Cellular and molecular analysis of barley leaf growth, in response to hormones and pathogen infection. / Krasauskas, Jovaras.

2020. 229 p.

Research output: ThesisDoctoral Thesis

Unpublished

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Abstract

Barley is a widely grown and agriculturally important crop along with maize, oat and wheat, and is well known as a model crop for plant breeding methodology, genetics, cytogenetics, pathology, virology and biotechnology studies. The leaf sheath forms a rigid structure by enveloping other leaf sheath and leaves, providing stability to keep the plant upright, while the leaves provide most of the photosynthetic output. Little is known about the process of proliferation and/or endoreduplication in the sheath and leaf. Barley, due its sessile nature, face constant challenges to biotic and abiotic stress. MeJA and GA treatment was used to examine the effects on sheath and leaf growth as well as if these phytohormones affect Blumeria graminis infection in the primary leaf in wild type and elongation barley mutants.
Flow cytometry and molecular analysis indicated a higher frequency of 4C to 2C nuclear DNA ratio, expression of G1/S- and G2/M-phase markers and smaller cells indicating cell proliferation in the sheath compared to the leaf, The leaf sheath also had a higher endoreduplication frequency, indicated by 8C nuclear DNA content, comparatively to the leaf. MeJA treatment reduced seedling growth and played a role in inhibition of the cell cycle by reducing the expression G1/S-and G2/M-phase cell cycle markers in the sheath and leaf. Flow cytometry indicated that MeJA negatively affected proliferation in the sheath with an increased 2C to 4C nuclear DNA content. Contrary to MeJA, GA treatment increased 4C and to some extent 8C nuclear DNA content in the leaf without affecting cell cycle marker expression. The elo mutants consisted of smaller cells with higher proliferation frequencies in the sheath and leaf, especially for elo3, and had a differential response to MeJA and GA treatment. Long-term (4 days) MeJA and continuous GA treatment may employ a similar pathway to regulate growth as indicated in the similarities in JA- and cell cycle-associated gene expression in 7-day old barley seedlings.
Quantification of Bgh infection indicated higher frequency of secondary hyphae formation in the distal leaf than in the proximal. Molecular analysis indicated an increased expression of G2/M-associated markers as well as JA-responsive genes in response to biotic stress. At later stages of infection, an increase in DNA synthesis through G1/S-phase with higher expression of CYCA3 and reduction of JA-associated gene expression were evident. An increase in epidermal cell nucleus size, next to Bgh appressoria, was observed with confocal microscopy which could suggest a link to increased CYCA3 expression following infection. MeJA and GA treatment reduced the frequency of secondary Bgh hyphae through potentially different pathways. Cell cycle inhibition and increased JA-associated and defence gene expression was observed with MeJA treatment and Bgh infection, suggesting a stress response to limit growth and induce resistance to Bgh. Comparatively, GA treatment followed by Bgh infection did not alter the expression of cell cycle markers, as well as JA-associated and defence genes at later stages of Bgh infection. In this study MeJA treatment may play a role to protect against biotroph infection, while GA treatment may synergise with SA signalling to reduce Bgh infection, but this would need to be determined in future studies. The elo mutants were more susceptible to Bgh infection, with more hyphae formed in the distal and proximal leaf blade in comparison to Himalaya. The mutation for elo mutant dwarfism has yet to be identified, nevertheless, in conjunction with other studies, higher levels of cellulose and lower pectin content could affect cell wall architecture and subsequently susceptibility to Bgh infection.
Barley embryos transformed with a recombinant CRISPR/Cas9 COI1 for the jasmonate receptor and the jasmonate inducible protein/s JIP60/JIP60-like binary plasmid were also analysed. Mutations in COI1 resulted in truncation of the Leucine Rich Repeat (LRR) motif which could attenuate JA responses. Mutations in the eIF4E C-terminal domain of JIP60/JIP60-like may have a role in defence responses.
Original languageEnglish
QualificationPh.D.
Awarding Institution
Supervisors/Advisors
Thesis sponsors
  • Biotechnology&BioSci Research BBSRC
Award date1 Jul 2020
Publication statusUnpublished - 2020

ID: 38188083