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Large scale proteomics in crops: Solanum lycopersicum LC-MSMS

ABSTRACT: Plants represent the nutritional basis of virtually all life on earth and protein-rich foods from crop plants are a global megatrend essential for sustaining an increasing human population and counteracting climate change. While the genomes of crops are increasingly elucidated, little is known about crop proteomes – the entirety of proteins that execute and control nearly every aspect of life. To address this shortcoming we optimized a protocol for mapping the proteome of different crops such as Solanum lycopersicum (tomato) fruit and included four technical replicates and three biological replicates from different tomato plants to demonstrate the robustness of the workflow.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Solanum Lycopersicum

TISSUE(S): Plant Cell, Fruit

SUBMITTER: Sarah Brajkovic

LAB HEAD: Bernhard Kuster

PROVIDER: PXD038945 | Pride | 2023-02-07

REPOSITORIES: Pride

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Project description:During the last decades, the use of plant growth promoting bacteria (PGPB) has been found to increase crop yield and quality and to confer abiotic and biotic stress tolerance. However, until now the PGPB mechanism to enhance plant performances is not clearly defined. Recently, our findings demonstrated that inoculations with both Kocuria rhizophila and Streptomyces violaceoruber, as well as their combination, determined an increase of tomato (Solanum lycopersicum) growth and development. In this study, through an advanced differential proteomic approach on tomato leaves, plant molecular mechanisms affected by both K. rhizophila and S. violaceoruber have been elucidated. To this aim, tomato plants were treated with K. rhizophila and/or Streptomyces violaceoruber cultures and grown on coconut fiber in greenhouse. In particular, PGPB treatments were conducted twice, on seed and after two weeks from the seedling by fertirrigation. Thus, the analyses have been performed at 14 days after sowing (DAS) (T1) and 42 DAS (T2). The results confirmed the growth stimulation ability of K. rhizophila/Streptomyces violaceoruber, showing shoot fresh and dry weight significantly improved at each time sampling. For the early phase (DAS-T1) comparative proteomics analysis of tomato plant leaves, 2 biological replicates were set up for the plants used as control (i.e. not subjected to treatment - samples I1 and I2-control I), 2 biological replicates for plants subjected to treatment with K. rhizophila (samples L1 and L2-treatment L), 2 biological replicates for plants subjected to treatment with S. violaceoruber (samples M1 and M2-treatment M), and 2 biological replicates for plants subjected to treatment with a mix of the two bacterial strains (samples N1 and N2-treatment N), for a total of 8 samples of leaf protein extracts. For the late phase (DAS-T2) comparative proteomics analysis of tomato plant leaves, 2 biological replicates were set up for the plants used as control (i.e. not treated - samples A1 and A2 - control A), 2 biological replicates for plants subjected to treatment with K. rhizophila (samples B1 and B2-treatment B), 2 biological replicates for plants subjected to treatment with S. violaceoruber (samples C1 and C2-treatment C), and 2 biological replicates for plants subjected to treatment with a mix of the two bacterial strains (samples D1 and D2-treatment D), for a total of 8 samples of leaf protein extracts. Proteomic analysis was able to identify 239 and 203 significantly differentially represented proteins (DRPs) at T1 and T2, respectively, comparing PGPB-treated vs. untreated control plants. KEGG Orthology (KO) identified DRP belonging to photosynthesis, biosynthesis of secondary metabolites, and carbon metabolism.

Project description:The tomato psyllid, Bactericera cockerelli Šulc (Hemiptera: Triozidae), is a pest of solanaceous crops such as tomato (Solanum lycopersicum L.) in the U.S. and vectors the disease-causing pathogen ‘Candidatus Liberibacter solanacearum’ (or Lso). Lso disease symptom severity is dependent on Lso haplotype: tomato plants infected with Lso haplotype B experience more severe symptoms and higher mortality compared to plants infected with Lso haplotype A. By characterizing the molecular differences in the tomato plant’s responses to Lso haplotypes, the key components of LsoB virulence can be identified and, thus, targeted for disease mitigation strategies. To characterize the tomato plant genes putatively involved in the differential immune responses to Lso haplotypes A and B, RNA was extracted from tomato ‘Moneymaker’ leaves three weeks after psyllid infestation. Gene expression levels were compared between uninfected tomato plants (i.e., controls and plants infested with Lso-free psyllids) and infected plants (i.e., plants infested with psyllids infected with either Lso haplotype A or Lso haplotype B). Furthermore, expression levels were compared between plants infected with Lso haplotype A and plants infected with Lso haplotype B. A whole transcriptome analysis identified 578 differentially expressed genes (DEGs) between uninfected and infected plants as well as 451 DEGs between LsoA- and LsoB-infected plants. These DEGs were primarily associated with plant defense against abiotic and biotic stressors, growth/development, plant primary metabolism, transport and signaling, and transcription/translation. These gene expression changes suggested that tomato plants traded off plant growth and homeostasis for improved defense against pathogens, especially when infected with LsoB. Consistent with these results, tomato plant growth experiments determined that LsoB-infected plants were significantly stunted and had impaired negative geotropism. However, it appeared that the defense responses mounted by tomatoes were insufficient for overcoming the disease symptoms and mortality caused by LsoB infection, while these defenses could compensate for LsoA infection. The transcriptomic analysis and growth experiments demonstrated that Lso-infected tomato plants underwent gene expression changes related to abiotic and biotic stressors, impaired growth/development, impaired plant primary metabolism, impaired transport and signaling transduction, and impaired transcription/translation. Furthermore, the transcriptomic analysis also showed that LsoB-infected plants, relative to LsoA-infected, experienced more severe stunting, had improved responses to some stressors and impaired responses to others, had poorer transport and signaling transduction, and had impaired carbohydrate synthesis and photosynthesis.

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Large scale proteomics in crops: Solanum lycopersicum LC-MSMS

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