Physiological and transcriptome analysis of Magnolia denudata leaf buds during long-term cold acclimation.

BACKGROUND: Magonlia denudata is an important perennial tree species of the Magnoliaceae family, known for its ornamental value, resistance to smoke pollution and wind, role in air purification, and robust cold tolerance. In this study, a high-throughput transcriptome analysis of leaf buds was performed, and gene expression following artificial acclimation 22 °C, 4 °C and 0 °C, was compared by RNA sequencing. RESULTS: Over 426 million clean reads were produced from three libraries (22 °C, 4 °C and 0 °C). A total of 74,503 non-redundant unigenes were generated, with an average length of 1173.7 bp (N50 = 1548). Based on transcriptional results, 357 and 235 unigenes were identified as being upregulated and downregulated under cold stress conditions, respectively. Differentially expressed genes were annotated using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analyses. The transcriptomic analysis focused on carbon metabolism and plant hormone signal transduction associated with cold acclimation. Transcription factors such as those in the basic helix-loop-helix and AP2/ERF families were found to play an important role in M. denudata cold acclimation. CONCLUSION: M. denudata exhibits responses to non-freezing cold temperature (4 °C) to increase its cold tolerance. Cold resistance was further strengthened with cold acclimation under freezing conditions (0 °C). Cold tolerance genes, and cold signaling transcriptional pathways, and potential functional key components for the regulation of the cold response were identified in M. denudata. These results provide a basis for further studies, and the verification of key genes involved in cold acclimation responses in M. denudata lays a foundation for developing breeding programs for Magnoliaceae species.

Cloning and expression analyses of a Pyrabactin Resistance 1 (PYR1) gene from Magnolia sieboldii K. Koch.

Magnolia sieboldii K. Koch is endemic to China and has high medicinal and ornamental values. However, its seed exhibits morphophysiological dormancy, and the molecular mechanisms of which are not clearly understood. To reveal the regulation mechanism of the ABA signal in seed dormancy, the M. sieboldii ABA receptor Pyrabactin Resistance 1 (PYR1) gene was cloned and analyzed. Analysis of the MsPYR1 sequence analysis showed that the full-length cDNA contained a complete open reading frame of 987 bp and encoded a predicted protein of 204 amino acid residues. The protein had a relative molecular weight of 22.661 kDa and theoretical isoelectric point of 5.01. The transcript levels of MsPYR1 were immediately upregulated at 16 DAI and then decreased at 40 DAI. The highest transcript level of MsPYR1 was found in the dry seeds, indicating that the MsPYR1 gene may play an important role in the regulation of dormancy. The MsPYR1 gene cDNA was successfully expressed in E. coli Rosetta (DE3), and the protein bands were consistent with the prediction. The Anti-MsPYR1antibody could detect the expression of MsPYR1 in M. sieboldii. The results provided a foundation for further study of the function of the MsPYR1 gene.ABBREVIATIONSABA: Abscisic acid; MPD: morphophysiological; PYR1: Pyrabactin Resistance1; PYL: Pyr1-Like; RCAR: Regulatory Components of Aba Receptors; PP2C: protein phosphatases 2C; SnRK2: sucrose non-fermenting1-related protein kinase2; DAI: day after imbibition; NCBI: National Center for Biotechnology Information; BCA: Bicinchoninic acid; CDD: Conserved Domains.

De novo transcriptome sequencing and gene expression profiling of Magnolia wufengensis in response to cold stress.

BACKGROUND: Magnolia wufengensis is a new species of Magnolia L. and has considerable ornamental and economic value due to its unique characteristics. However, because of its characteristic of poor low temperature resistance, M. wufengensis is hardly popularization and application in the north of China. Furthermore, the mechanisms of gene regulation and signaling pathways involved in the cold-stress response remained unclear in this species. In order to solve the above-mentioned problems, we performed de novo transcriptome assembly and compared the gene expression under the natural (25 °C) and cold (4 °C) conditions for M. wufengensis seedlings. RESULTS: More than 46 million high-quality clean reads were produced from six samples (RNA was extracted from the leaves) and were used for performing de novo transcriptome assembly. A total of 59,764 non-redundant unigenes with an average length of 899 bp (N50 = 1,110) were generated. Among these unigenes, 31,038 unigenes exhibited significant sequence similarity to known genes, as determined by BLASTx searches (E-value ≤1.0E-05) against the Nr, SwissProt, String, GO, KEGG, and Cluster of COG databases. Based on a comparative transcriptome analysis, 3,910 unigenes were significantly differentially expressed (false discovery rate [FDR] < 0.05 and |log2FC (CT/CK)| ≥ 1) in the cold-treated samples, and 2,616 and 1,294 unigenes were up- and down-regulated by cold stress, respectively. Analysis of the expression patterns of 16 differentially expressed genes (DEGs) by quantitative real-time RT-PCR (qRT-PCR) confirmed the accuracy of the RNA-Seq results. Gene Ontology and KEGG pathway functional enrichment analyses allowed us to better understand these differentially expressed unigenes. The most significant transcriptomic changes observed under cold stress were related to plant hormone and signal transduction pathways, primary and secondary metabolism, and photosynthesis. In addition, 113 transcription factors, including members of the AP2-EREBP, bHLH, WRKY, MYB, NAC, HSF, and bZIP families, were identified as cold responsive. CONCLUSION: We generated a genome-wide transcript profile of M. wufengensis and a de novo-assembled transcriptome that can be used to analyze genes involved in biological processes. In this study, we provide the first report of transcriptome sequencing of cold-stressed M. wufengensis. Our findings provide important clues not only for understanding the molecular mechanisms of cold stress in plants but also for introducing cold hardiness into M. wufengensis.

Reduced incompatibility in the production of second generation hybrids between two Magnolia species revealed by Bayesian gene dispersal modeling.

PREMISE OF THE STUDY: Hybrid zones are areas where gene flow between related species is currently occurring, so information on the compatibility between related species and their hybrids is essential for predicting the dynamics of such zones generated by introgressive hybridization. In this study, we quantified the compatibility among Magnolia stellata, M. salicifolia, and their hybrids in a hybrid zone using gene dispersal modeling. METHODS: After determining the genealogical classes of adult trees in the hybrid zone, the paternity of 574 open-pollinated seeds from 37 known maternal trees was analyzed with microsatellite markers. A neighborhood-based Bayesian gene dispersal model developed by us for estimating compatibility was then applied to the paternity data. KEY RESULTS: When M. stellata or M. salicifolia were mothers, interspecific mating to produce F1 hybrids yielded significant incompatibility, but backcrossing with F1 hybrids did not. Furthermore, when F1 hybrids became mothers, no significant incompatibility resulted from backcrossing to parental species or intra-F1 mating to produce F2 hybrids. The estimated proportion of F1 hybrids in the outcrossed seeds (1.7%) in the hybrid zone was much lower than that in the adult trees (14.0%). CONCLUSIONS: While it is difficult to obtain F1 hybrids, their low incompatibility makes it easy to produce advanced generation hybrids, once they have been successfully obtained. Although the production of F1 seeds is rare, heterosis and/or weak selection pressure in an empty niche between the parental species' niches may have contributed to the increased proportion of adult F1 hybrids in the hybrid zone.

The contribution of vascular and extra-vascular water pathways to drought-induced decline of leaf hydraulic conductance.

Drought stress can impair leaf hydraulic conductance (Kleaf), but the relative contribution of changes in the efficiency of the vein xylem water pathway and in the mesophyll route outside the xylem in driving the decline of Kleaf is still debated. We report direct measurements of dehydration-induced changes in the hydraulic resistance (R=1/K) of whole leaf (Rleaf), as well as of the leaf xylem (Rx) and extra-vascular pathways (Rox) in four Angiosperm species. Rleaf, Rx, and Rox were measured using the vacuum chamber method (VCM). Rleaf values during progressive leaf dehydration were also validated with measurements performed using the rehydration kinetic method (RKM). We analysed correlations between changes in Rx or Rox and Rleaf, as well as between morpho-anatomical traits (including dehydration-induced leaf shrinkage), vulnerability to embolism, and leaf water relation parameters. Measurements revealed that the relative contribution of vascular and extra-vascular hydraulic properties in driving Kleaf decline during dehydration is species-specific. Whilst in two study species the progressive impairment of both vascular and extra-vascular pathways contributed to leaf hydraulic vulnerability, in the other two species the vascular pathway remained substantially unaltered during leaf dehydration, and Kleaf decline was apparently caused only by changes in the hydraulic properties of the extra-vascular compartment.

Developing a Media Moisture Threshold for Nurseries to Reduce Tree Stress and Ambrosia Beetle Attacks.

Exotic ambrosia beetles are among the most damaging pests of trees grown in nurseries. The primary pests Xylosandrus crassiusculus Motschulsky and Xylosandrus germanus Blandford use ethanol to locate vulnerable trees. Research, primarily with X. germanus, has shown that flood-stressed trees emit ethanol and are preferentially attacked by ambrosia beetles. Our goal was to develop a media (also called potting soil) moisture threshold as an integrated pest management (IPM) tactic and assess grower practices that lead to ambrosia beetle attacks. Flooded Cornus florida L., Cornus kousa Burg., and Magnolia grandiflora L. trees incurred more attacks than unflooded trees that were not attacked. To determine optimal media moisture levels, we grew flood-tolerant Acer rubrum L. and flood-intolerant C. florida in containers with 10, 30, 50, 70, or 90% media moisture. No flooded or unflooded A. rubrum were attacked. However, C. florida grown in 70 or 90% moisture were attacked and died, whereas trees at 30 and 50% moisture were not attacked. Thus, we suggest an upper moisture threshold of 50% when growing C. florida and other flood-intolerant trees. However, during peak ambrosia beetle flight activity in spring 2013 and 2014, we found that media moisture levels in commercial nurseries were often between 50 and 90%. Implementing a media moisture threshold, as a new IPM tool, could reduce ambrosia beetle attacks and the need for insecticide applications, which is currently the only available management tactic. Future research should focus on how changes in substrates, irrigation, and other practices could help growers meet this threshold.

Arabinogalactan proteins mark stigmatic receptivity in the protogynous flowers of Magnolia virginiana (Magnoliaceae).

PREMISE OF THE STUDY: Factors affecting floral receptivity in angiosperms remain opaque, but recent studies suggest that the acquisition of stigmatic receptivity associated with cell-wall-related arabinogalactan proteins (AGPs) may be a widespread feature of flowering plants. Here, the time during which a stigma is receptive is evaluated and related to the secretion of AGPs in Magnolia virginiana, a protogynous member of an early-divergent angiosperm clade (magnoliids) with a clearly discernible female receptive phase. METHODS: Magnolia virginiana flower phenology was documented, and histochemical changes in the stigma before and after pollination were examined. Stigmatic receptivity was evaluated in relation to the secretion of AGPs detected in whole mounts and immunolocalized in sectioned stigmas. KEY RESULTS: Protogynous Magnolia flowers had a precise window of stigmatic receptivity, which is concomitant with the secretion of two AGPs labeled for different epitopes. After pollen germination and tube growth, these two AGPs could no longer be detected in the stigmas, suggesting that these AGPs interact with the growing male gametophytes and could be markers of stigmatic receptivity. CONCLUSIONS: These results show that the period of stigmatic receptivity is finely coordinated with the secretion of two arabinogalactan proteins on stigmas of flowers of M. virginiana. This first report of AGP presence in stigmatic tissues in a member of the magnoliids, together with recently described similar patterns in eudicots, monocots, and members of early-divergent lineages of flowering plants, suggests an ancient and widespread role for AGPs on stigmatic receptivity in angiosperms.

Thermogenesis, flowering and the association with variation in floral odour attractants in Magnolia sprengeri (Magnoliaceae).

Magnolia sprengeri Pamp. is an ornamentally and ecologically important tree that blooms at cold temperatures in early spring. In this study, thermogenesis and variation in the chemical compounds of floral odours and insect visitation in relation to flowering cycles were studied to increase our understanding of the role of floral thermogenesis in the pollination biology of M. sprengeri. There were five distinct floral stages across the floral cycle of this species: pre-pistillate, pistillate, pre-staminate, staminate and post-staminate. Floral thermogenesis during anthesis and consisted of two distinct peaks: one at the pistillate stage and the other at the staminate stage. Insects of five families visited M. sprengeri during the floral cycle, and sap beetles (Epuraea sp., Nitidulidae) were determined to be the most effective pollinators, whereas bees (Apis cerana, Apidae) were considered to be occasional pollinators. A strong fragrance was released during thermogenesis, consisting of 18 chemical compounds. Although the relative proportions of these compounds varied at different floral stages across anthesis, linalool, 1-iodo-2-methylundecane and 2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-ol were dominant. Importantly, we found that the floral blends released during the pistillate and staminate stages were very similar, and coincided with flower visitation by sap beetles and the two thermogenic episodes. Based on these results, we propose that odour acts as a signal for a reward (pollen) and that an odour mimicry of staminate-stage flowers occurs during the pistillate stage.

Pollen flow and effects of population structure on selfing rates and female and male reproductive success in fragmented Magnolia stellata populations.

BACKGROUND: Fragmentation of plant populations may affect mating patterns and female and male reproductive success. To improve understanding of fragmentation effects on plant reproduction, we investigated the pollen flow patterns in six adjacent local populations of Magnolia stellata, an insect-pollinated, threatened tree species in Japan, and assessed effects of maternal plant (genet) size, local genet density, population size and neighboring population size on female reproductive success (seed production rates), and effects of mating distance, paternal genet size, population size and separation of populations on male reproductive success. RESULTS: The seed production rate, i.e. the proportion of ovules that successfully turned into seeds, varied between 1.0 and 6.5%, and increased with increasing population size and neighboring population size, and with decreasing maternal genet size and local genet density. The selfing rate varied between 3.6 and 28.9%, and increased with increasing maternal genet size and with declining local genet density. Male reproductive success increased with increasing paternal genet size, and decreased with increasing mating distance and separation of population. Pollen flow between the populations was low (6.1%) and highly leptocurtic. CONCLUSIONS: Our results indicate that habitat fragmentation, separation and reduced size of populations, affected mating patterns and reproductive success of M. stellata. Local competition for pollinators and plant display size were likely to alter the reproductive success.

[Seed germinating characteristics of endangered plant Magnolia officinalis].

OBJECTIVE: To find out the optimal condition for the germination and cultivation condition on Magnolia officinalis, and offer the basis for standardized culture of M. officinalis. METHOD: The study was carried out with in-door experiment, using light, temperature, soil water content and seed soaking in different water temperature treatments, the effects of different treatments on seed germination percentage of M. officinalis were investigated. RESULT AND CONCLUSION: The germination percentage between light and dark treatments was significantly difference, it is indicated that the seed is light-sensitive. M. officinalis can germinate in a wide range of temperature, from 20 degrees C to 35 degrees C, and the suitable temperature was 25-30 degrees C, at which germination percentages were 59.2% and 54.6%. Germination percentage at 20/30 degrees C was 65%. The optimum soil water content was 25% for seed germination, at which germination percentages were 66.7%. The suitable soil water content was 20%-25%. In order to find out optimal temperature for soaking, seeds were soaked in water at the temperatures of 40, 60, 80, 90 degrees C for 10 minutes. The optimum water temperature was 60 degrees C for seed germination, and hard coated seed percentage was reduced. Seed germination rate can be inhibited under too high water temperature. Seed germination percentage was significantly difference in different germinating bed.

[Effects of tree ages and geographic area on quality of bark of Magnolia officinalis and M. officinalis var. biloba].

The effects of tree ages and geographic area on quality of bark of Magnolia officinalis (MO) and M. officinalis var. biloba (MOB) were studied. The volatile oils of samples extracted by a water stream distillation method were analyzed using a GC/MS method. The non-volatile ingredients of samples were determined by an HPLC method. Seven and twenty characteristic peaks were detected in the HPLC and GC fingerprint of MO, respectively, and the similar HPLC and GC fingerprint were showed in MOB. For samples collected in the same geographic area with different ages, the concentrations of active ingredients varied barely, whereas for those samples collected from different geographic area, differences on the concentrations of active ingredients were observed, for example, the concentrations of the amount of magnolol and honokiol in samples collected in Hubei province were higher than those collected in other areas. Big differences on the concentrations of active ingredients were found between MO and MOB, for MO, the ratio of honokiol/ magnolol was more than 0.7, whereas the ratio-was less than 0.7 in the case of MOB, and the concentrations of honokiol in MO were higher than those in MOB.

[Photosynthesis of five magnolia species in Nanjing City in winter and spring].

The photosynthesis and fluorescence characteristics of five magnolia ornamental species naturally distributed in various subtropical areas were studied in Nanjing City in winter and spring. The results showed that the diurnal changes of net photosynthetic rate (P(n)) and water use efficiency (WUE) of test species in winter were different from those in spring. The diurnal integral values of P(n), apparent quantum yield (AQY) and carboxylation efficiency (CE) were lower in winter than in spring, and the F(o) was higher, while the F(v)/F(m) F(v)/F(o) phi PS II, F(v)'/F(m)', ETR, qP and NPQ were lower in winter than in spring. The differences among the five species were remarkable, among which, Parakmeria lotungensis and Michelia platypetala mainly distributed in middle-subtropical area had higher diurnal integral values of P(n), AQY, CE and LSP, but lower value of LCP than other species in winter and spring. Their F(v)/F(m), F(v)/F(o),phi PS II, F(v)'/F(m)', ETR, qP and NPQ were also higher, indicating that they had higher photosynthetic capacity and wider ecological ranges of light adaptability. Manglietia insignis and Michelia wilsonii mainly distributed in southern subtropical area had lower photosynthetic capacity, and their fluorescence parameters were also lower in winter. Grey correlation analysis showed the main factors affecting the P(n) of test magnolia species in winter were T(a) and PAR.

Species diversity, structure and dynamics of two populations of an endangered species, Magnolia dealbata (Magnoliaceae)

Little is known about the ecology and demography of the genus Magnolia. Magnolia dealbata Zucc. is an endangered species endemic to Mexico. Two contrasting populations of M. dealbata (one from the grasslands and other from a secondary cloud forest) were studied. We asked the following questions: (a) Are size structure (diameter at breast height, DBH) and infrutescence production significantly different between the two populations? (b) What are the populations' growth rates (lambda) based on an initial 1987 study? (c) Are the associated species diversity indices of these M. dealbata populations significantly different? The results show no significant differences between the population size structure (p=.094); the growth rates of the populations were 0.992 in grassland and 1.053 in secondary cloud forest. The number of infrutescences produced in year 2001 and DBH relationship were significantly linear (p<.001) in both populations, and there was no significant difference (p>.01) between their slopes. The diversity indices were not significantly different (p>.05), and only 54% of the species were common to both sites. Our study suggests that both populations are relatively stable and that the management history could impact more on the species composition than on the diversity indices.

Linear energy transfer dependence of the effects of carbon ion beams on adventitious shoot regeneration from in vitro leaf explants of Saintpaulia ionahta.

PURPOSE: To determine the effects of carbon ion beams with five different linear energy transfer (LET) values on adventitious shoots from in vitro leaf explants of Saintpaulia ionahta Mauve cultivar with regard to tissue increase, shoots differentiation and morphology changes in the shoots. MATERIALS AND METHODS: In vitro leaf explant samples were irradiated with carbon ion beams with LET values in the range of 31 approximately 151 keV/microm or 8 MeV of X-rays (LET = 0.2 keV/microm) at different doses. Fresh weight increase, surviving fraction and percentage of the explants with regenerated malformed shoots in all the irradiated leaf explants were statistically analysed. RESULTS: The fresh weight increase (FWI) and surviving fraction (SF) decreased dramatically with increasing LET at the same doses. In addition, malformed shoots, including curliness, carnification, nicks and chlorophyll deficiency, occurred in both carbon ion beam and X-ray irradiations. The induction frequency with the former, however, was far more than that with the X-rays. CONCLUSIONS: This work demonstrated the LET dependence of the relative biological effectiveness (RBE) of tissue culture of Saintpaulia ionahta according to 50% FWI and 50% SF. After irradiating leaf explants with 5 Gy of a 221 MeV carbon ion beam having a LET value of 96 keV/microm throughout the sample, a chlorophyll-deficient (CD) mutant, which could transmit the character of chlorophyll deficiency to its progeny through three continuous tissue culture cycles, and plantlets with other malformations were obtained.

Leaf anatomy and light acclimation in woody seedlings after gap formation in a cool-temperate deciduous forest.

The photosynthetic light acclimation of fully expanded leaves of tree seedlings in response to gap formation was studied with respect to anatomical and photosynthetic characteristics in a natural cool-temperate deciduous forest. Eight woody species of different functional groups were used; two species each from mid-successional canopy species (Kalopanax pictus and Magnolia obovata), from late-successional canopy species (Quercus crispula and Acer mono), from sub-canopy species (Acer japonicum and Fraxinus lanuginosa) and from vine species (Schizophragma hydrangeoides and Hydrangea petiolaris). The light-saturated rate of photosynthesis (Pmax) increased significantly after gap formation in six species other than vine species. Shade leaves of K. pictus, M. obovata and Q. crispula had vacant spaces along cell walls in mesophyll cells, where chloroplasts were absent. The vacant space was filled after the gap formation by increased chloroplast volume, which in turn increased Pmax. In two Acer species, an increase in the area of mesophyll cells facing the intercellular space enabled the leaves to increase Pmax after maturation. The two vine species did not significantly change their anatomical traits. Although the response and the mechanism of acclimation to light improvement varied from species to species, the increase in the area of chloroplast surface facing the intercellular space per unit leaf area accounted for most of the increase in Pmax, demonstrating the importance of leaf anatomy in increasing Pmax.

Species diversity, structure and dynamics of two populations of an endangered species, Magnolia dealbata (Magnoliaceae).

Little is known about the ecology and demography of the genus Magnolia. Magnolia dealbata Zucc. is an endangered species endemic to Mexico. Two contrasting populations of M. dealbata (one from the grasslands and other from a secondary cloud forest) were studied. We asked the following questions: (a) Are size structure (diameter at breast height, DBH) and infrutescence production significantly different between the two populations? (b) What are the populations' growth rates (lambda) based on an initial 1987 study? (c) Are the associated species diversity indices of these M. dealbata populations significantly different? The results show no significant differences between the population size structure (p=.094); the growth rates of the populations were 0.992 in grassland and 1.053 in secondary cloud forest. The number of infrutescences produced in year 2001 and DBH relationship were significantly linear (p<.001) in both populations, and there was no significant difference (p>.01) between their slopes. The diversity indices were not significantly different (p>.05), and only 54% of the species were common to both sites. Our study suggests that both populations are relatively stable and that the management history could impact more on the species composition than on the diversity indices.

Effects of pollen shortage and self-pollination on seed production of an endangered tree, Magnolia stellata.

BACKGROUND AND AIMS: Pollen limitation is a significant determinant of seed production, and can result from both insufficient pollen quantity (pollen shortage) and quality (mainly relating to self-pollination). For animal-pollinated tree species with large floral displays, pollen limitation may be determined by a balance between increased pollen quantity due to increased attractiveness for pollinators, countered by increased self-pollination due to increased geitonogamy. The contributions of pollen shortage and self-pollination on seed production were quantitatively examined in the natural pollination of an insect-pollinated, dichogamous, endangered tree, Magnolia stellata, which has a large, showy floral display. METHODS: Manual self- and cross-pollinations were conducted to determine the effects of selfing on seed production. The outcrossing rate was measured using microsatellite analyses of open-pollinated seeds, and the embryo mortality rate caused by self-pollination was indirectly estimated. The frequency of ovule mortality due to pollen shortage was also inferred using the embryo mortality and ovule survival rates from natural pollination. KEY RESULTS: The average fruit set, seed set per fruit, and ovule survival rate per tree from hand cross-pollination were 1.37, 3.15, and 3.34 times higher than those from hand self-pollination, respectively, indicating that self-pollination causes inbreeding depression for fruit and seed set. The multilocus-outcrossing rate (t(m)) was intermediate, 0.632, and the primary selfing rate was 0.657. This indicates that frequent geitonogamous selfing occurs. The ovule mortality rate due to pollen shortage and the embryo mortality rate due to self-pollination were estimated to be 80.8 % and 45.9 %, respectively. CONCLUSIONS: It is concluded that seed production of M. stellata is strongly limited by both pollen shortage and self-pollination. Inefficient beetle-pollination and the automimicry system via asynchronous flowering might be responsible for the high level of pollen shortage and frequent geitonogamy. This is despite a large, showy floral display and the dichogamous system of the species.

[Studies on the megasporogenesis and microsporogenesis and the development of their female and male gametophyte in Magnolia biloba].

The anther of Magnolia biloba is tetrasporangiate with glandular tapetum, which consists of one or two layers of cells. Cytokinesis during meiosis of its microspore mother cell is modified simultaneous type, and the microspore tetrads are isobilateral. Mature pollen grains are two-celled. Tetrad cells and microspores are irregularly shaped during the microsporogenesis. There were two ovules on the ventral surface of unicarpellate ovary wall. Ovules were anatropous, bitegmnous and crassinucellar. Archesporial cell was one cell and differentiated from cell in the second layer beneath epidermis. The development of the embryo sac conformed to the polygonum type. The embryological characteristics of Magnolia biloba are very similar to those of other species in Magnoliaceae. The megasporogenesis and microsporogenesis and the development of their female and male gametophyte are partially abnormal. Abnormal phenomena in the process of reproduction of Magnolia biloba causing this species to be endangered was discussed.

Size distribution and genetic structure in relation to clonal growth within a population of Magnolia tomentosa Thunb. (Magnoliaceae).

To establish a baseline for conservation of a threatened clonal tree, Magnolia tomentosa, we investigated size distribution and genetic structure within a population, using six microsatellite markers. Within the study site, 1044 living ramets (stems) were distinguished into 175 genets (individuals). The mean number of ramets per genet was 5.97, and 76% of all genets had multiple ramets. Genets, which apparently produced new ramets through sprouting and layering, were generally composed of several large ramets and many small ramets. Spatial autocorrelation analysis of microsatellite alleles revealed positive autocorrelation over short distances for both ramets and genets. The Moran's I-value of ramets in the shortest distance class was 3.8 times larger than that of genets, reflecting the effect of clonal growth. To analyse the size-class differences in genetic structure, the 175 genets were separated into two size classes, small and large. The correlogram for the small genets exhibited positive spatial autocorrelation in the shortest distance class, but this was not the case for the correlogram for the large genets, indicating that genetic structure is weakened or lost through self-thinning as the genets grow. The FIS value over all loci for the small genets was positive and deviated significantly from zero, while the corresponding value for the large genets was close to zero. The excess homozygotes in the small genets may be the result of genetic substructuring and/or inbreeding, and the reduction in homozygote frequency from the small to large genets may be because of loss of genetic structure and/or inbreeding depression.

Variations in transpiration rate and leaf cell turgor maintenance in saplings of deciduous broad-leaved tree species common in cool temperate forests in Japan.

To clarify mechanisms underlying variation in transpiration rate among deciduous broad-leaved tree species, we measured diurnal changes in stomatal conductance (gs) and leaf water potential, and calculated the maximum transpiration rate (Emax), leaf-specific hydraulic conductance (K(s-l)) and difference between the soil water potential and the daily minimum leaf water potential (Psis - Psi(l,min)). Pressure-volume (P-V) measurements were made on leaves. Saplings of eight broad-leaved tree species that are common in Japanese cool temperate forests were studied. Maximum transpiration rate varied significantly among species. There was a statistically significant difference in Psis - Psi(l,min), but not in K(s-l). Species with large Emax also had large Psis - Psi(l,min) and gs. The results of the P-V analyses showed that species with a large Psis - Psi(l,min) maintained turgor even at low leaf water potentials. The similar daily minimum leaf pressure potentials (Psip) across all eight species indicate that Psip values below this minimum are critical. Based on these results, we suggest that the leaf cell capacity for turgor maintenance strongly affects Psis - Psi(l,min) and consequently Emax via stomatal regulation.