Combined in vitro and in vivo investigation of barite microcrystals in Spirogyra (Zygnematophyceae, Charophyta).

We have investigated the biomineralisation of barite ‒a useful proxy for reconstructing paleoproductivity‒ in a freshwater alga, Spirogyra, by combining in vitro and in vivo approaches to unveil the nature of its barite microcrystals. Scanning electron microscope (SEM) and energy-dispersive X-ray spectroscopy (EDXS) observations on simply dried samples revealed that the number and size of barite crystals were related to the barium concentration in the media. Additionally, their morphology showed a crystallographic face (011), which is not normally observed, suggesting the influence of organic molecules on the growth kinetics. The critical point drying method was used to preserve the internal and external structures of Spirogyra cells for SEM imaging. Crystals were found adjacent to the cytoplasmic membrane, near chloroplasts and fibrillary network. In vivo optical microscopy and Raman tweezer microspectroscopy in living cells showed that barite microcrystals are optically visible and follow cytoplasmic streaming. These results led us to propose that barite formation in Spirogyra occurs in the cytoplasm where barium and sulphate are both available: barium supplied non-selectively through the active transport of the divalent cations needed for actin polymerisation, and sulphate because necessary for amino acid biosynthesis in chloroplasts.

Differential Membrane Lipid Profiles and Vibrational Spectra of Three Edaphic Algae and One Cyanobacterium.

The membrane glycerolipids of four phototrophs that were isolated from an edaphic assemblage were determined by UPLC-MS after cultivation in a laboratory growth chamber. Identification was carried out by 18S and 16S rDNA sequencing. The algal species were Klebsormidium flaccidum (Charophyta), Oocystis sp. (Chlorophyta), and Haslea spicula (Bacillariophyta), and the cyanobacterium was Microcoleus vaginatus (Cyanobacteria). The glycerolipid profile of Oocystis sp. was dominated by monogalactosyldiacylglycerol (MGDG) species, with MGDG(18:3/16:4) accounting for 68.6%, whereas MGDG(18:3/16:3) was the most abundant glycerolipid in K. flaccidum (50.1%). A ratio of digalactosyldiacylglycerol (DGDG) species to MGDG species (DGDG/MGDG) was shown to be higher in K. flaccidum (0.26) than in Oocystis sp. (0.14). This ratio increased under high light (HL) as compared to low light (LL) in all the organisms, with its highest value being shown in cyanobacterium (0.38-0.58, LL-HL). High contents of eicosapentaenoic acid (EPA, C20:5) and hexadecenoic acid were observed in the glycerolipids of H. spicula. Similar Fourier transform infrared (FTIR) and Raman spectra were found for K. flaccidum and Oocystis sp. Specific bands at 1629.06 and 1582.78 cm-1 were shown by M. vaginatus in the Raman spectra. Conversely, specific bands in the FTIR spectrum were observed for H. spicula at 1143 and 1744 cm-1. The results of this study point out differences in the membrane lipid composition between species, which likely reflects their different morphology and evolutionary patterns.

Methods of Lipid Analyses for Microalgae: Charophytes, Eustigmatophytes, and Euglenophytes.

Algae are ecologically important organisms and are widely used for basic research, with a focus on for example photosynthesis, evolution, and lipid metabolism. Many biosynthetic pathways of algal lipids have been deciphered using available genomic information. Here we describe methods for lipid analyses from three representative algae, including Archaeplastida, the SAR lineage (Stramenopiles, Alveolata, Rhizaria), and Excavata. Archaeplastida acquired their plastids by primary endosymbiosis, and the others by secondary endosymbiosis with a Rhodophyceae-type plastid in SAR and a Chlorophyceae-type plastid in Excavata (Euglenozoa). Analytical methods for these algae are described for membrane lipids and neutral lipids including triacylglycerol and wax esters.

Expansion and innovation in auxin signaling: where do we grow from here?

The phytohormone auxin plays a role in almost all growth and developmental responses. The primary mechanism of auxin action involves the regulation of transcription via a core signaling pathway comprising proteins belonging to three classes: receptors, co-receptor/co-repressors and transcription factors. Recent studies have revealed that auxin signaling can be traced back at least as far as the transition to land. Moreover, studies in flowering plants have highlighted how expansion of the gene families encoding auxin components is tied to functional diversification. As we review here, these studies paint a picture of auxin signaling evolution as a driver of innovation.

Multi-genomic analysis of the cation diffusion facilitator transporters from algae.

Metal transport processes are relatively poorly understood in algae in comparison to higher plants and other eukaryotes. A screen of genomes from 33 taxonomically diverse algal species was conducted to identify members of the Cation Diffusion Facilitator (CDF) family of metal ion transporter. All algal genomes contained at least one CDF gene with four species having >10 CDF genes (median of 5 genes per genome), further confirming that this is a ubiquitous gene family. Phylogenetic analysis suggested a CDF gene organisation of five groups, which includes Zn-CDF, Fe/Zn-CDF and Mn-CDF groups, consistent with previous phylogenetic analyses, and two functionally undefined groups. One of these undefined groups was algal specific although excluded chlorophyte and rhodophyte sequences. The majority of sequences (22 out of 26 sequences) from this group had a putative ion binding site motif within transmembrane domain 2 and 5 that was distinct from other CDF proteins, such that alanine or serine replaced the conserved histidine residue. The phylogenetic grouping was supported by sequence cluster analysis. Yeast heterologous expression of CDF proteins from Chlamydomonas reinhardtii indicated Zn2+ and Co2+ transport function by CrMTP1, and Mn2+ transport function by CrMTP2, CrMTP3 and CrMTP4, which validated the phylogenetic prediction. However, the Mn-CDF protein CrMTP3 was also able to provide zinc and cobalt tolerance to the Zn- and Co-sensitive zrc1 cot1 yeast strain. There is wide diversity of CDF transporters within the algae lineage, and some of these genes may be attractive targets for future applications of metal content engineering in plants or microorganisms.

Comparison of UVB effects on growth and induction of UVB screening compounds in isolates of metaphytic algae from temperate zone streams and ponds.

Filaments in the surface layers of metaphytic mats are exposed to high photon flux densities of PAR and UVBR. We investigated the effect of UVBR exposure on growth of eight isolates of common metaphytic algae (Cladophora, Mougeotia, Oedogonium, Pithophora, Spirogyra, and Zygnema) acclimated to either high or low PAR levels prior to UVBR exposure. All isolates acclimated to low PAR exhibited significant reductions in growth rate caused by the UVBR exposure (P < 0.05). Acclimation to high PAR resulted in seven of the isolates being more tolerant of the UVB exposure. The two Zygnema isolates exhibited the most pronounced effect of high PAR acclimation with growth rates of UVB exposed treatments being equal to that of controls (P > 0.05). High PAR acclimation also protected chlorophyll a levels in the Zygnema isolates. Absorption of UVB by methanol extracts increased 322%-381% for the two Zygnema isolates when high PAR acclimated. The broad absorption peak at 270 nm suggests that phenolic compounds were responsible. Previous studies have shown that Zygnema isolates from extreme environments tolerate UVBR and contain UVB screening compounds, but our results extend these adaptions to Zygnema from typical temperate zone habitats. Although none of the other metaphytic algae produced UVB absorbing compounds, they all exhibited higher growth rates under UVBR exposure following high PAR acclimation. This suggests that the algae evaluated have inducible defenses against UVBR exposure that coupled with their mat structure would provide an adaption to the challenging light environment in shallow-water habitats.

Xyloglucan evolution and the terrestrialization of green plants.

Xyloglucan (XyG) is the major noncellulosic nonpectic matrix polysaccharide in cell walls of most land plants. Initially thought to be restricted to land plants, the last decade has seen the detection of XyG and the discovery of synthesis and modification/degradation genes in charophycean green algae (CGA). Recently, a totally new function of XyG was discovered as a potent soil aggregator released by roots and rhizoids of all major groups of land plants. In this Viewpoint, I show the presence of a complex XyG genetic machinery in most CGA groups. I discuss the context of XyG evolution in light of the terrestrialization of early CGA that gave rise to embryophytes and its possible role in early soil formation.

Embryophyte stress signaling evolved in the algal progenitors of land plants.

Streptophytes are unique among photosynthetic eukaryotes in having conquered land. As the ancestors of land plants, streptophyte algae are hypothesized to have possessed exaptations to the environmental stressors encountered during the transition to terrestrial life. Many of these stressors, including high irradiance and drought, are linked to plastid biology. We have investigated global gene expression patterns across all six major streptophyte algal lineages, analyzing a total of around 46,000 genes assembled from a little more than 1.64 billion sequence reads from six organisms under three growth conditions. Our results show that streptophyte algae respond to cold and high light stress via expression of hallmark genes used by land plants (embryophytes) during stress-response signaling and downstream responses. Among the strongest differentially regulated genes were those associated with plastid biology. We observed that among streptophyte algae, those most closely related to land plants, especially Zygnema, invest the largest fraction of their transcriptional budget in plastid-targeted proteins and possess an array of land plant-type plastid-nucleus communication genes. Streptophyte algae more closely related to land plants also appear most similar to land plants in their capacity to respond to plastid stressors. Support for this notion comes from the detection of a canonical abscisic acid receptor of the PYRABACTIN RESISTANCE (PYR/PYL/RCAR) family in Zygnema, the first found outside the land plant lineage. We conclude that a fine-tuned response toward terrestrial plastid stressors was among the exaptations that allowed streptophytes to colonize the terrestrial habitat on a global scale.

Primitive Auxin Response without TIR1 and Aux/IAA in the Charophyte Alga Klebsormidium nitens.

The phytohormone auxin regulates many aspects of growth and development in land plants, but the origin and evolution of auxin signaling and response mechanisms remain largely unknown. Indeed, it remains to be investigated whether auxin-related pathways diverged before the emergence of land plants. To address this knowledge deficit, we analyzed auxin responses in the charophyte alga Klebsormidium nitens NIES-2285, whose ancestor diverged from a green algal ancestor during the evolution of land plants. This strain is the same as Klebsormidium flaccidum NIES-2285, for which the draft genome was sequenced in 2014, and was taxonomically reclassified as K. nitens This genome sequence revealed genes involved in auxin responses. Furthermore, the auxin indole-3-acetic acid (IAA) was detected in cultures of K. nitens, but K. nitens lacks the central regulators of the canonical auxin-signaling pathway found in land plants. Exogenous IAA inhibited cell division and cell elongation in K. nitens Inhibitors of auxin biosynthesis and of polar auxin transport also inhibited cell division and elongation. Moreover, exogenous IAA rapidly induced expression of a LATERAL ORGAN BOUNDARIES-DOMAIN transcription factor. These results suggest that K. nitens has acquired the part of the auxin system that regulates transcription and cell growth without the requirement for the central players that govern auxin signaling in land plants.

How Embryophytic is the Biosynthesis of Phenylpropanoids and their Derivatives in Streptophyte Algae?

The origin of land plants from algae is a long-standing question in evolutionary biology. It is becoming increasingly clear that many characters that were once assumed to be 'embryophyte specific' can in fact be found in their closest algal relatives, the streptophyte algae. One such case is the phenylpropanoid pathway. While biochemical data indicate that streptophyte algae harbor lignin-like components, the phenylpropanoid core pathway, which serves as the backbone of lignin biosynthesis, has been proposed to have arisen at the base of the land plants. Here we revisit this hypothesis using a wealth of new sequence data from streptophyte algae. Tracing the biochemical pathway towards lignin biogenesis, we show that most of the genes required for phenylpropanoid synthesis and the precursors for lignin production were already present in streptophyte algae. Nevertheless, phylogenetic analyses and protein structure predictions of one of the key enzyme classes in lignin production, cinnamyl alcohol dehydrogenase (CAD), suggest that CADs of streptophyte algae are more similar to sinapyl alcohol dehydrogenases (SADs). This suggests that the end-products of the pathway leading to lignin biosynthesis in streptophyte algae may facilitate the production of lignin-like compounds and defense molecules. We hypothesize that streptophyte algae already possessed the genetic toolkit from which the capacity to produce lignin later evolved in vascular plants.

Three rings for the evolution of plastid shape: a tale of land plant FtsZ.

Nuclear-encoded plant FtsZ genes are derived from endosymbiotic gene transfer of cyanobacteria-like genes. The green lineage (Chloroplastida) and red lineage (Rhodophyta) feature FtsZ1 and FtsZ2 or FtsZB and FtsZA, respectively, which are involved in plastid division. These two proteins show slight differences and seem to heteropolymerize to build the essential inner plastid division ring. A third gene, encoding FtsZ3, is present in glaucophyte and charophyte algae, as well as in land plants except ferns and angiosperms. This gene was probably present in the last common ancestor of the organisms united by having a primary plastid (Archaeplastida) and was lost during vascular plant evolution as well as in the red and green algae. The presence/absence pattern of FtsZ3 mirrors that of a full set of Mur genes and the peptidoglycan wall encoded by them. Based on these findings, we discuss a role for FtsZ3 in the establishment or maintenance of plastid peptidoglycan shells.

Accumulation of copper in the cell compartments of charophyte Nitellopsis obtusa after its exposure to copper oxide nanoparticle suspension.

Cu accumulation in the internodal cell of charophyte Nitellopsis obtusa or its compartments was investigated after 3-h-exposure to lethal effective concentrations (8-day LC50) of CuO nanoparticle (nCuO) suspension or CuSO4 solution, i.e. 100 mg/L nCuO or 3.18 mg Cu/L as CuSO4. In both cases, the major part of Cu accumulated in the cell walls. The presence of CuO NPs in the cell wall and within the cell was visualized by scanning electron microscope images as well as confirmed by energy dispersive X-ray spectrum data. Although a threefold higher intracellular concentration of Cu was found after treatment with nCuO suspension, 3.18 mg Cu/L as CuSO4 induced fast and substantial depolarization of cell membrane potential contrary to that of 100 mg/L nCuO. A delayed effect of nCuO on the survival of the cells was also observed. This suggests that internally accumulated Cu was far less active and further supports the hypothesis of delayed toxicity of internalized nCuO NPs to charophyte cells.

Structural evolution of the 4/1 genes and proteins in non-vascular and lower vascular plants.

The 4/1 protein of unknown function is encoded by a single-copy gene in most higher plants. The 4/1 protein of Nicotiana tabacum (Nt-4/1 protein) has been shown to be alpha-helical and predominantly expressed in conductive tissues. Here, we report the analysis of 4/1 genes and the encoded proteins of lower land plants. Sequences of a number of 4/1 genes from liverworts, lycophytes, ferns and gymnosperms were determined and analyzed together with sequences available in databases. Most of the vascular plants were found to encode Magnoliophyta-like 4/1 proteins exhibiting previously described gene structure and protein properties. Identification of the 4/1-like proteins in hornworts, liverworts and charophyte algae (sister lineage to all land plants) but not in mosses suggests that 4/1 proteins are likely important for plant development but not required for a primary metabolic function of plant cell.

Evidence for land plant cell wall biosynthetic mechanisms in charophyte green algae.

BACKGROUND AND AIMS: The charophyte green algae (CGA) are thought to be the closest living relatives to the land plants, and ancestral CGA were unique in giving rise to the land plant lineage. The cell wall has been suggested to be a defining structure that enabled the green algal ancestor to colonize land. These cell walls provide support and protection, are a source of signalling molecules, and provide developmental cues for cell differentiation and elongation. The cell wall of land plants is a highly complex fibre composite, characterized by cellulose cross-linked by non-cellulosic polysaccharides, such as xyloglucan, embedded in a matrix of pectic polysaccharides. How the land plant cell wall evolved is currently unknown: early-divergent chlorophyte and prasinophyte algae genomes contain a low number of glycosyl transferases (GTs), while land plants contain hundreds. The number of GTs in CGA is currently unknown, as no genomes are available, so this study sought to give insight into the evolution of the biosynthetic machinery of CGA through an analysis of available transcriptomes. METHODS: Available CGA transcriptomes were mined for cell wall biosynthesis GTs and compared with GTs characterized in land plants. In addition, gene cloning was employed in two cases to answer important evolutionary questions. KEY RESULTS: Genetic evidence was obtained indicating that many of the most important core cell wall polysaccharides have their evolutionary origins in the CGA, including cellulose, mannan, xyloglucan, xylan and pectin, as well as arabino-galactan protein. Moreover, two putative cellulose synthase-like D family genes (CSLDs) from the CGA species Coleochaete orbicularis and a fragment of a putative CSLA/K-like sequence from a CGA Spirogyra species were cloned, providing the first evidence that all the cellulose synthase/-like genes present in early-divergent land plants were already present in CGA. CONCLUSIONS: The results provide new insights into the evolution of cell walls and support the notion that the CGA were pre-adapted to life on land by virtue of the their cell wall biosynthetic capacity. These findings are highly significant for understanding plant cell wall evolution as they imply that some features of land plant cell walls evolved prior to the transition to land, rather than having evolved as a result of selection pressures inherent in this transition.

Pectin metabolism and assembly in the cell wall of the charophyte green alga Penium margaritaceum.

The pectin polymer homogalacturonan (HG) is a major component of land plant cell walls and is especially abundant in the middle lamella. Current models suggest that HG is deposited into the wall as a highly methylesterified polymer, demethylesterified by pectin methylesterase enzymes and cross-linked by calcium ions to form a gel. However, this idea is based largely on indirect evidence and in vitro studies. We took advantage of the wall architecture of the unicellular alga Penium margaritaceum, which forms an elaborate calcium cross-linked HG-rich lattice on its cell surface, to test this model and other aspects of pectin dynamics. Studies of live cells and microscopic imaging of wall domains confirmed that the degree of methylesterification and sufficient levels of calcium are critical for lattice formation in vivo. Pectinase treatments of live cells and immunological studies suggested the presence of another class of pectin polymer, rhamnogalacturonan I, and indicated its colocalization and structural association with HG. Carbohydrate microarray analysis of the walls of P. margaritaceum, Physcomitrella patens, and Arabidopsis (Arabidopsis thaliana) further suggested the conservation of pectin organization and interpolymer associations in the walls of green plants. The individual constituent HG polymers also have a similar size and branched structure to those of embryophytes. The HG-rich lattice of P. margaritaceum, a member of the charophyte green algae, the immediate ancestors of land plants, was shown to be important for cell adhesion. Therefore, the calcium-HG gel at the cell surface may represent an early evolutionary innovation that paved the way for an adhesive middle lamella in multicellular land plants.

Heterotrimeric G proteins in green algae: an early innovation in the evolution of the plant lineage.

Heterotrimeric G-proteins (G-proteins, hereafter) are important signaling components in all eukaryotes. The absence of these proteins in the sequenced genomes of Chlorophyaceaen green algae has raised questions about their evolutionary origin and prevalence in the plant lineage. The existence of G-proteins has often been correlated with the acquisition of embryophytic life-cycle and/or terrestrial habitats of plants which occurred around 450 million years ago. Our discovery of functional G-proteins in Chara braunii, a representative of the Charophycean green algae, establishes the existence of this conserved signaling pathway in the most basal plants and dates it even further back to 1-1.5 billion years ago. We have now identified the sequence homologs of G-proteins in additional algal families and propose that green algae represent a model system for one of the most basal forms of G-protein signaling known to exist to date. Given the possible differences that exist between plant and metazoan G-protein signaling mechanisms, such basal organisms will serve as important resources to trace the evolutionary origin of proposed mechanistic differences between the systems as well as their plant-specific functions.

Allelopathic effects of microcystin-LR on the germination, growth and metabolism of five charophyte species and a submerged angiosperm.

Microcystins (MCs) are produced by cyanobacteria in aquatic environments and adversely affect macrophytes at very high concentrations. However, the effects of MC on macrophytes at concentrations of environmental relevance are largely unknown. The main objective of this study was to analyze the allelopathic effects of MC-LR at natural concentrations (1, 8 and 16 µg MC-LR/L) on five charophyte species (Chara aspera, C. baltica, C. hispida, C. vulgaris and Nitella hyalina) and the angiosperm Myriophyllum spicatum. Macrophyte specimens were obtained from a restored area located in Albufera de València Natural Park, a protected coastal Mediterranean wetland. Two different experiments were conducted involving (i) the addition of MC-LR to natural sediment to evaluate its effects on seed germination and (ii) the addition of MC-LR to water cultures of macrophytes to evaluate its effects on growth and metabolic functions. In water, the MC-LR concentration decreased by 84% in two weeks; the loss was not significant in sediment. The first seedlings (all C. hispida) emerged from the wetland sediment following a delay of a few days in the presence of MC-LR. The germination rates in 8 and 16 µg MC-LR/L treatments were 44% and 11% of that occurring in the absence of MC, but these differences disappeared over time. The final density was 6-7 germlings/dm(3). Final germling length was unaffected by MC-LR. Rotifers (Lecane spp.) emerging from the natural sediment during the experiment were favored by MC-LR; the opposite pattern was observed in the cladoceran Daphnia magna. The growth rates of C. vulgaris, C. baltica and N. hyalina were unaffected by MC exposure, whereas those of C. hispida and C. aspera were reduced in the MC treatments relative to the control treatment. The concentration of chlorophyll-a and the in vivo net photosynthetic rate were lower in the presence of MC-LR, even at the lowest concentration, for all of the characeans tested. M. spicatum was sensitive to the presence of MC-LR in the culture medium; the growth and chlorophyll-a concentrations were reduced. Therefore, environmentally relevant concentrations of MC might induce important changes in macrophyte meadows and the structure of the associated plankton community. Synchrony or delay in the processes evaluated here in response to environmentally relevant concentrations of cyanobacteria MC exudates can enhance understanding of the turning point to alternative states and the point of no return in eutrophicated shallow lakes.

Phytoremediation potential of charophytes: bioaccumulation and toxicity studies of cadmium, lead and zinc.

The ability for usage of common freshwater charophytes, Chara aculeolata and Nitella opaca in removal of cadmium (Cd), lead (Pb) and zinc (Zn) from wastewater was examined. C. aculeolata and N. opaca were exposed to various concentrations of Cd (0.25 and 0.5 mg/L), Pb (5 and 10 mg/L) and Zn (5 and 10 mg/L) solutions under hydroponic conditions for 6 days. C. aculeolata was more tolerant of Cd and Pb than N. opaca. The relative growth rate of N. opaca was drastically reduced at high concentrations of Cd and Pb although both were tolerant of Zn. Both macroalgae showed a reduction in chloroplast, chlorophyll and carotenoid content after Cd and Pb exposure, while Zn exposure had little effects. The bioaccumulation of both Cd and Pb was higher in N. opaca (1544.3 microg/g at 0.5 mg/L Cd, 21657.0 microg/g at 10 mg/L Pb) whereas higher Zn accumulation was observed in C. aculeolata (6703.5 microg/g at 10 mg/L Zn). In addition, high bioconcentration factor values (> 1000) for Cd and Pb were observed in both species. C. aculeolata showed higher percentage of Cd and Pb removal (> 95%) than N. opaca and seemed to be a better choice for Cd and Pb removal from wastewater due to its tolerance to these metals.

Phytoremediation of heavy metals by calcifying macro-algae (Nitella pseudoflabellata): implications of redox insensitive end products.

To evaluate the phytoremediation of heavy metals in water and understand the biochemistry of end products of calcifying macro algae (charophytes), an 84-wk laboratory experiment was conducted. Eighteen microcosms were maintained with and without plants. These were given different heavy metal treatments: no heavy metals, 0.2mgL(-1) Cr(6+) and 0.01mgL(-1) Cd. Accumulation observed to be 0.06% Cr by dry weight and for Cd it was 0.02%. The bioconcentration factors were 3000 and 25000 for Cr and Cd, respectively. Ratios of heavy metal accumulation in alkaline (i.e., calcified areas) to acidic areas of plants were 6 to 4 (for Cr) and 1 to 1 (for Cd). This elucidated an association between heavy metal accumulation and calcification. This was validated by sequential extraction of sediments. It was shown that in microcosms with plants, the heavy metals were mainly in redox insensitive and less bioavailable carbonate bound form (39-47%). This was followed by organic-bound form (23-34%). Carbonate bound end products will ensure long term storage of heavy metals and after plant senescence these will not re-enter the water column.

The catalytic domain CysPc of the DEK1 calpain is functionally conserved in land plants.

DEK1, the single calpain of land plants, is a member of the ancient membrane bound TML-CysPc-C2L calpain family that dates back 1.5 billion years. Here we show that the CysPc-C2L domains of land plant calpains form a separate sub-clade in the DEK1 clade of the phylogenetic tree of plants. The charophycean alga Mesostigma viride DEK1-like gene is clearly divergent from those in land plants, suggesting that a major evolutionary shift in DEK1 occurred during the transition to land plants. Based on genetic complementation of the Arabidopsis thaliana dek1-3 mutant using CysPc-C2L domains of various origins, we show that these two domains have been functionally conserved within land plants for at least 450 million years. This conclusion is based on the observation that the CysPc-C2L domains of DEK1 from the moss Physcomitrella patens complements the A. thaliana dek1-3 mutant phenotype. In contrast, neither the CysPc-C2L domains from M. viride nor chimeric animal-plant calpains complement this mutant. Co-evolution analysis identified differences in the interactions between the CysPc-C2L residues of DEK1 and classical calpains, supporting the view that the two enzymes are regulated by fundamentally different mechanisms. Using the A. thaliana dek1-3 complementation assay, we show that four conserved amino acid residues of two Ca²âº-binding sites in the CysPc domain of classical calpains are conserved in land plants and functionally essential in A. thaliana DEK1.