Comparative transcriptome analysis of the effect of different heat shock periods on the unfertilized ovule in watermelon(Citrullus lanatus)

In vitro gynogenesis is an important tool used in haploid or homozygous double-haploid plant breeding.However,because of low repeatability,embryoid induction rate and quality,the molecular mechanisms remain poorly understood.Heat shock treatment can promote the transformation of the gametophytic pathway into the sporophyte pathway,which induces the occurrence of haploid.In this study,unfertilized ovaries were heat shocked for 0 h(A0)before flowering and for 0 h(A1),4 h(A3),8 h(A5),12 h(A7),and 24 h(A8),respectively,at 37℃at the first day of the flowering stage.The ovule enlargement rate was increased from 0%at 25℃to 96.8%at 37℃(24 h treatment).Thus,we aimed to investigate the gene expression patterns in unfertilized ovules of watermelon after different periods of heat shock by using RNA-Seq technology.The results showed that compared with A3,A5,A7,and A8,the biosynthesis of amino acid,glycine,serine and threonine metabolic pathways in A1 has changed significantly.This indicated that heat shock treatment affected the synthesis and transformation of amino acids during ovule expansion.The transcriptome data suggested gene expressions of ovule growth were significantly changed by heat-specific influences.The results provide new information on the complex relationship between in vitro gynogenesis and temperature.This provides a basis for further study of the mechanism of heat shock affecting the expansion of watermelon ovule.

New observation on seed/ovule position in the fruit of Archaeanthus and its systematic implications

1.Introduction Angiosperms are the most important group of plants in the current earth ecosystem.However,little is known about the origin of angiosperms,which has been a headache for many botanists.The core question in the origin of angiosperms is the homology of carpels.Formerly,Arber EAN and Parkin J(1907)speculated that the carpels in angiosperms were derived from the former megasporophylls bearing ovules along their margins.

Interspecific Crossing between Yam Species (Dioscorea rotundata and Dioscorea bulbifera) through in Vitro Ovule Culture

In the present study, in vitro ovule culture technique was used to obtain interspecific cross combination of Dioscorea rotundata ufenyi and Dioscorea bulbifera wild. Ten days after pollination, ovules were excised and cultured onto 1/2 strength Murashige and Skoog (MS) medium (Basal salt mixture + Vitamins) supplemented with 6% sucrose, 0.7% agar and plant growth hormones such as GA3, BAP, Picrolam and TDZ. Cultured ovules were transferred on 1/2 MS medium with 3% sucrose and 0.7% agar after three weeks. 40 days after pollination, germination was observed from 7 months cultured ovule between D. rotundata ufenyi x D. bulbifera wild. Hybridity of the regenerated plant was checked by flow cytometric method. A close relation was observed between the fluorescence intensity of the obtained progeny with one of the parents’ fluorescence. The observed progeny can be closely correlated with an apomictic tissue from an ovule parent of D. rotundata ufenyi. Plantlets derived from ovule culture were proliferated through in vitro shoot multiplication with hormonal concentration (0.5 mg/l BAP) supplemented with 1/2 strength MS medium. Obtained ovule culture derived in vitro plantlets were successfully hardened, acclimatized and transferred to the field, where they survived and grew normally. In plant breeding, interspecific crossing is very important technique, enabling the time needed to produce homozygous lines to be shortened as compared to the conventional plant breeding techniques.

Ectopic expression of a male fertility gene,LOGL8,represses LOG and hinders panicle and ovule development

Grain number and seed-setting rate are components of crop yield.Cytokinin influences grain yield.However,emerging studies suggest that high cytokinin signals often lead to reduced branching or seed-setting rate,leading to reduced grain yield,although the mechanisms remain unclear.In this study,we identified and characterized the rice(Oryza sativa L.)gene LONELY GUY-LIKE 8(LOGL8),based on analysis of the LOGL8-pm(promoter mutant of LOGL8)mutant,which harbors a T-DNA insertion in the promoter of this gene.The mutation in LOGL8-pm causes ectopic hyperexpression of LOGL8 in inflorescence organs,resulting in plants with smaller panicles and defective ovules lacking archesporial cells and integuments.Knockout of LOGL8 caused pollen abortion,leading to a reduced seed-setting rate.LOGL8 encodes a putative cytokinin-activating enzyme.Our results showed that LOGL8 directly catalyzes the biosynthesis of bioactive cytokinins.Therefore,we propose that the ectopic expression of LOGL8 disrupts cytokinin spatiotemporal distribution and causes inhibition of LONELY GUY(LOG),which affects panicle branching and female organ development.These findings reveal the important role of LOGL8 in male development,and highlight the delicate balance of local cytokinin levels during panicle branching and female organ development.

Global Annotation of Small RNA and MicroRNA Mature Sequences from Developing Ovules of Gossypium hirsutum L.

The involvement of small RNAs in cotton fiber development is under explored.The objective of this work was to directly clone,annotate,and analyze small RNAs of developing ovules to reveal

BR Signal Influences Arabidopsis Ovule and Seed Number through Regulating Related Genes Expression by BZR1

Ovule and seed developments are crucial processes during plant growth, which are affected by different signaling pathways. In this paper, we demonstrate that the brassinosteroid （BR） signal is involved in ovule initiation and development. Ovule and seed numbers are significantly different when comparing BR-related mutants to wild-type con-trols. Detailed observation indicates that BR regulates the expression level of genes related to ovule development, includ-ing HLL, ANT, and AP2, either directly by targeting the promoter sequences or indirectly via regulation by BR-induced transcription factor BZR1. Also, Western blot demonstrates that the dephosphorylation level of BZR1 is consistent with ovule and seed number. The intragenic bzrl-lD suppressors bzs247 and bzs248 have much fewer ovules and seeds than bzrl-lD, which are similar to wild-type, suggesting that the phenotype can be rescued. The molecular and genetic experi-ments confirm that BZR1 and AP2 probably affect Arabidopsis ovule number determination antagonistically.

Pollen-Ovule Ratio and Gamete Investment in Pedicularis （Orobanchaceae）

The Pedicularis species provides ideal materials to study floral evolution because of their substantial flower variation based on a narrow genetic basis, even though they are almost exclusively pollinated by bumblebee. These traits allow us to detect the evolutionary trends of floral parameters without considering genetic background and the difference of pollination vectors. The pollen-ovule ratio is widely used to estimate the pattern of resource investment in two sexual functions in flowering plants. Forty species representing all of the corolla types in Pedicularls were used to study pollen-ovule ratio, gamete investment, and their correlations. Results show that pollen-ovule ratio does not differ among both different corolla types and taxonomic groups. It is therefore suggested that pollen-ovule ratio should be a parallel evolution. The correlations between pollen-ovule ratio and pollen size （-）, and ovule size （＋） can be successfully explained in terms of sex allocation theory. The biological significance of such relationships was also discussed. Additionally, we analyzed the pattern of resource investment into female gamete, which has been somewhat neglected, and found that plants have different patterns of gamete investment between the two sexual functions.

Comparative analysis of SIMILAR to RCD ONE(SRO)family from tetraploid cotton species and their diploid progenitors depict their significance in cotton growth and development

Background SRO(Similar to RCD1)genes family is largely recognized for their importance in the growth,develop-ment,and in responding to environmental stresses.However,genome-wide identification and functional characteri-zation of SRO genes from cotton species have not been reported so far.Results A total of 36 SRO genes were identified from four cotton species.Phylogenetic analysis divided these genes into three groups with distinct structure.Syntenic and chromosomal distribution analysis indicated uneven distribu-tion of GaSRO,GrSRO,GhSRO,and GbSRO genes on A2,D5 genomes,Gh-At,Gh-Dt,Gb-At,and Gb-Dt subgenomes,respectively.Gene duplication analysis revealed the presence of six duplicated gene pairs among GhSRO genes.In promoter analysis,several elements responsive to the growth,development and hormones were found in GhSRO genes,implying gene induction during cotton growth and development.Several miRNAs responsive to plant growth and abiotic stress were predicted to target 12 GhSRO genes.Organ-specific expression profiling demonstrated the roles of GhSRO genes in one or more tissues.In addition,specific expression pattern of some GhSRO genes dur-ing ovule development depicted their involvement in these developmental processes.Conclusion The data presented in this report laid a foundation for understanding the classification and functions of SRO genes in cotton.

Ovule initiation:the essential step controlling offspring number in Arabidopsis

Seed is the offspring of angiosperms.Plants produce large numbers of seeds to ensure effective reproduction and survival in varying environments.Ovule is a fundamentally important organ and is the precursor of the seed.In Arabidopsis and other plants characterized by multi-ovulate ovaries,ovule initiation determines the maximal ovule number,thus greatly affecting seed number per fruit and seed yield.Investigating the regulatory mechanism of ovule initiation has both scientific and economic significance.However,the genetic and molecular basis underlying ovule initiation remains unclear due to technological limitations.Very recently,rules governing the multiple ovules initiation from one placenta have been identified,the individual functions and crosstalk of phytohormones in regulating ovule initiation have been further characterized,and new regulators of ovule boundary are reported,therefore expanding the understanding of this field.In this review,we present an overview of current knowledge in ovule initiation and summarize the significance of ovule initiation in regulating the number of plant offspring,as well as raise insights for the future study in this field that provide potential routes for the improvement of crop yield.

A Comparative miRNAome Analysis Reveals Seven Fiber Initiation-Related and 36 Novel miRNAs in Developing Cotton Ovules

An increasing number of microRNAs （miRNAs） have been shown to play crucial regulatory roles in the process of plant development. Here, we used high-throughput sequencing combined with computational analysis to characterize miRNAomes from the ovules of wild-type upland cotton and a fiberless mutant during fiber initiation. Comparative miR- NAome analysis combined with northern blotting and RACE-PCR revealed seven fiber initiation-related miRNAs expressed in cotton ovules and experimentally validated targets of these miRNAs are involved in different cellular responses and metabolic processes, including transcriptional regulation, auxin and gibberellin signal transduction, actin bundles, and lignin biosynthesis. This paper describes a complex regulatory network consisting of these miRNAs expressed in cotton ovules to coordinate fiber initiation responses. In addition, 36 novel miRNAs and two conserved miRNAs were newly iden- tified, nearly doubling the number of known cotton miRNA families to a total of 78. Furthermore, a chromatin remodeling complex subunit and a pre-mRNA splicing factor are shown for the first time to be miRNA targets. To our knowledge, this study is the first systematic investigation of fiber initiation-related miRNAs and their targets in the developing cotton ovule, deepening our understanding of the important regulatory functions of miRNAs in cotton fiber initiation.

A Malvaceae-specific miRNA targeting the newly duplicated GaZIP1L to regulate Zn^(2+)ion transporter capacity in cotton ovules

MicroRNAs(miRNAs)play critical roles in regulating gene expression in plants,yet their functions underlying cultivated diploid Gossypium arboreum cotton ovule development are largely unknown.Here,we acquired small RNA profiles from G.arboreum ovules and fibers collected at different growth stages,and identified 46 novel miRNAs that accounted for 23.7%of all miRNAs in G.arboreum reported in the latest plant sRNA database.Through analysis of 84(including 38 conserved)differentially expressed G.arboreum miRNAs,we detected 215 putative protein-coding genes in 26 biological processes as their potential targets.A Malvaceae-specific novel miRNA named gar-miRN44 was found to likely regulate cotton ovule growth by targeting to a newly duplicated Zn^(2+)ion transporter gene GaZIP1L.During cotton ovule development,gar-miRN44 transcript level decreased sharply after 10 to 15 days post-anthesis(DPA),while that of the GaZIP1L increased significantly,with a concomitant increase of Zn^(2+)ion concentration in late ovule developmental stages.Molecular dynamics simulation and ion absorption analysis showed that GaZIP1L has stronger Zn2+ion binding ability than the original GaZIP1,indicating that the newly evolved GaZIP1L may be more suitable for maintaining high Zn2+ion transport capacity that is likely required for cotton ovule growth via enhanced cellulose synthase activities.Our systematic miRNA profiling in G.arboreum and characterization of gar-miRN44 not only contribute to the understanding of miRNA function in cotton,but also provide potential targets for plant breeding.

Interaction of brassinosteroid and cytokinin promotes ovule initiation and increases seed number per silique in Arabidopsis

Ovule initiation is a key step that strongly influences ovule number and seed yield.Notably,mutants with enhanced brassinosteroid(BR)and cytokinin(CK)signaling produce more ovules and have a higher seed number per silique(SNS)than wild-type plants.Here,we crossed BR-and CKrelated mutants to test whether these phytohormones function together in ovule initiation.We determined that simultaneously enhancing BR and CK contents led to higher ovule and seed numbers than enhancing BR or CK separately,and BR and CK enhanced each other.Further,the BR-response transcription factor BZR1 directly interacted with the CK-response transcription factor ARABIDOPSIS RESPONSE REGULATOR1(ARR1).Treatments with BR or BR plus CK strengthened this interaction and subsequent ARR1 targeting and induction of downstream genes to promote ovule initiation.Enhanced CK signaling partially rescued the reduced SNS phenotype of BR-deficient/insensitive mutants whereas enhanced BR signaling failed to rescue the low SNS of CK-deficient mutants,suggesting that BR regulates ovule initiation and SNS through CK-mediated and-independent pathways.Our study thus reveals that interaction between BR and CK promotes ovule initiation and increases seed number,providing important clues for increasing the seed yield of dicot crops.

The signals to trigger the initiation of ovule enlargement are from the pollen tubes: The direct evidence

In angiosperms, initiation of ovule enlargement represents the start of seed development, the molecular mechanism of which is not yet elucidated. It was previously reported that pollen tube contents, rather than double fertilization, can trigger ovule enlargement. However, it remains unclear whether the signal（s） to trigger the initiation of ovule enlargement are from the sperm cells or fromthe pollen tubes. Recently, we identified a mutant dropl- drop2-, which produces pollen tubes with no sperm cells. Taking advantage of this special genetic material, we conducted pollination assays, and found that the ovules pollinated with dropl- drop2- pollen could initiate the enlargement and exhibited significant enlarged sizes at 36h after pollination in comparison with those unpollinated ovules. However, the sizes of the ovules pollinated with drops- drop2- pollen are significantly smaller than those of the ovules pollinated with wildtype pollen. These results demonstrate that the pollen tube, rather than the sperm cells, release the signal to trigger the initiation of ovule enlargement, and that double fertilization is required for further enlargement of the seeds.

Proteo-transcriptomic profiles reveal key regulatory pathways and functions of LDHA in the ovulation of domestic chickens(Gallus gallus)

Background In poultry, the smooth transition of follicles from the preovulatory-to-postovulatory phase impacts egg production in hens and can benefit the poultry industry. However, the regulatory mechanism underlying follicular ovulation in avians is a complex biological process that remains unclear.Results Critical biochemical events involved in ovulation in domestic chickens(Gallus gallus) were evaluated by transcriptomics, proteomics, and in vitro assays. Comparative transcriptome analyses of the largest preovulatory follicle(F1) and postovulatory follicle(POF1) in continuous laying(CL) and intermittent laying(IL) chickens indicated the greatest difference between CL_F1 and IL_F1, with 950 differentially expressed genes(DEGs), and the smallest difference between CL_POF1 and IL_POF1, with 14 DEGs. Additionally, data-independent acquisition proteomics revealed 252 differentially abundant proteins between CL_F1 and IL_F1. Perivitelline membrane synthesis, steroid biosynthesis, lysosomes, and oxidative phosphorylation were identified as pivotal pathways contributing to ovulation regulation. In particular, the regulation of zona pellucida sperm-binding protein 3, plasminogen activator, cathepsin A, and lactate dehydrogenase A(LDHA) was shown to be essential for ovulation. Furthermore, the inhibition of LDHA decreased cell viability and promoted apoptosis of ovarian follicles in vitro.Conclusions This study reveals several important biochemical events involved in the process of ovulation, as well as crucial role of LDHA. These findings improve our understanding of ovulation and its regulatory mechanisms in avian species.

Usefulness of Newly Devised Clomiphene Citrate Administration Method Compared with the Conventional Method in Ovulation and Pregnancy

Objective: Since not all women wish to conceive a child through aggressive treatment, we investigated the usefulness of modified repeated intracyclic clomiphene citrate (CC) therapy (repeated CC therapy) as a newly devised administration method. Methods: We evaluated the effects of CC administration on menstrual cycle length and retrospectively compared ovulation and pregnancy in 220 women who received CC at our hospital. Patients in the conventional method group received 50 mg per day for five days, starting on the fifth day of menstruation (withdrawal bleeding). Groups with and without menstrual cycle shortening after conventional CC administration were compared. The repeated CC therapy group was also compared with the non-shortened group. Repeated CC therapy was administered for the first five days as in the conventional method, and a second five-day repeat treatment was administered after an interval of five to seven days. Pregnancy rates, including indirect pregnancies, were evaluated by three different methods. Results: Ovulation and pregnancy rates were significantly better in the shortened group than in the non-shortened group (P < 0.001 and P = 0.010, respectively). Even in the non-shortened group, ovulation and pregnancy rates including indirect pregnancies were significantly improved when ovulation was observed with repeated CC therapy (P < 0.001 and P = 0.022, respectively). Conclusions: For patients whose menstrual cycle was not improved or shortened, repeated CC therapy as the newly devised CC administration method is useful as the next step after the conventional CC administration method.

Nicotiana ovule extracts induce nuclear reconstitution of demembranated Xenopus sperm in cell-free system

Nicotiana tabaccum ovule extracts induced nuclear reconstitution of demembranated Xenopus leavis sperm in a cell-free system. Demembranated Xenopus sperm began to swell after 15 min of incubation with Nicotiana ovule extracts. Accompanying the process of incubation, Xenopus sperm decondensed and their shapes changed gradually from long and ellipse to round. The completely decondensed chromatin was surrounded with membrane structure, which was a mixture envelope of a double membrane and a single membrane. Nucleosome assembly was verified by means of micrococcal nuclease digestion to re-constituted nuclei and DNA electrophoresis. Nicotiana ovule extracts supplied one more experimental model and system. The new system could promote powerfully the research on mechanisms of cell division and cell cycle regulation.

Tobacco floral homeotic gene Nfbp6 is specifically expressed during pollen and ovule development

Nfbp6 was a floral homeotic gene isolated from tobacco. It showed high homology with other class C genes. Using in situ hybridization technique, the expression of Nfbp6 was investigated in various stages of flower development. The results showed that Nfbp6 was expressed at low level during the initiation period of stamen and carpel primordia. In the later stage of flower bud development, the transcription level of Nfbp6 gene increased especially during the formation of pollen and ovule. The Nfbp6 gene expression was also observed in the transmitting tissue of style, stomium and circular cell clusters of the anther.

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.

Clinical Study on the Therapeutic Effect of Combined Traditional Chinese Medicine with Acupoint Injection and Herbal Paste Application in Treating Ovulatory Dysfunction in Polycystic Ovary Syndrome (PCOS)

Objective: To observe the therapeutic effect of combined traditional Chinese medicine (TCM) therapy with acupoint injection and herbal patch application on patients with ovulatory dysfunction associated with polycystic ovary syndrome (PCOS). Methods: A total of 80 PCOS patients who received treatment at the First Affiliated Hospital of Guangxi University of Chinese Medicine between October 2022 and March 2024 were enrolled in this study. They were randomly divided into a control group (n = 40) and an observation group (n = 40). The control group received conventional Western medical treatments, while the observation group was administered TCM combined with acupoint injection and herbal patch application. Pre- and post-treatment conditions were compared between the two groups. Results: After treatment, the overall effective rate was significantly higher in the observation group compared to the control group (P 0.05). Following treatment, both ovulation and pregnancy rates increased in both groups, but the increase was more evident in the observation group, showing a statistically significant difference (P < 0.01). Conclusion: For patients with oligoovulation or anovulation due to PCOS, the combined use of TCM, acupoint injection, and herbal patch application appears to be effective in improving systemic symptoms and achieving notable clinical outcomes.