Location: Sustainable Agricultural Systems Laboratory
2023 Annual Report
Objectives
Objective 1: Identify metabolic pathways and nutrient molecules that are impacted by cold, heat, and drought stress in tomato. [NP301, C3, PS3A]
Objective 2: Determine the mineral and nutritional metabolite composition of field-grown legume and some non-legume cover crops, and determine how their constituents modify tomato stress tolerance – cold, heat, drought, and yield – using existing transgenic or mutant tomato lines. [NP301, C3, PS3A; C1, PS1A]
Objective 3: Determine how plant responses to cold, heat, and drought are modified at the transcript level in tomato by the hyperaccumulation of polyamines and/or the reduction of the stress hormones, ethylene and methyl jasmonate. [NP301, C3, PS3A]
Approach
Utilize previously developed genetically engineered tomato genotypes - two that have fruit ripening-specific accumulation of polyamines spermidine and spermine (Spd-Spm), other two that constitutively-express spermidine, one that is 50% reduced in fruit-ripening hormone ethylene (Eth-def), another that is deficient in the stress hormone methyl jasmonate (JAS-def), a cross between Eth-def and Spd-Spm, and a cross between JAS-def and Spd-Spm – and test them for tolerance against abiotic stresses such as drought, heat, and cold, and yield. Analyses for water use efficiency (WUE), gene medleys, nutrient content, yield, fruit quantity and quality, metabolic pathways, and gene networks will be defined. In addition, field-grown legume and non-legume cover crops will be analyzed for their levels of metabolites and biomolecules just before flowering time to provide a lead into their utilization for imparting field-based resistance against abiotic stresses in field-grown tomato genotypes.
Progress Report
Tomato is a major vegetable being cultivated on over 5 million hectares with an average annual production of 182 million tons. Tomato is an established model system for fleshy fruits for understanding the roles of ethylene and other hormones and important signal transduction pathways. The research on this project was on the combinatorial role(s) of plant hormones in enhancing nutrient load and stress tolerance in tomato.
Plant hormones are major cellular signaling molecules that modulate growth and development and respond to internal and external cues in plants. Nine plant hormones are recognized, and their perception, intra- and inter-cellular movement/communication, and interaction with receptors and gene regulators are becoming better understood. Intricate mechanistic details regarding plant hormones were discovered during research on this project. The polyamines spermidine, spermine, and T-spermine were implicated in regulating plant growth, inhibiting senescence, and stress responses of plants. Additionally, spermidine influenced floral organ identity and fruit set in tomato; involving the genetic program(s) that control the expression of homeotic genes likely via gibberellin (GA) signaling. These studies suggested a nexus between polyamines and developmental programs in plants. We also identified and characterized 13 gene members of lipoxygenase (LOX), an enzyme that is critical for the synthesis of methyl jasmonate, a stress hormone. Also, LOX genes that responded positively or negatively in tomato during drought and salt stress were identified. These data were found important for genetic manipulation and development of abiotic stress resistance in plants.
Numerous novel tomato heat shock protein (HSP) genes which are expressed at the transition of the mature green tomato fruit into the ripening process were characterized. Heat stress upregulates HSP gene expression, and it is now known that they are developmentally regulated. Two small HSP genes were identified, SlHSP17.7A and SlHSP17.7B, and localized to tomato chromosomes 6 and 9, respectively. It was also demonstrated that ethylene and the master regulator RIN (Ripening Inhibitor) transcription factor crosstalk to regulate these HSP genes. In addition, it was shown that tomato responses to abiotic stresses are unique to each stress. For example, genes impacted during the tomato response to heat were different than during the response to cold. Also, interactions between transcription factors and ethylene in regulating tomato responses to heat shock protein genes were determined. These novel findings are important for designing and developing new heat-resistant tomato germplasm.
Expression of polyamine anabolic and catabolic pathway genes including arginase (ARG) 1 and 2, arginine decarboxylase (ADC) 1 and 2, agmatine iminohydrolase/deiminase 1, N-carbamoyl putrescine amidase, two ornithine decarboxylases (ODC), three S-adenosylmethionine decarboxylases (SAMDC), two spermidine synthases (SPDS1 and 2), spermine synthase (SPMS), flavin-dependent polyamine oxidases (SlPAO4-like and SlPAO2) and copper-dependent amine oxidases (SlCuAO and SlCuAO-like) were characterized in tomato leaves during heat versus cold stress. Spatiotemporal transcript abundance analysis revealed the presence of these gene transcripts in all tissues examined, with higher transcript levels observed for SAMDC1, SAMDC2, and ADC2 in most tissues. Cellular levels of free and conjugated forms of putrescine and spermidine were found to decline during heat stress while they increased in response to cold stress. Transcript levels of ARG2, SPDS2, and PAO4-like increased in response to both heat and cold stresses. Transcript levels of ARG1/2, AIH1, CPA, SPDS1, and CuAO4 increased in response to heat while those of ARG2, ADC1, 2, ODC1, SAMDC1, 2, 3, PAO2, and CuPAO4-like increased in response to cold stress, respectively. Transcripts of ADC1 and 2, ODC1 and 2, and SPMS declined in response to heat stress while ODC2 transcripts declined under cold stress. These results demonstrated differential expression of polyamine metabolism genes under heat and cold stresses with more impairment clearly seen under heat stress. These data indicated a more pronounced role of polyamines in cold stress acclimation than under heat stress in tomato leaves. These novel findings should help in developing new strategies to produce tomato germplasm with prolonged postharvest shelf life along with resistance to heat/cold.
Ethylene-deficient and polyamine-accumulating tomato lines in response to drought stress were also assessed. The ethylene-deficient tomato line was found to have long-term drought tolerance until 14 days with lesser leaf wilting. Further, we determined interactions between transcription factors and ethylene in regulating tomato responses to heat shock protein genes. It was also demonstrated that catabolism of higher polyamine in an aquatic plant is diversified via tandem gene duplication while the putrescine (PUT) catabolic pathway is represented by a single gene. In the aquatic plant we identified a novel prokaryotic type arginine decarboxylase gene pathway. Multivariant analyses of drought and salinity stress polyamine kinetic data and analysis of polyamine biosynthesis gene regulation under these stresses indicated that polyamines are distinctly regulated under drought and salinity stress with different but specific homologs of polyamine biosynthesis and catabolic genes contributing to the accumulation of free, conjugated, and bound forms of polyamines. Datasets on gene expression suggest that a negative relationship exists between ethylene and polyamine synthesis during short-term drought. Subjecting higher polyamine transgenic tomato genotypes to drought revealed that such plants are tolerant to drought for 4 weeks with high tomato yield and root/shoot biomass. Further, subjecting ethylene-deficient germplasm to similar conditions revealed that they also had drought tolerance.
RNA sequencing was employed to identify novel metabolic pathways modulated in corn and tomato in response to the cover crop hairy vetch in comparison to non-vetch-grown corn and tomato plants. RNA sequencing data analysis revealed that phytohormones resulting from cover crop management hold the key for cover crop-based benefits to corn and tomato. Nontargeted metabolomic analysis was used to detect endogenous phytohormone metabolites, profiling approximately 30 hormones and their catabolites. Cover crop modulation of corn growth and phytohormone abundance was coordinated at transcriptome and gene expression levels and thereby cover crops modify the phenotype of whole plants. The transcriptome data indicated tweaking of more than 100 genes while at the same time helping to achieve hairy vetch benefits to corn. Targeted hormone metabolomics on these samples revealed that biosynthesis of specific hormone(s) is affected, including jasmonic acid at the vegetative growth stages. Interestingly, jasmonic acid signaling genes were found to be downregulated in transcriptome data at the same stage.
One major biogenetic process regulating transcription and processing of pre-mRNA complexes in the nucleus involves small nucleolar RNAs (snoRNAs). We cloned, sequenced, and identified a box C/D snoRNA cluster in tomato, namely, SlSnoR12, SlU24a, Slz44a, and Slz132b and determined the effect of spermidine and spermine on these processes. Like this snoRNA cluster housed on chromosome 6, two other noncoding C/D box genes, SlsnoR12.2 and SlU24b, with a 94% identity to those on chromosome 6 were found located on chromosome 3. RNASeq analysis of high spermidine/spermine transgenic tomatoes (579HO line) showed significant enrichment of RNA polymerases, ribosomal, and translational protein genes at the breaker+8 ripening stage as compared with the control. These results indicated that spermidine/spermine regulate snoRNA and rRNA expression directly or indirectly, in turn, affecting protein synthesis, metabolism, and other cellular activities in a positive manner.
Accomplishments
Review Publications
Koukounaras, A., Mellidou, I., Patelou, E., Kostas, S., Shukla, V., Engineer, C., Papaefthimiou, D., Amari, F., Chatzopoulos, D., Mattoo, A.K., Kanellis, A. 2022. Over-expression of GGP and GPP genes enhances ascorbate content and nutritional quality of tomato. Plant Physiology and Biochemistry. https://doi.org/10.1016/j.plaphy.2022.10.023.
Mattoo, A.K., Cavigelli, M.A., Misic, D., Gasic, U., Maksimovic, V., Kramer, M., Kaur, B., Matekalo, D., Nestorovic Zivkovic, J., Roberts, D.P. 2023. Maize metabolomics in relation to cropping system and growing year impacts. Frontiers in Sustainable Food Systems. https://doi.org/10.3389/fsufs.2023.113008910.3389/fsufs.2023.1130089.
Giardi, M.T., Antonacci, A., Touloupakis, E., Mattoo, A.K. 2022. Investigation of Photosystem II functional size in higher plants under physiological and stress conditions using radiation target analysis and sucrose gradient ultracentrifugation. Molecules. 27:5708. https://doi.org/10.3390/molecules27175708.
Arasimowicz-Jelonek, M., Jagodzi, A., Plóciennik, A., Sobieszczuk-Nowicka, E., Floryszak-Wieczorek, J., Mattoo, A.K., Polcyn, W. 2022. Dynamics of nitration phenomenon during dark-induced leaf senescence in Arabidopsis reveals proteins modified by tryptophan nitration. Journal of Experimental Botany. https://doi.org/10.1093/jxb/erac341.
