Laser capture microdissection-based spatiotemporal transcriptomes uncover regulatory networks during seed abortion in seedless Ponkan (Citrus reticulata).

Seed abortion is an important process in the formation of seedless characteristics in citrus fruits. However, the molecular regulatory mechanism underlying citrus seed abortion is poorly understood. Laser capture microdissection-based RNA-seq combined with Pacbio-seq was used to profile seed development in the Ponkan cultivars 'Huagan No. 4' (seedless Ponkan) (Citrus reticulata) and 'E'gan No. 1' (seeded Ponkan) (C. reticulata) in two types of seed tissue across three developmental stages. Through comparative transcriptome and dynamic phytohormone analyses, plant hormone signal, cell division and nutrient metabolism-related processes were revealed to play critical roles in the seed abortion of 'Huagan No. 4'. Moreover, several genes may play indispensable roles in seed abortion of 'Huagan No. 4', such as CrWRKY74, CrWRKY48 and CrMYB3R4. Overexpression of CrWRKY74 in Arabidopsis resulted in severe seed abortion. By analyzing the downstream regulatory network, we further determined that CrWRKY74 participated in seed abortion regulation by inducing abnormal programmed cell death. Of particular importance is that a preliminary model was proposed to depict the regulatory networks underlying seed abortion in citrus. The results of this study provide novel insights into the molecular mechanism across citrus seed development, and reveal the master role of CrWRKY74 in seed abortion of 'Huagan No. 4'.

Increased DNA methylation of the splicing regulator SR45 suppresses seed abortion in litchi.

The gene regulatory networks that govern seed development are complex, yet very little is known about the genes and processes that are controlled by DNA methylation. Here, we performed single-base resolution DNA methylome analysis and found that CHH methylation increased significantly throughout seed development in litchi. Based on the association analysis of differentially methylated regions and weighted gene co-expression network analysis (WGCNA), 46 genes were identified as essential DNA methylation-regulated candidate genes involved in litchi seed development, including LcSR45, a homolog of the serine/arginine-rich (SR) splicing regulator SR45. LcSR45 is predominately expressed in the funicle, embryo, and seed integument, and displayed increased CHH methylation in the promoter during seed development. Notably, silencing of LcSR45 in a seed-aborted litchi cultivar significantly improved normal seed development, whereas the ectopic expression of LcSR45 in Arabidopsis caused seed abortion. Furthermore, LcSR45-dependent alternative splicing events were found to regulate genes involved in seed development. Together, our findings demonstrate that LcSR45 is hypermethylated, and plays a detrimental role in litchi seed development, indicating a global increase in DNA methylation at this stage.

Seed abortion caused by the combination of two duplicate genes in the progeny from the cross between Oryza sativa and Oryza meridionalis.

Seed development is an essential phenomenon for all sexual propagative plant species. The functional allele at SEED DEVELOPMENT 1 (SDV1) or SEED DEVELOPMENT 2 (SDV2) loci is essential for seed development for Oryza sativa and Oryza meridionalis. In the present study, we performed fine mapping of SDV1, narrowing down the area of interest to 333kb on chromosome 6. Haplotype analysis around the SDV1 locus of O. meridionalis accessions indicated that they shared the DNA polymorphism, suggesting that they have a common abortive allele at the SDV1 locus. Linkage analysis of the candidate SDV2 gene showed that it was located on chromosome 4. The candidate SDV2 was confirmed using a population in which both the SDV1 and SDV2 genes were segregating. The chromosomal region covering the SDV1 gene was predicted to contain 30 protein-coding genes in O. sativa. Five of these genes have conserved DNA sequences in the chromosomal region of the SDV2 gene on chromosome 4, and not on chromosome 6, of O. meridionalis. These results suggest that these five genes could be candidates for SDV1, and that their orthologous genes located on chromosome 4 of O. meridionalis could be candidates for SDV2.

Identification of VvAGL Genes Reveals Their Network's Involvement in the Modulation of Seed Abortion via Responding Multi-Hormone Signals in Grapevines.

The formation of seedless traits is regulated by multiple factors. AGLs, which belong to the MADS-box family, were reported to be important regulators in this process; however, the underlying mechanism remains elusive. Here, we identified the VvAGL sub-family genes during the seed abortion process in seedless grapevine cv. 'JingkeJing' and found 40 differentially expressed VvAGL members and 1069 interacting proteins in this process. Interestingly, almost all members and their interacting proteins involved in the tryptophan metabolic pathway (K14486) and participated in the phytohormone signalling (KO04075) pathway, including the growth hormone (IAA), salicylic acid (SA), abscisic acid (ABA), cytokinin (CTK), and ethylene signalling pathways. The promoters of AGL sub-family genes contain cis-elements in response to hormones such as IAA, ABA, CTK, SA, and ETH, implying that they might respond to multi-hormone signals and involve in hormone signal transductions. Further expression analysis revealed VvAGL6-2, VvAGL11, VvAGL62-11, and VvAGL15 had the highest expression at the critical period of seed abortion, and there were positive correlations between ETH-VvAGL15-VvAGL6-2, ABA-VvAGL80, and SA-VvAGL62 in promoting seed abortion but negative feedback between IAA-VvAGL15-VvAGL6-2 and CTK-VvAGL11. Furthermore, many genes in the IAA, ABA, SA, CTK, and ETH pathways had a special expressional pattern in the seed, whereby we developed a regulatory network mediated by VvAGLs by responding to multihormonal crosstalk during grape seed abortion. Our findings provide new insights into the regulatory network of VvAGLs in multi-hormone signalling to regulate grape seed abortion, which could be helpful in the molecular breeding of high-quality seedless grapes.

Embryological observations on seed abortion in Hibiscus syriacus L. and physiological studies on nutrients, enzyme activity and endogenous hormones.

Under natural conditions, most Hibiscus syriacus L. individuals form very few mature seeds or the mature seeds that do form are of poor quality. As a result, seed yield is poor and seeds have low natural germinability. These phenomena strongly hinder utilization of the excellent germplasm resources of H. syriacus. The study has shown that pollen activity and stigma receptivity were high on the day of anthesis, and the pistils and stamens were fertile. Pollen release and stigma receptivity were synchronous. But in styles following self and cross-pollination, pollen tube abnormalities (distortion and twisting of the pollen tubes) and callose deposition were observed. Cross-pollinated pollen tubes elongated faster and fewer pollen tube abnormalities were observed compared with self-pollinated pollen tubes. And during embryo development, abnormalities during the heart-shaped embryo stage led to embryo abortion. Imbalance in antioxidant enzyme activities and low contents of auxin and cytokinin during early stages of embryo development may affect embryo development. Therefore, a low frequency of outcrossing and mid-development embryo abortion may be important developmental causes of H. syriacus seed abortion. Nutrient deficiencies, imbalance in antioxidant enzyme activities, and a high content of abscisic acid at advanced stages of seed development may be physiological causes of seed abortion.

Revealing Further Insights into Astringent Seeds of Chinese Fir by Integrated Metabolomic and Lipidomic Analyses.

Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.) stands as one of the pivotal afforestation tree species and timber resources in southern China. Nevertheless, the occurrence of seed abortion and a notably high proportion of astringent seeds significantly curtail the yield and quality of elite seeds, resulting in substantial economic losses. The development of astringent seeds is accompanied by significant physiological and biochemical alterations. Here, the first combined lipidomic and metabolomic analysis was performed to gain a comprehensive understanding of astringent seed traits. A total of 744 metabolites and 616 lipids were detected, of which 489 differential metabolites and 101 differential lipids were identified. In astringent seeds, most flavonoids and tannins, as well as proline and γ-aminobutyric acid, were more accumulated, along with a notable decrease in lipid unsaturation, indicating oxidative stress in the cells of astringent seeds. Conversely, numerous elemental metabolites were less accumulated, including amino acids and their derivatives, saccharides and alcohols, organic acids and nucleotides and their derivatives. Meanwhile, most lipid subclasses, mainly associated with energy storage (triglyceride and diglyceride) and cell membrane composition (phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine), also exhibited significant reductions. These results reflected a disruption in the cellular system or the occurrence of cell death, causing a reduction in viable cells within astringent seeds. Furthermore, only one lipid subclass, sphingosine phosphate (SoP), was more accumulated in astringent seeds. Additionally, lower accumulation of indole-3-acetic acid and more accumulation of salicylic acid (SA) were also identified in astringent seeds. Both SA and SoP were closely associated with the promotion of programmed cell death in astringent seeds. Collectively, our study revealed significant abnormal changes in phytohormones, lipids and various metabolites in astringent seeds, allowing us to propose a model for the development of astringent seeds in Chinese fir based on existing research and our findings. This work enriches our comprehension of astringent seeds and presents valuable bioindicators for the identification of astringent seeds.

Genome-scale transcriptomic insights into the gene co-expression network of seed abortion in triploid Siraitia grosvenorii.

BACKGROUND: Siraitia grosvenorii (Swingle) C. Jeffrey, also known as Luohanguo or monk fruit, is a famous traditional Chinese medicine ingredient with important medicinal value and broad development prospects. Diploid S. grosvenorii has too many seeds, which will increase the utilization cost of active ingredients. Thus, studying the molecular mechanism of seed abortion in triploid S. grosvenorii, identifying the abortion-related genes, and regulating their expression will be a new direction to obtain seedless S. grosvenorii. Herein, we examined the submicroscopic structure of triploid S. grosvenorii seeds during abortion. RESULTS: Upon measuring the endogenous hormone content, we found that abscisic acid (ABA) and trans-zeatin (ZR) levels were significantly downregulated after days 15 and 20 of flowering. RNA sequencing of triploid seeds at different developmental stages was performed to identify key genes regulating abortion in triploid S. grosvenorii seeds. Multiple genes with differential expression between adjacent stages were identified; seven genes were differentially expressed across all stages. Weight gene co-expression network analysis revealed that the enhancement of monoterpene and terpene metabolic processes might lead to seed abortion by reducing the substrate flow to ABA and ZR. CONCLUSIONS: These findings provide insights into the gene-regulatory network of seed abortion in triploid S. grosvenorii from different perspectives, thereby facilitating the innovation of the breeding technology of S. grosvenorii.

Locations of seed abortion in response to defoliation differ with pollen source in a native perennial legume herb.

PREMISE: In many flowering plants, flowers contain more ovules than fruits have seeds. What determines which ovules become seeds? When photosynthates are limited, as may happen when plants lose leaf area to herbivory, fewer fertilized ovules become seeds. METHODS: Greenhouse-grown ramets of distinct individuals of a perennial herbaceous legume were manually defoliated to various levels determined in the field, then self- or cross-pollinated. For each seed produced, we recorded its position in the fruit and its mass. From a subset of seeds from different treatments and positions in the fruits, we grew seedlings and measured their dry mass. RESULTS: Ovules were aborted more frequently in fruits from flowers that were self-pollinated and from those on plants with higher levels of defoliation. Ovules in the basal portion of the fruits were more likely to be aborted than those at the stigmatic end; this pattern was most pronounced for fruits after self-pollination with high levels of defoliation. Total number of seeds produced and seed mass per pod were greatest in cross-pollinated fruits after no or low levels of defoliation. Mean individual seed mass was greater for fruits with fewer seeds, indicating a trade-off between seed number and seed mass. Seedling dry mass (a measure of vigor) was greatest for seeds in the middle positions of fruit produced by cross-pollination after severe herbivory; no positional differences were seen for seeds from self-pollinated fruits. CONCLUSIONS: Observed locations of seed abortion may have been selected not only by defoliation, but in part by propensity for dispersal, while positional differences in seedling vigor may be related to seed size and differential maternal allocation based on pollination treatment and leaf area lost.

Transcriptomic Analysis Reveals Key Candidate Genes Related to Seed Abortion in Chinese Jujube (Ziziphus jujuba Mill).

Background: Seed abortion is a common phenomenon in Chinese jujube that seriously hinders the process of cross-breeding. However, the molecular mechanisms of seed abortion remain unclear in jujube. Methods: Here, we performed transcriptome sequencing using eight flower and fruit tissues at different developmental stages in Ziziphus jujuba Mill. 'Zhongqiusucui' to identify key genes related to seed abortion. Histological analysis revealed a critical developmental process of embryo abortion after fertilization. Results: Comparisons of gene expression revealed a total of 14,012 differentially expressed genes. Functional enrichment analyses of differentially expressed genes between various sample types uncovered several important biological processes, such as embryo development, cellular metabolism, and stress response, that were potentially involved in the regulation of seed abortion. Furthermore, gene co-expression network analysis revealed a suite of potential key genes related to ovule and seed development. We focused on three types of candidate genes, agamous subfamily genes, plant ATP-binding cassette subfamily G transporters, and metacaspase enzymes, and showed that the expression profiles of some members were associated with embryo abortion. Conclusion: This work generates a comprehensive gene expression data source for unraveling the molecular mechanisms of seed abortion and aids future cross-breeding efforts in jujube.

Abortion occurs during double fertilization and ovule development in Paeonia ludlowii.

Paeonia ludlowii (Stern & Taylor) D.Y.Hong, an endangered species, is indigenous to Tibet, China and propagated only by seed under natural conditions. Its natural reproduction is constrained by low fecundity. Excess seed abortion is a key factor restricting its natural reproduction, cultivation, introduction, and protection. Understanding the specific origin and occurrence of aborted ovules is important for the protection of offspring. Using serial sectioning analysis, we studied the process of pollination and fertilization of P. ludlowii and examined the characteristics of aborted ovules, developmental differences after flowering of normal and aborted ovules, and their ratios at different positions in P. ludlowii ovaries. During pollination, fertilization, and seed development, ovule abortion was frequent, with a random abortion position. There were three types of abortion, namely, abnormal pistil, sterile ovules, and embryo and endosperm abortions. Of these, embryo and endosperm abortions could be divided into early abortion and middle abortion. The early aborted ovules stopped growing on day 12, the endoblast and endosperm in the embryo sac aborted gradually. Furthermore, the shape of the embryo sac cavity changed. The volume of aborted ovules was significantly different from that of fertile ovules. At ripening, the external morphology of different types of aborted seeds was significantly different. The possible reasons for the abortion of the ovules are also discussed.

The co-expression of genes involved in seed coat and endosperm development promotes seed abortion in grapevine.

MAIN CONCLUSION: The seed coat gene VviAGL11 coordinates with endosperm development genes FIS2, PHERESE1 and IKU2 and functions as the key regulator in seed development and abortion processes in grapevine. Seed development is essential for the reproduction of flowering plants. Seed abortion is a specific characteristic that produces seedless berries and is often observed in cultivated grapevines. Although seedlessness is an important trait for table and dried grapevine production, the mechanism of seed abortion remains poorly understood. This research aimed to analyze the co-expression of the seed coat development gene VviAGL11 and the endosperm development genes FERTILIZATION INDEPENDENT SEED2 (FIS2), PHERESE1 and HAIKU2 (IKU2) that regulate seedless fruit development in grapevine. The transcript levels of VviAGL11, FIS2, PHERESE1 and IKU2 all decreased during seed abortion in the seedless grape 'Thompson Seedless' plants, compared to those of the seeded grape 'Pinot Noir'. The transcript levels of the salicylic acid (SA)-dependent defense response genes EDS1, NPR1, NDR1 and SID2 were higher in 'Thompson Seedless' than 'Pinot Noir' during seed development. Also, WRKY3, WRKY6 and WRKY52, which participate in the SA pathway, were higher expressed in 'Thompson Seedless' than in 'Pinot Noir', indicating that SA-dependent defense responses may regulate seed abortion. The genes related to synthesis and metabolism of gibberellic acid (GA) and abscisic acid (ABA) also showed differential expression between 'Thompson Seedless' and 'Pinot Noir'. Exogenous applications of plant growth regulators (PGRs) to inflorescences of three stenospermocarpy grapevines before flowering showed that GA3 was critical prominently in seed development. Therefore, the co-expression of seed coat and endosperm development-related genes, SA pathway genes, and genes for the synthesis and metabolism of GA3 together enhance seed abortion in seedless grapes.

Differential Response to Single and Combined Salt and Heat Stresses: Impact on Accumulation of Proteins and Metabolites in Dead Pericarps of Brassica juncea.

Dead organs enclosing embryos, such as seed coats and pericarps, are emerging as important maternally-derived components of the dispersal unit that affect seed performance and fate. In the face of climate change and increased incidents of heatwaves, we sought to investigate the effect of salinity (S), short episodes of high temperature (HS), and combination of S + HS (SHS), at the reproductive phase, on the properties of dead pericarps of Brassica juncea. Proteome and metabolome analyses revealed multiple proteins and metabolites stored in dead pericarps whose levels and composition were altered under single and combined stress conditions. The protein profile of SHS showed a higher correlation with salt than with HS indicating the dominant effect of salt over heat stress. On the other hand, the analysis of metabolites showed that the profile of SHS has better correlation with HS than with salt. The integration of metabolic and proteomic data showed that changes in TCA cycle intermediates and certain amino acids (e.g., proline) under salt treatments (S and SHS) are highly correlated with changes in proteins involved in their biosynthetic pathways. Thus, accumulation of proteins and metabolites in dead pericarps is differently affected by single and combination of salt and heat stresses. Salinity appears to dominate plant response to combined stresses at the protein level, while heat appears to be the major factor affecting metabolite accumulation in dead pericarps.

Multiple mechanisms explain how reduced KRP expression increases leaf size of Arabidopsis thaliana.

Although cell number generally correlates with organ size, the role of cell cycle control in growth regulation is still largely unsolved. We studied kip related protein (krp) 4, 6 and 7 single, double and triple mutants of Arabidopsis thaliana to understand the role of cell cycle inhibitory proteins in leaf development. We performed leaf growth and seed size analysis, kinematic analysis, flow cytometery, transcriptome analysis and mathematical modeling of G1/S and G2/M checkpoint progression of the mitotic and endoreplication cycle. Double and triple mutants progressively increased mature leaf size, because of elevated expression of cell cycle and DNA replication genes stimulating progression through the division and endoreplication cycle. However, cell number was also already increased before leaf emergence, as a result of an increased cell number in the embryo. We show that increased embryo and seed size in krp4/6/7 results from seed abortion, presumably reducing resource competition, and that seed size differences contribute to the phenotype of several large-leaf mutants. Our results provide a new mechanistic understanding of the role of cell cycle regulation in leaf development and highlight the contribution of the embryo to the development of leaves after germination in general.

Reduced expression of selected FASCICLIN-LIKE ARABINOGALACTAN PROTEIN genes associates with the abortion of kernels in field crops of Zea mays (maize) and of Arabidopsis seeds.

Abortion of fertilized ovaries at the tip of the ear can generate significant yield losses in maize crops. To investigate the mechanisms involved in this process, 2 maize hybrids were grown in field crops at 2 sowing densities and under 3 irrigation regimes (well-watered control, drought before pollination, and drought during pollination), in all possible combinations. Samples of ear tips were taken 2-6 days after synchronous hand pollination and used for the analysis of gene expression and sugars. Glucose and fructose levels increased in kernels with high abortion risk. Several FASCICLIN-LIKE ARABINOGALACTAN PROTEIN (FLA) genes showed negative correlation with abortion. The expression of ZmFLA7 responded to drought only at the tip of the ear. The abundance of arabinogalactan protein (AGP) glycan epitopes decreased with drought and pharmacological treatments that reduce AGP activity enhanced the abortion of fertilized ovaries. Drought also reduced the expression of AthFLA9 in the siliques of Arabidopsis thaliana. Gain- and loss-of-function mutants of Arabidopsis showed a negative correlation between AthFLA9 and seed abortion. On the basis of gene expression patterns, pharmacological, and genetic evidence, we propose that stress-induced reductions in the expression of selected FLA genes enhance abortion of fertilized ovaries in maize and Arabidopsis.

De novo transcriptome sequencing and gene expression analysis reveal potential mechanisms of seed abortion in dove tree (Davidia involucrata Baill.).

BACKGROUND: Dove tree (Davidia involucrata Baill.) is a rare and endangered species. Natural reproduction of dove tree is extremely difficult due to its low fecundity. Serious seed abortion is one of the key factors restraining its sexual reproduction. Understanding the inducements of seed abortion is critical for addressing the issue of offspring production and the survivability of such an endangered species. However, studies on the molecular mechanism of seed abortion in woody plants are lacking, and the dearth of genomic resources for dove tree restricts further research. RESULTS: In this study, using the Illumina platform, we performed de novo transcriptome sequencing of the fruit and seed in dove tree. A total of 149,099 transcripts were isolated and then assembled into 72,885 unigenes. Subsequently, differentially expressed genes (DEGs) between normal and abortive seeds were screened. Genes involved in response to stress, hormone signal transduction, programmed cell death, lignin biosynthesis, and secondary cell wall biogenesis showed significant different expression levels between normal and abortive seeds. CONCLUSION: Combined results indicated that the abortive seeds were under the adversity stress, which should be controlled by the maternal plant. Maternally controlled development of integument is assumed to be a critical process for abortion regulation. MYB and WRKY transcription factors, receptor kinase and laccase are considered to be important regulators in seed abortion. Moreover, mass sequence data facilitated further molecular research on this unique species.

Positive density-dependent reproduction regulated by local kinship and size in an understorey tropical tree.

BACKGROUND AND AIMS: Global pollinator declines and continued habitat fragmentation highlight the critical need to understand reproduction and gene flow across plant populations. Plant size, conspecific density and local kinship (i.e. neighbourhood genetic relatedness) have been proposed as important mechanisms influencing the reproductive success of flowering plants, but have rarely been simultaneously investigated. METHODS: We conducted this study on a continuous population of the understorey tree Miconia affinis in the Forest Dynamics Plot on Barro Colorado Island in central Panama. We used spatial, reproductive and population genetic data to investigate the effects of tree size, conspecific neighbourhood density and local kinship on maternal and paternal reproductive success. We used a Bayesian framework to simultaneously model the effects of our explanatory variables on the mean and variance of maternal viable seed set and siring success. KEY RESULTS: Our results reveal that large trees had lower proportions of viable seeds in their fruits but sired more seeds. We documented differential effects of neighbourhood density and local kinship on both maternal and paternal reproductive components. Trees in more dense neighbourhoods produced on average more viable seeds, although this positive density effect was influenced by variance-inflation with increasing local kinship. Neighbourhood density did not have significant effects on siring success. CONCLUSIONS: This study is one of the first to reveal an interaction among tree size, conspecific density and local kinship as critical factors differentially influencing maternal and paternal reproductive success. We show that both maternal and paternal reproductive success should be evaluated to determine the population-level and individual traits most essential for plant reproduction. In addition to conserving large trees, we suggest the inclusion of small trees and the conservation of dense patches with low kinship as potential strategies for strengthening the reproductive status of tropical trees.

Reproduction at the extremes: pseudovivipary, hybridization and genetic mosaicism in Posidonia australis (Posidoniaceae).

BACKGROUND AND AIMS: Organisms occupying the edges of natural geographical ranges usually survive at the extreme limits of their innate physiological tolerances. Extreme and prolonged fluctuations in environmental conditions, often associated with climate change and exacerbated at species' geographical range edges, are known to trigger alternative responses in reproduction. This study reports the first observations of adventitious inflorescence-derived plantlet formation in the marine angiosperm Posidonia australis, growing at the northern range edge (upper thermal and salinity tolerance) in Shark Bay, Western Australia. These novel plantlets are described and a combination of microsatellite DNA markers and flow cytometry is used to determine their origin. METHODS: Polymorphic microsatellite DNA markers were used to generate multilocus genotypes to determine the origin of the adventitious inflorescence-derived plantlets. Ploidy and genome size were estimated using flow cytometry. KEY RESULTS: All adventitious plantlets were genetically identical to the maternal plant and were therefore the product of a novel pseudoviviparous reproductive event. It was found that 87 % of the multilocus genotypes contained three alleles in at least one locus. Ploidy was identical in all sampled plants. The genome size (2 C value) for samples from Shark Bay and from a separate site much further south was not significantly different, implying they are the same ploidy level and ruling out a complete genome duplication (polyploidy). CONCLUSIONS: Survival at range edges often sees the development of novel responses in the struggle for survival and reproduction. This study documents a physiological response at the trailing edge, whereby reproductive strategy can adapt to fluctuating conditions and suggests that the lower-than-usual water temperature triggered unfertilized inflorescences to 'switch' to growing plantlets that were adventitious clones of their maternal parent. This may have important long-term implications as both genetic and ecological constraints may limit the ability to adapt or range-shift; this seagrass meadow in Shark Bay already has low genetic diversity, no sexual reproduction and no seedling recruitment.

Seed predation and climate impacts on reproductive variation in temperate forests of the southeastern USA.

Climatic effects on tree recruitment will be determined by the interactive effects of fecundity and seed predation. Evaluating how insect and vertebrate seed predators mediate tree reproductive responses to climate depends on long-term studies of seed production, development, and predation. In this study, our objectives were to (1) assess the effects of interannual climate variation on seed abortion rates, (2) assess the impact of seed density on predation rates, and (3) examine the degree to which density-dependent seed predation would amplify or dampen interannual variation in fecundity associated with seed abortion. We used a 19-year study of seed abortion and pre-dispersal predation rates by insects and vertebrates (birds and rodents) for five temperate tree species across forest plots from the North Carolina Piedmont to the Southern Appalachian Mountains in the southeastern USA. We found that rates of seed abortion and predation increased reproductive variation for oaks (Quercus species). Probability of seed abortion was greatest during years with cool, dry springs. Responses of seed predation on Quercus species to current year's seed density varied by species, but exhibited positive density-dependence to previous year's seed density consistent with numerical responses of seed predators. Seed abortion and predation rates for two drupe species responded little to variation in climate or seed density, respectively. Given that predation increased interannual variation in seed availability and the negative density-dependence to previous year's seed density, our results indicate that consistent numerical responses of oak seed predators may amplify interannual variation due to climate-mediated processes like seed abortion.

The influence of herbivory on pre- and postzygotic stages of reproduction following open, self, and outcross pollination.

PREMISE OF THE STUDY: Herbivory affects pollination success and reproductive output in plants. However, the different stages in the process from pollination to seed maturation have hardly been investigated within the context of herbivory. Herbivory might affect these stages via its effect on geitonogamous pollination and thereby the proportion of self pollen delivered to the stigma and/or via its effect on the nutritional capacity of the maternal plant. METHODS: Plants of monocarpic Cynoglossum officinale were experimentally subjected to root herbivory and exposed to natural open pollination in combination with self and outcross hand pollination. We quantified pollen germination, pollen tube competition intensity, pollen tube attrition, fruit set, and seed initiation, abortion, and maturation. KEY RESULTS: Although root herbivory did not affect pollen germination or pollen tube attrition, fruit set and seed initiation and maturation were negatively affected by herbivory, but for seed initiation only in the case of outcross- and open-pollinated flowers. The intensity of pollen tube competition positively affected seed initiation, but only in plants infested with the herbivore. CONCLUSIONS: Our study demonstrates that herbivory did not affect the early stages following pollination, but significantly impacted later postpollination stages such as fruit set and seed maturation and selection based on pollen tube competition intensity on zygote development. Our findings suggest that decreased nutritional capacity of the mother plant in response to root herbivory rather than herbivory effects on pollen quality was responsible for the lower fruit and seed production in infested plants.

DNA methylation-mediated ROS production contributes to seed abortion in litchi.

Although there is increasing evidence suggesting that DNA methylation regulates seed development, the underlying mechanisms remain poorly understood. Therefore, we aimed to shed light on this by conducting whole-genome bisulfite sequencing using seeds from the large-seeded cultivar 'HZ' and the abortive-seeded cultivar 'NMC'. Our analysis revealed that the 'HZ' seeds exhibited a hypermethylation level compared to the 'NMC' seeds. Furthermore, we found that the genes associated with differentially methylated regions (DMRs) and differentially expressed genes (DEGs) were mainly enriched in the reactive oxygen species (ROS) metabolic pathway. To investigate this further, we conducted nitroblue tetrazolium (NBT) and 2,7-Dichlorodihydrofluorescein (DCF) staining, which demonstrated a significantly higher amount of ROS in the 'NMC' seeds compared to the 'HZ' seeds. Moreover, we identified that the gene LcGPX6, involved in ROS scavenging, exhibited hypermethylation levels and parallelly lower expression levels in 'NMC' seeds compared to 'HZ' seeds. Interestingly, the ectopic expression of LcGPX6 in Arabidopsis enhanced ROS scavenging and resulted in lower seed production. Together, we suggest that DNA methylation-mediated ROS production plays a significant role in seed development in litchi, during which hypermethylation levels of LcGPX6 might repress its expression, resulting in the accumulation of excessive ROS and ultimately leading to seed abortion.