Progress 12/01/17 to 11/30/19
Outputs
Target Audience:The target audience of this research is the general scientific community that includes basic and applied scientists, as well as education and extension specialists. We are also committed to bringing plant biology to the general public, especially to young generations. We have developed a bilingual (English and Spanish) set of experimental modules in plant biology for kids (www.plants4kids.org) and are running monthly hands-on demonstrations at the North Carolina Museum of Natural Sciences and at local elementary schools. Our Plants4Kids web-based program consists of 14 experimental modules designed specifically for young children (ages 5 to 12). Each experimental module is divided into six sections: the question that will be addressed in the experiment, the materials that will be needed, the directions on how to perform the experiment, helpful suggestions, expected results, and follow-up questions. By providing step-by-step bilingual instructions for performing easy and inexpensive (and, most of the time, free) experiments, we hope to include children coming from low-income families that often have very limited resources and/or poor English language skills. Our goal is to stimulate interest in science among young people and to make basic science accessible to the underrepresented Hispanic community. To date, our program has reached thousands of kids over the course of over 120 live demos (ranging in duration from 2 to 8 hours each). To increase the range of our outreach program, we are currently producing videos where these different experimental modules are brought to life. All sections of the modules are recorded in English and Spanish by elementary school kids and the bilingual movies are available in YouTube. We feel that we are making a difference in our community, as we have kids returning to our museum demos to talk to us and share their personal experiences in plant biology inspired by our earlier demonstrations. Besides running monthly hands-on demonstrations every first Saturday of the month, we regularly participate in special events at the NC Museum of Natural Sciences (Darwin Day, Triangle Science Expo, Ground Hog Day, Final Friday, etc.). All personnel in the Alonso-Stepanova group takes part in the Plants4Kids activities and we have been able to attract several other members of the Program of Genetics and the Department of Plant and Microbial Biology (graduate and undergraduate students) to come out and speak with us to the public about plants and experimental research. Besides our Plants4Kids initiative, members of the lab are involved in other types of community outreach by judging school science fair projects, volunteering on local school STEM advisory boards, hosting visiting middle and high school students in our lab for tours and internships, teaching basic plant biology at Family Science Nights and Science Fairs at local elementary schools, meeting with NCSU undergraduate students during Speed Data-ing events where undergraduate students learn about research opportunities at NCSU, participating in high-school career fairs, giving talks at various community, science and outreach events, bringing science to the Scouts of America at Pack 398, making educational videos, and mentoring undergraduate students in research. Changes/Problems:Although we have not been able to complete all of the aims set in the original proposal due to the fact that we faced unanticipated problems with the quality of the ribosome footprints obtained from fruits at late ripening stages, we have been able to accomplish several key goals of the proposal during this limited time-frame. We have adapted the Ribo-seq protocols for tomato fruit samples at key developmental stages relevant to fruit ripening and implemented a reliable bioinformatics pipeline for tomato data analysis. We have also included a shotgun proteomics approach, an aim that was not in the original proposal, but that has proven very helpful from the data validation perspective. Our original protocols had been optimized for Arabidopsis and tomato seedlings, so the processing of tomato fruit samples that have a much higher water content needed some ratiometric adjustments. Although these adjustments allowed us to obtain high-quality ribosome footprints for fruits up to the BR+2 stage, the high content of pigments, sugars and acids present in the fruit after BR stage seemed to interfere with the isolation of high-quality ribosome footprints from later ripening stages. The experience gained during the troubleshooting of this problem suggests that an additional purification step of the polysomes, such as a sucrose cushion, would be necessary to alleviate if not solve this problem. Due to these problems, only three stages have been sequenced for the time-course experiment, and some candidate genes have been identified, as described above. On the other hand, our proteomics results, although not planed in the original proposal, have turned out to be very informative for validating the translational regulation of several ripening genes. This experience would be very valuable when designing the experiments for future grant proposal aiming to identify additional elements involved in the translational regulation of ripening as well in the plant response to biotic and abiotic stresses. Finally, our preliminary results with a previously identified regulatory cis-element that confers translational repression in response to ethylene indicate that the use of this elements represents and effective strategy to control fruit ripening and should serve as proof of principle in a future grant proposal aiming to control other aspects of fruit ripening or combat the detrimental effects of biotic and abiotic factors. What opportunities for training and professional development has the project provided?(Continued from above) In parallel, we have worked on adopting previously identified regulatory cis-elements that confer translational repression in response to ethylene to delay fruit softening. The 3'UTRs of the tomato EBF2 gene that is sufficient to downregulate translation of a reporter gene in the presence of ethylene was fused to the PECTATE LYASE (PCL) gene in the pseudo-genomic context of a Bacterial Artificial Chromosome (BAC) by recombineering (Brumos et al., 2020). The PCL gene Solyc03g111690 has been previously characterized as the major player contributing to the softening of the fruit during ripening (Uluisik et al., 2016). The fruits of PCL knockout and knockdown lines remain firm for a long period of time and do not experience large texture changes due to the reduced activity of the key enzyme, PCL (Uluisik et al., 2016). To synchronize fruit softening with the timing of the decline in ethylene production, the PCLp:PCL-3'EBF2 construct has been transformed into the pcl knockout lines of tomato along with the control construct, PCLp:PCL-3'PCL. The fruits of 8 hemizygous T0 plants for each construct have been phenotyped and the selected lines are being propagated to obtain T1 and T2-generation plants. The fruits of control lines harboring the PCL construct with the native 3'UTR of PCL exhibit firmness values similar to the ones of wild-type fruits, suggesting that the pcl mutation is fully complemented by our control recombineering construct. In contrast, the fruits of the PCL complementation lines with the 3' UTR of EBF2 display delayed fruit softening, as we anticipated. In these fruits, during the initial stages of fruit ripening (until the Breaker stage) when endogenous levels of ethylene are presumed to be high, the translation of the PCL transcripts should be repressed via the fused 3'UTR of EBF2. Only when the endogenous ethylene levels drop by the Red Ripe stage, translation of the PCL transcripts should be resumed, resulting in a delay of the fruit softening and thus, a significant gain in fruit shelf life. Importantly, keeping fruits at room temperature during shipping and storage should keep the transgenic fruits firm and delay softening due to the fruit endogenous ethylene production without affecting the global fruit ripening program or jeopardizing desirable organoleptic changes naturally associated with fruit ripening. Our current data indicate that specific aspects of fruit ripening can be manipulated by altering the translational regulation of key ripening genes. Thus, in a future grant proposal, we would like to focus on the identification and characterization of additional cis-regulatory elements driving translational regulation in response to various developmental or external signals (beyond ethylene) that could enable the development of novel strategies to improve plant stress-tolerance, nutritional value, and yield. Training: In year 1, the project has provided 70% of salary support to Dr. Brumos, a postdoctoral fellow in the Alonso-Stepanova lab who has been acting as a co-PI of the project. As a result of this project, Dr. Brumos has learned state-of-the-art technologies (such as Ribo-seq and recombineering) and received bioinformatics training in RNA-seq and Ribo-seq data analysis, skills that will help Dr. Brumos in his career progression and the upcoming job search (Dr. Brumos is on the job market this year looking for a faculty position to start his own research group). No salary support was provided to Dr. Stepanova or to Dr. Brumos in the 2nd (no-cost-extended) year of the project. In year two, Dr. Brumos has mastered tomato transformation and continued working on optimizing Ribo-seq protocols for ripe fruits. Drs. Stepanova and Brumos are also involved in training a talented group of graduate and undergraduate students, visiting scientists and high school volunteers. The list of past (https://alonsostepanova.wordpress.ncsu.edu/welcome/people/people-former-lab-members/) and current (https://alonsostepanova.wordpress.ncsu.edu/welcome/people/) trainees can be viewed online. How have the results been disseminated to communities of interest?Drs. Brumos and Stepanova presented a total of 36 talks (and 11 posters) over the course of this project Talks presented by Dr. Brumos: Brumos J. and Hellmann E. Social media and science communication. 31st International Conference on Arabidopsis Research (ICAR). Seattle, USA. July 6-10, 2020. Brumos J., Alonso J., Stepanova A. Dissecting plant responses to hormones: from root development to fruit ripening. XV Plant Molecular Biology Meeting. Torremolinos, Spain. June 3-5, 2020. Brumos J., Alonso J., and Stepanova A. Translation regulation in Tomato Fruit: It's Time to Ripen! Postdoctoral Research Symposium, NCSU. Raleigh May 29, 2020. Brumos J. Molecular insights into plant development regulation by local auxin biosynthesis and ethylene signaling. Plant Biology Seminar Series. University of North Carolina, Chapel Hill, March 17th, 2020. Brumos J. Dissecting plant responses to hormones: from root development to fruit ripening. Department of Biochemistry. University of Missouri. Columbia, Missouri, February 17th, 2020. Brumos J. Dissecting plant responses to hormones: from root development to fruit ripening. Department of Biological Sciences. Auburn University. Auburn, Alabama, February 10th, 2020. Brumos J. From transcription to translation, gene expression regulation in response to plant hormones. CALS Ideation Session. Wilson College Textiles, Raleigh, November 13th, 2019. Brumos J., Alonso J., and Stepanova A. Local auxin biosynthesis is a key regulator of plant development. Genetics & Genomics Initiative retreat, Park Alumni Center, Raleigh, August 19th, 2019. Brumos J., Alonso J., and Stepanova A. "Cautionary tales for plant biologists" Molecular stories of auxin and ethylene. Spanish National Centre for Biotechnology (CNB-CSIC), Madrid, Spain, July 22nd, 2019. Brumos J., Alonso J., and Stepanova A. "Translational genetics" New insights into fruit ripening. Novozymes 1, 2, 3 Research Competition, Durham, October 26th, 2018. Brumos J., Alonso J., and Stepanova A. New biotech applications to minimize food waste. Life Science Research Foundation Board meeting, Raleigh, September 10th, 2018. Brumos J., Zhao C., Alonso J., Stepanova A. Tailoring hormone responses in plants via synthetic signal integration devices. NSF PGRP Awardee Meeting, Alexandria, VA, September 6th, 2018. Brumos J., Alonso J., and Stepanova A. Translation regulation in Tomato Fruit: It's Time to Ripen! Beyond Arabidopsis: advantages and challenges of emerging plant models. 29th International Conference on Arabidopsis Research (ICAR). Turku, Finland. June 25-29, 2018. Brumos J., Alonso J., and Stepanova A. New strategies to control fruit ripening. Pop Talks. Visualization Lab, NCSU, Raleigh, March 23rd, 2018. Brumos J., Alonso J., and Stepanova A. Regulation of Translation in Tomato Fruit: It's Time to Ripen! PepsiCo Advanced Research Competition Finals. Raleigh, March 20th, 2018. Brumos J., Stepanova A., and Alonso J. New Insights into Ethylene Signaling and Auxin Biosynthesis. Institute for Plant Molecular and Cellular Biology (IBMCP), Valencia, Spain, December 21st, 2017. Brumos J., Alonso J., and Stepanova A. Dissecting plant responses to hormones: from fruit ripening to root development. XXV Avances en Biología Molecular por Jóvenes Investigadores en el Extranjero. Spanish National Centre for Biotechnology (CNB-CSIC), Madrid, Spain, December 19th-20th, 2017. Brumos J., Stepanova A., and Alonso J. Ethylene Signaling and Auxin Biosynthesis Regulation: Novel tools for crop bioengineering. Valencian Institute for Agricultural Research (IVIA), Moncada, Spain, December 18th, 2017. Talks presented by Dr. Stepanova: Building a synbio toolbox to monitor and control plant hormone activity, Plant Synthetic Biology in Crop Improvement Symposium, Plant Center, University of Georgia, February 10, 2020. Synthetic and molecular genetic approaches in plant hormone research, Special seminar, University of Malaga, Malaga, Spain, December 13, 2019 Tracking plant hormones, Symposium on "Small molecules in plant research", Valencia, Spain, December 10, 2019 Tailoring hormone responses in plants via synthetic signal integration devices, NSF PGRP Awardee Meeting, Alexandria, VA, September 6, 2019 Deciphering the role of local auxin and hormone homeostasis in plant development, The 23rd International Conference on Plant Growth Substances, Paris, France, June 26, 2019 Reaching out to K-12. NC Ag Tech Advisory Council Meeting, BASF, RTP, NC, June 19, 2019 Synthetic and molecular genetics approaches in plant hormone research, Syngenta Plant Expression Community Seminar Series, Syngenta, RTP, NC, April 16, 2019 Genetic approaches to studying hormone auxin in plants, Genetics and Genomics Seminar Series, NCSU, Raleigh, NC, January 7, 2019 Synthetic and molecular genetic approaches in plant hormone research, BASF-NCSU-VIB joint meeting, BASF, RTP, NC, December 4, 2018 Synthetic and molecular genetic approaches in plant hormone research, Novozymes-NCSU-VIB joint meeting, Novozymes, RTP, NC, December 4, 2018 Synthetic and molecular genetic approaches in plant hormone research, BASF Vegetables & Seeds visit, NCSU, Raleigh, NC, November 15, 2018 Reaching out to K-12, North American Arabidopsis Association Steering Committee workshop "Designing, Evaluating and Sharing Effective Outreach Programs in Plant Science", University of California, Davis, CA, November 8, 2018 All roads lead to auxin, Plant and Microbial Biology Departmental Seminar Series, NCSU, Raleigh, NC, October 2, 2018 Tailoring hormone responses in plants via synthetic signal integration devices. 9th Annual Fall Retreat, Program in Genetics, NCSU, Raleigh, NC, August 20, 2018 A single-locus biosensor for simultaneous monitoring of multiple plant hormones, XI International Symposium on the Plant Hormone Ethylene (Ethylene 2018), Chania, Crete, June 4, 2018 Reaching out to K-12, 29th International Conference on Arabidopsis Research; Workshop on "Communicating Science in the Age of Fake News: Broadening Your Impact", Turku, Finland, June 28, 2018 The role of local auxin biosynthesis and transport in plant development, Department of Molecular and Structural Biochemistry, NCSU, Raleigh, NC, April 5, 2018 Reaching out to K-12, Open Forum on "New opportunities for collaboration to build STEM Workforce", RTP, NC, February 28, 2018? What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Our Ribo-seq protocols have been adapted to tomato fruits and employed to identify sets of genes thatexperience changes in their transcript abundance or in translation efficiency during the ripening process. Side-by-side Ribo-seq and RNA-seq time-course experiments were designed to examine the contribution of translation regulation to the ripening process. Two identical and independent time-course experiments have been performed including seven key ripening stages: Immature Green, Mature Green, Breaker, Breaker+2 days, Breaker+5 days, Red Ripe, and Overripe, with four biological replicates per time point for a total of 28 samples per experiment. RNA-seq and Ribo-seq libraries from a select subset of these samples have been prepared and sequenced on the Illumina NextSeq platform. Mature Green (MG) and Breaker+2 days (BR+2) samples were chosen for the exploratory work due to the large differences reported between these two ripening stages at the transcriptional and physiological levels. For these two time points, 16 libraries have been sequenced on two NextSeq lanes. After applying our quality controls, only reads with unique alignments to the tomato genome (ITAG 4.0) were selected. For the eight Ribo-seq libraries, an average of 2.3 million reads were obtained, whereas for the RNA-seq libraries, an average of 17.2 million reads was achieved. The IG stage libraries have also been successfully prepared and their quality confirmed on a lower-scale Illumina MySeq platform. Although initial optimization allowed for the isolation of polysomes from fruits of all ripening stages, Ribo-seq library preparation has proven difficult in the later ripening-stage fruits, presumably due to their higher content of water, acids, sugars, and secondary metabolites. Future work should include a sucrose-cushion polysome isolation step that may provide the sample quality needed to prepare good Ribo-seq libraries. Between the MG and BR+2 stages, we have identified 491 genes induced in the RNA-seq and 922 genes in the Ribo-seq datasets, with 431 of these transcripts appearing in both datasets, indicating that 87% of all genes differentially regulated at the transcriptional level are also differentially represented in the footprint data. For down-regulated genes between the two time-points, there are 998 genes repressed in the RNA-seq and 1235 in the Ribo-seq data. 716 of these genes are present in both lists, comprising 71% of all transcriptionally downregulated genes. The large overlap in genes between the RNA-seq and Ribo-seq datasets indicates that for the majority of the genes differentially transcribed between MG and BR+2, transcript abundance dictates the level of translation (i.e., the scale of protein production). Our main interest is, however, to identify genes whose expression is regulated at the translational level. To this end, we examined the rate of translation per transcript or, in other words, transcript translation efficiency (TE) of each gene, calculated as the log2 of the relative expression in Ribo-seq/RNA-seq datasets. Employing the TE index, 54 genes have been identified to be translationally regulated (FDR<0.05) between MG and BR+2 stages. Among these, we have found that the translation efficiency of the three SlEBFs is severely reduced, as we had previously hypothesized. From prior work (Liu et al., 2015), we knew that in climacteric fruits at the onset of ripening (at the MG stage), a burst of respiration is observed that is accompanied by a large increase in ethylene production. At this stage, the transcripts of the negative regulators of ethylene response SlEBF1, SlEBF2, and SlEBF3 are known to begin to accumulate and reach their highest expression at BR+2 stage (Klee and Giovannoni, 2011; Liu et al., 2015). However, ethylene production and response are also the greatest in the fruit at the BR+2 stage (Barry et al., 1996; Barry et al., 2000; Tieman et al., 2001). The inhibitory effects of the EBFs on the ethylene signaling pathway and, more specifically, on the activity of EIN3-like proteins, SlEILs, are therefore somehow contained. Our new data show that although the transcription of three SlEBFs paralogues is induced by the endogenous ethylene produced by the fruit, their translation is inhibited, allowing for the tomato fruit to fully respond to the hormone. These findings are in agreement with our previous observations made in Arabidopsis (Merchante et al., 2015), where AtEBFs transcripts accumulate in response to ethylene but are poorly translated until the ethylene signal is withdrawn. Future work should focus on characterizing additional translationally regulated ripening genes beyond SlEBFs at different stages of fruit development. We anticipate that the detailed mechanistic understanding of the role of these genes in the ripening process will become the objective of future grant proposals to be submitted to USDA. A complementary proteomic analysis of the same samples collected for the RNA-seq and Ribo-seq assays has been pursued. This approach was not planned in the original proposal but we thought that proteomics is an excellent addition to the project. We ran a shotgun proteomics analysis on the MG and BR+2 samples and were able to identify 273 proteins that preferentially accumulate in the BR+2 stage and 326 proteins that are more abundant in the MG time-point. Importantly, of the 54 differentially translated transcripts in our Ribo-seq dataset, 46% are also identified as differentially translated by the proteomics approach, cross-validating these two inherently very different technologies. Combining the three different methods, we have been able to identify 11 genes whose transcript and protein accumulation show opposite trends (i.e., induced in RNA-seq but repressed in Ribo-seq, or vice versa). Using proteomics, RNA-seq, and Ribo-seq data, we have been able to confirm the results of previous reports on this group of 11 genes whose transcript levels identified by RNA-seq and protein accumulation analyzed by proteomics followed opposite trends (Szymanski et al., 2017). The differences can now be explained and attributed to the translation efficiency of the mRNAs. In future grant proposals, we would like to pursue the proteomics approach as a complement to Ribo-seq and implement a targeted-proteomics procedure for our top candidate genes. Both, shotgun and targeted proteomics could be performed side by side with RNA-seq and Ribo-seq assays to monitor the actual protein levels at different ripening stages and, hopefully, validate the results of RNA-based methodologies. (Continued below)
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Brumos J., Zhao C., Gong Y., Soriano D., Patel A.P., Perez-Amador M.A., Stepanova A.N., Alonso J.M. An improved recombineering toolset for plants. The Plant Cell. 2020, 32(1):100-122. doi: 10.1105/tpc.19.00431.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Zander M., Willige B., He Y., Nguyen T.A., Langford A.E., Nehring R., Howell E., McGrath R., Barlett A., Castanon R., Nery J., Chen H., Zhang Z., Jupe F., Stepanova A.N., Schmitz R.J., Lewsey M., Chory J, Ecker J.R. (2019) Epigenetic silencing of a multifunctional plant stress regulator. Elife 2019;8:e47835. doi: 10.7554/eLife.47835.001
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Stepanova A.N. and Alonso J.M. (2019) From Ethylene-Auxin Interactions to Auxin Biosynthesis and Signal Integration. Plant Cell 31: 13931394 doi: 10.1105/tpc.19.00339
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Perkins P., Mazzoni-Putman S., Stepanova A., Alonso J., Heber S. (2019) riboStreamR: a web application for quality control, analysis, and visualization of Ribo-seq data. BMC Genomics 20(Suppl 5):422. doi: 10.1186/s12864-019-5700-7
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Fernandez-Moreno J.P. and Stepanova A.N. (2019) Monitoring ethylene in plants: genetically encoded reporters and biosensors. Small Methods 2019: 1900260 doi: 10.1002/smtd.201900260
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Brumos J., Robles L.M., Yun J., Vu T.C., Jackson S., Alonso J.M., Stepanova A.N. (2018) Local auxin biosynthesis is a key regulator of plant development. Dev Cell Dev Cell 47, 306318. doi: https://doi.org/10.1016/j.devcel.2018.09.022
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Mazzoni-Putman S. and Stepanova A.N. (2018) A plant biologists toolbox to study translation. Front Plant Sci 9:873. doi:10.3389/fpls.2018.00873
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Bhosale R., Giri J., Pandey B.K., Giehl R.F.H., Hartmann A., Traini R., Truskina J., Leftley N., Hanlon M., Swarup K., Rashed A., Vo� U., Alonso J., Stepanova A., Yun J., Ljung K., , Brown K.M., Lynch J.P., Dolan L., , Vernoux T., Bishopp A., Wells D., von Wir�n N., Bennett M.J., Swarup R. (2018) A mechanistic framework for auxin dependent Arabidopsis root hair elongation to low external phosphate. Nature Commun 9(1):1409. doi: 10.1038/s41467-018-03851-3
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Ferrando A., Mar Castellano M., Lis�n P., Leister D., Stepanova A.N., Hanson J. (2017) Relevance of Translational Regulation on Plant Growth and Environmental Responses. Front Plant Sci 19. doi: 10.3389/fpls.2017.02170
- Type:
Journal Articles
Status:
Submitted
Year Published:
2020
Citation:
Brumos J., Bobay BG, Clark CA, Alonso JM, and Stepanova AN. Structure-function analysis of interallelic complementation in RTY transheterozygotes. BioRxiv. 2020.
doi: https://doi.org/10.1101/2020.01.02.893016
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Bagley M.C., Stepanova A.N., Ekel�f M., Alonso J.M., Muddiman D.C. (2019) Development of a relative quantification method for IR-MALDESI mass spectrometry imaging of Arabidopsis seedlings. Rapid Commun Mass Spectrom (preprint). doi: 10.1002/rcm.8616
|
Progress 12/01/17 to 11/30/18
Outputs
Target Audience:The target audience of this research is the general scientific community that includes basic and applied scientists, as well as education and extension specialists. We are also committed to bringing plant biology to the general public, especially to young generations. We have developed a bilingual (English and Spanish) set of experimental modules in plant biology for kids (www.plants4kids.org) and are running monthly hands-on demonstrations at the North Carolina Museum of Natural Sciences and at local elementary schools. Our Plants4Kids web-based program consists of 14 experimental modules designed specifically for young children (ages 5 to 12). Each experimental module is divided into six sections: the question that will be addressed in the experiment, the materials that will be needed, the directions on how to perform the experiment, helpful suggestions, expected results, and follow-up questions. By providing step-by-step bilingual instructions for performing easy and inexpensive (and, most of the time, free) experiments, we hope to include children coming from low-income families that often have very limited resources and/or poor English language skills. Our goal is to stimulate interest in science among young people and to make basic science accessible to the underrepresented Hispanic community. To date, our program has reached thousands of kids over the course of over 100 live demos (ranging in duration from 2 to 8 hours each). We feel that we make a difference in our community, as we have kids returning to our museum demos to talk to us and share their personal experiences in plant biology inspired by our earlier demonstrations. Besides running monthly hands-on demonstrations every first Saturday of the month, we regularly participate in special events at the NC Museum of Natural Sciences (Darwin Day, Triangle Science Expo, Ground Hog Day, Final Friday, etc.). All personnel in the Alonso-Stepanova group takes part in the Plants4Kids activities and we have been able to attract several other members of the Program of Genetics and the Department of Plant and Microbial Biology (graduate and undergraduate students) to come out and speak with us to the public about plants and experimental research. Besides our Plants4Kids initiative, members of the lab are involved in other types of community outreach by judging school science fair projects, volunteering on local school STEM advisory boards, hosting visiting middle and high school students in our lab for tours and internships, teaching basic plant biology at Family Science Nights and Science Fairs at local elementary schools, meeting with NCSU undergraduate students during Speed Data-ing events where undergraduate students learn about research opportunities at NCSU, participating in high-school career fairs, giving talks at various community, science and outreach events, bringing science to the Scouts of America at Pack 398, making educational YouTube videos, and mentoring undergraduate students in research. Changes/Problems:Although we have not been able to complete all of the initial aims set in the original proposal due to the fact that it took longer than anticipated to set up the Ribo-seq protocol for fruits, we have been able to accomplish several key goals of the proposal during this time-frame. We have adapted the Ribo-seq protocols for tomato fruit samples and implemented a reliable bioinformatics pipeline for tomato data analysis. We have also included a shotgun proteomics approach, an aim that was not in the original proposal, but is very helpful from the data validation prospective. Our original protocols have been optimized for Arabidopsis and tomato seedlings, so the processing of tomato fruit samples that have a much higher water content needed some ratio-metric adjustments. Likewise, pigments, sugars and acids present in the fruit after BR stage interfered with the purification and digestion of RNA, but protocol optimization of the Ribo-seq sample preparation has been able to solve those initial difficulties. Due to these delays, only two stages have been fully sequenced for the time-course experiment, and some candidate genes have been identified, as described above. Libraries for the remaining stages are being prepared and will be sequenced in the next months with the carried over funds. The planned expansion of the aforementioned proteomics analyses (to be done on the same samples as employed for Ribo-seq and RNA-seq in collaboration with Dr. Manuel Kleiner's proteomics laboratory at NCSU) will provide an additional layer of information (i.e., protein IDs and levels) to complement and validate our translation regulation work and will allow us to compare our results with previously reported data. Independently of the preceding aims, we have employed the previously identified regulatory cis-element that confers translational repression in response to ethylene with the goal of delaying fruit softening. The recombineering constructs utilizing BAC clones have been prepared and transferred to binary vectors to transform the pcl mutant. Selection and characterization of the transgenic T0 lines will be completed in the upcoming months. The results obtained in the present project will be an excellent foundation for future proposals. What opportunities for training and professional development has the project provided?The project has provided 70% of salary support to Dr. Brumos, a postdoctoral fellow in the Alonso-Stepanova lab who has been acting as a co-PI of the project. As a result of this project, Dr. Brumos has learned state-of-the-art technologies (such as Ribo-seq and recombineering) and received bioinformatics training in RNA-seq and Ribo-seq data analysis, skills that will help Dr. Brumos in his career progression and upcoming job search. Drs. Stepanova and Brumos are also involved in training a talented group of graduate and undergraduate students, visiting scientists and high school volunteers. The list of past (https://alonsostepanova.wordpress.ncsu.edu/welcome/people/people-former-lab-members/) and current (https://alonsostepanova.wordpress.ncsu.edu/welcome/people/) trainees can be viewed online. How have the results been disseminated to communities of interest?Talks presented by Dr. Brumos: 1. Brumos J., Alonso J., and Stepanova A. "Translational genetics" New insights into fruit ripening. Novozymes 1, 2, 3 Research Competition, Durham, October 26th, 2018. 2. Brumos J., Alonso J., and Stepanova A. New biotech applications to minimize food waste. Life Science Research Foundation Board meeting, Raleigh, September 10th, 2018. 3. Brumos J., Alonso J., and Stepanova A. Translation regulation in Tomato Fruit: It's Time to Ripen! Beyond Arabidopsis: advantages and challenges of emerging plant models. 29th International Conference on Arabidopsis Research. Turku, Finland. June 25-29, 2018. 4. Brumos J., Alonso J., and Stepanova A. New strategies to control fruit ripening. Pop Talks. Visualization Lab, NCSU, Raleigh, March 23rd, 2018. 5. Brumos J., Alonso J., and Stepanova A. Regulation of Translation in Tomato Fruit: It's Time to Ripen! PepsiCo Advanced Research Competition Finals. Raleigh, March 20th, 2018. 6. Brumos J., Stepanova A., and Alonso J. New Insights into Ethylene Signaling and Auxin Biosynthesis. Institute for Plant Molecular and Cellular Biology (IBMCP), Valencia, Spain, December 21st, 2017. 7. Brumos J., Alonso J., and Stepanova A. Dissecting plant responses to hormones: from fruit ripening to root development. XXV Avances en Biología Molecular por Jóvenes Investigadores en el Extranjero. Spanish National Centre for Biotechnology (CNB-CSIC), Madrid, Spain, December 19th-20th, 2017. 8. Brumos J., Stepanova A., and Alonso J. Ethylene Signaling and Auxin Biosynthesis Regulation: Novel tools for crop bioengineering. Valencian Institute for Agricultural Research (IVIA), Moncada, Spain, December 18th, 2017. Talks presented by Dr. Stepanova: 1. Synthetic and molecular genetic approaches in plant hormone research, BASF Vegetables & Seeds visit, NCSU, Raleigh, NC, November 15, 2018. 2. Reaching out to K-12, North American Arabidopsis Association Steering Committee workshop "Designing, Evaluating and Sharing Effective Outreach Programs in Plant Science", University of California, Davis, CA, November 8, 2018. 3. All roads lead to auxin, Plant and Microbial Biology Departmental Seminar Series, NCSU, Raleigh, NC, October 2, 2018. 4. Tailoring hormone responses in plants via synthetic signal integration devices. 9th Annual Fall Retreat, Program in Genetics, NCSU, Raleigh, NC, August 20, 2018. 5. A single-locus biosensor for simultaneous monitoring of multiple plant hormones, XI International Symposium on the Plant Hormone Ethylene (Ethylene 2018), Chania, Crete, June 4, 2018. 6. Reaching out to K-12, 29th International Conference on Arabidopsis Research; Workshop on "Communicating Science in the Age of Fake News: Broadening Your Impact", Turku, Finland, June 28, 2018. 7. The role of local auxin biosynthesis and transport in plant development, Department of Molecular and Structural Biochemistry, NCSU, Raleigh, NC, April 5, 2018. 8. Reaching out to K-12, Open Forum on "New opportunities for collaboration to build STEM Workforce", RTP, NC, February 28, 2018. Posters presented by Dr. Brumos: 1. Brumos J., Alonso J., Stepanova A. Local sources of auxin are essential for growth and development. Latin American Research Symposium, North Carolina State University, Raleigh, February 15th, 2019. 2. Brumos J., Alonso J., Stepanova A. Plant architecture is defined by local production of auxin. Genetics Symposium, North Carolina State University, Raleigh, February 8th, 2019. 3. Brumos J., Alonso J., and Stepanova A. Local production of the plant hormone auxin is sufficient to maintain the stem cell niche. Genetics Retreat, The Dorothy and Roy Park Alumni Center, Raleigh, August 20th, 2018. 4. Brumos J., Alonso J., Stepanova A. Local auxin biosynthesis is a key regulator of plant development. Salamanca, Spain. XIV Plant Molecular Biology Meeting. July 4-6, 2018. 5. Brumos J., Alonso J., Stepanova A. The role of local auxin biosynthesis and transport in plant development. 29th International Conference on Arabidopsis Research. Turku, Finland. June 25-29, 2018. 6. Brumos J. Scientific career as a Plant Geneticist in Academia. Career panel, Genetics Club. NCSU, Raleigh, April 10th, 2018. Posters presented by Dr. Stepanova: 1. Brumos J., Zhao C., Alonso J.M., Stepanova A.N. (2018) Tailoring hormone responses in plants via synthetic signal integration devices, NSF PGRP Awardee Meeting, Alexandria, VA. 2. Stepanova A.N., Fernandez Moreno J.P., Ascencio-Ibanez J., Alonso J.M. (2018) A single-locus biosensor for simultaneous monitoring of multiple plant hormones, 29th International Conference on Arabidopsis Research, Turku, Finland. In addition, a number of posters have been presented by the lab trainees. What do you plan to do during the next reporting period to accomplish the goals?During the next months, the remaining time-point samples will be processed and the libraries will be sequenced. Specific points in the protocol have been optimized to streamline the library preparation. Data will be analyzed and genes displaying translational regulation will be identified for the rest of the ripening stages. For the employment of specific regulatory cis-elements responsible for the translational repression in response to hormone ethylene to delay fruit softening, T0 plants will be screened and preliminary phenotyping assays will be completed before propagating the selected lines.
Impacts
What was accomplished under these goals?
Our Ribo-seq protocols have been adapted to tomato fruits and employed to identify sets of genes that experience changes in their transcript abundance or in translation efficiency during the ripening process. Side-by-side Ribo-seq and RNA-seq time-course experiments were designed to examine the contribution of translation regulation to the ripening process. Two identical and independent time-course experiments have been performed. Following the reviewers' recommendations, we incorporated an extra time point between Breaker and Breaker+5 days, thus collecting fruits at seven ripening stages for two time-course experiments: Immature Green, Mature Green, Breaker, Breaker+2 days, Breaker+5 days, Red, and Overripe, with four biological replicates per time point for a total of 28 samples per experiment. RNA-seq and Ribo-seq libraries either have or are still being prepared from each sample, resulting in a total of 56 libraries. Mature Green (MG) and Breaker+2 days (BR+2) were the initial samples processed due to the large differences reported between these two ripening stages at the transcriptional and physiological levels. For these two time points, 16 libraries have been sequenced on one lane of the Illumina Nextseq platform. After applying our quality controls, only reads with unique alignments to the tomato genome were selected. For the eight Ribo-seq libraries, an average of 2.3 million reads was obtained, whereas for the RNA-seq libraries an average of 5.2 million reads was achieved. The rest of the samples are currently being processed and will be sequenced in the next few months. Between the MG and BR+2 stages, we have identified 491 genes induced in the RNA-seq and 922 genes in the Ribo-seq datasets, with 431 of these transcripts appearing in both datasets, indicating that 87% of all genes differentially regulated at the transcriptional level are also differentially represented in the footprint data. For down-regulated genes between the two time-points, there are 998 genes repressed in the RNA-seq and 1235 in the Ribo-seq data. 716 of these genes are present in both lists, comprising 71% of all transcriptionally downregulated genes. The large overlap in genes between the RNA-seq and Ribo-seq datasets indicates that for the majority of the genes differentially transcribed between MG and BR+2, transcript abundance dictates the level of translation (i.e., the scale of protein production). Our main interest is, however, to find genes whose expression is regulated at the translational level. To this end, we examined the rate of translation per transcript or, in other words, transcript translation efficiency (TE) of each gene, calculated as the log2 of the relative expression in Ribo-seq/RNA-seq datasets. Employing the TE index, 54 genes have been identified to be translationally regulated (FDR<0.05) between MG and BR+2 stages. Among these, we have found that the translation efficiency of the SlEBFs is severely reduced, as we previously hypothesized. From prior work (Liu et al., 2015), we knew that in climacteric fruits at the onset of ripening (at the MG stage), a burst of respiration is observed that is accompanied by a large increase in ethylene production. At this stage, the transcripts of the negative regulators of ethylene response SlEBF1, SlEBF2, and SlEBF3 are known to begin to accumulate and reach their highest expression at Breaker+2 days stage (Klee and Giovannoni, 2011; Liu et al., 2015). However, ethylene production and response are also the greatest in the fruit at the Breaker+2 stage (Barry et al., 1996; Barry et al., 2000; Tieman et al., 2001). The inhibitory effects of the EBFs on the activity of EIN3-like proteins, SlEILs, are therefore somehow contained. Our new data demonstrate that although the transcription of these three SlEBFs is induced by the endogenous ethylene produced by the fruit, their translation is inhibited, allowing for the tomato fruit to fully respond to the hormone. These findings are in agreement with the observations made in Arabidopsis (Merchante et al., 2015), where AtEBF transcripts accumulate in response to ethylene but are poorly translated until the ethylene signal is withdrawn. We are in the process of characterizing additional translationally regulated ripening genes beyond SlEBFs at different stages of fruit development and anticipate that the detailed mechanistic understanding of the role of these genes in the ripening process will become the objective of future grant proposals. A complementary proteomic analysis of the samples collected for the RNA-seq and Ribo-seq assays has been pursued. This approach was not planned in the original proposal but we thought that proteomics is an excellent addition to the project. We ran a shotgun proteomics analysis on the MG and BR+2 samples and were able to identify 273 proteins that preferentially accumulate in the BR+2 stage and 326 proteins that are more abundant in the MG time-point. Importantly, 46% of the genes that code for these proteins are found among the 54 differentially translated transcripts in our Ribo-seq dataset. Combining the three different approaches, we have been able to identify 11 genes whose transcript and protein accumulation show the opposite trend (i.e., induced in RNA-seq but repressed in Ribo-seq, or the other way around). Using proteomics, RNA-seq and Ribo-seq data, we have been able to confirm the results of previous reports on this group of 11 genes whose transcript levels identified by RNA-seq and protein accumulation analyzed by proteomics followed opposite trends (Szymanski et al., 2017). The differences can now be explained and attributed to the translation efficiency of the RNAs. We plan to further pursue the proteomics approach as a complementary strategy that accompanies our RNA-seq and Ribo-seq assays by expanding the sample list to other fruit developmental stages and obtaining deeper coverage for all samples. In parallel, we have worked on adopting previously identified regulatory cis-elements that confer translational repression in response to ethylene to delay fruit softening. The 3'UTRs of Arabidopsis and tomato EBF2 genes that are sufficient to downregulate translation of a reporter gene in the presence of ethylene were fused to the softening-related PECTATE LYASE (PCL) gene in the pseudo-genomic context of a Bacterial Artificial Chromosome (BAC) by recombineering. To synchronize fruit softening with the timing of the decline in ethylene production, the PCLp:PCL-3'EBF2 construct is being transformed into the pcl knockout lines of tomato along with the control construct, PCLp:PCL-3'PCL. In the fruits of the 3'EBF2 lines, the high endogenous ethylene levels are expected to repress the translation of the PCL transcripts via the fused 3'UTR of EBF2 during the Breaker stages. Only when the ethylene levels drop at the Red stage, PCL transcripts should be translated leading to softening in the ripe fruit. References Barry CS, Llop-Tous MI, Grierson D. doi:10.1104/pp.123.3.979. Barry CS, Blume B, Bouzayen M, Cooper W, Hamilton AJ, Grierson D. doi:10.1046/j.1365-313X.1996.09040525.x Klee HJ, Giovannoni JJ. doi: 10.1146/annurev-genet-110410-132507. Liu M, Pirrello J, Chervin C, Roustan J, Bouzayen M. doi: 10.1104/pp.15.01361. Merchante C, Brumos J, Yun J, Hu Q, Spencer KR, Enríquez P, Binder BM, Heber S, Stepanova AN, Alonso JM. doi: 10.1016/j.cell.2015.09.036. Szymanski J, Levin Y, Savidor A, Breitel D, Chappell?Maor L, Heinig U, Töpfer N. and Aharoni A. doi:10.1111/tpj.13490. Tieman DM, Ciardi JA, Taylor MG, Klee HJ. doj: 10.1046/j.1365-313x.2001.01006.x
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Brumos J., Robles L.M., Yun J., Vu T.C., Jackson S., Alonso J.M., Stepanova A.N. (2018) Local auxin biosynthesis is a key regulator of plant development. Dev Cell Dev Cell 47, 306318. doi: https://doi.org/10.1016/j.devcel.2018.09.022
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Mazzoni-Putman S. and Stepanova A.N. (2018) A plant biologists toolbox to study translation. Front Plant Sci 9:873. doi:10.3389/fpls.2018.00873
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Bhosale R., Giri J., Pandey B.K., Giehl R.F.H., Hartmann A., Traini R., Truskina J., Leftley N., Hanlon M., Swarup K., Rashed A., Vo� U., Alonso J., Stepanova A., Yun J., Ljung K., , Brown K.M., Lynch J.P., Dolan L., , Vernoux T., Bishopp A., Wells D., von Wir�n N., Bennett M.J., Swarup R. (2018) A mechanistic framework for auxin dependent Arabidopsis root hair elongation to low external phosphate. Nature Commun 9(1):1409. doi: 10.1038/s41467-018-03851-3
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Ferrando A., Mar Castellano M., Lis�n P., Leister D., Stepanova A.N., Hanson J. (2017) Relevance of Translational Regulation on Plant Growth and Environmental Responses. Front Plant Sci 19. doi: 10.3389/fpls.2017.02170
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