FUNCTIONAL CHARACTERIZATION OF WD REPEAT PROTEINS, AtCstF50 AND AtFY IN CLEAVAGE AND POLYADENYLATION

Polyadenylation is an essential post-transcriptional modification resulting in a mature mRNA in eukaryotes. Three cis-elements the Far Upstream Element (FUE), Near Upstream Element (NUE), and Cleavage Site (CS) - guide the process of cleavage and polyadenylation with the help of multi-subunit protein complexes cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF) along with cleavage factors and poly(A) polymerase. Protein-protein interactions play an important role in the cleavage and polyadenylation process. WD repeat proteins play an important role in protein-protein interactions and have diverse functions in plant system. In the present study WD repeat proteins AtCstF50 and AtFY were studied for their role in polyadenylation process. Mammalian CstF50 is a WD repeat protein that is one of the subunit of CstF that aids in the cleavage step by associating with CPSF and cleavage factors. AtCstF50 was functionally characterized using T-DNA knock-out lines and by identifying the proteins that interacts with it in the process. Results shows that AtCstF50 is essential and was identified as part of CPSF complex, which is different from its mammalian counter part. CPSF was known to interact with Fip (factor interacting with PAP), Poly(A) polymerase and Poly(A) binding protein and AtCstF50 also interacts with these complexes. AtFY is a 3’ end processing factor which contains WD repeats is one of the subunits of the CPSF complex in Arabidopsis polyadenylation machinery. The AtFY interacts with FCA and promotes the alternative polyadenylation and also plays a role in polyadenylation site choice of FCA mRNA. We characterized the FY expression and localization of FY in the cell by fusing with RFP reporter. Results show that FY accumulates in the nucleus while FY with deleted calmodulin binding domain localizes both to the nucleus and outside the nucleus. The individual N-terminal and C-terminal domains also localized in the nucleus suggesting that they are multiple nuclear localization signals in FY and calmodulin might play a direct or indirect role in FY localization. Using a tethering assay we proved that AtFY is able to recruit the 3’ end processing complex in the proximal polyadenylation site choice of the reporter mRNA

Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling

BACKGROUND: The polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A) tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the cis-elements and produce the poly(A) tail. Here we report a comprehensive pairwise protein-protein interaction mapping and gene expression profiling of the mRNA polyadenylation protein machinery in Arabidopsis. RESULTS: By protein sequence homology search using human and yeast polyadenylation factors, we identified 28 proteins that may be components of Arabidopsis polyadenylation machinery. To elucidate the protein network and their functions, we first tested their protein-protein interaction profiles. Out of 320 pair-wise protein-protein interaction assays done using the yeast two-hybrid system, 56 (approximately 17%) showed positive interactions. 15 of these interactions were further tested, and all were confirmed by co-immunoprecipitation and/or in vitro co-purification. These interactions organize into three distinct hubs involving the Arabidopsis polyadenylation factors. These hubs are centered around AtCPSF100, AtCLPS, and AtFIPS. The first two are similar to complexes seen in mammals, while the third one stands out as unique to plants. When comparing the gene expression profiles extracted from publicly available microarray datasets, some of the polyadenylation related genes showed tissue-specific expression, suggestive of potential different polyadenylation complex configurations. CONCLUSION: An extensive protein network was revealed for plant polyadenylation machinery, in which all predicted proteins were found to be connecting to the complex. The gene expression profiles are indicative that specialized sub-complexes may be formed to carry out targeted processing of mRNA in different developmental stages and tissue types. These results offer a roadmap for further functional characterizations of the protein factors, and for building models when testing the genetic contributions of these genes in plant growth and development

Overexpression of an AP2/ERF Type Transcription Factor <i>OsEREBP1</i> Confers Biotic and Abiotic Stress Tolerance in Rice

<div><p>AP2/ERF–type transcription factors regulate important functions of plant growth and development as well as responses to environmental stimuli. A rice AP2/ERF transcription factor, <i>OsEREBP1</i> is a downstream component of a signal transduction pathway in a specific interaction between rice (<i>Oryza sativa</i>) and its bacterial pathogen, Xoo (<i>Xanthomonas oryzae</i> pv. <i>oryzae</i>). Constitutive expression of <i>OsEREBP1</i> in rice driven by maize <i>ubiquitin</i> promoter did not affect normal plant growth. Microarray analysis revealed that over expression of <i>OsEREBP1</i> caused increased expression of lipid metabolism related genes such as lipase and chloroplastic lipoxygenase as well as several genes related to jasmonate and abscisic acid biosynthesis. PR genes, transcription regulators and <i>Aldhs</i> (alcohol dehydrogenases) implicated in abiotic stress and submergence tolerance were also upregulated in transgenic plants. Transgenic plants showed increase in endogenous levels of α-linolenate, several jasmonate derivatives and abscisic acid but not salicylic acid. Soluble modified GFP (SmGFP)-tagged OsEREBP1 was localized to plastid nucleoids. Comparative analysis of non-transgenic and <i>OsEREBP1</i> overexpressing genotypes revealed that <i>OsEREBP1</i> attenuates disease caused by Xoo and confers drought and submergence tolerance in transgenic rice. Our results suggest that constitutive expression of <i>OsEREBP1</i> activates the jasmonate and abscisic acid signalling pathways thereby priming the rice plants for enhanced survival under abiotic or biotic stress conditions. <i>OsEREBP1</i> is thus, a good candidate gene for engineering plants for multiple stress tolerance.</p></div

<i>OsEREBP1-ox</i> plants show reduced susceptibility to bacterial pathogen Xoo.

<p>(A) Leaf lesion development: Six week old plants of Kitaake (control), 4–3 and 2–4 (T3 progeny of transgenic lines) were inoculated by clipping the leaves with a scissor dipped in the Xoo inoculum. Photos of leaves showing lesions 14 days post inoculation. (B) Leaf lesion length: Lesion lengths (in cms) were measured 7, 14 and 21 days post inoculation from 10 inoculated leaves. Values are means ± SD of three replicates. (C) Bacterial growth curves: Three leaves for each of the cultivars 3, 6, 9 and 12 days post inoculation were individually ground in water and plated at various dilutions to get suitable bacterial counts. Each data point represents average and standard deviation of three independent experiments.</p

Subcellular localization of OsEREBP1-SmGFP and Xb22a-SmGFP fusion proteins.

<p>Confocal micrographs of individual rice protoplast transformed with Ubi:<i>SmGFP</i> (SmGFP), Ubi:<i>Xb22a</i>-<i>SmGFP</i> (Xb22a) or Ubi:<i>OsEREBP1</i>-<i>SmGFP</i> (OsEREBP1) plasmids. The columns from left are: GFP-flourescence in green (false color), chlorophyll fluorescence in red (false color), differential interference contrast transmission (DIC) and the merged image.</p

Transcript levels of genes showing upregulation in <i>OsEREBP1-ox</i> plants.

<p>Total RNA was extracted from 4 week old non-transformed (Kit) and transgenic (4–3 and 2–4) plants and qPCR was performed with primers for genes which include (A) <i>NAC6</i>, <i>AP59</i>, <i>EREBP1 & WRKY</i> (transcription factors); (B) <i>Jmt</i>, <i>RERJ & OsLis</i> (Jasmonate biosynthesis and regulation); (C) <i>PBZ</i>, <i>PR10</i>, <i>Lox</i> (Pathogenicity related); (D) <i>CatB</i>, <i>HLH148 & Lea5</i> (ABA pathway). <i>Actin</i> was used as internal standard. Primer details are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127831#pone.0127831.s005" target="_blank">S1 Table</a>. Data representing mean±SE from three independent biological replicates was subjected to further statistical analysis by one way ANOVA using SigmaPlot Version 11.0. The asterix indicate <i>p</i> value of 0.05.</p

Fatty acid composition of total lipids extracted from the leaves of 2–3 week old non-transgenic (KIT) and <i>OsEREBP1-ox</i> (2–4 and 4–3) plants.

<p>Values are mol%± S.D (n = 3)</p><p>16:0, hexadecanoic acid (palmitic acid);</p><p>18:0, octadecanoic acid (stearic acid);</p><p>18:2, ∆ 9,12- octadecadienoic acid (linoleic acid);</p><p>18:3, ∆ 9,12,15-octadecatrienoic acid (α-linolenic acid)</p><p>Fatty acid composition of total lipids extracted from the leaves of 2–3 week old non-transgenic (KIT) and <i>OsEREBP1-ox</i> (2–4 and 4–3) plants.</p

Phenotypes and genotypes of transgenic rice plants.

<p>A) Photographs were taken of six week-old seedlings grown under greenhouse conditions. B) Pictures were taken of plants after the panicle initiation stage. <b>C</b>) Southern blot hybridization of <i>BamH</i>I-digested genomic DNA showed single independent insertions in progenies of 2–4 and 4–3 (transgenic lines), whereas no band was observed in non-transformed Kitaake (control) when probed with hygromycin gene obtained by PCR amplification of pC1300 plasmid using <i>HygS/AS</i> primer pair. D) PCR analysis of genomic DNA using a vector-specific (<i>UbiS</i>) and insert-specific (<i>AP2AS</i>) primer pair showed presence of transgene in the progenies of 2–4 and 4–3 and not in Kitaake. Ap2/pNC1300 plasmid DNA was used as positive control. E) RT-PCR of cDNA using <i>OsEREBP1</i> primers showed increased transcript in 2–4 and 4–3 as compared to Kitaake. <i>EF1a</i> was used to normalize the cDNA. All primer details are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127831#pone.0127831.s005" target="_blank">S1 Table</a>.</p

<i>OsEREBP1</i> confers submergence tolerance in rice.

<p>(A) Two-week old plants were removed from water after 7 days of submergence and photographed. (B) Plant height after submergence treatment. Two-week old plants were submerged for 7 days and plant height of 10 plants was measured at 0 and 7 days after submergence. The error bars represent means ± SD (n = 3) and asterisk indicates that the differences in length were significant (P<0.01) as analyzed by one way ANOVA using SigmaPlot Version 11.0. (C) Plant viability after submergence treatment. Two-week old seedlings of transgenic lines (4–3, 2–4) and Kitaake control were submerged for 14 days and allowed to recover under normal conditions. The plants were scored as viable if they produced new leaves. (D) Accumulation of reactive oxygen species upon submergence. Two-week old seedlings were submerged for 7 days and immediately after desubmergence, the leaves were stained with DAB or NBT to detect hydrogen peroxide or superoxide radicals, respectively.</p

<i>OsEREBP1-ox</i> plants show increased tolerance to drought.

<p>(A) Photos of transgenic rice plants (4–3 and 2–4) and non-transgenic control (Kit) plants subjected to drought stress for 26 days followed by 7 days of watering. (B) Drought tolerance at the reproductive stage. Drought was imposed by withdrawing water at panicle initiation stage till plants were completely brown before they were re-watered. Recovery was indicated by new tillers which formed panicles and grains following rewatering. (C) Percentage recovery of drought stress plants (DS1 and DS2) compared to well-watered ones (WW). Drought was imposed at four leaf stage (DS1) or panicle initiation or booting stage (DS2) till the plants showed complete browning of leaves. Pots were rewatered and percentage recovery was calculated based on the number of plants that produced new leaves out of the total number of plants per pot. Error bars represent mean of three independent experiments.</p