Separation and Identification of Differentially Expressed Proteins in Pistillate Flowers between Different Mulberry Cultivars

[Objective] This study aimed to explore the proteins related to pistillate flower development in different mulberry cultivars. [Method] The total proteins of the pistillate flowers of two mulberry cultivars Dal0 （Morus atropurpurea Roxb.） and SG01 （Morus muIticaulis Perr.） were extracted, separated and detected through two- dimensional electrophoresis （2-DE） and mass spectrometry. [Result] There was sig- nificant difference in the expression of proteins from the pistillate flowers of different mulberry cultivars. From the 2-DE images of Dal0 and SG01, 445_＋17 and 425_＋12 protein spots were respectively detected. The expression levels of 75 protein spots differed significantly. Thirteen spots those were expressed at high levels and well separated were analyzed by mass spectrometry, and nine of them were identified successfully. The nine proteins are involved in the glycometabolism, protein and amino acid metabolism and defense responses during the development of mulberry pistillate flower after they were pollinated. [Conclusion] The findings will provide reference for further study on the molecular mechanism of mulberry pistillate flower de- velopment.

Pistillate Flower Development and Stigma Receptivity of Euphorbia pulcherrima

Objective] This study aimed to investigate the relationship between pistil-late flower developmental morphology and stigma receptivity in Euphorbia pulcherri-ma. [Method] One-year-old adult plants of E. pulcherrima cultivar ‘Mil enium’ were used as experimental materials to analyze pistil ate flower development process by macroscopic and scanning electron microscope （SEM） observation. Stigma receptivity was detected with benzidine-hydrogen peroxide method and in vivo pol en germina-tion method. [Result] The pistil ate flower development process of E. pulcherrima was consisted of columnar stigma phase, stigma lobe slightly opening phase, Y-shape stigma lobe phase, inverse V-shaped stigma lobe phase and stigma curling phase. Pistil ate flower development and stigma receptivity of E. pulcherrima exhibit-ed certain correlation. The stigma receptive period lasted from stigma lobe slightly opening phase to inverse V-shaped stigma lobe phase; inverse V-shaped stigma lobe phase was appropriate for pol ination, lasting for 3-5 d, with V-shaped stigma lobe and a large amount of exudates on stigma surface according to SEM obser-vation. Stigma receptivity of E. pulcherrima detected with benzidine-hydrogen perox-ide method was consistent with that detected in vivo pol en germination method. [Conclusion] ln breeding practice, the optimal pol ination period of E. pulcherrima can be determined based on the developmental morphology of pistil ate flower.

Separation and Identification of Stage-specific Proteins in Pistillate Flowers of Mulberry

In order to excavate the function genes of stage-specific proteins in the development process of mulberry pistillate flowers, using fruit mulberry cuhivar ＇ Da 10＇ as experiment material, two-dimensional electrophoresis and mass-spectrometric technology were used to investigate specifically expressed proteins of mul- berry pistillate flowers in different flowering periods. The results showed that 471 ± 4,450 ± 15 and 446 ± 14 protein spots were determined in mulberry pistil- late flowers at full-bloom stage, initial flowering stage and terminal flowering stage respectively, including nine protein spots with characteristics of stage-specific ex- pression and clear electrophoretic bands.. By mass spectrometry analysis, database retrieval and bioinformatics analysis, five components were successfully identi- fied as lactoylglutathione lyase-like protein, perchloric acid soluble translation inhibitor protein, ubiquitin-conjugating enzyme 1 like protein, putative ethylene re- sponse protein and 3-hydroxyisobutyrate dehychngenase, which were involved in stress resistance reaction, protein catabolism, signal transduction, glycometabolism and other biological processes in plants, which indicated that these proteins might play an important biological function in the normal development and pollination fertilization of mulberry pistillate flowers.

Morphogenesis of Pistillate Flowers of Cercidiphyllum japonicum(Cercidiphyllaceae)

Floral morphogenesis and the development of Cercidiphyllumjaponicum Sieb. et Zucc. were observed by scanning electron microscopy （SEM）. The results showed that the pistillate inflorescences were congested spikes with the flowers arranged opposite. Great differences between the so-called ＂bract＂ and the vegetative leaf were observed both in morphogenesis and morphology. In morphogenesis, the ＂bract＂ primordium is crescent-shaped, truncated at the apex and not conduplicate, has no stipule primordium at the base but does have some inconspicuous teeth in the margin that are not glandular. The leaf primordium is triangular, cycloidal at the apex, conduplicate, has two stipule primordia at the base, has one gland-tooth at the apex occurring at first and some gland-teeth in the margin that occur later. In morphology, the ＂bract＂ is also different to the vegetative leaf in some characteristics that were also illustrated in the present paper. Based on the hypothesis that the bract is more similar to the vegetative leaf than the tepal, we considered that the so-called ＂bract＂ of C.japonicum might be the tepal of the pistillate flower in morphological nature. Therefore, each pistillate flower contains a tepal and a carpel. We did not find any trace of other floral organs in the morphogenesis of the pistillate flower. Therefore we considered that the unicarpellate status of extant Cercidiphyllum might be to highly reduce and advance characteristics that make the extant Cercidiphyllum isolated from both fossil Cercidiphyllum-like plants and its extant affinities.

Bulked Segregant RNA-Seq Analysis of Pollinated Pistils Reveals Genes Influencing Spikelet Fertility in Rice

Prezygotic isolation is important for successful fertilization in rice, significantly affecting yield. This study focused on F_(5:6) generation plants derived from inter-subspecific crosses(Nipponbare × KDML105) with low(LS) and high seed-setting rates(HS), in which normal pollen fertility was observed. However, LS plants showed a reduced number of pollen grains adhering to the stigma and fewer pollen tubes reaching the ovules at 4-5 h post-pollination, compared with HS plants. Bulked segregant RNA-Seq analysis of pollinated pistils from the HS and LS groups revealed 249 and 473 differentially expressed genes(DEGs), respectively. Kyoto Encyclopedia of Genes and Genomes analysis of the HS and LS-specific DEGs indicated enrichment in metabolic pathways, pentose and glucuronate interconversions, and flavonoid biosynthesis. Several of these DEGs exhibited co-expression with pollen development genes and formed extensive clusters of co-expression networks. Compared with LS pistils, enzyme genes controlling pectin degradation, such as OsPME35 and OsPLL9, showed similar expression patterns, with higher levels in HS pistils pre-pollination. Os02g0467600, similar to cinnamate 4-hydroxylase gene(CYP73), involved in flavonoid biosynthesis, displayed higher expression in HS pistils post-pollination. Our findings suggest that OsPME35, OsPLL9, and Os02g0467600 contribute to prezygotic isolation by potentially modifying the stigma cell wall(OsPME35 and OsPLL9) and controlling later processes such as pollen-stigma adhesion(Os02g0467600) genes. Furthermore, several DEGs specific to HS and LS were co-localized with QTLs and functional genes associated with spikelet fertility. These findings provide valuable insights for further research on rice spikelet fertility, ultimately contributing to the development of high-yielding rice varieties.

Flower morphology of Allium(Amaryllidaceae)and its systematic significance

Allium is a complicated genus that includes approximately 1000 species.Although its morphology is well studied,the taxonomic importance of many morphological traits,including floral traits,are poorly understood.Here,we examined and measured the floral characteristics of 87 accessions of 74 Allium taxa(belonging to 30 sections and nine subgenera)from Central to Eastern Asian countries.We then examined the taxonomic relationships between select flower characteristics and a phylogenetic tree based on ITS sequences.Our results confirm that floral morphology provides key taxonomic information to assess species delimitation in Allium.We found that perianth color is an important characteristic within the subg.Melanocrommyum,Polyprason,and Reticulatobulbosa.In subg.Allium,Cepa,and Rhizirideum,significant characteristics include ovary shape,perianth shape,and inner tepal apex.For species in subg.Angunium,the key taxonomic character is ovule number(only one ovule in per locule).In the subg.Allium,Cepa,Polyprason,and Reticulatobulbosa,which belong to the third evolutionary line of Allium,hood-like appendages occur in the ovary,although these do not occur in subg.Rhizirideum.Our results also indicated that the flower morphology of several species in some sections are not clearly distinguished,e.g.,sect.Sacculiferum(subg.Cepa)and sect.Tenuissima(subg.Rhizirideum).This study provides detailed photographs and descriptions of floral characteristics and information on general distributions,habitats,and phenology of the studied taxa.

A New Technique for Whole Clearing

[Objective] The research aimed to study a new whole clearing and preparation technology.[Method] After young seeds of Stephania sp.preserved in the alcohol solution,as well as the fresh pistil and nucellus of Sorghum bicolor were separated,they were soaked and cleared in the solution of 84 disinfectant.And the plant materials could be mounted by using three methods.[Result] The globular embryos and heart-shaped embryos at different stages of Stephania sp.as well as the pistil,nucellus,embryo sac,hypostase o...

Brassinosteroids mediate the effect of high temperature during anthesis on the pistil activity of photo-thermosensitive genetic male-sterile rice lines

Brassinosteroids(BRs)play critical roles in a wide range of plant developmental processes.However,it is unknown whether and how BRs mediate the effect of high temperature(HT)stress during anthesis on the pistil activity of photo-thermosensitive genetic male-sterile(PTSGMS)rice(Oryza sativa L.)lines.This study investigated the question.Three pot-grown PTSGMS rice lines were subjected to HT stress during anthesis.The contents of 24-epibrassinolide(24-EBL)and 28-homobrassinolide(28-HBL),the major forms of BR in rice plants,and levels of reactive oxygen species(ROS)or antioxidants(AOS),hydrogen peroxide(H2O2),1-aminocylopropane-1-carboxylic acid(ACC),ascorbic acid(AsA),and catalase activity in pistils,were determined.HT stress significantly reduced the contents of both 24-EBL and 28-EBL relative to those under normal temperatures,but the reduction varied by PTSGMS line.A line with higher BR contents under HT stress showed lower contents of ACC and H2O2,higher catalase activity and AsA content in pistils,and higher fertilization rate,seed-setting rate,and seed yield when the line was crossed with a restorer line,indicating that higher levels of BRs increase HT stress resistance.Applying 24-EBL,28-HBL or an inhibitor of BR biosynthesis confirmed the roles of BRs in response to HT stress.The results suggest that BRs mediate the effect of HT stress on pistil activity during anthesis and alleviate the harm of HT stress by increasing AOS and suppressing ROS generation.

Alterations of Panicle Antioxidant Metabolism and Carbohydrate Content and Pistil Water Potential Involved in Spikelet Sterility in Rice under Water-Deficit Stress

Two rice genotypes with different drought tolerance, namely Jin 23B （drought tolerant） and Zhenshan 97B （drought sensitive）, were used to study the antioxidant enzyme activities, soluble sugar and starch contents in spikelets, pistil water potential and pollen number on a stigma under water-deficit stress at the flowering stage, which were involved in the spikelet sterility. Compared with respective controls, drought stress induced more serious decreases of superoxide dismutase （SOD）, peroxidase （POD） and catalase （CAT） activities and more significant increase of malonaldehyde （MDA） content in spikekets of Zhenshan 97B than in Jin 23B on 9 and 12 days after water stress （DAWS）. The soluble sugar and starch contents increased significantly in spikelets of Jin 23B, but decreased significantly in spikelets of Zhenshan 97B during 9-12 DAWS. The pistil maintained higher water potential in Jin 23B than in Zhenshan 97B during 3-6 DAWS and 9-12 DAWS. In addition, water stress induced more significant decrease in the pollen number on a stigma as well as the percentage of unfilled grains in Zhenshan 97B than in Jin 23B. Thus, it is suggested that water stress induced spikelet sterility by damaging antioxidant enzyme activities, reducing carbohydrate content in spikelets and decreasing pistil water potential at the flowering stage in rice.

Effects of simulated acid rain on fertility of litchi

The regulatory role of calcium in fertility of pollen and pistil under simulated acid rain was investigated. The germination percentage of pollen treated with acid rain of pH 4.5 was 9.42% lower than that of control, and that of pH 3.5, pH 2.5 and pH 1.5 were 22.47%, 45.49% and 71.62%, respectively. Simultaneously, the injury character of pollen was obviously observed when flowers were treated with acid rain of pH 3.5. The difference in fruit setting rate between the female flower treated with acid rain of pH 4.0 and the control was significant at p 〈 0.05. Ca（NO3 ）5 of 0.2-0.4 mmol/L could promote pollen germination under the stress of acid rain. The beneficial function was reduced when calcium concentration surpassed 0.8 mmol/L. Spraying 2 mmol/L Ca（NO3 ）5 reduced the injury of acid rain to pistil and increased fruit-setting rate significantly. The physiological importance of calcium during pollen germination and pistil development was also discussed.

Effects of Exogenous Calcium and Calcium Ion Inhibitor on Cold Resistance of Pistil of Kernel Apricot

With a kernel apricot cultivar Youzhu 07-3 as the test material, the effects of exogenous calcium and calcium inhibitor on the cold resistance of pistils were investigated. In the bud germination and enlargement period, exogenous calcium and calcium ion inhibitor were sprayed to the kernel apdcot trees, respectively. At the full-bloom stage, a certain number of flower branches of kernel apricot were sam- pied and subjected to low-temperature stress indoor, and the soluble sugar content, MDA content, SOD activity and POD activity in the pistils were determined. The results showed that exogenous calcium increased the soluble sugar content, reduced the MDA content and improved the SOD and POD activity in pistils, and it significantly relieved the cold damage to pistils and improved the cold resistance of pistils; while exogenous calcium ion inhibitor showed the opposite effects.

Viability of Stigmas of Pistils in Winter Rape： Peculiarity of Samples with Different Origins

The researches have been carried out in 2011-2012, in the experimental fields of Yuryev Plant Production Institute Kharkiv, Ukraine. The forms of ＂00＂ type （a low content of erucic acid and glucosinolates） and self-pollinated lines--110M-10, 127M-10, 132M-10 were studied; the forms of＂0＋＂ type--selfed lines 124-10, 221-10, 305-10; the forms of＂＋0＂ type--cultivars Victor, Janus, Emerald; forms of ＂＋＋＂ type--cultivars Fyedorovskiy, Marens, Uspikh, as well as, self-incompatible forms 5C, 15C-10, 32C-10 and the original sterile form of mutant origin were used. Viability in stigmas of the pistil lasted 3-7 days, in the forms of ＂＋0＂, ＂＋＋＂ type, 7-15 days in the cultivars and forms of ＂0＋＂, ＂00＂ type, 14-21 days in the self-incompatible and sterile forms. A fast loss of sensitivity to pollen by pistil＇s stigma was observed in the forms of＂＋0＂ and ＂＋＋＂ types. On the contrary, in self-incompatible and sterile forms was observed a gradual increase of sensitivity of pistil＇s stigma to pollination and then its gradual fall. A maximum of sensitivity of a pistil＇s stigma to pollen in ＂00＂ and ＂0＋＂ forms was on the 3rd-6th day after castration.

Gene transformation by in situ pistil delivery method in Brassica

Agrobacterium-mediated gene transformation in Brassica crops is a time-con-suming and labor-cost method （tissue culture and plant regeneration） in a genotype-depen-dent way. Here we described a new method 7n situ pistil delivery （ISPDy method with no tissue culture and adaptive to three Brassica species. Briefly this method can be stated as placement of a liquid drop containing binary vector DNA onto the cut surface of emasculated and artificial pollinated Brassica stigma. Transformation rate （Basta-resistant seedlings per 100 pistils） of different species ranged from 0.4% to 16.3%. Factors affecting transforma-tion success included tinning and buffer type. PCR and Southern blot confirmed transgenic events. Reciprocal cross showed reporter gene steadily inherited in nuclear transformed pattern. GUS histochemical assay of transgenic immature seeds showed stained embroys and endosperm in the same seed, indicating DNA delivered through style and integrated into Brassica seed/genome after double fertilization.

Diversity of Sex Types and Seasonal Sexual Plasticity in a Cucumber Germplasm Collection

The sex type of a cucumber plant is determined by the proportion of male, female and hermaphrodite flowers that it bears and is an important factor that affects fruit yield. In this paper, the sex types and seasonal sexual stabilities of 322 accessions of cucumber germplasm were identified. This germplasm collection displayed a great variety of sex types. We used an updated 10-type sex classification system based on the flower types present and the proportion of nodes with pistillate flowers(PNPF). The PNPF ranges of all the accessions were 2.12%–100% in spring and 0–100% in autumn. A total of 81.37% of the accessions had PNPFs of 10%–50% in spring, but most(84.78%) accessions were reduced to 0–20% PNPF in autumn. The range of reduction of PNPF from spring to autumn was 0–67.91%. In other words, most of the germplasm was normal monoecious(31.68%) or subandroecious(62.73%) in spring, but 94.10% of the accessions were subandroecious in autumn. According to the statistical evaluation of the difference in PNPFs between the two seasons, each accession could be classified into one of three groups: seasonally stable, seasonally sensitive and highly seasonally sensitive, accounting for 10.56%, 20.50% and 68.94% of the accessions, respectively. With a few exceptions, the seasonal PNPF differences were positively correlated with the PNPFs in a given season for most accessions. These results provided useful information and materials for sex expression mechanism research and for breeding cucumbers with high and stable yields.

Influences of Cross Pollination on Pollen Tube Growth and Fruit Set in Zuili Plums (Prunus salicina)

Zuili plum （Prunus salicina L.） trees usually set fruit poorly, although they produce high quality fruit. To elucidate the causes of the poor fruit set, pollen tube growth into pistils and fruit set percentage were investigated after cross-, self- and open-pollination. Ovule development in Zuili pistils was also investigated. Pollen tube penetration into the ovules via the obturator and micropyle was best when Zuili pistils were pollinated by cv. Black Amber （P. domestica） pollen grains, although cross-pollinations with Hongxinli and Miili （P. salicina） pollen were more effective than self- and open-pollination. The fruit set percentage was also highest in pistils pollinated with Black Amber pollen grains. Morphological observation of Zuili pistils revealed that the trees produce ＂double pistils＂, developing two ovaries from a basal pistil, at a rate as high as 28%. In such abnormal pistils, most ovules were lacking an embryo sac or were entirely degenerated. The percentage of normally developed ovules was 24.3% and 8.9% in normal and double pistils, respectively. From these results, we conclude that the main causes of poor fruit set of Zuili plums are a lack of effective cross-pollination and the production of high percentages of double pistils in which normally developed ovules are scarcely formed.

Anatomical studies of ovule development in the post-bloom pistils of the 'Zuili' plum (Prunus salicina Lindl.)

In order to investigate the cause of poor fruit set in 'Zuili' plums,anatomical examinations of post-bloom pistils were conducted and the dates of young fruit drop were recorded during the growing seasons of 2008 and 2009.Pistils of cv.'Black Amber' were also examined as an abundant setting control.Two major dropping periods were detected in 'Zuili':one during the first 5 d after full bloom (DAF) and another between 10 and 17 DAF.Anatomical analyses of the pistils at the full bloom stage revealed that half of the ovules had not developed embryos,which may have caused their early drop.In most dropped pistils collected at 17 DAF,the micropyle had not been penetrated by a pollen tube,indicating that they were not fertilized.'Zuili' ovules initiated embryo division at 10-12 DAF,although thereafter embryo development was retarded when compared to the rates observed in 'Black Amber'.Ovule fertilization failure and inactive embryo development after ovule fertilization may be the major causes of the later fruit drop observed in 'Zuili' plum trees.

Analyses of Sexual Reproductive Success in Transgenic and/or Mutant Plants

The pistil, the female reproductive organ of plants, is a key player in the success of sexual plant reproduction. Ultimately, the production of fruits and seeds depends on the proper pistil development and function. Therefore, the identification and characterization of pistil expressed genes is essential for a better understanding and manipulation of the plant reproduction process. For studying the function of pistil expressed genes, transgenic and/or mutant plants for the genes of interest are used. The present article provides a review of methods already exploited to analyze sexual reproductive success. We intend to supply useful information and to guide future experiments in the study of genes affecting pistil development and function.