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.

Stress-related expression of the chloroplast EGY3 pseudoprotease and its possible impact on chloroplasts' proteome composition.

The EGY3 is a pseudoprotease, located in the thylakoid membrane, that shares homology with the family of site-2-proteases (S2P). Although S2P proteases are present in the cells of all living organisms, the EGY3 was found only in plant cells. The sequence of the pseudoprotease is highly conserved in the plant kingdom; however, little is known about its physiological importance. Results obtained with real-time PCR indicated that the expression of the EGY3 gene is dramatically induced during the first few hours of exposure to high light and high-temperature stress. The observed increase in transcript abundance correlates with protein accumulation level, which indicates that EGY3 participates in response to both high-temperature and high light stresses. The lack of the pseudoprotease leads, in both stresses, to lower concentrations of hydrogen peroxide. However, the decrease of chloroplast copper/zinc superoxide dismutase 2 level was observed only during the high light stress. In both analyzed stressful conditions, proteins related to RubisCO folding, glycine metabolism, and photosystem I were identified as differently accumulating in egy3 mutant lines and WT plants; however, the functional status of PSII during analyzed stressful conditions remains very similar. Our results lead to a conclusion that EGY3 pseudoprotease participates in response to high light and high-temperature stress; however, its role is associated rather with photosystem I and light-independent reactions of photosynthesis.

Contribution of Exogenous Proline to Abiotic Stresses Tolerance in Plants: A Review.

Abiotic stresses are the major environmental factors that play a significant role in decreasing plant yield and production potential by influencing physiological, biochemical, and molecular processes. Abiotic stresses and global population growth have prompted scientists to use beneficial strategies to ensure food security. The use of organic compounds to improve tolerance to abiotic stresses has been considered for many years. For example, the application of potential external osmotic protective compounds such as proline is one of the approaches to counteract the adverse effects of abiotic stresses on plants. Proline level increases in plants in response to environmental stress. Proline accumulation is not just a signal of tension. Rather, according to research discussed in this article, this biomolecule improves plant resistance to abiotic stress by rising photosynthesis, enzymatic and non-enzymatic antioxidant activity, regulating osmolyte concentration, and sodium and potassium homeostasis. In this review, we discuss the biosynthesis, sensing, signaling, and transport of proline and its role in the development of various plant tissues, including seeds, floral components, and vegetative tissues. Further, the impacts of exogenous proline utilization under various non-living stresses such as drought, salinity, high and low temperatures, and heavy metals have been extensively studied. Numerous various studies have shown that exogenous proline can improve plant growth, yield, and stress tolerance under adverse environmental factors.

Endogenous Polyamines and Ethylene Biosynthesis in Relation to Germination of Osmoprimed Brassica napus Seeds under Salt Stress.

Currently, seed priming is reported as an efficient and low-cost approach to increase crop yield, which could not only promote seed germination and improve plant growth state but also increase abiotic stress tolerance. Salinity represents one of the most significant abiotic stresses that alters multiple processes in plants. The accumulation of polyamines (PAs) in response to salt stress is one of the most remarkable plant metabolic responses. This paper examined the effect of osmopriming on endogenous polyamine metabolism at the germination and early seedling development of Brassica napus in relation to salinity tolerance. Free, conjugated and bound polyamines were analyzed, and changes in their accumulation were discussed with literature data. The most remarkable differences between the corresponding osmoprimed and unprimed seeds were visible in the free (spermine) and conjugated (putrescine, spermidine) fractions. The arginine decarboxylase pathway seems to be responsible for the accumulation of PAs in primed seeds. The obvious impact of seed priming on tyramine accumulation was also demonstrated. Moreover, the level of ethylene increased considerably in seedlings issued from primed seeds exposed to salt stress. It can be concluded that the polyamines are involved in creating the beneficial effect of osmopriming on germination and early growth of Brassica napus seedlings under saline conditions through moderate changes in their biosynthesis and accumulation.

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 on Water Status in Germinating Brassica napus Seeds in Relation to Priming-Improved Germination.

Seed priming is a pre-sowing method successfully used to improve seed germination. Since water plays a crucial role in germination, the aim of this study was to investigate the relationship between better germination performances of osmoprimed Brassica napus seeds and seed water status during germination. To achieve this goal, a combination of different kinds of approaches was used, including nuclear magnetic resonance (NMR) spectroscopy, TEM, and SEM as well as semi-quantitative PCR (semi-qPCR). The results of this study showed that osmopriming enhanced the kinetics of water uptake and the total amount of absorbed water during both the early imbibition stage and in the later phases of seed germination. The spin⁻spin relaxation time (T2) measurement suggests that osmopriming causes faster water penetration into the seed and more efficient tissue hydration. Moreover, factors potentially affecting water relations in germinating primed seeds were also identified. It was shown that osmopriming (i) changes the microstructural features of the seed coat, e.g., leads to the formation of microcracks, (ii) alters the internal structure of the seed by the induction of additional void spaces in the seed, (iii) increases cotyledons cells vacuolization, and (iv) modifies the expression pattern of aquaporin genes.

Molecular processes induced in primed seeds-increasing the potential to stabilize crop yields under drought conditions.

Environmental stress factors such as drought, salinity, temperature extremes and rising CO2 negatively affect crop growth and productivity. Faced with the scarcity of water resources, drought is the most critical threat to world food security. This is particularly important in the context of climate change and an increasing world population. Seed priming is a very promising strategy in modern crop production management. Although it has been known for several years that seed priming can enhance seed quality and the effectiveness of stress responses of germinating seeds and seedlings, the molecular mechanisms involved in the acquisition of stress tolerance by primed seeds in the germination process and subsequent plant growth remain poorly understood. This review provides an overview of the metabolic changes modulated by priming, such as the activation of DNA repair and the antioxidant system, accumulation of aquaporins and late embryogenesis abundant proteins that contribute to enhanced drought stress tolerance. Moreover, the phenomenon of "priming memory," which is established during priming and can be recruited later when seeds or plants are exposed to stress, is highlighted.

Different Modes of Hydrogen Peroxide Action During Seed Germination.

Hydrogen peroxide was initially recognized as a toxic molecule that causes damage at different levels of cell organization and thus losses in cell viability. From the 1990s, the role of hydrogen peroxide as a signaling molecule in plants has also been discussed. The beneficial role of H2O2 as a central hub integrating signaling network in response to biotic and abiotic stress and during developmental processes is now well established. Seed germination is the most pivotal phase of the plant life cycle, affecting plant growth and productivity. The function of hydrogen peroxide in seed germination and seed aging has been illustrated in numerous studies; however, the exact role of this molecule remains unknown. This review evaluates evidence that shows that H2O2 functions as a signaling molecule in seed physiology in accordance with the known biology and biochemistry of H2O2. The importance of crosstalk between hydrogen peroxide and a number of signaling molecules, including plant phytohormones such as abscisic acid, gibberellins, and ethylene, and reactive molecules such as nitric oxide and hydrogen sulfide acting on cell communication and signaling during seed germination, is highlighted. The current study also focuses on the detrimental effects of H2O2 on seed biology, i.e., seed aging that leads to a loss of germination efficiency. The dual nature of hydrogen peroxide as a toxic molecule on one hand and as a signal molecule on the other is made possible through the precise spatial and temporal control of its production and degradation. Levels of hydrogen peroxide in germinating seeds and young seedlings can be modulated via pre-sowing seed priming/conditioning. This rather simple method is shown to be a valuable tool for improving seed quality and for enhancing seed stress tolerance during post-priming germination. In this review, we outline how seed priming/conditioning affects the integrative role of hydrogen peroxide in seed germination and aging.

Lead-stress induced changes in the content of free, thylakoid- and chromatin-bound polyamines, photosynthetic parameters and ultrastructure in greening barley leaves.

The aim of this study was to determine the impact of lead (Pb) stress as 0.6mM Pb(NO3)2 on the content of free, thylakoid- and chromatin-bound polyamines (PAs) and diamine oxidase (DAO) activity in detached greening barley leaves. Additionally, photosynthetic-related parameters, generation of hydrogen peroxide (H2O2) and malondialdehyde (MDA) content and ultrastructural changes under Pb-stress were studied. The level of putrescine (Put) was reduced progressively to 56% at 24h of Pb stress, and it was correlated with 38% increase of DAO activity. Spermidine (Spd) content was not affected by Pb-stress, while the free spermine (Spm) level significantly increased by about 83% at 6h, and in that time the lowest level of H2O2 was observed. The exogenous applied Spm to Pb-treated leaves caused a decrease in the content of H2O2. In greening leaves exposed to Pb an accumulation of chlorophylls a and b was inhibited by about 39 and 47%, respectively, and photosynthetic parameters of efficiency of electron transport and photochemical reaction in chloroplasts as ΦPSII, ETR and RFd were lowered by about 23-32%. The level of thylakoid-bound Put decreased by about 22%. Moreover, thylakoids isolated from chloroplasts of Pb-treated leaves were characterized with lower Put/Spm ratio as compared to control leaves. In the presence of Pb the significant decrease in the number of thylakoids per granum and cap-shape invaginations of cytoplasmic material were noticed. In Pb-stressed leaves the level of chromatin-bound Spm increased by about 48% and sometimes condensed chromatin in nuclei was observed. We conclude that in greening barley leaves exposed to Pb-stress changes in free, thylakoid- and chromatin-bound PAs play some role in the functioning of leaves or plants in heavy metal stress conditions.

Enhanced expression of the proline synthesis gene P5CSA in relation to seed osmopriming improvement of Brassica napus germination under salinity stress.

Osmopriming is a pre-sowing treatment that enhances germination performance and stress tolerance of germinating seeds. Brassica napus seeds showed osmopriming-improved germination and seedling growth under salinity stress. To understand the molecular and biochemical mechanisms of osmopriming-induced salinity tolerance, the accumulation of proline, gene expression and activity of enzymes involved in proline metabolism and the level of endogenous hydrogen peroxide were investigated in rape seeds during osmopriming and post-priming germination under control (H2O) and stress conditions (100 mM NaCl). The relationship between gene expression and enzymatic activity of pyrroline-5-carboxylate synthetase (P5CS), ornithine-δ-aminotransferase (OAT) and proline dehydrogenase (PDH) was determined. The improved germination performance of osmoprimed seeds was accompanied by a significant increase in proline content. The accumulation of proline during priming and post-priming germination was associated with strong up-regulation of the P5CSA gene, down-regulation of the PDH gene and accumulation of hydrogen peroxide. The up-regulated transcript level of P5CSA was consistent with the increase in P5CS activity. This study shows, for the first time, the role of priming-induced modulation of activities of particular genes and enzymes of proline turnover, and its relationship with higher content of hydrogen peroxide, in improving seed germination under salinity stress. Following initial stress-exposure, the primed seeds acquired stronger salinity stress tolerance during post-priming germination, a feature likely linked to a 'priming memory'.

Deciphering priming-induced improvement of rapeseed (Brassica napus L.) germination through an integrated transcriptomic and proteomic approach.

Rape seeds primed with -1.2 MPa polyethylene glycol 6000 showed improved germination performance. To better understand the beneficial effect of osmopriming on seed germination, a global expression profiling method was used to compare, for the first time, transcriptomic and proteomic data for osmoprimed seeds at the crucial phases of priming procedure (soaking, drying), whole priming process and subsequent germination. Brassica napus was used here as a model to dissect the process of osmopriming into its essential components. A total number of 952 genes and 75 proteins were affected during the main phases of priming and post-priming germination. Transcription was not coordinately associated with translation resulting in a limited correspondence between mRNAs level and protein abundance. Soaking, drying and final germination of primed seeds triggered distinct specific pathways since only a minority of genes and proteins were involved in all phases of osmopriming while a vast majority was involved in only one single phase. A particular attention was paid to genes and proteins involved in the transcription, translation, reserve mobilization, water uptake, cell cycle and oxidative stress processes.

Lupine embryo axes under salinity stress. II. Mitochondrial proteome response.

Germination is the first step of plant growth in plant life cycle. An embryonic radicle protruding the seed coat is the first part of plant which has direct contact with external environment including salt-affected soil. In embryo axes, mitochondria are the main energy producer. To understand better salinity impact on mitochondria functioning, this study was focused on the effect of NaCl stress onto mitochondria proteome. Mitochondria were isolated from yellow lupine (Lupine luteus L. 'Mister') embryo axes cultured in vitro for 12 h with 250 and 500 mM NaCl. Two-dimensional gel electrophoresis of mitochondrial proteins isolated from NaCl-treated axes demonstrated significant changes in proteins abundances as a response to salinity treatment. Twenty-one spots showing significant changes in protein expression profiles both under 250 and 500 mM NaCl treatment were selected for tandem mass spectrometry identification. This approach revealed proteins associated with different metabolic processes that represent enzymes of tricarboxylic acid cycle, mitochondrial electron transport chain, enzymes and proteins involved in mitochondria biogenesis and stresses response. Among proteins involved in mitochondria biogenesis, mitochondrial import inner membrane translocase, subunit Tim17/22, mitochondrial-processing peptidase subunit alpha-1, mitochondrial elongation factor Tu and chaperonins CPN60 were revealed. Finally, formate dehydrogenase 1 was found to accumulate in lupine embryo axes mitochondria under salinity. The functions of identified proteins are discussed in relation to salinity stress response, including salinity-induced PCD.

A comparative study of water distribution and dehydrin protein localization in maturing pea seeds.

In this study, the distribution of water in pea seeds after harvesting at different seed stages was traced by magnetic resonance imaging (MRI). MRI visualized the process of water loss in maturing pea seeds. MR images showed local inhomogeneities of water distribution inside seeds. The intensity of the signal coming from water declined from the inner to the outer part of cotyledon tissue. This spatial inhomogeneity of water signals inside cotyledons may be correlated with the gradient of storage substances accumulation within cotyledons. Tissue localization of dehydrins showed the presence of dehydrin protein in the area of protovascular tissue of both the embryo axis and cotyledons. The temporal accumulation of two dehydrin proteins with molecular masses of 30 and 35kDa correlated well with seed desiccation. The pattern of dehydrin localization reflected the pattern of water distribution in the protovascular bundles region of maturing pea embryos, suggesting the involvement of these proteins in promoting water influx into the vascular bundles.

A comparative study of water distribution, free radical production and activation of antioxidative metabolism in germinating pea seeds.

The aim of this study was to investigate whether there is a relationship between hydration of the embryo axes and cotyledons and the resumption of the oxidative metabolism in both organs of germinating seeds of pea (Pisum sativum L. cv. Piast). Nuclear magnetic resonance ((1)H-NMR) spectroscopy and imaging were used to study temporal and spatial water uptake and distribution in pea seeds. The observations revealed that water penetrates into the seed through the hilum, micropyle and embryo axes, and cotyledons hydrate to different extents. Thus, inhomogeneous water distribution may influence the resumption of oxidative metabolism. Electron paramagnetic resonance (EPR) measurements showed that seed germination was accompanied by the generation of free radicals with g(1) and g(2) values of 2.0032 and 2.0052, respectively. The values of spectroscopic splitting coefficients suggest that they are quinone radicals. The highest content of free radicals was observed in embryo axes immediately after emergence of the radicle. Glutathione content decreased during the entire germination period in both embryo axes and cotyledons. A different profile was observed for ascorbate, with significant increases in embryo axes, coinciding with radicle protrusion. Electrophoretic analysis showed that superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2) were present in dry seeds and were activated later during germination, especially in embryo axes. The presence of all antioxidative enzymes as well as low molecular antioxidants in dry seeds allowed the antioxidative machinery to be active as soon as the enzymes were reactivated by seed imbibition. The observed changes in free radical levels, antioxidant contents and enzymatic activities in embryo axes and cotyledons appear to be more closely related to metabolic and developmental processes associated with preparation for germination, and do not correspond directly to the hydration of the tissues.