleolar GTPase 2 (OsNug2), a YlqF/YawG family GTPase, as having a role in pre-60S ribosomal subunit maturation. To investigate the potential factors involved in regulating OsNug2 function, yeast two-hybrid screens were performed using OsNug2 as bait. Rice serine/threonine kinase 1 (OsSTK1) was identified as a candidate interacting protein. OsSTK1 appeared to interact with OsNug2 both in vitro and in vivo. OsSTK1 was found to have no effect on the GTP-binding activity of OsNug2; however, the presence of recombinant OsSTK1 in OsNug2 assay reaction mixtures increased OsNug2 GTPase activity. A kinase assay showed that OsSTK1 had weak autophosphorylation activity and strongly phosphorylated serine 209 of OsNug2. Using yeast complementation testing, we identified a GAL::OsNug2(S209N) mutation-harboring yeast strain that exhibited a growth-defective phenotype on galactose medium at 39°C, which was divergent from that of a yeast strain harboring GAL::OsNug2. The intrinsic GTPase activity of OsNug2(S209N), which was found to be similar to that of OsNug2, was not fully enhanced upon weak binding of OsSTK1. Our findings indicate that OsSTK1 functions as a positive regulator of OsNug2 by enhancing OsNug2 GTPase activity. In addition, phosphorylation of OsNug2 serine 209 is essential for its complete function in biological functional pathway.

A cold-adapted carbohydrate esterase from the oil-degrading marine Bacterium Microbulbifer thermotolerans DAU221: gene cloning, purification, and characterization.

A cold-adapted carbohydrate esterase, CEST, belonging to the carbohydrate esterase family 6, was cloned from Microbulbifer thermotolerans DAU221. CEST was composed of 307 amino acids with the first 22 serving as a secretion signal peptide. The calculated molecular mass and isoelectric point of the mature enzyme were 31,244 Da and pH 5.89, respectively. The catalytic triad consisted of residues Ser37, Glu192, and His281 in the conserved regions: GQSNMXG, QGEX(D/N), and DXXH. The three-dimensional structure of CEST revealed that CEST belongs to the α/ß-class of protein consisted of a central six-stranded ß-sheet flanked by eight α-helices. The recombinant CEST was purified by His-tag affinity chromatography and the characterization showed its optimal temperature and pH were 15°C and 8.0, respectively. Specifically, CEST maintained up to 70% of its enzyme activity when preincubated at 50°C or 60°C for 6 h, and 89% of its enzyme activity when preincubated at 70°C for 1h . The results suggest CEST belongs to group 3 of the cold-adapted enzymes. The enzyme activity was increased by Na(+) and Mg(2+) ions but was strongly inhibited by Cu(+) and Hg(2+) ions, at all ion concentrations. Using p-nitrophenyl acetate as a substrate, the enzyme had a Km of 0.278 mM and a kcat of 1.9 s(-1). Site-directed mutagenesis indicated that the catalytic triad (Ser37, Glu192, and His281) and Asp278 were essential for the enzyme activity.

Epigenetic regulation by long noncoding RNAs in plants.

Many eukaryotes, including plants, produce a large number of long noncoding RNAs (lncRNAs).Growing number of lncRNAs are being reported to have regulatory roles in various developmental processes.Emerging mechanisms underlying the function of lncRNAs indicate that lncRNAs are versatile regulatory molecules. They function as potent cis- and trans-regulators of gene expression, including the formation of modular scaffolds that recruit chromatin-modifying complexes to target chromatin. LncRNAs have also been reported in plants. Here, we describe our current understanding on potential roles of lncRNA in plants.

Rice Rab11 is required for JA-mediated defense signaling.

Rab proteins play an essential role in regulating vesicular transport in eukaryotic cells. Previously, we characterized OsRab11, which in concert with OsGAP1 and OsGDI3 regulates vesicular trafficking from the trans-Golgi network (TGN) to the plasma membrane or vacuole. To further elucidate the physiological function of OsRab11 in plants, we performed yeast two-hybrid screens using OsRab11 as bait. OsOPR8 was isolated and shown to interact with OsRab11. A co-immunoprecipitation assay confirmed this interaction. The green fluorescent protein-OsOPR8 fusion product was targeted to the cytoplasm and peroxisomes of protoplasts from Arabidopsis thaliana. OsOPR8 exhibited NADPH-dependent reduction activity when 2-cyclohexen-1-one (CyHE) and 12-oxo-phytodienoic acid (OPDA) were supplied as possible substrates. Interestingly, NADPH oxidation by OsOPR8 was increased when wild-type OsRab11 or the constitutively active form of OsRab11 (Q78L) were included in the reaction mix, but not when the dominant negative form of OsRab11 (S28N) was included. OsRab11 was expressed broadly in plants and both OsRab11 and OsOPR8 were induced by jasmonic acid (JA) and elicitor treatments. Overexpressed OsRab11 transgenic plants showed resistance to pathogens through induced expression of JA-responsive genes. In conclusion, OsRab11 may be required for JA-mediated defense signaling by activating the reducing activity of OsOPR8.

Ca2+-dependent GTPase, extra-large G protein 2 (XLG2), promotes activation of DNA-binding protein related to vernalization 1 (RTV1), leading to activation of floral integrator genes and early flowering in Arabidopsis.

Heterotrimeric G proteins, consisting of Gα, Gß, and Gγ subunits, play important roles in plant development and cell signaling. In Arabidopsis, in addition to one prototypical G protein α subunit, GPA1, there are three extra-large G proteins, XLG1, XLG2, and XLG3, of largely unknown function. Each extra-large G (XLG) protein has a C-terminal Gα-like region and a ∼400 amino acid N-terminal extension. Here we show that the three XLG proteins specifically bind and hydrolyze GTP, despite the fact that these plant-specific proteins lack key conserved amino acid residues important for GTP binding and hydrolysis of GTP in mammalian Gα proteins. Moreover, unlike other known Gα proteins, these activities require Ca(2+) instead of Mg(2+) as a cofactor. Yeast two-hybrid library screening and in vitro protein pull-down assays revealed that XLG2 interacts with the nuclear protein RTV1 (related to vernalization 1). Electrophoretic mobility shift assays show that RTV1 binds to DNA in vitro in a non-sequence-specific manner and that GTP-bound XLG2 promotes the DNA binding activity of RTV1. Overexpression of RTV1 results in early flowering. Combined overexpression of XLG2 and RTV1 enhances this early flowering phenotype and elevates expression of the floral pathway integrator genes, FT and SOC1, but does not repress expression of the floral repressor, FLC. Chromatin immunoprecipitation assays show that XLG2 increases RTV1 binding to FT and SOC1 promoters. Thus, a Ca(2+)-dependent G protein, XLG2, promotes RTV1 DNA binding activity for a subset of floral integrator genes and contributes to floral transition.

Rice GDP dissociation inhibitor 3 inhibits OsMAPK2 activity through physical interaction.

GDP dissociation inhibitor (GDI) plays an essential role in regulating the state of bound nucleotides and subcellular localizations of Rab proteins. In our previous study, we showed that OsGDI3 facilitates the recycling of OsRab11 with a help of OsGAP1. In this study, we show that OsGDI3 complement the yeast sec19-1 mutant, a temperature-sensitive allele of the yeast GDI gene, suggesting that OsGDI3 is a functional ortholog of yeast GDI. To obtain further knowledge on the function of OsGDI3, candidate OsGDI3-interacting proteins were identified by yeast two-hybrid screens. OsMAPK2 is one of OsGDI3 interacting proteins from yeast two-hybrid screens and subject to further analysis. A kinase assay showed that the autophosphorylation activity of OsMAPK2 is inhibited by OsGDI3 in vitro. In addition, ectopic expressions of OsGDI3-in Arabidopsis cause reductions at the level of phosphorylated AtMPK in phosphorylation activity. Taken together, OsGDI3 functions as a negative regulator of OsMAPK2 through modulating its kinase activity.

Encoding memory of winter by noncoding RNAs.

In some plant species, prolonged exposure to low temperature during the winter season is necessary to acquire the competence to flower in the following spring. This process, known as vernalization, is an epigenetic change in that a mitotically stable change of the developmental potential of the meristem (competence to flower) is maintained even in the absence of the inducing signal (prolonged cold exposure). In Arabidopsis, vernalization results in stable epigenetic repression of a potent floral repressor, FLOWERING LOCUS C (FLC). Increased enrichment of Polycomb Repressive Complex 2 (PRC2) and trimethylated Histone H3 Lys 27 (H3K27me3) at FLC chromatin is necessary for the stable maintenance of FLC repression by vernalization. Recent recognition of long noncoding RNAs (ncRNAs) in vernalization response indicates that long ncRNAs are evolutionarily conserved components for PRC2-mediated repression in eukaryotes.

Nuclear/nucleolar GTPase 2 proteins as a subfamily of YlqF/YawG GTPases function in pre-60S ribosomal subunit maturation of mono- and dicotyledonous plants.

The YlqF/YawG families are important GTPases involved in ribosome biogenesis, cell proliferation, or cell growth, however, no plant homologs have yet to be characterized. Here we isolated rice (Oryza sativa) and Arabidopsis nuclear/nucleolar GTPase 2 (OsNug2 and AtNug2, respectively) that belong to the YawG subfamily and characterized them for pre-60S ribosomal subunit maturation. They showed typical intrinsic YlqF/YawG family GTPase activities in bacteria and yeasts with k(cat) values 0.12 ± 0.007 min(-1) (n = 6) and 0.087 ± 0.002 min(-1) (n = 4), respectively, and addition of 60S ribosomal subunits stimulated their activities in vitro. In addition, OsNug2 rescued the lethality of the yeast nug2 null mutant through recovery of 25S pre-rRNA processing. By yeast two-hybrid screening five clones, including a putative one of 60S ribosomal proteins, OsL10a, were isolated. Subcellular localization and pulldown assays resulted in that the N-terminal region of OsNug2 is sufficient for nucleolar/nuclear targeting and association with OsL10a. OsNug2 is physically associated with pre-60S ribosomal complexes highly enriched in the 25S, 5.8S, and 5S rRNA, and its interaction was stimulated by exogenous GTP. Furthermore, the AtNug2 knockdown mutant constructed by the RNAi method showed defective growth on the medium containing cycloheximide. Expression pattern analysis revealed that the distribution of AtNug2 mainly in the meristematic region underlies its potential role in active plant growth. Finally, it is concluded that Nug2/Nog2p GTPase from mono- and didicotyledonous plants is linked to the pre-60S ribosome complex and actively processed 27S into 25S during the ribosomal large subunit maturation process, i.e. prior to export to the cytoplasm.