In-Ja Song

The RAV1 transcription factor positively regulates leaf senescence in Arabidopsis

Leaf senescence is a developmentally programmed cell death process that constitutes the final step of leaf development and involves the extensive reprogramming of gene expression. Despite the importance of senescence in plants, the underlying regulatory mechanisms are not well understood. This study reports the isolation and functional analysis of RAV1, which encodes a RAV family transcription factor. Expression of RAV1 and its homologues is closely associated with leaf maturation and senescence. RAV1 mRNA increased at a later stage of leaf maturation and reached a maximal level early in senescence, but decreased again during late senescence. This profile indicates that RAV1 could play an important regulatory role in the early events of leaf senescence. Furthermore, constitutive and inducible overexpression of RAV1 caused premature leaf senescence. These data strongly suggest that RAV1 is sufficient to cause leaf senescence and it functions as a positive regulator in this process.

Spatiotemporal expression of duplicate AGAMOUS orthologues during floral development in Phalaenopsis

The AGAMOUS (AG) family of MADS-box genes plays important roles in controlling the development of the reproductive organs of flowering plants. To understand the molecular mechanisms behind the floral development in the orchid, we isolated and characterized two AG-like genes from Phalaenopsis that we denoted PhalAG1 and PhalAG2. Phylogenetic analysis indicated that PhalAG1 and PhalAG2 fall into different phylogenetic positions in the AG gene family as they belong to the C- and D-lineages, respectively. Reverse transcription-polymerase chair reaction (RT-PCR) analyses showed that PhalAG1 and PhalAG2 transcripts were detected in flower buds but not in vegetative organs. Moreover, in situ hybridization experiments revealed that PhalAG1 and PhalAG2 hybridization signals were observed in the lip, column, and ovule during the floral development of Phalaenopsis, with little difference between the expression patterns of the two genes. These results suggest that both AG-like genes in Phalaenopsis act redundantly with each other in floral development.

Characterization of TrcMADS1 gene of Trillium camtschatcense (Trilliaceae) reveals functional evolution of the SOC1/TM3-like gene family

Plant MADS-box genes encode transcriptional regulators that are critical for a number of developmental processes, such as the establishment of floral organ identity, flowering time, and fruit development. It appears that the MADS-box gene family has undergone considerable gene duplication and divergence within various angiosperm lineages. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1)/Tomato MADS-box gene 3 (TM3)-like genes are members of the MADS-box gene family and have undergone repeated duplication events. Here, we isolated and characterized the SOC1/TM3-like gene TrcMADS1 from Trillium camtschatcense (Trilliaceae) to infer the ancestral function of SOC1/TM3-like genes. The alignment of SOC1/TM3-like genes revealed the presence of a highly conserved region in the C-terminal of predicted protein sequences, designated the SOC1 motif. Phylogenetic analysis indicated that TrcMADS1 is at the basal position of the SOC1/TM3-like gene family. The TrcMADS1 mRNA was detected in both vegetative and reproductive organs by RT-PCR. Our results suggest that duplicated copies of SOC1/TM3-like gene evolved to become variously functionally specialized.

Molecular phylogeny and evolution of alcohol dehydrogenase (Adh) genes in legumes

BackgroundNuclear genes determine the vast range of phenotypes that are responsible for the adaptive abilities of organisms in nature. Nevertheless, the evolutionary processes that generate the structures and functions of nuclear genes are only now be coming understood. The aim of our study is to isolate the alcohol dehydrogenase (Adh) genes in two distantly related legumes, and use these sequences to examine the molecular evolutionary history of this nuclear gene.ResultsWe isolated the expressed Adh genes from two species of legumes, Sophora flavescens Ait. and Wisteria floribunda DC., by a RT-PCR based approach and found a new Adh locus in addition to homologues of the Adh genes found previously in legumes. To examine the evolution of these genes, we compared the species and gene trees and found gene duplication of the Adh loci in the legumes occurred as an ancient event.ConclusionThis is the first report revealing that some legume species have at least two Adh gene loci belonging to separate clades. Phylogenetic analyses suggest that these genes resulted from relatively ancient duplication events.

The structure and expression of SEPALLATA-like genes in Asparagus species (Asparagaceae)

MADS-box genes encode transcriptional regulators that are critical for a number of developmental processes. In the MADS-box gene family, the SEPALLATA (SEP) gene subfamily plays an important role in controlling the development of floral organs in flowering plants. To understand the molecular mechanisms of floral development in Asparagus, we isolated and characterized several SEP-like genes from dioecious Asparagus officinalis and hermaphrodite A. virgatus: AOMADS1, AOMADS2, AOMADS3, and AVMADS1, AVMADS2, AVMADS3, respectively. Through alignment of the predicted amino acid sequences of various SEP-like genes, we defined three characteristic motifs in the C-terminal region of the genes: SEP motif I, SEP/AGL6 motif, and SEP motif II. Of the genes we isolated, AOMADS3 and AVMADS3 had lost the SEP motif II. Phylogenetic analysis revealed that AOMADS1, AOMADS2, AVMADS1, and AVMADS2 were closely related to SEP3 from Arabidopsis, whereas AOMADS3 and AVMADS3 were classified in different clade which is far related to SEP3 gene. Northern hybridization and RT-PCR showed that three SEP-like genes in A. officinalis were specifically expressed in the flower buds. In addition, PCR RFLP showed that there was no significant difference in the amount of transcripts of AOMADS1 and AOMADS2. These results suggest that AOMADS1 and AOMADS2 may be redundant genes. In contrast, the expression of AOMADS3 was weaker than that of AOMADS1 or AOMADS2, suggesting that the function of AOMADS3 may be different than that of AOMADS1 or AOMADS2.

Expression of the protective antigen for PEDV in transgenic duckweed, Lemna minor

Duckweeds are small, floating aquatic plants with a number of useful characteristics, including edibility, fast-growing, and a clonal proliferation. Duckweed is also fed to animals as a diet complement because of its high nutritional value. Porcine epidemic diarrhea virus (PEDV) is a major causative agent of fatal diarrhea in piglets and is a serious problem in the hog-raising industry. In this study, we assessed the feasibility of producing a protective antigen for the PEDV spike protein 1 using duckweed, Lemna minor. Stably transformed Lemna were obtained by co-cultivation with A. tumefaciens EHA105 harboring the PEDV spike protein gene. Transgene integration and expression of the PEDV spike protein 1 gene were confirmed by genomic PCR and RT-PCR and western blot analysis of transgenic Lemna, respectively. This is the first report of the expression of a vaccine antigen against an animal infectious disease in duckweed.

Ginsenoside Production and Morphological Characterization of Wild Ginseng (Panax ginseng Meyer) Mutant Lines Induced by γ-irradiation ( 60 Co) of Adventitious Roots

With the purpose of improving ginsenoside content in adventitious root cultures of Korean wild ginseng (Panax ginseng Meyer), the roots were treated with different dosages of γ-ray (5, 10, 25, 50, 75, 100, and 200 Gy). The growth of adventitious roots was inhibited at over 100 Gy. The irradiated adventitious roots showed significant variation in the morphological parameters and crude saponin content at 50 to100 Gy. Therefore, four mutant cell lines out of the propagation of 35 cell lines treated with 50 Gy and 100 Gy were selected on the basis of phenotypic morphology and crude saponin contents relative to the wild type control. The contents of 7 major ginsenosides (Rg1, Re, Rb1, Rb2, Rc, Rf, and Rd) were determined for cell lines 1 and 3 from 100 Gy and lines 2 and 4 from 50 Gy treatments. Cell line 2 showed more secondary roots, longer length and superior growth rate than the root controls in flasks and bioreactors. Cell line 1 showed larger average diameter and the growth rate in the bioreactor was comparable with that of the control but greater in the flask cultured roots. Cell lines 1 and 2, especially the former, showed much more ginsenoside contents than the control in flasks and bioreactors. Therefore, we chose cell line 1 for further study of ginsenoside contents. The crude saponin content of line 1 in flask and bioreactor cultures increased by 1.4 and 1.8-fold, respectively, compared to the control. Total contents of 7 ginsenoside types (Rg1, Re, Rb1, Rb2, Rc, Rf, and Rd) increased by 1.8 and 2.3-fold, respectively compared to the control. Crude saponin and ginsenoside contents in the bioreactor culture increased by about 1.4-fold compared to that the flask culture.

Transgenic Turfgrasses Expressing Hyperactive Ser599Ala Phytochrome A Mutant Exhibit Abiotic Stress Tolerance

Turfgrasses are environmentally and recreationally valuable plants that are constantly subjected to various forms of stress in their artificial and natural habitats. Previously, it was shown that the transformation of a hyperactive mutant (Serine 599 Alanine, S599A) of oat phytochrome A in zoysia grass (Zoysia japonica) and creeping bentgrass (Agrostis stolonifera L.) resulted in superior quality turfgrass with improved shade tolerance response. We now examined the abiotic stress response of the transgenic turfgrasses expressing the hyperactive mutant S599A-PhyA. The transgenic S599A-PhyA plants subjected to high salinity and heavy metal toxicity stress exhibited higher chlorophyll content, lower hydrogen peroxide level, and higher proline accumulation than the controls. Furthermore, the anti-oxidative activities of four reactive oxygen species scavenging enzymes and the total biomass (above and below-ground) were higher in S599A-PhyA plants than in the controls under both the stress conditions. Moreover, higher photosynthetic efficiency (Fv/Fm) of S599A-PhyA plants indicated healthier growth than the controls under stress conditions. Results suggest that the hyperactive mutant of oat phytochrome A confers abiotic stress tolerance in plants, and can be used to efficiently develop abiotic stress tolerant crops in future.

Recent developments in biotechnological improvement of Zoysia japonica Steud.

Abstract Zoysiagrass ( Zoysia japonica Steud.), also called Korean or Japanese lawngrass, is the most popular warm- season turfgrass in Korea and is widely used for home lawns, parks, roadsides, golf courses and athletic fields. Its use is rapidly expanding in Korea and the other countries, due to its excellent characteristics which include tolerance to heat, drought and salinity. As the utilization area of this turfgrass increases, there is an increase in the demand for improved cultivars with disease and insect tolerance or with herbicide- tolerance or with extended greening periods. Conventional breeding methods have been used to improve the traits de-scribed above with limited success. However, with the advances in biotechnology, genetic transformation can be utilized for turfgrass improvement. In this paper, we review recent progress in biotechnological improvement of zoysiagrass and discuss future molecular breeding of this species. 서 론 들잔디 ( Zoysia japonica Steud.)는 한국을 포함한 극동아시아와 대부분의 온대지역에 분포하는 난지형 잔디 중의 하나로 초장이 짧아서 예초에 강하고 열, 가뭄 및 염분에 대해 내성이 뛰어나며 일반적으로 병충해에 강한 잔디이다. 그러나 몇몇 병충해에 대해서는 민감한 반응을 보이며 기온이 10℃ 이하가 되면 휴면에 들어가 지상부가 고사하여 녹색 유지기간이 한지형 잔디에 비해 짧고 음지에서 잘 자라지 못하는 단점이 있다 (Inokuma et al. 1996; Lee et al. 2004). 잔디는 경관보호, 토양 및 수자원 보호, 신변보호 등 여러 가지 목적으로 활용되고 있으며, 도로, 하천, 비행장의 토양침식 방지에서부터 주택, 공원, 정원, 골프장, 스포츠 경기장 등으로 그 이용범위가 나날이 확대되고 있다. 이로 인한 잔디의 급격한 수요 증가와 더불어 잔디관련 사업규모가 크게 확대되고 있어 병해충 저항성, 내한성, 제초제 저항성, 내음지성, 녹기 연장 등 다양한 형질을 가진 잔디품종의 개발이 요구되고 있다. 지금까지 들잔디의 품종육성은 전통적인 육종방법을 이용하고 있으나 타 작물의 경우와 마찬가지로 고전육종기술의 한계점들이 발견됨에 따라 최근에는 분자생물학적 방법을 이용한 신품종 개발이 시도되고 있다 (Inokuma et al. 1998; Rim et al. 2001; Kim et al. 2004; Zhang et al. 2007). 분자생물학적 방법을 이용한 들잔디의 신품종 개발을 위해서는 먼저 조직배양과 형질전환체계가 확립되어야 한다. 들잔디의 조직배양에 관한 연구는 원형질체로부터 식물체 재분화 (Asano 1989; Inokuma et al. 1996), 미성숙종자의 미숙배에서 유도한 캘러스로부터의 식물체 재분화 (Park et al. 1994; Noh et al. 1995; Park and Ahn 1998), 성숙종자 유래 캘러스로부터의 식물체 재분화 (Al-Khayri et al. 1989; Rim et al. 2001; Bae et al. 2001; Lee et al. 2004) 등이 보고 된 바 있다. 그러나 원형질체를 이용한 조직배양은 분리나 배양에 어려움이 있고 미성숙종자를 이용한 조직배양은 재료의 지속적인 확보가 어려워 그 활용이 크게 제한적이어서 최근의 들잔디 조직배양에 관한 연구에는 주로 성숙종자 유래의 캘러스가 이용되고 있다 (Liu et al. 2009; Song et al. 2010). 들잔디의 형질전환에 관한

Environmental risk assessment of genetically modified Herbicide-Tolerant zoysiagrass (Event: Jeju Green21)

Abstract Transgenic zoysiagrass ( Zoysia japonica Steud.) expressing the bar gene inserted in the plant genome has been generated previously through Agrobacterium tumefaciens - mediated transformation. The GM zoysiagrass (event: JG21) permits efficient management of weed control of widely cultivated zoysiagrass fields, reducing the frequency and cost of using various herbicides for weed control. Now we have carried out the environmental risk assessment of JG21 prior to applying to the governmental regulatory agency for the commercial release of the GM turf grass outside of test plots. The morphological phenotypes, molecular analysis, weediness and gene flow from each test plot of JG21 and wild-type zoysiagrasses have been evaluated by selectively analyzing environmental effects. There were no marked differences in morphological phenotypes between JG21 and wild-type grasses. The JG21 retained its stable integration in the host plant in T 1 generation, exhibiting a 3:1 segregation ratio according to the Mendelian genetics. We confirmed the copy number (1) of JG21 by using Southern blot analysis, as the transgenic plants were tolerant to ammonium glufosinate throughout the culture period. From cross-fertilization and gene flow studies, we found a 9% cross-pollination rate at the center of JG21 field and 0% at distances over 3 m from the field. The JG21 and wild-type zoysiagrass plants are not considered “weed” because zoysiagrasses generally are not dominant and do not spread into weedy areas easily. We assessed the horizontal gene transfer (HGT) of the transgene DNA to soil microorganisms from JG21 and wild-type plants. The bar gene was not detected from the total genomic DNA extracted from each rhizosphere soil of GM and non-GM Zoysia grass fields. Through the monitoring of JG21 transgene’s unintentional release into the environment, we found no evidence for either pollen mediated gene flow of zoysiagrass or seed dispersal from the test field within a 3 km radius of the natural habitat.