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Significance Early branching events in the diversification of land plants and closely related algal lineages remain fundamental and unresolved questions in plant evolutionary biology. Accurate reconstructions of these relationships are critical for testing hypotheses of character evolution: for example, the origins of the embryo, vascular tissue, seeds, and flowers. We investigated relationships among streptophyte algae and land plants using the largest set of nuclear genes that has been applied to this problem to date. Hypothesized relationships were rigorously tested through a series of analyses to assess systematic errors in phylogenetic inference caused by sampling artifacts and model misspecification. Results support some generally accepted phylogenetic hypotheses, while rejecting others. This work provides a new framework for studies of land plant evolution. Reconstructing the origin and evolution of land plants and their algal relatives is a fundamental problem in plant phylogenetics, and is essential for understanding how critical adaptations arose, including the embryo, vascular tissue, seeds, and flowers. Despite advances in molecular systematics, some hypotheses of relationships remain weakly resolved. Inferring deep phylogenies with bouts of rapid diversification can be problematic; however, genome-scale data should significantly increase the number of informative characters for analyses. Recent phylogenomic reconstructions focused on the major divergences of plants have resulted in promising but inconsistent results. One limitation is sparse taxon sampling, likely resulting from the difficulty and cost of data generation. To address this limitation, transcriptome data for 92 streptophyte taxa were generated and analyzed along with 11 published plant genome sequences. Phylogenetic reconstructions were conducted using up to 852 nuclear genes and 1,701,170 aligned sites. Sixty-nine analyses were performed to test the robustness of phylogenetic inferences to permutations of the data matrix or to phylogenetic method, including supermatrix, supertree, and coalescent-based approaches, maximum-likelihood and Bayesian methods, partitioned and unpartitioned analyses, and amino acid versus DNA alignments. Among other results, we find robust support for a sister-group relationship between land plants and one group of streptophyte green algae, the Zygnematophyceae. Strong and robust support for a clade comprising liverworts and mosses is inconsistent with a widely accepted view of early land plant evolution, and suggests that phylogenetic hypotheses used to understand the evolution of fundamental plant traits should be reevaluated.

Water loss has significant effects on physiological performance and survival rates of algae. However, despite the prominent presence of aeroterrestrial algae in terrestrial habitats, hardly anything is known about the molecular events that allow aeroterrestrial algae to survive harsh environmental conditions. We analyzed the transcriptome and physiology of a strain of the alpine aeroterrestrial alga Klebsormidium crenulatum under control and strong desiccation-stress conditions. For comparison we first established a reference transcriptome. The high-coverage reference transcriptome includes about 24,183 sequences (1.5 million reads, 636 million bases). The reference transcriptome encodes for all major pathways (energy, carbohydrates, lipids, amino acids, sugars), nearly all deduced pathways are complete or missing only a few transcripts. Upon strong desiccation, more than 7000 transcripts showed changes in their expression levels. Most of the highest up-regulated transcripts do not show similarity to known viridiplant proteins, suggesting the existence of some genus- or species-specific responses to desiccation. In addition, we observed the up-regulation of many transcripts involved in desiccation tolerance in plants (e.g. proteins similar to those that are abundant in late embryogenesis (LEA), or proteins involved in early response to desiccation ERD), and enzymes involved in the biosynthesis of the raffinose family of oligosaccharides (RFO) known to act as osmolytes). Major physiological shifts are the up-regulation of transcripts for photosynthesis, energy production, and reactive oxygen species (ROS) metabolism, which is supported by elevated cellular glutathione content as revealed by immunoelectron microscopy as well as an increase in total antiradical power. However, the effective quantum yield of Photosystem II and CO2 fixation decreased sharply under the applied desiccation stress. In contrast, transcripts for cell integrative functions such as cell division, DNA replication, cofactor biosynthesis, and amino acid biosynthesis were down-regulated. This is the first study investigating the desiccation transcriptome of a streptophyte green alga. Our results indicate that the cellular response is similar to embryophytes, suggesting that embryophytes inherited a basic cellular desiccation tolerance from their streptophyte predecessors.

Auxin conjugates are thought to play important roles as storage forms for the active plant hormone indole-3-acetic acid (IAA). In its free form, IAA comprises only up to 25% of the total amount of IAA, depending on the tissue and the plant species studied. The major forms of IAA conjugate are low molecular weight ester or amide forms, but there is increasing evidence of the occurrence of peptides and proteins modified by IAA. Since the discovery of genes and enzymes involved in synthesis and hydrolysis of auxin conjugates, much knowledge has been gained on the biochemistry and function of these compounds, but there is still much to discover. For example, recent work has shown that some auxin conjugate hydrolases prefer conjugates with longer-chain auxins such as indole-3-propionic acid and indole-3-butyric acid as substrate. Also, the compartmentation of these reactions in the cell or in tissues has not been resolved in great detail. The function of auxin conjugates has been mainly elucidated by mutant analysis in genes for synthesis or hydrolysis and a possible function for conjugates inferred from these results. In the evolution of land plants auxin conjugates seem to be connected with the development of certain traits such as embryo, shoot, and vasculature. Most likely, the synthesis of auxin conjugates was developed first, since it has been already detected in moss, whereas sequences typical of auxin conjugate hydrolases were found according to database entries first in moss ferns. The implications for the regulation of auxin levels in different species will be discussed.

The colonization of land by plants was a key event in the evolution of life. Here we report the draft genome sequence of the filamentous terrestrial alga Klebsormidium flaccidum (Division Charophyta, Order Klebsormidiales) to elucidate the early transition step from aquatic algae to land plants. Comparison of the genome sequence with that of other algae and land plants demonstrate that K. flaccidum acquired many genes specific to land plants. We demonstrate that K. flaccidum indeed produces several plant hormones and homologues of some of the signalling intermediates required for hormone actions in higher plants. The K. flaccidum genome also encodes a primitive system to protect against the harmful effects of high-intensity light. The presence of these plant-related systems in K. flaccidum suggests that, during evolution, this alga acquired the fundamental machinery required for adaptation to terrestrial environments. Plant colonization of land is an important evolutionary event. Here, the authors sequence the genome of a filamentous terrestrial alga and, through a comparative analysis with related algae and land plant species, provide insight into how aquatic algae adapted to terrestrial environments.

The metabolism of cytokinins (CKs) and auxins in vascular plants is relatively well understood, but data concerning their metabolic pathways in non-vascular plants are still rather rare. With the aim of filling this gap, 20 representatives of taxonomically major lineages of cyanobacteria and algae from Cyanophyceae, Xanthophyceae, Eustigmatophyceae, Porphyridiophyceae, Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Zygnematophyceae and Klebsormidiophyceae were analysed for endogenous profiles of CKs and auxins and some of them were used for studies of the metabolic fate of exogenously applied radiolabelled CK, [ H]trans-zeatin (transZ) and auxin ([ H]indole-3-acetic acid (IAA)), and the dynamics of endogenous CK and auxin pools during algal growth and cell division. Quantification of phytohormone levels was performed by high-performance or ultrahigh-performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-MS/MS, UHPLC-MS/MS). The dynamics of exogenously applied [ H]transZ and [ H]IAA in cell cultures were monitored by HPLC with on-line radioactivity detection. The comprehensive screen of selected cyanobacteria and algae for endogenous CKs revealed a predominance of bioactive and phosphate CK forms while O- and N-glucosides evidently did not contribute greatly to the total CK pool. The abundance of cis-zeatin-type CKs and occurrence of CK 2-methylthio derivatives pointed to the tRNA pathway as a substantial source of CKs. The importance of the tRNA biosynthetic pathway was proved by the detection of tRNA-bound CKs during the course of Scenedesmus obliquus growth. Among auxins, free IAA and its oxidation catabolite 2-oxindole-3-acetic acid represented the prevailing endogenous forms. After treatment with [ H]IAA, IAA-aspartate and indole-3-acetyl-1-glucosyl ester were detected as major auxin metabolites. Moreover, different dynamics of endogenous CKs and auxin profiles during S. obliquus culture clearly demonstrated diverse roles of both phytohormones in algal growth and cell division. Our data suggest the existence and functioning of a complex network of metabolic pathways and activity control of CKs and auxins in cyanobacteria and algae that apparently differ from those in vascular plants.

The green algal genus Klebsormidium (Klebsormidiophyceae, Streptophyta) is a typical member of biological soil crusts (BSCs) worldwide. Ecophysiological studies focused so far on individual strains and thus gave only limited insight on the plasticity of this genus. In the present study, 21 Klebsormidium strains (K. dissectum, K. flaccidum, K. nitens, K. subtile) from temperate BSCs in Central European grassland and forest sites were investigated. Photosynthetic performance under desiccation and temperature stress was measured under identical controlled conditions. Photosynthesis decreased during desiccation within 335–505 min. After controlled rehydration, most isolates recovered, but with large variances between single strains and species. However, all K. dissectum strains had high recovery rates (>69%). All 21 Klebsormidium isolates exhibited the capability to grow under a wide temperature range. Except one strain, all others grew at 8.5 °C and four strains were even able to grow at 6.2 °C. Twenty out of 21 Klebsormidium isolates revealed an optimum growth temperature >17 °C, indicating psychrotrophic features. Growth rates at optimal temperatures varied between strains from 0.26 to 0.77 µ day⁻¹. Integrating phylogeny and ecophysiological traits, we found no phylogenetic signal in the traits investigated. However, multivariate statistical analysis indicated an influence of the recovery rate and growth rate. The results demonstrate a high infraspecific and interspecific physiological plasticity, and thus wide ecophysiological ability to cope with strong environmental gradients. This might be the reason why members of the genus Klebsormidium successfully colonize terrestrial habitats worldwide.

Filamentous green algae of the genus Zygnema (Zygnematophyceae, Streptophyta) are key components of polar hydro-terrestrial mats where they face various stressors including UV irradiation, freezing, desiccation and osmotic stress. Their vegetative cells can develop into pre-akinetes, i.e. reserve-rich, mature cells. We investigated lipid accumulation and fatty acid (FA) composition upon pre-akinete formation in an Arctic and an Antarctic Zygnema strain using transmission electron microscopy and gas chromatography coupled with mass spectrometry. Pre-akinetes formed after 9 weeks of cultivation in nitrogen-free medium, which was accompanied by massive accumulation of lipid bodies. The composition of FAs was similar in both strains, and α-linolenic acid (C18:3) dominated in young vegetative cells. Pre-akinete formation coincided with a significant change in FA composition. Oleic (C18:1) and linoleic (C18:2) acid increased the most (up to 17- and 8-fold, respectively). Small amounts of long-chain polyunsaturated FAs were also detected, e.g. arachidonic (C20:4) and eicosapentaenoic (C20:5) acid. Pre-akinetes exposed to desiccation at 86% relative humidity were able to recover maximum quantum yield of photosystem II, but desiccation had no major effect on FA composition. The results are discussed with regard to the capability of Zygnema spp. to thrive in extreme conditions. Green algae Zygnema spp. survive in the Arctic and Antarctica as pre-akinetes, which are modified vegetative cells that accumulate lipids with oleic and linoleic acid being the main fatty acids. Graphical Abstract Figure. Green algae Zygnema spp. survive in the Arctic and Antarctica as pre-akinetes, which are modified vegetative cells that accumulate lipids with oleic and linoleic acid being the main fatty acids.

Two newly isolated strains of green algae from alpine regions were compared physiologically at different culture ages (1, 6, 9 and 15 months). The strains of Zygnema sp. were from different altitudes (‘Saalach’ (S), 440 m above sea level (a.s.l.), SAG 2419 and ‘Elmau-Alm’ (E-A), 1,500 m a.s.l., SAG 2418). Phylogenetic analysis of rbc L sequences grouped the strains into different major subclades of the genus. The mean diameters of the cells were 23.2 μm ( Zygnema S) and 18.7 μm ( Zygnema E-A) but were reduced significantly with culture age. The photophysiological response between the strains differed significantly; Zygnema S had a maximal relative electron transport rate ( rETR max ) of 103.4 μmol electrons m −2  s −1 , Zygnema E-A only 61.7 μmol electrons m −2  s −1 , and decreased significantly with culture age. Both strains showed a low-light adaption and the absence of strong photoinhibition up to 2,000 μmol photons m −2  s −1 . Photosynthetic oxygen production showed similar results ( P max Zygnema S, 527.2 μmol O 2  h −1  mg −1 chlorophyll (chl.) a , Zygnema E-A, 390.7 μmol O 2  h −1  mg −1 chl. a ); the temperature optimum was at 35 °C for Zygnema S and 30 °C for Zygnema E-A. Increasing culture age moreover leads to the formation of pre-akinetes, which accumulate storage products as revealed by light and transmission electron microscopy. Desiccation at 84 % relative air humidity (RH) lead to a reduction of the effective quantum yield of photosystem II (PSII) (Δ Fv /Fm′) to zero between 90 to 120 min ( Zygnema S) and between 30 to 60 min ( Zygnema E-A), depending on the culture age. A partial recovery of Δ Fv /Fm′ was only observed in older cultures. We conclude that pre-akinetes are crucial for the aeroterrestrial lifestyle of Zygnema .

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.

The mechanism by which plants cope with salt stress remains poorly understood. The goal of this study is to systematically investigate the contribution and distribution of inorganic ions and organic compounds to the osmotic adjustment (OA) in the halophyte species Halostachys caspica . The results indicate that 100–200 mM NaCl is optimal for plant growth; the water content and degree of succulence of the assimilating branches are higher in this treatment range than that in other treatments; parenchyma cells are more numerous with 100 mM NaCl treatment than they are in control. Inorganic ions (mainly Na + and Cl - ) may play a more important role than organic compounds in NaCl-induced OA and are the primary contributors in OA in H. caspica . The inorganic ions and organic solutes display a tissue-dependent distribution. Na + and Cl − are accumulated in the reproductive organs and within assimilating branches, which may represent a mechanism for protecting plant growth by way of salt ion dilution and organ abscission. Additionally, OA via increased accumulation of organic substances also protected plant growth and development. This finding provides additional evidence for plant tolerance to salinity stress which can be used for breeding new cultivars for stress tolerance.