Erratum: Publisher Correction: SnRK2 protein kinases represent an ancient system in plants for adaptation to a terrestrial environment.

[This corrects the article DOI: 10.1038/s42003-019-0281-1.].

SnRK2 protein kinases represent an ancient system in plants for adaptation to a terrestrial environment.

The SNF1-related protein kinase 2 (SnRK2) family includes key regulators of osmostress and abscisic acid (ABA) responses in angiosperms and can be classified into three subclasses. Subclass III SnRK2s act in the ABA response while ABA-nonresponsive subclass I SnRK2s are regulated through osmostress. Here we report that an ancient subclass III SnRK2-based signalling module including ABA and an upstream Raf-like kinase (ARK) exclusively protects the moss Physcomitrella patens from drought. Subclass III SnRK2s from both Arabidopsis and from the semiterrestrial alga Klebsormidium nitens, which contains all the components of ABA signalling except ABA receptors, complement Physcomitrella snrk2 - mutants, whereas Arabidopsis subclass I SnRK2 cannot. We propose that the earliest land plants developed the ABA/ARK/subclass III SnRK2 signalling module by recruiting ABA to regulate a pre-existing dehydration response and that subsequently a novel subclass I SnRK2 system evolved in vascular plants conferring osmostress protection independently from the ancient system.

NLR locus-mediated trade-off between abiotic and biotic stress adaptation in Arabidopsis.

Osmotic stress caused by drought, salt or cold decreases plant fitness. Acquired stress tolerance defines the ability of plants to withstand stress following an initial exposure1. We found previously that acquired osmotolerance after salt stress is widespread among Arabidopsis thaliana accessions2. Here, we identify ACQOS as the locus responsible for ACQUIRED OSMOTOLERANCE. Of its five haplotypes, only plants carrying group 1 ACQOS are impaired in acquired osmotolerance. ACQOS is identical to VICTR, encoding a nucleotide-binding leucine-rich repeat (NLR) protein3. In the absence of osmotic stress, group 1 ACQOS contributes to bacterial resistance. In its presence, ACQOS causes detrimental autoimmunity, thereby reducing osmotolerance. Analysis of natural variation at the ACQOS locus suggests that functional and non-functional ACQOS alleles are being maintained due to a trade-off between biotic and abiotic stress adaptation. Thus, polymorphism in certain plant NLR genes might be influenced by competing environmental stresses.

Structural characterization of highly branched glucan sheath from Ceriporiopsis subvermispora.

Wood rotting basidiomycetes produce extracellular mucilaginous sheaths interfacing fungal hyphae and plant biomass. While the versatility of these fungal sheaths has been addressed, sheaths generated by selective white-rot fungi remain poorly understood. To fill this gap, the sheath produced by the basidiomycete Ceriporiopsis subvermispora, which degrades lignin while inflicting limited cellulose damage, was analyzed in this study. Fluorescence and transmission electron microscopy revealed that the sheath formed three days after inoculation into a beech wood slice on an agar plate and was embedded at the interface between fungal hyphae and wood cell walls. The sheath's chemical structure was evaluated from fungus cultures in a liquid medium containing [U-13C6]-d-glucose and beech wood slices. Compositional analysis, methylation analysis, and 13C NMR demonstrated that the sheath mainly consisted of a comb-like ß-1,6-glucopyranose residue-branched ß-1,3-glucan, which is advantageous to retain water and extracellular secondary metabolites.

Large-scale proteome analysis of abscisic acid and ABSCISIC ACID INSENSITIVE3-dependent proteins related to desiccation tolerance in Physcomitrella patens.

Desiccation tolerance is an ancestral feature of land plants and is still retained in non-vascular plants such as bryophytes and some vascular plants. However, except for seeds and spores, this trait is absent in vegetative tissues of vascular plants. Although many studies have focused on understanding the molecular basis underlying desiccation tolerance using transcriptome and proteome approaches, the critical molecular differences between desiccation tolerant plants and non-desiccation plants are still not clear. The moss Physcomitrella patens cannot survive rapid desiccation under laboratory conditions, but if cells of the protonemata are treated by the phytohormone abscisic acid (ABA) prior to desiccation, it can survive 24 h exposure to desiccation and regrow after rehydration. The desiccation tolerance induced by ABA (AiDT) is specific to this hormone, but also depends on a plant transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3). Here we report the comparative proteomic analysis of AiDT between wild type and ABI3 deleted mutant (Δabi3) of P. patens using iTRAQ (Isobaric Tags for Relative and Absolute Quantification). From a total of 1980 unique proteins that we identified, only 16 proteins are significantly altered in Δabi3 compared to wild type after desiccation following ABA treatment. Among this group, three of the four proteins that were severely affected in Δabi3 tissue were Arabidopsis orthologous genes, which were expressed in maturing seeds under the regulation of ABI3. These included a Group 1 late embryogenesis abundant (LEA) protein, a short-chain dehydrogenase, and a desiccation-related protein. Our results suggest that at least three of these proteins expressed in desiccation tolerant cells of both Arabidopsis and the moss are very likely to play important roles in acquisition of desiccation tolerance in land plants. Furthermore, our results suggest that the regulatory machinery of ABA- and ABI3-mediated gene expression for desiccation tolerance might have evolved in ancestral land plants before the separation of bryophytes and vascular plants.

A simple and efficient dispersion correction to the Hartree-Fock theory (3): A comprehensive performance comparison of HF-Dtq with MP2 and DFT-Ds.

Accurate prediction of the intermolecular interaction energy (ΔEbind) has been a challenging and serious problem. Current in silico drug screening demands efficient and accurate evaluation of ΔEbind for ligands and their target proteins. It is desirable that ΔEbind including the dispersion interaction energy (Edisp) is calculated using a post-Hartree-Fock (HF) theory, such as the high-order coupled-cluster one, with a larger basis set. However, it remains computationally too expensive to apply such a one to large molecular systems. As another problem, it is necessary to consider the contribution of the basis set superposition error (BSSE) in calculation of ΔEbind. In Bioorg. Med. Chem. Lett. 2014 and 2015, we proposed simple and efficient corrections of dispersion and BSSE for the HF theory, which is not able to express the dispersion interaction energy correctly. The current Letter, as the final one in the series, aims to verify the HF theory enhanced by the dispersion correction (HF-Dtq) in the light of reproducibility of 'accurate' intermolecular ligand-protein interaction energy values, with comprehensive comparison with the MP2 and recently proposed various DFT-D theories. Taking ΔEbind calculated with the coupled-cluster theory coupled with a complete basis set as a reference, ΔEbind of over a hundred small sized noncovalent complexes as well as real ligand-protein complexes models was systematically examined in terms of accuracy and computational cost. The comprehensive comparison in the current work showed that HF-Dtq is a practical and reliable approach for in silico drug screening and quantitative structure-activity relationships.

A simple and efficient dispersion correction to the Hartree-Fock theory (2): Incorporation of a geometrical correction for the basis set superposition error.

One of the most challenging problems in computer-aided drug discovery is the accurate prediction of the binding energy between a ligand and a protein. For accurate estimation of net binding energy ΔEbind in the framework of the Hartree-Fock (HF) theory, it is necessary to estimate two additional energy terms; the dispersion interaction energy (Edisp) and the basis set superposition error (BSSE). We previously reported a simple and efficient dispersion correction, Edisp, to the Hartree-Fock theory (HF-Dtq). In the present study, an approximation procedure for estimating BSSE proposed by Kruse and Grimme, a geometrical counterpoise correction (gCP), was incorporated into HF-Dtq (HF-Dtq-gCP). The relative weights of the Edisp (Dtq) and BSSE (gCP) terms were determined to reproduce ΔEbind calculated with CCSD(T)/CBS or /aug-cc-pVTZ (HF-Dtq-gCP (scaled)). The performance of HF-Dtq-gCP (scaled) was compared with that of B3LYP-D3(BJ)-bCP (dispersion corrected B3LYP with the Boys and Bernadi counterpoise correction (bCP)), by taking ΔEbind (CCSD(T)-bCP) of small non-covalent complexes as 'a golden standard'. As a critical test, HF-Dtq-gCP (scaled)/6-31G(d) and B3LYP-D3(BJ)-bCP/6-31G(d) were applied to the complex model for HIV-1 protease and its potent inhibitor, KNI-10033. The present results demonstrate that HF-Dtq-gCP (scaled) is a useful and powerful remedy for accurately and promptly predicting ΔEbind between a ligand and a protein, albeit it is a simple correction procedure.

CSP41b, a protein identified via FOX hunting using Eutrema salsugineum cDNAs, improves heat and salinity stress tolerance in transgenic Arabidopsis thaliana.

Eutrema salsugineum (also known as Thellungiella salsuginea and formerly Thellungiella halophila), a species closely related to Arabidopsis thaliana, shows tolerance not only to salt stress, but also to chilling, freezing, and high temperatures. To identify genes responsible for stress tolerance, we conducted Full-length cDNA Over-eXpressing gene (FOX) hunting among a collection of E. salsugineum cDNAs that were stress-induced according to gene ontology analysis or over-expressed in E. salsugineum compared with A. thaliana. We identified E. salsugineum CSP41b (chloroplast stem-loop-binding protein of 41 kDa; also known as CRB, chloroplast RNA binding; named here as EsCSP41b) as a gene that can confer heat and salinity stress tolerance on A. thaliana. A. thaliana CSP41b is reported to play an important role in the proper functioning of the chloroplast: the atcsp41b mutant is smaller and paler than wild-type plants and shows altered chloroplast morphology and photosynthetic performance. We observed that AtCSP41b-overexpressing transgenic A. thaliana lines also exhibited marked heat tolerance and significant salinity stress tolerance. The EsCSP41b-overexpressing transgenic A. thaliana lines showed significantly higher photosynthesis activity than wild-type plants not only under normal growth conditions but also under heat stress. In wild-type plants, the expression levels of both EsCSP41b and AtCSP41b were significantly reduced under heat or salinity stress. We conclude that maintenance of CSP41b expression under abiotic stresses may alleviate photoinhibition and improve survival under such stresses.

Group A PP2Cs evolved in land plants as key regulators of intrinsic desiccation tolerance.

Vegetative desiccation tolerance is common in bryophytes, although this character has been lost in most vascular plants. The moss Physcomitrella patens survives complete desiccation if treated with abscisic acid (ABA). Group A protein phosphatases type 2C (PP2C) are negative regulators of abscisic acid signalling. Here we show that the elimination of Group A PP2C is sufficient to ensure P. patens survival to full desiccation, without ABA treatment, although its growth is severely hindered. Microarray analysis shows that the Group A PP2C-regulated genes exclusively overlap with genes exhibiting a high level of ABA induction. Group A PP2C disruption weakly affects ABA-activated kinase activity, indicating Group A PP2C action downstream of these kinases in the moss. We propose that Group A PP2C emerged in land plants to repress desiccation tolerance mechanisms, possibly facilitating plants propagation on land, whereas ABA releases the intrinsic desiccation tolerance from Group A PP2C regulation.

Arabidopsis sos1 mutant in a salt-tolerant accession revealed an importance of salt acclimation ability in plant salt tolerance.

An analysis of the salinity tolerance of 354 Arabidopsis thaliana accessions showed that some accessions were more tolerant to salt shock than the reference accession, Col-0, when transferred from 0 to 225 mM NaCl. In addition, several accessions, including Zu-0, showed marked acquired salt tolerance after exposure to moderate salt stress. It is likely therefore that Arabidopsis plants have at least two types of tolerance, salt shock tolerance and acquired salt tolerance. To evaluate a role of well-known salt shock tolerant gene SOS1 in acquired salt tolerance, we isolated a sos1 mutant from ion-beam-mutagenized Zu-0 seedlings. The mutant showed severe growth inhibition under salt shock stress owing to a single base deletion in the SOS1 gene and was even more salt sensitive than Col-0. Nevertheless, it was able to survive after acclimation on 100 mM NaCl for 7 d followed by 750 mM sorbitol for 20 d, whereas Col-0 became chlorotic under the same conditions. We propose that genes for salt acclimation ability are different from genes for salt shock tolerance and play an important role in the acquisition of salt or osmotic tolerance.

ABSCISIC ACID INSENSITIVE3 regulates abscisic acid-responsive gene expression with the nuclear factor Y complex through the ACTT-core element in Physcomitrella patens.

The phytohormone ABA and the transcription factor ABSCISIC ACID INSENSITIVE 3 (ABI3)/VIVIPAROUS 1 (VP1) function in protecting embryos during the desiccation stage of seed development. In a similar signaling pathway, vegetative tissue of the moss Physcomitrella patens survives desiccation by activating downstream genes (e.g. LEA1) in response to ABA and ABI3. We show that the PpLEA1 promoter responds to PpABI3 primarily through the ACTT-core element (5'-TCCACTTGTC-3'), while the ACGT-core ABA-responsive element (ABRE) appears to respond to ABA alone. We also found by yeast-two-hybrid screening that PpABI3A interacts with PpNF-YC1, a subunit of CCAAT box binding factor (CBF)/nuclear factor Y (NF-Y). PpNF-YC1 increased the activation of the PpLEA1 promoter when incubated with PpABI3A, as did NF-YB, NF-YC, and ABI3 from Arabidopsis. This new response element (ACTT) is responsible for activating the ABI3-dependent ABA response pathway cooperatively with the nuclear factor Y (NF-Y) complex. These results further define the regulatory interactions at the transcriptional level for the expression of this network of genes required for drought/desiccation tolerance. This gene regulatory set is in large part conserved between vegetative tissue of bryophytes and seeds of angiosperms and will shed light on the evolution of this pathway in the green plant lineage.

Purification and characterization of a soluble ß-1,4-glucan from bean (Phaseolus vulgaris L.)-cultured cells dehabituated to dichlobenil.

Bean cells habituated to grow in the presence of dichlobenil exhibited reduced cellulose and hemicellulose content and an increase in pectic polysaccharides. Furthermore, following the extraction of pectins and hemicelluloses, a large amount of neutral sugars was released. These sugars were found to be part of a soluble ß-1,4-glucan in a preliminary characterization, as reported by Encina et al. (Physiol Plant 114:182-191, 2002). When habituated cells were subcultured in the absence of the herbicide (dehabituated cells), the release of neutral sugars after the extraction of pectins and hemicelluloses was maintained. In this study, we have isolated a soluble ß-1,4-glucan from dehabituated cells by sonication of the wall residue (cellulose fraction) remaining after fractionation. Gel filtration chromatography revealed that its average molecular size was 14 kDa. Digestion of the sample with endocellulase revealed the presence of cellobiose, cellotriose, and cellotetraose. Methylation analysis showed that 4-linked glucose was the most abundant sugar residue, but 4,6-linked glucose, terminal arabinose and 4-linked galactose for xyloglucan, and arabinogalactan were also identified. NMR analysis showed that this 1,4-glucan may be composed of various kinds of substitutions along the glucan backbone together with acetyl groups linked to the OH group of sugar residues. Thus, despite its relatively high molecular mass, the ß-glucan remains soluble because of its unique configuration. This is the first time that a glucan with such characteristics has been isolated and described. The discovery of new molecules, as this ß-glucan with unique features, may help understand the composition and arrangement of the polymers within plant cell walls, contributing to a better understanding of this complex structure.

HsfA1d, a protein identified via FOX hunting using Thellungiella salsuginea cDNAs improves heat tolerance by regulating heat-stress-responsive gene expression.

Thellungiella salsuginea (formerly T. halophila), a species closely related to Arabidopsis (Arabidopsis thaliana), is tolerant not only to high salt levels, but also to chilling, freezing, and ozone. Here, we report that T. salsuginea also shows greater heat tolerance than Arabidopsis. We identified T. salsuginea HsfA1d (TsHsfA1d) as a gene that can confer marked heat tolerance on Arabidopsis. TsHsfA1d was identified via Full-length cDNA Over-eXpressing gene (FOX) hunting from among a collection of heat-stress-related T. salsuginea cDNAs. Transgenic Arabidopsis overexpressing TsHsfA1d showed constitutive up-regulation of many genes in the Arabidopsis AtHsfA1 regulon under normal growth temperature. In Arabidopsis mesophyll protoplasts, TsHsfA1d was localized in both the nucleus and the cytoplasm. TsHsfA1d also interacted with AtHSP90, which negatively regulates AtHsfA1s by forming HsfA1-HSP90 complexes in the cytoplasm. It is likely that the partial nuclear localization of TsHsfA1d induced the expression of the AtHsfA1d regulon in the transgenic plants at normal temperature. We also discovered that transgenic Arabidopsis plants overexpressing AtHsfA1d were more heat-tolerant than wild-type plants and up-regulated the expression of the HsfA1d regulon, as was observed in TsHsfA1d-overexpressing plants. We propose that the products of both TsHsfA1d and AtHsfA1d function as positive regulators of Arabidopsis heat-stress response and would be useful for the improvement of heat-stress tolerance in other plants.

A novel abi5 allele reveals the importance of the conserved Ala in the C3 domain for regulation of downstream genes and salt tolerance during germination in Arabidopsis.

Abscisic acid (ABA) signal transduction during Arabidopsis seed development and germination requires a Group A bZIP transcription factor encoded by ABA INSENSITIVE5 (ABI5). In addition to the basic leucine zipper DNA binding domain, Group A bZIPs are characterized by three N-terminal conserved regions (C1, C2 and C3) and one C-terminal conserved region (C4). These conserved regions are considered to play roles in ABI5 functions; however, except for the phosphorylation site, the importance of the highly conserved amino acids is unclear. Here, we report a novel abi5 recessive allele (abi5-9) that encodes an intact ABI5 protein with one amino acid substitution (A214G) in the C3 domain. The abi5-9 plants showed ABA insensitivity during germination and could germinate on medium containing 175 mM NaCl or 500 mM mannitol. Em1 and Em6--both encoding late embryogenesis abundant (LEA) proteins and directly targeted by ABI5 regulation--were expressed at very low levels in abi5-9 plants compared with the wild type. In yeast, the abi5-9 protein exhibited greatly reduced interaction with ABI3 compared with ABI5. These data suggest that Ala214 in ABI5 contributes to the function of ABI5 via its interaction with ABI3.

Functions of xyloglucan in plant cells.

While an increase in the number of xyloglucan tethers between the cellulose microfibrils in plant cell walls increases the walls' rigidity, the degradation of these tethers causes the walls to loosen. Degradation can occur either through the integration of xyloglucan oligosaccharides due to the action of xyloglucan endotransglucosylase or through direct hydrolysis due to the action of xyloglucanase. This is why the addition of xyloglucan and its fragment oligosaccharides causes plant tissue tension to increase and decrease so dramatically. Experiments involving the overexpression of xyloglucanase and cellulase have revealed the roles of xyloglucans in the walls. The degradation of wall xyloglucan in poplar by the transgenic expression of xyloglucanase, for example, not only accelerated stem elongation in the primary wall, but also blocked upright-stem gravitropism in the secondary wall. Overexpression of cellulase also reduced xyloglucan content in the walls as cellulose microfibrils were trimmed at their amorphous region, resulting in increased cell volume in Arabidopsis leaves and in sengon with disturbed leaf movements. The hemicellulose xyloglucan, in its function as a tether, plays a key role in the loosening and tightening of cellulose microfibrils: it enables the cell to change its shape in growth and differentiation zones and to retain its final shape after cell maturation.

Acceleration of cell growth by xyloglucan oligosaccharides in suspension-cultured tobacco cells.

The incorporation of xyloglucan oligosaccharide (XXXG) into the walls of suspension-cultured tobacco cells accelerated cell expansion followed by cell division, changed cell shape from cylindrical to spherical, decreased cell size, and caused cell aggregation. Fluorescent XXXG added to the culture medium was found to be incorporated into the surface of the entire wall, where strong incorporation occurred not only on the surface, but also in the interface walls between cells during cell division. Cell expansion was always greater in the transverse direction than in the longitudinal direction and then, immediately, expansion led to cell division in the presence of XXXG; this process might result in the high level of cell aggregation seen in cultured tobacco cells. We concluded that the integration of this oligosaccharide into the walls could accelerate not only cell expansion, but also cell division in cultured cells.

Potential role for purple acid phosphatase in the dephosphorylation of wall proteins in tobacco cells.

It is not yet known whether dephosphorylation of proteins catalyzed by phosphatases occurs in the apoplastic space. In this study, we found that tobacco (Nicotiana tabacum) purple acid phosphatase could dephosphorylate the phosphoryl residues of three apoplastic proteins, two of which were identified as alpha-xylosidase and beta-glucosidase. The dephosphorylation and phosphorylation of recombinant alpha-xylosidase resulted in a decrease and an increase in its activity, respectively, when xyloglucan heptasaccharide was used as a substrate. Attempted overexpression of the tobacco purple acid phosphatase NtPAP12 in tobacco cells not only decreased the activity levels of the glycosidases but also increased levels of xyloglucan oligosaccharides and cello-oligosaccharides in the apoplast during the exponential phase. We suggest that purple acid phosphatase controls the activity of alpha-xylosidase and beta-glucosidase, which are responsible for the degradation of xyloglucan oligosaccharides and cello-oligosaccharides in the cell walls.

Sucrose synthase is an integral component of the cellulose synthesis machinery.

Cellulose synthesis in plants is believed to be carried out by the plasma membrane-associated rosette structure which can be observed by electron microscopy. Despite decade-long speculation, it had not been demonstrated whether the rosette is the site of catalytic activity of cellulose synthesis. To determine the relationship between this structure and cellulose synthesis, we successfully isolated detergent-insoluble rosettes from the plasma membrane of bean epicotyls. However, the purified rosettes did not possess cellulose synthesis activity in vitro. Conversely, detergent-soluble granular particles of approximately 9.5-10 nm diameter were also isolated and exhibited UDP-glucose binding activity and possessed beta-1,4-glucan (cellulose) synthesis activity in vitro. The particle, referred to as the catalytic unit of cellulose synthesis, was enriched with a 78 kDa polypeptide which was verified as sucrose synthase like by mass spectrometry and immunoblotting. The catalytic units were able to bind to the rosettes and retained the cellulose synthesis activity in the presence of UDP-glucose or sucrose plus UDP when supplemented with magnesium. The incorporation of the catalytic unit into the rosette structure was confirmed by immunogold labeling with anti-sucrose synthase antibodies under an electron microscope. Our results suggest that the plasma membrane-associated rosette anchors the catalytic unit of cellulose synthesis to form the functional cellulose synthesis machinery.

Xyloglucan for generating tensile stress to bend tree stem.

In response to environmental variation, angiosperm trees bend their stems by forming tension wood, which consists of a cellulose-rich G (gelatinous)-layer in the walls of fiber cells and generates abnormal tensile stress in the secondary xylem. We produced transgenic poplar plants overexpressing several endoglycanases to reduce each specific polysaccharide in the cell wall, as the secondary xylem consists of primary and secondary wall layers. When placed horizontally, the basal regions of stems of transgenic poplars overexpressing xyloglucanase alone could not bend upward due to low strain in the tension side of the xylem. In the wild-type plants, xyloglucan was found in the inner surface of G-layers during multiple layering. In situ xyloglucan endotransglucosylase (XET) activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, began at the inner surface layers S1 and S2 and was retained throughout G-layer development, while the incorporation of xyloglucan heptasaccharide (XXXG) for wall loosening occurred in the primary wall of the expanding zone. We propose that the xyloglucan network is reinforced by XET to form a further connection between wall-bound and secreted xyloglucans in order to withstand the tensile stress created within the cellulose G-layer microfibrils.

Loosening xyloglucan accelerates the enzymatic degradation of cellulose in wood.

In order to create trees in which cellulose, the most abundant component in biomass, can be enzymatically hydrolyzed highly for the production of bioethanol, we examined the saccharification of xylem from several transgenic poplars, each overexpressing either xyloglucanase, cellulase, xylanase, or galactanase. The level of cellulose degradation achieved by a cellulase preparation was markedly greater in the xylem overexpressing xyloglucanase and much greater in the xylems overexpressing xylanase and cellulase than in the xylem of the wild-type plant. Although a high degree of degradation occurred in all xylems at all loci, the crystalline region of the cellulose microfibrils was highly degraded in the xylem overexpressing xyloglucanase. Since the complex between microfibrils and xyloglucans could be one region that is particularly resistant to cellulose degradation, loosening xyloglucan could facilitate the enzymatic hydrolysis of cellulose in wood.