The photosynthesis-induced accumulation of reactive oxygen species in chloroplasts can lead to oxidative stress, triggering changes in protein synthesis, degradation, and the assembly/disassembly of protein complexes. Using shot-gun proteomics, we identified methyl viologen-induced changes in protein abundance in wild-type Arabidopsis and oxidative stress-hypersensitive fsd1-1 and fsd1-2 knockout mutants, which are deficient in IRON SUPEROXIDE DISMUTASE 1 (FSD1). The levels of proteins that are localized in chloroplasts and the cytoplasm were modified in all lines treated with methyl viologen. Compared with the wild-type, fsd1 mutants showed significant changes in metabolic protein and chloroplast chaperone levels, together with increased ratio of cytoplasmic, peroxisomal, and mitochondrial proteins. Different responses in proteins involved in the disassembly of photosystem II-light harvesting chlorophyll a/b binding proteins were observed. Moreover, the abundance of PATELLIN 4, a phospholipid-binding protein enriched in stomatal lineage, was decreased in response to methyl viologen. Reverse genetic studies using patl4 knockout mutants and a PATELLIN 4 complemented line indicate that PATELLIN 4 affects plant responses to oxidative stress by effects on stomatal closure.

• Keywords
• Arabidopsis, IRON SUPEROXIDE DISMUTASE 1, PATELLIN 4, chloroplast, methyl viologen, oxidative stress, photosynthesis, plasma membrane, proteomics,
• MeSH
• Arabidopsis * metabolism   MeSH
• Chlorophyll A metabolism   pharmacology   MeSH
• Chloroplasts metabolism   MeSH
• Photosynthesis MeSH
• Oxidative Stress MeSH
• Paraquat pharmacology   metabolism   MeSH
• Arabidopsis Proteins * genetics   metabolism   MeSH
• Proteome metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Chlorophyll A MeSH
• Paraquat MeSH
• Arabidopsis Proteins * MeSH
• Proteome MeSH

The roles of mitogen-activated protein kinases (MAPKs) in plant-fungal pathogenic interactions are poorly understood in crops. Here, microscopic, phenotypic, proteomic, and biochemical analyses revealed that roots of independent transcription activator-like effector nuclease (TALEN)-based knockout lines of barley (Hordeum vulgare L.) MAPK 3 (HvMPK3 KO) were resistant against Fusarium graminearum infection. When co-cultured with roots of the HvMPK3 KO lines, F. graminearum hyphae were excluded to the extracellular space, the growth pattern of extracellular hyphae was considerably deregulated, mycelia development was less efficient, and number of appressoria-like structures and their penetration potential were substantially reduced. Intracellular penetration of hyphae was preceded by the massive production of reactive oxygen species (ROS) in attacked cells of the wild-type (WT), but ROS production was mitigated in the HvMPK3 KO lines. Suppression of ROS production in these lines coincided with elevated abundance of catalase (CAT) and ascorbate peroxidase (APX). Moreover, differential proteomic analysis revealed downregulation of several defense-related proteins in WT, and the upregulation of pathogenesis-related protein 1 (PR-1) and cysteine proteases in HvMPK3 KO lines. Proteins involved in suberin formation, such as peroxidases, lipid transfer proteins (LTPs), and the GDSL esterase/lipase (containing "GDSL" aminosequence motif) were differentially regulated in HvMPK3 KO lines after F. graminearum inoculation. Consistent with proteomic analysis, microscopic observations showed enhanced suberin accumulation in roots of HvMPK3 KO lines, most likely contributing to the arrested infection by F. graminearum. These results suggest that TALEN-based knockout of HvMPK3 leads to barley root resistance against Fusarium root rot.

• MeSH
• Fusarium * physiology   MeSH
• Hordeum * genetics   microbiology   MeSH
• Mitogen-Activated Protein Kinase 3 metabolism   MeSH
• Plant Diseases genetics   microbiology   MeSH
• Proteomics MeSH
• Reactive Oxygen Species metabolism   MeSH
• Transcription Activator-Like Effector Nucleases metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Mitogen-Activated Protein Kinase 3 MeSH
• Reactive Oxygen Species MeSH
• Transcription Activator-Like Effector Nucleases MeSH

The diverse roles of mitogen-activated protein kinases (MAPKs, MPKs) in plant development could be efficiently revealed by reverse genetic studies. In Arabidopsis, mpk6 knockout mutants complete the life cycle; however, ~40% of their embryos show defects in the development leading to abnormal phenotypes of seeds and seedlings' roots. Contrary to the Arabidopsis MPK6, the rice MPK6 (OsMPK6) is an essential gene as transfer DNA (T-DNA) insertion and CRISPR/Cas9 induced loss-of-function mutations in the OsMPK6 cause early embryo arrest. In this study, we successfully developed a viable transgenic barley line with the CRISPR/Cas9-induced heterozygous single base pair cytosine-guanine (CG) deletion [wild type (WT)/-1C] in the third exon of the HvMPK6 gene, a barley ortholog of the Arabidopsis and rice MPK6. There were no obvious macroscopic phenotype differences between the WT/-1C plants and WT plants. All the grains collected from the WT/-1C plants were of similar size and appearance. However, seedling emergence percentage (SEP) from these grains was substantially decreased in the soil in the T2 and T3 generation. The mutation analysis of the 248 emerged T2 and T3 generation plants showed that none of them was a biallelic mutant in the HvMPK6 gene, suggesting lethality of the -1C/-1C homozygous knockout mutation. In the soil, the majority of the -1C/-1C grains did not germinate and the minority of them developed into abnormal seedlings with a shootless phenotype and a reduced root system. Some of the -1C/-1C seedlings also developed one or more small chlorotic leaf blade-like structure/structures. The -1C/-1C grains contained the late-stage developed abnormal embryos with the morphologically obvious scutellum and root part of the embryonic axis but with the missing or substantially reduced shoot part of the embryonic axis. The observed embryonic abnormalities correlated well with the shootless phenotype of the seedlings and suggested that the later-stage defect is predetermined already during the embryo development. In conclusion, our results indicate that barley MPK6 is essential for the embryologically predetermined shoot formation, but not for the most aspects of the embryo and early seedling development.

• Keywords
• CRISPR/Cas9, Hordeum vulgare L., MPK6, abnormal embryo, barley, lethality, mitogen-activated protein kinase 6, shootless phenotype,
• Publication type
• Journal Article MeSH