Transcriptome sequencing and gene expression analysis revealed early ovule abortion of Paeonia ludlowii.

BACKGROUND: Paeonia ludlowii (Stern & G. Taylor D.Y. Hong) belongs to the peony group of the genus Paeonia in the Paeoniaceae family and is now classified as a "critically endangered species" in China. Reproduction is important for this species, and its low fruiting rate has become a critical factor limiting both the expansion of its wild population and its domestic cultivation. RESULTS: In this study, we investigated possible causes of the low fruiting rate and ovule abortion in Paeonia ludlowii. We clarified the characteristics of ovule abortion and the specific time of abortion in Paeonia ludlowii, and used transcriptome sequencing to investigate the mechanism of abortion of ovules in Paeonia ludlowii. CONCLUSIONS: In this paper, the ovule abortion characteristics of Paeonia ludlowii were systematically studied for the first time and provide a theoretical basis for the optimal breeding and future cultivation of Paeonia ludlowii.

Seedlessness Trait and Genome Editing-A Review.

Parthenocarpy and stenospermocarpy are the two mechanisms underlying the seedless fruit set program. Seedless fruit occurs naturally and can be produced using hormone application, crossbreeding, or ploidy breeding. However, the two types of breeding are time-consuming and sometimes ineffective due to interspecies hybridization barriers or the absence of appropriate parental genotypes to use in the breeding process. The genetic engineering approach provides a better prospect, which can be explored based on an understanding of the genetic causes underlying the seedlessness trait. For instance, CRISPR/Cas is a comprehensive and precise technology. The prerequisite for using the strategy to induce seedlessness is identifying the crucial master gene or transcription factor liable for seed formation/development. In this review, we primarily explored the seedlessness mechanisms and identified the potential candidate genes underlying seed development. We also discussed the CRISPR/Cas-mediated genome editing approaches and their improvements.

Combined Transcriptome Analysis Reveals the Ovule Abortion Regulatory Mechanisms in the Female Sterile Line of Pinus tabuliformis Carr.

Ovule abortion is a common phenomenon in plants that has an impact on seed production. Previous studies of ovule and female gametophyte (FG) development have mainly focused on angiosperms, especially in Arabidopsis thaliana. However, because it is difficult to acquire information about ovule development in gymnosperms, this remains unclear. Here, we investigated the transcriptomic data of natural ovule abortion mutants (female sterile line, STE) and the wild type (female fertile line, FER) of Pinus tabuliformis Carr. to evaluate the mechanism of ovule abortion during the process of free nuclear mitosis (FNM). Using single-molecule real-time (SMRT) sequencing and next-generation sequencing (NGS), 18 cDNA libraries via Illumina and two normalized libraries via PacBio, with a total of almost 400,000 reads, were obtained. Our analysis showed that the numbers of isoforms and alternative splicing (AS) patterns were significantly variable between FER and STE. The functional annotation results demonstrate that genes involved in the auxin response, energy metabolism, signal transduction, cell division, and stress response were differentially expressed in different lines. In particular, AUX/IAA, ARF2, SUS, and CYCB had significantly lower expression in STE, showing that auxin might be insufficient in STE, thus hindering nuclear division and influencing metabolism. Apoptosis in STE might also have affected the expression levels of these genes. To confirm the transcriptomic analysis results, nine pairs were confirmed by quantitative real-time PCR. Taken together, these results provide new insights into ovule abortion in gymnosperms and further reveal the regulatory mechanisms of ovule development.

Genome-Wide Characterization and Expression Profiling of GASA Genes during Different Stages of Seed Development in Grapevine (Vitis vinifera L.) Predict Their Involvement in Seed Development.

Members of the plant-specific GASA (gibberellic acid-stimulated Arabidopsis) gene family have multiple potential roles in plant growth and development, particularly in flower induction and seed development. However, limited information is available about the functions of these genes in fruit plants, particularly in grapes. We identified 14 GASA genes in grapevine (Vitis vinifera L.) and performed comprehensive bioinformatics and expression analyses. In the bioinformatics analysis, the locations of genes on chromosomes, physiochemical properties of proteins, protein structure, and subcellular positions were described. We evaluated GASA proteins in terms of domain structure, exon-intron distribution, motif arrangements, promoter analysis, phylogenetic, and evolutionary history. According to the results, the GASA domain is conserved in all proteins and the proteins are divided into three well-conserved subgroups. Synteny analysis proposed that segmental and tandem duplication have played a role in the expansion of the GASA gene family in grapes, and duplicated gene pairs have negative selection pressure. Most of the proteins were predicted to be in the extracellular region, chloroplasts, and the vacuole. In silico promoter analysis suggested that the GASA genes may influence different hormone signaling pathways and stress-related mechanisms. Additionally, we performed a comparison of the expression between seedless (Thompson seedless) and seeded (Red globe) cultivars in different plant parts, including the ovule during different stages of development. Furthermore, some genes were differentially expressed in different tissues, signifying their role in grapevine growth and development. Several genes (VvGASA2 and 7) showed different expression levels in later phases of seed development in Red globe and Thompson seedless, suggesting their involvement in seed development. Our study presents the first genome-wide identification and expression profiling of grapevine GASA genes and provides the basis for functional characterization of GASA genes in grapes. We surmise that this information may provide new potential resources for the molecular breeding of grapes.

Genomic Organization of the B3-Domain Transcription Factor Family in Grapevine (Vitis vinifera L.) and Expression during Seed Development in Seedless and Seeded Cultivars.

Members of the plant-specific B3-domain transcription factor family have important and varied functions, especially with respect to vegetative and reproductive growth. Although B3 genes have been studied in many other plants, there is limited information on the genomic organization and expression of B3 genes in grapevine (Vitis vinifera L.). In this study, we identified 50 B3 genes in the grapevine genome and analyzed these genes in terms of chromosomal location and syntenic relationships, intron-exon organization, and promoter cis-element content. We also analyzed the presumed proteins in terms of domain structure and phylogenetic relationships. Based on the results, we classified these genes into five subfamilies. The syntenic relationships suggest that approximately half of the genes resulted from genome duplication, contributing to the expansion of the B3 family in grapevine. The analysis of cis-element composition suggested that most of these genes may function in response to hormones, light, and stress. We also analyzed expression of members of the B3 family in various structures of grapevine plants, including the seed during seed development. Many B3 genes were expressed preferentially in one or more structures of the developed plant, suggesting specific roles in growth and development. Furthermore, several of the genes were expressed differentially in early developing seeds from representative seeded and seedless cultivars, suggesting a role in seed development or abortion. The results of this study provide a foundation for functional analysis of B3 genes and new resources for future molecular breeding of grapevine.

RNA-seq Analysis Reveals Gene Expression Profiling of Female Fertile and Sterile Ovules of PinusTabulaeformis Carr. during Free Nuclear Mitosis of the Female Gametophyte.

The development of the female gametophyte (FG) is one of the key processes of life cycle alteration between the haploid gametophyte and the diploid sporophytes in plants and it is required for successful seed development after fertilization. It is well demonstrated that free nuclear mitosis (FNM) of FG is crucial for the development of the ovule. However, studies of the molecular mechanism of ovule and FG development focused mainly on angiosperms, such as Arabidopsis thaliana and further investigation of gymnosperms remains to be completed. Here, Illumina sequencing of six transcriptomic libraries obtained from developing and abortive ovules at different stages during free nuclear mitosis of magagametophyte (FNMM) was used to acquire transcriptome data and gene expression profiles of Pinus tabulaeformis. Six cDNA libraries generated a total of 71.0 million high-quality clean reads that aligned with 63,449 unigenes and the comparison between developing and abortive ovules identified 7174 differentially expressed genes (DEGs). From the functional annotation results, DEGs involved in the cell cycle and phytohormone regulation were highlighted to reveal their biological importance in ovule development. Furthermore, validation of DEGs from the phytohormone signal transduction pathway was performed using quantitative real-time PCR analysis, revealing the dynamics of transcriptional networks and potential key components in the regulation of FG development in P. tabulaeformis were identified. These findings provide new insights into the regulatory mechanisms of ovule development in woody gymnosperms.

Emergence of apospory and bypass of meiosis via apomixis after sexual hybridisation and polyploidisation.

Hybridisation and polyploidy are major forces contributing to plant speciation. Homoploid (2x) and heteroploid (3x) hybrids, however, represent critical stages for evolution due to disturbed meiosis and reduced fertility. Apomixis--asexual reproduction via seeds--can overcome hybrid sterility, but requires several concerted alterations of developmental pathways to result in functional seed formation. Here, we analyse the reproductive behaviours of homo- and heteroploid synthetic hybrids from crosses between sexual diploid and tetraploid Ranunculus auricomus species to test the hypothesis that developmental asynchrony in hybrids triggers the shift to apomictic reproduction. Evaluation of male and female gametophyte development, viability and functionality of gametes shows developmental asynchrony, whereas seed set and germinability indicate reduced fitness in synthetic hybrids compared to sexual parents. We present the first experimental evidence for spontaneous apospory in most hybrids as an alternative pathway to meiosis, and the appearance of functional apomictic seeds in triploids. Bypassing meiosis permits these triploid genotypes to form viable seed and new polyploid progeny. Asynchronous development causes reduced sexual seed set and emergence of apospory in synthetic Ranunculus hybrids. Apomixis is functional in triploids and associated with drastic meiotic abnormalities. Selection acts to stabilise developmental patterns and to tolerate endosperm dosage balance shifts which facilitates successful seed set and establishment of apomictic lineages.

The Mechanism of Ovule Abortion in Self-Pollinated 'Hanfu' Apple Fruits and Related Gene Screening.

Apples exhibit S-RNase-mediated self-incompatibility and typically require cross-pollination in nature. 'Hanfu' is a cultivar that produces abundant fruit after self-pollination, although it also shows a high rate of seed abortion afterwards, which greatly reduces fruit quality. In this study, we investigated the ovule development process and the mechanism of ovule abortion in apples after self-pollination. Using a DIC microscope and biomicroscope, we found that the abortion of apple ovules occurs before embryo formation and results from the failure of sperm-egg fusion. Further, we used laser-assisted microdissection (LAM) cutting and sperm and egg cell sequencing at different periods after pollination to obtain the genes related to ovule abortion. The top 40 differentially expressed genes (DEGs) were further verified, and the results were consistent with switching the mechanism at the 5' end of the RNA transcript (SMART-seq). Through this study, we can preliminarily clarify the mechanism of ovule abortion in self-pollinated apple fruits and provide a gene reserve for further study and improvement of 'Hanfu' apple fruit quality.

Single-cell RNA-seq reveals a link of ovule abortion and sugar transport in Camellia oleifera.

Camellia oleifera is the most important woody oil crop in China. Seed number per fruit is an important yield trait in C. oleifera. Ovule abortion is generally observed in C. oleifera and significantly decreases the seed number per fruit. However, the mechanisms of ovule abortion remain poorly understood at present. Single-cell RNA sequencing (scRNA-seq) was performed using mature ovaries of two C. oleifera varieties with different ovule abortion rates (OARs). In total, 20,526 high-quality cells were obtained, and 18 putative cell clusters were identified. Six cell types including female gametophyte, protoxylem, protophloem, procambium, epidermis, and parenchyma cells were identified from three main tissue types of ovule, placenta, and pericarp inner layer. A comparative analysis on scRNA-seq data between high- and low-OAR varieties demonstrated that the overall expression of CoSWEET and CoCWINV in procambium cells, and CoSTP in the integument was significantly upregulated in the low-OAR variety. Both the infertile ovule before pollination and the abortion ovule producing after compatible pollination might be attributed to selective abortion caused by low sugar levels in the apoplast around procambium cells and a low capability of hexose uptake in the integument. Here, the first single-cell transcriptional landscape is reported in woody crop ovaries. Our investigation demonstrates that ovule abortion may be related to sugar transport in placenta and ovules and sheds light on further deciphering the mechanism of regulating sugar transport and the improvement of seed yield in C. oleifera.

KNOX transcription factor VvHB63 affects grape seed development by interacting with protein VvHB06.

The fast-growing demand for seedless table grapes has attracted the attention of scientists for the development of new seedless cultivars. Various genes and pathways have been identified which affect seedlessness. However, the detail of the mechanism(s) regulating seedless traits in grape is still unclear, and genes related to seedlessness in grape require further study. Transcriptomic and genomic analyses of Homeobox (HB) transcription factors have suggested the involvement of HB genes, especially of HB-KNOX members, in grape seed development. Here, we functionally characterize VvHB63 gene in grape and report its role in fruit and seed development. VvHB63 showed higher expressions levels in the chalaza and integument of ovules in seedless grapes, than in seeded ones. However, no differences were observed in the sequences of seedless and seeded grape cultivars. In situ hybridization (ISH) analysis showed that VvHB63 gene was expressed in the episperm cells and ovules of 'Thompson Seedless'. Conserved domains KNOX1 and KNOX2 were important for the interaction of VvHB63 with VvHB06. Heterologous over-expression of VvHB63 (35 S::VvHB63-OE) in tomato induced smaller fruits and seeds than in wild type or SlTkn1-KO. The synergistic cooperation between VvHB63 and related proteins play an important role in ovule development.

Horticultural Practices in Early Spring to Mitigate the Adverse Effect of Low Temperature on Fruit Set in 'Lapins' Sweet Cherry.

Yield of sweet cherry (Prunus avium L.) is determined by fruit set, a developmental stage sensitive to variable spring environmental conditions. To sustain fruit production and enhance crop climate resilience, it is important to understand the impacts of abiotic stresses and the effectiveness of horticultural mitigations in the spring on the critical developmental processes during fruit set. In this study, flowering phenology, pistil browning and percent fruit set of 'Lapins' were monitored at five sites of different elevation and frost risk in the Okanagan Valley, British Columbia, Canada, in 2019 and 2022. At Site 1 in Summerland Research and Development Centre ("SuRDC1"), where a 'Lapins' on Krymsk 5 planting was located in a frost pocket where the crops were exposed to high risk of cold damage in the spring, a series of experiments were conducted to investigate the floral organ viability and percent fruit set under low temperatures, and under the effects of four spring horticultural mitigation measures. Installation of polyethylene sleeves and FAME spray (fatty acid methyl esters-based plant growth regulator, WAIKEN, SST Australia) were implemented in 2019; boric acid spray and postponed irrigation were tested in 2022. Low fruit set at SuRDC1 in both years was associated with severe pistil browning after night temperature dropped below -4 °C in late April. In 2019, the semi-enclosure of polyethylene sleeves led to an increase in the surface temperature (Tsurfae) of floral buds by 2-4 °C, which prolonged the stage of first bloom, delayed petal fall and prevented frost damage on pistils, but led to the decrease in percent fruit set by 77%, due to ovule abortion or cessation of fruitlet development. The early and late sprays of FAME had no significant influence on either abundance of germinated pollen tubes or percent fruit set; however, the potential of late spray in improving pollen abundance and reducing pistil browning requires further investigation. In 2022, the spray of 0.01% boric acid solution led to a decrease in fruit set by 6.95%. Six-week postponement of irrigation starting from full bloom decreased soil moisture, but increased soil temperature and improved fruit set by 7.61%. The results improved our understanding about the damages of adverse spring air temperatures on pistils and ovules, and suggested the potential of irrigation adjustment in regulating soil moisture and temperature and improving fruit set in the cool and moist spring.

Integrative transcriptomics and proteomics analysis constructs a new molecular model for ovule abortion in the female-sterile line of Pinus tabuliformis Carr.

Ovule development is critical to plant reproduction and free nuclear mitosis of megagametophyte (FNMM) is vital for ovule development. However, most results of ovule development were based on the studies in angiosperms, and its molecular regulation remained largely unknown in gymnosperms, particularly, during FNMM. In this context, we studied the genome-wide difference between sterile line (SL) and fertile line (FL) ovules using transcriptomics and proteomics approaches in Pinus tabuliformis Carr. Comparative analyses revealed that genes involved in DNA replication, DNA damage repair, Cell cycle, Apoptosis and Energy metabolism were highlighted. Further results showed the low expressions of MCM 2-7, RRM1, etc. perhaps led to abnormal DNA replication and damage repair, and the significantly different expressions of PARP2, CCs1, CCs3, etc. implied that the accumulated DNA double-stranded breaks were failed to be repaired and the cell cycle was arrested at G2/M in SL ovules, potentially resulting in the occurrence of apoptosis. Moreover, the deficiency of ETF-QO might hinder FNMM. Consequently, FNMM stopped and ovule aborted in SL ovules. Our results suggested a selective regulatory mechanism led to FNMM half-stop and ovule abortion in P. tabuliformis and these insights could be exploited to investigate the molecular regulations of ovule development in woody gymnosperms.

Differential proteomic analysis revealing the ovule abortion in the female-sterile line of Pinus tabulaeformis Carr.

Ovule abortion affects the yield and quality of Pinus tabulaeformis Carr. seeds. Research into ovule abortion has importance for improving the seed setting rate and establishing artificial seed production techniques. Fertile line (FL) ovules (FL-E) and sterile line (SL) ovules (SL-E) in the early stage of free nuclear mitosis of megagametophyte (FNMM), FL ovules (FL-L) and SL ovules (SL-L) in the late stage of FNMM of P. tabulaeformis were collected as materials. 4192 proteins were identified by isobaric tags for relative and absolute quantitation (iTRAQ)-based analysis. Bioinformatics analysis implied that in SL ovules, substances and energy might be deficient, perhaps leading to abnormal DNA replication. Because the incomplete antioxidant system and the abnormal expression levels of enzymes involved in cell signal transduction, DNA DSBs probably occurs. Facing the abnormities of DNA replication and damage, the cell cycle was arrested and the DNA damage failed to be repaired, potentially resulting in the occurrence of PCD. Taken together, an inference can be drawn from our study - substance and energy deficiencies, reactive oxygen stress, and the failure of both cell cycle progression and DNA damage repair, which possibly hinder FNMM, leading to ovule abortion in the female-sterile line of P. tabulaeformis.

Self pollination and resource availability affect ovule abortion inCassia fasciculata (Caesalpiniaceae).

We examined the effects of pollen source and resource availability on ovule abortion in the annual legumeCassia fasciculata. Pollen source was controlled by hand-pollinating flowers with cross- or self-pollen. Resource availability to developing fruits was controlled by adjusting fruit loads (heavy versus light) on each plant and exposing plants to different photoperiod cycles (16 vs 12 h of light; short days favor fruit growth at the expense of vegetative growth). In mature fruits the proportion of ovules expanding (showing some development over virgin ovules) ranged from 89-95% and did not increase with resource availability, suggesting that unexpanded ovules were either unfertilized or obligately aborted shortly after fertilization. The proportion of expanded ovules maturing in mature fruits was near 97% for both self- and crosspollinations in the treatment with highest resource availability (light load, short days) and lower in the remaining treatments, where self-pollination resulted in up to 9% lower seed maturation than cross-pollination. In the latter three treatments a pollen source effect was dependent upon the maternal plant; in some plants selfing increased abortion and in others it did not. Collectively, the results suggest that (1) both pollen source and resource availability affect ovule abortion, (2) at least some abortion is facultative, and (3) when resources are limited, self-pollination increases abortion in some but not all maternal plants.

The metacaspase gene family of Vitis vinifera L.: characterization and differential expression during ovule abortion in stenospermocarpic seedless grapes.

In both plants and animals, programmed cell death (PCD) is an indispensable process that removes redundant cells. In seedless grapes (Vitis vinifera), abnormal PCD in ovule cells and subsequent ovule abortion play key roles in stenospermocarpy. Metacaspase, a type of cysteine-dependent protease, plays an essential role in PCD. To reveal the characteristics of the metacaspase (MC) gene family and the relationship between metacaspases and the seedless trait, we identified the 6 V. vinifera metacaspases VvMC1-VvMC6, from the grape genome, using BLASTN against the 9 known Arabidopsis metacaspases. We also obtained full-length cDNAs by RT-PCR. Each of the 6 grape metacaspases contains small (p10-like) and a large (p20-like) conserved structural domains. Phylogenetic analysis of 6 grape and 9 Arabidopsis metacaspases showed that all metacaspases could be grouped into two classes: Type I and Type II. Each phylogenetic branch shares a similar exon/intron structure. Furthermore, the putative promoters of the grape metacaspases contained cis-elements that are involved in grape endosperm development. Moreover, expression analysis of metacaspases using real-time quantitative PCR demonstrated that VvMC1 and VvMC2 were able to be detected in any tissue, and VvMC3, VvMC4, VvMC5 and VvMC6 exhibited tissue-specific expression. Lastly, in cv. Thompson seedless grapes VvMC1, VvMC3, and VvMC4 were significantly up-regulated at the 35 DAF during ovule development, roughly same stage as endosperm abortion. In addition, the expression trend of VvMC2 and VvMC5 was similar between cv. Pinot Noir and cv. Thompson grape ovule development and that of VvMC6 was sustained in a relatively low level except the expression of cv. Pinot Noir significantly up-regulated in 25 DAF. Our data provided new insights into PCD by identifying the grape metacaspase gene family and provide a useful reference for further functional analysis of metacaspases in grape.