Modifications in Tissue and Cell Ultrastructure as Elements of Immunity-Like Reaction in Chenopodium quinoa against Prune Dwarf Virus (PDV).

Prune dwarf virus (PDV) is a plant RNA viral pathogen in many orchard trees worldwide. Our knowledge about resistance genes or resistant reactions of plant hosts to PDV is scant. To fill in part of this gap, an aim of this study was to investigate reactions to PDV infection in a model host, Chenopodium quinoa. Our investigations concentrated on morphological and ultrastructural changes after inoculation with PDV strain 0599. It turned out that PDV infection can cause deformations in host cells but also induce changes in the organelles, such as chloroplasts in inoculated leaves. Moreover, we also demonstrated specific reactions/changes, which could be associated with both types of vascular tissue capable of effectively blocking the systemic spread of PDV to upper leaves. Furthermore, the relative amount of virus, P1 protein deposition, and movement protein (MP) gene expression consequently decreased in PDV-inoculated leaves.

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.

Mutual association of Broad bean wilt virus 2 VP37-derived tubules and plasmodesmata obtained from cytological observation.

The movement protein VP37 of broad bean wilt virus 2 (BBWV 2) forms tubules in the plasmodesmata (PD) for the transport of virions between cells. This paper reports a mutual association between the BBWV 2 VP37-tubule complex and PD at the cytological level as determined by transmission electron microscopy. The generation of VP37-tubules within different PD leads to a different occurrence frequency as well as different morphology lines of virus-like particles. In addition, the frequency of VP37-tubules was different between PD found at different cellular interfaces, as well as between single-lined PD and branched PD. VP37-tubule generation also induced structural alterations of PD as well as modifications to the cell wall (CW) in the vicinity of the PD. A structural comparison using three-dimensional (3D) electron tomography (ET), determined that desmotubule structures found in the center of normal PD were absent in PD containing VP37-tubules. Using gold labeling, modification of the CW by callose deposition and cellulose reduction was observable on PD containing VP37-tubule. These cytological observations provide evidence of a mutual association of MP-derived tubules and PD in a natural host, improving our fundamental understanding of interactions between viral MP and PD that result in intercellular movement of virus particles.

Cellular and molecular aspects of quinoa leaf senescence.

During leaf senescence, degradation of chloroplasts precede to changes in nuclei and other cytoplasmic organelles, RuBisCO stability is progressively lost, grana lose their structure, plastidial DNA becomes distorted and degraded, the number of plastoglobuli increases and abundant senescence-associated vesicles containing electronically dense particles emerge from chloroplasts pouring their content into the central vacuole. This study examines quinoa leaf tissues during development and senescence using a range of well-established markers of programmed cell death (PCD), including: morphological changes in nuclei and chloroplasts, degradation of RuBisCO, changes in chlorophyll content, DNA degradation, variations in ploidy levels, and changes in nuclease profiles. TUNEL reaction and DNA electrophoresis demonstrated that DNA fragmentation in nuclei occurs at early senescence, which correlates with induction of specific nucleases. During senescence, metabolic activity is high and nuclei endoreduplicate, peaking at 4C. At this time, TEM images showed some healthy nuclei with condensed chromatin and nucleoli. We have found that DNA fragmentation, induction of senescence-associated nucleases and endoreduplication take place during leaf senescence. This provides a starting point for further research aiming to identify key genes involved in the senescence of quinoa leaves.

Ricinosomes provide an early indicator of suspensor and endosperm cells destined to die during late seed development in quinoa (Chenopodium quinoa).

BACKGROUND AND AIMS: In mature quinoa (Chenopodium quinoa) seeds, the lasting endosperm forms a micropylar cone covering the radicle. The suspensor cells lie within the centre of the cone. During the final stage of seed development, the cells of the lasting endosperm accumulate protein and lipids while the rest are crushed and disintegrated. Both the suspensor and endosperm die progressively from the innermost layers surrounding the embryo and extending towards the nucellar tissue. Ricinosomes are endoplasmic reticulum-derived organelles that accumulate both the pro-form and the mature form of cysteine endopeptidase (Cys-EP), first identified in castor bean (Ricinus communis) endosperm during germination. This study sought to identify associations between the presence of ricinosomes and programmed cell death (PCD) hallmarks in suspensor and endosperm cells predestined to die during quinoa seed development. METHODS: A structural study using light microscopy and transmission electron microscopy was performed. To detect the presence of Cys-EP, both western blot and in situ immunolocalization assays were carried out using anti-R. communis Cys-EP antibody. A TUNEL assay was used to determine DNA fragmentation. RESULTS AND CONCLUSIONS: Except for the one or two cell layers that constitute the lasting endosperm in the mature seed, ricinosomes were found in suspensor and endosperm cells. These cells were also the site of morphological abnormalities, including misshapen and fragmented nuclei, vesiculation of the cytosol, vacuole collapse and cell wall disorganization. It is proposed that, in suspensor and endosperm cells, the early detection of Cys-EP in ricinosomes predicts the occurrence of PCD during late seed development.

Detection and subcellular localization of dehydrin-like proteins in quinoa (Chenopodium quinoa Willd.) embryos.

The aim of this study was to characterize the dehydrin content in mature embryos of two quinoa cultivars, Sajama and Baer La Unión. Cultivar Sajama grows at 3600-4000 m altitude and is adapted to the very arid conditions characteristic of the salty soils of the Bolivian Altiplano, with less than 250 mm of annual rain and a minimum temperature of -1 degrees C. Cultivar Baer La Unión grows at sea-level regions of central Chile and is adapted to more humid conditions (800 to 1500 mm of annual rain), fertile soils, and temperatures above 5 degrees C. Western blot analysis of embryo tissues from plants growing under controlled greenhouse conditions clearly revealed the presence of several dehydrin bands (at molecular masses of approximately 30, 32, 50, and 55 kDa), which were common to both cultivars, although the amount of the 30 and 32 kDa bands differed. Nevertheless, when grains originated from their respective natural environments, three extra bands (at molecular masses of approximately 34, 38, and 40 kDa), which were hardly visible in Sajama, and another weak band (at a molecular mass of approximately 28 kDa) were evident in Baer La Unión. In situ immunolocalization microscopy detected dehydrin-like proteins in all axis and cotyledon tissues. At the subcellular level, dehydrins were detected in the plasma membrane, cytoplasm and nucleus. In the cytoplasm, dehydrins were found associated with mitochondria, rough endoplasmic reticulum cisternae, and proplastid membranes. The presence of dehydrins was also recognized in the matrix of protein bodies. In the nucleus, dehydrins were associated with the euchromatin. Upon examining dehydrin composition and subcellular localization in two quinoa cultivars belonging to highly contrasting environments, we conclude that most dehydrins detected here were constitutive components of the quinoa seed developmental program, but some of them (specially the 34, 38, and 40 kDa bands) may reflect quantitative molecular differences associated with the adaptation of both cultivars to contrasting environmental conditions.