Land Plant Genomes Research Articles

Extensive import of nucleus-encoded tRNAs into chloroplasts of the photosynthetic lycophyte, Selaginella kraussiana

Over the course of evolution, land plant mitochondrial genomes have lost many transfer RNA (tRNA) genes and the import of nucleus-encoded tRNAs is essential for mitochondrial protein synthesis. By contrast, plastidial genomes of photosynthetic land plants generally possess a complete set of tRNA genes and the existence of plastidial tRNA import remains a long-standing question. The early vascular plants of the Selaginella genus show an extensive loss of plastidial tRNA genes while retaining photosynthetic capacity, and represent an ideal model for answering this question. Using purification, northern blot hybridization, and high-throughput tRNA sequencing, a global analysis of total and plastidial tRNA populations was undertaken in Selaginella kraussiana. We confirmed the expression of all plastidial tRNA genes and, conversely, observed that nucleus-encoded tRNAs corresponding to these plastidial tRNAs were generally excluded from the chloroplasts. We then demonstrated a selective and differential plastidial import of around forty nucleus-encoded tRNA species, likely compensating for the insufficient coding capacity of plastidial-encoded tRNAs. In-depth analysis revealed differential import of tRNA isodecoders, leading to the identification of specific situations. This includes the expression and import of nucleus-encoded tRNAs expressed from plastidial or bacterial-like genes inserted into the nuclear genome. Overall, our results confirm the existence of molecular processes that enable tRNAs to be selectively imported not only into mitochondria, as previously described, but also into chloroplasts, when necessary.

A land plant phylogenetic framework for GLABROUS INFLORESCENCE STEMS (GIS), SUPERMAN, JAGGED and allies plus their TOPLESS co-repressor

Members of the plant specific family of C1-1i zincfinger transcriptionfactors (ZF-TFs), such as SUPERMAN, JAGGED, KNUCKLES or GIS,regulatediversedevelopmental processes including sexual reproduction. C1-1is consist of one zinc-finger and one to two EAR domains, connected by large intrinsically disordered regions (IDR). While the role of C1-i1 ZF-TFs in development processes is well known for some genes in Arabidopsis, rice or tomatoa comprehensive and broadphylogenetic background is lacking, yet knowledge of orthology is a requirement for a better understanding of C1-1i-Zf-TFs diverse roles in plants. Here, we provide a fine-grained and land plant wide classification of C1-1i sub-families and their known co-repressors TOPLESS and TOPLESS RELATED. Our work combines the identification of orthologous groups with Maximum-Likelihood phylogeny reconstructions and digital gene expression analyses mining high quality land plant genomes and transcriptomes to generate a comprehensive framework of C1-1i ZF-TF evolution. We show that C1-1i’s are low to moderate copy genesand that orthologous genesonly partiallyhaveconserved sub-family and life cycle stage dependent expression pattern across land plants while others are highly diverged. Our workprovides the phylogenetic framework for C1-1i ZF-TFs, s and strengthen C1-1 ZF-TFs as a potential model for IDR-research in plants.

Post-transfer adaptation of HGT-acquired genes and contribution to guanine metabolic diversification in land plants.

Horizontal gene transfer (HGT) is a major driving force in the evolution of prokaryotic and eukaryotic genomes. Despite recent advances in distribution and ecological importance, the extensive pattern, especially in seed plants, and post-transfer adaptation of HGT-acquired genes in land plants remain elusive. We systematically identified 1150 foreign genes in 522 land plant genomes that were likely acquired via at least 322 distinct transfers from nonplant donors and confirmed that recent HGT events were unevenly distributed between seedless and seed plants. HGT-acquired genes evolved to be more similar to native genes in terms of average intron length due to intron gains, and HGT-acquired genes containing introns exhibited higher expression levels than those lacking introns, suggesting that intron gains may be involved in the post-transfer adaptation of HGT in land plants. Functional validation of bacteria-derived gene GuaD in mosses and gymnosperms revealed that the invasion of foreign genes introduced a novel bypass of guanine degradation and resulted in the loss of native pathway genes in some gymnosperms, eventually shaping three major types of guanine metabolism in land plants. We conclude that HGT has played a critical role in land plant evolution.

Mitochondrial RNA Helicases: Key Players in the Regulation of Plant Organellar RNA Splicing and Gene Expression.

Mitochondrial genomes of land plants are large and exhibit a complex mode of gene organization and expression, particularly at the post-transcriptional level. The primary organellar transcripts in plants undergo extensive maturation steps, including endo- and/or exo-nucleolytic cleavage, RNA-base modifications (mostly C-to-U deaminations) and both 'cis'- and 'trans'-splicing events. These essential processing steps rely on the activities of a large set of nuclear-encoded factors. RNA helicases serve as key players in RNA metabolism, participating in the regulation of transcription, mRNA processing and translation. They unwind RNA secondary structures and facilitate the formation of ribonucleoprotein complexes crucial for various stages of gene expression. Furthermore, RNA helicases are involved in RNA metabolism by modulating pre-mRNA maturation, transport and degradation processes. These enzymes are, therefore, pivotal in RNA quality-control mechanisms, ensuring the fidelity and efficiency of RNA processing and turnover in plant mitochondria. This review summarizes the significant roles played by helicases in regulating the highly dynamic processes of mitochondrial transcription, RNA processing and translation in plants. We further discuss recent advancements in understanding how dysregulation of mitochondrial RNA helicases affects the splicing of organellar genes, leading to respiratory dysfunctions, and consequently, altered growth, development and physiology of land plants.

Both-strand gene coding in a plastome-like mitogenome of an enoplid nematode.

The phylum Nematoda remains very poorly sampled for mtDNA, with a strong bias toward parasitic, economically important or model species of the Chromadoria lineage. Most chromadorian mitogenomes share a specific order of genes encoded on one mtDNA strand. However, the few sequenced representatives of the Dorylaimia lineage exhibit a variable order of mtDNA genes encoded on both strands. While the ancestral arrangement of nematode mitogenome remains undefined, no evidence has been reported for Enoplia, the phylum's third early divergent major lineage. We describe the first mitogenome of an enoplian nematode, Campydora demonstrans, and contend that the complete 37-gene repertoire and both-strand gene encoding are ancestral states preserved in Enoplia and Dorylaimia versus the derived mitogenome arrangement in some Chromadoria. The C. demonstrans mitogenome is 17,018 bp in size and contains a noncoding perfect inverted repeat with 2013 bp-long arms, subdividing the mitogenome into two coding regions. This mtDNA arrangement is very rare among animals and instead resembles that of chloroplast genomes in land plants. Our report broadens mtDNA taxonomic sampling of the phylum Nematoda and adds support to the applicability of cox1 gene as a phylogenetic marker for establishing nematode relationships within higher taxa.

Methods for assembling complex mitochondrial genomes in land plants.

The large size and complex structural rearrangements inherent in the mitochondrial genomes of land plants pose challenges for their sequencing. Originally, the assembly of these genomes required the cloning of mitochondrial DNA fragments followed by Sanger sequencing. Subsequently, the advent of next-generation sequencing significantly expedited the process. This review highlights examples of plant mitochondrial genome assembly employing various technologies, including 454 sequencing, Illumina short sequencing reads, and Pacific Biosciences or Oxford Nanopore Technology long sequencing reads. The combination of short and long reads in hybrid assembly has proven to be the most efficient approach for achieving reliable assemblies of land plant mitochondrial genomes.

SUPPRESSOR OF MAX2 1-LIKE (SMXL) homologs are MAX2-dependent repressors of Physcomitrium patens growth.

SUPPRESSOR OF MAX2 (SMAX)1-LIKE (SMXL) proteins are a plant-specific clade of type I HSP100/Clp-ATPases. SMXL genes are present in virtually all land plant genomes. However, they have mainly been studied in angiosperms. In Arabidopsis (Arabidopsis thaliana), 3 functional SMXL subclades have been identified: SMAX1/SMXL2, SMXL345, and SMXL678. Of these, 2 subclades ensure endogenous phytohormone signal transduction. SMAX1/SMXL2 proteins are involved in KAI2 ligand (KL) signaling, while SMXL678 proteins are involved in strigolactone (SL) signaling. Many questions remain regarding the mode of action of these proteins, as well as their ancestral roles. We addressed these questions by investigating the functions of the 4 SMXL genes in the moss Physcomitrium patens. We demonstrate that PpSMXL proteins are involved in the conserved ancestral MAX2-dependent KL signaling pathway and negatively regulate growth. However, PpSMXL proteins expressed in Arabidopsis cannot replace SMAX1 or SMXL2 function in KL signaling, whereas they can functionally replace SMXL4 and SMXL5 and restore root growth. Therefore, the molecular functions of SMXL proteins are conserved, but their interaction networks are not. Moreover, the PpSMXLC/D clade positively regulates SL signal transduction in P. patens. Overall, our data reveal that SMXL proteins in moss mediate crosstalk between the SL and KL signaling pathways.

Land plant peptide signaling: What we know—and don't know—about its evolution

AbstractThe availability of genome sequences from diverse algal and plant taxa combined with the refinement of comparative genomics tools has begun to reveal how land plant genomes were shaped through duplication, repeat expansion, and gene family gains and losses. Of particular note is a large increase in the complexity and variety of signaling systems in land plants. Among these, signaling through small peptide ligand‐receptor interactions has been considered one of the major innovations during land plant evolution. First discovered in angiosperms as mediators of various cell‐to‐cell communication processes, peptide signaling studies have been expanded to non‐angiosperms, including bryophytes. Recent studies point to both common and unique roles for peptide signaling in distantly related species, raising interesting questions about how peptide signaling systems evolved and diversified. While the origin of peptide signaling systems remains elusive, progress in sequencing algal genomes offers clues to understanding the evolution of peptide receptors. This article discusses recent studies of small peptide‐mediated signaling systems and highlights current gaps in our knowledge and new avenues for research, which could help us address how peptide signaling systems evolved and contributed to plant terrestrialization.

Conserved functions of chromatin regulators in basal Archaeplastida.

Chromatin is a dynamic network that regulates genome organization and gene expression. Different types of chromatin regulators are highly conserved among Archaeplastida, including unicellular algae, while some chromatin genes are only present in land plant genomes. Here, we review recent advances in understanding the function of conserved chromatin factors in basal land plants and algae. We focus on the role of Polycomb-group genes which mediate H3K27me3-based silencing and play a role in balancing gene dosage and regulating haploid-to-diploid transitions by tissue-specific repression of the transcription factors KNOX and BELL in many representatives of the green lineage. Moreover, H3K27me3 predominantly occupies repetitive elements which can lead to their silencing in a unicellular alga and basal land plants, while it covers mostly protein-coding genes in higher land plants. In addition, we discuss the role of nuclear matrix constituent proteins as putative functional lamin analogs that are highly conserved among land plants and might have an ancestral function in stress response regulation. In summary, our review highlights the importance of studying chromatin regulation in a wide range of organisms in the Archaeplastida.

TRIMETHYLGUANOSINE SYNTHASE1 mutations decanalize female germline development in Arabidopsis.

Here, we report the characterization of a plant RNA methyltransferase, orthologous to yeast trimethylguanosine synthase1 (Tgs1p) and whose downregulation was associated with apomixis in Paspalum grasses. Using phylogenetic analyses and yeast complementation, we determined that land plant genomes all encode a conserved, specific TGS1 protein. Next, we studied the role of TGS1 in female reproduction using reporter lines and loss-of-function mutants in Arabidopsis thaliana. pAtTGS1:AtTGS1 reporters showed a dynamic expression pattern. They were highly active in the placenta and ovule primordia at emergence but, subsequently, showed weak signals in the nucellus. Although expressed throughout gametophyte development, activity became restricted to the female gamete and was also detected after fertilization during embryogenesis. TGS1 depletion altered the specification of the precursor cells that give rise to the female gametophytic generation and to the sporophyte, resulting in the formation of a functional aposporous-like lineage. Our results indicate that TGS1 participates in the mechanisms restricting cell fate acquisition to a single cell at critical transitions throughout the female reproductive lineage and, thus, expand our current knowledge of the mechanisms governing female reproductive fate in plants.

Single-molecule Sequencing of an Animal Mitochondrial Genome Reveals Chloroplast-like Architecture and Repeat-mediated Recombination.

Recent advances in long-read sequencing technology have allowed for single-molecule sequencing of entire mitochondrial genomes, opening the door for direct investigation of the mitochondrial genome architecture and recombination. We used PacBio sequencing to reassemble mitochondrial genomes from two species of New Zealand freshwater snails, Potamopyrgus antipodarum and Potamopyrgus estuarinus. These assemblies revealed a ∼1.7 kb structure within the mitochondrial genomes of both species that was previously undetected by an assembly of short reads and likely corresponding to a large noncoding region commonly present in the mitochondrial genomes. The overall architecture of these Potamopyrgus mitochondrial genomes is reminiscent of the chloroplast genomes of land plants, harboring a large single-copy (LSC) region and a small single-copy (SSC) region separated by a pair of inverted repeats (IRa and IRb). Individual sequencing reads that spanned across the Potamopyrgus IRa-SSC-IRb structure revealed the occurrence of a "flip-flop" recombination. We also detected evidence for two distinct IR haplotypes and recombination between them in wild-caught P. estuarinus, as well as extensive intermolecular recombination between single-nucleotide polymorphisms in the LSC region. The chloroplast-like architecture and repeat-mediated mitochondrial recombination we describe here raise fundamental questions regarding the origins and commonness of inverted repeats in cytoplasmic genomes and their role in mitochondrial genome evolution.

The associated evolution among the extensive RNA editing, GC‐biased mutation, and PPR family expansion in the organelle genomes of Selaginellaceae

AbstractExtensive C‐to‐U editing has been reported from plastid genomes (plastomes) and mitochondrial genomes (mitogenomes) of spikemoss. While “reverse” U‐to‐C editing was recorded in other seed‐free plants such as hornworts, quillworts, and ferns, it was not observed in spikemosses. However, no comprehensive study on the association between RNA editing and other genomic features was conducted for the organelle genomes of spikemosses. Here, we report thousands of C‐to‐U editing sites from plastomes and mitogenomes of two species: 1767 and 2394 edits in Selaginella remotifolia, and 4091 and 2786 edits in Selaginella nipponica, respectively. Comparative analyses revealed two different editing frequencies among plastomes, but one similar frequency in mitogenomes. The different editing frequency in the Selaginella organelle genomes is related to the nonsynonymous substitution rate and the genome structural complexity. The high guanine and cytosine (GC) content caused by GC‐biased mutations in organelle genomes might be related to the absence of U‐to‐C editing in Selaginellaceae. Using RNA‐seq and whole‐genome data, we screened the pentatricopeptide repeat (PPR) family and discovered that the number of aspartic acid–tyrosine–tryptophan (DYW) domain‐containing PPR proteins corresponded roughly to the editing abundance in the Selaginella organelle genomes. Consequently, we hypothesize that associated evolution among RNA editing, GC‐biased mutation in organelle genomes, and the PPR protein family encoded in the nuclear genome, is probably triggered by the aberrant DNA repair system in Selaginellaceae. Our study provides new insights into the association between organelle and nuclear genomes in Selaginellaceae, which would contribute to understanding the evolution of post‐transcriptional modifications of organelle genomes in land plants.

DNA Sequence Analysis of an Inversion Hot Spot in Lobeliaceae Plastomes

The evolution of plastid genomes (plastomes) in land plants is typically conservative, with extensive structural rearrangements present in only a few groups. Early Southern blot analysis identified two Lobelia species that minimally required deletion of the plastid gene accD and five inversions to account for their plastome arrangement relative to the ancestral organization. Sixty alternative 5-step inversion scenarios could account for the observed arrangement, but only one scenario was consistent with the criterion of 'common cause' attributable to a putative rearrangement hot spot at the accD deletion-site. Plastome sequencing demonstrated that this previously hypothesized inversion order is historically accurate. Detailed reconstructions of the ancestral plastome organization before and after each inversion are presented herein. Stem-loop and disruption-rescue models were evaluated for each inversion. One inversion has an obvious stem-loop basis, but the other four inversions were primarily caused by serial insertion of foreign (extra-plastid) DNA bearing large open-reading frames that disrupted plastome organization at the accD deletion-site, and complete plastomes were rescued by seemingly arbitrary ligation or fortuitous recombination at the other inversion endpoint. Transposed copies of DNA segments from elsewhere in the plastome are frequently inserted at inversion junctions, and four junctions are consistent with the stem-loop ligation model.

Evolutionary analysis of the LORELEI gene family in plants reveals regulatory subfunctionalization.

A signaling complex comprising members of the LORELEI (LRE)-LIKE GPI-anchored protein (LLG) and Catharanthus roseus RECEPTOR-LIKE KINASE 1-LIKE (CrRLK1L) families perceive RAPID ALKALINIZATION FACTOR (RALF) peptides and regulate growth, reproduction, immunity, and stress responses in Arabidopsis (Arabidopsis thaliana). Genes encoding these proteins are members of multigene families in most angiosperms and could generate thousands of signaling complex variants. However, the links between expansion of these gene families and the functional diversification of this critical signaling complex as well as the evolutionary factors underlying the maintenance of gene duplicates remain unknown. Here, we investigated LLG gene family evolution by sampling land plant genomes and explored the function and expression of angiosperm LLGs. We found that LLG diversity within major land plant lineages is primarily due to lineage-specific duplication events, and that these duplications occurred both early in the history of these lineages and more recently. Our complementation and expression analyses showed that expression divergence (i.e. regulatory subfunctionalization), rather than functional divergence, explains the retention of LLG paralogs. Interestingly, all but one monocot and all eudicot species examined had an LLG copy with preferential expression in male reproductive tissues, while the other duplicate copies showed highest levels of expression in female or vegetative tissues. The single LLG copy in Amborella trichopoda is expressed vastly higher in male compared to in female reproductive or vegetative tissues. We propose that expression divergence plays an important role in retention of LLG duplicates in angiosperms.

Genomic insights into genetic diploidization in the homosporous fern Adiantum nelumboides.

Whole-genome duplication has been recognized as a major process in speciation of land plants, especially in ferns. Whereas genome downsizing contributes greatly to the post-genome shock responses of polyploid flowering plants, diploidization of polyploid ferns diverges by maintaining most of the duplicated DNA and is thus expected to be dominated by genic processes. As a consequence, fern genomes provide excellent opportunities to study ecological speciation enforced by expansion of protein families via polyploidy. To test the key predictions of this hypothesis, we reported the de novo genome sequence of Adiantum nelumboides, a tetraploid homosporous fern. The obtained draft genome had a size of 6.27 Gb assembled into 11,767 scaffolds with the contig N50 of 1.37 Mb. Repetitive DNA sequences contributed with about 81.7%, a remarkably high proportion of the genome. With 69,568, the number of predicted protein-coding genes exceeded those reported in most other land plant genomes. Intragenomic synteny analyses recovered 443 blocks with the average block size of 1.29 Mb and the average gene content of 16 genes. The results are consistent with the hypothesis of high ancestral chromosome number, lack of substantial genome downsizing, and dominance of genic diploidization. As expected in the calciphilous plants, a notable number of detected genes were involved in calcium uptake and transport. In summary, the genome sequence of a tetraploid homosporous fern not only provides access to a genomic resource of a derived fern, but also supports the hypothesis of maintenance of high chromosome numbers and duplicated DNA in young polyploid ferns.

Plastid phylogenomic analyses of the Selaginella sanguinolenta group (Selaginellaceae) reveal conflict signatures resulting from sequence types, outlier genes, and pervasive RNA editing

Different from the generally conserved plastomes (plastid genomes) of most land plants, the Selaginellaceae plastomes exhibit dynamic structure, high GC content and high substitution rates. Previous plastome analyses identified strong conflict on several clades in Selaginella, however the factors causing the conflictions and the impact on the phylogenetic inference have not been sufficiently investigated. Here, we dissect the distribution of phylogenetic signals and conflicts in Selaginella sanguinolenta group, the plastome of which is DR (direct repeats) structure and with genome-wide RNA editing. We analyzed the data sets including 22 plastomes representing all species of the S. sanguinolenta group, covering the entire geographical distribution from the Himalayas to Siberia and the Russian Far East regions. We recovered four different topologies by applying multispecies coalescent (ASTRAL) and concatenation methods (IQ-TREE and RAxML) on four data sets of PC (protein-coding genes), NC (non-coding sequences), PCN (the concatenated PC and NC), and RC (predicted RNA editing sites “C” were corrected by “T”), respectively. Six monophyletic clades, S. nummularifolia clade, S. rossii clade, S. sajanensis clade, S. sanguinolenta I clade, S. sanguinolenta II clade, and S. sanguinolenta III clade, were consistently resolved and supported by the characteristics of GC content, RNA editing frequency, and gene content. However, the relationships among these clades varied across the four topologies. To explore the underlying causes of the uncertainty, we compared the phylogenetic signals of the four topologies. We identified that the sequence types (coding versus non-coding), outlier genes (genes with extremely high |ΔGLS| values), and C-to-U RNA editing frequency in the protein-coding genes were responsible for the unstable phylogenomic relationship. We further revealed a significant positive correlation between the |ΔGLS| values and the variation coefficient of the RNA editing number. Our results demonstrated that the coalescent method performed better than the concatenation method in overcoming the problems caused by outlier genes and extreme RNA editing events. Our study particularly focused on the importance of exploring the plastid phylogenomic conflicts and suggested conducting concatenated analyses cautiously when adopting organelle genome data.

The supercomplex formation between the chloroplast NADH dehydrogenase-like complex and photosystem I

The chloroplast genomes of land plants encode 11 genes (ndhA–ndhK) for proteins similar to the subunits of respiratory complex I (NADH dehydrogenase) with some exceptions. It was a mystery why the chloroplast genomes encode the genes related to respiratory electron transport. We understand now that the genes encode the subunits of the chloroplast NADH dehydrogenase-like (NDH) complex mediating the antimycin A-resistant pathway of cyclic electron transport around photosystem I (Peltier et al., 2016).

Phylogeny and evolution of plant Phytochrome Interacting Factors (PIFs) gene family and functional analyses of PIFs in Brachypodium distachyon.

Plant PIFs have been characterized, WGDs contributed to the expansion of class II PIFs; BdPIFs localized in the nucleus; BdPIF4/5C most likely response to high temperature and light stress. Phytochrome interacting factors (PIFs) belong to a small subset of basic helix-loop-helix (bHLH) transcription factors (TFs). As cellular signaling hubs, PIFs integrate multiple external and internal signals to orchestrate the regulation of the transcriptional network, thereby actuating the pleiotropic aspects of downstream morphogenesis. Nevertheless, the origin, phylogeny and function of plant PIFs are not well understood. To elucidate their evolution history and biological function, the comprehensive genomic analysis of the PIF genes was conducted using 40 land plant genomes plus additionally four alga lineages and also performed their gene organizations, sequence features and expression patterns in different subfamilies. In this study, phylogenetic analysis displayed that 246 PIF gene members retrieved from all embryophytes could be divided into three main clades, which were further felled into five distinct classes (Class I-V). The duplications of Class II PIFs were associated specially with whole genome duplication (WGD) events during the plant evolution process. Sequence analysis showed that PIF proteins had a conserved APB motif, and its crucial amino acid residues were relatively high proportion in the average abundance. As expected, subcellular localization analysis revealed that all BdPIF proteins were localized to the nucleus. Especially, BdPIF4/5C showed the highest expression level at high temperature, and the most significant hypocotyl elongation phenotype of overexpression of BdPIFs in Arabidopsis, which was consistent with the function and phenotype of AtPIF4. In brief, our findings provide a novel perspective on the origin and evolutionary history of plant PIFs, and lays a foundation for further investigation on its functions in plant growth and development.

Evolution and functional diversification of R2R3-MYB transcription factors in plants.

R2R3-MYB genes (R2R3-MYBs) form one of the largest transcription factor gene families in the plant kingdom, with substantial structural and functional diversity. However, the evolutionary processes leading to this amazing functional diversity have not yet been clearly established. Recently developed genomic and classical molecular technologies have provided detailed insights into the evolutionary relationships and functions of plant R2R3-MYBs. Here, we review recent genome-level and functional analyses of plant R2R3-MYBs, with an emphasis on their evolution and functional diversification. In land plants, this gene family underwent a large expansion by whole genome duplications and small-scale duplications. Along with this population explosion, a series of functionally conserved or lineage-specific subfamilies/groups arose with roles in three major plant-specific biological processes: development and cell differentiation, specialized metabolism, and biotic and abiotic stresses. The rapid expansion and functional diversification of plant R2R3-MYBs are highly consistent with the increasing complexity of angiosperms. In particular, recently derived R2R3-MYBs with three highly homologous intron patterns (a, b, and c) are disproportionately related to specialized metabolism and have become the predominant subfamilies in land plant genomes. The evolution of plant R2R3-MYBs is an active area of research, and further studies are expected to improve our understanding of the evolution and functional diversification of this gene family.

Identification of Partner Proteins of the Algae Klebsormidium nitens NO Synthases: Toward a Better Understanding of NO Signaling in Eukaryotic Photosynthetic Organisms.

In animals, NO is synthesized from L-arginine by three isoforms of nitric oxide synthase (NOS) enzyme. NO production and effects have also been reported in plants but the identification of its sources, especially the enzymatic ones, remains one of the critical issues in the field. NOS-like activities have been reported, although there are no homologs of mammalian NOS in the land plant genomes sequenced so far. However, several NOS homologs have been found in algal genomes and transcriptomes. A first study has characterized a functional NOS in the chlorophyte Ostreococcus tauri and the presence of NOS homologs was later confirmed in a dozen algae. These results raise the questions of the significance of the presence of NOS and their molecular diversity in algae. We hypothesize that comparisons among protein structures of the two KnNOS, together with the identification of their interacting partner proteins, might allow a better understanding of the molecular diversification and functioning of NOS in different physiological contexts and, more generally, new insights into NO signaling in photosynthetic organisms. We recently identified two NOS homologs sequences in the genome of the streptophyte Klebsormidium nitens, a model alga in the study of plant adaptation to terrestrial life. The first sequence, named KnNOS1, contains canonical NOS signatures while the second, named KnNOS2, presents a large C-ter extension including a globin domain. In order to identify putative candidates for KnNOSs partner proteins, we draw the protein–protein interaction networks of the three human NOS using the BioGRID database and hypothesized on the biological role of K. nitens orthologs. Some of these conserved partners are known to be involved in mammalian NOSs regulation and functioning. In parallel, our methodological strategy for the identification of partner proteins of KnNOS1 and KnNOS2 by in vitro pull-down assay is presented.