Location: Sustainable Agricultural Systems Laboratory
2021 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 one of the major vegetables worldwide regarding consumption and production volume, with an annual yield surpassing 182 million tons. Ripe tomato fruit is a source of dietary micronutrients, vitamins, and antioxidants for daily human consumption. Tomato is also an established model system, especially for developing novel information on the ripening and senescence of fleshy fruits. In addition to unraveling mechanisms that increase fruit yield and longer shelf life, consumer recognition of fruits as sources of health-promoting nutrients for a healthier life has intensified research on tomato. Therefore, our research efforts have been directed toward understanding the genetics of the tomato ripening process, the role of plant hormones, particularly ethylene, and deciphering the signal transduction pathways. Tomato is also vulnerable to environmental extremes, including diverse abiotic and biotic stressors, impacting its growth, yield, and nutritional quality. Our goals include identifying genetic and biochemical determinants of the normal biology of tomato and unraveling the stress pathways so that novel tomato germplasm that can withstand abiotic stresses and minimize yield losses can be developed. Our research studies also focus on the combinatorial role(s) of plant hormones in enhancing nutrient load and stress tolerance in tomato. The salient features of hormone biology are important in understanding cross-talks between hormones as well as for developing stress-resistant germplasm. Plant hormones we are studying include ethylene, jasmonic acid, and a group of low molecular weight organic cations called polyamines, namely, putrescine (PUT), spermidine (SPD), spermine (SPM) and T-spermine (theo-SPM). These four polyamines have been implicated in regulating plant growth and inhibiting senescence and stress responses of plants.
Although a plethora of genes and proteins have been implicated in tomato fruit ripening, developing fruits to have longer shelf life without compromising the nutrient content has yet to be achieved. We have also undertaken a study to understand the novel dynamics involved in aquatic plants, which are not sessile and therefore are an excellent model to ascertain how these plants survive and regulate extreme environmental stresses.
For Objective 1 we characterized the expression of polyamine anabolic and catabolic pathway genes in tomato leaves under heat versus cold stress. The genes analyzed include 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). The spatiotemporal transcript abundances using qRT-PCR revealed their 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, revealing their differential responses. Transcript levels of ARG2, SPDS2, and PAO4-like increased in response to both heat and cold stresses. However, 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, 2, ODC1, 2, and SPMS declined in response to heat stress, while ODC2 transcripts declined under cold stress. These results showed differential expression of polyamine metabolism genes under heat and cold stresses, with more impairment seen under heat stress. These data indicate a more pronounced role of polyamines in cold stress acclimation compared to that under heat stress in tomato leaves. These findings are novel and should help develop new strategies to produce tomato germplasm with prolonged postharvest shelf life and resistance to heat or cold. Two manuscripts describing this work were written and were published.
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 SPD and SPM on these processes. Similar to this snoRNA cluster housed on chromosome (Chr.) 6, two other noncoding C/D box genes, SlsnoR12.2 and SlU24b, with a 94% identity to those on Chr. 6, were found located on Chr. 3. RNAseq analysis of high SPD/SPM transgenic tomatoes (579HO line) showed significant enrichment of RNA polymerases, ribosomal, and translational protein genes at the breaker+8 ripening stage compared with the 556AZ control. Thus, these results indicate that SPD/SPM regulates snoRNA and rRNA expression directly or indirectly, in turn, affecting protein synthesis, metabolism, and other cellular activities positively.
Inland plants polyamines increase water content, especially under stress conditions; this likely being a mechanism to help plants cope under water-deficit conditions. Further, we were able to show that catabolism of higher polyamine in an aquatic plant is diversified via tandem gene duplication while a single gene represents the putrescine (PUT) catabolic pathway. In the aquatic Spirodela we identified a novel prokaryotic type arginine decarboxylase gene pathway. Two publications describing this work are in the final stage of completion.
For Objective 2, fieldwork was impacted by the pandemic. The experimental samples generated from our fieldwork were analyzed for RNA sequencing to identify novel metabolic pathways modulated in response to the cover crop hairy vetch compared to non-vetch-grown tomato plants. The RNA sequencing data are currently being analyzed.
For Objective 3, we assessed ethylene-deficient and polyamine-accumulating tomato lines in response to drought stress. The differential ethylene-polyamine levels were found to provide a long-term drought tolerance until 14 days with lesser leaf wilting in the ethylene-silenced line. This study demonstrated that tomato responses to abiotic stresses are unique to each stress. For example, the tomato response to heat was different, in fact, opposite to the response to cold regarding the genes impacted. Further, we determined interactions between transcription factors and ethylene in regulating tomato responses to heat shock protein genes. Follow-up studies should help determine and unravel critical genetic switches for developing longer-lasting, nutrient-rich, and heat-tolerant tomato germplasm. One manuscript regarding this work is being drafted for publication.
Accomplishments
1. Heat shock protein gene cluster regulated by heat stress identified. ARS scientists in Beltsville, Maryland, identified and characterized a novel heat shock protein gene cluster that regulates heat stress in plants and is non-responsive to drought, salinity or cold. Now scientists and breeders can design and develop new heat-resistant tomato germplasm. This information is useful to molecular biologists and plant breeders developing heat-stress resistant tomatoes.
Review Publications
Upadhyay, R., Tucker, M., Mattoo, A.K. 2020. Ethylene and ripening inhibitor modulate expression of SlHSP17.7A, B class I small heat shock protein genes during tomato fruit ripening. Frontiers in Plant Science. 11:1-15.
Upadhyay, R.K., Fatima, T., Handa, A.K., Mattoo, A.K. 2020. Polyamines and their biosynthesis/catabolism genes are differentially modulated in response to heat versus cold stress in tomato leaves (Solanum lycopersicum L.). Cells. 9:1-19.
Mattoo, A.K. 2020. Historial Account: Duckweeds, photosynthesis and ethylene. Duckweed Forum. 8(3):56-93.
Upadhyay, R.K., Edelman, M., Mattoo, A.K. 2020. Identification, phylogeny,and comparative expression of the lipoxygenase gene family of the aquatic duckweed, Spirodela polyrhiza, during growth and in response to methyl jasmonate and salt. International Journal of Molecular Sciences. 21(24):9527. https://doi.org/10.3390/ijms21249527.
Shukla, V., Fatima, T., Goyal, R.K., Handa, A.K., Mattoo, A.K. 2020. Engineered ripening-specific accumulation of polyamines spermidine and spermine in tomato fruit upregulates clustered C/D box snoRNA gene transcripts in concert with ribosomal RNA biogenesis in the red-ripe fruit. Plants. 9:1710. https://doi.org/10.3390/plants9121710.
