Elucidating the function of a novel cysteine-rich peptide during flower-to-fruit transition in tomato

During fruit initiation, secreted peptides mediate communication between pollen and the female gametophyte, as well as the embryo sac and the surrounding female tissues. The cysteine-rich peptides (CRPs), a diverse class of secreted peptides that contain an N-terminal secretion signal and a C-terminal cysteine-rich region, play an important role in cell-cell signaling during flower-to-fruit transition. However, their role in tomato fertilization, fruit initiation, and seed formation is still unknown. Previous tissue-specific transcriptomic studies in our lab identified OVULE-SECRETED PROTEIN (OSP), a CRP highly expressed in the tomato ovule integument at anthesis. Based on this expression profile, we hypothesized that OSP may be involved in processes during flower-to-fruit transition, such as micropylar pollen tube guidance, ovule maturation, or seed formation. In this project, we conducted studies aimed at the functional characterization of OSP, which included phylogenetic analysis, a study of OSP spatiotemporal expression patterns, and the phenotypic characterization of OSP-RNAi plants. In addition, we designed and created molecular tools to generate osp knock-out mutants using CRISPR technology. Multiple sequence alignment and phylogeny tools demonstrated that the cysteine motif in OSP is unique to the cultivated tomato and its wild relatives. Using gOSP::YFP transgenic plants and confocal microscopy, we have observed that OSP is expressed in ovaries and fruit at stages surrounding anthesis. Our results suggest that OSP expression in the ovule inner integument beginning prior to anthesis. OSP-RNAi plants with reduced levels of OSP expression showed no abnormal phenotype when we examined seed weight, seed number per fruit, external seed and ovule morphology, and pollen tube guidance. We have therefore assembled a CRISPR vector that includes guide RNAs targeting the OSP coding region. We anticipate that osp knockout mutants will show a stronger phenotype that will help elucidate the function of OSP.

My Experience

The Plant Genome Research Program at BTI has been an invaluable experience. I felt immediately welcomed by my lab and the community at BTI. In addition to learning a variety of microscopy and molecular biology techniques, I had the opportunity to make experimental decisions and develop confidence as a researcher. Through working independently and with my mentor, I am better prepared for a research career. This internship has reinforced my intention to pursue plant science during graduate school, and I look forward to a continued relationship with my lab and the interns I have met this summer.

“Functional Characterization of the Tomato Sugar Transporter SWEET10”

Project Summary:

Carbohydrates produced from photosynthesis are crucial to development, growth, and signaling in plants. To maintain optimal plant function, sugars must be moved from the leaf to other areas of the plant by transporters. A novel class of sugar transporters, named Sugar Will Eventually be Exported Transporters or SWEETs has been shown to play a key role in sugar efflux during several physiological processes, however little is known about their role in fruit development. It has been hypothesized that the action of SWEET transporters is needed in specific fruit tissues and stages, allowing efficient utilization of sucrose during fruit and seed development. However, direct molecular genetic evidence of this hypothesis is still lacking. My project aims to elucidate the role of the tomato SWEET transporter, SlSWEET10, in regulating sugar transport during early fruit development. SlSWEET10 is specifically expressed during early fruit development, and mostly in vascular rich tissues such as the columella and the placenta. The SWEETs gene family is split into four clades, with SlSWEET10 belonging to Clade III. In Arabidopsis, several Clade III SWEETs are known to transport sucrose, therefore we hypothesize that SlSWEET10 is involved in the transport of sucrose from the sieve elements into the fruit parenchyma apoplast. In this work, we have analyzed the effect of variable SWEET10 expression on sugar accumulation and fruit weight using transgenic tomato plants. We have identified the subcellular localization of the SWEET10 protein in fruit, and have compared the expression of SWEET10 and its closest homologs in domesticated tomatoes to their wild relatives. Our results suggest that SlSWEET10 plays an important role in sugar transport during early fruit development, a novel, so far unreported function for SWEET transporters. This research will generate insights into the regulation of fruit development and help identify important genes contributing to crop quality.

My Experience:

Through my time at BTI I have become a more confident researcher. Working independently improved my problem-solving skills and critical thinking, while working together with my lab towards one common project has helped to refine my skills in communication and time management. I felt like I was a member of a wider community of plant biologists whose enthusiasm for their work is infectious and inspiring. Through these experiences I have been able to better define my own research interests and have also been able to speak with Cornell professors about the research being conducted in these areas. I have learned more about life as a graduate student, and the process of applying to Cornell PhD programs. Overall, I believe that this summer has been one of the most transformative and educational in my academic career, and I now look even more forward to pursuing a career in plant biology.

Functional characterization of the tomato sugar transporter SWEET10

Project Summary

Tomatoes are one of the most important fleshy fruit crops of modern agriculture. The sugary globe’s versatile nature has led it to become a staple in the dishes of countless cultures across the world. It is this sugar content that differentiates a refreshingly sweet tomato from an unpalatably bitter one. Although a new family of sucrose transporters called SWEETs has been recently discovered in Arabidopsis and rice, little is known of their actions with regards to sugar acquisition in fleshy fruits in general and tomato fruit in particular. Preliminary RNA-seq data has shown that SWEET10, a member of the SWEET family, is highly expressed during early tomato fruit development in the placenta. Due to this early insight, SWEET10 was selected as a suitable candidate for preliminary functional characterization. Comparison of fruit sugar content in wild-type plants and in transgenic RNAi plants in which SWEET10 expression is down-regulated revealed an altered sugar accumulation pattern in the SWEET10 RNAi lines. Using quantitative RT-PCR we showed that auxin regulates the expression of SWEET10 and some of its closest homologs, a finding which may shed light on its expression, and on the regulation of fruit sugar content during ripening.

Our results not only aim to characterize SWEET genes in tomato, but may also provide insight for breeders hoping to harness genetic information to restore the sweeter characteristics of tomatoes that were lost during domestication.

My Experience

My experiences in the Plant Genome Research Program at BTI will be tremendously helpful in my future scientific endeavors. Not only will the laboratory techniques and skills learned be of great assistance, but the very experience of having spent time at a world-class plant research institute will prove valuable. By observing other researchers collaborate between labs and even between institutes I have gained an understanding of the way science is accomplished. The program has also exposed me to the entire spectrum of plant biology research. Having been shown to such a wide array of the dynamic field of plant biology I feel as though I can make more informed decisions for graduate school and beyond. It is due to the aforementioned traits that my summer at BTI has impressed upon me lessons which will aid me at every step of my future.