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A comparative proteomics approach to detect unintended effects in transgenic Arabidopsis

Journal of Genetics and Genomics 2009年第10期36卷 629-639页

作者： Yanfei Ren Jun Lv Hua Wang linchuan li Yufa Peng li-jia qu National Key Laboratory of Protein Engineering and Plant Genetic Engineering College of Life Sciences Peking University Beijing 100871 China lnstitute of Plant Protection Chinese Academy of Agricultural Sciences Beijing 100193 China

Recently, as part of biosafety assessments, unintended effects have been given much attention. In this study, we applied a proteomics approach to elucidate the unintended effects of random T-DNA insertion in transgenic plants. Separated proteins extracted from 12 transgenic Arabidopsis thaliana with different T-DNA insertion sites and from wild-type （ecotype Col-o） were analyzed. In the transgenic plants, 102 significantly altered protein spots were detected, in which 59 were up-regulated and 43 down-regulated. MALDI-TOF MS analysis showed that most of these expression level-altered proteins were involved in energy transfer, oxidative respiration and photosynthesis. However, none of these proteins was a toxic protein or allergen. Using plants with or without cold treatment, a natural environmental stress, as controls, we found that the number of the altered proteins was even less in those transgenic plants than those triggered by the cold treatment, suggesting that the transgenic events had a weaker impact on the plants than the environmental stresses. Interestingly, the phosphinothricin acetyl transferase （PAT）, the BAR-encoded protein, was detected in nine out of twelve different T-DNA insertion lines at five different insertion sites. These data suggest that the most significant impact of transgenic events on the host plants is from the transgene itself, i.e., from the predictable intended effects, rather than unintended effects. This study also suggests that the proteomics approach has the potential to detect the unintended effects in transgenic plants.

关键词： biosafety proteomics Arabidopsis thaliana 2-D PAGE MALDI-TOF unintended effect

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Distinguishing transgenic from non-transgenic Arabidopsis plants by ~1H NMR-based metabolic fingerprinting

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Journal of Genetics and Genomics 2009年第10期36卷 621-628页

作者： Yanfei Ren Tao Wang Yufa Peng Bin Xia li-jia qu National Key Laboratory of Protein Engineering and Plant Genetic Engineering College of Life Sciences Peking University Beijing 100871 China Beijing NMR Center College of Life Sciences Peking University Beijing 100871 China Institute of Plant Protection Chinese Academy of Agricultural Sciences Beijing 100193 China

We have recently reported the construction of an nuclear magnetic resonance （NMR）-based metabonomics study platform, Automics. To examine the application of Automics in transgenic plants, we performed metabolic fingerprinting analysis, i.e., 1H NMR spectroscopy and multivariate analysis, on wild-type and transgenic Arabidopsis. We found that it was possible to distinguish wild-type from four transgenic plants by PLS-DA following application of orthogonal signal correction （OSC）. Scores plot following OSC clearly demonstrates significant variation between the transgenic and non-transgenic groups, suggesting that the metabolic changes among wild-type and transgenic lines are possibly associated with transgenic event, We also found that the major contributing metabolites were some specific amino acids （i.e., threonine and alanine）, which could correspond to the insertion of the selective marker BAR gene in the transgenic plants. Our data suggests that NMR-based metabonomics is an efficient method to distinguish fingerprinting difference between wild-type and transgenic plants, and can potentially be applied in the bio-safety assessment of transgenic plants.

关键词： Arabidopsis metabonomics nuclear magnetic resonance principal component analysis orthogonal signal correction

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Overexpression of a New Putative Membrane Protein Gene AtMRB1 Results in Organ Size Enlargement in Arabidopsis

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Journal of Integrative Plant Biology 2009年第2期51卷 130-139页

作者： Hua Guan Dingming Kang Min Fan Zhangliang Chen li-jia qu College of Biological Sciences China Agricultural University Beijing 100094 China College of Agronomy and Biotechnology China Agricultural University Beijing 100094 China National Laboratory of Protein Engineering and Plant Genetic Engineering College of Life Sciences Peking University Beijing 100871 China

Arabidopsis AtMRB1 is predicted to encode a novel protein of 432 amino acid residues in length, with four putative transmembrane domains. In the present study, characterization of AtMRB1 is conducted. Green fluorescent protein （GFP） fusion protein assay showed that AtMRB1 was located in the plasma membrane. Transgenic lines overexpressing AtMRB1 driven by a CaMV 35S promoter were generated. Statistic analysis showed that, during the seedling stage, the organ sizes of the transgenic lines including hypocotyl length, root length and root weight were significantly larger than those of the wild type plants under both light and dark conditions. In the adult plant stage, the AtMRB1 overexpressor plants were found to have larger organ sizes in terms of leaf length and width, and increased number of cauline leaves and branches when bolting. Further observation indicated that the larger leaf size phenotype was due to a larger number of mesophyll cells, the size of which was not altered. quantitative real-time polymerase chain reaction analysis showed that the transcription of ANT, ROT3 and GRF5 were upregulated in the *** plants. These data suggest that AtMRB1 is possibly a positive regulator of organ size development in Arabidopsis, mainly through cell number control.

关键词： Arabidopsis AtMRB1 cell number overexpressor lines organ size.

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FERONIA and reactive oxygen species:regulators in the self‑incompatibility responseand in interspecific pollination

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Molecular Horticulture 2023年第1期3卷 280-282页

作者： Zihan Song Sheng Zhong li‑jia qu State Key Laboratory for Protein and Plant Gene Research Peking‑Tsinghua Center for Life SciencesNew Cornerstone Science LaboratoryCollege of Life SciencesPeking UniversityBeijing 100871P.R.China

In plants,the stigma represents the initial site of pollenpistil communication and serves as a crucial location for rejection of incompatible *** self-compatible(SC)plants,such as Arabidopsis thaliana from the Brassicaceae family,pollen coat protein B(PCP-B)peptides were reported to compete with stigmatic RAPID ALKAliNIZATION FACTOR 33(RALF33)peptides for binding to the FERONIA/ANJEA(FER/ANJ)receptors in the stigma to promote pollen hydration via inhibition of reactive oxygen species(ROS)production(liu et al.2021).

关键词： reactive regulators RAPID

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Active role of small peptides in Arabidopsis reproduction: Expression evidence

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Journal of Integrative Plant Biology 2015年第6期57卷 518-521页

作者： Qingpei Huang Thomas Dresselhaus Hongya Gu li-jia qu State Key Laboratory of Protein and Plant Gene Research Peking-Tsinghua Center for Life SciencesSchool of Life SciencesPeking University Cell Biology and Plant Biochemistry Biochemie-Zentrum RegensburgUniversity of Regensburg

Dear Editor, Successful sexual reproduction in plants requires exten- sive communication between male and female gametophytes, their gametes, and with the surrounding sporophytic tissues （Dresselhaus and Franklin-Tong 2013）. Over the past two decades, many peptides including small cysteine-rich peptides （***） and non-CRPs were found to play key roles in plant reproduction, mainly acting as signaling cues in this male- female communication （Marshall et al.2011）.

关键词： gene Figure Expression evidence CRP

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Plant Hormones:Metabolism,Signaling and Crosstalk

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Journal of Integrative Plant Biology 2011年第6期53卷 410-411页

作者： li-jia qu Issue Editor Yunde Zhao Issue Editor School of life Sciences Peking University Beijing China Section of Cell and Developmental Biology University of California at San Diego USA

Plants synthesize various hormones in response to environ- mental cues and developmental signals to ensure their proper growth and development. Elucidation of the molecular mech- anisms by which plant hormones control growth and devel- opment contributes to our understanding of fundamental plant biology and provides tools to improve crops. Because of their critical roles in plant growth and development, plant hormones have been studied extensively since the early days of plant biology.

关键词： Metabolism Signaling Crosstalk

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TRANSLUCENT GREEN, an ERF Family Transcription Factor, Controls Water Balance in Arabidopsis by Activating the Expression of Aquaporin Genes

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Molecular Plant 2014年第4期7卷 601-615页

作者： Danling Zhu Zhe Wu Guangyu Cao Jigang li jia Wei Tomohiko Tsuge Hongya Gua Takashi Aoyama li-jia qu State Key Laboratory of Protein and Plant Gene Research Peking-Tsinghua Center for Life Sciences College of Life Sciences Peking University Beijing 100871 China State Key Laboratory of Plant Physiology and Biochemistry College of Biological Sciences China Agricultural University Beijing 100193 China Institute for Chemical Research Kyoto University Gokasho Uji Kyoto 611-0011 Japan The National Plant Gene Research Center (Beijing) Beijing 100101 China

Water is the most abundant molecule in almost all living organisms. Aquaporins are channel proteins that play critical roles in controlling the water content of cells. Here, we report the identification of an AP2/EREBP family transcription factor in Arabidopsis thaliana, TRANSLUCENT GREEN （TG）, whose overexpression in transgenic plants gave enhanced drought tolerance and vitrified leaves. TG protein is localized in the nucleus, binds DRE and GCC elements in vitro, and acts as a transcriptional activator in yeast cells. Microarray analysis revealed a total of 330 genes regulated by TG, among which five genes encode aquaporins. A transient expression assay showed that TG directly binds to the promoters of three aquaporin genes, such as AtTIP1;1, AtTIP2;3, and AtPIP2;2, indicating that TG directly regulates the expression of these genes. Moreover, overexpression of AtTIP1;1 resulted in vitrified phenotypes in transgenic Arabidopsis plants, similar to those observed in TG overexpression lines. Water injection into wild-type leaves recapitulated the vitrified leaf phenotypes, which was reversed by cutting off the water supply from vascular bundles. Taken together, our data support that TG controls water balance in Arabidopsis through directly activating the expression of aquaporin genes.

关键词： AP2/EREBP TRANSLUCENT GREEN vitrified leaf transcriptional regulation aquaporin.

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Novel DYW-type pentatricopeptide repeat(PPR) protein BLX controls mitochondrial RNA editing and splicing essential for early seed development of Arabidopsis

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Journal of Genetics and Genomics 2018年第3期45卷 155-168页

作者： Yan Sun jiaying Huang Sheng Zhong Hongya Gu Shan He li-jia qu State Key Laboratory for Protein and Plant Gene Research Peking-Tsinghua Center for Life Sciences at College of Life Sciences Peking University Beijing 100871 China The National Plant Gene Research Center (Beijing) Beijing 100101 China

In plants, RNA editing is a post-transcriptional process that changes specific cytidine to uridine in both mitochondria and plastids. Most pentatricopeptide repeat（PPR） proteins are involved in organelle RNA editing by recognizing specific RNA sequences. We here report the functional characterization of a PPR protein from the DYW subclass, Baili Xi（BLX）, which contains five PPR motifs and a DYW domain. BLX is essential for early seed development, as plants lacking the BLX gene was embryo lethal and the endosperm failed to initiate cellularization. BLX was highly expressed in the embryo and endosperm, and the BLX protein was specifically localized in mitochondria, which is essential for BLX function. We found that BLX was required for the efficient editing of 36 editing sites in mitochondria. Moreover, BLX was involved in the splicing regulation of the fourth intron of nad1 and the first intron of nad2. The loss of BLX function impaired the mitochondrial function and increased the reactive oxygen species（ROS） level. Genetic complementation with truncated variants of BLX revealed that, in addition to the DYW domain, only the fifth PPR motif was essential for BLX function. The upstream sequences of the BLX-targeted editing sites are not conserved, suggesting that BLX serves as a novel and major mitochondrial editing factor（MEF） via a new non-RNA-interacting manner. This finding provides new insights into how a DYW-type PPR protein with fewer PPR motifs regulates RNA editing in plants.

关键词： Arabidopsis PPR RNA editing RNA splicing Embryo and endosperm development

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GAMT2 Encodes a Methyltransferase of Gibberellic Acid That is Involved in Seed Maturation and Germination in Arabidopsis

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Journal of Integrative Plant Biology 2007年第3期49卷 368-381页

作者： Shufan Xing Genji Qin Yan Shi Zhangliang Chen Hongya GU li-jia qu National Laboratory for Protein Engineering and Plant Genetic Engineering Peking-Yale Joint Research Center for Plant Molecular Genetics and AgroBiotechnology College of Life Sciences Peking University Beijing 100871 China National Plant Gene Research Center (Beijing) Beijing 100101 China

Salicylic acid methyltransferase （SAMT）, benzoic acid methyltransferase （BAMT） and theobromine methyltransferase （TH）（henceforth, SABATH） family proteins belong to a unique class of mehtyltransferase that can methylate small molecular compounds Including indole-3-acidic acid （IAA）, salicylic acid （SA） and jasmonic acid （JA）, in plants. Here we report that the GAMT2 protein, which has 34.2% similarity with IAMT1 in the amino acid sequence, can methylate gibberellic acid （GA）. Biolnformatics analysis suggests that GAMT2 may be able to methylate one molecule larger than SA. GAMT2 is predominantly expressed in the developing seed embryo and endosperm in Arabidopsis. During seed germination, the expression of GAMT2 decreases until the cotyledons expand out of the seed coat. Overexpression of GAMT2 in Arabidopsis resulted in multiple phenotypes, including dwarfism, retarded growth, late flowering, and reduced fertility, which are similar to the phenotypes of GA-deficient mutants. Seed germination assay showed that GAMT2 overexpression in plants was hypersensitive to GA biosynthesis inhibitor （ancymidol） and abscisic acid （ABA） treatments, whereas the GAMT2 null mutant （SALK_075450） was slightly Insensitive to such treatments, suggesting that GAMT2 may methylate GA or ABA. Enzyme activity analysis indicated that GAMT2 was able to methylate GA3 into Methyi-GA3 in vitro, but could not methylate ABA. Microarray analysis on GAMT2 overexpression plants suggested that Methyl-GA may be an Inactive form of GA in Arabidopsis. These data suggest that GAMT2 Is Involved in seed maturation and germination by modulating GA activity.

关键词： GAMT2 GA-deficient seed germination seed maturation Me-GA methyl acceptors SABATH methyltransferase

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Overexpression of the Wounding-Responsive Gene AtMYB15 Activates the Shikimate Pathway in Arabidopsis

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Journal of Integrative Plant Biology 2006年第9期48卷 1084-1095页

作者： Yanhui Chen Xiangbo Zhang Wei Wu Zhangliang Chen Hongya Gu li-jia qu National Laboratory for Protein Engineering and Plant Genetic Engineering Peking-Yale Joint Research Center for Plant Molecular Genetics and AgroBiotechnology College of Life Sciences Peking University Beijing 100871 China The National Plant Gene Research Center （Beijing） Beijing 100101 China

The MYB transcription factor genes play important roles in many developmental processes and various defense responses of plants. The shikimate pathway is a major biosynthetic pathway for the production of three aromatic amino acids and other aromatic compounds that are involved in multiple responses of plants, including protection against UV and defense. Herein, we describe the characterization of the R2R3-MYB gene AtMYB15as an activator of the shikimate pathway in Arabidopsis. The AtMYB15 protein is nuclear localized and a transcriptional activation domain is found in its C-terminal portion. Northern blots showed that AtMYB15 is an early wounding-inducible gene. Resutls of microarray analysis, confirmed using quantitative real-time polymerase chain reaction, showed that overexpression of AtMYB15 in transgenic plants resulted in elevated expression of almost all the genes involved in the shikimate pathway. Bioinformatics analysis showed that one or more AtMYB15-binding AC elements were detected in the promoters of these upregulated genes. Furthermore, these genes in the shikimate pathway were also found to be induced by wounding. These data suggest an important role of AtMYB15as a possible direct regulator of the Arabidopsis shikimate pathway in response to wounding.

关键词： MYB transcription factor shikimate pathway transcriptional activation domain wounding inducible

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