The axe-txe type II toxin-antitoxin (TA) system is characterized by a complex and multilayered mode of gene expression regulation. Precise and tight control of this process is crucial to keep the toxin in an appropriate balance with the cognate antitoxin until its activation is needed for the cell. In this report, we provide evidence that a minigene encoded within the axe-txe operon influences translation of the Txe toxin. This is the first example to date of such a regulatory mechanism identified in the TA modules. Here, in a series of genetic studies, we employed translational reporter gene fusions to establish the molecular basis of this phenomenon. Our results show that translation of the two-codon mini-ORF displays an in cis mode of action, and positively affects the expression of txe, possibly by increasing its mRNA stability through protection from an endonuclease attack. Moreover, we established that the reading frame in which the two cistrons are encoded, as well as the distance between them, are critical parameters that affect the level of such regulation. In addition, by searching for two-codon ORFs we found sequences of several potential minigenes in the leader sequences of several other toxins belonging to the type II TA family. These findings suggest that this type of gene regulation may not only apply for the axe-txe cassette, but could be more widespread among other TA systems.

Bone morphogenetic protein 15 (BMP15) is secreted by the mammalian oocytes and is indispensable for ovarian follicular development, ovulation, and fertility. To determine the regulation mechanism of BMP15 gene, the regulatory sequence of porcine BMP15 was investigated in this study. The cloned BMP15 promoter retains the cell-type specificity, and is activated in cells derived from ovarian tissue. The luciferase assays in combination with a series of deletion of BMP15 promoter sequence show that the -427 to -376 bp region of BMP15 promoter is the primary regulatory element, in which there are a number of transcription factor binding sites, including LIM homeobox 8 (LHX8), newborn ovary homeobox gene (NOBOX), and paired-like homeodomain transcription factor 1 (PITX1). Determination of tissue-specific expression reveals that LHX8, but not PITX1 and NOBOX, is exclusively expressed in pig ovary tissue and is translocated into the cell nuclei. Overexpression of LHX8 in Chinese hamster ovary (CHO) cells could significantly promote BMP15 promoter activation. This study confirms a key regulatory element that is located in the proximal region of BMP15 promoter and is regulated by the LHX8 factor.

Many proteins are localized at the vacuolar membrane, but most of them are still poorly described, due to the inaccessibility of this membrane from the extracellular environment. This work focused on the characterization of the CAT2 transporter from S. lycopersicum (SlCAT2) that was previously overexpressed in E. coli and reconstituted in proteoliposomes for transport assay as [³H]Arg uptake. The orientation of the reconstituted transporter has been attempted and current data support the hypothesis that the protein is inserted in the liposome in the same orientation as in the vacuole. SlCAT2 activity was dependent on the pH, with an optimum at pH 7.5. SlCAT2 transport activity was stimulated by the increase of internal osmolality from 0 to 175 mOsmol while the activity was inhibited by the increase of external osmolality. K⁺, Na⁺, and Mg2+ present on the external side of proteoliposomes at physiological concentrations, inhibited the transport activity; differently, the cations had no effect when included in the internal proteoliposome compartment. This data highlighted an asymmetric regulation of SlCAT2. Cholesteryl hemisuccinate, included in the proteoliposomal membrane, stimulated the SlCAT2 transport activity. The homology model of the protein was built using, as a template, the 3D structure of the amino acid transporter GkApcT. Putative substrate binding residues and cholesterol binding domains were proposed. Altogether, the described results open new perspectives for studying the response of SlCAT2 and, in general, of plant vacuolar transporters to metabolic and environmental changes.

Protein inhibition is a natural regulatory process to control cellular metabolic fluxes. PII-family signal-transducing effectors are in this matter key regulators of the nitrogen metabolism. Their interaction with their various targets is governed by the cellular nitrogen level and the energy charge. Structural studies on GlnK, a PII-family inhibitor of the ammonium transporters (Amt), showed that the T-loops responsible for channel obstruction are displaced upon the binding of 2-oxoglutarate, magnesium and ATP in a conserved cleft. However, GlnK from Methanocaldococcus jannaschii was shown to bind 2-oxoglutarate on the tip of its T-loop, causing a moderate disruption to GlnK-Amt interaction, raising the question if methanogenic archaea use a singular adaptive strategy. Here we show that membrane fractions of Methanothermococcus thermolithotrophicus released GlnKs only in the presence of Mg-ATP and 2-oxoglutarate. This observation led us to structurally characterize the two GlnK isoforms apo or in complex with ligands. Together, our results show that the 2-oxoglutarate binding interface is conserved in GlnKs from Methanococcales, including Methanocaldococcus jannaschii, emphasizing the importance of a free carboxy-terminal group to facilitate ligand binding and to provoke the shift of the T-loop positions.

Phytochrome-interacting factors (PIFs), a subfamily of basic helix-loop-helix (bHLH) transcription factors (TFs), play critical roles in regulating plant growth and development. The resurrection plant Myrothamnus flabellifolia possesses a noteworthy tolerance to desiccation, but no PIFs related to the response to abiotic stress have been functionally studied. In this study, a dehydration-inducible PIF gene, MfPIF1, was cloned and characterized. Subcellular localization assay revealed that MfPIF1 is localized predominantly in the nucleus. Overexpression of MfPIF1 in Arabidopsis thaliana led to enhanced drought and salinity tolerance, which was attributed to higher contents of chlorophyll, proline (Pro), soluble protein, and soluble sugar, activities of antioxidant enzymes as well as lower water loss rate, malondialdehyde (MDA) content, and reactive oxygen species (ROS) accumulation in transgenic lines compared with control plants. Moreover, MfPIF1 decreased stomatal aperture after drought and abscisic acid (ABA) treatment, and increased expression of both ABA biosynthesis and ABA-responsive genes including NCED3, P5CS, and RD29A. Overall, these results indicated that MfPIF1 may act as a positive regulator to drought and salinity responses, and therefore could be considered as a potential gene for plant genetic improvement of drought and salinity tolerance.

Self-incompatibility (SI) is a widespread mechanism in angiosperms that prevents inbreeding by rejecting self-pollen. However, the regulation of the SI response in Brassica napus is not well understood. Here, we report that the M-locus protein kinase (MLPK) BnaMLPKs, the functional homolog of BrMLPKs in Brassica rapa, controls SI in B. napus. We identified four paralogue MLPK genes in B. napus, including BnaA3.MLPK, BnaC3.MLPK, BnaA4.MLPK, and BnaC4.MLPK. Two transcripts of BnaA3.MLPK, BnaA3.MLPKf1 and BnaA3.MLPKf2, were generated by alternative splicing. Tissue expression pattern analysis demonstrated that BnaA3.MLPK, especially BnaA3.MLPKf2, is highly expressed in reproductive organs, particularly in stigmas. We subsequently created RNA-silencing lines and CRISPR/Cas9-induced quadruple mutants of BnaMLPKs in B. napus SI line S-70. Phenotypic analysis revealed that SI response is partially suppressed in RNA-silencing lines and is completely blocked in quadruple mutants. These results indicate the importance of BnaMLPKs in regulating the SI response of B. napus. We found that the expression of SI positive regulators S-locus receptor kinase (SRK) and Arm-Repeat Containing 1 (ARC1) are suppressed in bnmlpk mutant, whereas the self-compatibility (SC) element Glyoxalase I (GLO1) maintained a high expression level. Overall, our findings reveal a new regulatory mechanism of MLPK in the SI of B. napus.

Desaturases are essentially required for unsaturated fatty acid (UFA) biosynthesis. We identified 10 genes encoding putative desaturases in the transcriptome database of the brown planthopper (BPH), Nilaparvata lugens. These include eight First Desaturase family genes, one cytochrome b5 fused desaturase gene (Nlug-Cytb5r) and one Sphingolipid Desaturase gene (Nlug-ifc). Transcript level profiling revealed significant variation in the expression patterns of these genes across tissues and developmental stages, which occur in a gene-specific manner. Interestingly, their expression was also modulated by the insect food source: the mRNA levels of Nlug-desatC and Nlug-Cytb5r were down-regulated, but the expression level of Nlug-desatA1-b and Nlug-desatA1-c were elevated in the BPH fed on the resistant rice variety Babawee as compared to the non-resistant variety Taichun Native 1 (TN1). Silencing Nlug-desatA1-b, Nlug-desatA1-c, or Nlug-Ifc reduced fatty acid composition and abundance in female BPH 1-d-old-adults compared to controls. Whereas, single knockdown of all ten desaturase genes significantly increased mortality of BPH nymphs compared with controls. Of the ten desaturase genes, knockdown of Nlug-desatA1-b and Nlug-desatA2 caused the highest mortality in BPH (91% and 97%, respectively). Our findings offer a base for expression and functional characterization of newly identified desaturase genes in BPH, and may contribute to RNA interference-based pest management strategies.

The recently introduced semi-orthogonal system of nucleic acid imaging offers a greatly improved method of identifying DNA sequences that are capable of adopting noncanonical structures. This paper uses our newly developed G-QINDER tool to identify specific repeat sequences that adopt unique structural motifs in DNA: TG and AG repeats. The structures were found to adopt a left-handed G-quadruplex form under extreme crowding conditions and a unique tetrahelical motif under certain other conditions. The tetrahelical structure likely consists of stacked AGAG-tetrads but, unlike G-quadruplexes, their stability does not appear to be dependent on the type of monovalent cation present. The occurrence of TG and AG repeats in genomes is not rare, and they are also found frequently in the regulatory regions of nucleic acids, so it is reasonable to assume that putative structural motifs, like other noncanonical forms, could play an important regulatory role in cells. This hypothesis is supported by the structural stability of the AGAG motif; its unfolding can occur even at physiological temperatures since the melting temperature is primarily dependent on the number of AG repeats in the sequence.

Recent studies have demonstrated that strigolactones (SLs) also participate in the regulation of stress adaptation; however, the regulatory mechanism remains elusive. In this study, the homolog of More Axillary Branches 2, which encodes a key component in SL signaling, in the perennial oil plant Sapium sebiferum was identified and functionally characterized in Arabidopsis. The results showed that the expression of SsMAX2 in S. sebiferum seedlings was stress-responsive, and SsMAX2 overexpression (OE) in Arabidopsis significantly promoted resistance to drought, osmotic, and salt stresses. Moreover, SsMAX2 OE lines exhibited decreased chlorophyll degradation, increased soluble sugar and proline accumulation, and lower water loss ratio in response to the stresses. Importantly, anthocyanin biosynthesis and the activities of several antioxidant enzymes, such as superoxide dismutase (SOD), peroxidase (POD), and ascorbate peroxidase (APX), were enhanced in the SsMAX2 OE lines, which further led to a significant reduction in hydrogen peroxide levels. Additionally, the SsMAX2 OE lines exhibited higher expression level of several abscisic acid (ABA) biosynthesis genes, suggesting potential interactions between SL and ABA in the regulation of stress adaptation. Overall, we provide physiological and biochemical evidence demonstrating the pivotal role of SsMAX2 in the regulation of osmotic, drought, and salt stress resistance and show that MAX2 can be a genetic target to improve stress tolerance.

Glucose 6-phosphate dehydrogenase (G6PD) (EC 1.1.1.363) is a crucial regulatory enzyme in the oxidative pentose phosphate pathway that provides reductive potential in the form of NADPH, as well as carbon skeletons for the synthesis of macromolecules. In this study, we report the cloning, expression, and characterization of G6PD (SpG6PD1) from a lichen-associated psychrophilic bacterium Sphingomonas sp. PAMC 26621. SpG6PD1 was expressed in Escherichia coli as a soluble protein, having optimum activity at pH 7.5⁻8.5 and 30 °C for NADP⁺ and 20 °C for NAD⁺. SpG6PD1 utilized both NADP⁺ and NAD⁺, with the preferential utilization of NADP⁺. A high Km value for glucose 6-phosphate and low activation enthalpy (ΔH‡) compared with the values of mesophilic counterparts indicate the psychrophilic nature of SpG6PD1. Despite the secondary structure of SpG6PD1 being maintained between 4⁻40 °C, its activity and tertiary structure were better preserved between 4⁻20 °C. The results of this study indicate that the SpG6PD1 that has a flexible structure is most suited to a psychrophilic bacterium that is adapted to a permanently cold habitat.

Short-term (3 h) treatment of embryos isolated from dormant apple (Malus domestica Borkh.) seeds with NO donors stimulates their transition from dormancy to germination. Seed dormancy is maintained by ABA, while germination is controlled mainly by gibberellins (GAs) and jasmonic acid (JA). NO-induced dormancy removal correlates with low ABA concentration in embryonic axes and reduced embryo sensitivity to ABA. We analyzed the expression of genes encoding key enzymes of ABA degradation (CYP707A1, CYP707A2), biosynthesis (NCED3, NCED9), and elements of the ABA transduction pathway (PYL1, PYL2, RCAR1, RCAR3, PP2CA, ABI1, ABI2, SNRK2, ABI5, AREB3, ABF). A role for JA in the regulation of germination led us to investigate the expression of genes encoding enzymes of JA biosynthesis (AOS1, JMT, JAR1) and the transduction pathway (COI1, MYC2, JAZ3, JAZ12). The expression profiles of the genes were estimated in embryonic axes isolated from dormant or NO fumigated apple embryos. The analyzed genes were differentially regulated during dormancy alleviation, the main modifications in the transcription level were detected for NCED3, NCED9, CYP707A2, RCAR1, ABF, AOS1, JMT, JAR1 and JAZ3. A regulatory role of NO in the removal of seed dormancy is associated with the stimulation of expression of genes related to ABA degradation, down-regulation of genes responsible for ABA synthesis, an increase of expression level of genes engaged in JA synthesis and modification of the expression of genes engaged in signaling pathways of the hormones. To confirm a signaling role of NO during dormancy breakage, an increased RNA nitration level in embryonic axes was demonstrated.

Auxin Response Factors (ARFs) mediate auxin signaling and govern diverse biological processes. However, a comprehensive analysis of the ARF gene family and identification of their key regulatory functions have not been conducted in Melastoma dodecandrum, leading to a weak understanding of further use and development for this functional shrub. In this study, we successfully identified a total of 27 members of the ARF gene family in M. dodecandrum and classified them into Class I-III. Class II-III showed more significant gene duplication than Class I, especially for MedARF16s. According to the prediction of cis-regulatory elements, the AP2/ERF, BHLH, and bZIP transcription factor families may serve as regulatory factors controlling the transcriptional pre-initiation expression of MedARF. Analysis of miRNA editing sites reveals that miR160 may play a regulatory role in the post-transcriptional expression of MeARF. Expression profiles revealed that more than half of the MedARFs exhibited high expression levels in the stem compared to other organs. While there are some specific genes expressed only in flowers, it is noteworthy that MedARF16s, MedARF7A, and MedARF9B, which are highly expressed in stems, also demonstrate high expressions in other organs of M. dodecandrum. Further hormone treatment experiments revealed that these MedARFs were sensitive to auxin changes, with MedARF6C and MedARF7A showing significant and rapid changes in expression upon increasing exogenous auxin. In brief, our findings suggest a crucial role in regulating plant growth and development in M. dodecandrum by responding to changes in auxin. These results can provide a theoretical basis for future molecular breeding in Myrtaceae.

The mitogen-activated protein kinase-activated protein kinase MK5 is a substrate of the mitogen-activated protein kinases p38, ERK3 and ERK4. Cell culture and animal studies have demonstrated that MK5 is involved in tumour suppression and promotion, embryogenesis, anxiety, cell motility and cell cycle regulation. In the present study, homology models of MK5 were used for molecular dynamics (MD) simulations of: (1) MK5 alone; (2) MK5 in complex with an inhibitor; and (3) MK5 in complex with the interaction partner p38α. The calculations showed that the inhibitor occupied the active site and disrupted the intramolecular network of amino acids. However, intramolecular interactions consistent with an inactive protein kinase fold were not formed. MD with p38α showed that not only the p38 docking region, but also amino acids in the activation segment, αH helix, P-loop, regulatory phosphorylation region and the C-terminal of MK5 may be involved in forming a very stable MK5-p38α complex, and that p38α binding decreases the residual fluctuation of the MK5 model. Electrostatic Potential Surface (EPS) calculations of MK5 and p38α showed that electrostatic interactions are important for recognition and binding.

WRKY proteins are a large family of transcription factors that are involved in diverse developmental processes and abiotic stress responses in plants. However, our knowledge of the regulatory mechanisms of WRKYs participation in protein-protein interactions is still fragmentary, and such protein-protein interactions are fundamental in understanding biological networks and the functions of proteins. In this study, we report that a WRKY protein from Tamarix hispida, ThWRKY4, can form both homodimers and heterodimers with ThWRKY2 and ThWRKY3. In addition, ThWRKY2 and ThWRKY3 can both bind to W-box motif with binding affinities similar to that of ThWRKY4. Further, the expression patterns of ThWRKY2 and ThWRKY3 are similar to that of ThWRKY4 when plants are exposed to abscisic acid (ABA). Subcellular localization shows that these three ThWRKY proteins are nuclear proteins. Taken together, these results demonstrate that ThWRKY4 is a dimeric protein that can form functional homodimers or heterodimers that are involved in abiotic stress responses.

Nitric oxide (NO) is a regulator of growth, development, and stress responses in living organisms. Plant nitrate reductases (NR) catalyze the reduction of nitrate to nitrite or, alternatively, to NO. In plants, NO action and its targets remain incompletely understood, and the way NO regulates its own homeostasis remains to be elucidated. A significant transcriptome overlapping between NO-deficient mutant and NO-treated wild type plants suggests that NO could negatively regulate its biosynthesis. A significant increase in NO content was detected in transgenic plants overexpressing NR1 and NR2 proteins. In turn, NR protein and activity as well as NO content, decreased in wild-type plants exposed to a pulse of NO gas. Tag-aided immunopurification procedures followed by tandem mass spectrometry allowed identifying NO-triggered post-translational modifications (PTMs) and ubiquitylation sites in NRs. Nitration of tyrosine residues and S-nitrosation of cysteine residues affected key amino acids involved in binding the essential FAD and molybdenum cofactors. NO-related PTMs were accompanied by ubiquitylation of lysine residues flanking the nitration and S-nitrosation sites. NO-induced PTMs of NRs potentially inhibit their activities and promote their proteasome-mediated degradation. This auto-regulatory feedback loop may control nitrate assimilation to ammonium and nitrite-derived production of NO under complex environmental conditions.

FTO is an N6-methyladenosine demethylase removing methyl groups from nucleic acids. Several studies indicate the creation of FTO complexes with other proteins. Here, we looked for regulatory proteins recognizing parts of the FTO dioxygenase region. In the Calmodulin (CaM) Target Database, we found the FTO C-domain potentially binding CaM, and we proved this finding experimentally. The interaction was Ca2+-dependent but independent on FTO phosphorylation. We found that FTO-CaM interaction essentially influences calcium-binding loops in CaM, indicating the presence of two peptide populations-exchanging as CaM alone and differently, suggesting that only one part of CaM interacts with FTO, and the other one reminds free. The modeling of FTO-CaM interaction showed its stable structure when the half of the CaM molecule saturated with Ca2+ interacts with the FTO C-domain, whereas the other part is disconnected. The presented data indicate calmodulin as a new FTO interactor and support engagement of the FTO protein in calcium signaling pathways.

The activity of membrane proteins depends strongly on the surrounding lipid environment. Here, we characterize the lipid stimulation of the plant plasma membrane H+-ATPase Arabidopsis thaliana H+-ATPase isoform 2 (AHA2) upon purification and reconstitution into liposomes of defined lipid compositions. We show that the proton pumping activity of AHA2 is stimulated by anionic phospholipids, especially by phosphatidylserine. This activation was independent of the cytoplasmic C-terminal regulatory domain of the pump. Molecular dynamics simulations revealed several preferential contact sites for anionic phospholipids in the transmembrane domain of AHA2. These contact sites are partially conserved in functionally different P-type ATPases from different organisms, suggesting a general regulation mechanism by the membrane lipid environment. Our findings highlight the fact that anionic lipids play an important role in the control of H+-ATPase activity.

The pink stem borer, Sesamia inferens (Walker), is a major pest of rice and is endemic in China and other parts of Asia. Small heat shock proteins (sHSPs) encompass a diverse, widespread class of stress proteins that have not been characterized in S. inferens. In the present study, we isolated and characterized three S. inferens genes that encode members of the α-crystallin/sHSP family, namely, Sihsp21.4, Sihsp20.6, and Sihsp19.6. The three cDNAs encoded proteins of 187, 183 and 174 amino acids with calculated molecular weights of 21.4, 20.6 and 19.6 kDa, respectively. The deduced amino acid sequences of the three genes showed strong similarity to sHSPs identified in other lepidopteran insects. Sihsp21.4 contained an intron, but Sihsp20.6 and Sihsp19.6 lacked introns. Real-time quantitative PCR analyses revealed that Sihsp21.4 was most strongly expressed in S. inferens heads; Whereas expression of Sihsp20.6 and Sihsp19.6 was highest in eggs. The three S. inferens sHSP genes were up-regulated during low temperature stress. In summary, our results show that S. inferens sHSP genes have distinct regulatory roles in the physiology of S. inferens.

Arabidopsis thaliana (Arabidopsis) has been used extensively as a heterologous system for molecular manipulation to genetically characterize both dicotyledonous and monocotyledonous plant species. Here, we report on Arabidopsis transformant lines molecularly manipulated to over-accumulate the small regulatory RNA microRNA397 (miR397) from the emerging C4 monocotyledonous grass model species Setaria viridis (S. viridis). The generated transformant lines, termed SvMIR397 plants, displayed a range of developmental phenotypes that ranged from a mild, wild-type-like phenotype, to a severe, full dwarfism phenotype. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR)-based profiling of the SvMIR397 transformant population revealed a strong correlation between the degree of miR397 over-accumulation, repressed LACCASE (LAC) target gene expression, reduced lignin content, and the severity of the developmental phenotype displayed by SvMIR397 transformants. Further, exposure of SvMIR397 transformants to a 7-day regime of salt stress revealed the SvMIR397 transformant lines to be more sensitive to the imposed stress than were wild-type Arabidopsis plants. Taken together, the findings reported here via the use of Arabidopsis as a heterologous system show that the S. viridis miR397 small regulatory RNA is able to repress the expression of three Arabidopsis LAC genes which led to reduced lignin content and increased salt stress sensitivity.

Abiotic stresses restrict the growth and yield of crops. Plants have developed a number of regulatory mechanisms to respond to these stresses. WRKY transcription factors (TFs) are plant-specific transcription factors that play essential roles in multiple plant processes, including abiotic stress response. At present, little information regarding drought-related WRKY genes in maize is available. In this study, we identified a WRKY transcription factor gene from maize, named ZmWRKY40. ZmWRKY40 is a member of WRKY group II, localized in the nucleus of mesophyll protoplasts. Several stress-related transcriptional regulatory elements existed in the promoter region of ZmWRKY40. ZmWRKY40 was induced by drought, high salinity, high temperature, and abscisic acid (ABA). ZmWRKY40 could rapidly respond to drought with peak levels (more than 10-fold) at 1 h after treatment. Overexpression of ZmWRKY40 improved drought tolerance in transgenic Arabidopsis by regulating stress-related genes, and the reactive oxygen species (ROS) content in transgenic lines was reduced by enhancing the activities of peroxide dismutase (POD) and catalase (CAT) under drought stress. According to the results, the present study may provide a candidate gene involved in the drought stress response and a theoretical basis to understand the mechanisms of ZmWRKY40 in response to abiotic stresses in maize.