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This study investigated the influence of cadmium (Cd) and copper (Cu) heavy metals on germination, metabolism, and growth of zucchini seedlings (Cucurbita pepo L.). Zucchini seeds were subjected to two concentrations (100 and 200 µM) of CdCl2 and CuCl2. Germination parameters, biochemical and phytochemical attributes of embryonic axes were assessed. Results revealed that germination rate remained unaffected by heavy metals (Cd, Cu). However, seed vigor index (SVI) notably decreased under Cd and Cu exposure. Embryonic axis length and dry weight exhibited significant reductions, with variations depending on the type of metal used. Malondialdehyde and H2O2 content, as well as catalase activity, did not show a significant increase at the tested Cd and Cu concentrations. Superoxide dismutase activity decreased in embryonic axis tissues. Glutathione S-transferase activity significantly rose with 200 µM CdCl2, while glutathione content declined with increasing Cd and Cu concentrations. Total phenol content and antioxidant activity increased at 200 µM CuCl2. In conclusion, Cd and Cu heavy metals impede zucchini seed germination efficiency and trigger metabolic shifts in embryonic tissue cells. Response to metal stress is metal-specific and concentration-dependent. These findings contribute to understanding the intricate interactions between heavy metals and plant physiology, aiding strategies for mitigating their detrimental effects on plants.

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Modifications of DNA nucleobases are present in all forms of life. The purpose of these modifications in eukaryotic cells, however, is not always clear. Although the role of 5-methylcytosine (m5C) in epigenetic regulation and the maintenance of stability in plant genomes is becoming better understood, knowledge pertaining to the origin and function of oxidized nucleobases is still scarce. The formation of 5-hydroxymetylcytosine (hm5C) in plant genomes is especially debatable. DNA modifications, functioning as regulatory factors or serving as DNA injury markers, may have an effect on DNA structure and the interaction of genomic DNA with proteins. Thus, these modifications can influence plant development and adaptation to environmental stress. Here, for the first time, the changes in DNA global levels of m5C, hm5C, and 8-oxo-7,8-dihydroguanine (8-oxoG) measured by ELISA have been documented in recalcitrant embryonic axes subjected to desiccation and accelerated aging. We demonstrated that tissue desiccation induces a similar trend in changes in the global level of hm5C and 8-oxoG, which may suggest that they both originate from the activity of reactive oxygen species (ROS). Our study supports the premise that m5C can serve as a marker of plant tissue viability whereas oxidized nucleobases, although indicating a cellular redox state, cannot.

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Seeds are one of the preferable and most used sources of germplasm for the ex situ preservation of plant genetic resources. They are generally stored dry at -20 °C in seed banks following international standards. However, some seeds do not tolerate drying and/or storage at -20 °C, or present short lifespans at these conditions. For them cryopreservation is indicated for long-term preservation. When seeds tolerate desiccation (i.e., orthodox seeds), they can be dried at about 32 ± 3% relative humidity at 18 °C and stored in the vapor phase of liquid nitrogen. This is the method followed in the Millennium Seed Bank of the Royal Botanic Gardens, Kew, for wild species with short lifespans in the standard conditions of seed banks. When seeds do not tolerate desiccation (i.e., recalcitrant seeds) or their tolerance to desiccation and/or -20 °C storage is limited (i.e., intermediate seeds), drying and cooling procedures must be adjusted, and often, cryoprotection is also required. Some methods are detailed for diverse species of temperate and tropical origin.

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A long standing axiom in the field of teratology states that the teratogenic period, when most birth defects are produced, occurs during the third to eighth weeks of development post-fertilization. Any insults prior to this time are thought to result in a slowing of embryonic growth from which the conceptus recovers or death of the embryo followed by spontaneous abortion. However, new insights into embryonic development during the first two weeks, including formation of the anterior-posterior, dorsal-ventral, and left-right axes, suggests that signaling pathways regulating these processes are prime targets for genetic and toxic insults. Establishment of the left-right (laterality) axis is particularly sensitive to disruption at very early stages of development and these perturbations result in a wide variety of congenital malformations, especially heart defects. Thus, the time for teratogenic insults resulting in birth defects should be reset to include the first two weeks of development.

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Seed imbibition and radicle emergence are generally less affected by salinity in soybean than in other crop plants. In order to unveil the mechanisms underlying this remarkable salt tolerance of soybean at seed germination, a comparative label-free shotgun proteomic analysis of embryonic axes exposed to salinity during germination sensu stricto (GSS) was conducted. The results revealed that the application of 100 and 200 mmol/L NaCl stress was accompanied by significant changes (>2-fold, P<0.05) of 97 and 75 proteins, respectively. Most of these salt-responsive proteins (70%) were classified into three major functional categories: disease/defense response, protein destination and storage and primary metabolism. The involvement of these proteins in salt tolerance of soybean was discussed, and some of them were suggested to be potential salt-tolerant proteins. Furthermore, our results suggest that the cross-protection against aldehydes, oxidative as well as osmotic stress, is the major adaptive response to salinity in soybean.

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BACKGROUND AND AIMS: Conservation of the genetic diversity afforded by recalcitrant seeds is achieved by cryopreservation, in which excised embryonic axes (or, where possible, embryos) are treated and stored at temperatures lower than -180 °C using liquid nitrogen. It has previously been shown that intracellular ice forms in rapidly cooled embryonic axes of Acer saccharinum (silver maple) but this is not necessarily lethal when ice crystals are small. This study seeks to understand the nature and extent of damage from intracellular ice, and the course of recovery and regrowth in surviving tissues. METHODS: Embryonic axes of A. saccharinum, not subjected to dehydration or cryoprotection treatments (water content was 1·9 g H2O g(-1) dry mass), were cooled to liquid nitrogen temperatures using two methods: plunging into nitrogen slush to achieve a cooling rate of 97 °C s(-1) or programmed cooling at 3·3 °C s(-1). Samples were thawed rapidly (177 °C s(-1)) and cell structure was examined microscopically immediately, and at intervals up to 72 h in vitro. Survival was assessed after 4 weeks in vitro. Axes were processed conventionally for optical microscopy and ultrastructural examination. KEY RESULTS: Immediately following thaw after cryogenic exposure, cells from axes did not show signs of damage at an ultrastructural level. Signs that cells had been damaged were apparent after several hours of in vitro culture and appeared as autophagic decomposition. In surviving tissues, dead cells were sloughed off and pockets of living cells were the origin of regrowth. In roots, regrowth occurred from the ground meristem and procambium, not the distal meristem, which became lethally damaged. Regrowth of shoots occurred from isolated pockets of surviving cells of peripheral and pith meristems. The size of these pockets may determine the possibility for, the extent of and the vigour of regrowth. CONCLUSIONS: Autophagic degradation and ultimately autolysis of cells following cryo-exposure and formation of small (0·2-0·4 µm) intracellular ice crystals challenges current ideas that ice causes immediate physical damage to cells. Instead, freezing stress may induce a signal for programmed cell death (PCD). Cells that form more ice crystals during cooling have faster PCD responses.

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Plant nucleases are involved in nucleic acid degradation associated to programmed cell death processes as well as in DNA restriction, repair and recombination processes. However, the knowledge about the function of plant nucleases is limited. A major nuclease activity was detected by in-gel assay with whole embryonic axes of common bean by using ssDNA or RNA as substrate, whereas this activity was minimal in cotyledons. The enzyme has been purified to electrophoretic homogeneity from embryonic axes. The main biochemical properties of the purified enzyme indicate that it belongs to the S1/P1 family of nucleases. This was corroborated when this protein, after SDS-electrophoresis, was excised from the gel and further analysis by MALDI TOF/TOF allowed identification of the gene (PVN1) that codes this protein. The gene that codes the purified protein was identified. The expression of PVN1 gene was induced at the specific moment of radicle protrusion. The inclusion of inorganic phosphate to the imbibition media reduced the level of expression of this gene and the nuclease activity suggesting a relationship with the phosphorous status in French bean seedlings.

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BACKGROUND AND AIMS: Cryopreservation is the only long-term conservation strategy available for germplasm of recalcitrant-seeded species. Efforts to cryopreserve this form of germplasm are hampered by potentially lethal intracellular freezing events; thus, it is important to understand the relationships among cryo-exposure techniques, water content, structure and survival. METHODS: Undried embryonic axes of Acer saccharinum and those rapidly dried to two different water contents were cooled at three rates and re-warmed at two rates. Ultrastructural observations were carried out on radicle and shoot tips prepared by freeze-fracture and freeze-substitution to assess immediate (i.e. pre-thaw) responses to cooling treatments. Survival of axes was assessed in vitro. KEY RESULTS: Intracellular ice formation was not necessarily lethal. Embryo cells survived when crystal diameter was between 0·2 and 0·4 µm and fewer than 20 crystals were distributed per µm(2) in the cytoplasm. Ice was not uniformly distributed within the cells. In fully hydrated axes cooled at an intermediate rate, the interiors of many organelles were apparently ice-free; this may have prevented the disruption of vital intracellular machinery. Intracytoplasmic ice formation did not apparently impact the integrity of the plasmalemma. The maximum number of ice crystals was far greater in shoot apices, which were more sensitive than radicles to cryo-exposure. CONCLUSIONS: The findings challenge the accepted paradigm that intracellular ice formation is always lethal, as the results show that cells can survive intracellular ice if crystals are small and localized in the cytoplasm. Further understanding of the interactions among water content, cooling rate, cell structure and ice structure is required to optimize cryopreservation treatments without undue reliance on empirical approaches.

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The plant cell wall is a dynamic structure whose constant modification is necessary for plant cells to grow and divide. In the cell walls of chickpea (Cicer arietinum) there are at least four ß-galactosidases, whose presence and location in embryonic axes during the first 48 h of seed imbibition are discussed in this paper. We examined their roles as cell wall-modifying enzymes in germinative and/or post-germinative events. At the start of germination, only ßV-Gal, and to a lesser extent ßIV-Gal, appear in the axes before rupture of the testa, suggesting they are related to germination sensu stricto. Once the testa has broken, the four ß-galactosidases are involved in growth and differentiation of the axes. Immunolocation of the different proteins in axes, which in part confirms previous results in seedlings and plants, allows assignment of post-germinative roles to ßI-Gal and ßIII-Gal as cell wall modifiers in vascular tissue elements. ßIV-Gal and ßV-Gal participate in the initial events of germination in which cell walls are involved: ßV-Gal in cell proliferation, detachment of root cap cells and initial vascular tissue differentiation; both of them in xylem maturation; and ßIV-Gal in thickening of the primary cell wall. Together with other cell wall-modifying enzymes, such as expansins and XTH, chickpea galactosidases might function in a sequential order in turnover of the primary cell wall, allowing the elongation of embryonic axes during seed germination.

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A cell-free translation system was prepared from 3- to 5-day-old embryonic axes of gherkin (Cucumis sativus L.). The system was optimized for Mg2+ , K+ , NH+4 , high speed supernatants, tRNA mixture from wheat germ, time and temperature. The system translates efficiently both endogenous mRNA (using a 30000 g supernatant) and polyuridylic acid (using either a 30000 g supernatant or a 100000 g supernatant supplemented with purified ribosomes). Translation by gherkin ribosomes was inhibited by several well-known eukaryotic inhibitors, antibiotics and ribosome-inactivating proteins. A translational inhibitory activity found in Cucumis sativus L. dry seeds acted on polypeptide synthesis carried out by cell-free systems from several mammals and plants, including gherkin embryonic axes. Our results indicate that the inhibitor is located in the seed bark and cotyledons, and is either blocked or absent in the embryonic axes, thus allowing the isolation of active gherkin ribosomes. The presence of the putative inhibitor appeared to be unevenly distributed in developing plants.