Source: VIRGINIA POLYTECHNIC INSTITUTE submitted to NRP
EXPEDITING ROOTSTOCK AND SCION BREEDING FOR COLD AND DROUGHT TOLERANCE BY PREDICTING HORMONE SIGNALING INTERACTIONS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1027979
Grant No.
2022-67013-36293
Cumulative Award Amt.
$299,441.00
Proposal No.
2021-07687
Multistate No.
(N/A)
Project Start Date
Dec 1, 2021
Project End Date
Nov 30, 2024
Grant Year
2022
Program Code
[A1141]- Plant Health and Production and Plant Products: Plant Breeding for Agricultural Production
Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
Biological Systems Engineering
Non Technical Summary
Grafting, transplanting the roots of one variety of a plant species with the above-ground leaf and fruit bearing (scion) tissue of another variety, provides a unique opportunity to "engineer" desirable traits in crops. Particularly for fruit trees grafting is essential, as the root variety can control the overall size and growth pattern of the above-ground portion of the plant, regardless of the typical size of above-ground variety. In this way grafting can increase yields and ease harvesting. Root varieties can also enhance the tolerance of the above ground variety to stresses such as late frosts. However little is known about how the root varieties and above-ground varieties interact to enhance stress tolerance and other traits. Late frost events, frosts after fruit trees are in bloom, can cause nearly complete loss of fruit tree crops, a $10 billion dollar industry in the US. As full bloom dates aremoving earlier and earlier due to climate change, late frost events will become more and more frequent and catastrophic.To better understand and improve our ability to incorporate frost tolerance in fruit trees through grafting strategies we aim to identify the signals coming from root varieties that are responsible for these valuable traits such as frost tolerance. To do this we will (1) Identify the signals associated with frost tolerance in a panel of apple root varieties grafted with the same fruit variety through paired measurements of gene expression and chemical signals in the two tissues, and (2) develop methods to rapidly measure the ability of different root varieties and above-ground varieties to send and receive the signals responsible for frost tolerance. Together these results will provide insights into rootstock variety selection for stakeholders such as orchards, nursuries, and home gardners. This work will also inform plant breeders ability to create even more stress tolerant varieties and provide a proof-of-concept for applying similar approaches to enhance other traits through selective breeding.
Animal Health Component
10%
Research Effort Categories
Basic
80%
Applied
10%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20611101060100%
Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
1110 - Apple;

Field Of Science
1060 - Biology (whole systems);
Goals / Objectives
The most productive fruit trees result from grafting well-adapted, disease resistant, and stress-resilient rootstock with high-bearing, desirable scion (shoot tissue). In many casesgrafted combinations are even more than the sum of their parts, as strong synergy between rootstock and scion can result from hormone signals transmitted between the tissues, which have different genetic makeups.Our major goal is to identify the mechanisms and breeding targets throughwhich apple rootstock tissues can provide apple scion tissue with the ability to resist late frosts, which result in significant reductions in deciduous fruit productivity. We have identified several rootstocks that increased the frost resilience of two scion varieties in the field in 2020.Our objectives are to (1) identify hormone-mediated interactions between rootstock and scion through paired transcriptomics and hormonal profiling of a panel of rootstock-scion pairs, and (2) validate high throughput molecular assays for functional characterization of hormone perception in deciduous fruits. Through this pairing of systems and synthetic biology we will identify and validate breeding targets and grafting strategies which will increase the resilience of fruit trees to osmotic stress events such as frost and drought.
Project Methods
Objective 1: Identify hormone-mediated interactions between rootstock and scion through paired transcriptomics and hormonal profiling of a panel of rootstocksMethod: To determine the network of genes and hormonal responses mediating frost resistance between six rootstock genotypes of different cold-hardiness and common scion genotypes we will correlate frost bud damage with differential expression of RNA transcripts and hormone metabolite accumulation of simulated frost treated plants and greenhouse grown control plants (6 rootstocks x 3 biological replicates x 2 treatments x 2 tissue types = 72 samples).Task 1- Plant material preparation: Two-year-old trees consisting of fuji scions grafted to a panel of six rootstock varieties (B.9, B.10, M.9, M.26, G.11, and G.41) will be grown in root bags so they are amenable to frost simulation. Ten plants of each rootstock will be grown to ensure three replicates of each frost and control treatment are achieved. The trees will be placed in the greenhouse until they reach full bloom. Trees will be photographed twice weekly and the fraction of buds at each stage will quantified.Task 2- Plant treatments and bud viability: The day each tree blooms they will be exposed to freezing temperature (21°F) for 1 h, by moving it to an environmental chamber programmed to mimic a temperature profile of a frost night with temperatures starting at 38°F by 12:00 am and dropping gradually to 21°F at 6:00 am. At 7:00 AM we will collect rootstock bark tissue from just below the graft junction and scion bark tissue from just below the lowest buds of the lowest lateral branch. Samples will be snap frozen in liquid nitrogen and stored at -80°C. Simultaneously, branches with flowering buds will be collected from treated trees and placed in a growth chamber for six hours before assessing the freeze damage. Flowers will be dissected using a razor blade and those with brown/dark pistils will be considered dead.Task 3- Transcriptome and hormonal analysis: For transcriptome analysis, total RNA will be extracted from each sample and quality will be checked at the Virginia Tech Frailin Genomics Sequencing Center prior to library preparation and sequencing. Raw reads will be processed using a high accuracy pipeline [24] and transcript reads will be mapped to the Malus domestica reference genome for expression quantification, and single nucleotide polymorphism and insertion/deletion analysis. For hormonal analysis, auxins, cytokinins, ABAs, gibberelins, and ethylene precursors will be quantified from the most and least frost resistant rootstock genotypes. Transcripts and hormones which are differentially expressed/accumulated in each tissue in frost treatment relative to control treatment will be identified. Principle component, hierarchical clustering, GO term, KEGG, and weighted gene co-expression network analyses [25] will be performed to identify genes, networks, and metabolic pathways associated with frost tolerance.Objective 2: Functional characterization of hormone perception in deciduous fruits using high throughput molecular assaysMethod: To translate the knowledge we gain about the key signaling networks involved in frost tolerance, we will develop functional assays of these networks as well as of individual genes. These high-throughput in vitro assays will allow us to rapidly screen rootstock and scion varieties for their sensitivity to key hormones involved in frost tolerance, and to identify potential genetic mechanisms for this variation in hormone sensitivity. This quantitative and mechanistic knowledge will help to advance breeding programs and grafting strategies. The evidence presented above suggests that ABA is responsible for frost tolerance. However, if other hormone pathways are equally correlated with frost resistance in Objective 1, we will adjust Objective 2 to incorporate development of similar assays for these additional pathways.Task 1a- Measure response of hormone responsive synthetic promoters in apple: We will initially test the existing hormone responsive synthetic promoter reporters in apple, by transforming these reporter plasmids driving erYFP:firefly luciferase fusion into calli cultures or whole leaves via agrobacterium infiltration. A similar reporter with mutant promoter driving erBFP:click beetle luciferase fusion will be included in the plasmid as an internal transformation control. Transformed tissues will be treated with hormone or vehicle control and reporter expression will be visualized using fluorescent microscopy/luminescent imaging and measured using a fluorescent/luminescent plate reader.Pitfall mitigation: Hormone response element sequences are generally highly conserved, however should these synthetic promoters not respond to hormone treatment, we will perform motif enrichment of hormone induced genes using existing transcriptome datasets to identify apple candidates that can then be tested in the above assay.Task 1b- Measure the variation in hormone responses between rootstock and scion varieties. We hypothesize that upregulated transport/biosynthesis and increased sensitivity to ABA are associated with frost resistance. To test this, we will compare the expression of the validated ABRE reporter between the different rootstock varieties, via infiltration of bud leaves. A linear regression model will be used to determine the degree to which ABRE reporter expression is able to predict frost damage of buds for the 6 rootstock genotypes.Field application: Critically, we will compare the responses of these hormone reporters between our greenhouse-grown and field-grown plants of the same genotypes to validate the in-the-field relevance of these tools.Task 2a- Reconstitute the core apple ABA signaling pathway in yeast: The core ABA signaling, biosynthesis, and transport pathway components from Arabidopsis have recently been expressed in yeast and shown to recapitulate similar signaling behavior to assays in plant cells. We will similarly clone and express the Malus × domestica proteins of this pathway in yeast and test their function. Gene sequences of the core ABA signaling components will be obtained from the Malus domestica reference genome and codon optimized for expression in yeast prior to DNA synthesis. Genes will be cloned using Golden Gate cloning and integrated into the yeast genome using standard methods. An ABRE reporter promoter as above driving a fluorescent response gene will be used to measure ABA responsiveness of this synthetic recapitulation of the apple ABA signaling pathway.Task 2b- Measure the functional variations in ABA signaling between rootstock varieties.The validation of Task 2a will allow us to compare the functional effects of genetic variants that exist between rootstock and scion varieties by incorporating these genetic variants into the yeast integrating plasmids. We will identify genetic variants in core ABA signaling genes of the 6 rootstock varieties from the transcriptome analysis in Aim 1 as well as from available apple cultivar resequencing data and introduce these variants into the reference sequences of the yeast plasmids. Activity of variant containing strains will be compared to activity of the genome reference proteins in their ability to activate an ABRE-reporter to identify variants using a 3-parameter dose response model fit using maximum likelihood.

Progress 12/01/21 to 11/30/24

Outputs
Target Audience:The target audience for this project are pomme fruit horticulturists and scientists as well as growers and breeders. The target audience was reached through scientific publication and conference presentations as listed in the Products sections. Growers, breeders, and horticulturists were also reached through presentations at the 99th Annual CUmberland-Shenandoah Fruit Workers Conference in Winchester VA as well as Tree Fruit Schools at Virginia Tech's Also H. Smith Jr. Agricultural Research and Extension Center on February 12th 2024, at Rappahannock-Madison Virginina on February 13th 2024, and at Central Virginia Lovingston on February 14th 2024. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A total of 7 mentees have been involved in the project including 1 post doc, 4 graduate students, and 2 undergraduates. These mentees have been trained in bioinformatics, molecular biology, and plant physiology techniques through this project in addition to written and oral science communication to a variety of audiences. Most of these students have presented their work on this project at a conference and/or extension event. Several students have also been involved in manuscript and grant writing related to this project. In the past year, onegraduate student has passed their preliminary exam and two others have completed and defended their dissertations. How have the results been disseminated to communities of interest?The results of the project have been disseminated to the communities of interest through presentations at scientific conferences as well as fruit growers conferences, field days, and extension events. A manuscript describing our RNAseq analysis is under review at BMC Plant Biology and the data has been submitted toNCBI Sequence Read Archive (SRA) BioProject ID: PRJNA112256. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Over the past year we have further validatedB.9as a frost-tolerant rootstockconferringsuperior adaptation to frost stress, in contrast to M.26,which exhibits highbud mortality. This finding highlights the critical role of rootstock selection in mitigating frost damage in deciduous fruit trees. To examine the gene networks that may be responsible for the resilience of B.9we performed RNAseq on scion and sucker leavesand flowers of field grown trees with Gala scions and either B.9 or M.26 rootstocks both before and after a natural frost event in May 2022. Our analysis highlighted increase activation of cold responsive pathways, abscisic acid signaling, lipid biosynthesis and transport, and stress responses in B.9's frost resilience. This analysis has also allowed us to identify over 192 million SNPs and indels between B.9, M.26, and Gala and define phylogenies of gene families across Rosaceae and Brassicaceae that we initially expected to be involved in frost tolerance. To further validate the effects of these gene networks we have pursued CRISPR/Cas gene editing of apple tissue cultures as well as transient overexpression and synthetic biology approaches. Agroinfiltration remains a challenge in apple leaf tissue for transient studies. Ternary vectors have shown promise for improving agroinfiltration in other crops over the binary vectors we have used to date. However, transformation of apple cell suspension cultures has proven less recalcitrant, supporting our gene editing and reporter gene efforts. We are still in the process of recapitulating apple abscisic acid signaling in yeast, but have plasmids sequenced and are currently building yeast strains for initial tests. These plasmids and strains will allow us to map the functional variation across the SNPs we have identified in abscisic acid signaling genes.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Chisholm S, Bryant JA, Waller C, Sherif S, Wright RC. Autoluminescence and apples  Developing a high-throughput screening tool for freeze tolerance in apple. 2024 Virginia Tech Translational Plant Science Mini-symposium, 2024 February 2, Blacksburg, VA, USA.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Neres DF, Wright RC (2024) Pleiotropy, a feature or a bug? Toward co-ordinating plant growth, development, and environmental responses through engineering plant hormone signaling. Current Opinion in Biotechnology, 88: 103151. https://doi.org/10.1016/j.copbio.2024.103151 (2022 IF = 7.7)
  • Type: Other Journal Articles Status: Submitted Year Published: 2025 Citation: Saini AK, Jahid K, Neres DF, Wright RC, Sherif S. Decoding frost tolerance of apple rootstock cultivars through the lens of transcriptome analysis. BMC Plant Biology. Submitted 21 August 2024
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Saini, AK (2024) Investigating the Physiological and Molecular Aspects of Frost Tolerance in Apple Trees across Different Rootstocks. 2024 Virginia Tech Translational Plant Science Mini-symposium, 2024 February 2, Blacksburg, VA, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: 4. Saini, A.K. (2024). Investigating Frost Response, Rootstock-Dependent Cold Tolerance, and Floral Bud Mortality in Apple Cultivars through Transcriptomic Insights  Southeast Professional Fruit Workers Conference, Auburn University, Auburn, Oct 23rd- 25th, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: 5. Saini, A.K. (2024). Rootstock Driven Frost Tolerance and Bud Mortality in Apple: Gene Identification for future germplasm development  100th Cumberland Shenandoah Fruit Workers Conference, Martinsburg, WV, Dec 5th- 6th, 2024.


Progress 12/01/22 to 11/30/23

Outputs
Target Audience:This project has been presented to growers, the general public,and scientists spanning breeders, horticulture, plant physiology, and engineering fields through our groups presentations at more than 10 different conferences, symposia, conventions and outreach events during this year. We have also communicated these results and discussed how our collaboration between an expert in horticulture and plant physiology and a synthetic biologist and engineer was spawned from a day-trip to visit an agricultural research station at two events Virginia Tech College of Agriculture and Life Science Fall 2023 New Faculty Orientationand theCenter for Advanced Innovation in Agriculture Fall 2023 Kickoff Event. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has supported training in systems and synthetic biology for 1 postdoc, 6graduate students (2 recently onboarded), and 1 undergraduate. Regular whole project group meetings (once every three weeks) have helped train these students spanning disciplines of bioinformatics, plant physiology, engineering, and molecular biology. Each of these trainees has contributed to manuscript writing and presented results at local, regional, and/or national meetings during this period. How have the results been disseminated to communities of interest?Results have been disseminated through publications and presentations listed in Products as well as grower meetings facilitate by Dr. Sherif and his mentees. What do you plan to do during the next reporting period to accomplish the goals?We are currenlty meeting regularly to finalize publication of our systems biology analysis of frost tolerancevs frost sensitivity conferred by different rootstocks and also our methods for apple leaf infiltration and transient expression. These two manuscripts will be submitted in the next reporting period.

Impacts
What was accomplished under these goals? This year we have made great progress on the project particularly towards developing and understanding of the major drivers of frost tolerance via our systems biology approach. We have identified carbohydrate and reactive oxygen accumulation as primarymetabolic differences between frost-sensitive and resistant rootstocks. Our transcriptomic analysis has revealed several enzymes involved in oxidative and alkaline stress resistance andhormone biosynthesis (gibberelin) as well as a few transcription factors (ethylene responsive AP-2 type and MADs-box) and several uncharacterized protein transcripts are differentially regulated in scion tissues between B9 and M26 rootstocks. We are currently revising themanuscript describing this dataset and our conclusions and plan to submits in the next month or 2.We have simultaneously developed a bioinformatics pipeline for calling variants in B9, M26 and Gala derived transcriptswhich we will use to follow up on the genetic diversity of these potential candidate genes as well as upstream regulators of these genes. We have also recruited a Master's student in Electrical and Computer Engineering co-advised by an expert in machine-learing analysis of graph networks to assist in integrated analaysis of these datasets to facilitate predictions of the molecular mechanism of the differences we have observed. We have developed plasmids capable of expressing fluorescent proteins in apple leaves, but have found that typical abscisic acid responsivepromoters from arabidopsis are not visibly responsive in apples, however constitutively expressed controls are clearly visible compared to controls. We have also found that fungal bioluminescence pathways (which allow autoluminescence via complete synthesis of luciferin and luciferase enzymes in plants) are not functional in apples possibly due to insufficient expression of one of the 5 enzymes for luciferin biosynthesis. We have a new PhD student continuing this work and building out synthetic assays of gene function for the pathways we identify, as the previous student will graduate in May.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Jahed KR, Saini AK, Sherif SM (2023) Coping with the cold: unveiling cryoprotectants, molecular signaling pathways, and strategies for cold stress resilience. Frontiers in Plant Science, 14:1246093. https://doi.org/10.3389/fpls.2023.1246093
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Bryant JA, Longmire C, Sridhar S, Janousek S, Kellinger M, Wright RC (2023) TidyTron: Reducing lab waste using validated wash-and-reuse protocols for common plasticware in Opentrons OT-2 lab robots. SLAS Technology, :100107. https://doi.org/10.1016/j.slast.2023.08.007
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Chaisupa P, Wright RC (2023) State-of-the-art in engineering small molecule biosensors and their applications in metabolic engineering. SLAS Technology, https://doi.org/10.1016/j.slast.2023.10.005
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Bryant JAG, Taylor J, Bargmann BOR, Wright RC. Blazing a trail towards orthogonal auxin transcriptional regulation. Plant Biology 2023, 2023 August 59; Savanah, GA, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Neres DFG, Rahman MP, Wright RC. Understanding Gene Epistasis through Heterologous Systems. 2023 AICHE Annual Conference, 2023 November 8; Orlando, FL, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Chisholm S, Bryant JA, Waller C, Sherif S, Wright RC. Autoluminescence and Apples  Developing a high-throughput screening tool for freeze tolerance in Apple. 2024 Virginia Tech Translational Plant Science Mini-symposium, 2024 February 2, Blacksburg, VA, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Bryant JA, Wright RC. Build Your Own Biofoundry. 2024 Mid-Atlantic Synthetic Biology Research Coordination Network Symposium, 2024 January 1112; Durham, NC, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Waller C, Bryant JA, Sherif S, Wright RC. Fungal Bioluminescence Pathway for Gene Reporters in Apples. 2023 Summer research Symposium at Virginia Tech, 2023 July 27; Blacksburg, VA, USA.


Progress 12/01/21 to 11/30/22

Outputs
Target Audience:The results of the study are disseminated via the extension program of Virginia Tech's Alson H. Smith Jr. AREC. Sherif, the Co-PI, normally participates in seven in-orchard meetings, five in-class sessions, and two evening meetings annually. The PI also attends and participates in Winter Fruit Schools, which are conducted yearly during the second week of February at five different places across the state. Through these fruit-themed educational programs, the PI aims to educate and inspire thoughtful decision-making. He lectures and distributes educational materials on such topics as crop load management, frost mitigation strategies and recommended rootstocks for apples and peaches. There are more than 300 regular attendees at fruit schools including producers, extension workers, agricultural consultants, and corporate representatives. There are orchard and classroom programs from March through June. At these gatherings, Sherif educates fruit growers on the latest research findings, new chemical ingredients, and recommended rates and application timings of various PGRs. The Co-PI also manages a Facebook page called "Virginia Tech Tree Fruit Extension and Outreach," which has over 1100 followers, and a blog called "Tree Fruit Horticulture Updates," which has over 320 blog subscribers. The findings of this project will be disseminated to growers, extension agents, tree fruit experts, and other stakeholders through presentations at local and regional professional gatherings (such the Mid-Atlantic fruit and vegetable convention and the Cumberland-Shenandoah fruit works conference), as well as written extension materials. Extension meetings where the results of this research were disseminated: - Sherif, S.M. (2022). Use of Ethephon for Bloom Delay in Peaches and Cherries & Effects of Rootstocks on Apple Tolerance to Frost. New Jersey Commercial Fruit Growers' Meetings - Virtual- Invited - Sherif, S.M. (2022). Rootstocks and plant growth regulators for apple. Presented 5X: Winchester Regional Commercial Tree Fruit School, Rappahannock-Madison Area Fruit School, Central Virginia Commercial Tree Fruit Production School, Roanoke-Botetourt Orchard Fruit School and Carroll - Patrick Fruit Growers School. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students have been trained in systems and synthetic biology through this project. They have developed experimental designs and protocols, prepared presentations for a variety of audiences, and drafted manuscripts for scientific publications under the mentorship of the PD and co-PD as described above. How have the results been disseminated to communities of interest?Results thus far have been presented at conferences and extension meetings and published as a preprint as listed above in the Products and Target Audience sections.Additionally, the applications of synthetic biology in agriculture were introduced to 90 high school students participating in the Virginia Governor's School for Agriculture Summer Camp, through hands-on lab actvities. See the results here: https://sites.google.com/vt.edu/wrightlab/outreach? What do you plan to do during the next reporting period to accomplish the goals? Build de novo transcriptomes of B.9 and M.26 rootstocks from transcriptome data and identify SNPs between rootstock genotypes. Identify differentially expressed genes between B.9 and M.26 rootstock leaves before and after frost treatment. Identify differentially expressed genes in Fuji-scion leaves and flowers from plants grafted on B.9 or M.26 rootstocks, before and after frost treatment. Build transcriptional reporters for differentially expressed genes that are associated with frost tolerance and test their response in scion tissues of B.9 and M.26 rootstock trees in response to frost and simulated frost. Build yeast strains for testing molecular function of SNPs that are associated with frost tolerance by synthesizing highly expressed genes in the identified pathways and recapitulating the function of these pathways in yeast. Repeat our experiment comparing frost protection conferred by rootstocks in response to late frosts in spring 2023. Submit another grant proposal leveraging the results from this year and next to develop a rootstock breeding program towards introgressing the abiotic stress tolerance trait we are aiming to define with biotic stress tolerance of Geneva rootstocks.

Impacts
What was accomplished under these goals? Under Objective 1 we have repeated the experiment used as preliminary data in our proposal, providing further confirmation that the B.9 rootstock provides frost protection. This has allowed us to collect sufficient tissue samples to perform, the currently in progress, transcriptome sequencing and metabolomics analyses as described above. Preliminary sugar profiling of previous samples has pointed to accumulation of glucose and fructose in B.9-grafted-fuji-scion leaves and flower, respectively, as a potential mechanism for this frost protection. We have also identified genes comprising the signaling pathways for abscisic acid, auxin, ethylene, gibberelic acid, and sugars in apple, as well as other deciduous fruits, based on their orthology with well-studied Arabidopsis thaliana pathways. Under Objective 2 we have established robotic protocols for development of DNA plasmids for molecular assays for functional characterization of hormone perception and signaling in deciduous fruits. This will reduce human errors in our work this coming year, building plasmids and yeast strains recapitulating the mechanisms of abscisic acid, auxin, ethylene, gibberelic acid, and sugar signaling from apples. We have also established a protocol for transient transformation of intact apple leaves, which will allow us to test transcriptional reporters of genes that are associated with frost tolerance. We have also received yeast strains and plasmids for a previous recapitulation of abscisic acid signaling from Arabidopsis thaliana in yeast.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Bryant, JA, *Kellinger M, *Longmire C, Miller R, and Wright RC (2022). AssemblyTron: Flexible automation of DNA assembly with Opentrons OT-2 lab robots. In revision, resubmitted. BioRxiv: https://doi.org/10.1101/2022.09.29.510219 (*undergraduate mentee author)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Bryant JA, DNtegrAte: An Open-Source Python Package for Performing DNA Assemblies with the OT-2 Liquid Handling Robot (Rapid Fire Talk). 2022 Synthetic Biology: Engineering, Evolution, and Design (SEED), 2022 May 2nd5th; Washington, DC. ID: 641420.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Saini, A.K. (2022). How soluble sugars influence the tolerance of apple trees (Malus domestica Borkh.) to late spring frost damage  Annual SPES Graduate Student Poster Show Competition, Virginia Tech, Blacksburg October 6 th 2022.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Saini, A.K. (2022). How soluble sugars influence the tolerance of apple trees (Malus domestica Borkh.) to late spring frost damage  Southeastern Professional Fruit workers conference, University of Florida, Citrus Research & Education Center November, Florida 14th  November 16th 2022.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Saini, A.K. (2022). How soluble sugars influence the tolerance of apple trees (Malus domestica Borkh.) to late spring frost damage  Cumberland-Shenandoah Fruit Workers Conference, Winchester December 1 st 2022.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Neres D.F., Wright R.C., Phylogenetic and expression analysis of auxin regulatory genes in soybean (Glycine max). 2022 Mid-Atlantic Section of American Society of Plant Biology and University of Maryland Plant Minisymposium. 2022 May 25. College Park, MD, USA.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Neres D.F., Bryant J.A., Wright R.C., Phylogenetic and expression analysis of auxin regulatory genes in soybean (Glycine max). 2022 Virginia Tech Translational Plant Science Center Symposium. 2022 April 29. Blacksburg, VA, USA.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Chaisupa P., Rahman M., Wright R.C., Fungal auxin biosynthesis variation and dynamics revealed by a noise-tolerant, whole-cell auxin biosensor. Annual Meeting of the Phytochemical Society of North America 2022. 24th28th July 2022. Blacksburg, VA, USA.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Neres D.F., Bryant J.A., Wright R.C., Phylogenetic and expression analysis of auxin regulatory genes in soybean (Glycine max). Plant Biology 2022, 9th13th July 2022. Portland, Oregon, USA.