Ultrastructure of Protein Bodies and Plastids in Cotyledon of Nelumbo nucifera Gaertn.at Seed Germination

The present article deals mainly with the formation and dissolution of protein bodies and development of plastids in cotyledon cells of Nelumbo nucifera during seed germination. Electron microscopic studies reveal that protein bodies are formed after imbibition of the cotyledons before germination. They are produced through accumulation of protein material in small vacuoles delivered from the exudates of endoplasmic reticulum or by fragmentation of endoplasmic reticulum itself. In the period of germination, most of the material in the protein bodies dissolute and they coalesce with each other forming large vacuoles. The protein residue of the vacuoles condenses into small blocks with high electron density adhering to the tonoplast or freely floating in the vacuole. Thus, it suggests that the protein bodies of the germinating N. nucifera cotyledons are originated from vacuoles formed by endoplasmic reticulum. Part of the plastids found in cotyledonous cells of mature N. nucifera seeds exists as proplastids. They develop continuously after imbibition of the cotyledons. During the period of seed germination, many concentric lamellae are developed along the plastid membrane on which they later coalesce with the neighboring concentric lameUae forming loosely organized prolamellar bodies which condense into paracrystalline lattices. No ribosomes are present in the inter spaces of paracrystatline lattice. One to several prolamellar bodies can be developed in one plastid.     

<Emphasis Type="Italic">NnHSP17.5</Emphasis>, a cytosolic class II small heat shock protein gene from <Emphasis Type="Italic">Nelumbo nucifera</Emphasis>, contributes to seed germination vigor and seedling thermotolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

In plants, small heat shock proteins (sHSPs) are unusually abundant and diverse proteins involved in various abiotic stresses, but their functions in seed vigor remain to be fully explored. In this study, we report the isolation and functional characterization of a sHSP gene, NnHSP17.5, from sacred lotus (Nelumbo nucifera Gaertn.) in seed germination vigor and seedling thermotolerance. Sequence alignment and phylogenetic analysis indicate that NnHSP17.5 is a cytosolic class II sHSP, which was further supported by the cytosolic localization of the NnHSP17.5-YFP fusion protein. NnHSP17.5 was specifically expressed in seeds under normal conditions, and was strongly up-regulated in germinating seeds upon heat and oxidative stresses. Transgenic Arabidopsis seeds ectopically expressing NnHSP17.5 displayed enhanced seed germination vigor and exhibited increased superoxide dismutase activity after accelerated aging treatment. In addition, improved basal thermotolerance was also observed in the transgenic seedlings. Taken together, this work highlights the importance of a plant cytosolic class II sHSP both in seed germination vigor and seedling thermotolerance.     

Cytoplasmic HSP70 homologues of pea: differential expression in vegetative and embryonic organs

Eukaryotes express several cytoplasmic HSP70 genes, and their encoded proteins participate in diverse cellular processes. Three cDNAs encoding highly expressed cytoplasmic HSP70 homologues from Pisum sativum were cloned and characterized. They were designated PsHSP71.2, PsHSC71.0, and PsHSP70b. These HSP70 genes have different expression profiles in leaves: PsHSP71.2 is observed only in response to heat stress, PsHSC71.0 is present constitutively, and PsHSP70b is weakly constitutively expressed, but induced strongly in response to heat stress. In addition to being heat induced, the PsHSP71.2 mRNA is also expressed in zygotic, but not maternal organs of developing pea seeds, while PsHSC71.0 and PsHSP70b mRNAs are present in maternal and zygotic organs throughout seed development. Immunoblot analysis of parallel protein samples detects a 70 kDa polypeptide in all samples, and a 72 kDa polypeptide that corresponds to the PsHSP71.2 gene product is observed in cotyledons beginning at mid-maturation and in axes beginning between late maturation and desiccation. This polypeptide is not detected in the seed coat. The 72 kDa polypeptide remains abundant in both cotyledons and axes through germination, but declines substantially between 48 and 72 h after the onset of imbibition. Differential control of HSP70 expression during heat stress, seed maturation, and germination is consistent with the hypothesis that there are functional distinctions between cytoplasmic HSP70s.     

Changes in late embryogenesis abundant proteins and a small heat shock protein during storage of beech (Fagus sylvatica L.) seeds

Beech seed physiology, including the effect of stress proteins like late embryogenesis abundant (LEA) and small heat shock proteins (sHSP) on viability during storage, is not fully understood. Four lots of beech (Fagus sylvatica L.) seeds have been stored for 1, 4, 6 and 8 years at −10 °C and 8–9% moisture content (MC). Under these conditions, the germination capacity ranges from 81.5% to 100% in the youngest seeds. However, the seeds decrease in vigour with prolonged time of storage. Dehydrins and dehydrin-like proteins were identified both in cotyledons and embryonic axes of the dry stored seeds. In general, decreased contents of LEA proteins as well as reduced content of total soluble protein were detected during prolonged storage. The contents of soluble proteins in embryonic axes and nearly all detected dehydrins and dehydrin-like proteins were correlated with germination capacity. Moreover a sHSP with molecular mass of approximately 22 kDa was identified. The largest content of this protein was observed in the oldest seeds, especially in embryonic axes. The proteins identified may play a protective role during water deficit and storage.     

Gibberellin-like activity in cotyledons and embryonic axes during pea seed germination

Endogenous gibberellin-like activity was determined in dry pea seeds (Pisum sativum cv. Bördi), in cotyledons and axes of germinating pea seeds and also in excised cotyledons and axes. During the first two days of pea seed germination, neither the embryonic axes nor the cotyledons show a mutual influence on gibberellin activity, but this appears after 72–96 h of germination. The gibberellin-like activity m cotyledons and axes of germinating seeds increased during the same period, but it decreased in isolated axes and excised cotyledons.     

The diversification of plant cytosolic small heat shock proteins preceded the divergence of mosses.

A cDNA library was constructed with mRNA isolated from heat-stressed cell cultures of Funaria hygrometrica (Bryophyta, Musci, Funariaceae). cDNA clones encoding six cytosolic small heat shock proteins (sHSPs) were identified using differential screening. Phylogenetic analysis of these sHSP sequences with other known sHSPs identified them as members of the previously described higher plant cytosolic class I and II families. Four of the F. hygrometrica sHSPs are members of the cytosolic class I family, and the other two are members of the cytosolic class II family. The presence of members of the cytosolic I and II sHSP families in a bryophyte indicates that these gene families are ancient, and evolved at least 450 MYA. This result also indicates that the plant sHSP gene families duplicated much earlier than did the well-studied phytochrome gene family. Members of the cytosolic I and II sHSP families are developmentally regulated in seeds and flowers in higher plants. Our findings show that the two cytosolic sHSP families evolved before the appearance of these specialized structures. Previous analysis of angiosperm sHSPs had identified class- or family-specific amino acid consensus regions and determined that rate heterogeneity exists among the different sHSP families. The analysis of the F. hygrometrica sHSP sequences reveals patterns and rates of evolution distinct from those seen among angiosperm sHSPs. Some, but not all, of the amino acid consensus regions identified in seed plants are conserved in the F. hygrometrica sHSPs. Taken together, the results of this study illuminate the evolution of the sHSP gene families and illustrate the importance of including representatives of basal land plant lineages in plant molecular evolutionary studies.     

Immunomodulation of function of small heat shock proteins prevents their assembly into heat stress granules and results in cell death at sublethal temperatures

The conformational dynamism and aggregate state of small heat shock proteins (sHSPs) may be crucial for their functions in thermoprotection of plant cells from the detrimental effects of heat stress. Ectopic expression of single chain fragment variable (scFv) antibodies against cytosolic sHSPs was used as new tool to generate sHSP loss-of-function mutants by antibody-mediated prevention of the sHSP assembly in vivo . Anti-sHSP scFv antibodies transiently expressed in heat-stressed tobacco protoplasts were not only able to recognize the endogenous sHSPs but also prevented their assembly into heat stress granula (HSGs). Constitutive expression of the same scFv antibodies in transgenic plants did not alter their phenotype at normal growth temperatures, but their leaves turned yellow and died after prolonged stress at sublethal temperatures. Structural analysis revealed a regular cytosolic distribution of stress-induced sHSPs in mesophyll cells of stress-treated transgenic plants, whereas extensive formation of HSGs was observed in control cells. After prolonged stress at sublethal temperatures, mesophyll cells of transgenic plants suffered destruction of all cellular membranes and finally underwent cell death. In contrast, mesophyll cells of the stressed controls showed HSG disintegration accompanied by appearance of polysomes, dictyosomes and rough endoplasmic reticulum indicating normalization of cell functions. Apparently, the ability of sHSPs to assemble into HSGs as well as the HSG disintegration is a prerequisite for survival of plant cells under continuous stress conditions at sublethal temperatures.     

Changes in extreme high-temperature tolerance and activities of antioxidant enzymes of sacred lotus seeds

Sacred lotus (Nelumbo nucifera Gaertn. ‘Tielian’) seed is long-lived and extremely tolerant of high temperature. Water content of lotus and maize seeds was 0.103 and 0.129 g H2O [g DW] ?1, respectively. Water content, germination percentage and fresh weight of seedlings produced by surviving seeds gradually decreased with increasing treatment time at 100℃. Germination percentage of maize (Zea mays L. ‘Huangbaogu’) seeds was zero after they were treated at 100℃ for 15 min and that of lotus seeds was 13.5% following the treatment at 100℃ for 24 h. The time in which 50% of lotus and maize seeds were killed by 100℃ was about 14.5 h and 6 min, respectively. With increasing treatment time at 100℃, relative electrolyte leakage of lotus axes increased significantly, and total chlorophyll content of lotus axes markedly decreased. When treatment time at 100℃ was less than 12 h, subcellular structure of lotus hypocotyls remained fully intact. When treatment time at 100℃ was more than 12 h, plasmoly-sis gradually occurred, endoplasmic reticulum became unclear, nuclei and nucleoli broke down, most of mitochondria swelled, lipid granules accumulated at the cell periphery, and organelles and plas-molemma collapsed. Malondialdehyde (MDA) content of lotus axes and cotyledons decreased during 0-12 h of the treatment at 100℃ and then increased. By contrast, the MDA content of maize embryos and endosperms increased during 5-10 min of the treatment at 100℃ and then decreased slightly. For lotus seeds: (1) activities of superoxide dismutase (SOD) and glutathione reductase (GR) of axes and cotyledons and of catalase (CAT) of axes increased during the early phase of treatment at 100℃ and then decreased; and (2) activities of ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) of axes and cotyledons and of CAT of cotyledons gradually decreased with increasing treat-ment time at 100℃. For maize seeds: (1) activities of SOD and DHAR of embryos and endosperms and of GR of embryos increased during the early phase of the treatment at 100℃ and then decreased; and (2) activities of APX and CAT of embryos and endosperms and of GR of endosperms rapidly decreased with increasing treatment time at 100℃. With decrease in seed germination, activities of SOD, APX, CAT, GR and DHAR of axes and cotyledons of lotus seeds decreased slowly, and those of embryos and endosperms of maize seeds decreased rapidly.     

Changes in extreme high-temperature tolerance and activities of antioxidant enzymes of sacred lotus seeds

Sacred lotus (Nelumbo nucifera Gaertn. ‘Tielian’) seed is long-lived and extremely tolerant of high temperature. Water content of lotus and maize seeds was 0.103 and 0.129 g H₂O [g DW] ⁻¹, respectively. Water content, germination percentage and fresh weight of seedlings produced by surviving seeds gradually decreased with increasing treatment time at 100°C. Germination percentage of maize (Zea mays L. ‘Huangbaogu’) seeds was zero after they were treated at 100°Cfor 15 min and that of lotus seeds was 13.5% following the treatment at 100°C for 24 h. The time in which 50% of lotus and maize seeds were killed by 100°C was about 14.5 h and 6 min, respectively. With increasing treatment time at 100°C, relative electrolyte leakage of lotus axes increased significantly, and total chlorophyll content of lotus axes markedly decreased. When treatment time at 100°C was less than 12 h, subcellular structure of lotus hypocotyls remained fully intact. When treatment time at 100°C was more than 12 h, plasmolysis gradually occurred, endoplasmic reticulum became unclear, nuclei and nucleoli broke down, most of mitochondria swelled, lipid granules accumulated at the cell periphery, and organelles and plasmolemma collapsed. Malondialdehyde (MDA) content of lotus axes and cotyledons decreased during 0 −12 h of the treatment at 100°C and then increased. By contrast, the MDA content of maize embryos and endosperms increased during 5–10 min of the treatment at 100°C and then decreased slightly. For lotus seeds: (1) activities of superoxide dismutase (SOD) and glutathione reductase (GR) of axes and cotyledons and of catalase (CAT) of axes increased during the early phase of treatment at 100°C and then decreased; and (2) activities of ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) of axes and cotyledons and of CAT of cotyledons gradually decreased with increasing treatment time at 100°C. For maize seeds: (1) activities of SOD and DHAR of embryos and endosperms and of GR of embryos increased during the early phase of the treatment at 100°C and then decreased; and (2) activities of APX and CAT of embryos and endosperms and of GR of endosperms rapidly decreased with increasing treatment time at 100°C. With decrease in seed germination, activities of SOD, APX, CAT, GR and DHAR of axes and cotyledons of lotus seeds decreased slowly, and those of embryos and endosperms of maize seeds decreased rapidly.     

Biogenesis of Protein Bodies in Embryonic Axes of Soybean Seeds (Glycine max. cv. Enrei)

Protein bodies in embryonic axes of soybean seeds have inclusion structures containing phytin globoids. Biogenesis of the protein bodies during seed development was examined by transmission electron microscopy. Protein bodies in embryonic axes originated from central vacuoles. The central vacuole in embryonic axes subdivided into smaller vacuoles with internal membranous structure. Then the subdivided vacuoles were directly associated with rough endoplasmic reticulum (rER), and were filled with proteinaceous matrix from the peripheral region. The increase of matrix was simultaneous with accumulation of β-conglycinin estimated by SDS-polyacrylamide gel electrophoresis. Glycinin-rich granules that had been found in developing cotyledons were not observed in embryonic axes. After proteinaceous matrix filled the protein bodies, electron-transparent regions presumably surrounded by a single membrane appeared in the matrix. Phytin globoids were constructed in this internal structures of protein bodies as the final step of protein body formation.     

ZmHSP16.9, a cytosolic class I small heat shock protein in maize (Zea mays), confers heat tolerance in transgenic tobacco

Various organisms produce HSPs in response to high temperature and other stresses. The function of heat shock proteins, including small heat shock protein (sHSP), in stress tolerance is not fully explored. To improve our understanding of sHSPs, we isolated ZmHSP16.9 from maize. Sequence alignments and phylogenetic analysis reveal this to be a cytosolic class I sHSP. ZmHSP16.9 expressed in root, leaf and stem tissues under 40 °C treatment, and was up-regulated by heat stress and exogenous H?O?. Overexpression of ZmHSP16.9 in transgenic tobacco conferred tolerance to heat and oxidative stresses by increased seed germination rate, root length, and antioxidant enzyme activities compared with WT plants. These results support the positive role of ZmHSP16.9 in response to heat stress in plant. KEY MESSAGE: The overexpression of ZmHSP16.9 enhanced tolerance to heat and oxidative stress in transgenic tobacco.     

Endoplasmic Reticulum of Mung Bean Cotyledons: ACCUMULATION DURING SEED MATURATION AND CATABOLISM DURING SEEDLING GROWTH

Homogenates of mung bean cotyledons were subjected to equilibrium density centrifugation on linear sucrose gradients and the positions of the various organelles determined by assay of marker enzymes. Measurement of phospholipid distribution on such gradients showed that the major peak of phospholipid at a density of 1.11 to 1.13 grams per cubic centimeter coincided with the position of the endoplasmic reticulum (ER), confirming ultrastructural evidence that storage parenchyma cells are rich in ER. Germination and seedling growth were accompanied by a rapid decline in ER-associated phospholipid but a marked increase in the ER marker enzyme NADH cytochrome c reductase. Similar experiments with developing seeds indicated that the amount of ER-associated phospholipid increases during cotyledon expansion reaching a maximum during seed maturation. There was no subsequent decline during seed desiccation, instead ER-associated phospholipid levels were maintained in the dry seed until germination when catabolism was initiated 12 to 24 hours after the start of imbibition. This timing indicates that the observed ER breakdown is not an expression of the overall senescence of the cotyledons, but may represent the dismantling of the extensive rough ER used for reserve protein synthesis during cotyledon development.     

A comparative study of water distribution, free radical production and activation of antioxidative metabolism in germinating pea seeds

The aim of this study was to investigate whether there is a relationship between hydration of the embryo axes and cotyledons and the resumption of the oxidative metabolism in both organs of germinating seeds of pea (Pisum sativum L. cv. Piast). Nuclear magnetic resonance (¹H-NMR) spectroscopy and imaging were used to study temporal and spatial water uptake and distribution in pea seeds. The observations revealed that water penetrates into the seed through the hilum, micropyle and embryo axes, and cotyledons hydrate to different extents. Thus, inhomogeneous water distribution may influence the resumption of oxidative metabolism. Electron paramagnetic resonance (EPR) measurements showed that seed germination was accompanied by the generation of free radicals with g₁ and g₂ values of 2.0032 and 2.0052, respectively. The values of spectroscopic splitting coefficients suggest that they are quinone radicals. The highest content of free radicals was observed in embryo axes immediately after emergence of the radicle. Glutathione content decreased during the entire germination period in both embryo axes and cotyledons. A different profile was observed for ascorbate, with significant increases in embryo axes, coinciding with radicle protrusion. Electrophoretic analysis showed that superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2) were present in dry seeds and were activated later during germination, especially in embryo axes. The presence of all antioxidative enzymes as well as low molecular antioxidants in dry seeds allowed the antioxidative machinery to be active as soon as the enzymes were reactivated by seed imbibition. The observed changes in free radical levels, antioxidant contents and enzymatic activities in embryo axes and cotyledons appear to be more closely related to metabolic and developmental processes associated with preparation for germination, and do not correspond directly to the hydration of the tissues.     

Analysis of Chaperone Function and Formation of Hetero-oligomeric Complexes of Hsp18.1 and Hsp17.7, Representing Two Different Cytoplasmic sHSP Classes in Pisum sativum

Small heat shock proteins (sHSPs) are the most abundant stress proteins in plants. Usually not expressed under permissive conditions, they can accumulate to more than 2% of the total cellular protein content during heat stress. At present several points of evidence indicate that these proteins act as molecular chaperones by keeping partially denatured proteins in a folding-competent state. In plants sHSPs are encoded by a multigene family, which can be segregated into several classes according to their subcellular position and/or sequence homology. Curiously, two different classes appear in the cytoplasm. Their specific role during heat shock remains elusive. Here we present some evidence that both classes of sHSPs enhance recovery of reporter protein activity in the presence of HSP70. Applying peptide arrays prepared by SPOT synthesis and in situ analysis by confocal laser scanning microscopy, we could further show that the two classes of sHSP are attached to each other and are able to interact with non-native proteins both in vivo and in vitro. Although both of the sHSPs act similarly as molecular chaperones, immunohistochemistry experiments support the hypothesis that the two have different cellular functions in the development of heat-induced cytoplasmic heat shock granules under elevated temperatures. Daniela Wagner Deceased 24 Feburary 2004.     

Differential response of antioxidative enzymes in embryonic axes and cotyledons of germinating lupine seeds

Germination of lupine (Lupinus luteus L.) seeds was accompanied by an increase in concentration of free radicals with g ₁ and g ₂ values of 2.0056 ± 0.0003 and 2.0033 ± 0.0005, respectively. The highest intensity of free radical signal was observed in embryo axes immediately after radicle protruded through the seed coat. Hydrogen peroxide accumulated in embryonic axes and cotyledons during imbibition before the onset of germination in the seed population. The activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) rose progressively in embryo axes. In cotyledons SOD activity did not change significantly, while that of CAT increased during germination. The enhancement of Cu, Zn-SODs and Mn-SOD isoforms in embryonic axes was observed. A new isoform of catalase was synthesized, suggesting that it plays a relevant role during germination. SOD and CAT activities were detected in dry seeds. Free radical generation and response of antioxidative enzymes differed between embryo axes and cotyledons during the germination timecourse.