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Differential expression patterns of arabinogalactan proteins in Arabidopsis thaliana reproductive tissues.

ABSTRACT: Arabinogalactan proteins (AGPs) are heavily glycosylated proteins existing in all members of the plant kingdom and are differentially distributed through distinctive developmental stages. Here, we showed the individual distributions of specific Arabidopsis AGPs: AGP1, AGP9, AGP12, AGP15, and AGP23, throughout reproductive tissues and indicated their possible roles in several reproductive processes. AGP genes specifically expressed in female tissues were identified using available microarray data. This selection was confirmed by promoter analysis using multiple green fluorescent protein fusions to a nuclear localization signal, ?-glucuronidase fusions, and in situ hybridization as approaches to confirm the expression patterns of the AGPs. Promoter analysis allowed the detection of a specific and differential presence of these proteins along the pathway followed by the pollen tube during its journey to reach the egg and the central cell inside the embryo sac. AGP1 was expressed in the stigma, style, transmitting tract, and the chalazal and funiculus tissues of the ovules. AGP9 was present along the vasculature of the reproductive tissues and AGP12 was expressed in the stigmatic cells, chalazal and funiculus cells of the ovules, and in the septum. AGP15 was expressed in all pistil tissues, except in the transmitting tract, while AGP23 was specific to the pollen grain and pollen tube. The expression pattern of these AGPs provides new evidence for the detection of a subset of specific AGPs involved in plant reproductive processes, being of significance for this field of study. AGPs are prominent candidates for male-female communication during reproduction.

SUBMITTER: Pereira AM

PROVIDER: S-EPMC4400541 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Differential expression patterns of arabinogalactan proteins in Arabidopsis thaliana reproductive tissues.

Pereira Ana Marta AM Masiero Simona S Nobre Margarida Sofia MS Costa Mário Luís ML Solís María-Teresa MT Testillano Pilar S PS Sprunck Stefanie S Coimbra Sílvia S

Journal of experimental botany 20140722 18

Arabinogalactan proteins (AGPs) are heavily glycosylated proteins existing in all members of the plant kingdom and are differentially distributed through distinctive developmental stages. Here, we showed the individual distributions of specific Arabidopsis AGPs: AGP1, AGP9, AGP12, AGP15, and AGP23, throughout reproductive tissues and indicated their possible roles in several reproductive processes. AGP genes specifically expressed in female tissues were identified using available microarray data ...[more]

PMID: 25053647

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Project description:Arabinogalactan proteins (AGPs) are hydroxyproline-rich glycoproteins containing a high proportion of carbohydrates, widely distributed in the plant kingdom and ubiquitously present in land plants. AGPs have long been suggested to play important roles in plant reproduction and there is already evidence that specific glycoproteins are essential for male and female gametophyte development, pollen tube growth and guidance, and successful fertilization. However, the functions of many of these proteins have yet to be uncovered, mainly due to the difficulty to study individual AGPs. In this work, we generated molecular tools to analyze the expression patterns of a subgroup of individual AGPs in different Arabidopsis tissues, focusing on reproductive processes. This study focused on six AGPs: four classical AGPs (AGP7, AGP25, AGP26, AGP27), one AG peptide (AGP24) and one chimeric AGP (AGP31). These AGPs were first selected based on their predicted expression patterns along the reproductive tissues from available RNA-seq data. Promoter analysis using β-glucuronidase fusions and qPCR in different Arabidopsis tissues allowed to confirm these predictions. AGP7 was mainly expressed in female reproductive tissues, more precisely in the style, funiculus, and integuments near the micropyle region. AGP25 was found to be expressed in the style, septum and ovules with higher expression in the chalaza and funiculus tissues. AGP26 was present in the ovules and pistil valves. AGP27 was expressed in the transmitting tissue, septum and funiculus during seed development. AGP24 was expressed in pollen grains, in mature embryo sacs, with highest expression at the chalazal pole and in the micropyle. AGP31 was expressed in the mature embryo sac with highest expression at the chalaza and, occasionally, in the micropyle. For all these AGPs a co-expression analysis was performed providing new hints on its possible functions. This work confirmed the detection in Arabidopsis male and female tissues of six AGPs never studied before regarding the reproductive process. These results provide novel evidence on the possible involvement of specific AGPs in plant reproduction, as strong candidates to participate in pollen-pistil interactions in an active way, which is significant for this field of study.

Project description:BackgroundThe fasciclin-like arabinogalactan proteins (FLAs) belong to the arabinogalactan protein (AGP) superfamily and are known to play different physiological roles in plants. This class of proteins was shown to participate in plant growth, development, defense against abiotic stresses and, notably, cell wall biosynthesis. Although some studies are available on the characterization of FLA genes from different species, both woody and herbaceous, no detailed information is available on the FLA family of textile hemp (Cannabis sativa L.), an economically important fibre crop.ResultsBy searching the Cannabis genome and EST databases, 23 CsaFLAs have been here identified which are divided into four phylogenetic groups. A real-time qPCR analysis performed on stem tissues (isolated bast fibres and shivs sampled at three heights), hypocotyls (6-9-12-15-17-20 days-old), whole seedlings, roots, leaves and female/male flowers of the monoecious fibre variety Santhica 27, indicates that the identified FLA genes are differentially expressed. Interestingly, some hemp FLAs are expressed during early phases of fibre growth (elongation), while others are more expressed in the middle and base of the stem and thus potentially involved in secondary cell wall formation (fibre thickening). The bioinformatic analysis of the promoter regions shows that the FLAs upregulated in the younger regions of the stem share a conserved motif related to flowering control and regulation of photoperiod perception. The promoters of the FLA genes expressed at higher levels in the older stem regions, instead, share a motif putatively recognized by MYB3, a transcriptional repressor belonging to the MYB family subgroup S4.ConclusionsThese results point to the existence of a transcriptional network fine-tuning the expression of FLA genes in the older and younger regions of the stem, as well as in the bast fibres/shivs of textile hemp. In summary, our study paves the way for future analyses on the biological functions of FLAs in an industrially relevant fibre crop.

Project description:RNA-seq is currently the only method that can provide a comprehensive landscape of circular RNA (circRNAs) in the whole organism and its particular organs. Recent years have brought an increasing number of RNA-seq-based reports on plant circRNAs. Notably, the picture they revealed is questionable and depends on the applied circRNA identification and quantification techniques. In consequence, little is known about the biogenesis and functions of circRNAs in plants. In this work, we tested two experimental and six bioinformatics procedures of circRNA analysis to determine the optimal approach for studying the profiles of circRNAs in Arabidopsis thaliana. Then using the optimized strategy, we determined the accumulation of circular and corresponding linear transcripts in plant seedlings and organs. We observed that only a small fraction of circRNAs was reproducibly generated. Among them, two groups of circRNAs were discovered: ubiquitous and organ-specific. The highest number of circRNAs with significantly increased accumulation in comparison to other organs/seedlings was found in roots. The circRNAs in seedlings, leaves and flowers originated mainly from genes involved in photosynthesis and the response to stimulus. The levels of circular and linear transcripts were not correlated. Although RNase R treatment enriches the analyzed RNA samples in circular transcripts, it may also have a negative impact on the stability of some of the circRNAs. We also showed that the normalization of NGS data by the library size is not proper for circRNAs quantification. Alternatively, we proposed four other normalization types whose accuracy was confirmed by ddPCR. Moreover, we provided a comprehensive characterization of circRNAs in A. thaliana organs and in seedlings. Our analyses revealed that plant circRNAs are formed in both stochastic and controlled processes. The latter are less frequent and likely engage circRNA-specific mechanisms. Only a few circRNAs were organ-specific. The lack of correlation between the accumulation of linear and circular transcripts indicated that their biogenesis depends on different mechanisms.

Project description:Arabinogalactan protein 31 (AGP31) is a remarkable plant cell-wall protein displaying a multi-domain organization unique in Arabidopsis thaliana: it comprises a predicted signal peptide (SP), a short AGP domain of seven amino acids, a His-stretch, a Pro-rich domain and a PAC (PRP-AGP containing Cys) domain. AGP31 displays different O-glycosylation patterns with arabinogalactans on the AGP domain and Hyp-O-Gal/Ara-rich motifs on the Pro-rich domain. AGP31 has been identified as an abundant protein in cell walls of etiolated hypocotyls, but its function has not been investigated thus far. Literature data suggest that AGP31 may interact with cell-wall components. The purpose of the present study was to identify AGP31 partners to gain new insight into its function in cell walls.Nitrocellulose membranes were prepared by spotting different polysaccharides, which were either obtained commercially or extracted from cell walls of Arabidopsis thaliana and Brachypodium distachyon. After validation of the arrays, in vitro interaction assays were carried out by probing the membranes with purified native AGP31 or recombinant PAC-V5-6xHis. In addition, dynamic light scattering (DLS) analyses were carried out on an AGP31 purified fraction.It was demonstrated that AGP31 interacts through its PAC domain with galactans that are branches of rhamnogalacturonan I. This is the first experimental evidence that a PAC domain, also found as an entire protein or a domain of AGP31 homologues, can bind carbohydrates. AGP31 was also found to bind methylesterified polygalacturonic acid, possibly through its His-stretch. Finally, AGP31 was able to interact with itself in vitro through its PAC domain. DLS data showed that AGP31 forms aggregates in solution, corroborating the hypothesis of an auto-assembly.These results allow the proposal of a model of interactions of AGP31 with different cell-wall components, in which AGP31 participates in complex supra-molecular scaffolds. Such scaffolds could contribute to the strengthening of cell walls of quickly growing organs such as etiolated hypocotyls.

Project description:The SeedGenes database (www.seedgenes.org) contains information on more than 400 genes required for embryo development in Arabidopsis. Many of these EMBRYO-DEFECTIVE (EMB) genes encode proteins with an essential function required throughout the life cycle. This raises a fundamental question. Why does elimination of an essential gene in Arabidopsis often result in embryo lethality rather than gametophyte lethality? In other words, how do mutant (emb) gametophytes survive and participate in fertilization when an essential cellular function is disrupted? Furthermore, why do some mutant embryos proceed further in development than others? To address these questions, we first established a curated dataset of genes required for gametophyte development in Arabidopsis based on information extracted from the literature. This provided a basis for comparison with EMB genes obtained from the SeedGenes dataset. We also identified genes that exhibited both embryo and gametophyte defects when disrupted by a loss-of-function mutation. We then evaluated the relationship between mutant phenotype, gene redundancy, mutant allele strength, gene expression pattern, protein function, and intracellular protein localization to determine what factors influence the phenotypes of lethal mutants in Arabidopsis. After removing cases where continued development potentially resulted from gene redundancy or residual function of a weak mutant allele, we identified numerous examples of viable mutant (emb) gametophytes that required further explanation. We propose that the presence of gene products derived from transcription in diploid (heterozygous) sporocytes often enables mutant gametophytes to survive the loss of an essential gene in Arabidopsis. Whether gene disruption results in embryo or gametophyte lethality therefore depends in part on the ability of residual, parental gene products to support gametophyte development. We also highlight here 70 preglobular embryo mutants with a zygotic pattern of inheritance, which provide valuable insights into the maternal-to-zygotic transition in Arabidopsis and the timing of paternal gene activation during embryo development.

Project description:BackgroundNatural accessions of Arabidopsis thaliana are a well-known system to measure levels of intraspecific genetic variation. Leaf starch content correlates negatively with biomass. Starch is synthesized by the coordinated action of many (iso)enzymes. Quantitatively dominant is the repetitive transfer of glucosyl residues to the non-reducing ends of ?-glucans as mediated by starch synthases. In the genome of A. thaliana, there are five classes of starch synthases, designated as soluble starch synthases (SSI, SSII, SSIII, and SSIV) and granule-bound synthase (GBSS). Each class is represented by a single gene. The five genes are homologous in functional domains due to their common origin, but have evolved individual features as well. Here, we analyze the extent of genetic variation in these fundamental protein classes as well as possible functional implications on transcript and protein levels.FindingsIntraspecific sequence variation of the five starch synthases was determined by sequencing the entire loci including promoter regions from 30 worldwide distributed accessions of A. thaliana. In all genes, a considerable number of nucleotide polymorphisms was observed, both in non-coding and coding regions, and several amino acid substitutions were identified in functional domains. Furthermore, promoters possess numerous polymorphisms in potentially regulatory cis-acting regions. By realtime experiments performed with selected accessions, we demonstrate that DNA sequence divergence correlates with significant differences in transcript levels.ConclusionsExcept for AtSSII, all starch synthase classes clustered into two or three groups of haplotypes, respectively. Significant difference in transcript levels among haplotype clusters in AtSSIV provides evidence for cis-regulation. By contrast, no such correlation was found for AtSSI, AtSSII, AtSSIII, and AtGBSS, suggesting trans-regulation. The expression data presented here point to a regulation by common trans-regulatory transcription factors which ensures a coordinated action of the products of these four genes during starch granule biosynthesis. The apparent cis-regulation of AtSSIV might be related to its role in the initiation of de novo biosynthesis of granules.

Dataset Information

Differential expression patterns of arabinogalactan proteins in Arabidopsis thaliana reproductive tissues.

Publications

Differential expression patterns of arabinogalactan proteins in Arabidopsis thaliana reproductive tissues.

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