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Sweet proteins are the natural alternative to the artificial sweeteners as well as flavor enhancers. Among other sweet protein, thaumatin protein was isolated from Thaumatococcus daniellii Benth plant fruit. In this study, pinII Ti plasmid vector was constructed with thaumatin gene, where thaumatin was placed under the control of the duel cauliflower mosaic virus 35S promoter into rice (Oryza sativa L. var. japonica cv. ‘Dongjinbyeo’) by Agrobacterium-mediated transformation to generate transgenic plants. Thirteen plant lines were regenerated and the transgenic rice lines were confirmed by different molecular analysis. The genomic PCR result revealed that all of the plant lines were transgenic. The single copy and intergenic plant lines were selected by Taqman PCR analysis and FST analysis, respectively. Expression of thaumatin gene in transgenic rice resulted in the accumulation of thaumatin protein in the leave. Thaumatin protein was also accumulated in leave of T1 generation. Sensory analysis result suggested that the thaumatin protein expressing transgenic lines exerted sweet tasting activity. These results demonstrated that thaumatin was expressed in transgenic rice plants.

We generated an orange-colored (OC) rice callus line by targeted mutagenesis of the orange gene (OsOr) using the CRISPR-Cas9 system. The OC line accumulated more lutein, β-carotene, and two β-carotene isomers compared to the WT callus line. We also analyzed the expression levels of carotenoid biosynthesis genes by qRT-PCR. Among the genes encoding carotenoid metabolic pathway enzymes, the number of transcripts of the PSY2, PSY3, PDS, ZDS and β-LCY genes were higher in the OC line than in the WT line. In contrast, transcription of the ε-LCY gene was downregulated in the OC line compared to the WT line. In addition, we detected increases in the transcript levels of two genes involved in carotenoid oxidation in the OC lines. The developed OC lines also showed increased tolerance to salt stress. Collectively, these findings indicate that targeted mutagenesis of the OsOr gene via CRISPR/Cas9-mediated genome editing results in β-carotene accumulation in rice calli. Accordingly, we believe that this type of genome-editing technology could represent an effective alternative approach for enhancing the β-carotene content of plants.

Transgenic rice overexpressing PLCP attenuated the virulence of Xanthomonas oryzae pv. oryzae through extensive activation of transduction signal and transcription activities that orchestrate downstream responses including the biosynthesis of secondary metabolites and up-regulation of several pathogenesis-related proteins. High-throughput transcriptome investigations of plant immunity highlight the complexity of gene networks leading to incompatible interaction with the pathogen. Accumulating findings implicate papain-like cysteine proteases (PLCPs) as a central hub in plant defense. While diverse roles of PLCPs in different pathosystems have become more evident, information on gene networks and signaling pathways necessary to orchestrate downstream responses are lacking. To understand the biological significance of cysteine protease against Xanthomonas oryzae pv. oryzae, PLCP overexpression and knockout rice lines were generated. The pathogenicity test revealed the attenuation of Xanthomonas oryzae pv. oryzae race K3a virulence in transgenic lines which is ascribed to high hydrogen peroxide and free salicylic acid accumulation. Next-generation sequencing of RNA from transgenic and wild-type plants identified 1597 combined differentially expressed genes, 1269 of which were exclusively regulated in the transgenic libraries. It was found that PLCP aids rice to circumvent infection through the extensive activation of transduction signal and transcription factors that orchestrate downstream responses, including up-regulation of multiple pathogenesis-related proteins and biosynthesis of secondary metabolites.

Cysteine protease activity comprises the majority of proteolytic activities in plants. They are involved in almost every facet of the plant’s development. Accumulating evidence indicates multiple roles of this protease type in response to biotic and abiotic stress. To understand the regulations and functions of cysteine protease in rice, its evolutionary and structural evidence was uncovered in this study. Using MEROPS, a peptidase database, the 74 rice cysteine proteases belonging to six families were queried. Each of these families represents distinct proteolytic enzyme; C1 is a papain-like protease, C2 is a calpain-2-type, C12 is an ubiquitinyl hydrolase-L1 enzyme, C13 is legumain, C14 is a caspase-1 type, and C15 is a pyroglutamyl peptidase 1 enzyme type. Evolutionary expansion attributed to gene duplication and diversification was particularly evident in C1 family which showed the highest number (n = 53) of members, most of which contained the highest number and most variable introns and motifs, whereas families C13, C14, and C15 had only a few members which all contained lesser number and variation of intron and motif. Out of 74 total cysteine protease gene members, 73 were globular proteins and 55 were predicted as stable proteins. Spatial expression assay of selected C1 members showed that LOC_Os01g73980 and LOC_Os05g01810 were highly expressed in the stem and leaves, while LOC_Os02g27030 was constitutively expressed in all tissues. The expression of LOC_Os01g73980 and LOC_Os05g01810 was also highly activated by salinity stress, while LOC_Os02g27030 was activated by both salinity and heat. LOC_Os05g01810 overexpression transgenic rice exhibited moderate tolerance to salinity stress, which provides interesting clues on biological functions of these genes in rice.

Genome editing offers great advantages in identifying gene function and generating agronomical important mutations in crops. Here, we report the development of edited lines with reduced seed dormancy by knockout viviparous-1 (OsVP1) gene known as a transcription factor that regulates key aspects of plant seed development and ABA signaling in rice. Thirty-three genetic edited lines out of 55 T0 rice plants were generated using CRISPR/Cas9 system. Sequencing analysis showed that the plants had four different mutation types at the target site of OsVP1, the mutations were found to be transmitted to the succeeding generations. Stable transmission of CRISPR/Cas9-mediated mutant lines without the transferred DNA (T-DNA) was confirmed by segregation in the T1 generation. Regarding many investigated agronomic trait, there are no significant differences between homozygous mutants and wildtype plants under field’s growth conditions. Especially in RT-PCR analysis of ABA/GA signaling genes, the expression of OsNCED2, OsGA20ox1, OsGA20ox2, OsGA20ox3 genes in homozygous mutants was increased compared to wildtype plants. Results of this study exemplified the effectiveness of CRISPR/Cas9 as a gene editing tool in broke down the seed dormancy in rice.

Altering a trait by CRISPR-Cas9-targeted mutagenesis offers great advantages in identifying gene function and crop improvement. In the present study, three genes (OsF3′H, OsDFR and OsLDOX) in the anthocyanin biosynthesis pathway were successfully edited on the Heugseonchal or Sinmyungheugchal variety using the CRISPR/Cas9 system. As a result, the ratio of the edited plants in the transformed early generation was 56.7%. These edited mutant lines were observed with the changes of seed color and anthocyanin content. All mutations were stably inherited to the T2 progeny. In addition, we could select edited homozygous mutant lines lacking the T-DNA already in the first offspring generation. Also the insertion of vector backbone sequences in f3′h-9, dfr-4 and ldox-16 lines was not detected in the whole genome resequencing. These results demonstrated that the CRISPR/Cas9 system can induce clearly gene-specific mutations with a high efficiency in rice and null plants selected from these mutants cannot be distinguished from non-GMO plants even under strict GMO regulation.

The primary goals of rice breeding programs are grain quality and yield potential improvement. With the high demand for rice varieties of premium cooking and eating quality, we developed low-amylose content breeding lines crossed with Samgwang and Milkyqueen through the marker-assisted backcross (MABc) breeding program. Trait markers of the SSIIIa gene referring to low-amylose content were identified through an SNP mapping activity, and the markers were applied to select favorable lines for a foreground selection. To rapidly recover the genetic background of Samgwang (recurrent parent genome, RPG), 386 genome-wide markers were used to select BC1F1 and BC2F1 individuals. Seven BC2F1 lines with targeted traits were selected, and the genetic background recovery range varied within 97.4–99.1% of RPG. The amylose content of the selected BC2F2 grains ranged from 12.4–16.8%. We demonstrated the MABc using a trait and genome-wide markers, allowing us to efficiently select lines of a target trait and reduce the breeding cycle effectively. In addition, the BC2F2 lines confirmed by molecular markers in this study can be utilized as parental lines for subsequent breeding programs of high-quality rice for cooking and eating.

The tight regulation of local auxin homeostasis and signalling maxima in xylem precursor cells specifies the organising activity of the vascular cambium and consequently promotes xylem differentiation and wood formation. However, the molecular mechanisms underlying the local auxin signalling maxima in the vascular cambium are largely unknown. Here, we reveal that brassinosteroid (BR)-activated WALLS ARE THIN1 (WAT1) facilitates wood formation by enhancing local auxin signalling in the vascular cambium in Solanum lycopersicum. Growth defects and low auxin signalling readouts in the BR-deficient tomato cultivar, Micro-Tom, were associated with a novel recessive allele, Slwat1-copi, created by the insertion of a retrotransposon in the last exon of the SlWAT1 locus. Molecular and genetic studies by generating the gain-of-function and loss-of-function tomato mutants revealed that SlWAT1 is a critical regulator for fine tuning local auxin homeostasis and signalling outputs in vascular cambium to facilitate secondary growth. Finally, we discovered that BR-regulated SlBZR1/2 directly activated downstream auxin responses by SlWAT1 upregulation in xylem precursor cells to facilitate xylem differentiation and subsequent wood formation. Our data suggest that the BR-SlBZR1/2-WAT1 signalling network contributes to the high level of auxin signalling in the vascular cambium for secondary growth.

The utilization of male sterility into hybrid seed production reduces its cost and ensures high purity of tomato varieties because it does not produce pollen and has exserted stigmas. Here, we report on the generation of gene edited lines into male sterility phenotype by knockout of SlMS10 gene (Solyc02g079810) encoding the bHLH transcription factor that regulates meiosis and cell death of the tapetum during microsporogenesis in the tomato. Twenty-eight gene edited lines out of 60 transgenic plants were selected. Of these, eleven different mutation types at the target site of the SlMS10 gene were selected through deep sequencing analysis. These mutations were confirmed to be transmitted to subsequent generations. The null lines without the transferred DNA (T-DNA) were obtained by segregation in the T1 and T2 generations. In addition, we showed that the cr-ms10-1-4 mutant line exhibited dysfunctional meiosis and abnormal tapetum during flower development, resulting in no pollen production. RT-PCR analysis showed that the most genes associated with pollen and tapetum development in tomatoes had lower expression in the cr-ms10-1-4 mutant line compared to wild type. We demonstrate that modification of the SlMS10 gene via CRISPR/Cas9-mediated genome editing results in male sterility of tomato plants. Our results suggest an alternative approach to generating male sterility in crops.

In plants, starch is synthesized in leaves during the day-time from fixed carbon through photosynthesis and is mobilized at night to support continued respiration, sucrose export, and growth in the dark. The main crops where starch is biosynthesized and stored are corn, rice, wheat, and potatoes, and they are mainly used as food resources for humankind. There are many genes that are involved in starch biosynthesis from cytosol to storage organs in plants. ADP-glucose, UDP- glucose, and glucose-6-phosphate are synthesized catalyzed by UDP-invertase, AGPase, hexokinase, and P- hexose-isomerase in cytosol. Starch composed of amylopectin and amylose is synthesized by starch synthase, granule bound starch synthase, starch-branching enzyme, debranching enzyme, and pullulanase, which is primarily responsible for starch production in storage organs. Recently, it has been uncovered that structural genes are controlled by proteins derived from other genes such as transcription factors. To obtain more precise information on starch metabolism, the functions of genes and transcription factors need to be studied to understand their roles and functions in starch biosynthesis in plants. However, the roles of genes related to starch biosynthesis are not yet clearly understood. The papers of this special issue contain reviews and research articles on these topics and will be a useful resource for researchers involved in the quality improvement of starch storage crops.

The altered rice leaf color based on the knockout of CAO1 gene generated using CRISPR/Cas9 technology plays important roles in chlorophyll degradation and ROS scavenging to regulate both natural and induced senescence in rice. Rice chlorophyllide a oxygenase (OsCAO1), identified as the chlorophyll b synthesis under light condition, plays a critical role in regulating rice plant photosynthesis. In this study, the development of edited lines with pale green leaves by knockout of OsCAO1 gene known as a chlorophyll synthesis process is reported. Eighty-one genetically edited lines out of 181 T0 plants were generated through CRISPR/Cas9 system. The edited lines have short narrow flag leaves and pale green leaves compared with wild-type ‘Dongjin’ plants (WT). Additionally, edited lines have lower chlorophyll b and carotenoid contents both at seedling and mature stages. A transcriptome analysis identified 580 up-regulated and 206 downregulated genes in the edited lines. The differentially expressed genes (DEGs) involved in chlorophyll biosynthesis, magnesium chelatase subunit (CHLH), and glutamate-1-semialdehyde2, 1-aminomutase (GSA) metabolism decreased significantly. Meanwhile, the gel consistency (GC) levels of rice grains, chalkiness ratios and chalkiness degrees (CD) decreased in the edited lines. Thus, knockout of OsCAO1 influenced growth period, leaf development and grain quality characters of rice. Overall, the result suggests that OsCAO1 also plays important roles in chlorophyll degradation and ROS scavenging to regulate both natural and induced rice senescence.

Rice grain quality is usually observed by its chalkiness and is affected by genetic effects of endosperm, cytoplasm and maternal plant. Controlling the chalkiness in rice can be a very challenging task because it is affected by genotype and environmental factors. The present study aimed to introduce 5-methyl tryptophan (5MT) resistance from the 5MT resistant mutants into Samgwang, a high grain and eating quality Korean variety by introgression of, resulting to elevated tryptophan content in grains. The progenies generated from single crosses of two different cross combinations were phenotyped based on agronomic traits and by 5MT growth inhibition test. Through direct PCR sequencing, the inheritance of single base mutation (F124V) in OsASA was selected among the progenies. The latter generations were used to analyze the grain and eating quality of the selected lines. Inbred lines (S4-10, S4-28, and S5-11) carrying the point mutation in OsASA and with reduced chalkiness plus good eating qualities were successfully generated. Tryptophan content in the milled grains of the selected lines showed 2 - 4 times higher (mg/100 mg) than the maternal parent. The three selected lines, S4-10, S4-28, and S5-11, were later renamed as GolSam-1, GolSam-2, and GolSam-3 respectively.

Meiosis is a specialized cell division, essential in most reproducing organisms to halve the number of chromosomes, thereby enabling the restoration of ploidy levels during fertilization. A key step in meiosis is homologous recombination, which promotes homologous pairing and generates crossovers (COs) to connect homologous chromosomes until their separation at anaphase I. These CO sites, seen cytologically as chiasmata, represent a reciprocal exchange of genetic information between two homologous non-sister chromatids. RAD51, the eukaryotic homolog of the bacterial RecA recombinase, plays a central role in homologous recombination (HR) in yeast and animals. Loss of RAD51 function causes lethality in the flowering plant, Arabidopsis thaliana, suggesting that RAD51 has a meiotic stage-specific function that is different from homologous pairing activity.

Brazzein은 열대식물인 P. brazzeana Baillon의 과실에서 분리된 가장 작은 감미단백질로 토착민들의 단맛원료로 사용되어 왔다. Brazzein은 sucrose보다 분자량 기준으로 500 ~ 2000배, 몰 기준으로 9500배 당도가 높아 감미료로써 매우 높은 평가를 받고 있다. 그러나 이 감미단백질은 재배가 어렵고 생산 비용이 높아서 brazzein 단백질의 이용 가능성을 높이기 위한 대체 생산 시스템으로 형질전환 식물체 육성 하고자 하였다. 본 연구에서는 brazzein 관련 유전자를 벼에 도입하기 위하여 식물형질전환용 Ti-plasmid에 2 x CaMV 35S 프로모터에 의해 지배되어 발현하도록 하고, 선발 마커로 bar 유전자가 삽입된 식물발현 벡터를 구축하여 A. tumefaciens EHA105에 형질전환시켜 17개의 재분화 식물체를 육성하였다. 17개 재분화 식물체는 PCR 및 RT-PCR 분석을 통하여 유전자 도입 및 발현을 확인하였으며, TaqMan PCR을 통해 single copy로 도입된 T0 세대 9개체를 선발하였다. 또한 FST 분석을 통하여 도입 유전자가 intergenic으로 삽입된 개체 5개를 선발하였다. 이들 5개체를 이용하여 western blot 분석에 의해 단백질 발현량을 분석한 결과 선발된 모든 개체에서 발현 밴드를 확인하였다. 그 중 brazzein 단백질의 발현량이 높은 개체를 TG11으로 계통화하여 후대 종자를 육성하였다. TG11 계통은 천연 감미료 brazzein을 생산하는 새로운 벼 품종을 개발하기 위한 육종 소재로 활용 가능하다고 시사된다.

본 연구에서는 국내외에서 수집한 벼 294개 유전자원 핵심집단을 대상으로 벼의 지엽각 특성에 대한 조사를 수행하였고, GWAS를 이용하여 지엽각 연관 유전자를 추출 및 분석하였다. 표현형 데이터를 이용한 GWAS의 Manhattan plot 결과 분석을 통해, 각 집단에서 염색체를 대상으로 표현형과 통계적 유의성을 나타내 연관성을 보이는 SNP를 발굴하였다. 지엽각 관련 특성에 대하여 선행 연구된 QTL region과의 비교를 통하여 본 연구에서 발굴된 SNP간의 유의성을 조사한 결과, 지엽각과 유의성이 있는 SNP (S8-19815442)가 이미 확인된 QTL region에 위치하는 것으로 나타났으며, 후보유전자 Os08g31950 대해 연관 유전자 변이를 관찰하기 위해서 형질 특이적 품종군 간의 염기서열을 비교한 결과 1개의 지역에서 단일염기변이가 검출되었다. Os08g31950의 조직별 RNA의 상대적 발현량 수준을 비교한 결과, Os08g31950 유전자는 모든 조직에서 높은 발현량을 확인할 수 있었으며 조직별로 다양한 발현 양상을 관찰할 수 있었다. 또한, 모두 직립형 품종군에서 상대적으로 발현량이 높게 나타났으며 뿌리보다 잎에서의 발현율이 높게 나타났다. 본 연구를 통해 동정된 지엽각 연관 후보유전자 Os08g31950는 벼 생육 및 수량 증대에 이용할 수 있는 마커제작 및 육종의 기초자료가 될 것으로 기대된다.

Plants adapt to hostile environmental condition through specialized cellular processes which require coordinated regulation of multiple transport mechanisms. Low-affinity cation transporter (LCT) 1 is a membrane transporter gene exclusive only to members of the grass family, and the rice genome contained only one copy of the gene. Accumulating evidence highlighted the important role of this gene in the regulation of various cations transport into the plant cells including heavy metals. To further characterize the role of this membrane transporter in planta, we overexpressed the OsLCT1 in japonica rice using CaMV 35S and screened the transgenic plants for high salt toxicity at the seedling stage. A striking difference in the phenotype of OsLCT1-overexpression plants and the wild-type was observed at seven days after treatment, where transgenic plants exhibited moderate tolerance reaction to salinity stress. Our findings suggest that OsLCT1 gene can be useful to develop new resistant varieties against salinity stress.

MicroRNAs are small, endogenous, non-coding RNAs found in plants, animals, and in some viruses, which negatively regulate the expression of genes by promoting the degradation of target mRNAs or by translation inhibition. Ever since the discovery of miRNAs, its biology, mechanisms, and functions were extensively studied in the past two decades. Plant and animal miRNAs both regulate target mRNAs, but they differ in scope of complementarity to their target mRNA. Plant microRNAs are known to play essential roles in a wide array of plant development. To date, there are many studies giving evidence that the regulation of miRNA levels can reprogram plant responses to abiotic (physical environment) and biotic stresses (pathogen and herbivore). Most of these studies were first carried out in the model plant Arabidopsis thaliana. Recently, the trend of miRNA research is furthering its role in crop breeding and its evolutionary origin. In this review, we presented the dynamic biogenesis of microRNAs, the diverse functions of miRNAs in plants, and experimental designs used in studying microRNAs in plants, and most importantly, we presented the applications of microRNA-based technology to improve the resistance of crops in abiotic and biotic stresses.

벼 품종의 DNA profiling을 위하여 SSR 마커를 활용하여 한국 벼의 품종판별 기술 확립을 위하여 국내에서 육성된 벼 243개 품종에 대하여 최종 선발된 7개의 SSR 마커(RM21, RM257, HsSSR01-52, RM333, RM580, RM1306, RM157)를 이용하여 판별하였다. 판별용으로 이용된 SSR 마커 7개의 총 대립인자 수는 130개였으며, 대립인자 수의 범위는 10 ~ 32개이었고, 평균 대립인자 수는 18.57이었다. PIC 값은 0.679(HsSSR01-52) ~ 0.895 (RM333)의 범위이었으며, 평균 PIC 값은 0.774이었다. SSR 마커 7개의 조합으로 6단계의 판별을 통해 총 243개 품종 중 243개 모든 품종의 구별이 가능하였다. 이와 같은 결과, 본 연구에 이용된 SSR 7개 마커는 한국 벼 품종의 판별과 순도유지에 매우 효율적으로 이용할 수 있을 것으로 여겨진다.

식물의 유일한 활성 스테로이드 호르몬인 Brassinosteroid (BR)는 다양한 내재적 또는 외부 신호 전달 경로와의 통합적인 결합을 통해 식물의 생장 및 발달 과정에서 중요한 기능을 하는 것으로 알려져 있다. 최근 식물학 연구들은 종자의 발아와 초기 발달과정에서 BR과 ABA 사이의 필수적인 상호 작용 메커니즘이 존재하고 있음을 보고하고 있다. 하지만 이들 두 호르몬의 중요한 신호전달 상호작용에 대한 분자 메커니즘은 거의 알려지지 않았다. 식물의 초기 발달과정에서 BR에 의해 매개되는 ABA 신호전달과의 기능학적, 생물학적 상호작용 네트워크를 이해하기 위해 Agilent Arabidopsis 4 x 44K 올리고 칩을 사용하여 비교 전사체 분석을 수행하였다. ABA에 반응하지 않는 bes1-D 돌연변이체에서의 ABA 처리에 따른 다양한 유전자의 발현 패턴을 야생형 식물과 비교 분석하였다. 그 결과 발현의 변화가 발생하는 유전자(DEGs) 2,353 개를 확인하였다. GO 분석을 통해 ABA 신호전달 및 대사에 관여하는 유전자들이 BR 신호전달 경로에 의해 하향 조절되는 것으로 확인되었다. 뿐만 아니라, BR 신호전달 경로는 다양한 비생물학적/생물학적 스트레스, 오옥신 및 ROS 등 다양한 신호전달 체계와 밀접하게 연관되어 있음을 확인하였다. 본 연구를 통해 BR 신호전달의 활성화는 ABA 신호전달에 관여하는 다양한 유전자들의 발현을 억제함을 확인하였다. 또한 본 연구는 다양한 신호 경로 사이의 상호 작용이 다양한 환경요인에 대한 식물의 적응 반응에 중요하게 작용할 수 있음을 보여주고 있다.

Implementation of crop improvement programs relies on genetic diversity. To overcome the limited occurrence of natural mutations, researchers and breeders applied diverse methods, ranging from conventional crossing to classical bio-technologies. Earlier generations of knockout and gain-of-function technologies often result in incomplete gene disruption or random insertions of transgenes into plant genomes. The newly developed editing tool, CRISPR/Cas9 system, not only provides a powerful platform to efficiently modify target traits, but also broadens the scope and prospects of genome editing. Customized Cas9/guide RNA (gRNA) systems suitable for efficient genomic modification of mammalian cells or plants have been reported. Following successful demonstration of this technology in mammalian cells, CRISPR/Cas9 was suc-cessfully adapted in plants, and accumulating evidence of its feasibility has been reported in model plants and major crops. Recently, a modified version of CRISPR/Cas9 with added novel functions has been developed that enables programmable direct irreversible conversion of a target DNA base. In this review, we summarized the milestone applications of CRISPR/ Cas9 in plants with a focus on major crops. We also present the implications of an improved version of this technology in the current plant breeding programs.

Male sterility is an important trait and plays a key role for hybrid crop production in agricultural industry field. Different genes and enzymes are involved with male sterile traits. Dysfunction of these genes in any stage of male reproductive system often results in male sterility i.e., the inability to produce functional pollen. Functional male sterility can be utilized in the production of hybrid seed. During plant breeding for hybrid crop production, a lot of genetic diversities are created. Plant breeding is enhanced by the availability of molecular markers for rapid screening and selection in populations. Molecular markers are useful for a variety of purposes relevant to crop improvement. Functional markers that detect the functional mutations causing phenotypic changes offer a precise method for genetic identification. Various DNA markers are available now in plant breeding sectors. Among all of these markers, the new generation molecular markers called single nucleotide polymorphisms (SNPs) are most abundant, robust and feasible because of its availability in whole genome and that they play a key role in the induction of phenotypic variations like male sterility. In this review, we described the classification and mechanism of male sterility in crops, the genes responsible for male sterility, and application of SNP markers for selection of male sterile plants.

To study the transcript levels of epigenetically regulated genes in tobacco, we have developed a transgenic line OX1 overexpressing NtROS2a gene encoding cytosine DNA demethylation and a RNAi plant line RNAi13. It has been reported that salt- and -stress tolerance of these transgenic lines are enhanced with various phenotypic characters (Lee et al. 2015). In this paper, we conducted microarray analysis with Agilent Tobacco 4 x 44K oligo chip by using overexpression line OX1, RNAi plant line RNAi 13, and wild type plant WT. Differentially expressed genes (DEGs) related to metabolism, nutrient supply, and various stressed were up-regulated by approximately 1.5- to 80- fold. DEGs related to co-enzymes, metabolism, and methylation functional genes were down-regulated by approximately 0.03- to 0.7- fold. qRT-PCR analysis showed that the transcript levels of several candidate genes in OX1 and RNAi lines were significantly (p

The completion of the tomato genome sequence and recent advances in DNA sequencing technology allow in-depth characterization of genetic variation present in the tomato genome. Cultivated tomato lines show low molecular but high phenotypic diversity. We resequenced the genomes of four elite tomato lines with an Illumina HiSeq 2000 sequencer. We generated, on average, 84 million 100-bp paired-end reads per line. Mapped reads covered 89.0-93.4% of the mapped S. lycopersicum reference genome (782 Mb). On average, 0.42 single nucleotide polymorphisms (SNPs)/kb and 0.05 short insertions and deletions (InDels)/kb were found in the four elite lines. The highest number of SNPs was 558,526 found in tomato line 13-1084. We identified many SNPs and InDels, derived from 1012 genes, in region 7-62 Mb of chromosome 5 in the four elite lines. The same pattern of multiple SNPs was detected on chromosome 5 in lines 13-1151 and 10-3321, deriving from genes in region 0-2 Mb, and many homozygous SNPs and InDels were detected on chromosome 1 of these two lines. The same pattern of multiple SNPs and InDels derived from genes located between regions 53-60 Mb of chromosomes 4 and 11 was found in the four elite lines. The SNPs and InDels identified in this resequencing study will serve as useful genetic tools and candidate polymorphisms in the search for DNA variations associated with valuable phenotypic diversity.

Single nucleotide polymorphism (SNP) is an abundant form of genetic variation within individuals of species. DNA polymorphism can arise throughout the whole genome at different frequencies in different species. SNP may cause phenotypic diversity among individuals, such as individuals with different color of plants or fruits, fruit size, ripening, flowering time adaptation, quality of crops, grain yields, or tolerance to various abiotic and biotic factors. SNP may result in changes in amino acids in the exon of a gene (asynonymous). SNP can also be silent (present in coding region but synonymous). It may simply occur in the noncoding regions without having any effect. SNP may influence the promoter activity for gene expression and finally produce functional protein through transcription. Therefore, the identification of functional SNP in genes and analysis of their effects on phenotype may lead to better understanding of their impact on gene function for varietal improvement. In this mini-review, we focused on evidences revealing the role of functional SNPs in genes and their phenotypic effects for the purpose of crop improvements.

UDP-glucose 4-epimerase (UGE) catalyzes the reversible conversion of UDP-glucose to UDP-galactose. To understand the biological function of UGE from Brassica rapa, the gene BrUGE1 was cloned and introduced into the genome of wild type rice 'Gopum' using the Agrobacterium-mediated transformation method. Four lines which carried a single copy gene were selected and forwarded to T3 generation. Agronomic traits evaluation of the transgenic T3 lines (CB01, CB03, and CB06) under optimal field conditions revealed enriched biomass production particularly in panicle length, number of productive tillers, number of spikelets per panicle, and filled spikelets. These remarkably improved agronomic traits were ascribed to a higher photosynthetic rate complemented with higher CO2 assimilation. Transcripts of BrUGE1 in transgenic lines continuously accumulated at higher levels after the 20% PEG6000 treatment, implying its probable role in drought stress regulation. This was paralleled by rapid accumulation of soluble sugars which act as osmoprotectants, leading to delayed leaf rolling and drying. Our findings suggest the potential of BrUGE1 in improving rice growth performance under optimal and water deficit conditions.

Brassica rapa cysteine protease 3 (BrCP3) is an endopeptidase member of C1A family (papain-like) and CA clan of cysteine protease. Members of papain-like cysteine protease (CP) have emerged to be key enzymes involved in cell death in response to biotic and environmental stresses. Recent progress of CP research features its diverse and overlapping role in basal and effector-triggered immunity. This study was carried out to characterize BrCP3 gene in Chinese cabbage and explore its function in rice - Xanthomonas oryzae pv. oryzae pathosystem. Phylogenetic analysis and BLASTP search of its amino acid sequence to the Arabidopsis Protein TAIR 10 database identified BrCP3 as the closest ortholog of Arabidopsis RD21a. The full-length cDNA of BrCP3 was cloned and transformed into the genome of japonica rice cv. 'Gopum'. Constitutive overexpression of the transgene is driven by Ubi-1 promoter in pFLCIII vector. Regenerated single copy overexpression rice lines were inoculated with three Xoo Korean isolates. Regulation of rice resistance through cysteine protease activity is evident in the overexpression lines which showed enhanced immunity to three isolates as depicted by reduced lesion length compared with the wild type. Our initial results implicate an interesting biological function of BrCP3 in rice system that has never been reported before.

Anthranilate synthase (AS) is a key enzyme in the biosynthesis of tryptophan (Trp), which is the precursor of bioactive metabolites like indole-3-acetic acid and other indole alkaloids. Alpha anthranilate synthase 2 (OsASA2) plays a critical role in the feedback inhibition of tryptophan biosynthesis. In this study, two vectors with single (F124V) and double (S126F/L530D) point mutations of the OsASA2 gene for feedback-insensitive subunit of rice anthranilate synthase were constructed and transformed into wildtype Dongjinbyeo by Agrobacterium-mediated transformation. Transgenic single and double mutant lines were selected as a single copy using TaqMan PCR utilized nos gene probe. To select intergenic lines, the flanking sequence of RB or LB was digested with a BfaI enzyme. Four intergenic lines were selected using a flanking sequence tagged (FST) analysis. Expression in rice (Oryza sativa L.) of the transgenes resulted in the accumulation of tryptophan (Trp), indole-3-acetonitrile (IAN), and indole-3-acetic acid (IAA) in leaves and tryptophan content as a free amino acid in seeds also increased up to 30 times relative to the wildtype. Two homozygous event lines, S-TG1 and D-TG1, were selected for characterization of agronomic traits and metabolite profiling of seeds. Differentially expressed genes (DEGs), related to ion transfer and nutrient supply, were upregulated and DEGs related to co-enzymes that work as functional genes were down regulated. These results suggest that two homozygous event lines may prove effective for the breeding of crops with an increased level of free tryptophan content.

The calcineurin B-like protein (CBL)-CBL-interacting protein kinase (CIPK) pathway is emerging as a major signaling pathway in plants. To understand the function of CIPK, the gene named BrCIPK1 from Brassica rapa were introduced into rice. Characterization of BrCIPK1 gene showed a 1982 bp, containing 1509 bp coding region and 502 amino acids. Green fluorescent protein (GFP)-tagged BrCIPK1 was observed exclusively in the cytoplasmic and peripheral regions in the plant cell. Gene expression showed that its messenger RNA (mRNA) transcription in B. rapa was differentially accumulated in the presence of cold, salinity, and drought, indicating its biological roles in multiple stress response pathways in plants. Furthermore, Ubi-1::BrCIPK1 rice lines showed significantly higher biomass, water content, and proline and free sugar content relative to those in the wild-type Gopum. The BrCIPK1 interacted with rice calcineurin B-like protein 1 and 5 (OsCBL1, OsCBL5), suggesting that it is activated by Ca2+-bound CBLs in the cytosol by calcium spiking and regulates its downstream target proteins in these regions to increase abiotic stress tolerance. The results imply that BrCIPK1 gene may be involved in stress adaptations through the activation of pyrroline-5-carboxylate synthase in the proline biosynthetic pathway. In this paper, a hypothetical mechanism of elevated tolerance to cold, drought, and salinity is presented.

UDP-glucose 4-epimerase (UGE; EC 5.1.3.2) is an enzyme that plays an essential role in the interconverts UDP-d-glucose (UDP-Glc) and UDP-Dgalactose (UDP-Gal). Five members of the Chinese cabbage (Brassica rapa) UGE gene family, designated BrUGE1 to BrUGE5, have been cloned and characterized. Quantitative PCR shows that the BrUGE1and BrUGE4 mRNA are most abundant among other BrUGE genes, accounting for more than 55 % of total BrUGE transcripts in most of the tissues examined. All genes showed organ-specific expression pattern, two of which (BrUGE1 and 4) actively responded after Pectobacterium carotovorum subsp. carotovorum infection, while four genes (BrUGE-1, -3, -4, and -5)were shown to respond considerably against salt, drought and abscisic acid treatments. To better understand the function of the UGE gene, we constructed a recombinant pART vector carrying the BrUGE1 gene under the control of the CaMV 35S promoter and nos terminator and transformed using Agrobacterium tumefaciens. We then investigated BrUGE1 overexpressing rice lines at the physiological and molecular levels under biotic and abiotic stress conditions. Bioassay of T3 progeny lines of the transgenic plants in Yoshida solution containing 120 mM NaCl for 2 weeks, confirmed that the BrUGE1 enhances salt tolerance to transgenic rice plants. Also T3 progeny lines of the transgenic plants, when exposed to infection caused by Xanthomonas oryzae pv. oryzae, showed tolerance to bacterial blight. These results showed that BrUGE1 can be used as potential genetic resource for engineering Brassica with multiple stress resistance.

본 연구는 다양한 LED광 처리가 상추의 생장상, 플라보노이드 생합성 관련 유전자들의 발현양상 및 플라보노이드 함량에 미치는 영향을 분석하였다. 상추의 생장상 분석에서 혼합광 처리가 단일 처리구보다 잎생장이 월등히 우수하였으며, 단일광내에서 청색광이 적색광보다 생체중이 높게 나타났다. LED광질에 따른 플라보노이드 생합성 관련 8개 유전자들의 발현을 분석한 결과 LsF3H와 LsDFR 유전자에서 혼합광 처리한 적상추에서 청상추보다 높게 나타났다. 또한 혼합광 처리가 단일광 처리보다 Gallic acid, Chlorogenic acidn, Quercetin 및 안토시아닌의 함량이 가장 높게 나타났다. 이상의 결과로부터 LED 광질에 따라 상추 생장과 발육차이를 보이며, 유전자의 발현 및 기능성 물질의 축적에 중요한 요인으로 작용하며, 기능성 물질의 축적을 유도하기 위해서는 적색과 청색의 혼합처리가 유용하리라 생각된다.

Plants have adapted the ability to respond to various abiotic stresses such as high salinity, osmotic stress, high and low temperatures, and drought in order to survive. Small heat shock proteins (sHsps) play important and extensive roles in plant defenses against abiotic stresses. Herein, we cloned an sHsp gene from the rice, which we named Oshsp16.9 based on the molecular weight of the protein. Real-time PCR analysis showed that expression of the Oshsp16.9 gene was rapidly and strongly induced by stresses including high-salinity (250 mM NaCl), osmotic stress (300 mM mannitol), 100 μM ABA, cold (4°C) and heat (45°C). Subcellular localization assay indicated that Oshsp16.9 was localized specifically in the cytoplasm. In addition, overexpression of Oshsp16.9 in rice conferred tolerance of transgenic plants to salt and drought stress. Taken together, these results suggest that the Oshsp16.9 gene is an important determinant of stress response in plants.

An increasing preference for good eating quality of rice among consumers has become one of the important considerations in rice breeding. Amylose content is a leading factor affecting eating quality of rice. Amylose composition is determined by the relative activity of soluble starch synthase (SSS) and granule-bound starch synthase (GBSS). This study focused on modifying the expression of SSSI gene which is responsible for amylopectin and amylose synthesis in rice by using RNA interference (RNAi) technology. The transgenic rice plants showed various amylose contents (11-17%) in rice grains. Favorable rice lines were selected according to genomic PCR, transgene expression and amylose contents analysis. A semi-quantitative RT-PCR was carried out to determine the expression level of SSSI gene after flowering of transgenic rice and wild type. Down-regulation of SSSI gene in transgenic plants was evident in the decreasing expression in rice grains. Accordingly, scanning electron microscopy (SEM) analysis revealed uniform size with smooth curves starch granules in down-regulation rice lines, in contrast with the non-uniform granules in wild type. Results indicated that RNAi-SSSI transgenic lines produced low amylose contents that fell between glutinous and non-glutinous rice. This study showed that down-regulation of endogenous SSSI may improve the eating quality in rice.

A large number of antimicrobial peptides (AMPs) from different organisms have been characterized to date. AMPs are small molecular weight proteins with broad spectrum antimicrobial activity against bacteria, viruses, and fungi. Several diseases in plants could result in serious losses and decreasing quality and safety of agricultural products. Being an agent for plant defense, AMPs kill target cells through diverse mechanisms once in a target microbial membrane. Cathelicidins, defensins and thionins are the three major groups of epidermal AMPs in human and plants. Plant AMPs are structurally and functionally diverse and can be directed against other organisms, like herbivorous insects. The biological activity of plant AMPs primarily depends on interactions with membrane lipids. Several antimicrobial peptides have been expressed in transgenic plants to confer disease protection. Antimicrobial peptides are interesting compounds that can be efficiently exploited for disease control in plants in a way that complies with the strict regulations on the efficacy and safety of disease control strategy.

The novel BrRZFPs genes encoding C3HC4- type RING zinc finger protein were identified from FOX (full length cDNA over-expressing) library of Brassica rapa . Ten full-length cDNAs obtained from the library encode zinc-finger protein containing 346 amino acids, designated BrRZFPs. These genes were classified into four groups by phylogenic analysis showing conserved protein sequences at both termini. The tissue distribution of BrRZFPs trans- cription was examined by qRT-PCR revealing ubiquitous expression pattern. However, each gene was strongly ex- pressed in the specific tissue. Transcriptional analysis showed that those acquired 10 genes were inducible under abiotic stresses. Likewise, the transcript of BrRZFP3 was strongly induced (~12-folds) by exogenous abscisic acid, whereas the transcripts of BrRZFP1, BrRZFP2 and BrRZFP3 were (> 9-folds) induced by cold. We suggest that these BrRZFPs that function as signal or response to abiotic stress are useful for crop improvement.

C3H4-type RING zinc finger protein (BrRZP1) from Brassica rapa under abiotic stresses. To elucidate the role of the BrRFP1 transcription factor in gene regulation, we transformed tobacco plants with the BrRZFP1 gone. Plants were regenerated from 82 independently transformed callus lines of tobacco and analysed for transgene expression. Trans-gene integration and expression was confirmed by Southern and RT-PCR analyses, respectively. T2 plants displayed more tolerance to the bacterial pathogens Pectobacterium carotovorum and Ralstonia solanacearum, and the tolerance levels were correlated with BrRZFP1 expression levels. These results suggest that the transcription factor BrRZFP1 is an important determinant of stress response in plants and its overexpression in plants could increase biotic stress resistance.