Sugar Starvation Disrupts Lipid Breakdown by Inducing Autophagy in Embryonic Axes of Lupin (Lupinus spp.) Germinating Seeds.

Under nutrient deficiency or starvation conditions, the mobilization of storage compounds during seed germination is enhanced to primarily supply respiratory substrates and hence increase the potential of cell survival. Nevertheless, we found that, under sugar starvation conditions in isolated embryonic axes of white lupin (Lupinus albus L.) and Andean lupin (Lupinus mutabilis Sweet) cultured in vitro for 96 h, the disruption of lipid breakdown occurs, as was reflected in the higher lipid content in the sugar-starved (-S) than in the sucrose-fed (+S) axes. We postulate that pexophagy (autophagic degradation of the peroxisome-a key organelle in lipid catabolism) is one of the reasons for the disruption in lipid breakdown under starvation conditions. Evidence of pexophagy can be: (i) the higher transcript level of genes encoding proteins of pexophagy machinery, and (ii) the lower content of the peroxisome marker Pex14p and its increase caused by an autophagy inhibitor (concanamycin A) in -S axes in comparison to the +S axes. Additionally, based on ultrastructure observation, we documented that, under sugar starvation conditions lipophagy (autophagic degradation of whole lipid droplets) may also occur but this type of selective autophagy seems to be restricted under starvation conditions. Our results also show that autophagy occurs at the very early stages of plant growth and development, including the cells of embryonic seed organs, and allows cell survival under starvation conditions.

Identification and Potential Participation of Lipases in Autophagic Body Degradation in Embryonic Axes of Lupin (Lupinus spp.) Germinating Seeds.

Autophagy is a fundamental process for plants that plays a crucial role in maintaining cellular homeostasis and promoting survival in response to various environmental stresses. One of the lesser-known stages of plant autophagy is the degradation of autophagic bodies in vacuoles. To this day, no plant vacuolar enzyme has been confirmed to be involved in this process. On the other hand, several enzymes have been described in yeast (Saccharomyces cerevisiae), including Atg15, that possess lipolytic activity. In this preliminary study, which was conducted on isolated embryonic axes of the white lupin (Lupinus albus L.) and Andean lupin (Lupinus mutabilis Sweet), the potential involvement of plant vacuolar lipases in the degradation of autophagic bodies was investigated. We identified in transcriptomes (using next-generation sequencing (NGS)) of white and Andean lupin embryonic axes 38 lipases with predicted vacuolar localization, and for three of them, similarities in amino acid sequences with yeast Atg15 were found. A comparative transcriptome analysis of lupin isolated embryonic axes cultured in vitro under different sucrose and asparagine nutrition, evaluating the relations in the levels of the transcripts of lipase genes, was also carried out. A clear decrease in lipase gene transcript levels caused by asparagine, a key amino acid in lupin seed metabolism which retards the degradation of autophagic bodies during sugar-starvation-induced autophagy in lupin embryonic axes, was detected. Although the question of whether lipases are involved in the degradation of autophagic bodies during plant autophagy is still open, our findings strongly support such a hypothesis.

Cyclophilins and Their Functions in Abiotic Stress and Plant-Microbe Interactions.

Plants have developed a variety of mechanisms and regulatory pathways to change their gene expression profiles in response to abiotic stress conditions and plant-microbe interactions. The plant-microbe interaction can be pathogenic or beneficial. Stress conditions, both abiotic and pathogenic, negatively affect the growth, development, yield and quality of plants, which is very important for crops. In contrast, the plant-microbe interaction could be growth-promoting. One of the proteins involved in plant response to stress conditions and plant-microbe interactions is cyclophilin. Cyclophilins (CyPs), together with FK506-binding proteins (FKBPs) and parvulins, belong to a big family of proteins with peptidyl-prolyl cis-trans isomerase activity (Enzyme Commission (EC) number 5.2.1.8). Genes coding for proteins with the CyP domain are widely expressed in all organisms examined, including bacteria, fungi, animals, and plants. Their different forms can be found in the cytoplasm, endoplasmic reticulum, nucleus, chloroplast, mitochondrion and in the phloem space. They are involved in numerous processes, such as protein folding, cellular signaling, mRNA processing, protein degradation and apoptosis. In the past few years, many new functions, and molecular mechanisms for cyclophilins have been discovered. In this review, we aim to summarize recent advances in cyclophilin research to improve our understanding of their biological functions in plant defense and symbiotic plant-microbe interactions.

In situ approaches show the limitation of the spoilage potential of Juniperus phoenicea L. essential oil against cold-tolerant Pseudomonas fluorescens KM24.

The present study aimed to elucidate the effect of subinhibitory concentrations (sub-MICs) of juniper essential oil (EO), α-pinene, and sabinene on the quorum-sensing (QS)-mediated proteolytic and lipolytic properties of Pseudomonas fluorescens KM24. These activities were verified under in situ conditions, in which sub-MICs of the agents altered the morphology of KM24 cells. RNA-Seq studies revealed key coding sequences (CDSs)/genes related to QS and the proteolytic/lipolytic activities of pseudomonads. In this work, all the examined agents decreased autoinducer synthesis and influenced the mRNA expression of the encoding acyltransferase genes lptA, lptD, and plsB. The highest reduction on the 3rd and 5th days of cultivation was observed for the genes lptD (-5.5 and -5.61, respectively) and lptA (-3.5 and -4.0, respectively) following treatment with EO. Inhibition of the lptA, lptD, and plsB genes by singular constituents of EO was on average, from -0.4 to -0.7. At 5 days of cultivation the profile of AHLs of the reference P. fluorescens KM24 strain consisted of 3-oxo-C14-HSL, 3-oxo-C6-HSL, C4-HSL, and N-[(RS)-3-hydroxybutyryl]-HSL, the concentrations of which were 0.570, 0.018, 3.744, and 0.554 µg ml-1, respectively. Independent of the incubation time, EO, α-pinene, and sabinene also suppressed the protease genes prlC (-1.5, -0.5, and -0.5, respectively) and ctpB (-1.5, -0.7, and -0.4, respectively). Lipolysis and transcription of the lipA/lipB genes were downregulated by the agents on average from -0.3 to -0.6. α-Pinene- and sabinene-rich juniper EO acts as an anti-quorum-sensing agent and can repress the spoilage phenotype of pseudomonads. KEY POINTS: Juniper EO, α-pinene, sabinene exhibited anti-QS potential toward KM24. RNA-Seq revealed key CDSs/genes related to QS/proteolytic/lipolytic activities of KM24. Agents at sub-MIC levels influenced the mRNA expression of QS/lipase/protease genes.

What nature separated, and human joined together: About a spontaneous hybridization between two allopatric dogwood species (Cornus controversa and C. alternifolia).

In this study, possible hybridization between two allopatric species, Cornus controversa and Cornus alternifolia, was explored using molecular and morphological approaches. Scanning electron microscope analyses of the adaxial and the abaxial leaf surfaces yielded a few new not yet described characters typical for the particular species and intermediate for hybrids. With the use of 14 Random Amplified Polymorphic DNA and 5 Amplified Fragment Length Polymorphism primer combinations, 44 fragments species specific to C. controversa and 51 species specific to C. alternifolia were obtained. Most of these bands were also found in putative hybrids. All clustering analyses based on binary data combined from both methods confirmed a separate and intermediate status of the hybrids. Hybrid index estimates for hybrids C1-C5 indicated that all were the first generation of offspring (F1). Chloroplast intergenic spacers (trnF-trnL and psbC-trnS) were used to infer the hybridization direction. Based on the assumption of maternal inheritance of chloroplast DNA, C. controversa seems to be the maternal parent of the hybrid. Internal transcribed spacer sequences of the five hybrids analyzed here indicated higher similarity with the sequences of C. controversa (all shared the majority of its single nucleotide polymorphisms). Sequence analysis of PI-like genes fully confirmed the hybrid origin of C1-C5 hybrids. Our results also showed that two specimens in the C. alternifolia group, A1 and A3, are not free of introgression. They are probably repeated backcrosses toward C. alternifolia. Furthermore, molecular data seem to point not only to unidirectional introgression toward C. controversa (the presence of hybrids) but to bidirectional introgression as well, since the presence of markers specific for C. controversa in the profiles of C. alternifolia specimen A3 was observed.

[Cyclophilins ­ proteins with many functions]. / Cyklofiliny - bialka o wielu funkcjach

Cyclophilins together with FK-506-binding proteins and parvulins, belong to a group of proteins that have peptidyl-prolyl cis-trans isomerase activity. They also belong to proteins that are collectively known as immunophilins. Cyclophilins are found in all cells of all organisms studied, in both prokaryotes and eukaryotes. The first member of the cyclophilins to be identified in mammals, cyclophilin A, is the major cellular target for the immunosuppressive drug cyclosporin A. This review discusses recently available data about proteins with cyclophilin domain (CBD). Recent studies have implicated a diverse array of additional cellular functions for cyclophilins, including roles as chaperones and in cell signalling as well as in several human diseases.

Regulation of proline biosynthesis and resistance to drought stress in two barley (Hordeum vulgare L.) genotypes of different origin.

Drought is considered the main abiotic stress factor that inhibits growth of crop plants (including barley), limiting yield in many regions worldwide. Predicted climate changes show that in future the frequency and intensity of drought events will rise, so crops that are resistant to this stress are in demand. One of the adaptive metabolic responses to drought is the accumulation of proline. The aim of this study was to examine the effect of 10-day drought on tissue dehydration and proline biosynthesis in leaves as well as in roots of barley genotypes of different origin: the Syrian breeding line Cam/B1/CI and the German cultivar Maresi. The involvement of Δ1 pyrroline-5-carboxylate synthetase (P5CS), the expression of the P5CS gene and ABA in proline synthesis under drought were also studied. Finally, we examined the resistance of tested genotypes to applied drought using chlorophyll fluorescence parameters and above-ground dry matter accumulation. Drought caused a gradual decrease of water content and an increase of proline and ABA content in roots and leaves of both genotypes. A statistically significant positive correlation between proline accumulation and activity of P5CS was also revealed. The skyrocketing increase of P5CS activity and proline accumulation was proceeded by transcriptional up-regulation of P5CS. The relationships between changes in P5CS expression, P5CS activity and ABA content show that the latter compound is involved in drought-induced proline synthesis at the transcription and enzyme activity level. The examined barley genotypes were equally resistant to applied moderate drought stress regardless of the differences in the level of proline accumulated.

Exogenous adenosine 5'-phosphoramidate behaves as a signal molecule in plants; it augments metabolism of phenylpropanoids and salicylic acid in Arabidopsis thaliana seedlings.

Cells contain various congeners of the canonical nucleotides. Some of these accumulate in cells under stress and may function as signal molecules. Their cellular levels are enzymatically controlled. Previously, we demonstrated a signaling function for diadenosine polyphosphates and cyclic nucleotides in Arabidopsis thaliana and grape, Vitis vinifera. These compounds increased the expression of genes for and the specific activity of enzymes of phenylpropanoid pathways resulting in the accumulation of certain products of these pathways. Here, we show that adenosine 5'-phosphoramidate, whose level can be controlled by HIT-family proteins, induced similar effects. This natural nucleotide, when added to A. thaliana seedlings, activated the genes for phenylalanine:ammonia lyase, 4-coumarate:coenzyme A ligase, cinnamate-4-hydroxylase, chalcone synthase, cinnamoyl-coenzyme A:NADP oxidoreductase and isochorismate synthase, which encode proteins catalyzing key reactions of phenylpropanoid pathways, and caused accumulation of lignins, anthocyanins and salicylic acid. Adenosine 5'-phosphofluoridate, a synthetic congener of adenosine 5'-phosphoramidate, behaved similarly. The results allow us to postulate that adenosine 5'-phosphoramidate should be considered as a novel signaling molecule.

Both cyclic-AMP and cyclic-GMP can act as regulators of the phenylpropanoid pathway in Arabidopsis thaliana seedlings.

Cyclic nucleotides (cAMP and cGMP) are important signaling molecules that control a range of cellular functions and modulate different reactions. It is known that under abiotic or biotic stress plant cells synthesize these nucleotides and that they also enhance the activity of the phenylpropanoid pathway. Wondering what is the relation between these two facts, we investigated how the exogenously applied membrane-permeable derivatives, 8-Br-cAMP or 8-Br-cGMP, which are believed to act as the original cyclic nucleotides, affect the expression of the genes for and the specific activity of three enzymes of the phenylpropanoid pathway in Arabidopsis thaliana seedlings. We found that the expression of the genes of phenylalanine ammonia-lyase (PAL2), 4-coumarate:coenzyme A ligase (4CL1) and chalcone synthase (CHS), and the specific activities of PAL (EC 4.3.1.5), 4CL (EC 6.2.1.12) and CHS (EC 2.3.1.74) were induced in the same way by either of these cyclic nucleotides used at 5 µM concentration. None of the possible cAMP and cGMP degradation products (AMP, GMP, adenosine or guanosine) evoked such effects. Expression of PAL1, 4CL2 and 4CL3 were practically not affected. Although the investigated nucleotides induced rapid expression of the aforementioned enzymes, they did not affect the level of anthocyanins within the same period. We discuss the effects exerted by the exogenously administered cyclic nucleotides, their relation with stress and the role which the phenylpropanoid pathways the cyclic nucleotides may play in plants.

Sucrose controls storage lipid breakdown on gene expression level in germinating yellow lupine (Lupinus luteus L.) seeds.

This study revealed that cytosolic aconitase (ACO, EC 4.2.1.3) and isocitrate lyase (ICL, EC 4.1.3.1, marker of the glyoxylate cycle) are active in germinating protein seeds of yellow lupine. The glyoxylate cycle seems to function not only in the storage tissues of food-storage organs, but also in embryonic tissue of growing embryo axes. Sucrose (60mM) added to the medium of in vitro culture of embryo axes and cotyledons decreased activity of lipase (LIP, EC 3.1.1.3) and activity of glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2). The opposite effect was caused by sucrose on activity of cytosolic ACO, ICL as well as NADP(+)-dependent (EC 1.1.1.42) and NAD(+)-dependent (EC 1.1.1.41) isocitrate dehydrogenase (NADP-IDH and NAD-IDH, respectively); activity of these enzymes was clearly stimulated by sucrose. Changes in the activity of LIP, ACO, NADP-IDH, and NAD-IDH caused by sucrose were based on modifications in gene expression because corresponding changes in the enzyme activities and in the mRNA levels were observed. The significance of cytosolic ACO and NADP-IDH in carbon flow from storage lipid to amino acids, as well as the peculiar features of storage lipid breakdown during germination of lupine seeds are discussed.

5'-methylthioadenosine nucleosidase from yellow lupine (Lupinus luteus): molecular characterization and mutational analysis.

This is report of mutational analysis of higher plant 5'-methylthioadenosine nucleosidase (MTAN). We identified and characterized the gene encoding yellow lupine (Lupinus luteus) MTAN (LlMTAN). The role of active site amino acids residues Glu24, Phe134, Glu188 and Asp211 was analyzed by site-directed mutagenesis. The Glu24Gln and Asp211Asn substitutions completely abolished the enzyme activity. The Glu188Gln mutant showed only trace activity toward 5'-methylthioadenosine. These results indicate that these three amino acid residues are necessary for enzyme activity. Furthermore, as the result of replacement of Phe134 by less bulky leucine, LlMTAN acquired the ability to bind and hydrolyze S-adenosylhomocysteine. We also analyzed the sequence of the LlMTAN promoter region. It appeared that there may be a direct link between LlMTAN expression regulation and sulfate metabolism.

Diadenosine polyphosphates (Ap3A and Ap4A) behave as alarmones triggering the synthesis of enzymes of the phenylpropanoid pathway in Arabidopsis thaliana.

It is known that cells under stress accumulate various dinucleoside polyphosphates, compounds suggested to function as alarmones. In plants, the phenylpropanoid pathways yield metabolites protecting these organisms against various types of stress. Observations reported in this communication link these two phenomena and provide an example of a metabolic "addressee" for an "alarm" signaled by diadenosine triphosphate (Ap3A) or diadenosine tetraphosphate (Ap4A). In response to added Ap3A or Ap4A, seedlings of Arabidopsis thaliana incubated in full nutrition medium increased both the expression of the genes for and the specific activity of phenylalanine ammonia-lyase and 4-coumarate:coenzyme A ligase, enzymes that control the beginning of the phenylpropanoid pathway. Neither adenine mononucleotides (AMP, ADP or ATP) nor adenosine evoked such effects. Reactions catalyzed in vitro by these enzymes were not affected by Ap3A or Ap4A.

Yellow lupine cyclophilin interacts with nucleic acids.

To investigate properties of yellow lupine cytosolic cyclophilin, an expression vector pET15CYP was constructed. The CyP cDNA (GenBank accession no.Y16088) reveals an open reading frame of 172 amino acids with the conserved tryptophan residue at position 128 and an insertion of seven amino acids spanning positions 48-54. Yellow lupine cyclophilin, purified after expression in E. coli cells, exhibits peptidyl-prolyl cis/trans isomerase activity when assayed with a synthetic oligopeptide. We have demonstrated that the recombinant cyclophilin is able to interact with nucleic acids, both single and double stranded DNA fragments as well as RNA.

[Recombinant protein production in Escherichia coli]. / Produkcja rekombinowanych bialek w Escherichia coli.

Growing needs for efficient recombinant production pose new challenges; starting from cell growth optimization under overexpression conditions, improving vectors, gene and protein sequence to suit them to protein biosynthesis machinery of the host, through extending the knowledge of protein folding, fusion protein construction, and coexpression systems, to improvements in protein purification and renaturation technologies. Hitherto Escherichia coli is the most defined and the cheapest protein biosynthesis system. With its wealth of available mutants tested is the best suited to economically test new gene constructs and to scale up the recombinant protein production.

Lupin nad9 and nad6 genes and their expression: 5' termini of the nad9 gene transcripts differentiate lupin species.

The mitochondrial nad9 and nad6 genes were analyzed in four lupin species: Lupinus luteus, Lupinus angustifolius, Lupinus albus and Lupinus mutabilis. The nucleotide sequence of these genes confirmed their high conservation, however, higher number of nucleotide substitution was observed in the L. albus genes. Southern hybridizations confirmed the presence of single copy number of these genes in L. luteus, L. albus and L. angustifolius. The expression of nad9 and nad6 genes was analyzed by Northern in different tissue types of analyzed lupin species. Transcription analyses of the two nad genes displayed single predominant mRNA species of about 0.6 kb in L. luteus and L. angustifolius. The L. albus transcripts were larger in size. The nad9 and nad6 transcripts were modified by RNA editing at 8 and 11 positions, in L. luteus and L. angustifolius, respectively. The gene order, rps3-rpl16-nad9, found in Arabidopsis thaliana is also conserved in L. luteus and L. angustifolius mitochondria. L. luteus and L. angustifolius showed some variability in the sequence of the nad9 promoter region. The last feature along with the differences observed in nad9 mRNA 5' termini of two lupins differentiate L. luteus and L. angustifolius species.