Temporal changes in soil bacterial and archaeal communities with different fertilizers in tea orchards.

It is important to understand the effects of temporal changes in microbial communities in the acidic soils of tea orchards with different fertilizers. A field experiment involving organic fertilizer (OF), chemical fertilizer (CF), and unfertilized control (CK) treatments was arranged to analyze the temporal changes in the bacterial and archaeal communities at bimonthly intervals based on the 16S ribosomal RNA (rRNA) gene using terminal restriction fragment length polymorphism (T-RFLP) profiling. The abundances of total bacteria, total archaea, and selected functional genes (bacterial and archaeal amoA, bacterial narG, nirK, nirS, and nosZ) were determined by quantitative polymerase chain reaction (qPCR). The results indicate that the structures of bacterial and archaeal communities varied significantly with time and fertilization based on changes in the relative abundance of dominant T-RFs. The abundancy of the detected genes changed with time. The total bacteria, total archaea, and archaeal amoA were less abundant in July. The bacterial amoA and denitrifying genes were less abundant in September, except the nirK gene. The OF treatment increased the abundance of the observed genes, while the CF treatment had little influence on them. The soil temperature significantly affected the bacterial and archaeal community structures. The soil moisture was significantly correlated with the abundance of denitrifying genes. Of the soil chemical properties, soil organic carbon was the most important factor and was significantly correlated with the abundance of the detected genes, except the nirK gene. Overall, this study demonstrated the effects of both temporal alteration and organic fertilizer on the structures of microbial communities and the abundance of genes involved in the nitrogen cycle.

Virus-induced gene silencing of PEAM4 affects floral morphology by altering the expression pattern of PsSOC1a and PsPVP in pea.

pea-MADS4 (PEAM4) regulates floral morphology in Pisum sativum L., however, its molecular mechanisms still remain unclear. Virus-induced gene silencing (VIGS) is a recently developed reverse genetic approach that facilities an easier and more rapid study of gene functions. In this study, the PEAM4 gene was effectively silenced by VIGS using a pea early browning virus (PEBV) in wild type pea JI992. The infected plants showed abnormal phenotypes, as the floral organs, especially the sepals and petals changed in both size and shape, which made the corolla less closed. The petals changed in morphology and internal symmetry with, the stamens reduced and carpel dehisced. Larger sepals and longer tendrils with small cauline leaves appeared, with some sepals turning into bracts, and secondary inflorescences with fused floral organs were formed, indicating a flower-to-inflorescence change. The infected plants also displayed a delayed and prolonged flowering time. The PEAM4-VIGS plants with altered floral morphology were similar to the pim (proliferating inflorescence meristem) mutant and also mimicked the phenotypes of ap1 mutants in Arabidopsis. The expression pattern of the homologous genes PsSOC1a and PsSVP, which were involved in flowering time and florescence morphological control downstream of PEAM4, were analyzed by real-time RT-PCR and mRNA in situ hybridization. PsSOC1a and PsSVP were ectopically expressed and enhanced in the floral meristems from PEAM4-silenced plants. Our data suggests that PEAM4 may have a similar molecular mechanism as AtAP1, which inhibits the expression of PsSOC1a and PsSVP in the floral meristem from the early stages of flower development. As such, in this way PEAM4 plays a crucial role in maintaining floral organ identity and flower development in pea.

Overexpression of the cucumber LEAFY homolog CFL and hormone treatments alter flower development in gloxinia (Sinningia speciosa).

Leafy (LFY) and LFY-like genes control the initiation of floral meristems and regulate MADS-box genes in higher plants. The Cucumber-FLO-LFY (CFL) gene, a LFY homolog in Cucumis sativus L. is expressed in the primordia, floral primordia, and each whirl of floral organs during the early stage of flower development. In this study, functions of CFL in flower development were investigated by overexpressing the CFL gene in gloxinia (Sinningia speciosa). Our results show that constitutive CFL overexpression significantly promote early flowering without gibberellin (GA(3)) supplement, suggesting that CFL can serve functionally as a LFY homolog in gloxinia. Moreover, GA(3) and abscisic acid (ABA) treatments could modulate the expression of MADS-box genes in opposite directions. GA(3) resembles the overexpression of CFL in the expression of MADS-box genes and the regeneration of floral buds, but ABA inhibits the expression of MADS-box genes and flower development. These results suggest that CFL and downstream MADS-box genes involved in flower development are regulated by GA(3) and ABA.

[Studies on the megasporogenesis and microsporegenesis and the development of the female gametophyte and male gametophyte in Camptotheca acuminate].

The ovarium is hypostasy in Camptotheca acuminate Decne.. It has a locule and an ovule. The ovule is pendulous, anatropous andunitegmic. The ovule of Camptotheca acuminate Decne. is pseudocrassinucellate ovule. The development of embryo sac is polygonum type. Cytokinesis during the meiosis of microspore mother cells is of simultaneous type. The arrangement of microspores in tetrad is tetrahedral and isobilateral. One-nucleate microspore is triangle. Maturity pollen is triangle, circular and square. This paper mainly studied the megasporogenesis and microsporogenesis, and studied the development of their female and male gametophyte in Camptotheca acuminate Decne., and preliminarily discussed the cause of the part pistil abortion in Camptotheca acuminate Decne.

[Morphological studies on direct regeneration of floral buds from in vitro cultures of sepal segments in Sinningia speciosa hiern].

The morphological changes in the cultures of sepal segments in Sinningia speciosa Hiern were observed with Zeiss Stemi 2000-C Stereomicroscope from 0 to 65 days after culture in vitro. The light yellow globular protuberances were observed on the cut edge and the surface of sepal segments after culture for 24 days. Then the globular protuberances grew bigger gradually. A lot of floral buds on the surface of sepal segments formed after culture for 60 days. The morphological changes in the cultures of sepal segments were studied with light microscopy after culture for 0 to 30 days as well. The cells of tissues of sepal segments arranged regularly and no dividing cell was observed on the first day culture. There appeared some small meristematic centers of dividing cells on cut edge and the lower epidermis on the 7th day after culture. To the 20th day culture in vitro, the floral organ primordia were differentiated on the cut edge. On the 30th day culture, floral buds with petal, stamen primordia were observed. In addition, the morphological changes in the cultures of sepal segment were studied during the 14 to 30 days culture with scanning microscopy as well.

[In vitro flowering of cultures from a hybrid of Cymbidium goeringii and C. hybridium].

Wild-type female spring orchid (Cymbidium goeringii) was crossed with male Cymbidium hybridium. Over eight hundred protocorm clones were obtained from hybrid offsprings. Among them, one protocorm clone was identified to differentiate visible floral buds two months after subculture in vitro (Plate I). The protocorms and shoots derived from this clone were further used in studying the effects of abscisic acid (ABA) and paclobutrazol (PP333) pretreatment as well as different concentrations of 6-benzyladenine (6-BA) on floral bud differentiation. The optimum combination of hormones in floral bud induction was 6-BA 1.0 mg/L and NAA 0.1 mg/L, and total frequency of floral bud formation was up to 31% (Table 1). The optimum length of shoots used in floral bud induction was 1-2 cm, and the frequency of floral bud formation was 19% (Table 1). The increase in total frequency was not significant in floral bud induction from protocorms and shoots with length of 1-2 cm or 2-4 cm cultured on MS medium containing 6-BA 1.0 mg/L and NAA 0.1 mg/L after pretreatment on MS medium supplemented with ABA 0.5 mg/L and PP333 0.5 mg/L for 35 d (Table 2).

Wrinkled petals and stamens 1, is required for the morphogenesis of petals and stamens in Lotus japonicus.

Although much progress has been made in understanding how floral organ identity is determined during the floral development, less is known about how floral organ is elaborated in the late floral developmental stages. Here we describe a novel floral mutant, wrinkled petals and stamens1 (wps1), which shows defects in the development of petals and stamens. Genetic analysis indicates that wps1 mutant is corresponding to a single recessive locus at the long arm of chromosome 3. The early development of floral organs in wps1 mutant is similar to that in wild type, and the malfunction of the mutant commences in late developmental stages, displaying a defect on the appearance of petals and stamens. In the mature flower, petals and stamen filaments in the mutant are wrinkled or folded, and the cellular morphology under L1 layer of petals and stamen filaments is abnormal. It is found that the expression patterns of floral organ identity genes are not affected in wps1 mutants compared with that of wild type, consistent with the unaltered development of all floral organs. Furthermore, the identities of epidermal cells in different type of petals are maintained. The histological analysis shows that in wps1 flowers all petals are irregularly folded, and there are knotted structures in the petals, while the shape and arrangement of inner cells are malformed and unorganized. Based on these results, we propose that Wps1 acts downstream to the class B floral organ identity genes, and functions to modulate the cellular differentiation during the late flower developmental stages.

[Hairy root induced by wild-type agrobacterium rhizogenes K599 in soybean, cucumber and garden balsam in vivo].

The plantlets of soybean, cucumber and garden balsam were inoculated by wild-type Agrobacterium rhizogenes K599, and hairy root was induced on inoculated sites in vivo. The frequencies of hairy root induction from wound cotyledons of soybean, cucumber and garden balsam were 100%, 65% and 91%, respectively. Moreover, hairy root was induced from healthy cucumber axillary bud with frequency of 10%. PCR analysis of hairy root DNA was conducted using the primers from rolC gene. The PCR results showed that all hairy root lines contained T-DNA. The established system should be ideal for studying soybean and cucumber nematode and garden balsam breeding of flower dwarf architecture.

[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.

[Effect of gibberellin on direct regeneration of floral buds from young flower buds in Sinningia speciosa Hiern].

The phenomena of direct regeneration of floral buds on young flower bud and sepal cultures in vitro were observed in Sinningia speciosa Hiern. The gibberellin concentration in medium was very important for inducing the direct regeneration of floral buds. No direct floral bud was regenerated when the concentration of gibberellin (GA) supplemented in medium was equal to or less than 0.5 mg/L. The young floral buds and sepals inoculated on modified Murashige and Skoog (MS) medium supplemented with 1.0-1.5 mg/L GA and 0.1 mg/L 6-benzylaminopurine (BA) for 40 days produced floral buds directly on the surface of the sepals. There were two types of the floral buds regenerated directly: one was only with floral buds, which was up to 14.3%; the other was with both floral and vegetative buds, which was up to 28.6%. The effect of exogenous GA on the formation of floral buds in Sinningia cultured in vitro is discussed.

[Regulation network and biological roles of LEAFY in Arabidopsis thaliana in floral development].

Recent research progress on regulation network and biological roles of LFY gene in Arabidopsis thaliana and its homologue genes in floral development are reviewed emphatically in the present paper. LFY gene expresses widely in both vegetative and reproductive tissues in different higher plants, therefore investigation on role of LFY gene on flowering is of general significance. LFY gene plays an important role to promote flower formation by interaction and coordination with other genes,such as TFL, EMF, AP1, AP2, CAL, FWA, FT, AP3, PI, AG, UFO, CO, LD, GA1 etc, and a critical level of LFY expression is essential. LFY gene not only controls flowering-time and floral transition,but also plays an important role in inflorescence and floral organ development. It was situated at the central site in gene network of flowering regulation,positively or negatively regulates the level or activities of flowering-related genes. Some physiological factors, such as carbon sources, phytohormones, affect directly or indirectly the expression and actions of LFY gene. This indicates that level of LFY expression can also be regulated with physiological methods. It is probable that we can explain the principal mechanism of flowering by regulation network of LFY gene.

[Expression of CFL gene during differentiation of floral and vegetative buds in cucumber cotyledonary nodes cultured in vitro].

CFL gene, a LFY homologue, was cloned from cucumber (Cucumis sativus L.). In this paper, in situ hybridization was performed to analyze the expression pattern of CFL gene at the stage of floral and vegetative buds differentiation in cucumber cotyledonary nodes cultured in vitro. The results showed that at the stage of floral differentiation, CFL gene was strongly expressed in primordia, floral organ primordia, and each whirl of floral organs at the early stage of their formation, but weakly expressed or not expressed in floral organs after their formation (Fig. 2). At the stage of vegetative bud differentiation, CFL gene was strongly expressed in meristem, leaf primordium and young leaves, and no apparent expression signal was detected in mature tissues (Fig. 3). The results suggest that the expression of CFL gene be necessary for the differentiation and formation of floral and vegetative primordias, and it plays an important role in floral and vegetative development in cucumber. The results also indicate that CFL gene involving in mitosis initiation, mitosis controlling, and transformation of vegetative meristem to floral meristem.

[Floral homeotic variants of in vitro seedlings in Sinningia speciosa Hiern].

Six types of floral homeotic variants of in vitro seedlings were observed in doubleflower sinningia. Type I, red and green mosaic petals exist in the outermost whorl of petal-whorls, 2.38%. Type II, the outermost whorl of petal-whorls exhibit green petals with thin yellow edge, 25.0%. Type III, green petals exist in the innermost side of normal red petal whorls, 1.78%. Type IV, multiple whorls of green petals exist in the inner side of normal sepals, no stamen and carpel, 1.67%. Type V, it exhibits duplicated whorls of sepals in the outermost, 7.14%. Type VI, it exists multiple whorls of green sepals, no petal, stamen and carpel, 0.12%. The total percentage of all types of floral homeotic variants is up to 38.1%. The distribution of nodal site of homeotic flowers were analyzed, and the results showed that the homeotic flower occurred mainly at the fourth and fifth nodes.

[Relationship between floral differentiation and endogenous hormones and polyamines contents in cucumber cotyledonary nodes cultured in vitro].

Changes of endogenous hormones and polyamines(PAs) contents during floral differentiation (0-6 day) in cucumber cotyledonary nodes cultured in vitro were determined by using HPLC. The results showed that all four endogenous hormones decreased obviously within 0-2 day and increased slightly within 4-5 day after culture. This indicated that low levels of indole-3-acetic acid (IAA), gibberelline(GA3) and abscisin acid(ABA) during 0-2 day were favorable to floral primordia differentiation, while high level of zeatin(ZT) during 3-5 day was favorable to floral organ primordia differentiation. Contents of spermine(Spm), spermidine(Spd) and cadavarine(Cad) were decreased within 0-1 day, but increased within 1-4 day, and then decreased again within 4-5 day after culture. On the contrary, content of putrescine(Put) was increased significantly on the first day and then declined during 1-6 day. Changes of PAs contents indicated that high levels of PAs and Put were favorable to floral primordia differentiation, the increasing of Spm after the second day was favorable to floral organ primordia differentiation, and change of Cad content may be one of the characteristic symbols between floral and vegetative bud differentiation.

[Studies on calcium sensitive period during floral differentiation of cucumber cotyledonary node cultures in vitro].

Cotyledonary nodes of cucumber cultured on calcium-free medium for 0, 1, 2, 3, 4, 5, 6d respectively, were transferred to medium with 6.0 mmol/L CaCl2 for 24h, then returned to calcium-free medium. Cotyledonary nodes cultured on calcium-free or 6.0 mmol/L CaCl2 medium for all time, were taken as controls. Results showed that cotyledonary nodes were transferred to 6.0 mmol/L CaCl2 medium for 24h during 0-3d after the beginning of culture, percentage of floral bud formation at cotyledonary nodes was increased significantly. Transferring cotyledonary nodes on the 3d day after the beginning of culture was achieved best effect, percentage of floral bud formation was up to 34.3%. We deduced that the calcium sensitive period during floral differentiation of cucumber cotyleddonary node cultured in vitro may be 0-4d after the beginning of culture.