Irrigation affects characteristics of narrow-leaved lupin (Lupinus angustifolius L.) seeds.

MAIN CONCLUSION: While plant irrigation usually increases yield, irrigation also affects seed characteristics with respect to endoreplication level, chemical composition, number of carbonyl bands, and cuticular wax profiles. Seeds of sweet varieties of the narrow-leaved lupin have good nutritional properties; however, these plants are sensitive to water deficit. Irrigation improves lupin yield, but can affect seed characteristics. The purpose of the study was to evaluate irrigation influence on lupin seed features and their chemical composition. Morphological analyses showed worse quality of seeds from the irrigated plants, with regard to their size and weight. This was confirmed by cytophotometric analyses which revealed a lower DNA content in the nuclei of cells from the apical and basal regions of the irrigated seeds. The lower degree of polyploidy of the nuclei entails lower cell sizes and limited space for storage components. Fourier transform infrared spectroscopic analysis demonstrated that protein and cuticular wax profiles of the irrigated seeds were different from the control. The electrophoretic analyses indicated differences in protein profiles including changes in the proportion of lupin storage proteins. Among the various studied elements, only the nitrogen content decreased in the embryo axis of irrigated plants. Although germination dynamics of the irrigated seeds was higher, the seedlings' development rate was slightly lower than in the control. The hydrogen peroxide level in root meristem cells was higher during germination in the control suggesting its regulatory role in seed metabolism/signaling. Our study indicated that irrigation of lupin plant affected seed features and composition.

Confocal laser scanning microscopy elucidation of the micromorphology of the leaf cuticle and analysis of its chemical composition.

Electron microscopy techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM) have been invaluable tools for the study of the micromorphology of plant cuticles. However, for electron microscopy, the preparation techniques required may invariably introduce artefacts in cuticle preservation. Further, there are a limited number of methods available for quantifying the image data obtained through electron microscopy. Therefore, in this study, optical microscopy techniques were coupled with staining procedures and, along with SEM were used to qualitatively and quantitatively assess the ultrastructure of plant leaf cuticles. Leaf cryosections of Triticum aestivum (wheat), Zea mays (maize), and Lupinus angustifolius (lupin) were stained with either fat-soluble azo stain Sudan IV or fluorescent, diarylmethane Auramine O and were observed under confocal laser scanning microscope (CLSM). For all the plant species tested, the cuticle on the leaf surfaces could be clearly resolved in many cases into cuticular proper (CP), external cuticular layer (ECL), and internal cuticular layer (ICL). Novel image data analysis procedures for quantifying the epicuticular wax micromorphology were developed, and epicuticular waxes of L. angustifolius were described here for the first time. Together, application of a multifaceted approach involving the use of a range of techniques to study the plant cuticle has led to a better understanding of cuticular structure and provides new insights into leaf surface architecture.

Diversity of selected Lupinus angustifolius L. genotypes at the phenotypic and DNA level with respect to microscopic seed coat structure and thickness.

The paper investigates seed coat characteristics (as a percentage of overall seed diameter) in Lupinus angustifolius L., a potential forage crop. In the study ten L. angustifolius genotypes, including three Polish cultivars, two Australian cultivars, three mutants originated from cv. 'Emir', and one Belarusian and one Australian breeding line were evaluated. The highest seed coat percentage was recorded in cultivars 'Sonet' and 'Emir'. The lowest seed coat thickness percentage (below 20%) was noted for breeding lines 11257-19, LAG24 and cultivar 'Zeus' (17.87%, 18.91% 19.60%, respectively). Despite having low seed weight, the Australian line no. 11257-19 was characterized by a desirable proportion of seed coat to the weight of seeds. In general, estimation of the correlation coefficient indicated a tendency that larger seeds had thinner coats. Scanning Electron Microscopy images showed low variation of seed coat sculpture and the top of seeds covered with a cuticle. Most of the studied genotypes were characterized by a cristatepapillate seed coat surface, formed by elongated polygonal cells. Only breeding line no. 11267-19 had a different shape of the cells building the surface layer of the coat. In order to illustrate genetic diversity among the genotypes tested, 24 ISSR primers were used. They generated a total of 161 polymorphic amplification products in 10 evaluated narrow-leaved lupin genotypes.

Copper microlocalisation and changes in leaf morphology, chloroplast ultrastructure and antioxidative response in white lupin and soybean grown in copper excess.

The microlocalisation of Cu was examined in the leaves of white lupin and soybean grown hydroponically in the presence of 1.6 (control) or 192 µM (excess) Cu, along with its effect on leaf morphology, (ultra)structure and the antioxidative response. The 192 µM dose led to a reduction in the total leaf area and leaf thickness in both species, although more strongly so in white lupin. In the latter species it was also associated with smaller spongy parenchyma cells, and smaller spaces between them, while in the soybean it more strongly reduced the size of the palisade parenchyma and epidermal cells. Energy-dispersive X-ray microanalysis showed that under Cu excess the metal was mainly localised inside the spongy parenchyma cells of the white lupin leaves, and in the lower epidermis cell walls in those of the soybean. Cu excess also promoted ultrastructural chloroplast alterations, reducing the photosynthetic capacity index and the green area of the leaves, especially in the soybean. Despite this, soybean appeared to be more tolerant to Cu excess than white lupin, because soybean displayed (1) lower accumulation of Cu in the leaves, (2) enhanced microlocalisation of Cu in the cell walls and (3) greater levels of induced total -SH content and superoxide dismutase and catalase activities that are expected for better antioxidative responses.

The perichromatin region of the plant cell nucleus is the area with the strongest co-localisation of snRNA and SR proteins.

The spatial organisation of the splicing system in plant cells containing either reticular (Allium cepa) or chromocentric (Lupinus luteus) nuclei was studied by immunolabelling of SR proteins, snRNA, and the PANA antigen, known markers for interchromatin granule clusters in mammalian cells. Electron microscope results allowed us to determine the distribution of these molecules within the structural domains of the nucleus. Similar to animal cells, in both plant species SR proteins were localised in interchromatin granules, but contrary to animal cells contained very small amounts of snRNA. The area with the strongest snRNA and SR protein co-localisation was the perichromatin region, which may be the location of pre-mRNA splicing in the plant cell nuclei. The only observable differences in the organisation of reticular and chromocentric nuclei were the size of the speckles and the number of snRNA pools in the condensed chromatin. We conclude that, despite remarkable changes in the nuclear architecture, the organisation of the splicing system is remarkably similar in both types of plant cell nuclei.

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.

Alterations induced by glyphosate on lupin photosynthetic apparatus and nodule ultrastructure and some oxygen diffusion related proteins.

The effects of glyphosate on protein metabolism, mesophyll cell ultrastructure and nodule ultrastructure and functioning of Lupinus albus cv. Multolupa inoculated with Bradyrhizobium sp. (Lupinus) were investigated. Young leaves and nodules were especially affected because these organs act as sinks of the herbicide. The alterations on nodular and chloroplast ultrastructure varied depending on herbicide concentration and time of exposure. After 3 days of 2.5 mM glyphosate application some toxic effects were detected. The most important alterations on nodules were the progressive cellular degradation of plant and bacteroidal cytosol and the rupture of bacteroidal membrane, whilst the peribacteroid membrane of the symbiosomes was preserved. This is the first report on the effect of glyphosate on legume-nodule ultrastructure. Glyphosate inhibited B. sp. (Lupinus) growth at concentrations higher than 62.5 microM. In the mesophyll cells, gradual disorganization of grana and intergrana was observed, loosing the parallel alignment with the chloroplast axis. As in nodules, degradation of membrane systems was observed, with the deformation, and even the rupture, of the tonoplast. These progressive effects were similar to those described in senescence processes. The adverse effects produced on infected zone can be due both to a direct effect of the herbicide on microsymbiont and to an indirect effect of glyphosate action on photosynthetic apparatus. Glyphosate produced changes in nodule cytosol and bacteroid proteins content and polypeptide pattern of leaves and nodules. With respect to proteins related to the oxygen diffusion mechanism, a large decrease in leghemoglobin and glycoproteins (recognized by antibodies MAC236 and MAC265) content was detected, which suggests that the oxygen diffusion mechanisms were also affected by glyphosate.

Distribution of chlorophyll-bearing organelles in the shoot apex of a range of dicotyledonous plants.

Confocal laser scanning microscopy was used to study the distribution of the smallest detectable autofluorescing, chlorophyll-bearing structures in fresh, 40 microm thick longitudinal sections of the shoot apex of four dicotyledonous plants (Arabidopsis thaliana, Nicotiana glauca, Lupinus alba, and Spinacia oleracea). In all species, the smallest chlorophyll-bearing particles were found in the outermost cell layers (L1 and L2) of the shoot apex. Their distribution between these layers differed in each species. The smallest such particles were about 0.5-1.0 microm in maximum dimension, approximating the size of a single granum in the developing leaf. Their size and abundance increased with increasing cell age and distance from the peak of the apex. Immediately beneath the L1 and L2 layers was a zone largely devoid of these particles. Below this nonfluorescing zone, in the region where the derivatives of the meristematic zone differentiate into cells of the central pith region, the size and abundance of the chlorophyll-bearing particles increased progressively with increasing distance from the nonfluorescing zone. The presence of these small autofluorescing particles in the L1 and L2 cell layers of the shoot apex places the development of photosystem II fluorescence at an earlier stage of leaf development than previously observed. The use of confocal laser scanning microscopy to study unfixed sections provides another useful metabolic marker for mapping patterns of differentiation and development in the cells of the shoot apex.

Metabolic and ultrastructural responses of lupine embryo axes to sugar starvation.

Embryo axes isolated from germinating lupine seeds were cultivated in vitro for 24-96 h over media containing either 60 mmol/L sucrose or no sucrose. Ultrastructural studies showed that large vacuoles were accumulating in a central region of primary parenchyma cells in sucrose starved lupine embryo axes, whereas cytoplasm along with organelles were forced to a periphery of the cells. We suggest that the autolysis of cytoplasmic proteins contributes to the accumulation of the vacuoles and this suggestion is consistent with the results of the characterisation of protein content. The level of cytosolic proteins was reduced by 50% and the activity of cytosolic marker enzyme, PEP carboxylase, was reduced by 46% in starved embryos as compared to control. The mitochondria from starved tissues were not degraded. The level of mitochondrial proteins was reduced by only 10% and the activity of mitochondrial NAD-isocitrate dehydrogenase decreased by 8% as a result of starvation. As demonstrated by the results of Percoll density gradient centrifugation, sucrose starvation caused an increase of 49% in many of the higher density mitochondria fractions, whereas many of the lower density mitochondria fractions were decreased by 33%. The samples of mitochondria from starved embryo axes were determined to have higher respiration activity in the presence of glutamate and malate as compared to control samples. EPR-based analyses of free radicals showed the presence of free radicals with a signal at g = 2.0060 in embryo axes. The level of the radical was two times higher in sucrose-starved embryo axes than in control (the level of this radical increased in senescing plant tissues as well). The results of EPR-based quantitation of Mn2+ ions revealed that the level was a few times higher in starved material than in control. Starved embryo axes, however, do possess a number of adaptive mechanisms protecting them from oxidative damage. Densitometric analyses of gels revealed an increase in the activity of SOD in sugar-starved embryos, whereas CAT and POX activities were lower in axes grown without sucrose as compared to control. Superoxide dismutase, catalase and peroxidase zymogram analyses showed that synthesis of new isoforms was not induced by sugar starvation. An accumulation of phytoferritin was found in plastids of sucrose starved embryos. These results are discussed in relation to the metabolic changes observed in senescing plant tissues.