The Plasma Membrane Purinoreceptor P2K1/DORN1 Is Essential in Stomatal Closure Evoked by Extracellular Diadenosine Tetraphosphate (Ap4A) in Arabidopsis thaliana.

Dinucleoside polyphosphates (NpnNs) are considered novel signalling molecules involved in the induction of plant defence mechanisms. However, NpnN signal recognition and transduction are still enigmatic. Therefore, the aim of our research was the identification of the NpnN receptor and signal transduction pathways evoked by these nucleotides. Earlier, we proved that purine and pyrimidine NpnNs differentially affect the phenylpropanoid pathway in Vitis vinifera suspension-cultured cells. Here, we report, for the first time, that both diadenosine tetraphosphate (Ap4A) and dicytidine tetraphosphate (Cp4C)-induced stomatal closure in Arabidopsis thaliana. Moreover, we showed that plasma membrane purinoreceptor P2K1/DORN1 (does not respond to nucleotide 1) is essential for Ap4A-induced stomata movements but not for Cp4C. Wild-type Col-0 and the dorn1-3 A. thaliana knockout mutant were used. Examination of the leaf epidermis dorn1-3 mutant provided evidence that P2K1/DORN1 is a part of the signal transduction pathway in stomatal closure evoked by extracellular Ap4A but not by Cp4C. Reactive oxygen species (ROS) are involved in signal transduction caused by Ap4A and Cp4C, leading to stomatal closure. Ap4A induced and Cp4C suppressed the transcriptional response in wild-type plants. Moreover, in dorn1-3 leaves, the effect of Ap4A on gene expression was impaired. The interaction between P2K1/DORN1 and Ap4A leads to changes in the transcription of signalling hubs in signal transduction pathways.

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.

Vacuolar Processing Enzymes in Plant Programmed Cell Death and Autophagy.

Vacuolar processing enzymes (VPEs) are plant cysteine proteases that are subjected to autoactivation in an acidic pH. It is presumed that VPEs, by activating other vacuolar hydrolases, are in control of tonoplast rupture during programmed cell death (PCD). Involvement of VPEs has been indicated in various types of plant PCD related to development, senescence, and environmental stress responses. Another pathway induced during such processes is autophagy, which leads to the degradation of cellular components and metabolite salvage, and it is presumed that VPEs may be involved in the degradation of autophagic bodies during plant autophagy. As both PCD and autophagy occur under similar conditions, research on the relationship between them is needed, and VPEs, as key vacuolar proteases, seem to be an important factor to consider. They may even constitute a potential point of crosstalk between cell death and autophagy in plant cells. This review describes new insights into the role of VPEs in plant PCD, with an emphasis on evidence and hypotheses on the interconnections between autophagy and cell death, and indicates several new research opportunities.

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.

Nucleoside 5'-Phosphoramidates Control the Phenylpropanoid Pathway in Vitis vinifera Suspension-Cultured Cells.

It is known that cells contain various uncommon nucleotides such as dinucleoside polyphosphates (NpnN's) and adenosine 5'-phosphoramidate (NH2-pA) belonging to nucleoside 5'-phosphoramidates (NH2-pNs). Their cellular levels are enzymatically controlled. Some of them are accumulated in cells under stress, and therefore, they could act as signal molecules. Our previous research carried out in Arabidopsis thaliana and grape (Vitis vinifera) showed that NpnN's induced the expression of genes in the phenylpropanoid pathway and favored the accumulation of their products, which protect plants against stress. Moreover, we found that NH2-pA could play a signaling role in Arabidopsis seedlings. Data presented in this paper show that exogenously applied purine (NH2-pA, NH2-pG) and pyrimidine (NH2-pU, NH2-pC) nucleoside 5'-phosphoramidates can modify the expression of genes that control the biosynthesis of both stilbenes and lignin in Vitis vinifera cv. Monastrell suspension-cultured cells. We investigated the expression of genes encoding for phenylalanine ammonia-lyase (PAL1), cinnamate-4-hydroxylase (C4H1), 4-coumarate:coenzyme A ligase (4CL1), chalcone synthase (CHS1), stilbene synthase (STS1), cinnamoyl-coenzyme A:NADP oxidoreductase (CCR2), and cinnamyl alcohol dehydrogenase (CAD1). Each of the tested NH2-pNs also induced the expression of the trans-resveratrol cell membrane transporter VvABCG44 gene and caused the accumulation of trans-resveratrol and trans-piceid in grape cells as well as in the culture medium. NH2-pC, however, evoked the most effective induction of phenylpropanoid pathway genes such as PAL1, C4H1, 4CL1, and STS1. Moreover, this nucleotide also induced at short times the accumulation of N-benzoylputrescine (BenPut), one of the phenylamides that are derivatives of phenylpropanoid and polyamines. The investigated nucleotides did not change either the lignin content or the cell dry weight, nor did they affect the cell viability throughout the experiment. The results suggest that nucleoside 5'-phosphoramidates could be considered as new signaling molecules.

Completing Autophagy: Formation and Degradation of the Autophagic Body and Metabolite Salvage in Plants.

Autophagy is an evolutionarily conserved process that occurs in yeast, plants, and animals. Despite many years of research, some aspects of autophagy are still not fully explained. This mostly concerns the final stages of autophagy, which have not received as much interest from the scientific community as the initial stages of this process. The final stages of autophagy that we take into consideration in this review include the formation and degradation of the autophagic bodies as well as the efflux of metabolites from the vacuole to the cytoplasm. The autophagic bodies are formed through the fusion of an autophagosome and vacuole during macroautophagy and by vacuolar membrane invagination or protrusion during microautophagy. Then they are rapidly degraded by vacuolar lytic enzymes, and products of the degradation are reused. In this paper, we summarize the available information on the trafficking of the autophagosome towards the vacuole, the fusion of the autophagosome with the vacuole, the formation and decomposition of autophagic bodies inside the vacuole, and the efflux of metabolites to the cytoplasm. Special attention is given to the formation and degradation of autophagic bodies and metabolite salvage in plant cells.

New Insight into Plant Signaling: Extracellular ATP and Uncommon Nucleotides.

New players in plant signaling are described in detail in this review: extracellular ATP (eATP) and uncommon nucleotides such as dinucleoside polyphosphates (NpnN's), adenosine 5'-phosphoramidate (NH2-pA), and extracellular NAD+ and NADP+ (eNAD(P)+). Recent molecular, physiological, and biochemical evidence implicating concurrently the signaling role of eATP, NpnN's, and NH2-pA in plant biology and the mechanistic events in which they are involved are discussed. Numerous studies have shown that they are often universal signaling messengers, which trigger a signaling cascade in similar reactions and processes among different kingdoms. We also present here, not described elsewhere, a working model of the NpnN' and NH2-pA signaling network in a plant cell where these nucleotides trigger induction of the phenylpropanoid and the isochorismic acid pathways yielding metabolites protecting the plant against various types of stresses. Through these signals, the plant responds to environmental stimuli by intensifying the production of various compounds, such as anthocyanins, lignin, stilbenes, and salicylic acid. Still, more research needs to be performed to identify signaling networks that involve uncommon nucleotides, followed by omic experiments to define network elements and processes that are controlled by these signals.

Purine and pyrimidine dinucleoside polyphosphates differentially affect the phenylpropanoid pathway in Vitis vinifera L. cv. Monastrell suspension cultured cells.

It is known that the concentration of dinucleoside polyphosphates (NpnN's) in cells increases under stress and that adverse environmental factors induce biosynthesis of phenylpropanoids, which protect the plant against stress. Previously, we showed that purine NpnN's such as Ap3A and Ap4A induce both the activity of enzymes of the phenylpropanoid pathway and the expression of relevant genes in Arabidopsis seedlings. Moreover, we showed that Ap3A induced stilbene biosynthesis in Vitis vinifera cv. Monastrell suspension cultured cells. Data presented in this paper show that pyrimidine-containing NpnN's also modify the biosynthesis of stilbenes, affecting the transcript level of genes encoding key enzymes of the phenylpropanoid pathway and of these, Up4U caused the most effective accumulation of trans-resveratrol in the culture media. Similar effect was caused by Ap3A and Gp3G. Other pyrimidine NpnN's, such as Cp3C, Cp4C, and Ap4C, strongly inhibited the biosynthesis of stilbenes, but markedly (6- to 8-fold) induced the expression of the cinnamoyl-CoA reductase gene that controls lignin biosynthesis. Purine counterparts also clearly induced biosynthesis of trans-resveratrol and trans-piceid, but only slightly induced the expression of genes involved in lignin biosynthesis. In cells, Up3U caused a greater accumulation of trans-resveratrol and trans-piceid than did Up4U. Each of the NpnN's studied induced expression of the gene encoding the resveratrol transporter VvABCG44, which operates within the Vitis vinifera cell membrane. AMP, GMP, UMP, and CMP, potential products of NpnN degradation, did not affect the accumulation of stilbenes. The results obtained strongly support that NpnN's play a role as signaling molecules in plants.

Autophagic Machinery of Plant Peroxisomes.

Peroxisomes are cell organelles that play an important role in plants in many physiological and developmental processes. The plant peroxisomes harbor enzymes of the ß-oxidation of fatty acids and the glyoxylate cycle; photorespiration; detoxification of reactive oxygen and nitrogen species; as well as biosynthesis of hormones and signal molecules. The function of peroxisomes in plant cells changes during plant growth and development. They are transformed from organelles involved in storage lipid breakdown during seed germination and seedling growth into leaf peroxisomes involved in photorespiration in green parts of the plant. Additionally, intensive oxidative metabolism of peroxisomes causes damage to their components. Therefore, unnecessary or damaged peroxisomes are degraded by selective autophagy, called pexophagy. This is an important element of the quality control system of peroxisomes in plant cells. Despite the fact that the mechanism of pexophagy has already been described for yeasts and mammals, the molecular mechanisms by which plant cells recognize peroxisomes that will be degraded via pexophagy still remain unclear. It seems that a plant-specific mechanism exists for the selective degradation of peroxisomes. In this review, we describe the physiological role of pexophagy in plant cells and the current hypotheses concerning the mechanism of plant pexophagy.

Melatonin redirects carbohydrates metabolism during sugar starvation in plant cells.

Recent studies have shown that melatonin is an important molecule in plant physiology. It seems that the most important is that melatonin efficacy eliminates oxidative stress (direct and indirect antioxidant) and moreover induce plant stress reaction and switch on different defence strategies (preventively and interventively actions). In this report, the impact of exogenous melatonin on carbohydrate metabolism in Nicotiana tabacum L. line Bright Yellow 2 (BY-2) suspension cells during sugar starvation was examined. We analysed starch concentration, α-amylase and PEPCK activity as well as proteolytic activity in culture media. It has been shown that BY-2 cell treatment with 200 nM of melatonin improved viability of sugar-starved cells. It was correlated with higher starch content and phosphoenolpyruvate carboxykinase (PEPCK) activity. The obtained results revealed that exogenous melatonin under specific conditions (stress) can play regulatory role in sugar metabolism, and it may modulate carbohydrate concentration in etiolated BY-2 cells. Moreover, our results confirmed the hypothesis that if the starch is synthesised even in sugar-starved cells, it is highly probable that melatonin shifts the BY-2 cell metabolism on gluconeogenesis pathway and allows for synthesis of carbohydrates from nonsugar precursors, that is amino acids. These points to another defence strategy that was induced by exogenous melatonin applied in plants to overcome adverse environmental conditions.

Nitrate simultaneously enhances lipid and protein accumulation in developing yellow lupin cotyledons cultured in vitro, but not under field conditions.

The research was conducted on yellow lupin (Lupinus luteus L.) mature seeds, developing cotyledons, developing pods, and seedlings. The main storage compound in yellow lupin seeds is protein, whose content may reach up to 45%. Oil content in seeds of yellow lupin is about 6%. In such protein-storing seeds there is a strong negative relationship between accumulation of storage lipid and protein. An increase in protein content causes a decrease in lipid level, and vice versa. However, simultaneous increase in lipid and protein content is possible in developing lupin cotyledons (the main storage organs of lupin seeds) cultured in vitro. Such an effect was obtained by feeding the cotyledons with nitrate (35mM). The same positive relationship in storage lipid and protein accumulation was also obtained in developing lupin pods fed with nitrate (35mM), detached from the mother plant, and maintained under quasi in vitro conditions. Fertilization of lupin plants with nitrate under field conditions (40 or 80kgNha-1 applied before sowing, at the nodulation stage or at the flowering and pod formation stage) did not cause significant changes in lipid and protein contents in mature seeds. Experiments performed on lupin seedlings cultivated hydroponically showed that nitrate added to the medium was accumulated mainly in roots, and at a remarkably lower level in shoots. We hypothesize that the lack of stimulatory effect of nitrate on storage lipid and protein accumulation in seeds under field conditions is due to inefficient transport of nitrate from the root to developing pods in lupin plants. This causes that the level of nitrate inside the developing lupin seeds is not elevated under field conditions.

Asparagine slows down the breakdown of storage lipid and degradation of autophagic bodies in sugar-starved embryo axes of germinating lupin seeds.

The research was conducted on embryo axes of yellow lupin (Lupinus luteus L.), white lupin (Lupinus albus L.) and Andean lupin (Lupinus mutabilis Sweet), which were isolated from imbibed seeds and cultured for 96h in vitro under different conditions of carbon and nitrogen nutrition. Isolated embryo axes were fed with 60mM sucrose or were sugar-starved. The effect of 35mM asparagine (a central amino acid in the metabolism of germinating lupin seeds) and 35mM nitrate (used as an inorganic kind of nitrogen) on growth, storage lipid breakdown and autophagy was investigated. The sugar-starved isolated embryo axes contained more total lipid than axes fed with sucrose, and the content of this storage compound was even higher in sugar-starved isolated embryo axes fed with asparagine. Ultrastructural observations showed that asparagine significantly slowed down decomposition of autophagic bodies, and this allowed detailed analysis of their content. We found peroxisomes inside autophagic bodies in cells of sugar-starved Andean lupin embryo axes fed with asparagine, which led us to conclude that peroxisomes may be degraded during autophagy in sugar-starved isolated lupin embryo axes. One reason for the slower degradation of autophagic bodies was the markedly lower lipolytic activity in axes fed with asparagine.

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.

Storage lipids as a source of carbon skeletons for asparagine synthesis in germinating seeds of yellow lupine (Lupinus luteus L.).

The (14)C-acetate metabolism and regulatory functions of sucrose and sodium fluoride (NaF) were examined in embryo axes and cotyledons isolated from yellow lupine seeds and grown in vitro. After 15 min of incubating organs in solutions of labeled acetate, more radioactivity was found in amino acids (particularly in glutamate, asparagine and glutamine) than in sugars. After 120 min of incubation, (14)C was still localized mainly in amino acids (particularly in asparagine and glutamate). The (14)C atoms from position C-1 of acetate were mostly localized in the liberated (14)CO(2), whereas those from position C-2 were incorporated chiefly into amino acids, sugars and the insoluble fraction of the studied organs. The addition of NaF caused a decrease in the amount of (14)C incorporated into amino acids and in the insoluble fraction. The influence of NaF on incorporation of (14)C into sugars differed between organs. In embryo axes, NaF inhibited this process, but in cotyledons it stimulated (14)C incorporation into glucose. The release of (14)CO(2) with the C-1 and C-2 carbon atoms from acetate was more intensive in embryo axes and cotyledons grown on a medium without sucrose. This process was markedly limited by NaF, which inhibits glycolysis and gluconeogenesis. Alternative pathways of carbon flow from fatty acids to asparagine are discussed.

Lipid and protein accumulation in developing seeds of three lupine species: Lupinus luteus L., Lupinus albus L., and Lupinus mutabilis Sweet.

A comparative study was carried out on the dynamics of lipid accumulation in developing seeds of three lupine species. Lupine seeds differ in lipid content; yellow lupine (Lupinus luteus L.) seeds contain about 6%, white lupine (Lupinus albus L.) 7-14%, and Andean lupine (Lupinus mutabilis Sweet) about 20% of lipids by dry mass. Cotyledons from developing seeds were isolated and cultured in vitro for 96 h on Heller medium with 60 mM sucrose (+S) or without sucrose (-S). Each medium was additionally enriched with 35 mM asparagine or 35 mM NaNO3. Asparagine caused an increase in protein accumulation and simultaneously decreased the lipid content, but nitrate increased accumulation of both protein and lipid. Experiments with [1-14C]acetate and [2-14C]acetate showed that the decrease in lipid accumulation in developing lupine seeds resulted from exhaustion of lipid precursors rather than from degradation or modification of the enzymatic apparatus. The carbon atom from the C-1 position of acetate was liberated mainly as CO2, whereas the carbon atom from the C-2 position was preferentially used in anabolic pathways. The dominant phospholipid in the investigated lupine seed storage organs was phosphatidylcholine. The main fatty acid in yellow lupine cotyledons was linoleic acid, in white lupine it was oleic acid, and in Andean lupine it was both linoleic and oleic acids. The relationship between stimulation of lipid and protein accumulation by nitrate in developing lupine cotyledons and enhanced carbon flux through glycolysis caused by the inorganic nitrogen form is discussed.

A transfer of carbon atoms from fatty acids to sugars and amino acids in yellow lupine (Lupinus luteus L.) seedlings.

The metabolism of 14C-acetate was investigated during the in vitro germination of yellow lupine seeds. Carbon atoms (14C) from the C-2 position of acetate were incorporated mainly into amino acids: aspartate, glutamate, and glutamine and into sugars: glucose, sucrose, and fructose. In contrast to this, 14C from the C-1 position of acetate was released mainly as 14CO2. Incorporation of 1-14C and 2-14C from acetate into amino acids and sugars in seedling axes was more intense when sucrose was added to the medium. However, in cotyledons where lipids are converted to carbohydrates, this process was inhibited by exogenous sucrose. Since acetate is the product of fatty acid beta-oxidation, our results indicate that, at least in lupine, seed storage lipids can be converted not only to sucrose, but mainly to amino acids. Inhibitory effects of sucrose on the incorporation of 14C from acetate into amino acids and sugars in cotyledons of lupine seedlings may be explained as the effect of regulation of the glyoxylate cycle by sugars.