How do environmental factors regulate cytoskeleton and cell wall synthesis in rice seedlings?

This joint PhD project will be based at the Shanghai Jiao Tong University with a minimum 12 month stay at The University of Melbourne. Please note the deadlines associated with SJTU admission eligibility and requirements.

Project description
Rice is a global staple food crop. Knowledge for improvement of growth is therefore critical to agriculture. Cellulose biosynthesis is fundamental for plants and fiber-based industries. It is affected by external cues including diurnal cycles and cell cytoskeletons, although mechanisms remain unclear. We revealed that a rice cytoskeleton protein RMD regulates actin and microtubule dynamics and photomorphorgenesis in rice. We showed that one light-responsive ERF transcription factor directly regulates expression of RMD and cellulose producing Cellulose Synthase (CESA) genes. Here we will reveal how diurnal changes impact the cytoskeleton and cellulose deposition in rice seedlings through the ERF protein.

Hypothesis

The light-responsive ERF-X transcription factor regulates plant morphogenesis by controlling the expression of cellulose biosynthesis and cytoskeleton genes.

Key questions

  1. How does RMD-mediated cytoskeleton changes impact cellulose biosynthesis and CESA distribution?
  2. How does diurnal change impact the process in 1?
  3. How does the ERF-X sense light and regulate photomorphogenesis?

Scope of work

The student will investigate how the ERF-X responds to light and  regulates the expression of RMD and  CESAs and how such changes control cell wall biosynthesis and photomorphogenesis. These analyses will focus on the materials of rice seedlings and cell walls using rmd and the CRISPR-CAS9 mutants and over-expression lines of ERF-X.

Firstly, to address  how  RMD-mediated  cytoskeleton affects cellulose biosynthesis and distribution: i) advanced light and electron microscopy will be used to visualize cell wall thickness of rmd mutant seedling shoots and mature stems:

  1. Updegraff method will be used to measure the cellulose content of rmd mutant seedlings;
  2. Spinning disc microscopy will be used to observe cellulose arrangement stained by S4B, or use atomic force microscopy to observe cellulose microfibrils arrangement in rmd background.

Secondly, to address the role of RMD-mediated cytoskeletal changes in regulating the light-associated cellulose biosynthesis, the student will:

  • cross the actin fluorescent marker line into rmd background;
  1. observe the actin dynamics under light and dark by spinning disc microscopy;
  2. check RMD expression level in wild type with light stimulation;
  3. compare the cellulose content and cellulose microfibrils arrangement of rmd mutant in light and dark, respectively.

Thirdly, to understand how the ERF-X senses light signal and regulate photomorphogenesis, the student will conduct:

  • yeast one-hybrid screening for the motifs within the promoters of RMD, CESA1, CESA3, CESA5, CESA6 and CESA8 that is recognized by the ERF-X protein;
  • transient dual-luciferase assay in rice protoplast system to confirm the binding of ERF-X to the promoter regions;
  1. overexpression of the ERF-X driven by ubiquitin promoter and knock-out of ERF-X, CESA1, CESA3, CESA5, CESA6 and CESA8 in rice by our efficient CRISPR-Cas9 system, as well as crossing the mutants of RMD, ERF-X, CESA1, CESA3, CESA5, CESA6 and CESA8 with our recent established marker lines of F-actin and microtubules;
  2. phenotypic analysis including changes in development and cellulose of genetic materials;
  3. analysis of cytoskeleton dynamics, cellulose content, cellulose microfibrils arrangement of ERF-X genetic lines and compare with rmd mutant;
  4. assessment of wall anisotropy by investigating cytoskeleton organization and cellulose synthesis in light and dark.

The project will be complemented by the project How does a root change its ability to grow through different layers of soil? and the collaboration will ensure a successful completion of the project.

Supervision team:

Professor Dabing Zhang (Shanghai Jiao Tong University)

Professor Michelle Watt (The University of Melbourne)

How to apply

If you are interested in this opportunity, read the application guidelines before contacting the lead supervisor.