Large-scale top-down proteomics of the Arabidopsis thaliana leaf and chloroplast proteomes.

We present a large-scale top-down proteomics (TDP) study of plant leaf and chloroplast proteins, achieving the identification of over 4700 unique proteoforms. Using capillary zone electrophoresis coupled with tandem mass spectrometry analysis of offline size-exclusion chromatography fractions, we identify 3198 proteoforms for total leaf and 1836 proteoforms for chloroplast, with 1024 and 363 proteoforms having post-translational modifications, respectively. The electrophoretic mobility prediction of capillary zone electrophoresis allowed us to validate post-translational modifications that impact the charge state such as acetylation and phosphorylation. Identified modifications included Trp (di)oxidation events on six chloroplast proteins that may represent novel targets of singlet oxygen sensing. Furthermore, our TDP data provides direct experimental evidence of the N- and C-terminal residues of numerous mature proteoforms from chloroplast, mitochondria, endoplasmic reticulum, and other sub-cellular localizations. With this information, we suggest true transit peptide cleavage sites and correct sub-cellular localization signal predictions. This large-scale analysis illustrates the power of top-down proteoform identification of post-translational modifications and intact sequences that can benefit our understanding of both the structure and function of hundreds of plant proteins.

A stress-inducible protein regulates drought tolerance and flowering time in Brachypodium and Arabidopsis.

To cope with environmental stresses and ensure maximal reproductive success, plants have developed strategies to adjust the timing of their transition to reproductive growth. This has a substantial impact on the stress resilience of crops and ultimately on agricultural productivity. Here, we report a previously uncharacterized, plant-specific gene family designated as Regulator of Flowering and Stress (RFS). Overexpression of the BdRFS gene in Brachypodium distachyon delayed flowering, increased biomass accumulation, and promoted drought tolerance, whereas clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated knockout mutants exhibited opposite phenotypes. A double T-DNA insertional mutant in the two Arabidopsis (Arabidopsis thaliana) homologs replicated the effects on flowering and water deprivation seen in the B. distachyon CRISPR knockout lines, highlighting the functional conservation of the family between monocots and dicots. Lipid analysis of B. distachyon and Arabidopsis revealed that digalactosyldiacylglycerol (DGDG) and phosphatidylcholine (PC) contents were significantly, and reciprocally, altered in overexpressor and knockout mutants. Importantly, alteration of C16:0-containing PC, a Flowering Locus T-interacting lipid, associated with flowering phenotype, with elevated levels corresponding to earlier flowering. Co-immunoprecipitation analysis suggested that BdRFS interacts with phospholipase Dα1 as well as several other abscisic acid-related proteins. Furthermore, reduction of C18:3 fatty acids in DGDG corresponded with reduced jasmonic acid metabolites in CRISPR mutants. Collectively, we suggest that stress-inducible RFS proteins represent a regulatory component of lipid metabolism that impacts several agronomic traits of biotechnological importance.

MtSSPdb: The Medicago truncatula Small Secreted Peptide Database.

A growing number of small secreted peptides (SSPs) in plants are recognized as important regulatory molecules with roles in processes such as growth, development, reproduction, stress tolerance, and pathogen defense. Recent discoveries further implicate SSPs in regulating root nodule development, which is of particular significance for legumes. SSP-coding genes are frequently overlooked, because genome annotation pipelines generally ignore small open reading frames, which are those most likely to encode SSPs. Also, SSP-coding small open reading frames are often expressed at low levels or only under specific conditions, and thus are underrepresented in non-tissue-targeted or non-condition-optimized RNA-sequencing projects. We previously identified 4,439 SSP-encoding genes in the model legume Medicago truncatula To support systematic characterization and annotation of these putative SSP-encoding genes, we developed the M. truncatula Small Secreted Peptide Database (MtSSPdb; https://mtsspdb.noble.org/). MtSSPdb currently hosts (1) a compendium of M. truncatula SSP candidates with putative function and family annotations; (2) a large-scale M. truncatula RNA-sequencing-based gene expression atlas integrated with various analytical tools, including differential expression, coexpression, and pathway enrichment analyses; (3) an online plant SSP prediction tool capable of analyzing protein sequences at the genome scale using the same protocol as for the identification of SSP genes; and (4) information about a library of synthetic peptides and root and nodule phenotyping data from synthetic peptide screens in planta. These datasets and analytical tools make MtSSPdb a unique and valuable resource for the plant research community. MtSSPdb also has the potential to become the most complete database of SSPs in plants.