Structure, ultrastructure and cation accumulation in quinoa epidermal bladder cell complex under high saline stress.

Quinoa is a facultative halophyte with excellent tolerance to salinity. In this study, the epidermal bladder cell complex (EBCc) of quinoa leaves was studied to determine their cellular characteristics and involvement in salt tolerance. We used light microscopy, confocal RAMAN microscopy, confocal fluorescence microscopy, transmission electron microscopy, and environmental scanning electron microscopy complemented by energy dispersive X-ray analysis. Ionic content was quantified with flame atomic absorption spectroscopy and with flame emission photometry. Results show that: (i) the number of EBCcs remains constant but their density and area vary with leaf age; (ii) stalk cells store lipids and exhibit thick walls, bladder cells present carotenes in small vesicles, oxalate crystals in vacuoles and lignin in their walls and both stalk and bladder cells have cuticles that differ in wax and cutin content; (iii) chloroplasts containing starch can be found on both stalk and bladder cells, and the latter also presents grana; (iv) plasmodesmata are observed between the stalk cell and the bladder cell, and between the epidermal cell and the stalk cell, and ectodesmata-like structures are observed on the bladder cell. Under high salinity conditions, (v) there is a clear tendency to accumulate greater amounts of K+ with respect to Na+ in the bladder cell; (vi) stalk cells accumulate similar amounts of K+ and Na+; (vii) Na+ accumulates mainly in the medullary parenchyma of the stem. These results add knowledge about the structure, content, and role of EBCc under salt stress, and surprisingly present the parenchyma of the stem as the main area of Na+ accumulation.

Snapshot of four mature quinoa (Chenopodium quinoa) seeds: a shotgun proteomics analysis with emphasis on seed maturation, reserves and early germination.

Chenopodium quinoa Willd. is a crop species domesticated over 5000 years ago. This species is highly diverse, with a geographical distribution that covers more than 5000 km from Colombia to Chile, going through a variety of edaphoclimatic conditions. Quinoa grains have great nutritional quality, raising interest at a worldwide level. In this work, by using shotgun proteomics and in silico analysis, we present an overview of mature quinoa seed proteins from a physiological context and considering the process of seed maturation and future seed germination. For this purpose, we selected grains from four contrasting quinoa cultivars (Amarilla de Maranganí, Chadmo, Sajama and Nariño) with different edaphoclimatic and geographical origins. The results give insight on the most important metabolic pathways for mature quinoa seeds including: starch synthesis, protein bodies and lipid bodies composition, reserves and their mobilization, redox homeostasis, and stress related proteins like heat-shock proteins (HSPs) and late embryogenesis abundant proteins (LEAs), as well as evidence for capped and uncapped mRNA translation. LEAs present in our analysis show a specific pattern of expression matching that of other species. Overall, this work presents a complete snapshot of quinoa seeds physiological context, providing a reference point for further studies. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01295-8.

Beyond Arabidopsis: Differential UV-B Response Mediated by UVR8 in Diverse Species.

Ultraviolet-B radiation (UV-B, 280-315 nm) is an important environmental signal that regulates growth and development in plants. Two dose-dependent UV-B response pathways were described in plants: a specific one, mediated by UVR8 (the specific UV-B receptor) and an unspecific one, activated by the oxidative damage produced by radiation. The constitutively expressed receptor appears inactive as a dimer, with the two monomers dissociating upon UV-B irradiation. The monomer then interacts with COP1, an ubiquitin ligase, hindering its ability to poly-ubiquitinate transcriptional factor HY5, thus averting its degradation and activating the photomorphogenic response. HY5 induces the synthesis of proteins RUP1 and RUP2, which interact with UVR8, releasing COP1, and inducing the re-dimerization of UVR8. This mechanism has been thoroughly characterized in Arabidopsis, where studies have demonstrated that the UVR8 receptor is key in UV-B response. Although Arabidopsis importance as a model plant many mechanisms described in this specie differ in other plants. In this paper, we review the latest information regarding UV-B response mediated by UVR8 in different species, focusing on the differences reported compared to Arabidopsis. For instance, UVR8 is not only induced by UV-B but also by other agents that are expressed differentially in diverse tissues. Also, in some of the species analyzed, proteins with low homology to RUP1 and RUP2 were detected. We also discuss how UVR8 is involved in other developmental and stress processes unrelated to UV-B. We conclude that the receptor is highly versatile, showing differences among species.

Deterioration of willow seeds during storage.

Willow (Salix spp.) seeds are able to tolerate desiccation, but differ from typical orthodox seeds in that they lose viability in a few days at room temperature, and in that the chloroplasts in embryo tissues do not dedifferentiate during maturation drying, thus retaining chlorophyll and maintaining intact their thylakoid membranes. In the present study, we investigated the damage generated in willow seeds during storage under appropriate conditions to exclude the eventual generation of reactive oxygen species by photooxidation. To this end, we measured different indicators of molecular damage, such as changes in the fatty acid profile, protein degradation, nuclease activities, and DNA damage, and evaluated normal germination and total germination in seeds stored for one, ten and sixteen years. We found: (i) a decrease in the fraction of unsaturated fatty acids; (ii) changes in the protein profile due to a decrease in protein solubility; (iii) activation of nucleases; and (iv) DNA fragmentation. Taken together, our findings identified programmed cell death as a key mechanism in seed deterioration during storage. We also found that, although the seeds maintained high percentages of total germination, the death program had already started in the seeds stored for ten years and was more advanced in those stored for sixteen years.

Death of embryos from 2300-year-old quinoa seeds found in an archaeological site.

In the 1970s, during excavations at Los Morrillos, San Juan, Argentina, quinoa seeds were found within ancient pumpkin crocks protected from the light and high temperatures, and preserved in the very dry conditions of the region. The radiocarbon dates confirmed the age of these seeds at around 2300 years. Sectioning of some of these seeds showed reddish-brown embryos, different from the white embryos of recently harvested quinoa seeds. The ancient seeds did not germinate. The structure of the embryo cells was examined using light and transmission electron microscopy; proteins were analyzed by electrophoresis followed by Coomassie blue and periodic acid Schiff staining and fatty acids by gas chromatography. The state of nuclear DNA was investigated by TUNEL assay, DAPI staining, ladder agarose electrophoresis and flow cytometry. Results suggest that, although the embryo tissues contained very low water content, death occurred by a cell death program in which heterochromatin density was dramatically reduced, total DNA was degraded into small fragments of less than 500bp, and some proteins were modified by non-enzymatic glycation, generating Maillard products. Polyunsaturated fatty acids decreased and became fragmented, which could be attributable to the extensive oxidation of the most sensitive species (linolenic and linoleic acids) and associated with a collapse of lipid bodies.

Immunoanalysis of dehydrins in Araucaria angustifolia embryos.

The aim of this study was to describe the dehydrin content of mature Araucaria angustifolia embryos, a species of endangered and economically important conifers, native to southern Brazil, northeastern Argentina, and eastern Paraguay. The A. angustifolia seeds have been categorized as recalcitrant. Dehydrins were studied by western blot analysis and in situ immunolocalization microscopy using antibodies raised against the K segment, a highly conserved lysine-rich 15-amino acid sequence extensively used to recognize proteins immunologically related to the dehydrin family. Western blot analysis of the heat-stable protein fraction, as estimated by 15 % SDS-PAGE, revealed three main bands of approximately 20-, 26-, and 29-kDa; when 17.5 % SDS-PAGE was used, each band resolved into two other bands. Two thermosensitive dehydrin bands of around 16 and 35 kDa were common to the axis and cotyledons, and another thermosensitive band, with molecular mass of approximately 10 kDa, was present in the cotyledons only. Following alkaline phosphatase (AP) treatment, a gel mobility shift was detected for each one of the four main bands that can be due to phosphorylation. Dehydrins were detected in all axis and cotyledon tissues using in situ immunolocalization microscopy. At the subcellular level, dehydrins were immunolocalized in the nuclei, protein bodies, and microbodies. In the nucleus, dehydrins were found to be associated with chromatin. We concluded that the gel mobility shift for the four main bands (probably due to phosphorylation), the presence of thermosensitive bands, and the specific localizations in nuclei and protein bodies provide key starting points to understand the function of dehydrins in the embryo cells of this species.

Accumulation pattern of dehydrins during sugarcane (var. SP80.3280) somatic embryogenesis.

UNLABELLED: The objective of the present study was to determine dehydrin protein levels in sugarcane var. SP80-3280 during somatic embryogenesis. Dehydrins from embryogenic and non-embryogenic cell cultures were analyzed using western blot and in situ immunolocalization microscopy. Both techniques employ antibodies raised against a highly conserved lysine-rich 15-amino acid sequence termed the K-domain, which is extensively used to recognize proteins immunologically related to the dehydrin family. In embryogenic cultures, western blot analysis of the heat-stable protein fraction revealed eleven major bands ranging from 52 to 17 kDa. They were already visible on the first days, gradually increasing until reaching peak values around day 14, when organogenesis begins, to later decrease in concurrence with the appearance of green plantlets (around day 28). These fluctuations indicate that this pattern of accumulation is under developmental control. Dehydrins were mainly immunolocalized in the nuclei. A phosphatase treatment of protein extracts caused a mobility shift of the 52, 49, and 43 kDa dehydrin bands suggesting a putative modulation mechanism based on protein phosphorylation. In sugarcane embryogenic cultures, presence of dehydrins is a novel finding. Dehydrins were absent in non-embryogenic cultures. The novel findings regarding accumulation, nuclear localization, and phosphorylation of dehydrins provide a starting point for further research on the role of these proteins in the induction and/or maintenance of embryogenesis. KEY MESSAGE: The novel findings regarding accumulation, nuclear localization, and phosphorylation of dehydrins provide a starting point for further research on the role of these proteins in the induction and/or maintenance of embryogenesis.

On the nature and origin of the oxalate package in Solanum sisymbriifolium anthers.

This is a detailed study carried out in Solanum sisymbriifolium Lam. on the development of the circular cell cluster (CCC) during crystal deposition, as well as the composition of the crystals. Light microscopy and scanning and transmission electron microscopy (TEM) were used to characterize tissue throughout anther development. Energy dispersive X-ray analysis (EDAX) allowed the determination of the elemental composition of crystals that form in the CCC region, and infrared and x-ray diffraction analysis were used to specify the crystal salt composition. TEM analysis revealed that the crystals originated simultaneously within the vacuoles in association with a paracrystalline protein. Prior to the appearance of protein within vacuoles, protein paracrystals were visible in both rough endoplasmic reticulum and vesicles with ribosomes on their membranes. In vacuoles, paracrystals constitute nucleation sites for druse crystals formation. EDAX revealed that C, O, and Ca were the main elements, and K, Cl, Mg, P, S, and Si, the minor elements. X-ray powder diffraction of crystals detected the predominant presence of calcium oxalate, but also vestiges of calcite, quartz, and sylvite. The calcium oxalate coexisted in the three chemical forms, that is, whewellite, weddellite, and caoxite. Infrared spectrophotometry identified bands that characterize O-C-O, H-O, C-H bonds, all of calcium oxalate, and Si-O-Si, of quartz. These results were compared with studies of anthers carried out in other Solanaceae genera.