UMD Theses and Dissertations
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Item GENOMICS ENABLED GENE DISCOVERY IN DIPLOID AND POLYPLOID WHEAT(2024) Yadav, Inderjit Singh; Tiwari, Vijay; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hexaploid bread wheat (Triticum aestivum) is one of the most important staple food crops for humans. Sustainable genetic improvement in wheat is critical for ensuring global food security and requires the introduction of new genes and alleles into elite wheat cultivars. The progenitor species and wild wheat relatives are a reservoir of genetic diversity for wheat improvement. This doctoral thesis demonstrates the application of genomic resources and bioinformatics pipelines to characterize the wild germplasm and to streamline the gene discovery pipeline using five diverse species involving wheat progenitor, wild, and related species. Genomics-assisted characterization of the genetic diversity present in gene banks is a major step towards the systematic utilization of unexploited germplasm to ensure the sustainable development of new varieties. Toward this end, we used genomics datasets to curate wild and related accessions of tetraploid wheat from two distinct species Triticum turgidum and Triticum timopheevii. Using Genotyping by sequencing (GBS) data and a unique similarity matrix and powercore analysis, a set of 102 accessions were identified as the core set accessions that represent 20 and 35 percent of the total accessions of the WGRC tetraploid wheat collection of T. turgidum and T. timopheevii, respectively. Further, three distinct centers of rich genetic diversity were identified for wild and domesticated emmer and T. timopheevii in the Fertile Crescent. GWAS analysis of the genotypic and phenotypic dataset identified a novel QTL for leaf rust resistance on chromosome 2B in T. timopheevii. Triticale is a man-made cereal derived from a cross between tetraploid and hexaploid wheat with diploid rye. There are large numbers of triticale germplasm available in different gene banks; however, in many cases, the ploidy information is not accurate and affects the quality of work with large triticale germplasm. In this work, using the low-cost GBS datasets, a pipeline was developed to detect contamination in the UMD triticale collection and facilitated the accurate classification of ploidy, ensuring the purity of the triticale germplasm. This approach identified contamination of 11 wheat accessions and enabled the correct classification of 236 hexaploid and 12 octoploid triticale, these results were further confirmed through GISH experiments. Wild and related germplasms are considered as the goldmine of genetic diversity for wheat improvement. The modern wheat cultivars have gone through several rounds of heavy selections for yield related traits and have lost the genetic diversity against several abiotic and biotic stresses. On the other hand, wild relatives of wheat have been growing naturally without any substantial artificial selection pressure and it allowed them to preserve their genetic diversity. This study investigates the genetic diversity of a selected set of genes to visualize the differences in wild wheat relatives and polyploid wheat cultivars. To study these differences, group 5 chromosome of Aegilops geniculata and Aegilops umbellulata, belonging to the tertiary gene pool, were assembled. Comparative analysis revealed a higher rate of pseudogenization in bread wheat compared to these two wild relatives, primarily due to the difference in exon/intron length between the genes, rendering these genes non-functional. Diploid einkorn wheat (Triticum monococcum), with inherent disease resistance, offers a valuable resource for wheat improvement. To facilitate its proper utilization, two of the reference genomes-one wild (T. monococcum ssp. aegilopoides) and one domesticated (T. monococcum ssp. monococcum) were assembled in the study. Kmer-GWAS identified seven novel QTLs associated with powdery mildew resistance, three for leaf rust resistance, and two for stem rust resistance. These QTLs harbor diverse gene classes encoding for resistance gene analogs, cysteine-rich receptor kinases, transcription factors, and G-type lectins. Overall, the knowledge and resources developed in this research would contribute to the characterization of vast germplasm and the development of climate-resilient wheat.Item INTEGRATING FORWARD AND REVERSE GENETIC TOOLS FOR FAST FORWARDING WHEAT IMPROVEMENT(2024) Schoen, Adam William; Tiwari, Vijay K; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bread wheat (Triticum aestivum) provides roughly 20% of the human daily caloric intake and is an important crop for global food security. Changing climatic conditions as well as biotic and abiotic stresses are threatening wheat production worldwide. Sustainable and continuous improvement of wheat using novel genes and alleles is critical to tackle wheat’s imminent challenges. Recent advances in wheat genomics have allowed researchers now to fast-track gene discovery pipelines by implementing strategies first developed in less complex model species. This thesis explores the use of forward and reverse genetic approaches to efficiently discover, map, and validate genes controlling important agronomic traits in bread wheat as well as describes a robust protocol to reduce the generation time in winter wheat. Speed breeding is an important tool that utilizes an increased photoperiod and growing temperature to increase vegetative growth and reduce the time from sowing to harvest. Chapter 1 of this thesis outlines a reproducible method to significantly reduce the generation time in winter wheat from over 120 days based on what has been previously reported to 93 days regardless of vernalization requirements or photoperiod sensitivity and provide a useful tool to increase the pace of the genetic gains in the winter wheat breeding programs. Tillering in wheat directly influences the major yield-related trait, spikes per unit area. Using the forward genetics approach, chapter 2 of thesis reports the identification of a novel tiller inhibition gene (tin6) to a small physical region of 2.1 Mb region on chromosome 2DS. This was the first example of using a genome coming from the pan-genome of wheat to perform MutMap. Using reverse genetics also has the potential to improve the end-use properties of wheat by knocking out genes which result in an increase of the nutritional value of the flour. Chapter 3 of this thesis, TILLING was used to identify knockouts in all three homeologous copies of the starch synthase gene SSIIa, which has been shown to increase the amount resistant starch in the endosperm of wheat which is known to have health benefits in humans. The grains coming from triple knockouts of SSIIa contained 118% higher resistant starch, and though they showed a decrease in thousand kernel weight, they did not have a shriveled phenotype which had been seen in other ssiia mutants. Chapter 4 of the thesis demonstrate reference genome enabled positional cloning of a tiller inhibition gene (tin3) in diploid wheat species Triticum monococcum. A MutMap population generated from a cross between tin3 and wildtype T. monococcum resulted in the identification of a single candidate gene, encoding a BLADE-ON-PETIOLE-Like protein, containing a splice-variant mutation. To show the power of using a diploid species for translational research in hexaploid wheat, the reverse genetics approach TILLING (Targeting Induced Local Lesions IN Genomes) was used to identify mutations in all three homeologous copies of tin3 in the Jagger mutant population. The full null mutant for the tin3 locus in wheat showed significantly reduced tillering in comparison to wildtype providing concrete evidence that genetic discoveries that are found in diploid wheat can be effectively translated to hexaploid wheat. There are some genes and QTLs have been identified that increase spike length, spikelets per spike, and grain size, very few studies have focused on increasing the number of grains per floret. Chapter 5 of the thesis was focused on positional cloning of the Mov-1 locus which is the underlying gene responsible for the multiovary (MOV) phenotype. The Mov-1 locus dominantly expresses as three ovaries per wheat floret, each of which develop into a grain. Using high resolution genetic mapping with the MOV-reference genome and gene expression data, we identified a single candidate gene that was localized to a small 144kb region on the Mov-1 physical region. To validate the role of the Mov-1 candidate gene in the MOV phenotype, ethyl methanesulfonate (EMS) and gamma radiation mutagenesis was performed to create deleterious point and deletions mutations, respectively. Using 5 independent TILLING and 5 deletion mutants this study demonstrate that Mov-1 candidate gene is required for the MOV phenotype in wheat. It is an exciting time to work in wheat research as the growing wheat genomic toolbox allows for researchers to efficiently identify and validate genes that have potential to improve wheat performance. The methods and findings in this body of work provide a breadth of knowledge that can be implemented in additional genetic studies in wheat in order to fast-track gene and trait discovery for the benefit of wheat geneticists and breeders alike.Item MAKING APPLES BLUSH: UNDERSTANDING HOW THE COMBINED USE OF REFLECTIVE GROUNDCOVERS AND PLANT GROWTH REGULATORS IMPACT RED SKIN COLORATION AND QUALITY OF ‘HONEYCRISP’ APPLES IN THE MID-ATLANTIC US(2024) Miah, Md Shipon; Farcuh, Macarena; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Apples are among the most valuable fruits globally, with 'Honeycrisp' ranking as the top sales-producing cultivar in the US. However, challenges such as insufficient red skin coloration and increased preharvest fruit drop significantly diminish their market value. Reflective groundcovers have been reported to enhance apple skin coloration, while the application of the plant growth regulator AVG reduces fruit drop but may negatively impact skin coloration. Research on the impacts of these practices in mid-Atlantic US-grown apples remains limited. In this two years study, our aims were 1) to evaluate the effect of reflective groundcovers on solar radiation (PPFD, UV) distribution; 2) to assess the combined effect of reflective groundcovers and ethylene inhibitor (AVG) on preharvest fruit drop, ethylene production, red blush percentage, and overall fruit quality; 3) to investigate the combined effect of reflective groundcovers and ethylene inhibitor (AVG) on expression level of key anthocyanin and ethylene biosynthesis related genes; 4) to determine the combined effect of reflective groundcovers and ethylene inhibitors (AVG) in the accumulation of total anthocyanin. Apples underwent four treatment combinations of reflective groundcover (Extenday) and AVG (130 mg L−1). Our findings revealed that Extenday significantly enhanced skin coloration (>75% blush) through increased reflectance of PPFD and UV radiation, along with increased IEC, while also accelerating fruit maturity, i.e., overripening. In fact, Extenday-only treated fruit exhibited the highest upregulation of ethylene and anthocyanin biosynthetic-related genes, as well as total anthocyanins. Conversely, AVG notably reduced fruit drop and decreased IEC, delaying fruit maturity while significantly diminishing red coloration (30–48% blush). AVG treated fruit significantly suppressed the expression of key ethylene and anthocyanin biosynthetic structural and regulatory genes, as well as total anthocyanins. The combined application of Extenday and AVG synergistically decreased fruit drop while enhancing skin coloration (>50% blush), but without inducing overripening. This combination fine-tuned the transcript accumulation of ethylene and anthocyanin biosynthetic-related genes, as well as total anthocyanins, enabling 'Honeycrisp' fruit to exceed 50% blush, while moderately increasing IEC (compared to Extenday-only and control fruit), thus enhancing fruit economic value. Therefore, combining Extenday and AVG can boost the market value for 'Honeycrisp' apples in the mid-Atlantic US.Item Refining the understanding of the crosstalk between auxin and indolic defense compound metabolism in Arabidopsis thaliana to improve transferability of the model system to major crops(2024) House, Lillyanna; Peer, Wendy A; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The main natural auxin, indole-3-acetic acid (IAA), modulates a diversity of plant growth and developmental responses through the tight regulation of auxin biosynthesis and inactivation, transport, and signaling. Evidence that analogous auxin metabolic pathways contributing to programmed and plastic development exist amongst different plant species, points to the necessity for refinement of the current understanding of auxin metabolism in Arabidopsis thaliana to improve its transferability as a model system to major crops. Evaluation of the current understanding of auxin biology in plants and analytical tools available for the visualization and quantification of auxin reveal several gaps of knowledge. This work seeks to address this by 1.) elucidating the specific roles of DIOXYGENASE FOR AUXIN OXIDATION 1 and 2 (DAO1 and DAO2) in the oxidative catabolism of IAA and IA-conjugates and functionality of oxidized IAA (oxIAA), 2.) clarifying the function of ATP-BINDING CASSETTE subfamily B (ABCB) transporters and the interplay of auxin, brassinolide, and ethylene in apical hook development and 3.) investigating the possibility of an analogous auxin metabolic pathway involving the natural auxin, phenylacetic acid (PAA), in Oryza sativa. Herein, it is shown that DAO2 activity is similar to DAO1 and assists in the regulation of auxin homeostasis under high auxin conditions and DAO1 is both soluble and plasma membrane associated. Additionally, oxIAA functions as a weak “anti-auxin” to modulate processes like apical hook development, where a subset of ABCB transporters is found to function in auxin mobilization downstream of ethylene signaling and brassinolide transport. The detection of PAA was achieved using LC-MS, however further method development is required for accurate quantification of PAA in rice tissue samples. Application of previous and new knowledge pertaining to the crosstalk between auxin and indolic defense metabolism in Arabidopsis revealed a seemingly analogous pathway in rice whereby phenyl acetonitrile (PAN) is converted to PAA, likely via nitrolases. The results presented herein, refine the understanding of auxin metabolism in Arabidopsis and exemplify the use of a model system in elucidating similar pathways in agriculturally significant crops.Item POSITIONAL CLONING OF BROAD-SPECTRUM LEAF RUST RESISTANCE GENE, LR57, FROM AEGILOPS GENICULATA, A TERTIARY GENE POOL MEMBER OF WHEAT(2023) Schulden, Taylor Francis; Rawat, Nidhi; Erwin, John; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The tertiary gene pool of wheat includes wild relatives like Aegilops geniculata (UUMM, 2n=4x=28) that are valuable genetic reservoirs for novel abiotic and biotic resistance genes. However, modern wheat varieties share limited genomic commonality with these gene pool members presenting barriers to recombination and genetic mapping of desirable genes. We mapped a broad-spectrum leaf rust resistance gene Lr57 located on chromosome 5Mg of Ae. geniculata using a simple but powerful methodology for high-resolution genetic mapping in tertiary gene pool members of wheat. Five gene candidates were revealed all with possible defense related functions. Strategic application of differential expression analysis, Virus-Induced Gene Silencing, and mutagenesis analysis reduced the candidate gene of interest to a singular and novel ID-NLR resistance gene containg a protein kinase, NB-Arc, and LRR domain. Using multiple strategies, validation of Lr57 candidate was completed. Gene complementation by transformation of Lr57 candidate is currently being conducted.Item SPRAY STRATEGIES AND SELECTION FOR FUNGICIDE RESISTANCE: FENHEXAMID RESISTANCE IN BOTRYTIS CINEREA AS A CASE STUDY(2023) Boushell, Stephen Carl; Hu, Mengjun; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Fungicide resistance is a limiting factor in sustainable crop production. Despite the wide adoption of general resistance management strategies by growers, the recent rate of resistance development in important fungal pathogens is concerning. In this study, Botrytis cinerea and the high-risk fungicide fenhexamid were used to determine the effects of fungicide dose, tank mixture, and application timing on resistance selection across varied frequencies of resistance via both detached fruit assays and greenhouse trials. The results showed that application of doses lower than the fungicide label dose, mixture with the low-risk fungicide captan, and application post-infection seem to be the most effective management strategies in our experimental settings. In addition, even a small resistant B. cinerea population can lead to a dramatic reduction of disease control efficacy. Our findings were largely consistent with the recent modeling studies which favored the use of the lowest possible fungicide dose for improved resistance management.Item DEVELOPING AND EXPANDING CRISPR-CAS PLANT GENOME ENGINEERING SYSTEMS(2022) Malzahn, Aimee Alyssa; Qi, Yiping; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In order to advance plant biology and speed up crop breeding, researchers have used genome engineering tools in their research. Genome engineering with CRISPR has revolutionized agriculture by providing an easy, fast, and accessible tool to induce desirable mutations. This thesis works on addressing problems in the application of CRISPR for plant genome engineering. CRISPR systems are adopted from bacterial immune systems and consists of a Cas endonuclease and a guide RNA (or crRNA). Cas variants have different characteristics and exploring natural variants can provide systems with enhanced or new applications. The first aim in this thesis is to demonstrate novel LbCas12a for genome editing in Arabidopsis. To overcome the temperature sensitivity of LbCas12a, a heat treatment regime was developed. In order to expand LbCas12a use beyond genome editing, a transcription repression system was developed and used successfully for multiplexed repression of two homologs of EDS1. Two crRNA processing systems were compared, and results suggest that either can be used successfully in Arabidopsis. The second aim is to improve Cas9 and Cas12a editing outcomes by creating Cas-effector fusions. Cas9 and Cas12a were fused with six different exonucleases and compared at three targets in rice protoplasts. Several Cas-exonuclease fusions resulted in an increase in editing efficiency and the production of larger deletions. The Cas-exonuclease fusions’ editing efficiency differed between Cas9 and Cas12a, along with the deletion profile. Additionally, chromatin modulating peptides were fused to Cas9, which resulted in higher editing efficiency without altering deletion profiles. These engineered Cas proteins can be used to create unique editing outcomes, and paired with an increased editing efficiency, could be used to target difficult-to-edit target sites for gene knockout and cis-regulatory elements for fine-tuning gene expression. In summary, this work explored new Cas variant LbCas12a, developed multiplex gene repression systems, and compared engineered fusion Cas9 and Cas12a proteins for increased editing efficiency and larger deletions. The developed and improved CRISPR systems expand the number of available targets, improve efficiency, demonstrate novel editing outcomes, and enable multiplexed transcriptional regulation in plants.Item DEVELOPING AND IMPROVING CRISPR-BASED GENOME EDITING TECHNOLOGIES IN PLANTS(2022) Sretenovic, Simon; Qi, Yiping; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Earlier genome editing technologies were developed based on programmable nucleases including zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), both requiring tedious protein engineering. By contrast, clustered regularly interspaced short palindromic repeats (CRISPR) systems, such as CRISPR-Cas9, has revolutionized the genome editing field in the past decade due to ease of guiding Cas9 endonuclease to the target site by programmable guide RNAs. However, not every target site can be edited due to Cas9 endonucleases’ recognition of so-called protospacer adjacent motif (PAM) when binding to the target site. For example, the PAM for the widely used SpCas9 is NGG (N=A, C, T or G). This drastically limits targeting scope in the genomes. Thus, researchers have developed engineered Cas9 variants recognizing more relaxed PAMs and tested them in mammalian cell lines. Repair of Cas9 endonuclease-induced double strand breaks through non-homologous end joining (NHEJ) DNA repair pathway typically generates unspecified insertions and deletions, which is a missed opportunity for introducing precise edits. To confer precise genome editing, CRISPR-Cas9 derived base editors and prime editors have been developed. In this work, expanding the plant genome editing scope with engineered Cas9 variants, improving precise cytosine and adenine base editing in plants as well as demonstrating prime editing in plant cells were pursued. The technologies were tested in the model crop, rice, in transiently transformed protoplasts and stably transformed T0 lines. Findings suggest that engineered Cas9 variants can drastically expand the targeting scope for targeted mutagenesis and base editing in plants. Additionally, newer genome editing technologies such as base editors and prime editors can be applied in plants to achieve precise genome editing with varying efficiencies. These validated and useful CRISPR-Cas9 genome editing toolkits have been deposited to the public vector depository, Addgene. Adoption of these genome editing technologies by plant scientists and breeders will enable basic research discoveries and accelerate breeding of next generation crops, ensuring global food security amidst climate change and increasing global population.Item INVESTIGATING MOLECULAR MECHANISMS SPECIFYING DIVERSE ROSACEAE FRUIT TYPES THROUGH COMPARATIVE TRANSCRIPTOMIC ANALYSIS(2022) Li, Muzi; Liu, Zhongchi; Mount, Stephen; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Rosaceae is a plant family with over 3,000 species including a number of economically important fruit-bearing species. Although plants in Rosaceae family have similar basic flower structure, their fruit flesh comes from distinct floral tissues. In drupe fruit, such as peach and plum, the ovary wall becomes enlarged and fleshy. In pome fruit, such as apple and pear, the fruit fleshy is mainly derived from the hypanthium that encases the ovary. In drupetum fruit, such as raspberry, numerous unfused ovaries each grow into a fleshy drupelet. In achenetum fruit, such as strawberry, the numerous unfused ovaries eventually dry up, but the receptacle, the stem tip that supports these ovaries, instead develops into the fruit flesh. By investigating and comparing the transcriptomes from these four Rosaceae fruits, peach (Prunus persica), apple (Malus x domestica), strawberry (Fragaria vesca), and raspberry (Rubus idaeus), at the earliest stages of fruit development, we gain important insights into the genetic mechanisms underlying fleshy fruit diversity. The expression of B class MADS-box genes, PISTILLATA, APETALA3 and TM6, shows negative correlation with the ability to form fleshy fruit tissues. Based on RNA transcript and phylogenetic analysis, FBP9, a MADS-box gene related to the E class, appears to be necessary but insufficient for flesh formation. In addition to the regulatory roles MADS-box genes play in fruit identity specification, extensive lignification of the strawberry ovary wall may contribute to the inability of strawberry ovary to become fleshy. Finally, a database (ROsaceae Fruit Transcriptome database, ROFT) is established for researchers to query for orthologous genes and their expression patterns during fruit development in the four species as well as to query for the tissue-specific and tissue- and stage-specific genes. Together, these findings provide the framework for functional investigations of fruit type specification and insights into the evolution of diverse fruit types in the Rosaceae family. The knowledge gained will advance our understanding in the evolution of fleshy fruits, a defining feature of angiosperm, and enable the creation of new fruit types for consumers.Item MECHANISM OF DREAM COMPONENT TSO1 IN PLANT STEM CELL REGULATION(2022) Wang, Fuxi; Liu, Zhongchi ZL; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Plants are important for human survival and the environment. They provide oxygen, food, medicine and fuel. Understanding the development of plants has been a fundamental research question. Among all the plant tissues, the most important ones are the meristems. Sitting at the tip of the shoot and the root are the shoot apical meristem (SAM) and the root apical meristem (RAM). The shoot apical meristem gives rise to the above-ground organs like leaves and flowers while the root apical meristem produces all the root tissues that help to anchor the plants and transport water and nutrients. As the meristem is capable of producing new organs throughout the lifespan of a plant, the study of meristem maintenance and development provides the key to the understanding of plant development.Arabidopsis transcription factor TSO1 plays an essential role for the proper development of shoot apical meristem and root apical meristem. TSO1 encodes a protein with a cysteine-rich repeats domain and TSO1 is a potential component of a cell cycle regulating complex, the DREAM complex. The tso1-1 mutant has fasciated SAM due to shoot meristem cell over-proliferation and complete sterility due to lack of differentiated female and male floral organs. Interestingly, the tso1-1 mutant also produces shorter root than the wild type, presumably caused by early differentiation of the cells in the RAM. A prior mutagenesis screen identified two major suppressors of tso1-1. Characterization of these tso1-1 suppressor mutations provides important insights to the understanding of TSO1-regulatory pathways. My dissertation project focuses on analyzing one of these suppressors that was shown to be a mutated type-A cyclin gene named CYCA3;4. Mutations in CYCA3;4 suppress the shoot phenotype but not the root phenotype of tso1-1. The suppressed plants can produce normal floral organs and become partially fertile. Using transgenic method, I showed that the expression of CYCA3;4 was increased in the tso1-1 SAM, and overexpression of CYCA3;4 in the tso1-3 mutant enhanced the fertility defect, suggesting that overexpression of CYCA3;4 partially mediates the tso1-1 shoot phenotype. In addition, I provided evidence supporting that TSO1 likely represses CYCA3;4 gene expression indirectly through MYB3R1, whose mutations also suppress tso1-1 mutants. My dissertation provides an important link between TSO1, a potential cell cycle regulatory complex component and meristem regulator, and cyclin A, a protein directly involved in cell cycle regulation. This link provides an important mechanistic insight into how plant meristems maintain their identity by limiting their cell division activity. To further investigate the mechanism of TSO1 action in the root, I collaborated with two other scientists to profile the gene expression in the tso1-1 root at single cell level. I compared the single cell RNA sequencing data of tso1-1 and wild type roots and identified molecular defects in the tso1-1 root vasculature. Correspondingly, the known regulators of vasculature development, the HD-ZIP III genes, are ectopically expressed in some of the vascular cells in the tso1-1 root. It suggests that the defects of root vasculature may be attributed to mis-expressed HD-ZIP III genes in the tso1-1 mutant. The HD-ZIPIII function was previously linked to their regulation of cytokinin biosynthesis genes, which were ectopically expressed in tso1-1 roots as revealed by our scRNA-seq data. Together, our data suggest that the over-production of cytokinin might be the cause of tso1-1 short root phenotype. In summary, my dissertation research revealed previously unknown links between TSO1 and cell cycle regulation in the shoot and root meristems as well as the molecular mechanisms of TSO1 function in the root vascular development at single cell level. These findings have furthered our understanding of how cell cycle regulation is integrated with plant development.