Morphological and metabolomics profiling of intraspecific Arabidopsis hybrids in relation to biomass heterosis.

Heterosis contributes greatly to the worldwide agricultural yield. However, the molecular mechanism underlying heterosis remains unclear. This study took advantage of Arabidopsis intraspecific hybrids to identify heterosis-related metabolites. Forty-six intraspecific hybrids were used to examine parental effects on seed area and germination time. The degree of heterosis was evaluated based on biomass: combinations showing high heterosis of F1 hybrids exhibited a biomass increase from 6.1 to 44% over the better parent value (BPV), whereas that of the low- and no-heterosis hybrids ranged from - 19.8 to 9.8% over the BPV. Metabolomics analyses of F1 hybrids with high heterosis and those with low one suggested that changes in TCA cycle intermediates are key factors that control growth. Notably, higher fumarate/malate ratios were observed in the high heterosis F1 hybrids, suggesting they provide metabolic support associated with the increased biomass. These hybrids may produce more energy-intensive biomass by speeding up the efficiency of TCA fluxes. However, the expression levels of TCA-process-related genes in F1 hybrids were not associated with the intensity of heterosis, suggesting that the post-transcriptional or post-translational regulation of these genes may affect the productivity of the intermediates in the TCA cycle.

Synchronization of Arabidopsis flowering time and vegetative growth stage via FT overexpression can reveal inherent heterosis due to heterozygosity in intraspecific hybrids.

Intraspecific hybrids of Arabidopsis sometimes display heterosis. However, allelic variation of flowering repressor genes causes late flowering in F1, which might distort the potential heterosis effect due to prolonged vegetative growth. Here, overexpression of flowering gene FT synchronized flowering and eliminated growth differentials between parental and F1. These findings indicate the possibility of quantitatively demonstrating the inherent heterosis caused by heterozygosity.

Exploring Novel Polytubey Reproduction Pathways Utilizing Cumulative Genetic Tools.

In the anthers and ovaries of flowers, pollen grains and embryo sacs are produced with uniform cell compositions. This stable gametogenesis enables elaborate interactions between male and female gametophytes after pollination, forming the highly successful sexual reproduction system in flowering plants. As most ovules are fertilized with a single pollen tube, the resulting genome set in the embryo and endosperm is determined in a single pattern by independent fertilization of the egg cell and central cell by two sperm cells. However, if ovules receive four sperm cells from two pollen tubes, the expected options for genome sets in the developing seeds would more than double. In wild-type Arabidopsis thaliana plants, around 5% of ovules receive two pollen tubes. Recent studies have elucidated the abnormal fertilization in supernumerary pollen tubes and sperm cells related to polytubey, polyspermy, heterofertilization and fertilization recovery. Analyses of model plants have begun to uncover the mechanisms underlying this new pollen tube biology. Here, we review unusual fertilization phenomena and propose several breeding applications for flowering plants. These arguments contribute to the remodeling of plant reproduction, a challenging concept that alters typical plant fertilization by utilizing the current genetic toolbox.

Removal of the endoplasma membrane upon sperm cell activation after pollen tube discharge.

In pollen and pollen tubes, immotile sperm cells are enclosed by an inner vegetative plasma membrane (IVPM), a single endomembrane originating from the vegetative-cell plasma membrane. It is widely believed that sperm cells must be removed from the IVPM prior to gamete associations and fusions; however, details of the timing and morphological changes upon IVPM dissociation remain elusive. Here, we report a rapid IVPM breakdown immediately before double fertilization in Arabidopsis thaliana. The IVPM was stably observed in coiling pollen tubes when pollen tube discharge was prevented using lorelei mutant ovules. In contrast, a semi-in vivo fertilization assay in wild-type ovules demonstrated fragmented IVPM around sperm nuclei 1 min after pollen tube discharge. These observations revealed the dynamic alteration of released sperm cells and provided new insights into double fertilization in flowering plants. With a summary of recent findings on IVPM lipid composition, we discussed the possible physiological signals controlling IVPM breakdown.

F-actin regulates the polarized secretion of pollen tube attractants in Arabidopsis synergid cells.

Pollen tube attraction is a key event of sexual reproduction in flowering plants. In the ovule, two synergid cells neighboring the egg cell control pollen tube arrival via the active secretion of attractant peptides such as AtLURE1 and XIUQIU from the filiform apparatus (FA) facing toward the micropyle. Distinctive cell polarity together with longitudinal F-actin and microtubules are hallmarks of the synergid cell in various species, though the functions of these cellular structures are unclear. In this study, we used genetic and pharmacological approaches to indicate the roles of cytoskeletal components in FA formation and pollen tube guidance in Arabidopsis thaliana. Genetic inhibition of microtubule formation reduced invaginations of the plasma membrane but did not abolish micropylar AtLURE1.2 accumulation. By contrast, the expression of a dominant-negative form of ACTIN8 induced disorganization of the FA and loss of polar AtLURE1.2 distribution toward the FA. Interestingly, after pollen tube reception, F-actin became unclear for a few hours in the persistent synergid cell, which may be involved in pausing and resuming pollen tube attraction during early polytubey block. Our data suggest that F-actin plays a central role in maintaining cell polarity and in mediating male-female communication in the synergid cell.

Possible molecular mechanisms of persistent pollen tube growth without de novo transcription.

The vegetative cell nucleus proceeds ahead of a pair of sperm cells located beneath the pollen tube tip during germination. The tip-localized vegetative nucleus had been considered to play a pivotal role in the control of directional pollen tube growth and double fertilization. However, we recently reported the female-targeting behavior of pollen tubes from mutant plants, of which the vegetative nucleus and sperm nuclei were artificially immotile. We showed that the apical region of the mutant pollen tubes became physiologically enucleated after the first callose plug formation, indicating the autonomously growing nature of pollen tubes without the vegetative nucleus and sperm cells. Thus, in this study, we further analyzed another Arabidopsis thaliana mutant producing physiologically enucleated pollen tubes and discussed the mechanism by which a pollen tube can grow without de novo transcription from the vegetative nucleus. We propose several possible molecular mechanisms for persistent pollen tube growth, such as the contribution of transcripts before and immediately after germination and the use of persistent transcripts, which may be important for a competitive race among pollen tubes.

Integrated transcript and metabolite profiling reveals coordination between biomass size and nitrogen metabolism in Arabidopsis F1 hybrids.

Heterosis refers to the improved agronomic performance of F1 hybrids relative to their parents. Although this phenomenon is widely employed to increase biomass, yield, and stress tolerance of plants, the underlying molecular mechanisms remain unclear. To dissect the metabolic fluctuations derived from genomic and/or environmental differences contributing to the improved biomass of F1 hybrids relative to their parents, we optimized the growth condition for Arabidopsis thaliana F1 hybrids and their parents. Modest but statistically significant increase in the biomass of F1 hybrids was observed. Plant samples grown under the optimized condition were also utilized for integrated omics analysis to capture specific changes in the F1 hybrids. Metabolite profiling of F1 hybrids and parent plants was performed using gas chromatography-mass spectrometry. Among the detected 237 metabolites, 2-oxoglutarate (2-OG) and malate levels were lower and the level of aspartate was higher in the F1 hybrids than in each parent. In addition, microarray analysis revealed that there were 44 up-regulated and 12 down-regulated genes with more than 1.5-fold changes in expression levels in the F1 hybrid compared to each parent. Gene ontology (GO) analyses indicated that genes up-regulated in the F1 hybrids were largely related to organic nitrogen (N) process. Quantitative PCR verified that glutamine synthetase 2 (AtGLN2) was upregulated in the F1 hybrids, while other genes encoding enzymes in the GS-GOGAT cycle showed no significant differences between the hybrid and parent lines. These results suggested the existence of metabolic regulation that coordinates biomass and N metabolism involving AtGLN2 in F1 hybrids.

Association analysis of phenotypic and metabolomic changes in Arabidopsis accessions and their F1 hybrids affected by different photoperiod and sucrose supply.

Photoperiod and sucrose (Suc) assimilation play important roles in the regulation of plant growth and development. However, it remains unclear how natural variation of plants could contribute to metabolic changes under various growth conditions. Here, we investigated the developmental and metabolomic responses of two natural accessions of Arabidopsis thaliana, Columbia (Col) and C24, and their reciprocal F1 hybrids grown under four carbon source regimens, i.e., two different photoperiods and the presence or absence of exogenous Suc supply. The effect of exogenous Suc clearly appeared in the growth of Col and the F1 hybrid but not in C24, whereas long-day conditions had significant positive effects on the growth of all lines. Comparative metabolite profiling of Col, C24, and the F1 hybrid revealed that changes in metabolite levels, particularly sugars, were highly dependent on genotype-specific responses rather than growth conditions. The presence of Suc led to over-accumulation of seven metabolites, including four sugars, a polyamine, and two amino acids in C24, whereas no such accumulation was observed in the profiles of Col and the F1 hybrid. Thus, the comparative metabolite profiling revealed that the two parental lines of the hybrid show a distinct difference in sugar metabolism.