Welcome to MOLE KNIGHTS a web based tool for the exploration of our multiomics analysis ( Transcriptomic and Metabolomic ) of the response to 3 hours of high light treatment in the fascinating charophyte microalga Klebsormidium nitens. We observed an activation of different protection mechanisms at the molecular and celular level. Here, we present an integrative analysis to characterize the molecular mechanisms involved in the response to high light in Klebsormidium and relate them to the mechanisms used by land plants or Embryophyta.

For more information, select from the navigation panel on the left the type of exploration in which you are interested and follow the instructions. In addition, you can watch our video tutorial on how to use MOLEKNIGTS. Our code for this web app is available on Github. If you find this work interesting and useful in your research, please cite us.

The evolutionary history of the green lineage or Viridiplantae splits into two different clades Chlorophyta and Streptophyta. Chlorophyta are primarily constituted by marine and freshwater green microalgae although multicellular organisms are also present and terrestralization events have taken place. In turn, Streptophyta are divided into two different clades Charophyta and Embryophyta . Whereas Embryophyta comprises mainly land plants, Charophyta are still considered algae with a preference for freshwater and with some facultative terrestrial species. Present-day Charophyta are generally accepted as the extant algal species most closely related to the aquatic ancestors of land plants or Embryophyta (Fig. 1) . Accordingly, the molecular systems that potentially allowed this group of photosynthetic organisms to evolve towards terrestrial land plants are under intense analysis.

During this transition, the evolution of response molecular systems to terrestrial environmental stresses was critical. Some terrestrial physiological adaptations, such as desiccation resistance and tolerance to UV radiation are present in Charophyta from which current land plant mechanisms supposedly evolved. Other system found in Embryophyta such as auxin transport, photoprotective capacity and adaptation to transient light changes have been identified in Charophyta as Zygnema circumcarinatum. Whereas these studies focus mainly on genomic data, the lack of multi-omic data such as transcriptomic and metabolomic data for Charophyta under specific conditions relevant to the terrestralization process is preventing the full characterization of the molecular systems that promoted the transition to the first land plants.

Figure 1. Another key event in evolution. Inspired by the classical ‘Great moments in evolution’ cartoon by Gary Larson, this figure illustrates some of the key features that probably enabled early plants to thrive on land: the rigid cell wall, several plastids per cell, phenylpropanoids enabling protection to ultraviolet-B radiation; artwork by Debbie Maizels (Rensing, 2018).

In this study, we have chosen the freshwater facultative terrestrial Charophyta Klebsormidium nitens as model organism to study the transcriptomic and metabolomic response to high light irradiance recreating at least one of the most critical environmental changes faced by plants during terrestralization. K. nitens cultures consist of multicellular and non-branching filaments without specialized cells with a single chloroplast. Many Klebsormidium species are cosmopolitan distributed in terrestrial environments as soil crusts and rocks as well as freshwater habitats like streams and rivers.

Their presence in these environments expose cells to extreme conditions including high light irradiance. Physiological studies under such conditions have been carried out reporting photosynthetic resistance against intense light meditated by the presence of photoprotective mechanisms dissipating energy as heat (non- photochemical quenching, NPQ) and/or by the activation of alternative electron routes to reduce reactive oxygen species (ROS) production. Several comparative genomic analyses have been carried out providing evidence about Klebsormidium possessing fundamental molecular mechanisms required for the adaptation and survival in terrestrial environments including wax-related genes, phytohormone signaling and transcription factors involved in resistance to high light and UV radiation. Nonetheless, there are very few transcriptomic studies integrating gene expression with physiological data aiming at the characterization of Klebsormidium responses to abiotic stresses.

Klebsormidium nitens (strain NIES-2285) was obtained from the National Institute for Environmental Studies (Japan). Cells were grown photoautotrophically in Bold’s Basal Medium using photobioreactors containing 0.8 L of cell suspension and bubbled with air supplemented with 1% (v/v) CO2 as carbon source. Photobioreactors were continuously illuminated with white light lamps at 50 μE m -2 s -1 and maintained at 20ºC. Defoamer (Antifoam 204) was added to avoid the contamination of the aeration systems. Cultures at exponential phase with 45 μg/ml chlorophyll content were used in our experiments. Control cultures were kept under a control light irradiance of 50 μE m -2 s -1 whereas high light cultures were illuminated for three hours with an irradiance of 1500 μE m -2 s -1. Six independent biological replicates were considered for low and high light irradiance metabolomic data generation. Cells were collected, washed with PBS and stored at -80ºC.

Using these cells, RNA extraction was performed to obtein purified RNA and the computational pipeline MARACAS was used to determine differentially expressed genes according to a log2FC of ± 1 and a adjusted p-value or FDR (False Discobery Rate) threshold of 0.05. The software tool AlgaeFun was used to perform functional enrichment analysis based on Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways over the sets of differentially expressed genes.

For metabolite content determination (primary metabolite, phytohormone and carotenoids) , cell pellets were lyophilized and the determination was carried out by ultra high performance liquid chromatography system coupled with mass spectrometry (UPLC/MS), or HPLC (High-performance liquid chromatography) coupled to an UV-visible scanning spectrophotometer in case of carotenoids.

One of the main mechanisms responding to environmental changes is differntial gene expression. Accordingly, RNA-seq was developed as a massive cDNA sequencing technique obtained through RNA extraction using high-performance sequencing. In this study we focus on RNA-seq applied to eukaryotic coding mRNA.

Mathematical/computational analysis was carried out using the automatic pipeline implemented in MARACAS.

Figure 2. Boxplot before and after normalization. Please, hover over the plot for more information.

A quick method to visualize the variability between samples is PCA (Principal Component Analysis).

MARACAS produces lists of differentially expressed genes . In our study, expression was detected for 68.4 % of the 17290 genes in the current Klebsormidium genome annotation. According to a log2FC of ± 1 and a q-value or FDR (False Discobery Rate) threshold of 0.05 7.84 % of the entire Klebsormidium genome was significant differentially expressed after three hours of high light treatment. Specifically, we identified 667 activates and 678 repressed genes (Fig. 3).

Figure 3. Volcano plot of DEGs. Please, hover over the plot for more information and select any point of your interest.

The software tool AlgaeFUN was used to perform functional enrichment analysis based on Gene Ontology (GO) terms, to identify the cellular components and biological processes significantly affected by high light, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways over the sets of differentially expressed genes Fig. 4.

The proteins encoded by differentially expressed genes, both activated and repressed genes , were significantly localized in the chloroplast thylakoid membranes indicating the initiation of a major chloroplast reprogramming.

Figure 4. GO terms enrichment.

Specifically, proteins encoded by repressed genes were significantly associated with photosystems and cellular structures present during cell division such as condensed nuclear chromosomes and microtubules Fig. 4. . Accordingly, photosynthesis, hexose biosynthesis, cell cycle and DNA metabolism were significantly enriched processes in the repressed genes. This points to an arrest in the photosynthetic machinery and cell cycle progression as response to high light.

Proteins encoded by activated genes are, in turn, significantly localized in cellular structures involved in de novo protein biosybthesis such as preribosomes and translation initiation factor 3’ complex Fig. 4. In particular, categories encompassing ribosome biogenesis, cytoplasmic translation initiation and protein folding were significantly enriched in the activated genes. Moreover, response to oxidative stress, response to high light intensity, tetraterpenoid and carotenoid metabolism were identified as significantly activated processes. This suggests an activation of repair and protective mechanisms to damages caused by high light.

In the Global Transcriptomic Statistics tab, a global exploration of the transcriptomic data is performed. Here the expression of individual genes can be represented with a barplot. Enter the gene identifier in the box below and a barplot with gene expression measured as FPKM under low light (LL, blue) and high light (HL, red) will be represented.

Metabolomics is understood as the discipline that analyzes the metabolites of a living organism and tries to find the interaction between metabolic pathways as well as quantify the largest amount of metabolites present. In the omics context, it offers an additional view on the characteristics of a metabolism, giving an idea of points of regulation and association of functions for unknown genes. In this case, a metabolic profile was performed where interrelated compounds were studied, generating a specific metabolome for that sample using mass spectrometry.

Analogously to the Global Transcriptomic Statistics section, the metabolic data were normalized. Specifically, from the data obtained by the mass spectrometer, the amount of metabolite present was determined according to the area under the curve of the peak obtained and assigned to each metabolite. They were relativized based on the total weight of the sample and the presence of a standard compound: paracetamol.

Six independent biological replicates were considered for both, high and low light conditions. We detected 69 different primary and secondary metabolites including most amino acids and some phytohormones. Significant differentially abundant metabolites were identified by performing the non-parametric Wilcoxon test using a p-value threshold of 0.05. We found 12 significantly more abundant and 8 less abundant metabolites under high light when compared to low light (Figure 5). For instance, under high light, we detected significant changes in specific carotenoids, accumulation of the amino acid tryptophan and the phytohormone indole-3- acetic acid (IAA)

Figure 5. Volcano plot of Metabolites. Please, hover over the plot for more information and select any point of your interest.

In the Global Metabolomic Statistics tab, a global exploration of the metabolomic data is performed. Users can also study individual metabolites entering the metabolite name in the box below to obtain a barplot with their abundance under high light irradiance with respect to low light.

Until now we have explored information from different omics separately, but the real insight lies in the integration of both. Bioinformatics offers the possibility of obtaining a more complete view of the behavior of biological systems, as will be seen in the following results. If you are interested in more details, you can get it in our complete article.

Here, we present an integrated transcriptomic and metabolomic analysis of this specific photoprotective response to high light in Klebsormidium (Figure 6) . As can be seen, carotenoid biosynthesis is favored for the generation of beta-carotenoid by the activation of several enzymes of the pathway, while the ε-branch leading to lutein is repressed. Although, β–carotene content was similar under low and high light conditions, the cycle of xanthophylls towards the formation of zeaxanthin from violaxanthin is favored. Violaxanthin content decreased 4.73 fold whereas antheraxanthin and zeaxanthin contents were increased 3.44 and 41.5 fold respectively under high light when compared to low light. Accordingly, the gene encoding the enzyme involved in the xanthophyll cycle, violaxanthin de-epoxidase (VDE, kfl00604_0070) converting violaxanthin into antheraxanthin and zeaxanthin was activated 1.86 fold. Furthermore, the gene encoding zeaxanthin epoxidase (ZEP, kfl00092_0060) that catalyzes the synthesis of violaxanthin from zeaxanthin and antheraxanthin was 3.84 fold repressed under high light.

Figure 6. Gene expression level and relative carotenoid content in the carotenoid biosynthesis pathway in Klebsormidium under high light (HL) and low light (LL)

In the xanthophyll cycle, the interconversion of violaxanthin into antheraxanthin and zeaxanthin, constitutes one of the major photoprotective mechanism in Embryophyta and Chlorophyta. High light induces the mobilization of violaxanthin to zeaxanthin whereas low light or darkness produce the reverse reaction (Goss and Jakob, 2010; Latowski et al., 2011). De-epoxidation of violaxanthin to zeaxanthin enhances dissipation of excess excitation energy (non-photochemical quenching, NPQ) in the photosystem II (PSII) antenna, thereby preventing inactivation and damage to the photosynthetic apparatus. NPQ is considered a fundamental mechanism for Streptophyta adaptation to terrestrial habitats (Pierangelini et al., 2017). Here, we specifically show that the xanthophyll cycle is part of the early transcriptomic and metabolomic response to high light intensity in the Charophyta Klebsormidium.

Under high light conditions exceeding photosynthetic capacity, production of harmful reactive oxygen species (ROS) is unavoidable associated with electron transport in the photosystems. Excess electron leakage to molecular oxygen and incomplete water oxidation produce singlet oxygen, superoxide, hydrogen peroxide and hydroxyl radical (Pospíšil, 2016). This triggers a signaling cascade communicating the chloroplast state to the nucleus termed retrograde signaling that ultimately induces the expression of nuclear genes. The evolution of this system has played a central role in plant terrestralization (Zhao et al., 2019; Calderon and Strand, 2021).

Indeed, response to oxidative stress was one of the most significant GO term in our functional enrichment analysis over the activated genes in a response to high light treatment in Klebsormidium Figure 7. Under these conditions proteins suffer oxidative damage specifically but not limited to the active thiol groups of cysteine residues, which are oxidized to disulfide bonds (Cejudo et al., 2021). This produces major modifications in protein structure that can lead to misfolding and loss of function. The accumulation in the chloroplast of aberrant misfolded proteins also contributes to initiate retrograde signaling (Dogra et al., 2019a). Moreover, we found the activation of multiple chloroplast targeted chaperones, co-chaperones and chaperonins that would contribute to restore misfolded proteins.

Figure 7. Gene expression level for enzymes involved in retrograde signaling triggered by high light oxidative stress inducing gene activation of protein repair mechanisms and de novo protein synthesis.

Concomitant to the activation of protein repair mechanisms we found significant activation of ribosome biogenesis and cytoplasmic translation initiation. These strongly activated processes are required for de novo protein synthesis and, together with the previously described protein repair mechanisms, constitute part of the response to high light in Klebsormidium, contributing to maintain proteome homeostasis under this stress. Besides, the retrograde signaling pathways induced by ROS and aberrant misfolded proteins discussed above, there exists another pathway regulated by the accumulation of 3′- phosphoadenosine-5′-phosphate (PAP). The inositol polyphosphate 1-phosphatase SAL1 removes PAP preventing its accumulation. The gene encoding this enzyme kfl00096_0240 was 2 fold repressed indicating a possible accumulation of PAP and an activation of the SAL1-PAP retrograde signaling pathway, as a response to high light intensity in Klebsormidium.

MOLE KNIGHTS, is entirely developed using the R package shiny. The source code is released under MIT License and is hosted at GitHub. If you experience any problem using MOLE KNIGHTS please create an issue in GitHub and we will address it.

MOLE KNIGHTS at GitHub

Here we present some of the other main programs that we have used in this exploratory tool:

AlgaeFUN, is entirely developed using the R package shiny. The source code is released under GNU General Public License v3.0 and is hosted at GitHub.

AlgaeFUN at GitHub

MARACAS, is developed using bash scripting and several bioconductor R packages. The source code is released under GNU General Public License v3.0 and is hosted at GitHub.

MARACAS at GitHub

Recently we published our work in a journal if you find this information useful in your research we would be most grateful if you cite our GitHub repository with a, DOI as follows: