Conserved and non-conserved RNA-target modules in plants: lessons for a better understanding of Marchantia development.

A wide variety of functional regulatory non-coding RNAs (ncRNAs) have been identified as essential regulators of plant growth and development. Depending on their category, ncRNAs are not only involved in modulating target gene expression at the transcriptional and post-transcriptional levels but also are involved in processes like RNA splicing and RNA-directed DNA methylation. To fulfill their molecular roles properly, ncRNAs must be precisely processed by multiprotein complexes. In the case of small RNAs, DICER-LIKE (DCL) proteins play critical roles in the production of mature molecules. Land plant genomes contain at least four distinct classes of DCL family proteins (DCL1-DCL4), of which DCL1, DCL3 and DCL4 are also present in the genomes of bryophytes, indicating the early divergence of these genes. The liverwort Marchantia polymorpha has become an attractive model species for investigating the evolutionary history of regulatory ncRNAs and proteins that are responsible for ncRNA biogenesis. Recent studies on Marchantia have started to uncover the similarities and differences in ncRNA production and function between the basal lineage of bryophytes and other land plants. In this review, we summarize findings on the essential role of regulatory ncRNAs in Marchantia development. We provide a comprehensive overview of conserved ncRNA-target modules among M. polymorpha, the moss Physcomitrium patens and the dicot Arabidopsis thaliana, as well as Marchantia-specific modules. Based on functional studies and data from the literature, we propose new connections between regulatory pathways involved in Marchantia's vegetative and reproductive development and emphasize the need for further functional studies to understand the molecular mechanisms that control ncRNA-directed developmental processes.

Biogenesis, conservation, and function of miRNA in liverworts.

MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules, 18-24 nucleotides long, that control multiple gene regulatory pathways via post-transcriptional gene silencing in eukaryotes. To develop a comprehensive picture of the evolutionary history of miRNA biogenesis and action in land plants, studies on bryophyte representatives are needed. Here, we review current understanding of liverwort MIR gene structure, miRNA biogenesis, and function, focusing on the simple thalloid Pellia endiviifolia and the complex thalloid Marchantia polymorpha. We review what is known about conserved and non-conserved miRNAs, their targets, and the functional implications of miRNA action in M. polymorpha and P. endiviifolia. We note that most M. polymorpha miRNAs are encoded within protein-coding genes and provide data for 23 MIR gene structures recognized as independent transcriptional units. We identify M. polymorpha genes involved in miRNA biogenesis that are homologous to those identified in higher plants, including those encoding core microprocessor components and other auxiliary and regulatory proteins that influence the stability, folding, and processing of pri-miRNAs. We analyzed miRNA biogenesis proteins and found similar domain architecture in most cases. Our data support the hypothesis that almost all miRNA biogenesis factors in higher plants are also present in liverworts, suggesting that they emerged early during land plant evolution.

The identification of differentially expressed genes in male and female gametophytes of simple thalloid liverwort Pellia endiviifolia sp. B using an RNA-seq approach.

MAIN CONCLUSION: This study shows differences in gene expression between male and female gametophytes of the simple thalloid liverwort with a distinction between the vegetative and reproductive phases of growth. Pellia endiviifolia is a simple thalloid liverwort that, together with hornworts and mosses, represents the oldest living land plants. The limited taxon sampling for genomic and functional studies hampers our understanding of processes governing evolution of these plants. RNA sequencing represents an attractive way to elucidate the molecular mechanisms of non-model species development. In the present study, RNA-seq was used to profile the differences in gene expression between P. endiviifolia male and female gametophytes, with a distinction between the vegetative and reproductive phases of growth. By comparison of the gene expression profiles from individuals producing sex organs with the remaining thalli types, we have determined a set of genes whose expression might be important for the development of P. endiviifolia reproductive organs. The selected differentially expressed genes (DEGs) were categorized into five main pathways: metabolism, genetic information processing, environmental information processing, cellular processes, and organismal systems. A comparison of the obtained data with the Marchantia polymorpha transcriptome resulted in the identification of genes exhibiting a similar expression pattern during the reproductive phase of growth between members of the two distinct liverwort classes. The common expression profile of  87 selected genes suggests a common mechanism governing sex organ development in both liverwort species. The obtained RNA-seq results were confirmed by RT-qPCR for the DEGs with the highest differences in expression level. Five Pellia-female-specific and two Pellia-male-specific DEGs showed enriched expression in archegonia and antheridia, respectively. The identified genes are promising candidates for functional studies of their involvement in liverwort sexual reproduction.

mirEX 2.0 - an integrated environment for expression profiling of plant microRNAs.

BACKGROUND: MicroRNAs are the key post-transcriptional regulators of gene expression in development and stress responses. Thus, precisely quantifying the level of each particular microRNA is of utmost importance when studying the biology of any organism. DESCRIPTION: The mirEX 2.0 web portal ( http://www.combio.pl/mirex ) provides a comprehensive platform for the exploration of microRNA expression data based on quantitative Real Time PCR and NGS sequencing experiments, covering various developmental stages, from wild-type to mutant plants. The portal includes mature and pri-miRNA expression levels detected in three plant species (Arabidopsis thaliana, Hordeum vulgare and Pellia endiviifolia), and in A. thaliana miRNA biogenesis pathway mutants. In total, the database contains information about the expression of 461 miRNAs representing 268 families. The data can be explored through the use of advanced web tools, including (i) a graphical query builder system allowing a combination of any given species, developmental stages and tissues, (ii) a modular presentation of the results in the form of thematic windows, and (iii) a number of user-friendly utilities such as a community-building discussion system and extensive tutorial documentation (e.g., tooltips, exemplary videos and presentations). All data contained within the mirEX 2.0 database can be downloaded for use in further applications in a context-based way from the result windows or from a dedicated web page. CONCLUSIONS: The mirEX 2.0 portal provides the plant research community with easily accessible data and powerful tools for application in multi-conditioned analyses of miRNA expression from important plant species in different biological and developmental backgrounds.

The liverwort Pellia endiviifolia shares microtranscriptomic traits that are common to green algae and land plants.

Liverworts are the most basal group of extant land plants. Nonetheless, the molecular biology of liverworts is poorly understood. Gene expression has been studied in only one species, Marchantia polymorpha. In particular, no microRNA (miRNA) sequences from liverworts have been reported. Here, Illumina-based next-generation sequencing was employed to identify small RNAs, and analyze the transcriptome and the degradome of Pellia endiviifolia. Three hundred and eleven conserved miRNA plant families were identified, and 42 new liverwort-specific miRNAs were discovered. The RNA degradome analysis revealed that target mRNAs of only three miRNAs (miR160, miR166, and miR408) have been conserved between liverworts and other land plants. New targets were identified for the remaining conserved miRNAs. Moreover, the analysis of the degradome permitted the identification of targets for 13 novel liverwort-specific miRNAs. Interestingly, three of the liverwort microRNAs show high similarity to previously reported miRNAs from Chlamydomonas reinhardtii. This is the first observation of miRNAs that exist both in a representative alga and in the liverwort P. endiviifolia but are not present in land plants. The results of the analysis of the P. endivifolia microtranscriptome support the conclusions of previous studies that placed liverworts at the root of the land plant evolutionary tree of life.

Female-specific gene expression in dioecious liverwort Pellia endiviifolia is developmentally regulated and connected to archegonia production.

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.

Comparative analysis of SPL transcription factors from streptophyte algae and embryophytes reveals evolutionary trajectories of SPL family in streptophytes.

SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) genes encode plant-specific transcription factors which are important regulators of diverse plant developmental processes. We took advantage of available genome sequences of streptophyte algae representatives to investigate the relationships of SPL genes between freshwater green algae and land plants. Our analysis showed that streptophyte algae, hornwort and liverwort genomes encode from one to four SPL genes which is the smallest set, in comparison to other land plants studied to date. Based on the phylogenetic analysis, four major SPL phylogenetic groups were distinguished with Group 3 and 4 being sister to Group 1 and 2. Comparative motif analysis revealed conserved protein motifs within each phylogenetic group and unique bryophyte-specific motifs within Group 1 which suggests lineage-specific protein speciation processes. Moreover, the gene structure analysis also indicated the specificity of each by identifying differences in exon-intron structures between the phylogenetic groups, suggesting their evolutionary divergence. Since current understanding of SPL genes mostly arises from seed plants, the presented comparative and phylogenetic analyzes from freshwater green algae and land plants provide new insights on the evolutionary trajectories of the SPL gene family in different classes of streptophytes.

Developmentally regulated expression and complex processing of barley pri-microRNAs.

BACKGROUND: MicroRNAs (miRNAs) regulate gene expression via mRNA cleavage or translation inhibition. In spite of barley being a cereal of great economic importance, very little data is available concerning its miRNA biogenesis. There are 69 barley miRNA and 67 pre-miRNA sequences available in the miRBase (release 19). However, no barley pri-miRNA and MIR gene structures have been shown experimentally. In the present paper, we examine the biogenesis of selected barley miRNAs and the developmental regulation of their pri-miRNA processing to learn more about miRNA maturation in barely. RESULTS: To investigate the organization of barley microRNA genes, nine microRNAs - 156g, 159b, 166n, 168a-5p/168a-3p, 171e, 397b-3p, 1120, and 1126 - were selected. Two of the studied miRNAs originate from one MIR168a-5p/168a-3p gene. The presence of all miRNAs was confirmed using a Northern blot approach. The miRNAs are encoded by genes with diverse organizations, representing mostly independent transcription units with or without introns. The intron-containing miRNA transcripts undergo complex splicing events to generate various spliced isoforms. We identified miRNAs that were encoded within introns of the noncoding genes MIR156g and MIR1126. Interestingly, the intron that encodes miR156g is spliced less efficiently than the intron encoding miR1126 from their specific precursors. miR397b-3p was detected in barley as a most probable functional miRNA, in contrast to rice where it has been identified as a complementary partner miRNA*. In the case of miR168a-5p/168a-3p, we found the generation of stable, mature molecules from both pre-miRNA arms, confirming evolutionary conservation of the stability of both species, as shown in rice and maize. We suggest that miR1120, located within the 3' UTR of a protein-coding gene and described as a functional miRNA in wheat, may represent a siRNA generated from a mariner-like transposable element. CONCLUSIONS: Seven of the eight barley miRNA genes characterized in this study contain introns with their respective transcripts undergoing developmentally specific processing events prior to the dicing out of pre-miRNA species from their pri-miRNA precursors. The observed tendency to maintain the intron encoding miR156g within the transcript, and preferences in splicing the miR1126-harboring intron, may suggest the existence of specific regulation of the levels of intron-derived miRNAs in barley.

Recent Insights into Plant miRNA Biogenesis: Multiple Layers of miRNA Level Regulation.

MicroRNAs are small RNAs, 20-22 nt long, the main role of which is to downregulate gene expression at the level of mRNAs. MiRNAs are fundamental regulators of plant growth and development in response to internal signals as well as in response to abiotic and biotic factors. Therefore, the deficiency or excess of individual miRNAs is detrimental to particular aspects of a plant's life. In consequence, the miRNA levels must be appropriately adjusted. To obtain proper expression of each miRNA, their biogenesis is controlled at multiple regulatory layers. Here, we addressed processes discovered to influence miRNA steady-state levels, such as MIR transcription, co-transcriptional pri-miRNA processing (including splicing, polyadenylation, microprocessor assembly and activity) and miRNA-encoded peptides synthesis. MiRNA stability, RISC formation and miRNA export out of the nucleus and out of the plant cell also define the levels of miRNAs in various plant tissues. Moreover, we show the evolutionary conservation of miRNA biogenesis core proteins across the plant kingdom.

Novel candidate genes AuxRP and Hsp90 influence the chip color of potato tubers.

Potato (Solanum tuberosum L.) tubers exhibit significant variation in reducing sugar content directly after harvest, cold storage and reconditioning. Here, we performed QTL analysis for chip color, which is strongly influenced by reducing sugar content, in a diploid potato mapping population. Two QTL on chromosomes I and VI were detected for chip color after harvest and reconditioning. Only one region on chromosome VI was linked with cold-induced sweetening. Using the RT-PCR technique, we showed differential expression of the auxin-regulated protein (AuxRP) gene. The AuxRP transcript was presented in light chip color parental clone DG 97-952 and the RNA progeny of the bulk sample consisting of light chip color phenotypes after cold storage. This amplicon was absent in dark chip parental clone DG 08-26/39 and the RNA bulk sample of dark chip progeny. Genetic variation of AuxRP explained up to 16.6 and 15.2 % of the phenotypic variance after harvest and 3 months of storage at 4 °C, respectively. Using an alternative approach, the RDA-cDNA method was used to recognize 25 gene sequences, of which 11 could be assigned to potato chromosome VI. One of these genes, Heat-shock protein 90 (Hsp90), demonstrated higher mRNA and protein expression in RT-qPCR and western blotting assays in the dark chip color progeny bulk sample compared with the light chip color progeny bulk sample. Our study, for the first time, suggests that the AuxRP and Hsp90 genes are novel candidate genes capable of influencing the chip color of potato tubers.

Novel genes specifically expressed during the development of the male thalli and antheridia in the dioecious liverwort Pellia endiviifolia.

In bryophytes (lower plants), sex determination is manifested in the gametophyte generation by the production of egg- and sperm-forming gametangia. We identified four genes specifically expressed in the male thalli of dioecious liverwort Pellia endiviifolia species B using RDA-cDNA method. These are: PenB_TUA1 coding for an α-tubulin family protein, PenB_Raba1/11 coding for a Rab family protein, PenB_HMG-box coding for an HMG-box family protein and PenB_MT coding for an unknown transcript that contains an ORF of 295 amino acid residues. The expression of identified genes shows developmental and environmental regulation. PenB_TUA1 and PenB_Raba1/11 are expressed in the male thalli, regardless of whether they develop antheridia. PenB_HMG-box and PenB_MT are exclusively expressed in the male thalli-producing antheridia while growing in the field. Moreover, two genes PenB_TUA1 and PenB_Raba1/11 are encoded only in the male genome of P. endiviifolia sp B. Our studies show for the first time the specific contribution of identified genes in the liverwort male gametophyte development. In higher plants, correct regulation of α-tubulin and Rab family genes activity is essential for tip-focused membrane trafficking and growth of the male gametophyte. Thus these genes are critical to the reproductive success of these plants. Plant HMG-box proteins bind DNA and may affect chromatin structure, promoting the assembly of nucleoprotein complexes that control DNA-dependent processes including transcription. Our results show that genes connected with the gametogenesis processes are evolutionarily conserved from the liverworts - the oldest living land plants, to higher plants.