Post-germination Stages Research Articles

Freezing Stress Influences Emergence of Germinated Perennial Grass Seeds

In sagebrush rangelands perennial bunchgrasses are typically seeded in fall and a high proportion of planted seeds germinate prior to winter onset but fail to emerge in spring. Our objectives were to evaluate freezing tolerance of germinated but nonemergent bluebunch wheatgrass seeds under laboratory conditions. We used data from a 2-yr pilot study to determine overwinter freezing temperature and duration for soils in southeastern Oregon. We then conducted two experiments to assess freezing tolerance. In experiment 1, bluebunch wheatgrass seeds were planted in control pots and compared to seeds planted at early, mid, or late postgermination stages. Pots from each treatment were placed in a grow room maintained at 12h 40min light/11h 20min dark photoperiod, with a constant temperature of 22°C for 30 d either immediately or following a 30-d freeze. In experiment 2, germinated bluebunch wheatgrass seeds were planted in pots that were left nonfrozen or were frozen for a specified duration prior to a 30-d period in the grow room. Emergence density and tillers · seedling−1 were quantified for both experiments. The number of days per year for freezing soil conditions in the pilot study ranged yearly from 25 to 51; maximum duration of continuous freezing was 16.5 and 11.2 d. Freezing reduced or eliminated seedling emergence at all postgermination stages (P < 0.001) and tiller density was reduced by at least 50% (P < 0.001). Maximum reduction in seedling density (P < 0.001) was realized within 4 d of initiation of freezing and tillers · seedling−1 were reduced 30–70% with > 6 d of freezing (P = 0.001). Our data indicate that freezing-associated mortality of germinated but nonemergent bluebunch wheatgrass seedlings can be extremely high and suggest that management practices to reduce prewinter germination of seeds could improve subsequent emergence and seeding success.

Cold Tolerance of an Inbred Line Population of Rice (Oryza sativa L) at Different Growth Stages

Indica rice cultivar Hokuriku142, japonica rice cultivar Hyogokithanishiki and a recombinant inbred line population involving these cultivars were evaluated for cold tolerance at germination, post-germination and seedling stages. For each experiment 10 replicates were arranged with 20-40 seeds per replicate. At germination stage, cold stress was applied at 20°C and 15°C and the number of germinated seeds was counted. Post-germination stage cold tolerance was evaluated on 4-day germinated seeds by holding them at 4°C for 1-12 days and gained hypocotyl length was measured after a 4-day recovery period at normal growth conditions. To assess the seedling stage cold tolerance 1-week-old seedlings were maintained at 4°C for 1-7 days and green plant height was measured after a 5-day recovery period. In all growth stages Hyogokithanishiki showed higher degree of cold tolerance than Hokuriku. Recombinant inbred line population showed normal distribution curves for germination and post-germination stage cold tolearance with transgressive segregants for both higher and lower levels than the parents. At the seedling stage, the inbred populations showed a skew towards the susceptible cultivar but there were two transgressive segregants for greater cold tolerance than Hyogokithanishiki. Identification of such significant differences in the two parental rice cultivars and distribution of the character across the range of different tolerance levels with transgressive segregation indicates that this population is useful for the development of cold tolerant rice cultivars and to understand the basis of the cold regulation of rice using molecular tools. DOI: http://dx.doi.org/10.4038/tare.v14i2.4838 Tropical Agricultural Research & Extension 14(2): 2011 24-33

COMPARATIVE ANALYSIS OF SOME PHYSIOLOGICAL RESPONSES OF RICE SEEDLINGS TO COLD, SALT, AND DROUGHT STRESSES

Rice (Oryza sativa L.) is one of the most sensitive crops to drought, salt and cold stresses, particularly at post germination stage. The effects of these stresses on some physiological responses of two (a salt tolerant and a sensitive) rice cultivars ‘FL478’ and ‘IR29’ were investigated in this study. Two-day seedlings were transferred to MS media complemented with iso-osmotic concentrations of sodium chloride (NaCl; 0, 50, 100, and 150 mM) or mannitol (0, 100, 180, and 275 mM) at 25°C or four and 15°C for 10 days. Experiments were carried out based on completely randomized design, with at least three replicates. All three stresses decreased shoot growth, chlorophylls, carotenoids and root starch while increased shoot soluble sugars. The effect of exerted cold stress on growth, hydrogen peroxide (H2O2) and malonyldialdehyde levels, electrolyte leakage, chlorophylls and carotenoids contents was more than or comparable with drought, but greater than salinity. The results also indicated higher tolerance of ‘‘FL478’’ not only to salinity but also to drought compared to ‘‘IR29’’. Therefore, a mechanism for osmotic stress adjustment is probable in ‘‘FL478’’ in addition to low sodium (Na+) to potassium (K+) ratio in shoot tissues under salinity.

Induction and Repression of Gene Expression Mediating Ethylene Biosynthesis and Sodium/Proton Exchange in Rice Seedlings Under Submergence Stress

ABSTRACTElongation escape mediated by seedling vigor with fast shoot elongation under submergence is considered to be an important strategy for rice particularly in direct seeding cultivation systems. We compared the expression profiles of two key genes in rice for ethylene biosynthesis—1-aminocyclopropane-1-carboxylate synthase (OsACS) and oxidase (OsACO) genes, and five Na+/H+ exchanger (OsNHX) paralogs under normal and submerged conditions. The amount of transcripts was studied by RT-PCR using seedlings of one japonica and three indica cultivars with different levels of seedling vigor under submergence. In both types, submergence induced marked accumulation of transcripts of all paralogs of OsACS and OsACO except for OsACS1, which showed complete repression under the stress in non-vigorous indica cultivars suggesting its key role in seedling vigor under submergence. OsNHX expression was also enhanced under submergence, and the levels of OsNHX1 and OsNHX5 transcripts agreed well with those of seedling vigor under submergence. The observed mode of OsACS, OsACO and OsNHX expression under submergence suggests that these genes can be potential targets for understanding the mechanism regulating seedling vigor under submergence at the post-germination stage in rice.

A novel rice calmodulin-like gene, OsMSR2, enhances drought and salt tolerance and increases ABA sensitivity in Arabidopsis

Many abiotic stimuli, such as drought and salt stresses, elicit changes in intracellular calcium levels that serve to convey information and activate adaptive responses. Ca²⁺ signals are perceived by different Ca²⁺ sensors, and calmodulin (CaM) is one of the best-characterized Ca²⁺ sensors in eukaryotes. Calmodulin-like (CML) proteins also exist in plants, but their functions at the physiological and molecular levels are largely unknown. In this report, we present data on OsMSR2 (Oryza sativa L. Multi-Stress-Responsive gene 2), a novel calmodulin-like protein gene isolated from rice Pei'ai 64S (Oryza sativa L.). Expression of OsMSR2 was strongly up-regulated by a wide spectrum of stresses, including cold, drought, and heat in different tissues at different developmental stages of rice, as revealed by both microarray and quantitative real-time RT-PCR analyses. Analysis of the recombinant OsMSR2 protein demonstrated its potential ability to bind Ca²⁺ in vitro. Expression of OsMSR2 conferred enhanced tolerance to high salt and drought in Arabidopsis (Arabidopsis thaliana) accompanied by altered expression of stress/ABA-responsive genes. Transgenic plants also exhibited hypersensitivity to ABA during the seed germination and post-germination stages. The results suggest that expression of OsMSR2 modulated salt and drought tolerance in Arabidopsis through ABA-mediated pathways.

MicroRNA Gene Regulation Cascades During Early Stages of Plant Development

MicroRNAs (miRNAs) regulate various developmental programs of plants. This review focuses on miRNA involvement in early events of plant development, such as seed germination, seedling development and the juvenile to adult phase transition. miR159 and miR160 are involved in the regulation of seed germination through their effects on the sensitivity of seeds to ABA. miR156 and miR172 play critical roles in the emergence of vegetative leaves at post-germinative stages, which is important for the transition to autotrophic growth. The phase transition from the juvenile to adult stage in both monocots and dicots is also regulated by miR156 and miR172. In these early developmental processes, there are miRNA gene regulation cascades where the miR156 pathway acts upstream of the miR172 pathway. Moreover, targets of miR156 and miR172 exert positive feedback on the expression of MIR genes that suppress themselves. The early events of plant development appear to be controlled by complex mechanisms involving sequential expression of different miRNA pathways and feedback loops among miRNAs and their target genes.

Progesterone and β-Estradiol Stimulate Seed Germination in Chickpea by Causing Important Changes in Biochemical Parameters

Effects of progesterone and beta-estradiol on morphologic (germination velocity, root and shoot length) and biochemical (activities of alpha-amylase, superoxide dismutase, peroxidase and catalase, H2O2 content, lipid peroxidation) parameters during germination and post-germination stages of chickpea seeds were studied. The seeds germinated at various hormone concentrations (10(-4), 10(-9), 10(-9), 10(-12), 10(-15) M) were harvested at the end of the 1st, 3rd, and 5th day. With comparison to the control, these hormones caused an increment in the number of germinating seeds at the end of days 1 and 3 by accelerating the seed germination. Root and shoot lengths were augmented by both hormones at all hormone concentrations tested. The highest elongation was recorded in 10(-6) M progesterone and 10(-9)-10(-12) M beta-estradiol. Similarly, activities of a-amylase and superoxide dismutase were increased by all concentrations of both hormones, and maximum increases were obtained with 10(-6) M progesterone and 10(-1)-10(-12) M beta-estradiol. In the case of superoxide dismutase activity, not only the H2O2 content but also the peroxidase and catalase activities increased. Lipid peroxidation decreased depending on an increase in the antioxidant enzyme activities. In the present study, it was demonstrated that progesterone and beta-estradiol even at low concentrations increase the germination velocity and resistance to stress conditions by changing the activities of some biochemical pathways.

The regulation of post-germinative transition from the cotyledon- to vegetative-leaf stages by microRNA-targeted SQUAMOSA PROMOTER-BINDING PROTEIN LIKE13 in Arabidopsis

AbstractGermination and early seedling development are critical for successful stand establishment of plants. Following germination, the cotyledons, which are derived from embryonic tissue, emerge from the seed. Arabidopsis seedlings at post-germinative stages are supported mainly by the supply of nutrition from the cotyledons until vegetative leaves emerge and initiate photosynthesis. The switch to autotrophic growth is a significant transition at the post-germinative stage. Here, we provide evidence that down-regulation of SQUAMOSA PROMOTER-BINDING PROTEIN LIKE13 (SPL13) by microRNA156 (miR156) plays an important role in the regulation of the post-germinative switch from the cotyledon stage to the vegetative-leaf stage. Silent mutations created in the SPL13 sequence in the region that is complementary to the miR156 sequence caused the deregulation of the mutant form of SPL13 (mSPL13) mRNA from miR156. Mutant seedlings over-accumulated miRNA-resistant messages and exhibited a delay in the emergence of vegetative leaves compared to wild-type seedlings. The delay was not observed in control transgenic plants expressing non-mutated SPL13, indicating that the phenotype was caused specifically by the silent mutations and deregulation of SPL13 from miR156. Characterization of the SPL13 promoter indicated that this gene is expressed mainly in the hypocotyl and affects leaf primordium development. These results suggest that the repression of SPL13 by miR156 is essential for normal post-germinative growth in Arabidopsis.

The microRNA156 and microRNA172 gene regulation cascades at post-germinative stages inArabidopsis

AbstractMicroRNAs (miRNAs) are involved in developmental programmes of plants, including seed germination and post-germination. Here, we provide evidence that two different miRNA pathways, miR156 and miR172, interact during the post-germination stages inArabidopsis. Mutant seedlings expressing miR156-resistantSQUAMOSA PROMOTER-BINDING PROTEIN-LIKE13(mSPL13), which has silent mutations in the miR156 complementary sequence, over-accumulatedSPL13mRNA and exhibited a delay in seedling development. Microarray analysis indicated thatSCHNARCHZAPFEN(SNZ), anAP2-like gene targeted by miR172, was down-regulated in these mutants. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and miRNA gel blot analyses showed that theMIR172genes were up-regulated inmSPL13mutants. These results suggest that the miRNA regulation cascades (miR156⊣SPL13 → miR172⊣SNZ) play a critical role during the post-germination developmental stages inArabidopsis.

Soil efficacy of fipronil to early stage pests of sugarcane, and its effect on development on Chilo infuscatellus Snellen (Crambidae: Lepidoptera)

Fipronil soil applications of 75 and 90 g a.i./ha at pre- and post-germination stages of sugarcane improved plant germination and cane yield without affecting juice quality. Fipronil was highly toxic against the stem boring moth C. infuscatellus, and termites (Odontotermes spp.). Pre- and post-germination application of fipronil 75 days after planting (DAP), resulted in 65 and 31% reductions in dead-hearts and termite incidence, respectively. Fipronil-treated cane shoots fed to C. infuscatellus larvae resulted in 64.6–73.6% and 39–54.1% mortality at 15 and 30 days post-insecticide treatment, respectively. Fipronil treatment resulted in reduced larval and pupal weight, as well as an increase in pupal duration time. Fipronil at 90 g a.i./ha applied at 0 or 45 DAP did not reduce C. infuscatellus between-season population carry-over in stubbles.

Investigation on cell death in the megagametophyte of Araucaria bidwillii Hook. post-germinated seeds

The megagametophyte of the Araucaria bidwillii seed is a storage tissue that surrounds and feeds the embryo. When all its reserves are mobilized, the megagametophyte degenerates as a no longer needed tissue. In this work we present a biochemical and a cytological characterization of the megagametophyte cell death. The TUNEL assay showed progressive DNA fragmentation throughout the post-germinative stages, while DNA electrophoretic analysis highlighted a smear as the predominant pattern of DNA degradation and internucleosomal DNA cleavage only for a minority of cells at late post-germinative stages. Cytological investigations at these stages detected profound changes in the size and morphology of the megagametophyte nuclei. By using in vitro assays, we were able to show a substantial increase in proteolytic activities, including caspase-like protease activities during the megagametophyte degeneration. Among the caspase-like enzymes, caspase 6- and 1-like proteases appeared to be the most active in the megagametophyte with a preference for acidic pH. On the basis of our results, we propose that the major pathway of cell death in the Araucaria bidwillii megagametophyte is necrosis; however, we do not exclude that some cells undergo developmental programmed cell death.

Mapping of Quantitative Trait Loci Associated with Cold Tolerance at the Post-Germination Stage in Rice

ABSTRACTCold tolerance at the post-germination stage in rice was studied using gained shoot length (GSL) as a new parameter, which was measured as increased shoot length during cold stress at 4°C followed by a 4-day recovery period at 35°C. GSL decreased until 4th day in both japonica and indica cultivar, while a marked increase occurred in a japonica cultivar after 5 days of cold stress, indicating its higher ability of cold acclimation. Quantitative trait loci (QTLs) associated with cold tolerance were identified using recombinant inbred lines derived from these parents on a linkage map constructed with 102 simple sequence repeat (SSR) markers. By the interval analysis, one and five putative QTLs were identified after 5 and 11 days of cold stress, respectively. The single point analysis indicated that these QTLs explained 16% and 54% of the phenotypic variance after 5 and 11 days of cold stress, respectively. A cold tolerant japonica parent contributed all QTLs, among which one on the long arm of chromosome 7 was common for the two cold stress periods. Based on gained root length one QTL was identified on chromosome 12 explaining 11% of the variance.

Temporal and Spatial Expression of 2S Albumin in Castor (Ricinus communis L.)

We studied the temporal and spatial expression of the 2S albumin in castor (Ricinus communis L.) during seed development, germination, post-germination, and plant development. Quantitative polymerase chain reaction analysis showed that the 2S albumin transcript accumulated to a maximum level at the middle of seed development, showing a bell-shaped temporal pattern. Residual levels of the transcript were present in the mature seed and degraded rapidly upon germination. Immunodetection analysis was performed using an anti-2S albumin antibody under reducing conditions. During seed development, the 2S albumin precursor pro-protein began to be synthesized at 26 days after pollination (DAP); the pro-protein was thereafter processed to mature proteins at 40 DAP, suggesting that the post-translation modification of 2S albumin takes place during this time period. Both the 2S albumin precursor pro-protein and the mature proteins accumulated throughout seed maturation and desiccation stages. During seed germination, both forms of the 2S albumin proteins were present in endosperm and cotyledon until the completion of germination and degraded rapidly afterwards. However, the antibody also detected a group of proteins/peptides in endosperm and cotyledon when the seeds progressed to germination and post-germination stages. A 14 kDa protein in the leaves of fully developed seedlings and mature plants also reacted to the anti-2S albumin antibody. The identity of the proteins accumulated in germinating seed and leaf remains unknown.

Firing of transcription and compartmentalization of splicing factors in tomato radicle nuclei during germination1

Germination is a well-characterized process in which embryo cells of seeds experience a programmed transition from quiescence to proliferation. For this reason they constitute a very good system to analyse nuclear evolution from a dehydrated practically inactive state until the steady state of proliferation. We analysed the temporal and spatial organization of transcription and splicing factors in nuclei of tomato radicle cells during germination. To address this issue we performed in situ immunodetection of several markers of these processes: the Z-DNA stretches forming behind the active RNA polymerases, the splicing proteins U2B'' and Sm, and the trimethyl guanosin cap of small nuclear RNA. The concomitant structural changes of the different nuclear compartments were studied in meristematic nuclei by electron microscopy and high-resolution cytochemistry for DNA and ribonucleoproteins. In quiescent cells practically no Z-DNA stretches were detected and splicing components localized mainly to one or two Cajal bodies associated to the nucleolus. In early germination, a massive de-condensation of chromatin and nucleolar Z-DNA conformation stretches were first detected, followed by the relocation of scarce splicing components to the small interchromatin spaces. Nucleoplasmic Z-DNA stretches were not detected until 4 h of imbibition and were accompanied by an important increase of splicing components in this nuclear domain. Soon after the post-germination stage, transcription and splicing topology and nuclear organization in meristematic nuclei resemble those in steady state growing tomato roots. Our results demonstrate that, in tomato, dormant nuclei splicing factors are stored in nucleolar Cajal bodies. In early germination, RNA polymerase I transcription is first activated, whereas mRNA transcription is fired later and is accompanied by a massive de-condensation of chromatin and accumulation of splicing factors in the interchromatin domains. Nucleoplasmic Cajal bodies appear later in germination.

Developmental and embryo axis regulation of gibberellin biosynthesis during germination and young seedling growth of pea.

The expression patterns of five genes (PsGA20ox1, PsGA20ox2, PsGA3ox1, PsGA2ox1, and PsGA2ox2) encoding five regulatory gibberellin (GA) biosynthesis enzymes (two GA 20-oxidases, a GA 3beta-hydroxylase, and two GA 2beta-hydroxylases) were examined to gain insight into how these genes coordinate GA biosynthesis during germination and early postgermination stages of the large-seeded dicotyledonous plant pea (Pisum sativum). At the time the developing embryo fills the seed coat, high mRNA levels of PsGA20ox2 (primarily responsible for conversion of C20-GAs to GA(20)), PsGA2ox1 (primarily responsible for conversion of GA(20) to GA(29)), and PsGA2ox2 (primarily responsible for conversion of GA(1) to GA(8)) were detected in the seeds, along with high GA(20) and GA(29) levels, the enzymatic products of these genes. Embryo maturation was accompanied by a large reduction in PsGA20ox2 and PsGA2ox1 mRNA and lower GA(20) and GA(29) levels. However, PsGA2ox2 transcripts remained high. Following seed imbibition, GA(20) levels in the cotyledons decreased, while PsGA3ox1 mRNA and GA(1) levels increased, implying that GA(20) was being used for de novo synthesis of GA(1). The presence of the embryo axis was required for stimulation of cotyledonary GA(1) synthesis at the mRNA and enzyme activity levels. As the embryo axis doubled in size, PsGA20ox1 and PsGA3ox1 transcripts increased, both GA(1) and GA(8) were detectable, PsGA2ox2 transcripts decreased, and PsGA2ox1 transcripts remained low. Cotyledonary-, root-, and shoot-specific expression of these GA biosynthesis genes and the resultant endogenous GA profiles support a key role for de novo GA biosynthesis in each organ during germination and early seedling growth of pea.

Identification of Individual Powdery Mildew Fungi Infecting Leaves and Direct Detection of Gene Expression by Single Conidium Polymerase Chain Reaction

ABSTRACT Greenhouse-grown tomato seedlings were inoculated naturally with two genera of powdery mildew conidia forming appressorial germ tubes that could not be differentiated by length alone. For direct identification, single germinated conidia were removed from leaves by means of a glass pipette linked to the manipulator of a high-fidelity digital microscope. This microscope enabled in vivo observation of the fungi without leaf decoloration or fungal staining. The isolated conidia were subjected to PCR amplification of the 5.8S rDNA and its adjacent internal transcribed spacer sequences followed by nested PCR to attain sensitivity high enough to amplify target nucleotide sequences (PCR/nested PCR). Target sequences from the conidia were completely coincident with those of the pathogen Oidium neolycopersici or Erysiphe trifolii (syn. Microsphaera trifolii), which is nonpathogenic on tomato. Using RT-PCR/nested PCR or multiplex RT-PCR/nested PCR, it was possible to amplify transcripts expressed in single conidia. Conidia at pre- and postgermination stages were removed individually from tomato leaves, and two powdery mildew genes were monitored. The results indicated that the beta-tubulin homolog TUB2-ol was expressed at pre- and postgermination stages and the cutinase homolog CUT1-ol was only expressed postgermination. Combining digital microscopic micromanipulation and two-step PCR amplification is thus useful for investigation of individual propagules on the surface of plants.

Heterogeneity of times required for germination and outgrowth from single spores of nonproteolytic Clostridium botulinum.

Knowledge of the distribution of growth times from individual spores and quantification of this biovariability are important if predictions of growth in food are to be improved, particularly when, as for Clostridium botulinum, growth is likely to initiate from low numbers of spores. In this study we made a novel attempt to determine the distributions of times associated with the various stages of germination and subsequent growth from spores and the relationships between these stages. The time to germination (t(germ)), time to emergence (t(emerg)), and times to reach the lengths of one (t(C1)) and two (t(C2)) mature cells were quantified for individual spores of nonproteolytic C. botulinum Eklund 17B using phase-contrast microscopy and image analysis. The times to detection for wells inoculated with individual spores were recorded using a Bioscreen C automated turbidity reader and were compatible with the data obtained microscopically. The distributions of times to events during germination and subsequent growth showed considerable variability, and all stages contributed to the overall variability in the lag time. The times for germination (t(germ)), emergence (t(emerg) - t(germ)), cell maturation (t(C1) - t(emerg)), and doubling (t(C2) - t(C1)) were not found to be correlated. Consequently, it was not possible to predict the total duration of the lag phase from information for just one of the stages, such as germination. As the variability in postgermination stages is relatively large, the first spore to germinate will not necessarily be the first spore to produce actively dividing cells and start neurotoxin production. This information can make a substantial contribution to improved predictive modeling and better quantitative microbiological risk assessment.

Identification of spatial specifically expressed genes in rice (Oryza sativa) seedlings using cDNA microarray

AbstractMembranous cDNA microarrays containing 2200 unique rice transcripts were designed for screening the characteristics of spatially expressed genes in post-germination rice seedlings. By comparing the profiles obtained, 31 genes were identified as expressed specifically in the plumule, 36 in the mesocotyl and 73 in the radicle. Several genes, such aspolyubiquitin,UDP-glucose pyrophosphorylase,sucrose synthaseandphosphoglycerate kinase, which encode components of the carbohydrate or protein metabolic reaction cascades, were expressed specifically in the mesocotyl, indicating that degradation reactions of the endospermous reserve starch and proteins occur mainly in the mesocotyl during the post-germination stage. A number of genes involved in defence mechanisms or in the processes of replication, transcription and translation were identified as expressed specifically in the plumule or radicle. Among plumule specifically expressed genes,translation initiation factor 5a,40s ribosomal protein s28andribosomal protein 136are considered to have a critical role in protein biosynthesis; whileallergenic protein, β-D-glucan exohydrolaseandactin 11are genes with defending functions. Among the catalogue of radicle specifically expressed genes,EF-1a,Tat binding protein,replication protein A2,histone h3.2,ribosomal protein s29aand40s ribosomal protein s19are genes that function in the process of replication, transcription or translation; whereasglycine-rich protein,wound-induced basic protein,Bowman-Birk proteinase inhibitorandlipid transfer protein-2are genes involved in defence responses. Results of this experiment have provided insight into post-germination molecular physiology at the genomic level of gene expression.

Hypocotyl features of Myrtus communis (Myrtaceae): a many-sided strategy for possible enhancement of seedling establishment in the Mediterranean environment

Seed germination and seedling development were studied in Myrtus communis L. (Myrtaceae), an evergreen shrub widespread in Mediterranean woodlands, maquis and garrigues, to investigate the presence of morphological and anatomical adaptations that enhance successful seedling establishment under biotic and abiotic stresses. Seeds were germinated under controlled conditions and were observed daily to identify different post-germination stages of development. The anatomy of developing hypocotyls and emerging radicles was studied through light and epi-fluorescence microscopy using both cross and longitudinal sections. Tegument rupture was caused by hypocotyl elongation, which, when water availability in the substrate was limited, was accompanied by a dense ring of hairs in the lower region. Absorption tests showed that the hypocotyl hairs were functional in water uptake. They might also enhance the anchorage of the seedling to the substrate. Further investigations showed the early formation of a suberized layer of cells (exodermis) in the radicle and the occurrence of phenolics in the hypocotyl cells of cortical parenchyma. In conclusion, our results show that seedlings of M. communis have developed a strategy of establishment that: (a) regulates water uptake through hypocotyl hairs and the exodermis; (b) might allow mechanical support through hypocotyl hairs; and (c) accumulates phenolics that might defend the plant against animal predation.

Proteases associated with programmed cell death of megagametophyte cells after germination of white spruce (Picea glauca) seeds.

During post-germinative seedling growth, the major storage organ of the white spruce (Picea glauca) seed, the megagametophyte, undergoes programmed cell death (PCD). Protease activities in megagametophyte cells that arise post-germinatively were investigated. The accumulation of protease activities can be divided into two phases: the first phase correlated with degradation of storage proteins while the second phase was temporally associated with cell death, although some of the early proteases were also active during the later phase. Proteases induced during PCD were mainly serine and cysteine proteases. One of the PCD-associated cysteine proteases had homology to Cys-EP, a PCD-related cysteine protease of the castor bean endosperm. Transcripts encoding a Cys-EP-related protein were not present in megagametophytes when seeds were imbibed, nor were they present during germination and early post-germinative growth (radicle length ca. 2-5 mm). At a later post-germinative stage (i.e when the seed's radicle was ca. 15 mm), the Cys-EP-related transcripts (ca. 1.3 kb) became abundant and, at this time, the 48 kDa proform of the enzyme first appeared. The mature form of the Cys-EP (ca. 38 kDa) was predominant at a very late stage of post-germinative growth. Immunocytochemistry showed that the Cys-EP-related protein was localized to spherical organelles (ca. 2 microm) that may be equivalent to the 'ricinosomes' of castor bean endosperm cells. Caspase-like protease (CLP) activities were first detected 3 days after germination with the caspase-specific substrate Ac-DEVD-AMC; maximum activities occurred when the seed's radicle was ca. 20-25 mm. When germinated seeds were treated with a caspase-3 inhibitor, both the peak of CLP activities and the death of megagametophyte cells were delayed. We propose that the Cys-EP-related protein and CLP activity are involved in PCD of white spruce megagametophyte cells.