Location: Plant Gene Expression Center
2023 Annual Report
Objectives
A major bottleneck to research progress and the rate of crop genetic improvement is lengthy periods (months to years) of vegetative growth preceding initiation of reproductive maturity, thereby extending generational times. Methods for controlling and accelerating the transition to flowering, and reducing dormancy time, can dramatically compress this time scale, giving breeders and researchers a significant time advantage for identifying and introgressing desirable alleles for crop traits. Such accelerated crop breeding methods are widely used in annual crops but remain to be established for most woody crop species. A promising opportunity for compressing the generational time lies in manipulation of Flowering locus T (FT) gene expression or by mutating the homologous gene, Terminal Flower (TFL), which is a floral repressor. Manipulation of FT to reduce flowering time has been achieved through genetic engineering, grafting, and viral induction-based approaches. TFL can be edited to achieve a similar early flowering. Controlling the time to flowering and reproductive maturity might also be achieved by decreasing juvenility through manipulation of evolutionarily conserved microRNA (miRNAs). This project will integrate cutting-edge technologies, such as new transformation strategies, genome editing, grafting, and computational biology to develop and implement accelerated crop breeding strategies for California woody horticultural crops such as pistachio, almonds, walnuts, and/or grapes. The deliverables include research and breeding tools and information for controlling flowering, reproductive maturity, and decreasing generation time for woody horticultural crops.
Specific Objectives of this project are:
Objective 1: Develop a suite of research and breeding tools to accelerate time to flowering and reproductive maturity in woody horticultural crops through modification of florigen or miR156 activity.
Objective 2: Enlist and apply the approaches of transcriptomics, network analysis and comparative genomics to discover novel genetic mechanisms that operate across woody horticultural crops that control initiation and maturation of the reproductive phase.
Approach
Objective 1, Hypothesis: Expression of FT in the desired crop will shorten time to flowering. The first step is to identify FT orthologs in the selected woody horticultural crop by sequence homology-based searches and domain-based gene phylogenetic approaches, using existing genome sequences for related tree species, published RNA-seq transcriptome assemblies, and resources from Objective 2. Next, FT gene activity will be assessed by testing for complementation of the late flowering phenotype of the Arabidopsis ft-10 mutant. Flowering time of Arabidopsis transformants overexpressing the candidate FT will be assayed under long day conditions, where the ft-10 phenotype is most severe, based on rosette leaf number. FT ortholog(s) found to accelerate flowering will be moved to the next phase to test these FT constructs in the crop of interest as first generation proof-of-principle transgenics. Initial transformation attempts will employ epicotyl transformation with Agrobacterium. Whole genome sequencing will confirm transgene location and verify that transformation caused no other genome changes. Long term goals will be identifying inducible promoters and possible virus-inducible systems to control FT expression. Data from Objective 2 is expected to help guide the decision of which FT to use in the final round of tree transformation.
Objective 2, Hypothesis: Discover novel genetic mechanisms that control initiation and maturation of the reproductive phase. A quantitative RT-PCR (qPCR) experiment will document the 24-hour, or diurnal, expression pattern for the FT genes identified in Objective 1. Testing samples taken during a summer month (April-June) and a winter month (November-February) will reveal where seasonal regulation occurs for these FT genes. This effort is expected to give insight into potential functions of each FT paralog. Transcriptional profiling by RNA-seq of the same temporal and seasonal samples will identify differentially expressed genes by pairwise comparisons (leaf vs flower, summer vs winter, etc.) and assess temporal expression pattern (diurnal change over 72 hours, season). This analysis will determine what other genes are expressed in dynamic, coordinated networks with FT genes. To catalogue the transcript splice forms that exist across seasons and tissues and to further refine available reference transcriptomes of the selected tree species, a full-length transcriptome will be constructed using Pacific Biosciences Single Molecule Real-Time sequencing. This full-length transcriptome will help to identify the total number and sequence similarity of FT orthologues, design primers for cDNA cloning or qPCR, and profile isoform-level gene expression in RNA-seq projects. Also, this resource will be of use to future genomics research and breeding efforts in the woody crop species of interest.
Progress Report
This is the final report for project 2030-21000-054-000D, which expired in March 2023 and was replaced by project 2030-21210-001-000D.
In year one of this project a job search for a Molecular Biologist (Plants) SY position was conducted and the selectee from that search began the position in spring of fiscal year (FY) 2022. The new researcher is building on many of the findings from the life of this project (2030-21000-054-000D), including the analyses of the gene regulatory networks upstream of FLOWERING LOCUS T (FT) in Populus and Citrus in the new project (2030-21210-001-000D).
In the first year of this project, for Objectives 1 and 2, public genome sequence databases were searched for amino acid sequences similar to well-known FT proteins (from the model plant Arabidopsis thaliana and the cereal crop rice) in five citrus species: Kumquat, Citron, Pomelo, Mandarin, and Meyer lemon. This effort identified two citrus FT proteins present in all five species that were highly similar amongst one another and to the known FT proteins. The conclusion was that these FT proteins, and the genes encoding them, are likely to have an important role in regulating flowering in citrus. Oligonucleotide primers with sequences from these FT genes were designed and purchased to amplify the FT transcript sequences. The same approach also was employed to identify genes encoding UBIQUITIN (UBQ) proteins. Expression of these UBQ genes are controls when monitoring FT gene expression in Objective 2. Leaf samples were taken from adult kumquat and Meyer lemon trees in the summer to provide a source of transcripts to amplify the citrus FT sequences for Objective 1. Samples were taken in the morning and evening in the event expression was time-of-day dependent. The next step for Objective 1 is to test these two citrus FT homologs for their capacity to functionally substitute for the Arabidopsis FT gene. The DNA sequences for the citrus FT transcripts will be put into a transgenic expression construct suitable for transformation of both Arabidopsis and citrus, which was identified in literature searches.
For Objective 2, the morning and evening time points were used to evaluate how gene expression varies throughout the day and night. The levels of FT and UBQ transcripts were assessed by quantitative reverse transcriptase-polymerase chain reaction (qPCR). In the kumquat leaves, expression levels of the two FT genes were higher in the evening. In contrast, expression levels of the two FT genes in Meyer lemon leaves were higher in the morning. These observations indicate that while these two types of citrus have highly similar FT genes, the daily regulation of them is different. This indicates flowering regulatory networks in these closely related trees may be somewhat different. It is possible these differences may impact strategies for promotion of flowering.
In year two of this project, ARS researchers in Albany, California, made progress toward the goal of Objective 1 to design and develop constructs for testing FT gene homologs for promoting flowering in Arabidopsis thaliana (Arabidopsis) and woody crops. One of the unknowns associated with this work is which promoter DNA sequence will produce the best results, given the diversity of woody plant species that will be transformed, the potential variation in each FT gene’s effectiveness at promoting flowering, and the degree to which promoting flowering alters other important plant traits. A modular system that makes it easy to test different promoters in combination with different FT homologs was developed to streamline these tests. To this end, the Golden Gate method of combining DNA sequences was used to rapidly test promoters in diverse transformation systems. In these experiments, the coding sequence of the FT1 gene from poplar (Populus tremula x alba) and two different strong, constitutive promoter sequences (double 35S and Arabidopsis UBI10) were combined with the octaline synthase terminator to transform wildtype Arabidopsis. Both promoters were able to induce early flowering, with the 35S promoter generating earlier bolting than the UBI10 promoter. These promoter-FT1 DNA sequence combinations were tested with two different DNA sequences for selection of transgenic plants based on resistance to either hygromycin or kanamycin. These successful experiments made a system that will enable planned experimental tests of citrus FT homologs in a citrus transformation system. Additionally, the researchers cloned a number of other promoters into the Golden Gate system to allow fine tuning of the expression level and tissue-specific expression of FT homologs.
For Objective 2, which aims to identify genetic mechanisms controlling initiation and maturation of the reproductive phase, progress was made toward the goal of generating gene models from woody crops. The researchers took advantage of the many recent publicly available transcriptome data sets for this purpose. A data set from mature shoot apices of Key Lime that was generated to examine the developmental mechanisms of thorn production by comparing thornless and thorned varieties, and also includes samples of young leaf tissue and apical meristems, was used to generate de novo transcriptomes assemblies. This effort identified 5,310 unique expressed genes across the thornless and thorned datasets, and 62 genes differentially expressed between varieties. Going forward, this pipeline will be used to identify genes differentially expressed between citrus samples differing in degree of maturation or across floral initiation to identify novel factors controlling these processes.
In year three of this project, ARS researchers in Albany, California, made progress toward the goal of Objective 1 to test FT gene homologs for promoting flowering in Arabidopsis thaliana and woody crops. Transformations using Populus FT1 where completed in Arabidopsis using a strong (35S) and weak (UBI10) constitutive promoter. The UBI10 promoter was found to induce flowering earlier and more consistently across independent transformed plants. Additionally, transformation systems were established in citrus using Carrizo citrange epicotyls and Populus tremula x alba leaf explants and will be used for testing FT gene homologs and promoter combinations identified in years one and two of this project.
Progress was also made for Objective 2, where the aim is to identify novel genetic mechanisms regulating maturation. Bioinformatic analyses were applied to a dataset of woody Acacia species with samples differing in developmental age. This work identified homologs of SHORT VEGETATIVE PHASE as having a role in the promotion of maturation, a finding that is opposite of their known functions in Arabidopsis and may offer novel gene products for manipulating maturation in other species. Additionally, the genetic regulatory network controlling FT1 and FT2 expression in Populus was found to be regulated by levels of MIR156 a finding that is central to the new project.
Accomplishments
