Characterization of Peanut Germin-Like Proteins, <i>AhGLPs</i> in Plant Development and Defense

<div><p>Background</p><p>Germin-like superfamily members are ubiquitously expressed in various plant species and play important roles in plant development and defense. Although several <i>GLPs</i> have been identified in peanut (<i>Arachis hypogaea</i> L.), their roles in development and defense remain unknown. In this research, we study the spatiotemporal expression of <i>AhGLPs</i> in peanut and their functions in plant defense.</p><p>Results</p><p>We have identified three new <i>AhGLP</i> members (<i>AhGLP3b</i>, <i>AhGLP5b</i> and <i>AhGLP7b</i>) that have distinct but very closely related DNA sequences. The spatial and temporal expression profiles revealed that each peanut <i>GLP</i> gene has its distinct expression pattern in various tissues and developmental stages. This suggests that these genes all have their distinct roles in peanut development. Subcellular location analysis demonstrated that AhGLP2 and 5 undergo a protein transport process after synthesis. The expression of all <i>AhGLPs</i> increased in responding to <i>Aspergillus flavus</i> infection, suggesting <i>AhGLPs'</i> ubiquitous roles in defense to <i>A. flavus.</i> Each <i>AhGLP</i> gene had its unique response to various abiotic stresses (including salt, H₂O₂ stress and wound), biotic stresses (including leaf spot, mosaic and rust) and plant hormone stimulations (including SA and ABA treatments). These results indicate that <i>AhGLPs</i> have their distinct roles in plant defense. Moreover, <i>in vivo</i> study of <i>AhGLP</i> transgenic <i>Arabidopsis</i> showed that both <i>AhGLP2</i> and <i>3</i> had salt tolerance, which made transgenic <i>Arabidopsis</i> grow well under 100 mM NaCl stress.</p><p>Conclusions</p><p>For the first time, our study analyzes the <i>AhGLP</i> gene expression profiles in peanut and reveals their roles under various stresses. These results provide an insight into the developmental and defensive roles of <i>GLP</i> gene family in peanut.</p></div

Venn diagram representing the expression profiles of <i>AhGLP</i> genes commonly or specifically regulated by various environmental stimuli, including plant hormones, abiotic stress and/or biotic stress in peanut leaves.

<p>1, 2, 3, 4, 5, 6, 7, 8 were AhGLP1, AhGLP1, AhGLP2, AhGLP3, AhGLP4, AhGLP5, AhGLP6, AhGLP7 and AhGLP8, respectively. The underline numbers indicated down-regulated genes, and the numbers without underlines were up-regulated genes.</p

Overexpression of <i>AhGLP</i>s and salt tolerance analysis in transgenic <i>Arabidopsis thaliana</i>.

<p>WT: wild type; control: The modified pCAMBIA1301 inserted with 35S only was used as negative control. (A): Diagrams of the constructs (the pCAMBIA1301-35S- <i>AhGLP1</i>, <i>2</i>, <i>3</i>, <i>4</i>, <i>5</i> and <i>7</i>) used for <i>Agrobacterium tumefaciens</i>-mediated transformation of <i>Arabidopsis</i>. (B): Effects of different NaCl content (0, 50 and 100 mM) on the germination of transgenic <i>Arabidopsis</i> seeds for 5 days after germination; (C): Comparison of germination rates and percentages of seedlings with green cotyledons between transgenic lines and wild-type plants under 100 mM NaCl stress. (D): The seeding cultivated on 1/2 MS agar plate containing 50 mM NaCl for 15 days. (E): Transcript analysis of <i>AhGLPs</i>-activated defense related genes (DFR, CHS, 3GT, and AtPR3, 4 and 5) in transgenic <i>Arabidopsis</i> plants.</p

The length of trimmed EST sequence (cDNA length after removal of vector sequence and low quality sequences) submitted to clustering

<p><b>Copyright information:</b></p><p>Taken from "Peanut gene expression profiling in developing seeds at different reproduction stages during infection"</p><p>http://www.biomedcentral.com/1471-213X/8/12</p><p>BMC Developmental Biology 2008;8():12-12.</p><p>Published online 4 Feb 2008</p><p>PMCID:PMC2257936.</p><p></p> The number of EST within different categories of trimmed sequence length is presented on the Y-axis. The number on the X-axis represent ranges of trimmed sequence lengths (101–200, 201–300, 301–400 bp, etc, respectively)

Subcellular localization of AhGLPs-GFP proteins in onion epidermal cells.

<p>Localization of AhGLPs-GFP fusion protein. Control: fluorescence of onion epidermal cells under empty vector. 35S-smGFP: onion epidermal cells expressing the <i>GFP</i> gene only driven by he 35S promoter. GFP fluorescence and differential interference contrast images and Visible/GFP merged images are shown from left to right.</p

Difference of closely related multiple <i>AhGLPs</i>.

a<p><i>AhGLP3a</i> (GU457419.1), <i>AhGLP5a</i> (GU457421.1) and <i>AhGLP7a</i> (GU457423.1) were the known genes as reported by Chen et al (2011) in NCBI genebank, three newfound genes were designated AhGLP3b, AhGLP5b and AhGLP7b distinguished from AhGLP3a, AhGLP5a and AhGLP7a respectively.</p>b<p>un-know bases in the nucleotide sequence of AhGLP5a and AhGLP7a with indefinable amino acids (*).</p

Functional classification of peanut unique ESTs by comparison to Arabidopsis Sequencing Project functional categories

<p><b>Copyright information:</b></p><p>Taken from "Peanut gene expression profiling in developing seeds at different reproduction stages during infection"</p><p>http://www.biomedcentral.com/1471-213X/8/12</p><p>BMC Developmental Biology 2008;8():12-12.</p><p>Published online 4 Feb 2008</p><p>PMCID:PMC2257936.</p><p></p> A: functional categories of 'GT-C20' unique EST sequences; B: functional categories of 'Tifrunner' unique ESTs

Expression of <i>AhGLPs</i> in response to <i>A. flavus</i> infection in pre - and post- harvested peanut seeds.

<p>A: Con: control; Dt: drought stress; Ad: <i>A. flavus</i> infection under drought stress condition; B: Changes of the expression of <i>AhGLP</i> family genes in damp-dry peanut seed with 20% RH (relative humidity) under <i>A. flavus</i> infection. Con: control; A. f: <i>A. flavus</i> infection.</p

Hierarchical clustering analysis of differentially expressed transcripts for 'GT-C20' and 'Tifrunner'

<p><b>Copyright information:</b></p><p>Taken from "Peanut gene expression profiling in developing seeds at different reproduction stages during infection"</p><p>http://www.biomedcentral.com/1471-213X/8/12</p><p>BMC Developmental Biology 2008;8():12-12.</p><p>Published online 4 Feb 2008</p><p>PMCID:PMC2257936.</p><p></p> TCs with R > 4 (84 in total) were used for hierarchical clustering analysis

Data_Sheet_7_Genome Sequencing and Analysis of the Peanut B-Genome Progenitor (Arachis ipaensis).XLSX

<p>Peanut (Arachis hypogaea L.), an important leguminous crop, is widely cultivated in tropical and subtropical regions. Peanut is an allotetraploid, having A and B subgenomes that maybe have originated in its diploid progenitors Arachis duranensis (A-genome) and Arachis ipaensis (B-genome), respectively. We previously sequenced the former and here present the draft genome of the latter, expanding our knowledge of the unique biology of Arachis. The assembled genome of A. ipaensis is ~1.39 Gb with 39,704 predicted protein-encoding genes. A gene family analysis revealed that the FAR1 family may be involved in regulating peanut special fruit development. Genomic evolutionary analyses estimated that the two progenitors diverged ~3.3 million years ago and suggested that A. ipaensis experienced a whole-genome duplication event after the divergence of Glycine max. We identified a set of disease resistance-related genes and candidate genes for biological nitrogen fixation. In particular, two and four homologous genes that may be involved in the regulation of nodule development were obtained from A. ipaensis and A. duranensis, respectively. We outline a comprehensive network involved in drought adaptation. Additionally, we analyzed the metabolic pathways involved in oil biosynthesis and found genes related to fatty acid and triacylglycerol synthesis. Importantly, three new FAD2 homologous genes were identified from A. ipaensis and one was completely homologous at the amino acid level with FAD2 from A. hypogaea. The availability of the A. ipaensis and A. duranensis genomic assemblies will advance our knowledge of the peanut genome.</p