Carotenoids play an important role in many physiological processes in plants and the phytoene desaturase gene (PDS3) encodes one of the important enzymes in the carotenoid biosynthesis pathway. Here we report the identification and analysis of a T-DNA insertion mutant of PDS3 gene. Functional complementation confirmed that both the albino and dwarf phenotypes of the pds3 mutant resulted from functional disruption of the PDS3 gene. Chloroplast development was arrested at the proplastid stage in the pds3 mutant. Further analysis showed that high level of phytoene was accumulated in the pds3 mutant. Addition of exogenous GA3 could partially rescue the dwarf phenotype, suggesting that the dwarf phenotype of the pds3 mutant might be due to GA deficiency. Microarray and RT-PCR analysis showed that disrupting PDS3 gene resulted in gene expression changes involved in at least 20 metabolic pathways, including the inhibition of many genes in carotenoid, chlorophyll, and GA biosynthesis pathways. Our data suggest that the accumulated phytoene in the pds3 mutant might play an important role in certain negative feedbacks to affect gene expression of diverse cellular pathways.

Disruption of Phytoene Desaturase Gene Results in Albino and Dwarf Phenotypes in Arabidopsis by Impairing ChlorophyII,Carotenoid,and Gibberellin Biosynthesis

Cyclocarya paliurus (Batal.) Iljinskaja, a unique species growing in southern China, is a multi-function tree species with medicinal, healthcare, material, and ornamental values. So far, sexual reproduction is the main method for extensive cultivation of C. paliurus plantations, but this is limited by low seed plumpness resulted from the character of heterodichogamy. Phenological observations have revealed the asynchronism of flower development in this species. However, its molecular mechanism remains largely unknown. To reveal molecular mechanism of heterodichogamy in C. paliurus, transcriptome of female (F) and male (M) buds from two mating types (protandry, PA; protogyny, PG) at bud break stage were sequenced using Illumina Hiseq 4000 platform. The expression patterns of both 32 genes related to flowering and 58 differentially expressed transcription factors (DETFs) selected from 6 families were divided four groups (PG-F, PG-M, PA-F, and PA-M) into two categories: first flowers (PG-F and PA-M) and later flowers (PA-F and PG-M). The results indicated that genes related to plant hormones (IAA, ABA, and GA) synthesis and response, glucose metabolism, and transcription factors (especially in MIKC family) played significant roles in regulating asynchronism of male and female flowers in the same mating type. The expression of DETFs showed two patterns. One contained DETFs up-regulated in first flowers in comparison to later flowers, and the other was the reverse. Nine genes related to flowering were selected for qRT-PCR to confirm the accuracy of RNA-seq, and generally, the RPKM values of these genes were consistent with the result of qRT-PCR. The results of this work could improve our understanding in asynchronism of floral development within one mating type in C. paliurus at transcriptional level, as well as lay a foundation for further study in heterodichogamous plants.

Morphological Characterization of Flower Buds Development and Related Gene Expression Profiling at Bud Break Stage in Heterodichogamous Cyclocarya paliurus (Batal.) lljinskaja

Sesame harbors a large diversity in root morphological and anatomical traits and a high root biomass improves the plant aboveground biomass as well as the seed yield. Sesame provides one of the most nutritious and healthy vegetable oils, sparking an increasing demand of its seeds. However, with the low yield and productivity of sesame, there is still a huge gap between the seed demand and supply. Improving the root system has a high potential to increase crop productivity, but information on the diversity of the sesame root systems is still lacking. In this study, 40 diverse sesame varieties were grown in soil and hydroponics systems and the diversity of the root system was investigated. The results showed that sesame holds a large root morphological and anatomical diversity, which can be harnessed in breeding programmes. Based on the clustering of the genotypes in hydroponics and soil culture systems, we found that similar genotypes were commonly clustered either in the small-root or in the big-root group, indicating that the hydroponics system can be employed for a large-scale root phenotyping. Our results further revealed that the root biomass positively contributes to increased seed yield in sesame, based on multi-environmental trials. By comparing the root transcriptome of two contrasting genotypes, 2897 differentially expressed genes were detected and they were enriched in phenylpropanoid biosynthesis, starch and sucrose metabolism, stilbenoid, diarylheptanoid and gingerol biosynthesis, flavonoid biosynthesis, suggesting that these pathways are crucial for sesame root growth and development. Overall, this study sheds light on the diversity of sesame root system and offers the basis for improving root traits and increasing sesame seed yield.

Root diversity in sesame (Sesamum indicum L.): insights into the morphological, anatomical and gene expression profiles.

Flower induction and development in saffron: Timing and hormone signalling pathways

Polyploid induction is of utmost importance in horticultural plants for the development of new varieties with desirable morphological and physiological traits. Polyploidy may occur naturally due to the formation of unreduced gametes or can be artificially induced by doubling the number of chromosomes in somatic cells. In this experiment, a protocol for in vitro polyploid induction of highbush blueberry (Vaccinium corymbosum L.) leaf tissues was studied by using different concentrations of colchicine and oryzalin. Oryzalin was found to be highly toxic to this species, while the adventitious shoot organogenesis media enriched with 25 and 250 µM colchicine was able to induce polyploidization, with significant differences among the treatments used. Higher concentrations of both antimitotic agents led to the browning and death of the leaf tissues. The polyploids obtained showed several morphological differences when compared with the diploid shoots. Flow cytometry analysis was used to confirm the ploidy level of the regenerated shoots, demonstrating that a total of 15 tetraploids and 34 mixoploids were obtained. The stomatal sizes (length and width) of the tetraploids were larger than those of the diploids, but a reduced stomatal density was observed as compared to the controls. These shoots will be acclimatized and grown until they reach the reproductive phase in order to test their potential appeal as new varieties or their use for breeding and genetic improvement.

/pdf/in-vitro-polyploid-induction-of-highbush-blueberry-through-1vfndvax.pdf

In Vitro Polyploid Induction of Highbush Blueberry through De Novo Shoot Organogenesis

Gibberellins (GAs) and their regulator DELLA are involved in many aspects of plant growth and development and most of our current knowledge in the DELLA-facilitated GA signaling was obtained from the studies of annual species. To understand GA-DELLA signaling in perennial species, we created ten GA-insensitive transgenic grapevines carrying a DELLA mutant allele (Vvgai1) in the background of Vitis vinifera ‘Thompson Seedless’ and conducted comprehensive analysis of their RNA expression profiles in the shoot, leaf and root tissues. The transgenic lines showed varying degrees of dwarf stature and other typical DELLA mutant phenotypes tightly correlated with the levels of Vvgai1 expression. A large number of differentially expressed genes (DEGs) were identified in the shoot, leaf and root tissues of the transgenic lines and these DEGs were involved in diverse biological processes; many of the DEGs showed strong tissue specificity and about 30% them carried a DELLA motif. We further discovered unexpected expression patterns of several key flowering induction genes VvCO, VvCOL1 and VvTFL1. Our results not only confirmed many previous DELLA study findings in annual species, but also revealed new DELLA targets and responses in grapevine, including the roles of homeodomain transcription factors as potential co-regulators with DELLA in controlling the development of grapevine which uniquely possess both vegetative and reproductive meristems at the same time. The contrasting responses of some key flowering induction pathway genes provides new insights into the divergence of GA-DELLA regulations between annual and perennial species in GA-DELLA signaling.

/pdf/rna-seq-reveals-new-della-targets-and-regulation-in-3cwvhrk23o.pdf

RNA-Seq reveals new DELLA targets and regulation in transgenic GA-insensitive grapevines

To develop an effective genome editing tool for blueberry breeding, CRISPR-Cas9 and CRISPR-Cas12a were evaluated for their editing efficiencies of a marker gene, beta-glucuronidase (gusA), which was previously introduced into two blueberry cultivars each a single-copy transgene. Four expression vectors were built, with CRISPR-Cas9 and CRISPR-Cas12a each driven by a 35S promoter or AtUbi promoter. Each vector contained two editing sites in the gusA. These four vectors were respectively transformed into the leaf explants of transgenic gusA blueberry and the resulting transgenic calli were induced under hygromycin selection. GUS staining showed that some small proportions of the hygromycin-resistant calli had non-GUS stained sectors, suggesting some possible occurrences of gusA editing. We sequenced GUS amplicons spanning the two editing sites in three blueberry tissues and found about 5.5% amplicons having editing features from the calli transformed with the 35S-Cas9 vector. Further, we conducted a second round of shoot regeneration from leaf explants derived from the initial Cas9- and Cas12a-containing calli (T0) and analyzed amplicons of the target editing region. Of the newly induced shoots, 15.5% for the 35S-Cas9 and 5.3% for the AtUbi-Cas9 showed non-GUS staining, whereas all of the shoots containing the Cas12a vectors showed blue staining. Sanger sequencing confirmed the editing-induced mutations in two representative non-GUS staining lines. Clearly, the second round of regeneration had enriched editing events and enhanced the production of edited shoots. The results and protocol described will be helpful to facilitating high-precision breeding of blueberries using CRISPR Cas technologies. A second round of regeneration enriched editing events and enhanced the production of edited blueberry shoots. The new protocol described facilitates high-precision breeding of blueberries using CRISPR Cas technologies.

https://link.springer.com/content/pdf/10.1007/s11240-021-02177-1.pdf

Yingzhen Yang

Papers

RNA-Seq reveals new DELLA targets and regulation in transgenic GA-insensitive grapevines

CRISPR Cas9- and Cas12a-mediated gusA editing in transgenic blueberry