Fungal association and root morphology shift stepwise during ontogenesis of orchid Cremastra appendiculata towards autotrophic nutrition

The chlorophyllous, terrestrial orchid Cremastra appendiculata from East Asia is unique concerning its fungal mycorrhiza partners. The initially mycoheterotrophic protocorms exploit rather specialized non-rhizoctonia saprotrophic Psathyrellaceae. Adult individuals of this orchid species are either linked to Psathyrellaceae being partially mycoheterotrophic or form mycorrhiza with fungi of the ubiquitous saprotrophic rhizoctonia group. This study provides new insights on nutrition mode, subterranean morphology and fungal partners across different life stages of C. appendiculata. We compared different development stages of C. appendiculata to surrounding autotrophic reference plants based on multi-element natural abundance stable isotope analyses (δ(13)C, δ(15)N, δ(2)H, δ(18)O) and total N concentrations. Site- and sampling-time-independent enrichment factors of stable isotopes were used to reveal trophic strategies. We determined mycorrhizal fungi of C. appendiculata protocorm, seedling and adult samples using high-throughput DNA sequencing. We identified saprotrophic non-rhizoctonia Psathyrellaceae as dominant mycorrhizal fungi in protocorm and seedling rhizomes. In contrast, the roots of seedlings and mature C. appendiculata were mainly colonized with fungi belonging to the polyphyletic assembly of rhizoctonia (Ceratobasidium, Thanatephorus and Serendipitaceae). Mature C. appendiculata did not differ in isotopic signature from autotrophic reference plants suggesting a fully autotrophic nutrition mode. Characteristic of orchid specimens entirely relying on fungal nutrition, C. appendiculata protocorms were enriched in (15)N, (13)C and (2)H compared to reference plants. Seedlings showed an intermediate isotopic signature, underpinning the differences in the fungal community depending on their subterranean morphology. In contrast to the suggestion that C. appendiculata is a partially mycoheterotrophic orchid species, we provide novel evidence that mature C. appendiculata with rhizoctonia mycobionts can be entirely autotrophic. Besides an environmentally driven variability among populations, we suggest high within-individual flexibility in nutrition and mycobionts of C. appendiculata, which is subject to the ontogenetic development stage

Plant regeneration from protocorm-derived callus of Cyripedium formosanum

Summary Totipotent callus of Cypripedium formosanum, an endangered slipper orchid species, was induced from seed-derived protocorm segments on a quarter-strength Murashige and Skoog medium containing 4.52 mM 2,4-dichlorophenoxyacetic acid and 4.54 mM 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (thidiazuron). This callus proliferated well and was maintained by subculturing on the same medium. On average, 13 protocorm-like bodies could be obtained from a piece of 4 mm callus after being transferred to the medium with 4.44 mM N 6 -benzyladenine after 8 wk of culture. The regenerated protocormlike bodies formed shoots and roots on medium containing 1 g l 21 activated charcoal and 20 g l 21 potato homogenate. After 24 wk of culture on this medium, well-developed plantlets ready for potting were established

Taxonomic placement of Paphiopedilum rungsuriyanum (Cypripedioideae; Orchidaceae) based on morphological, cytological and molecular analyses

Additional file 9: Table S4. The comparison of main significant traits between subgenera and sections of Paphiopedilum by Gorniak et al. (2014) and the present study

Increased Expression of 9-Cis-Epoxycarotenoid Dioxygenase, PtNCED1, Associated With Inhibited Seed Germination in a Terrestrial Orchid, Phaius tankervilliae

The phytohormone abscisic acid (ABA) is involved in regulating seed dormancy and germination. A crucial step of ABA biosynthesis in higher plants is the oxidative cleavage of cis-epoxycarotenoids by 9-cis-epoxycarotenoid dioxygenase (NCED). Seed development in orchids is unusual because the embryos are minute in size, without obvious histodifferentiation, and lack endosperm. To understand the regulation of ABA biosynthesis in orchid seeds, we isolated and characterized a full-length cDNA encoding an NCED homolog, PtNCED1, from developing seeds of an ornamental orchid, Phaius tankervilliae. Germination percentage was high at 90 days after pollination (DAP), when a globular embryo proper with a degenerating suspensor was evident. After 90 DAP, seed maturation was accompanied by a decrease in water content and a concomitant increase in ABA content and PtNCED1 mRNA level along with a marked decrease in germination percentage. Mature seeds pretreated with NaOCl solution lowered ABA content and improved seed germination. Moreover, after seed germination, developing protocorms could respond to dehydration stress. Dehydration of protocorms stimulated an increase in PtNCED1 level along with ABA content. Our results provide evidence of the involvement of PtNCED1 in regulating endogenous ABA content in developing seeds and protocorms. The accumulation of endogenous ABA content in orchid seeds may have a critical role in seed dormancy and the protocorm response to water stress after seed germination

Embryology of a Lady’s Slipper Orchid, Paphiopedilum spicerianum and Cytokinin Requirements for Seed Germination and Protocorm Development

In this study, we document the primary structural changes that occur during the seed development of Paphiopedilum spicerianum (Rchb.f.) Pfitzer, an endangered species with high horticultural value. Within a defined timeline, our results offer insights into the connection between these structural changes in seeds and their germination percentage. The optimum germination was recorded for immature seeds collected at 180 to 210 days after pollination (DAP), during which the embryos are in the late globular stage and the suspensor begins to degenerate. As seeds continued to mature by 240 DAP, there was a gradual decline in germination. Histochemical staining of mature seeds reveals that only the inner seedcoat and the surface of the embryo exhibit positive reactions to the Nile red stain, suggesting a relatively weak coat-imposed dormancy. This weaker dormancy may contribute to the higher germination observed in mature seeds of P. spicerianum compared with other challenging-to-germinate species. Of the cytokinins examined, 6-(γ,γ-dimethylallylamino)purine (2iP), kinetin (KN), and 6-benzylaminopurine (BA) exhibited a stimulating effect on germination, concurrently enhancing the formation of amorphous protocorms

Expression Profiles of Phosphoenolpyruvate Carboxylase and Phosphoenolpyruvate Carboxylase Kinase Genes in Phalaenopsis, Implications for Regulating the Performance of Crassulacean Acid Metabolism

Phalaenopsis is one of the most important potted plants in the ornamental market of the world. Previous reports implied that crassulacean acid metabolism (CAM) orchids at their young seedling stages might perform C3 or weak CAM photosynthetic pathways, but the detailed molecular evidence is still lacking. In this study, we used a key species in white Phalaenopsis breeding line, Phalaenopsis aphrodite subsp. formosana, to study the ontogenetical changes of CAM performance in Phalaenopsis. Based on the investigations of rhythms of day/night CO2 exchange, malate contents and phosphoenolpyruvate carboxylase (PEPC) activities, it is suggested that a progressive shift from C3 to CAM occurred as the protocorms differentiated the first leaf. To understand the role of phosphoenolpyruvate carboxylase kinase (PEPC kinase) in relation to its target PEPC in CAM performance in Phalaenopsis, the expression profiles of the genes encoding PEPC (PPC) and PEPC kinase (PPCK) were measured in different developmental stages. In Phalaenopsis, two PPC isogenes were constitutively expressed over a 24-h cycle similar to the housekeeping genes in all stages, whereas the significant day/night difference in PaPPCK expression corresponds to the day/night fluctuations in PEPC activity and malate level. These results suggest that the PaPPCK gene product is most likely involved in regulation of CAM performance in different developmental stages of Phalaenopsis seedlings

What is the Difference Between ABNORMAL CLIMATE and EXTREME CLIMATE that Cause Yield Damage to Silage Maize (Zea mays L.)?

This study aimed to compare the characteristics by estimating the impacts of abnormal climate and extreme climate on silage maize. In the maize-climate database, the variables were dry matter yield (DMY, kg/ha), mean temperature (MT, °C), lowest temperature (LT, °C), highest temperature (HT, °C), maximum precipitation (MP, mm/hr), accumulated precipitation (AP, mm), maximum wind speed (MW, m/s), mean wind speed (WS, m/s), sunshine duration (SD, hr). To define the abnormal climate, favorable condition was confirmed using the principal component analysis against a normal climate. In order to define the extreme climate, firstly, the median-interquartile method was used instead of the mean-standard deviation method. As a result, in mid-June, the characteristics of early monsoons without typhoons were confirmed as poor. In particular, MP in abnormal climate (20.54 mm/hr) was three times greater than that in normal climate (6.61 mm/hr). The damage in DMY caused by the abnormal climate in mid-June was estimated to be 1,155.9 kg/ha. For the extreme climate in mid-June, the low-extreme LT, high-extreme AP, and high-extreme MW were detected in the trends. However, the year records were different for each point. Thus, the damage in DMY was estimated based on high-extreme AP, as a representative scenario. As a result, the damage in DMY caused by the extreme climate in mid-June was estimated to be 927.2 kg/ha. Hence, the magnitude of yield damage to silage maize caused by extreme climate and abnormal climate, as well as characteristics of high precipitation, were similar

Domain Walls and Flux Tubes in N=2 SQCD: D-Brane Prototypes

This paper could have been entitled "D branes and strings from flesh and blood." We study field theoretic prototypes of D branes/strings. To this end we consider (2+1)-dimensional domain walls in (3+1)-dimensional N=2 SQCD with SU(2) gauge group and two quark flavors in the fundamental representation. This theory is perturbed by a small mass term of the adjoint matter which, in the leading order in the mass parameter, does not break N=2 supersymmetry, and reduces to a (generalized) Fayet-Iliopoulos term in the effective low-energy N=2 SQED. We find 1/2 BPS-saturated domain wall solution interpolating between two quark vacua at weak coupling, and show that this domain wall localizes a U(1) gauge field. To make contact with the brane/string picture we consider the Abrikosov-Nielsen-Olesen magnetic flux tube in one of two quark vacua and demonstrate that it can end on the domain wall. We find an explicit 1/4 BPS-saturated solution for the wall/flux tube junction. We verify that the end point of the flux tube on the wall plays the role of an electric charge in the dual (2+1)-dimensional SQED living on the wall. Flow to N=1 theory is discussed. Our results lead us to a conjecture regarding the notorious "missing wall" in the solution of Kaplunovsky et al.Comment: 41 pages, 5 figures, Sect. 9.3 expanded, typos correcte

Genetic Elimination of Connective Tissue Growth Factor in the Forebrain Affects Subplate Neurons in the Cortex and Oligodendrocytes in the Underlying White Matter

Connective tissue growth factor (CTGF) is a secreted extracellular matrix-associated protein, which play a role in regulating various cellular functions. Although the expression of CTGF has been reported in the cortical subplate, its function is still not clear. Thus, to explore the significance of CTGF in the brain, we created a forebrain-specific Ctgf knockout (FbCtgf KO) mouse model. By crossing Ctgffl/fl mice with Emx1-Cre transgenic mice, in which the expression of Cre is prenatally initiated, the full length Ctgf is removed in the forebrain structures. In young adult (2–3 months old) FbCtgf KO mice, subplate markers such as Nurr1 and Cplx3 are still expressed in the cortical layer VIb; however, the density of the subplate neurons is increased. Interestingly, in these mutants, we found a reduced structural complexity in the subplate neurons. The distribution patterns of neurons and glial cells, examined by immunohistochemistry, are comparable between genotypes in the somatosensory cortex. However, increased densities of mature oligodendrocytes, but not immature ones, were noticed in the external capsule underneath the cortical layer VIb in young adult FbCtgf KO mice. The features of myelinated axons in the external capsule were then examined using electron microscopy. Unexpectedly, the thickness of the myelin sheath was reduced in middle-aged (>12 months old), but not young adult FbCtgf KO mice. Our results suggest a secretory function of the subplate neurons, through the release of CTGF, which regulates the density and dendritic branching of subplate neurons as well as the maturation and function of nearby oligodendrocytes in the white matter