Three novel complexes of deprotonated diflunisal (dif) with neocuproine (neo) were synthesized and characterized via elemental, spectral (UV-vis, FTIR, fluorescence, and mass spectrometry), and single-crystal X-ray diffraction analyses. Although the compounds shared a similar composition of [MCl(dif)(neo)], where M represents Zn(II) (1), Co(II) (2) and Cu(II) (3), only 1 and 2 were isostructural, while 3 differed in both the molecular and supramolecular structures. In all three complex molecules, the central atom is coordinated by two nitrogen atoms of neo in a bidentate chelate mode, and one chlorido ligand and dif is bonded in either a monodentate mode via one oxygen atom of the carboxylate in 1 and 2 or in a bidentate chelate mode via both carboxylate oxygen atoms in 3. All three compounds demonstrated remarkable antiproliferative activity against human prostate (PC-3), colon (HCT116) and breast (MDA-MB-468) cancer cell lines with IC50 values in the nanomolar range, with the lowest values observed in the case of PC-3 and MDA-MB-468 with 2 (20.0 nM) and 3 (31.1 nM), respectively. Moreover, complex 2, as the most active, was further investigated for its potential to induce perturbations in the cell cycle of PC-3 cells. The results indicated an induction of caspase-independent apoptosis. The interaction of the complexes with genomic DNA isolated from the respective cancer cell lines was evaluated for the intercalative mode, with binding strength correlated with the antiproliferative activity against PC-3 and MDA-MB-468 cancer cell lines.

Arabidopsis primary root growth response to phosphate (Pi) deficiency is mainly controlled by changes in apoplastic iron (Fe). Upon Pi deficiency, apoplastic Fe deposition in the root apical meristem activates pathways leading to the arrest of meristem maintenance and inhibition of cell elongation. Here, we report that a member of the uncharacterized cytochrome b561 and DOMON domain (CYBDOM) protein family, named CRR, promotes iron reduction in an ascorbate-dependent manner and controls apoplastic iron deposition. Under low Pi, the crr mutant shows an enhanced reduction of primary root growth associated with increased apoplastic Fe in the root meristem and a reduction in meristematic cell division. Conversely, CRR overexpression abolishes apoplastic Fe deposition rendering primary root growth insensitive to low Pi. The crr single mutant and crr hyp1 double mutant, harboring a null allele in another member of the CYDOM family, shows increased tolerance to high-Fe stress upon germination and seedling growth. Conversely, CRR overexpression is associated with increased uptake and translocation of Fe to the shoot and results in plants highly sensitive to Fe excess. Our results identify a ferric reductase implicated in Fe homeostasis and developmental responses to abiotic stress, and reveal a biological role for CYBDOM proteins in plants.

The genomes of many plants, animals, and fungi frequently comprise dispensable B chromosomes that rely upon various chromosomal drive mechanisms to counteract the tendency of non-essential genetic elements to be purged over time. The B chromosome of rye – a model system for nearly a century – undergoes targeted nondisjunction during first pollen mitosis, favouring segregation into the generative nucleus, thus increasing their numbers over generations. However, the genetic mechanisms underlying this process are poorly understood. Here, using a newly-assembled, ~430 Mb-long rye B chromosome pseudomolecule, we identify five candidate genes whose role as trans-acting moderators of the chromosomal drive is supported by karyotyping, chromosome drive analysis and comparative RNA-seq. Among them, we identify DCR28, coding a microtubule-associated protein related to cell division, and detect this gene also in the B chromosome of Aegilops speltoides. The DCR28 gene family is neo-functionalised and serially-duplicated with 15 B chromosome-located copies that are uniquely highly expressed in the first pollen mitosis of rye.

Methylerythritol cyclodiphosphate (MEcPP) is an intermediate in the biosynthesis of isoprenoids in plant plastids and in bacteria, and acts as a stress signal in plants. Here, we show that MEcPP regulates biofilm formation in Escherichia coli K-12 MG1655. Increased MEcPP levels, triggered by genetic manipulation or oxidative stress, inhibit biofilm development and production of fimbriae. Deletion of fimE, encoding a protein known to downregulate production of adhesive fimbriae, restores biofilm formation in cells with elevated MEcPP levels. Limited proteolysis-coupled mass spectrometry (LiP-MS) reveals that MEcPP interacts with the global regulatory protein H-NS, which is known to repress transcription of fimE. MEcPP prevents the binding of H-NS to the fimE promoter. Therefore, our results indicate that MEcPP can regulate biofilm formation by modulating H-NS activity and thus reducing fimbriae production. Further research is needed to test whether MEcPP plays similar regulatory roles in other bacteria.

For the development of anticancer drugs with higher activity and reduced toxicity, two approaches were combined: preparation of platinum(IV) complexes exhibiting higher stability compared to their platinum(II) counterparts and loading them into mesoporous silica SBA-15 with the aim to utilise the passive enhanced permeability and retention (EPR) effect of nanoparticles for accumulation in tumour tissues. Three conjugates based on a cisplatin scaffold bearing the anti-inflammatory drugs naproxen, ibuprofen or flurbiprofen in the axial positions (1, 2 and 3, respectively) were synthesised and loaded into SBA-15 to afford the mesoporous silica nanoparticles (MSNs) SBA-15|1, SBA-15|2 and SBA-15|3. Superior antiproliferative activity of both free and immobilised conjugates in a panel of four breast cancer cell lines (MDA-MB-468, HCC1937, MCF-7 and BT-474) with markedly increased cytotoxicity with respect to cisplatin was demonstrated. All compounds exhibit highest activity against the triple-negative cell line MDA-MB-468, with conjugate 1 being the most potent. However, against MCF-7 and BT-474 cell lines, the most notable improvement was found, with IC50 values up to 240-fold lower than cisplatin. Flow cytometry assays clearly show that all compounds induce apoptotic cell death elevating the levels of both early and late apoptotic cells. Furthermore, autophagy as well as formation of reactive oxygen species (ROS) and nitric oxide (NO) were elevated to a similar or greater extent than with cisplatin.

Phosphorus is an essential nutrient taken up by organisms in the form of inorganic phosphate (Pi). Eukaryotes have evolved sophisticated Pi sensing and signaling cascades, enabling them to stably maintain cellular Pi concentrations. Pi homeostasis is regulated by inositol pyrophosphate signaling molecules (PP-InsPs), which are sensed by SPX domain-containing proteins. In plants, PP-InsP-bound SPX receptors inactivate Myb coiled-coil (MYB-CC) Pi starvation response transcription factors (PHRs) by an unknown mechanism. Here we report that a InsP8–SPX complex targets the plant-unique CC domain of PHRs. Crystal structures of the CC domain reveal an unusual four-stranded anti-parallel arrangement. Interface mutations in the CC domain yield monomeric PHR1, which is no longer able to bind DNA with high affinity. Mutation of conserved basic residues located at the surface of the CC domain disrupt interaction with the SPX receptor in vitro and in planta, resulting in constitutive Pi starvation responses. Together, our findings suggest that InsP8 regulates plant Pi homeostasis by controlling the oligomeric state and hence the promoter binding capability of PHRs via their SPX receptors.

Cullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5 and their AUX/IAA targets perceive the phytohormone auxin. The F-box protein TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, by adopting biochemical, structural proteomics and in vivo approaches we unveil how flexibility in AUX/IAAs and regions in TIR1 affect their conformational ensemble allowing surface accessibility of degrons. We resolve TIR1·auxin·IAA7 and TIR1·auxin·IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) in the degron’s vicinity, cooperatively position AUX/IAAs on TIR1. We identify essential residues at the TIR1 N- and C-termini, which provide non-native interaction interfaces with IDRs and the folded PB1 domain of AUX/IAAs. We thereby establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation, and might provide conformational flexibility for a multiplicity of functional states.

Auxin is a small molecule morphogen that bridges SCFTIR1/AFB-AUX/IAA co-receptor interactions leading to ubiquitylation and proteasome-dependent degradation of AUX/IAA transcriptional repressors. Here, we systematically dissect auxin sensing by SCFTIR1-IAA6 and SCFTIR1-IAA19 co-receptor complexes, and assess IAA6/IAA19 ubiquitylation in vitro and IAA6/IAA19 degradation in vivo. We show that TIR1-IAA19 and TIR1-IAA6 have distinct auxin affinities that correlate with ubiquitylation and turnover dynamics of the AUX/IAA. We establish a system to track AUX/IAA ubiquitylation in IAA6 and IAA19 in vitro and show that it occurs in flexible hotspots in degron-flanking regions adorned with specific Lys residues. We propose that this signature is exploited during auxin-mediated SCFTIR1-AUX/IAA interactions. We present evidence for an evolving AUX/IAA repertoire, typified by the IAA6/IAA19 ohnologues, that discriminates the range of auxin concentrations found in plants. We postulate that the intrinsic flexibility of AUX/IAAs might bias their ubiquitylation and destruction kinetics enabling specific auxin responses.

Crop yield loss due to flooding is a threat to food security. Submergence-induced hypoxia in plants results in stabilization of group VII ETHYLENE RESPONSE FACTORs (ERF-VIIs), which aid survival under these adverse conditions. ERF-VII stability is controlled by the N-end rule pathway, which proposes that ERF-VII N-terminal cysteine oxidation in normoxia enables arginylation followed by proteasomal degradation. The PLANT CYSTEINE OXIDASEs (PCOs) have been identified as catalysts of this oxidation. ERF-VII stabilization in hypoxia presumably arises from reduced PCO activity. We directly demonstrate that PCO dioxygenase activity produces Cys-sulfinic acid at the N terminus of an ERF-VII peptide, which then undergoes efficient arginylation by an arginyl transferase (ATE1). This provides molecular evidence of N-terminal Cys-sulfinic acid formation and arginylation by N-end rule pathway components, and a substrate of ATE1 in plants. The PCOs and ATE1 may be viable intervention targets to stabilize N-end rule substrates, including ERF-VIIs, to enhance submergence tolerance in agriculture.

Most Gram-negative phytopathogenic bacteria inject type III effector (T3E) proteins into plant cells to manipulate signaling pathways to the pathogen’s benefit. In resistant plants, specialized immune receptors recognize single T3Es or their biochemical activities, thus halting pathogen ingress. However, molecular function and mode of recognition for most T3Es remains elusive. Here, we show that the Xanthomonas T3E XopH possesses phytase activity, i.e., dephosphorylates phytate (myo-inositol-hexakisphosphate, InsP6), the major phosphate storage compound in plants, which is also involved in pathogen defense. A combination of biochemical approaches, including a new NMR-based method to discriminate inositol polyphosphate enantiomers, identifies XopH as a naturally occurring 1-phytase that dephosphorylates InsP6 at C1. Infection of Nicotiana benthamiana and pepper by Xanthomonas results in a XopH-dependent conversion of InsP6 to InsP5. 1-phytase activity is required for XopH-mediated immunity of plants carrying the Bs7 resistance gene, and for induction of jasmonate- and ethylene-responsive genes in N. benthamiana.