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Female-specific Gene Expression in Dioecious Liverwort Pellia Endiviifolia Is Developmentally Regulated and Connected to Archegonia Production

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Female-specific Gene Expression in Dioecious Liverwort Pellia Endiviifolia Is Developmentally Regulated and Connected to Archegonia Production

Izabela Sierocka et al. BMC Plant Biol.

Abstract

Background: In flowering plants a number of genes have been identified which control the transition from a vegetative to generative phase of life cycle. In bryophytes representing basal lineage of land plants, there is little data regarding the mechanisms that control this transition. Two species from bryophytes - moss Physcomitrella patens and liverwort Marchantia polymorpha are under advanced molecular and genetic research. The goal of our study was to identify genes connected to female gametophyte development and archegonia production in the dioecious liverwort Pellia endiviifolia species B, which is representative of the most basal lineage of the simple thalloid liverworts.

Results: The utility of the RDA-cDNA technique allowed us to identify three genes specifically expressed in the female individuals of P.endiviifolia: PenB_CYSP coding for cysteine protease, PenB_MT2 and PenB_MT3 coding for Mysterious Transcripts1 and 2 containing ORFs of 143 and 177 amino acid residues in length, respectively. The exon-intron structure of all three genes has been characterized and pre-mRNA processing was investigated. Interestingly, five mRNA isoforms are produced from the PenB_MT2 gene, which result from alternative splicing within the second and third exon. All observed splicing events take place within the 5'UTR and do not interfere with the coding sequence. All three genes are exclusively expressed in the female individuals, regardless of whether they were cultured in vitro or were collected from a natural habitat. Moreover we observed ten-fold increased transcripts level for all three genes in the archegonial tissue in comparison to the vegetative parts of the same female thalli grown in natural habitat suggesting their connection to archegonia development.

Conclusions: We have identified three genes which are specifically expressed in P.endiviifolia sp B female gametophytes. Moreover, their expression is connected to the female sex-organ differentiation and is developmentally regulated. The contribution of the identified genes may be crucial for successful liverwort sexual reproduction.

Figures

Figure 1
Figure 1
RDA-cDNA amplicon analysis on 1.5% agarose gels. (A) cDNA of the TESTER (F-female) and the DRIVER (M-male) amplicons are shown. 1 kb marker is on the right of the gel. (B) Difference products (DP) after first (DPI), second (DPII), third (DPIII), and fourth (DPIV) round of subtractive hybridization are presented.100 bp marker is on the left of the gels.
Figure 2
Figure 2
Quantitative analyses of three cDNA fragments identified in RDA-cDNA experiment, representing fragments of three P.endiviifolia sp B genes: cysteine protease ( PenB_CYSP ), mysterious transcripts 2 and 3 ( PenB_MT2, PenB_MT3 ). (A) Semi-quantitative RT-PCR and (B) qPCR analysis using RNA isolated from the female (lane 1 and light bars, respectively) and the male (lane 2 and dark bars, respectively) thalli producing sex organs. The results of PCR reaction without template are shown in lane 3. The 100 bp ladder is on the right of the gels. All transcript levels were normalized against ACTIN1. Calculation shows the mean ± SD from three technical replicates. PenB_TUA1 gene encoding α-tubulin was used as a control of male specifically expressed gene.
Figure 3
Figure 3
The full-length genes (lanes 1 in panels A-C) and their transcripts (lanes 2 in panels A-C) analyzed on 1% agarose gels. (A)PenB_CYSP, (B)PenB_MT2 and (C)PenB_MT3. The PCR reaction without template is shown in lanes 3. 1 kb + ladder is on the right of the gels.
Figure 4
Figure 4
PenB_CYSP gene and its transcript structure and PenB_CYSP protien homology based model. (A) Schematic representation of the PenB_CYSP gene and its transcript. Exons are represented by boxes, introns by lines; dark grey boxes denote 5′ and 3′ UTRs; light grey boxes denote the coding sequence. The black triangle indicates a bioinformatically identified polyadenylation signal. (B) Homology based model of PenB_CYSP protein. Model was build using I-Tasser server based on 7PCK template (left side). First 92 N terminal residues (orange) represent procathepsin variable region (thus this part of the model is the least reliable, mostly modeled on secondary structure and transmembrane helix restrains). This region contains putative signal peptide and transmembrane domain. Next, there is propeptide inhibitor domain I29 (cyan), residues 93–157. The rest of the protein constitute cathepsin peptidase C1 domain (grey). Boxed part of C1 domain enlarged on right contains catalytic dyad with important residues (red) and residues of S2 pocket (green) which is responsible for substrate specific binding.
Figure 5
Figure 5
PenB_MT2 gene, its mRNA isoform structures, RT-qPCR analysis of mRNA isoforms abundance, and a model of the Pen_MT2 protein secondary structure. (A) Schematic representation of the PenB_MT2 gene and its five mRNA isoforms. All designations are the same as in Figure 4. Real-time PCR analysis for the quantification of five alternatively spliced isoforms of PenB_MT2 gene in P. endiviifolia sp B female thalli-producing archegonia (B). Material was collected in the third season (2008) from the natural habitat. Calculation shows mean ± SD from three technical replicates. (C) Amino acid sequence of predicted PenB_MT2 protein. Transmembrane (tmm) and secondary structure (ss) prediction represent consensus prediction from GeneSilico metaserver; black barrels represent α-helises and white arrows represent β-sheets. Intrinsically unstructured residues (disorder) were predicted by GeneSilico MetaDisorder.
Figure 6
Figure 6
PenB_MT3 gene, its mRNA structures, and a model of the Pen_MT3 protein secondary structure. (A) Schematic representation of the PenB_MT3 gene and its transcript. All designations are the same as in Figure 4. (B) Amino acid sequence of predicted PenB_MT3 protein. Transmembrane (tmm) and secondary structure (ss) prediction represent consensus prediction from GeneSilico metaserver; black barrels represent α-helises and white arrows represent β-sheets. Intrinsically unstructured residues (disorder) were predicted by GeneSilico MetaDisorder.
Figure 7
Figure 7
Transcript levels of PenB_CYSP, PenB_MT2 and PenB_MT3 genes are regulated by growth conditions. (A) Transcripts levels of PenB_CYSP, PenB_MT2 analyzed using semi-quantitative RT-PCR. RNA was isolated in an independent experiment from: the female thalli without archegonia cultivated in vitro (lane 1), the female thalli producing archegonia collected from the natural habitat (lane 2), the male thalli without antheridia cultivated in vitro (lane 3), and the male thalli producing antheridia collected from the natural habitat (lane 4). Specific expression of three identified genes in the female thalli was analyzed by semi-quantitative RT-PCR. Negative control is shown in lane 5 and 100 bp ladder is on the right of the gels. (B) RT-qPCR analyses of three studied genes expression show different transcripts levels in the female thalli. All transcript levels were normalized against ACTIN1. All transcript levels were normalized against ACTIN1. As a control of comparable gene expression in the tested female thalli primers amplifying fragment of histone H4 were used. Calculation shows the mean ± SD from three technical replicates.
Figure 8
Figure 8
Transcripts levels of PenB_CYSP, PenB_MT2 and PenB_MT3 genes are connected to with archegonia development. Quantitative real-time PCR analyses of three studied genes show different transcripts levels in the vegetative and archegonia bearing parts of female gametophytes grown in natural habitat. All transcript levels were normalized against ACTIN1. As a control of comparable gene expression in the tested female thalli primers amplifying fragment of histone H4 were used. Calculation shows the mean ± SD from three technical replicates.

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