B‐GATA factors are required to repress high‐light stress responses in Marchantia polymorpha and Arabidopsis thaliana

GATAs are evolutionarily conserved zinc-finger transcription factors from eukaryotes. In plants, GATAs can be subdivided into four classes, A–D, based on their DNA-binding domain, and into further subclasses based on additional protein motifs. B-GATAs with a so-called leucine-leucine-methionine (LLM)-domain can already be found in algae. In angiosperms, the B-GATA family is expanded and can be subdivided in to LLM- or HAN-domain B-GATAs. Both, the LLM- and the HAN-domain are conserved domains of unknown biochemical function. Interestingly, the B-GATA family in the liverwort Marchantia polymorpha and the moss Physcomitrium patens is restricted to one and four family members, respectively. And, in contrast to vascular plants, the bryophyte B-GATAs contain a HAN- as well as an LLM-domain. Here, we characterise mutants of the single B-GATA from Marchantia polymorpha. We reveal that this mutant has defects in thallus growth and in gemma formation. Transcriptomic studies uncover that the B-GATA mutant displays a constitutive high-light (HL) stress response, a phenotype that we then also confirm in mutants of Arabidopsis thaliana LLM-domain B-GATAs, suggesting that the B-GATAs have a protective role towards HL stress.Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659Peer Reviewe

Conserved redox-dependent DNA binding of ROXY glutaredoxins with TGA transcription factors

peer-reviewedThe Arabidopsis thaliana CC‐type glutaredoxin (GRX) ROXY1 and the bZIP TGA transcription factor (TF) PERIANTHIA (PAN) interact in the nucleus and together regulate petal development. The CC‐type GRXs exist exclusively in land plants, and in contrast to the ubiquitously occurring CPYC and CGFS GRX classes, only the CC‐type GRXs expanded strongly during land plant evolution. Phylogenetic analyses show that TGA TFs evolved before the CC‐type GRXs in charophycean algae. MpROXY1/2 and MpTGA were isolated from the liverwort Marchantia polymorpha to analyze regulatory ROXY/TGA interactions in a basal land plant. Homologous and heterologous protein interaction studies demonstrate that nuclear ROXY/TGA interactions are conserved since the occurrence of CC‐type GRXs in bryophytes and mediated by a conserved ROXY C‐terminus. Redox EMSA analyses show a redox‐sensitive binding of MpTGA to the cis‐regulatory as‐1‐like element. Furthermore, we demonstrate that MpTGA binds together with MpROXY1/2 to this motif under reducing conditions, whereas this interaction is not observed under oxidizing conditions. Remarkably, heterologous complementation studies reveal a strongly conserved land plant ROXY activity, suggesting an ancestral role for CC‐type GRXs in modulating the activities of TGA TFs. Super‐resolution microscopy experiments detected a strong colocalization of ROXY1 with the active form of the RNA polymerase II in the nucleus. Together, these data shed new light on the function of ROXYs and TGA TFs and the evolution of redox‐sensitive transcription regulation processes, which likely contributed to adapt land plants to novel terrestrial habitats

The N-Terminus of the Floral Arabidopsis TGA Transcription Factor PERIANTHIA Mediates Redox-Sensitive DNA-Binding.

The Arabidopsis TGA transcription factor (TF) PERIANTHIA (PAN) regulates the formation of the floral organ primordia as revealed by the pan mutant forming an abnormal pentamerous arrangement of the outer three floral whorls. The Arabidopsis TGA bZIP TF family comprises 10 members, of which PAN and TGA9/10 control flower developmental processes and TGA1/2/5/6 participate in stress-responses. For the TGA1 protein it was shown that several cysteines can be redox-dependently modified. TGA proteins interact in the nucleus with land plant-specific glutaredoxins, which may alter their activities posttranslationally. Here, we investigated the DNA-binding of PAN to the AAGAAT motif under different redox-conditions. The AAGAAT motif is localized in the second intron of the floral homeotic regulator AGAMOUS (AG), which controls stamen and carpel development as well as floral determinacy. Whereas PAN protein binds to this regulatory cis-element under reducing conditions, the interaction is strongly reduced under oxidizing conditions in EMSA studies. The redox-sensitive DNA-binding is mediated via a special PAN N-terminus, which is not present in other Arabidopsis TGA TFs and comprises five cysteines. Two N-terminal PAN cysteines, Cys68 and Cys87, were shown to form a disulfide bridge and Cys340, localized in a C-terminal putative transactivation domain, can be S-glutathionylated. Comparative land plant analyses revealed that the AAGAAT motif exists in asterid and rosid plant species. TGA TFs with N-terminal extensions of variable length were identified in all analyzed seed plants. However, a PAN-like N-terminus exists only in the rosids and exclusively Brassicaceae homologs comprise four to five of the PAN N-terminal cysteines. Redox-dependent modifications of TGA cysteines are known to regulate the activity of stress-related TGA TFs. Here, we show that the N-terminal PAN cysteines participate in a redox-dependent control of the PAN interaction with a highly conserved regulatory AG cis-element, emphasizing the importance of redox-modifications in the regulation of flower developmental processes

Evolution of the N-terminal PAN extension and the <i>AAGAAT</i> motif.

<p>Cladogram of evolutionary informative species from seed plants indicating <i>AAGAAT</i> motifs and N-terminal extensions of the respective PAN and <i>AG</i> homologs. Orange boxes indicate presence of an <i>AAGAAT</i> motif comprising the AAGAAT sequence and core TGA binding site. Beige boxes label partial <i>AAGAAT</i> motifs, which possess only the AAGAAT sequence and lack the core TGA binding sequence TGACG. Schematic representation of PAN protein homologs shows N-terminal extensions with significant (grey bar) or low sequence homology (striped bar) to the PAN N-terminus or its absence. Presence (C) or absence (X) of cysteines equivalent to the five N-terminal PAN cysteines is depicted. Stars indicate that the definition of a magnoliid PAN homolog start codon was not possible and that no genome sequencing data were available.</p

Analysis of posttranslational PAN modifications.

<p>(A) Non-reducing SDS-PAGE analysis of MBP-PAN and MBP-PAN6xCysmut proteins. Proteins were incubated with 20 mM DTT, 2 mM diamide or with 2 mM diamide followed by 20 mM DTT. Triangles indicate MBP-PAN (~90 kDa) and MBP-PAN6xCysmut (~90 kDa) proteins and the asterisk marks MBP (~43 kDa). Dimerized and oligomerized fusion proteins are labeled with full circles. (B) Non-reducing SDS-PAGE analysis for identification of posttranslational cysteine modifications. MBP-PAN protein was incubated under different conditions (20 mM DTT, 2 mM diamide, 10 mM GSSG and 5 mM GSH/5 mM H₂O₂) and interesting bands, marked with an asterisk, were further processed for MS analysis. (C) Posttranslational PAN cysteine modifications as determined by the difference between predicted, unmodified and detected MS mass values.</p

Analysis of redox-dependent PAN DNA-binding.

<p>(A) <i>Arabidopsis</i> wild-type flowers are composed of four sepals, four petals, six stamens and two fused carpels. Flowers of <i>pan</i> mutants form three pentameric whorls with five sepals, five petals, five stamens and two normal carpels. Scale bar 1 mm. (B) Sequence of the palindromic core TGA DNA-binding motif is shown on the top and the three used binding sites comprising the core motif below. The <i>as-1</i>-like motif from the Arabidopsis <i>PR1</i> promoter comprises two TGA binding sites. The <i>AAGAAT</i> motif from the second intron of <i>AG</i> encompasses one central TGA binding site with one nucleotide exchange, the name-giving 5’ <i>AAGAAT</i> motif is underlined. Mutagenesis of two nucleotides (indicated in red) abolishes binding to the <i>ΔbZIP</i> motif. (C) EMSA analysis of the PAN interaction with the <i>AAGAAT</i>, <i>as-1</i>-like and <i>ΔbZIP</i> motifs. Radioactive-labeled DNA probes were incubated with PAN protein or with a mock translation under reducing conditions (0.9 mM DTT). The arrow indicates DNA-protein complex formation and the asterisk marks free DNA probe. D) Analysis of redox-dependent binding of PAN to the <i>AAGAAT</i> motif. For comparison of reducing and oxidizing conditions, PAN protein was incubated prior to DNA-binding with 0.9 mM or 20 mM DTT and with 2 mM diamide, respectively. Reversibility of the redox-dependent DNA-binding was analyzed by the addition of 20 mM DTT after a 2 mM diamide treatment. The control reaction was conducted without protein addition.</p

Redox-dependent DNA-binding of PAN is mediated via the N-terminal cysteines.

<p>Redox EMSA analyses to investigate the influence of single (A) and multiple Cys-to-Ser substitutions (B and C) and the effect of a N-terminal PAN truncation (D). Schematic illustrations of the PAN variants indicate the bZIP domain in orange and the glutamine-rich regions in blue boxes. Cys-to-Ser substitutions are labeled in red. PAN proteins were incubated in 0.9 mM DTT (red) or 2 mM diamide (ox) prior to addition of the <i>AAGAAT</i> motif. Asterisks mark unbound free probe.</p

Redox-dependent binding of selected Arabidopsis TGA TFs to the <i>AAGAAT</i> motif.

<p>(A) Cladogram of Arabidopsis TGA TFs indicating clade memberships. (B) Schematic representation of selected TGA TF family members and respective cysteine residue positions. Orange boxes mark the bZIP domain and blue boxes two glutamine-rich regions. Cys340, Cys260 and Cys301 (red) are localized in a conserved position in the first glutamine-rich region. (C) Gel retardation analysis of selected TGA proteins incubated with the <i>AAGAAT</i> probe under reducing (0.9 mM DTT; red) or oxidizing (2 mM diamide; ox) conditions. Asterisk marks unbound <i>AAGAAT</i> probe.</p