Study of the Relationship between Leaf Color Formation and Anthocyanin Metabolism among Different Purple Pakchoi Lines.

Purple pakchoi (Brassica rapa ssp. Chinensis) is particularly appreciated due to its high edible quality and ornamental value, but there are few studies on the underlying mechanisms of leaf color formation. To comprehensively assess the differences in purple formation in pakchoi, four lines of pakchoi with different purple leaves were used in this experiment to determine the pigment content and to investigate the distribution and components of anthocyanin using LCMS (Liquid Chromatography Mass Spectrometry) and leaf cross-sections. Moreover, the expression levels of anthocyanin synthesis-related genes in four lines were calculated by qRT-PCR. The results showed that three new purple lines rich in anthocyanin and of high-quality were bred, and the anthocyanin were mainly distributed in both the upper epidermis and lower epidermis of leaves. Thirteen anthocyanin components were separated and identified, all the anthocyanins were acylated and glycosylated cyanidins; the main anthocyanins in purple pakchoi were a diacylated form of cyanidin 3-trans-(feruloyl)diglucoside-5-(malonyl)glucoside. Both the ratio of non-aromatic acylated cyanidin to aromatic acylated cyanidin and the ratio of anthocyanin content to chlorophyll content were responsible for the color formation in different purple pakchoi lines. When the ratio was high, the leaf appeared reddish purple, and when the ratio was low, the leaf appeared deep purple, even blackish purple. The expression level of BrF3H was significantly correlated with the content of anthocyanin through the correlation coefficient, which was speculated to be the main anthocyanin synthesis-related gene resulting in color differences among the four purple pakchoi lines. These results will enhance our understanding for the cultivation of new purple pakchoi varieties.

AgNAC1, a celery transcription factor, related to regulation on lignin biosynthesis and salt tolerance.

The NAC transcription factor participates in various biotic and abiotic stress responses and plays a critical role in plant development. Lignin is a water-insoluble dietary fiber, but it is second only to cellulose in abundance. Celery is the main source of dietary fiber, but its quality and production are limited by various abiotic stresses. Here, AgNAC1 containing the NAM domain was identified from celery. AgNAC1 was found to be a nuclear protein. Transgenic Arabidopsis thaliana plants hosting AgNAC1 have longer root lengths and stomatal axis lengths than the wide type (WT). The evidence from lignin determination and expression levels of lignin-related genes indicated that AgNAC1 plays a vital role in lignin biosynthesis. Furthermore, the results of the physiological characterization and the drought and salt treatments indicate that AgNAC1-overexpressing plants are significantly resistive to salt stress. Under drought and salt treatments, the AgNAC1 transgenic Arabidopsis thaliana plants presented increased superoxide dismutase (SOD) and peroxidase (POD) activities and decreased malondialdehyde (MDA) content and size of stomatal apertures relatively to the WT plants. The AgNAC1 served as a positive regulator in inducing the expression of stress-responsive genes. Overall, the overexpressing AgNAC1 enhanced the plants' resistance to salt stress and played a regulatory role in lignin accumulation.

Identification and Expression Analysis of GRAS Transcription Factors to Elucidate Candidate Genes Related to Stolons, Fruit Ripening and Abiotic Stresses in Woodland Strawberry (Fragaria vesca).

The cultivated strawberry (Fragaria × ananassa), an allo-octoploid with non-climacteric fleshy fruits, is a popular Rosaceae horticultural crop worldwide that is mainly propagated via stolons during cultivation. Woodland strawberry (Fragaria vesca), one of the four diploid progenitor species of cultivated strawberry, is widely used as a model plant in the study of Rosaceae fruit trees, non-climacteric fruits and stolons. One GRAS transcription factor has been shown to regulate stolon formation; the other GRAS proteins in woodland strawberry remain unknown. In this study, we identified 54 FveGRAS proteins in woodland strawberry, and divided them into 14 subfamilies. Conserved motif analysis revealed that the motif composition of FveGRAS proteins was conserved within each subfamily, but diverged widely among subfamilies. We found 56 orthologous pairs of GRAS proteins between woodland strawberry and Arabidopsis thaliana, 47 orthologous pairs between woodland strawberry and rice and 92 paralogous pairs within woodland strawberry. The expression patterns of FveGRAS genes in various organs and tissues, and changes therein under cold, heat and GA3 treatments, were characterized using transcriptomic analysis. The results showed that 34 FveGRAS genes were expressed with different degrees in at least four organs, including stolons; only a few genes displayed organ-specific expression. The expression levels of 16 genes decreased, while that of four genes increased during fruit ripening; FveGRAS54 showed the largest increase in expression. Under cold, heat and GA3 treatments, around half of the FveGRAS genes displayed increased or decreased expression to some extent, suggesting differing functions of these FveGRAS genes in the responses to cold, heat and GAs. This study provides insight into the potential functions of FveGRAS genes in woodland strawberry. A few FveGRAS genes were identified as candidate genes for further study, in terms of their functions in stolon formation, fruit ripening and abiotic stresses.

A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants.

Developing CRISPR/Cas9-mediated non-transgenic mutants in asexually propagated perennial crop plants is challenging but highly desirable. Here, we report a highly useful method using an Agrobacterium-mediated transient CRISPR/Cas9 gene expression system to create non-transgenic mutant plants without the need for sexual segregation. We have also developed a rapid, cost-effective, and high-throughput mutant screening protocol based on Illumina sequencing followed by high-resolution melting (HRM) analysis. Using tetraploid tobacco as a model species and the phytoene desaturase (PDS) gene as a target, we successfully created and expediently identified mutant plants, which were verified as tetra-allelic mutants. We produced pds mutant shoots at a rate of 47.5% from tobacco leaf explants, without the use of antibiotic selection. Among these pds plants, 17.2% were confirmed to be non-transgenic, for an overall non-transgenic mutation rate of 8.2%. Our method is reliable and effective in creating non-transgenic mutant plants without the need to segregate out transgenes through sexual reproduction. This method should be applicable to many economically important, heterozygous, perennial crop species that are more difficult to regenerate.

An AGAMOUS intron-driven cytotoxin leads to flowerless tobacco and produces no detrimental effects on vegetative growth of either tobacco or poplar.

Flowerless trait is highly desirable for poplar because it can prevent pollen- and seed-mediated transgene flow. We have isolated the second intron of PTAG2, an AGAMOUS (AG) orthologue from Populus trichocarpa. By fusing this intron sequence to a minimal 35S promoter sequence, we created two artificial promoters, fPTAG2I (forward orientation of the PTAG2 intron sequence) and rPTAG2I (reverse orientation of the PTAG2 intron sequence). In tobacco, expression of the ß-glucuronidase gene (uidA) demonstrates that the fPTAG2I promoter is non-floral-specific, while the rPTAG2I promoter is active in floral buds but with no detectable vegetative activity. Under glasshouse conditions, transgenic tobacco plants expressing the Diphtheria toxin A (DT-A) gene driven by the rPTAG2I promoter produced three floral ablation phenotypes: flowerless, neuter (stamenless and carpel-less) and carpel-less. Further, the vegetative growth of these transgenic lines was similar to that of the wild-type plants. In field trials during 2014 and 2015, the flowerless transgenic tobacco stably maintained its flowerless phenotype, and also produced more shoot and root biomass when compared to wild-type plants. In poplar, the rPTAG2I::GUS gene exhibited no detectable activity in vegetative organs. Under field conditions over two growing seasons (2014 to the end of 2015), vegetative growth of the rPTAG2I::DT-A transgenic poplar plants was similar to that of the wild-type plants. Our results demonstrate that the rPTAG2I artificial promoter has no detectable activities in vegetative tissues and organs, and the rPTAG2I::DT-A gene may be useful for producing flowerless poplar that retains normal vegetative growth.

Changes of cytosine methylation induced by wide hybridization and allopolyploidy in Cucumis.

We previously demonstrated that allopolyploidization could induce phenotypic variations and genome changes in a newly synthesized allotetraploid in Cucumis. To explore the molecular involvement of epigenetic phenomena, we investigated cytosine methylation in Cucumis by using methylation-sensitive amplified polymorphism (MSAP). Results revealed a twofold difference in the level of cytosine methylation between the reciprocal F1 hybrids and the allotetraploid. Analysis of the methylation pattern indicated that methylation changed at 2.0% to 6.4% of total sites in both the F1 hybrids and the allotetraploid compared with their corresponding parents. Furthermore, 68.2% to 80.0% of the changed sites showed an increase in cytosine methylation and a majority of the methylated sites were from the maternal parent. Observations in different generations of the allotetraploid found that the extent of change in cytosine methylation pattern between the S1 and S2 was significantly higher than that between the S2 and S3, suggesting stability in advanced generations. Analysis of 7 altered sequences indicated their similarity to known functional genes or genes involved in regulating gene expression. Reverse transcription - polymerase chain reaction analysis suggested that at least two of the methylation changes might be related to gene expression changes, which further supports the hypothesis that DNA methylation plays a significant role in allopolyploidization.

Cytological diploidization and rapid genome changes of the newly synthesized allotetraploids Cucumis x hytivus.

We used a newly synthesized allotetraploid between C. sativus (2n = 2x = 14, n gametic chromosome number, x haploid chromosome number) and C. hystrix (2n = 2x = 24) to study the genomic events in its early generations. Results from cytological characterization of the F(1) and the allotetraploid progenies showed that the rate of bivalents in meiotic metaphase I of the F(1) was greatly improved by chromosome doubling, and further improved during the selfing process of allopolyploid resulting into relatively diploid-like meiosis. Extensive genomic changes were detected by amplified fragment length polymorphism analysis. The changes mainly involved loss of parental restriction fragments and gaining of novel fragments. The total detectable changes were from 11.1 to 32.1%, and the frequency of losing parental fragments was much higher than that of gaining novel fragments. Some of the changes were initiated as early as in the F(1) hybrid, whereas others occurred after chromosome doubling (polyploid formation). No significant differences were detected in the reciprocal F(1) hybrids and S(0) generations. But the data showed that the frequency of sequence losing in C. sativus was about two times higher than in the C. hystrix. Our results demonstrated that the sequence elimination was the major event of genomic changes, and it might provide the physical basis for the diploid-like meiotic behavior in the diploidization of the newly formed allopolyploids. Moreover, the results suggest that the sequence elimination was not caused by cytoplasmic factors, and might relate to genomic recombination and to the numbers of parental chromosome.