Sara Maldonado

Patterns of protein and carbohydrate accumulation during somatic embryogenesis of Acca sellowiana

Abstract –The aim of this work was to quantify the protein, starch and total sugars levels during histodifferentiation and development of somatic embryos of Acca sellowiana Berg. For histological observations, the samples were dehydrated in a battery of ethanol, embedded in historesin and stained with toluidine blue (morphology), coomassie blue (protein bodies) and periodic acid–Schiff (starch). Proteins were extracted using a buffer solution, precipitated using ethanol and quantified using the Bradford reagent. Total sugars were extracted using a methanol-chloroform-water (12:5:3) solution and quantified by a reaction with anthrone at0.2%. Starch was extracted using a 30% perchloric acid solution and quantified by a reaction with anthrone at0.2%. During the somatic embryogenesis’ in vitro morphogenesis and differentiation processes, the total protein levels decreased and the soluble sugars levels increased during the first 30days in culture and remained stable until the 120 th day. On the other hand, total protein levels increased according to the progression in the developmental stages of the somatic embryos. The levels of total sugars and starch increased in the heart and cotyledonary stages, and decreased in the torpedo and pre-cotyledonary stages. These compounds play a central role in the development of somatic embryos of

Detection of dehydrin-like proteins in embryos and endosperm of mature Euterpe edulis seeds

Summary.Euterpe edulis Martius, a tropical palm species characterized as highly recalcitrant, accumulated dehydrin proteins in both the endosperm and the embryo of the mature seed, as detected by Western blot analysis and immunogold electron microscopy. Three major bands at molecular masses of approximately 16, 18, and 24 kDa were identified in both samples analysed. Immunogold electron microscopy studies detected the presence of dehydrins in the embryo and endosperm. In both cases, dehydrins were immunolocalized in cytoplasm and chromatin. No labelling associated with either membranes or organelles was detected. It is known that dehydrins are produced as part of the developmental program of orthodox seeds and are also present in some recalcitrant seeds of temperate regions. The constitutive presence of dehydrins in embryos of extremely recalcitrant species of tropical origin has not been previously reported.

Genomic and functional characterization of StCDPK1.

StCDPK1 is a calcium dependent protein kinase expressed in tuberizing potato stolons and in sprouting tubers. StCDPK1 genomic sequence contains eight exons and seven introns, the gene structure is similar to Arabidopsis, rice and wheat CDPKs belonging to subgroup IIa. There is one copy of the gene per genome and it is located in the distal portion of chromosome 12. Western blot and immunolocalization assays (using confocal and transmission electron microscopy) performed with a specific antibody against StCDPK1 indicate that this kinase is mainly located in the plasma membrane of swelling stolons and sprouting tubers. Sucrose (4–8%) increased StCDPK1 protein content in non-induced stolons, however the amount detected in swelling stolons was higher. Transgenic lines with reduced expression of StCDPK1 (β7) did not differ from controls when cultured under multiplication conditions, but when grown under tuber inducing conditions some significant differences were observed: the β7 line tuberized earlier than controls without the addition of CCC (GA inhibitor), developed more tubers than wild type plants in the presence of hormones that promote tuberization in potato (ABA and BAP) and was more insensitive to GA action (stolons were significantly shorter than those of control plants). StCDPK1 expression was induced by GA, ABA and BAP. Our results suggest that StCDPK1 plays a role in GA-signalling and that this kinase could be a converging point for the inhibitory and promoting signals that influence the onset of potato tuberization.

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

Summary.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 °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 °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.

High salinity induces dehydrin accumulation in Chenopodium quinoa Willd. cv. Hualhuas embryos

Background and AimsChenopodium quinoa can grow at altitudes of 3,600–4,000 masl and is adapted to the highly arid conditions typical of the salty soils in the South American Altiplano, with less than 250xa0mm of annual rain and temperatures below 0°C. The aim of the study was to investigate the effect of salinity on the dehydrin content of mature embryos harvested from salt-stressed Chenopodium quinoa cv. Hualhuas plants grown at 100 to 500xa0mM NaCl. To date, no studies exist on the dehydrins of seeds from salt-stressed plants, although dehydrins in the root, stems and leaves have been reported as an adaptation to water deficit produced by salinity.MethodsDehydrin-like protein detection was carried out with an antiserum raised against a highly-conserved lysine-rich 15-amino acid sequence known as the K-segment, which is capable of recognizing proteins immunologically related to the dehydrin family.ResultsDehydrins were analyzed in embryos by both western blot and in situ immunolocalization. Western blot analysis detected at least four dehydrins (55, 50, 34, and 30xa0kDa) in seeds harvested from quinoa salt-stressed plants treated under a wide range of salinities. The 30xa0kDa dehydrin increased its accumulation in both 300 and 500xa0mM NaCl growth conditions as revealed by densitometric analyses. Dehydrin subcellular localization was mostly nuclear at 500xa0mM of NaCl. A phosphatase treatment of protein extracts caused a mobility shift of the 34 and 30xa0kDa dehydrin bands suggesting a putative modulation mechanism based on protein phosphorylation.ConclusionsWe propose that these novel observations regarding dehydrin accumulation, subcellular localization and phosphorylation state are related to the high salt stress tolerant phenotype previously reported on this cultivar.

Tomato leaf spatial expression of stress-induced Asr genes

Asr1 and Asr2 are water stress-inducible genes belonging to the Asr gene family, which transcriptionally regulate a sugar transporter gene, at least in grape. Using an inxa0situ RNA hybridization methodology, we determined that, in basal conditions, expression of Asr2 in tomato leaves is detected in the phloem tissue, particularly in companion phloem cells. When plants are exposed to water stress, Asr2 expression is contained in companion cells but expands occasionally to mesophyll cells. In contrast, Asr1 transcript localization seems to be sparse in leaf vascular tissue under both non-stress and stress conditions. The occurrence of Asr transcripts precisely in companion cells is in accordance with the cell type specificity reported for hexose-transporter protein molecules in grape encoded by the only Asr-target gene known to date. The results are discussed in light of the reported scarcity of plasmodesmata between companion cells and the rest of leaf tissue in the family Solanaceae.

Toward establishing a morphological and ultrastructural characterization of proembryogenic masses and early somatic embryos of Araucaria angustifolia (Bert.) O. Kuntze

Somatic embryogenesis is a morphogenetic route useful for the study of embryonic development, as well as the large-scale propagation of endangered species, such as the Brazilian pine (Araucaria angustifolia). In the present study, we investigated the morphological and ultrastructural organization of A. angustifolia somatic embryo development by means of optical and electron microscopy. The proembryogenic stage was characterized by the proliferation of proembryogenic masses (PEMs), which are cellular aggregates composed of embryogenic cells (ECs) attached to suspensor-like cells (SCs). PEMs proliferate through three developmental stages, PEM I, II, and III, by changes in the number of ECs and SCs. PEM III-to-early somatic embryo (SE) transition was characterized by compact clusters of ECs growing out of PEM III, albeit still connected to it by SCs. Early SEs showed a dense globular embryonic mass (EM) and suspensor region (SR) connected by embryonic tube cells (TCs). By comparison, early somatic and zygotic embryos showed similar morphology. ECs are round with a large nucleus, nucleoli, and many cytoplasmic organelles. In contrast, TCs and SCs are elongated and vacuolated with cellular dismantling which is associated with programmed cell death of SCs. Abundant starch grains were observed in the TCs and SCs, while proteins were more abundant in the ECs. Based on the results of this study, a fate map of SE development in A. angustifolia is, for the first time, proposed. Additionally, this study shows the cell biology of SE development of this primitive gymnosperm which may be useful in evolutionary studies in this area.

Programmed Cell Death in Seeds of Angiosperms

During the diversification of angiosperms, seeds have evolved structural, chemical, molecular and physiologically developing changes that specially affect the nucellus and endosperm. All through seed evolution, programmed cell death (PCD) has played a fundamental role. However, examples of PCD during seed development are limited. The present review examines PCD in integuments, nucellus, suspensor and endosperm in those representative examples of seeds studied to date.

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 15xa0% SDS-PAGE, revealed three main bands of approximately 20-, 26-, and 29-kDa; when 17.5xa0% SDS-PAGE was used, each band resolved into two other bands. Two thermosensitive dehydrin bands of around 16 and 35xa0kDa were common to the axis and cotyledons, and another thermosensitive band, with molecular mass of approximately 10xa0kDa, 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.

The barley plastome mutant CL2 affects expression of nuclear and chloroplast housekeeping genes in a cell-age dependent manner

The barley plastome mutant CL2 (cytoplasmic line 2) carries a point mutation in the infA gene, a homologue of the bacterial gene for the conserved translation initiator factor 1 (IF1). The function of infA in plastids is not known. The mutation in CL2 leads to a temporal chlorophyll deficiency in the primary leaf blade that is normalised in the basal and middle parts during further development. We have compared the expression of selected nuclear and plastid genes in different parts of primary leaves of CL2 and wild-type and found no indication for an adverse effect of the mutation on plastidial transcription. We observed an enhanced expression of RpoTp (encoding the phage-type nuclear-encoded plastid RNA polymerase) suggested to be caused by retrograde plastid signalling. Decreased amounts of plastid rRNA in basal and top sections are in agreement with the idea that the mutation in infA leads to a time- and position-dependent defect of plastid translation that causes a delay in plastid development. The normalisation of the phenotype in the middle section of CL2 leaves correlates with wild-type levels of chloroplast 16S rRNA and RbcL and increased expression of plastid housekeeping genes. The normalisation was not observed in cells at the tip of CL2 leaves suggesting different ways of regulating chloroplast development in cells at the tip of primary barley leaves as compared with other leaf sections.