Melatonin Inhibits Peroxide Production in Plant Mitochondria.

The effect of melatonin on respiration and production (release) of hydrogen peroxide during succinate oxidation in mitochondria isolated from lupine cotyledons and epicotyls of pea seedlings was studied. It was shown for the first time that melatonin (10-7-10-3 M) had a significant inhibitory effect on the production of peroxide by plant mitochondria, which was characterized by concentration dependence and species specificity. At the same time, melatonin (at a concentration of up to 100 µM) had virtually no effect on mitochondrial respiration rate and respiratory control coefficient. The results confirm the antioxidant function of melatonin and indicate that it is involved in the regulation of ROS levels and maintenance of redox balance in plant mitochondria.

Paths of water entry and structures involved in the breaking of seed dormancy of Lupinus.

Physical dormancy is the water impermeability of the seed coat caused by one or more palisade cell layer(s) called macrosclereids. The specialised structure for water entry sites is the water gap, which serves as a detector of environmental cues for germination. In Fabaceae, the water gap is the lens, although another seed structure for water entry could exist. In this study, we identified the initial site of water entry, observed the hydration of a cushion-like structure near the radicle, described the anatomy of the water gap, and analysed the association of anatomical seed traits with the initial site of water entry and the imbibition velocity of six species of Lupinus from the state of Jalisco, Mexico. Dye tracking with a toluidine blue solution was used to identify the initial site of water entry. The anatomical description was performed using conventional microtechnique and a light microscope. The entry of the toluidine solution into seeds of L. montanus was observed after 6h, followed by L. exaltatus and L. mexicanus after 18h and L. elegans, L. reflexus and L. rotundiflorus after 48h. The site of water entry was the lens in L. elegans, L. exaltatus, L. reflexus and L. rotundiflorus and the micropyle in L. mexicanus and L. montanus. The cushion-like structure was responsible for water accumulation in embryo imbibition. Significant differences among anatomical seed traits such as thickness in the hilar region, the counter-palisade layer, cushion-like structure, epidermis, hypodermis, and innermost parenchyma layer were found among the species.

Poly(A) RNAs including coding proteins RNAs occur in plant Cajal bodies.

The localisation of poly(A) RNA in plant cells containing either reticular (Allium cepa) or chromocentric (Lupinus luteus, Arabidopsis thaliana) nuclei was studied through in situ hybridisation. In both types of nuclei, the amount of poly(A) RNA was much greater in the nucleus than in the cytoplasm. In the nuclei, poly(A) RNA was present in structures resembling nuclear bodies. The molecular composition as well as the characteristic ultrastructure of the bodies containing poly(A) RNA demonstrated that they were Cajal bodies. We showed that some poly(A) RNAs in Cajal bodies code for proteins. However, examination of the localisation of active RNA polymerase II and in situ run-on transcription assays both demonstrated that CBs are not sites of transcription and that BrU-containing RNA accumulates in these structures long after synthesis. In addition, it was demonstrated that accumulation of poly(A) RNA occurs in the nuclei and CBs of hypoxia-treated cells. Our findings indicated that CBs may be involved in the later stages of poly(A) RNA metabolism, playing a role storage or retention.

Effects of endogenous signals and Fusarium oxysporum on the mechanism regulating genistein synthesis and accumulation in yellow lupine and their impact on plant cell cytoskeleton.

The aim of the study was to examine cross-talk interactions of soluble sugars (sucrose, glucose and fructose) and infection caused by Fusarium oxysporum f.sp. lupini on the synthesis of genistein in embryo axes of Lupinus luteus L.cv. Juno. Genistein is a free aglycone, highly reactive and with the potential to inhibit fungal infection and development of plant diseases. As signal molecules, sugars strongly stimulated accumulation of isoflavones, including genistein, and the expression of the isoflavonoid biosynthetic genes. Infection significantly enhanced the synthesis of genistein and other isoflavone aglycones in cells of embryo axes of yellow lupine with high endogenous sugar levels. The activity of ß-glucosidase, the enzyme that releases free aglycones from their glucoside bindings, was higher in the infected tissues than in the control ones. At the same time, a very strong generation of the superoxide anion radical was observed in tissues with high sugar contents already in the initial stage of infection. During later stages after inoculation, a strong generation of semiquinone radicals was observed, which level was relatively higher in tissues deficient in sugars than in those with high sugar levels. Observations of actin and tubulin cytoskeletons in cells of infected embryo axes cultured on the medium with sucrose, as well as the medium without sugar, showed significant differences in their organization.

Uptake and cellular distribution, in four plant species, of fluorescently labeled mesoporous silica nanoparticles.

KEY MESSAGE: We report the uptake of MSNs into the roots and their movement to the aerial parts of four plant species and their quantification using fluorescence, TEM and proton-induced x - ray emission (micro - PIXE) elemental analysis. Monodispersed mesoporous silica nanoparticles (MSNs) of optimal size and configuration were synthesized for uptake by plant organs, tissues and cells. These monodispersed nanoparticles have a size of 20 nm with interconnected pores with an approximate diameter of 2.58 nm. There were no negative effects of MSNs on seed germination or when transported to different organs of the four plant species tested in this study. Most importantly, for the first time, a combination of confocal laser scanning microscopy, transmission electron microscopy and proton-induced X-ray emission (micro-PIXE) elemental analysis allowed the location and quantification MSNs in tissues and in cellular and sub-cellular locations. Our results show that MSNs penetrated into the roots via symplastic and apoplastic pathways and then via the conducting tissues of the xylem to the aerial parts of the plants including the stems and leaves. The translocation and widescale distribution of MSNs in plants will enable them to be used as a new delivery means for the transport of different sized biomolecules into plants.

The alternative respiratory pathway mediates carboxylate synthesis in white lupin cluster roots under phosphorus deprivation.

Plant adaptations associated with a high efficiency of phosphorus (P) acquisition can be used to increase productivity and sustainability in a world with a growing population and decreasing rock phosphate reserves. White lupin (Lupinus albus) produces cluster roots that release carboxylates to efficiently mobilize P from P-sorbing soils. It has been hypothesized that an increase in the activity of the alternative oxidase (AOX) would allow for the mitochondrial oxidation of NAD(P)H produced during citrate synthesis in cluster roots at a developmental stage when there is a low demand for ATP. We used the oxygen-isotope fractionation technique to study the in vivo respiratory activities of the cytochrome oxidase pathway (COP) and the alternative oxidase pathway (AOP) in different root sections of white lupins grown hydroponically with and without P. In parallel, AOX protein levels and internal carboxylate concentrations were determined in cluster and non-cluster roots. Higher in vivo AOP activity was measured in cluster roots when malate and citrate concentrations were also high, thus confirming our hypothesis. AOX protein levels were not always correlated with in vivo AOP activity, suggesting post-translational regulation of AOX.

Where do roots take up water? Neutron radiography of water flow into the roots of transpiring plants growing in soil.

Where and how fast does water flow from soil into roots? The answer to this question requires direct and in situ measurement of local flow of water into roots of transpiring plants growing in soil. We used neutron radiography to trace the transport of deuterated water (D2O) in lupin (Lupinus albus) roots. Lupins were grown in aluminum containers (30 × 25 × 1 cm) filled with sandy soil. D2O was injected in different soil regions and its transport in soil and roots was monitored by neutron radiography. The transport of water into roots was then quantified using a convection-diffusion model of D2O transport into roots. The results showed that water uptake was not uniform along roots. Water uptake was higher in the upper soil layers than in the lower ones. Along an individual root, the radial flux was higher in the proximal segments than in the distal segments. In lupins, most of the water uptake occurred in lateral roots. The function of the taproot was to collect water from laterals and transport it to the shoot. This function is ensured by a low radial conductivity and a high axial conductivity. Lupin root architecture seems well designed to take up water from deep soil layers.

Nitric oxide implication in cadmium-induced programmed cell death in roots and signaling response of yellow lupine plants.

The sequence of events leading to the programmed cell death (PCD) induced by heavy metals in plants is still the object of extensive investigation. In this study we showed that roots of 3-day old yellow lupine (Lupinus luteus L.) seedlings exposed to cadmium (Cd, 89µM CdCl(2)) resulted in PCD starting from 24h of stress duration, which was evidenced by TUNEL-positive reaction. Cd-induced PCD was preceded by a relatively early burst of nitric oxide (NO) localized mainly in the root tips. Above changes were accompanied by the NADPH-oxidase-dependent superoxide anion (O(2)(·-)) production. However, the concomitant high level of both NO and O(2)(·-) at the 24th h of Cd exposure did not provoke an enhanced peroxynitrite formation. The treatment with the NADPH-oxidase inhibitor and NO-scavenger significantly reduced O(2)(·-) and NO production, respectively, as well as diminished the pool of cells undergoing PCD. The obtained data indicate that boosted NO and O(2)(·-) production is required for Cd-induced PCD in lupine roots. Moreover, we found that in roots of 14-day old lupine plants the NO-dependent Cd-induced PCD was correlated with the enhanced level of the post-stress signals in leaves, including distal NO cross-talk with hydrogen peroxide.

The reaction of Lupinus angustifolius L. root meristematic cell nucleoli to lead.

The effect of 2-48 h treatment of Lupinus angustifolius L. roots with lead nitrate at the concentration of 10(-4) M on the nucleoli in meristematic cells was investigated. In the lead presence the number of ring-shaped as well as segregated nucleoli increased especially after 12-48 h of treatment, while spindle-shaped nucleoli appeared after 24 h and 48 h. Lead presence also increased the frequency of cells with silver-stained particles in the nucleus and the number of these particles especially from the 12th hour of treatment. It was accompanied by significant decline of nucleolar area. Analysis of these cells in transmission electron microscope confirmed the presence of ring-shaped and segregated nucleoli. Moreover, electron microscopy revealed compact structure nucleoli without granular component. Additionally, one to three oval-shaped fibrillar structures attached to nucleolus or lying free in the nucleoplasm were visible. The possible mechanism of lead toxicity to the nucleolus is briefly discussed.

Self-consistent equation of plant cell growth.

We introduce a transcendental equation, describing the subsequent stages of plant cell/organ growth. Starting from empirically verified conclusions originating from the central limit theorem we also insert the influence of temperature on elongation growth to receive a time-dependent equation of growth parameterized by temperature. This self-consistent equation evolves with time using three cardinal parameters: t0, T0 and V0. They represent the time of maximum expansion rate, the growth optimum temperature and the corresponding volume, respectively. Experimental determination of these cardinal values enables evaluation of the dynamic extensibility coefficient Phi=PhiT(t) in time and temperature domain.

Conformation of cytoskeletal elements during the division of infected Lupinus albus L. nodule cells.

Lupin nodule cells maintain their ability to divide for several cycles after being infected by endosymbiotic rhizobia. The conformation of the cytoskeletal elements of nodule cells was studied by fluorescence labelling, immunocytochemistry, and laser confocal and transmission electron microscopy. The dividing infected cells showed the normal microtubule and actin patterns of dividing plant cells. The clustered symbiosomes were tethered to the spindle-pole regions and moved to the cell poles during spindle elongation. In metaphase, anaphase, and early telophase, the symbiosomes were found at opposite cell poles where they did not interfere with the spindle filaments or phragmoplast. This symbiosome positioning was comparable with that of the organelles (which ensures organelle inheritance during plant cell mitosis). Tubulin microtubules and actin microfilaments appeared to be in contact with the symbiosomes. The possible presence of actin molecular motor myosin in nodules was analysed using a monoclonal antibody against the myosin light chain. The antigen was detected in protein extracts of nodule and root cytosol as bands of approximately 20 kDa (the size expected). In the nodules, an additional polypeptide of 65 kDa was found. Immunogold techniques revealed the antigen to be localized over thin microfilaments linked to the cell wall, as well as over the thicker microfilament bundles and surrounding the symbiosomes. The pattern of cytoskeleton rearrangement in dividing infected cells, along with the presence of myosin antigen, suggests that the positioning of symbiosomes in lupin nodule cells might depend on the same mechanisms used to partition genuine plant cell organelles during mitosis.

Subcellular compartmentalisation of cadmium in white lupins determined by energy-dispersive X-ray microanalysis.

The microlocalisation of cadmium (Cd) at the tissue-cellular level in Lupinus albus L. cv. Multolupa was determined by energy-dispersive X-ray microanalysis (EDXMA). Experimental plants were grown on Cd-treated (0 and 150 microM) perlite for 35 days. In leaves, Cd was found inside cells (cytoplasm or vacuoles), especially in the vascular bundle cells. Cd-induced damage of the chloroplast structure was also detected. EDXMA of the roots showed the cell wall to be the main area of Cd binding at the cellular level; only a small amount of Cd was found in the vacuoles. At the tissue level, a decreasing Cd gradient was seen from the outer to the inner root cortical parenchyma. Cd and S were found co-localised in the vascular cylinder.

Nuclear DNA endoreduplication and expression of the mitotic inhibitor Ccs52 associated to determinate and lupinoid nodule organogenesis.

Lotus japonicus determinate nodules differ greatly from indeterminate nodules in their organogenesis and morphological characteristics, whereas Lupinus albus lupinoid nodules share features of determinate and indeterminate nodules. The mitotic inhibitor Ccs52A is essential for endoreduplication and ploidy-dependent cell enlargement during symbiotic cell differentiation in Medicago truncatula indeterminate nodules. ccs52A homolog genes were isolated from lupin and lotus nodules; the deduced Ccs52A proteins showed high sequence similarity with other Cdh-1-type activators of the anaphase-promoting complex and were grouped with A-type Ccs52 proteins from different plants. In lupin, ccs52A expression was restricted to the earlier stages of nodule development, whereas ccs52A transcripts accumulated in lotus nodule primordia and, to a lesser extent, in mature nodules. Nodule development in Lupinus albus involved a progressive increase in nuclear and cellular size and ploidy level; similarly, Lotus japonicus nodules contained polyploid nuclei and enlarged cells in the infected zone. Nevertheless, in situ hybridization experiments showed the highest ccs52A expression in the inner cortex cells of the lupin nodule primordium, probably associated to the increased size of these cells in mature nodules. In view of our results, Ccs52A-mediated endoreduplication appears to be a universal mechanism required for nodule cell differentiation during the establishment of nitrogen-fixing symbioses.

Transgenic proteoid roots of white lupin: a vehicle for characterizing and silencing root genes involved in adaptation to P stress.

White lupin (Lupinus albus L.) has become an illuminating model for the study of plant adaptation to phosphorus (P) deficiency. It adapts to -P stress with a highly coordinated modification of root development and biochemistry resulting in short, densely clustered secondary roots called proteoid (or cluster) roots. In order to characterize genes involved in proteoid root formation and function in a homologous system, we have developed an Agrobacterium rhizogenes-based transformation system for white lupin roots that allows rapid analysis of reporter genes as well as RNA interference (RNA(i))-based gene silencing. We used this system to characterize a lupin multidrug and toxin efflux (Lupinus albus MULTIDRUG AND TOXIN EFFLUX, LaMATE) gene previously shown to have enhanced expression under -P stress. Here, we show that LaMATE had high expression in proteoid roots not only under -P, but also under -Fe, -N, -Mn and +Al stress. A portion containing the putative LaMATE promoter was fused to GUS and enhanced green fluorescence protein (EGFP) reporter genes, and a translational LaMATE::EGFP fusion was constructed under control of the LaMATE promoter. The LaMATE promoter directed P-dependent GUS and EGFP expression to proteoid roots. Confocal microscopy in white lupin and Arabidopsis point to the plasma membrane as the likely location of the LaMATE protein. LaMATE displayed homology to FRD3 in Arabidopsis, but did not complement an Arabidopsis ferric reductase defective 3 (FRD3) mutant. RNA(i)-based gene silencing was shown to effectively reduce LaMATE expression in transformed white lupin roots. LaMATE RNAi-silenced plants displayed an about 20% reduction in dry weight.

A systemic small RNA signaling system in plants.

Systemic translocation of RNA exerts non-cell-autonomous control over plant development and defense. Long-distance delivery of mRNA has been proven, but transport of small interfering RNA and microRNA remains to be demonstrated. Analyses performed on phloem sap collected from a range of plants identified populations of small RNA species. The dynamic nature of this population was reflected in its response to growth conditions and viral infection. The authenticity of these phloem small RNA molecules was confirmed by bioinformatic analysis; potential targets for a set of phloem small RNA species were identified. Heterografting studies, using spontaneously silencing coat protein (CP) plant lines, also established that transgene-derived siRNA move in the long-distance phloem and initiate CP gene silencing in the scion. Biochemical analysis of pumpkin (Cucurbita maxima) phloem sap led to the characterization of C. maxima Phloem SMALL RNA BINDING PROTEIN1 (CmPSRP1), a unique component of the protein machinery probably involved in small RNA trafficking. Equivalently sized small RNA binding proteins were detected in phloem sap from cucumber (Cucumis sativus) and lupin (Lupinus albus). PSRP1 binds selectively to 25-nucleotide single-stranded RNA species. Microinjection studies provided direct evidence that PSRP1 could mediate the cell-to-cell trafficking of 25-nucleotide single-stranded, but not double-stranded, RNA molecules. The potential role played by PSRP1 in long-distance transmission of silencing signals is discussed with respect to the pathways and mechanisms used by plants to exert systemic control over developmental and physiological processes.