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Recent Submissions

  • ZAXINONE SYNTHASE 2 regulates growth and arbuscular mycorrhizal symbiosis in rice

    Ablazov, Abdugaffor; Votta, Cristina; Fiorilli, Valentina; Wang, Jian You; Aljedaani, Fatimah R.; Jamil, Muhammad; Balakrishna, Aparna; Balestrini, Raffaella; Liew, Kit Xi; Rajan, Chakravarthy; Berqdar, Lamis; Blilou, Ikram; Lanfranco, Luisa; Al-Babili, Salim (Cold Spring Harbor Laboratory, 2022-07-23) [Preprint]
    Carotenoid cleavage, catalyzed by CAROTENOID CLEAVAGE DIOXYGENASES (CCDs), provides signaling molecules and precursors of plant hormones. Recently, we showed that zaxinone, a novel apocarotenoid metabolite formed by the CCD Zaxinone Synthase (ZAS), is a growth regulator required for normal rice growth and development. The rice genome encodes three OsZAS homologs, called here OsZAS1b, OsZAS1c, and OsZAS2, with unknown functions. Here, we investigated the enzymatic activity, expression pattern, and subcellular localization of OsZAS2, and generated and characterized loss-of-function CRISPR/Cas9-Oszas2 mutants. We show that OsZAS2 formed zaxinone in vitro. OsZAS2 is a plastid-localized enzyme mainly expressed in the root cortex under phosphate starvation. Moreover, OsZAS2 expression increased during mycorrhization, specifically in arbuscule-containing cells. Oszas2 mutants contained lower zaxinone content in roots and exhibited reduced root and shoot biomass, less productive tiller, and higher strigolactone (SL) levels. Exogenous zaxinone application repressed SL biosynthesis and partially rescued the growth retardation of Oszas2 mutant. Consistent with the OsZAS2 expression pattern, Oszas2 mutants displayed a lower frequency of AM colonization. In conclusion, OsZAS2 encodes a further zaxinone-forming enzyme that determines rice growth and architecture and strigolactone content and is required for optimal mycorrhization.
  • Identification of novel, bacteria assisted, iron acquisition strategies in Arabidopsis thaliana

    García Ramírez, Gabriel Xicoténcatl (2022-07) [Thesis]
    Advisor: Hirt, Heribert
    Committee members: Wulff, Brande B. H.; Rosado, Alexandre S.
    Iron is abundant in most agricultural systems, however it is also one of the three most limiting nutrients for crop growth. This can be attributed to iron’s low solubility in aerobic and alkaline conditions, rendering it non-bioavailable for plant uptake. With around 1/3 of the world arable land presenting conditions that limit iron solubility, deciphering the plant machinery behind iron uptake and identifying microbial benefits to iron deficiency are of major interest. In this work, 33 endophytic bacterial isolates previously isolated from three regions in Jordan were tested for iron stress alleviation of Arabidopsis plants with the goal of identifying novel interaction mechanisms. On media with a low concentration of bioavailable iron, 11 isolates were found to be beneficial, while 15 isolates behaved in a pathogenic manner, reducing plant growth in both control as well as limited iron conditions. Beneficial isolates were then tested in plant assays with non-bioavailable iron, we concluded that our strains are also beneficial in these conditions. To further characterize the interaction between the beneficial strains and Arabidopsis plants, gene expression was assessed for plants colonized with select strains. The highest expression of iron-deficiency response genes was at day 6. The increase in expression was also met with an increase in colonization of plants at day 6. Mutant studies revealed that the beneficial effect by the bacterial isolates is dependent on the coumarin pathway, with mutants in FERULOYL-COA 6-HYDROXYLASE 1, f6’h1, showing a drastic decrease in fresh weight compared to wild type counterparts. We also discovered the phytohormone abscisic acid as an important contributor to iron stress alleviation by the beneficial isolates. Colonization assays as well as additional mutant studies will be necessary to further assess the effect of f6’h1 mutants on plant-microbe interaction as well as ABA’s role in plants under iron deficient conditions.
  • Multiple Alr genes exhibit allorecognition-associated variation in the colonial cnidarian Hydractinia

    Rodriguez-Valbuena, Henry; Gonzalez-Muñoz, Andrea; Cadavid, Luis F (Immunogenetics, Springer Science and Business Media LLC, 2022-06-27) [Article]
    The genetics of allorecognition has been studied extensively in inbred lines of Hydractinia symbiolongicarpus, in which genetic control is attributed mainly to the highly polymorphic loci allorecognition 1 (Alr1) and allorecognition 2 (Alr2), located within the Allorecognition Complex (ARC). While allelic variation at Alr1 and Alr2 can predict the phenotypes in inbred lines, these two loci do not entirely predict the allorecognition phenotypes in wild-type colonies and their progeny, suggesting the presence of additional uncharacterized genes that are involved in the regulation of allorecognition in this species. Comparative genomics analyses were used to identify coding sequence differences from assembled chromosomal intervals of the ARC and from genomic scaffold sequences between two incompatible H. symbiolongicarpus siblings from a backcross population. New immunoglobulin superfamily (Igsf) genes are reported for the ARC, where five of these genes are closely related to the Alr1 and Alr2 genes, suggesting the presence of multiple Alr-like genes within this complex. Complementary DNA sequence evidence revealed that the allelic polymorphism of eight Igsf genes is associated with allorecognition phenotypes in a backcross population of H. symbiolongicarpus, yet that association was not found between parental colonies and their offspring. Alternative splicing was found as a mechanism that contributes to the variability of these genes by changing putative activating receptors to inhibitory receptors or generating secreted isoforms of allorecognition proteins. Our findings demonstrate that allorecognition in H. symbiolongicarpus is a multigenic phenomenon controlled by genetic variation in at least eight genes in the ARC complex.
  • Evaluation of the Biostimulant Activity of Zaxinone Mimics (MiZax) in Crop Plants

    Wang, Jian You; Jamil, Muhammad; Hossain, Md. Golap; Chen, Guan-Ting Erica; Berqdar, Lamis; Ota, Tsuyoshi; Blilou, Ikram; Asami, Tadao; Al-Solimani, Samir Jamil; Mousa, Magdi Ali Ahmed; Al-Babili, Salim (Frontiers in Plant Science, Frontiers Media SA, 2022-06-16) [Article]
    Global food security is a critical concern that needs practical solutions to feed the expanding human population. A promising approach is the employment of biostimulants to increase crop production. Biostimulants include compounds that boost plant growth. Recently, mimics of zaxinone (MiZax) were shown to have a promising growth-promoting effect in rice (Oryza sativa). In this study, we investigated the effect of MiZax on the growth and yield of three dicot horticultural plants, namely, tomato (Solanum lycopersicum), capsicum (Capsicum annuum), and squash (Cucurbita pepo) in different growth environments, as well as on the growth and development of the monocot date palm (Phoenix dactylifera), an important crop in the Middle East. The application of MiZax significantly enhanced plant height, flower, and branch numbers, fruit size, and total fruit yield in independent field trials from 2020 to 2021. Importantly, the amount of applied MiZax was far less than that used with the commercial compound humic acid, a widely used biostimulant in horticulture. Our results indicate that MiZax have significant application potential to improve the performance and productivity of horticultural crops.
  • The Arabidopsis D27-like1 is a cis/cis/trans-β-carotene Isomerase that Contributes to Strigolactone Biosynthesis and Negatively Impacts Abscisic Acid Level

    Yang, Yu; Abuauf, Haneen W.; Song, Shanshan; Wang, Jian You; Alagoz, Yagiz; Moreno, Juan C; Mi, Jianing; Ablazov, Abdugaffor; Jamil, Muhammad; Ali, Shawkat; Zheng, Xiongjie; Balakrishna, Aparna; Blilou, Ikram; Al-Babili, Salim (Cold Spring Harbor Laboratory, 2022-06-09) [Preprint]
    The enzyme DWARF27 (D27) catalyzes the reversible isomerization of all-trans- into 9-cis-?-carotene, initiating strigolactone (SL) biosynthesis. Genomes of higher plants encode two D27-homologs, D27-like1 and -like2, with unknown functions. Here, we investigated the enzymatic activity and biological function of the Arabidopsis D27-like1. In vitro enzymatic assays and Expression in Synechocystis sp. PCC6803 revealed a yet not reported 13-cis/15-cis/9-cis- and a 9-cis/all-trans-β-carotene isomerization. Although disruption of AtD27-like1 did not cause SL deficiency phenotypes, overexpression of AtD27-like1 in the Atd27 mutant restored the more-branching phenotype, indicating a contribution of AtD27-like1 to SL biosynthesis. Accordingly, generated Atd27 Atd27like1 double mutants showed more pronounced branching phenotype, compared to Atd27. The contribution of AtD27-like1 to SL biosynthesis is likely due to its formation of 9-cis-β-carotene that was present at higher levels in AtD27-like1 overexpressing lines . In contrast, AtD27-like1 expression correlated negatively with the content of 9-cis-violaxanthin, a precursor of abscisic acid (ABA), in shoots. Consistently, ABA levels were higher in shoots and also in dry seeds of the Atd27like1 and Atd27 Atd27like1 mutants. Transgenic lines expressing β-glucuronidase (GUS) driven by the AtD27LIKE1 promoter and transcript analysis performed with hormone-treated Arabidopsis seedlings unraveled that AtD27LIKE1 is expressed in different tissues and regulated ABA and auxin. Taken together, our work revealed a cis/cis-b-carotene isomerase activity that affects the content of both cis-carotenoid derived plant hormones ABA and SLs.
  • Unconventional R proteins in the botanical tribe Triticeae

    Athiyannan, Naveenkumar; Aouini, Lamia; Wang, Yajun; Krattinger, Simon G. (Essays in biochemistry, 2022-06-07) [Article]
    Plant immunity is triggered following the perception of pathogen-derived molecules by plant receptor proteins. Two protein families, membrane-localized receptor-like kinases (RLK) and intracellular nucleotide-binding leucine-rich repeat (NLR) receptors, play key roles in pathogen perception and in the initiation of downstream signaling cascades that lead to defense responses. In addition to RLKs and NLRs, recent research has identified additional protein families that function as plant resistance (R) proteins. In particular, the botanical tribe Triticeae, which includes the globally important crop species wheat and barley, has played a significant role in the discovery of 'unconventional' R proteins. In this review, we will summarize the current knowledge on unconventional R genes in Triticeae and the proteins they encode. The knowledge on unconventional R proteins will not only broaden our understanding of plant-pathogen interactions but also have great implications for disease resistance breeding in crops.
  • A New Approach to the Study of Plastidial Stress Granules: The Integrated Use of Arabidopsis thaliana and Chlamydomonas reinhardtii as Model Organisms

    Rafique, Fareena; Lauersen, Kyle J.; Chodasiewicz, Monika; Figueroa, Nicolás E. (Plants, MDPI AG, 2022-05-30) [Article]
    The field of stress granules (SGs) has recently emerged in the study of the plant stress response, yet these structures, their dynamics and importance remain poorly characterized. There is currently a gap in our understanding of the physiological function of SGs during stress. Since there are only a few studies addressing SGs in planta, which are primarily focused on cytoplasmic SGs. The recent observation of SG-like foci in the chloroplast (cpSGs) of Arabidopsis thaliana opened even more questions regarding the role of these subcellular features. In this opinion article, we review the current knowledge of cpSGs and propose a workflow for the joint use of the long-established model organisms Chlamydomonas reinhardtii and A. thaliana to accelerate the evaluation of individual plant cpSGs components and their impact on stress responses. Finally, we present a short outlook and what we believe are the significant gaps that need to be addressed in the following years.
  • Oat genome — sequence of a superfood

    Krattinger, Simon G.; Keller, Beat (Nature Plants, Springer Science and Business Media LLC, 2022-05-26) [Article]
    The analysis of the 11-gigabase, hexaploid oat genome reveals a mosaic chromosome structure with complex rearrangements related to polyploidization. The high-quality oat reference genome will facilitate the molecular identification of health food traits and the implementation of genomics-based oat breeding.
  • Protocol for characterizing strigolactones released by plant roots

    Wang, Jian You; Chen, Guan-Ting Erica; Jamil, Muhammad; Braguy, Justine; Sioud, Salim; Liew, Kit Xi; Balakrishna, Aparna; Al-Babili, Salim (STAR protocols, Elsevier BV, 2022-05-18) [Article]
    The plant hormone strigolactones (SLs) are secreted by plant roots to act as rhizospheric signals. Here, we present a protocol for characterizing plant-released SLs. We first outline all necessary steps required for collection, processing, and analysis of plant root exudates using the C18 column for SL extraction, followed by liquid chromatography-mass spectrometry (LC-MS) for SL quantification. We then describe image processing by SeedQuant, an open-source artificial-intelligence-based software, for measuring the biological activity of SLs in inducing root parasitic plant seed germination.
  • Striga hermonthica Suicidal Germination Activity of Potent Strigolactone Analogs: Evaluation from Laboratory Bioassays to Field Trials

    Jamil, Muhammad; Wang, Jian You; Yonli, Djibril; Ota, Tsuyoshi; Berqdar, Lamis; Traore, Hamidou; Margueritte, Ouedraogo; Zwanenburg, Binne; Asami, Tadao; Al-Babili, Salim (Plants, MDPI AG, 2022-04-12) [Article]
    The obligate hemiparasite Striga hermonthica is one of the major global biotic threats to agriculture in sub-Saharan Africa, causing severe yield losses of cereals. The germination of Striga seeds relies on host-released signaling molecules, mainly strigolactones (SLs). This dependency opens up the possibility of deploying SL analogs as “suicidal germination agents” to reduce the accumulated seed bank of Striga in infested soils. Although several synthetic SL analogs have been developed for this purpose, the utility of these compounds in realizing the suicidal germination strategy for combating Striga is still largely unknown. Here, we evaluated the efficacy of three potent SL analogs (MP3, MP16, and Nijmegen-1) under laboratory, greenhouse, and farmer’s field conditions. All investigated analogs showed around a 50% Striga germination rate, equivalent to a 50% reduction in infestation, which was comparable to the standard SL analog GR24. Importantly, MP16 had the maximum reduction of Striga emergence (97%) in the greenhouse experiment, while Nijmegen-1 appeared to be a promising candidate under field conditions, with a 43% and 60% reduction of Striga emergence in pearl millet and sorghum fields, respectively. These findings confirm that the selected SL analogs appear to make promising candidates as simple suicidal agents both under laboratory and real African field conditions, which may support us to improve suicidal germination technology to deplete the Striga seed bank in African agriculture
  • Diversifying the menu for crop powdery mildew resistance

    Brown, James K.M.; Wulff, Brande B.H. (Cell, Elsevier BV, 2022-03-03) [Article]
    Powdery mildew, a potentially severe crop disease, can be controlled by mlo mutations, which suppress fungal proliferation but typically also reduce yield. Li et al. (2022) demonstrate that productivity can be restored by overexpressing a host sugar transporter, thus offering a new option for economically and environmentally benign disease control.
  • Towards Understanding the Biological Background of Strigolactone Diversity

    Braguy, Justine (2021-10) [Dissertation]
    Advisor: Al-Babili, Salim
    Committee members: Zurbriggen, Matias; Blilou, Ikram; Bouwmeester, Harro J.; Lauersen, Kyle J.
    Strigolactones (SLs) are a class of plant hormones regulating several aspects of plant growth and development according to nutrient availability, particularly the modulation of root and shoot architectures. Under nutrient deficiency, SLs are abundantly released into the soil to recruit a plant-beneficial partner, arbuscular mycorrhizal fungi (AMF), and establish plant-AMF symbiosis that provides the plant with minerals and water. However, released SLs are also seed germination signals for the root parasitic plants Orobanchacea family and pave their way to the host plants’ roots. “New comers” in the field of plant hormones, their large structural variety intrigues and led to ask why plants produce many different types of SLs. In this work, we generated tools that can help to link the SL structural diversity with their biological function(s). The most common way to evaluate SL activity is based on their ability to be parasitic seeds’ germination stimulants. Despite being the most sensitive assay for SL quantification, parasitic seed-based bioassays are laborious and time-consuming as performed usually manually. Therefore, we developed a detection algorithm, SeedQuant, which identifies and counts germinated and non-germinated seeds 600 times faster than a trained human; thus, reducing the data processing from days down to minutes. To gain quantitative insights in SL perception, depending on the structural diversity, we developed a precise and detailed protocol for the use of a genetically encoded biosensor in Arabidopsis protoplast, StrigoQuant. StrigoQuant takes advantage of the SL-dependent degradation of the repressor protein AtSMXL6 coupled with luciferase reporter proteins. We also tried to adapt this molecular sensor to the rice repressor protein D53, but the use of rice protoplasts made it very challenging. Moreover, to better understand the later steps in SL biosynthesis in vivo, we generated CRISPR/Cas9-based rice mutants and shed light on the biological function of different SLs at the organismal level. MAX1-900 mutants defined the minor role of the canonical SL 4-deoxyorobanchol (4DO) - a major plant SL - in determining rice architecture, while being a crucial contributor to rhizospheric interactions. Finally, we reviewed other strategies to decipher plant signaling pathways in general.
  • Layered double hydroxide (LDH)-mediated topical delivery of dsRNA for protection against Tomato yellow leaf curl virus (TYLCV) in Nicotiana benthamiana

    Hernandez, Edith Sanchez (2021-04) [Thesis]
    Advisor: Mahfouz, Magdy M.
    Committee members: Habuchi, Satoshi; Ghaffour, NorEddine
    Cell wall is the major barrier in the delivery of biomolecules such as nucleic acids into the plant cell. Biological (bacteria or viruses) and biolistic (particle-based) methods are used to deliver nucleic acids into the plant cell. However, these methods have significant limitations when it comes to species range, scalability, and field assays. In this work, we report the use of layered double hydroxide (LDH) topically applied to deliver RNA molecules into the plant cell. LDH were assembled by methanol-based co-precipitation of magnesium and aluminum nitrate solution with sodium hydroxide and finally dispersed in deionized water. The assembled LDH were physically characterized by AFM, zeta-sizer and their binding to RNA was confirmed by gel electrophoresis. LDH complexed with double stranded RNA (dsRNA) was topically applied to Nicotiana benthamiana leaves. As a model system, virus specific dsRNA-LDH complexes were used to activate cellular RNAi machinery against Tomato Yellow leaf Curl Virus (TYLCV) in N. benthamiana plants. Our results demonstrated that topical application of the TYLCV specific dsRNA-LDH complexes reduce viral genome accumulation and viral symptoms development. Similarly, dsRNA-LDH protected plants produce typical leaves, flowers, and seeds, confirming efficient virus resistance compared unprotected TYLCV infected plants. Topical application and noninvasive delivery of nucleic acid has several advantages, as these methods are specie independent, easy to scale up, applied with low-pressure spray, requires no tissue culture and no sophisticated equipment. The LDH based noninvasive delivery of nucleic acids has the capability to overcome the cell wall barrier limitations and will open new opportunities to exploit the full potential of cellular machinery to produce resilient plants and insure sustainable food production.
  • Involvement of Beneficial Microbe-derived Cyclodipeptides (CDPs) in Promoting Plant Tolerance to Abiotic Stresses

    abdulhakim, fatimah (2020-07) [Thesis]
    Advisor: Hirt, Heribert
    Committee members: Merzaban, Jasmeen; Al-Babili, Salim; Saad, Maged M.; Abulfaraj, Alaa
    Cyclodipeptides (CDPs) are the smallest, most stable cyclic peptides that are synthesized as secondary metabolites by bacteria. The aim of this study was to investigate the effect of the Pseudomonas argentinensis (SA190) and four (CDPs), named as cis-cyclo-(Pro-Phe) (Cyclo2), cis-cyclo-(Pro-Leu) (Cyclo3), cis-cyclo-(Pro-Tyr) (Cyclo4) and cis-cyclo-(Pro-Val) (Cyclo5), with three concentrations (1µM, 100nM, and 10nM), on the growth of Arabidopsis thaliana under normal plant growth conditions [1/2MS media], salt conditions [125 mM NaCl] and drought conditions [25% PEG]. Moreover, we determined the most effective CDPs with optimal concentration. It was found that cis-cyclo-(Pro-Tyr) (Cyclo4) at a concentration of 100nM had an effect on the plant growth and can mimic the effect of SA190 under normal [1/2MS media] conditions. Also, cis-cyclo-(Pro-Tyr) (Cyclo4) at a concentration of 1µM can mimic the effect of SA190 under salt conditions [125mM NaCl]. Finally, cis-cyclo-(Pro-Val) (Cyclo5) at a concentration of 1µM can mimic the effect of SA190 under drought conditions [25% PEG].
  • Dissecting the genetic architecture of salt tolerance in the wild tomato Solanum pimpinellifolium

    Morton, Mitchell (2019-10) [Dissertation]
    Advisor: Tester, Mark A.
    Committee members: Krattinger, Simon G.; McCabe, Matthew; Schafleitner, Roland
    Salt stress severely constrains plant performance and global agricultural productivity. 5% of arable land, 20% of irrigated areas and 98% of water reserves worldwide are saline. Improving the salt tolerance of major crop species could help attenuate yield losses and expand irrigation opportunities and provide in situ relief in areas where poverty, food and water scarcity are prevalent. Increasing the salt tolerance of crops with high commercial and nutritional value, such as tomato (Solanum lycopersicum L.), would provide particularly significant economic and health benefits. However, salt tolerance is a complex trait with a limited genetic repertoire in domesticated crop varieties, including tomato, frustrating attempts to breed and engineer tolerant crop varieties. Here, a genome-wide association study (GWAS) was undertaken, leveraging the rich genetic diversity of the wild, salt tolerant tomato Solanum pimpinellifolium and the latest phenotyping technologies to identify traits that contribute to salt tolerance and the genetic basis for variation in those traits. A panel of 220 S. pimpinellifolium accessions was phenotyped, focusing on image-based high-throughput phenotyping over time in controlled and field conditions in young and mature plants. Results reveal substantial natural variation in salt tolerance over time across many traits. In particular, the use of unmanned aerial vehicle (UAV)-based remote sensing in the field allowed high-resolution RGB, thermal and hyperspectral mapping that offers new insights into plant performance in the field, over time. To empower our GWAS and facilitate the identification of candidate genes, a new S. pimpinellifolium reference genome was generated, 811Mb in size, N50 of ~76kb, containing 25,970 annotated genes. Analysis of this reference genome highlighted potential contributors to salt tolerance, including enrichments in genes with stress response functions and a high copy number of the salt tolerance-associated gene inositol- 3-phosphate synthase (I3PS). A recently completed full genome re-sequencing of the panel, along with a newly available pseudomolecule-level assembly of the S. pimpinellifolium genome with N50 of ~11Mb, will serve to drive a GWAS to identify loci associated with traits that contribute to salt tolerance. Further research including gene validation, breeding, genetic modification and gene editing experiments will drive the development of new salt tolerant tomato cultivars.
  • The role of NAC transcription factors in responses of plants to heat and salt stresses

    Alshareef, Nouf Owdah Hameed (2019-08) [Dissertation]
    Advisor: Tester, Mark A.
    Committee members: Blilou, Ikram; Pain, Arnab; Balazadeh, Salma
    Soil salinity and heat stress are two major abiotic stresses affecting plant growth and yield. Transcription factors (TFs) are key regulators in stress responses. They link stress sensing with many tolerance mechanisms by translating stress signals into changes in gene expression that ultimately contribute to stress tolerance. The NAC (NAM, ATAF and CUC) TF family have been found to be involved in responses to biotic and abiotic stresses. In this PhD project, the role of NAC TFs in response to heat and salt stress was studied in the model system Arabidopsis thaliana (Arabidopsis), and in two agriculturally relevant species, Solanum lycopersicum (tomato) and Chenopodium quinoa (quinoa). Plants have the ability to acquire thermotolerance if they are pre-exposed to a mild, non-lethal high temperature. The maintenance of acquired thermotolerance for several days is known as thermomemory. Here we investigated the role of NAC TFs in thermotolerance. The expression profiles of 104 Arabidopsis NAC TFs were measured and compared between primed and unprimed plants. Some NACs with a distinctive expression pattern in response to thermopriming were selected for further phenotypic analysis. Knock-out (KO) mutants of the ATAF1 gene showed an enhanced thermomemory phenotype compared with wild type plants (WT) and from this work, the functions of the ATAF1 gene were studied further. RNAseq co-expression analyses of ATAF1 overexpressor and ataf1 KO plants found that ANAC055 expression was co-regulated with that of ATAF1. JUBGBRUNNEN1 (JUB1) is another NAC TF involved in responses to heat, drought and salinity. In this study, the role of AtJUB1 overexpression in salinity was investigated in tomato plants. AtJUB1 overexpression resulted in higher proline levels and improved maintenance of water content and biomass in AtJUB1-overexpressing plants grown hydroponically under salinity compared with WT plants. Quinoa has recently gained much attention because of its high nutritional value and high tolerance to several stresses including drought and salinity. NAC TFs are hypothesized to play a major role in quinoa’s tolerance to abiotic stresses. In this study, the NAC TFs family were identified and investigated in the genome of quinoa. 107 NAC TF genes were identified and their transcriptional responses to different stresses including salt, drought and heat were investigated.
  • Genetics of Salinity Tolerance in Rice

    Al Tamimi, Nadia (2019-05) [Dissertation]
    Advisor: Tester, Mark A.
    Committee members: Al-Babili, Salim; Gojobori, Takashi; McCouch, Susan
    For more than half of the world’s population, rice (Oryza sativa L.), the most saltsensitive cereal, is a dietary staple. Soil salinity is a major constraint to rice production worldwide. Thus, to feed 9 billion people by 2050, we need to increase rice production while facing the challenges of rapid global environmental changes. To meet some of these challenges, there is a vital requirement to significantly increase rice production in salinized land and improve photosynthetic efficiency. Exposure of plants to soil salinity rapidly reduces their growth and transpiration rates (TRs) due to the ‘osmotic component’ of salt stress (sensu Munns and Tester), which is hypothesized to be related to sensing and signaling mechanisms. Over time, toxic concentrations of Na+ and Cl− accumulate in the cells of the shoot, known as the ‘ionic component’ of salt stress, which causes premature leaf senescence. Both osmotic and ionic components of salinity stress are likely to impact yield. Despite significant advances in our understanding of the ionic components of salinity tolerance, little is known about the early responses of plants to salinity stress. In my PhD project, the aim was to analyze naturally occurring variation in salinity tolerance of rice and identify key genes related to higher salinity tolerance using high-throughput phenomics and field trials. I used a forward genetics approach, with two rice diversity panels (indica and aus) and recently published sequencing data (McCouch et al., 2017). Indica and aus were phenotyped under controlled conditions, while the indica diversity panel was also further studied under field conditions for salinity tolerance. I also examined previously unexplored traits associated with salinity tolerance, in particular the effects of salinity on transpiration and transpiration use efficiency. The non-destructive high-throughput experiments conducted under controlled conditions gave insights into the understudied shoot ion-independent component of salinity tolerance. In parallel, the field experiments increased our understanding of the genetic control of further components of salinity tolerance, including the maintenance of yield under saline conditions. Importantly, this project also aimed to improve the current association methods of GWAS by exploring and testing novel Mixed Linear Models. One major benefit of this Ph.D. project was the development of a more holistic approach that recognizes the complexity of the genotype–phenotype interaction. The purpose of my work was to shed more light on the genetic mechanisms of salinity tolerance in rice and discover genes associated with traits contributing to higher photosynthetic activity under both controlled and field conditions. This will ultimately lead to further exploration of the genetic diversity present in the PRAY indica panel, in order to develop higher yielding rice varieties.
  • How does light affect the heat stress response in Arabidopsis?

    Kim, Eunje (2018-11) [Thesis]
    Advisor: Tester, Mark A.
    Committee members: Hirt, Heribert; Voolstra, Christian R.
    Light and temperature are two of the most important environmental factors regulating plant development. Although heat stress has been well studied, little is known about the interaction between light and temperature. In this study, we performed phenotypic assays comparing seedling responses to heat under light and dark conditions. Seedlings exposed to heat in the dark show lower survival rates than seedlings stressed in the light. To identify transcriptional changes underlying light-dependent heat tolerance, we used RNA-sequencing. The light-dependent heat stress responses involved a plethora of genes which could be potential candidate genes for light-induced heat tolerance, including transcription factors (bHLH) and genes commonly associated with biotic stress. By using the latest high-throughput phenotyping facility, we found that the light-dependent heat tolerance is reflected more on the maintenance of photosynthetic capacity, rather than leaf temperature. These results provide insights into how light increases heat stress tolerance in Arabidopsis seedlings and suggest its underlying mechanisms.
  • The Role of Chromatin Associated Proteins in Plant Innate Immunity and Jasmonic Acid Signaling

    Jarad, Mai (2018-11) [Dissertation]
    Advisor: Hirt, Heribert
    Committee members: Blilou, Ikram; Arold, Stefan T.; Gust, Andrea
    Pathogen-associated molecular pattern (PAMP) recognition occurs by plasma membrane located receptors that induce among other processes nuclear gene expression. The plant FLS2-BAK1 receptor complex binds the bacterial PAMP, flg22 and induces a series of defense responses. The resulting signal transduction events occur through the activation of two MAPK signaling cascades, which trigger a rapid and strong activation of MPK3, MPK4 and MPK6. Cellular responses to pathogens are regulated by the activated MAPKs, which lead to the eventual phosphorylation of cytoplasmic and nuclear substrates. These MAPK substrates in turn respond to phosphorylation by reprogramming the expression of defense genes. A large scale phosphoproteomics screen of nuclear proteins in wild type and mpk mutant plants in response to flg22 revealed several novel putative targets of MAP kinases. This thesis is aimed at identifying the role of two of these chromatin associated proteins in plant immunity and their signaling mechanisms. The chromatin associated proteins we chose to study here are LITTLE NUCLEI/CROWDED NUCLEI (LINC/CRWN), LINC1 and the AT-HOOK MOTIF CONTANING NUCLEAR LOCALIZED 13 (AHL13) proteins. We demonstrate that these two chromatin associated proteins play a positive regulatory role in jasmonic acid signaling and immunity. Knock out mutants for both genes exhibit impairment in early and late innate immune reposes to both PAMP and hemibiotrophic pathogen strains. We also demonstrate that these mutants are compromised in regulating the expression of genes involved in jasmonic acid (JA) signaling and responses and genes involved in the biosynthesis both the indole and aliphatic glucosinolate (GS) pathways. Moreover, Pst DC3000 hrcC triggers JA and JAIle accumulation in these mutants, whereas salicylic acid (SA) levels are unchanged. We were also able to identify and validate two novel MAPK targeted phosphosites in AHL13 that affect the protein stability of AHL13 and we establish its role as a MPK6 substrate that affects jasmonic acid biosynthesis and PTI responses. Together this work identifies two novel signaling components involved in the regulation of jasmonic acid homeostasis and immunity.
  • In-field characterization of salt stress responses of chlorophylls a and b and carotenoid concentrations in leaves of Solanum pimpinellifolium

    Ilies, Dragos-Bogdan (2018-10) [Thesis]
    Advisor: Tester, Mark A.
    Committee members: Al-Babili, Salim; McCabe, Matthew
    Food security is a major concern of the 21st century, given climate change and population growth. In addition, high salt concentrations in soils affect ~20% of irrigated land and cause a substantial reduction in crop yield. Cultivating salt-tolerant crops could enable the use of salt-affected agricultural land, reduce the use of fresh water and alleviate yield losses. Innovative methods need to be developed to study traditional and novel traits that contribute to salinity tolerance and accurately quantify them. These studies would eventually serve for developing new salt tolerant crops, adapted to the harsh arid and semi-arid climate conditions. A study of 200 accessions of the wild tomatoes (Solanum pimpinellifolium) was conducted in field conditions with phenotyping using an unmanned aerial vehicle (UAV)-mounted hyperspectral camera. Six genotypes with different levels of salt tolerance were sampled for leaf pigment analyses, revealing a clear pattern for the high salt tolerant accession M007, where pigment content in the salt-treated plants significantly increased compared to their control counterparts only in harvesting campaigns 3 and 6, each performed two days after the first and second salt stress application events. Moreover, the light harvesting capacity was found to be better maintained under salt stress in the medium (M255) and highly salt tolerant (M007 and M061) accessions. Pigment quantitation data will contribute towards the groundtruthing of hyperspectral imaging for the development of remote sensing-based predictive pigment mapping methods. This work establishes a reliable quantification protocol for correlating pigment content with vegetation indices. Hence, pigment content captured by imaging techniques and validated using biochemical analysis would serve in developing a high-throughput method for pigment quantitation in the field using UAV-based hyperspectral imaging. This would serve as a tool for measuring pigment content in large number of genotypes in the field which would eventually lead to new salt-tolerant genes.

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