Cellular pathways, genetic analysis and molecular biology of phi thickening induction in Brassica roots

'Akau'ola Aleamotu’a, Maketalena

Title: Cellular pathways, genetic analysis and molecular biology of phi thickening induction in Brassica roots
Creator: 'Akau'ola Aleamotu’a, Maketalena
Relation: University of Newcastle Research Higher Degree Thesis
Resource Type: thesis
Date: 2022
Description: Research Doctorate - Doctor of Philosophy (PhD)
Description: The Earth's population is predicted to increase to almost ten billion by 2050, and feeding this population will only become more challenging as climate change increases rainfall variability. Because of Australia’s susceptibility to drought and salinity, Australian plant science research has always had a strong focus on understanding how plants cope with water and salt stress, and in developing crops for dryland agriculture that use water more efficiently. Such research benefits not just Australia, but warm climate regions worldwide. However, it has been argued that the role of plant roots, the so-called “hidden half” is too often ignored in plant stress biology. To start unravelling one component of this hidden half, this thesis investigated the cellular pathways and genetic analysis of phi thickening induction in roots of the genus Brassica which includes several important crop species. Phi thickenings are specialized bands of secondary cell wall (SCW) found in the cortical cells in roots of diverse species. These thickenings typically exist as a single band of lignified secondary wall material, and their deposition in the radial wall of neighbouring cortical cells are normally spatially co-ordinated, thus resembling the Greek letter phi (Φ) when seen in cross-section. Although first described in the late 1800s, phi thickenings remain poorly understood and very little is known about their function, although it has been proposed that they can mechanically stabilise the root during growth and stress responses. However, directed research into phi thickening development and function(s) has been lacking. Thus, this project has investigated this ignored area of root biology, identifying links between phi thickenings and abiotic and mechanical stresses responses as well as various phytohormones. Thus, for the first time in 150 years since their discovery, the triggers for phi thickening development can be studied. Furthermore, this project expanded to investigate the genomics of phi thickening development, taking advantage of genetic variations from several millennia of selective crop breeding Brassica, to identify novel genetic loci linked to stress-induced phi thickening development. This thesis consists of five chapters including a literature review as the general introduction (Chapter 1), three experimental aims (Chapters 2-4) and a general discussion and conclusion (Chapter 5). A version of the literature review covering a comprehensive analysis of phi thickenings was published under the title “Phi thickenings in roots: novel secondary wall structures responsive to biotic and abiotic stresses” (Aleamotuʻa et al., 2019). The introduction surveys more than a century of phi thickenings research, and highlights numerous deficiencies in our basic understanding of phi thickening development and function. In summary, phi thickenings are defined as Type I, II or III depending on their cellular location within cortical tissue. Moreover, thickenings can also display variations in structure such as a fine reticulate networks of wall material extending laterally from the central band of thickening as seen in the Brassicaceae. Phi thickenings occur in many gymnosperm species and widely in angiosperms, particularly in the Brassicaceae, although interestingly, they have not been reported in the model species Arabidopsis thaliana. Phi thickenings are composed of cellulose microfibrils running parallel with the length of the thickening, and become heavily lignified as during development. Unlike secondary wall thickenings seen in xylem elements, however, the deposition and maturation of phi thickening is not correlated with programmed cell death and thus thickenings are found in living cortical phi cells. Phi thickenings have been proposed to perform roles in mechanical strengthening of the root, acting as a permeability barrier to regulate lateral solute movement in the root, and possibly regulation of fungal interactions. These possibilities have not, however, been experimentally confirmed because of the lack of tractable experimental systems in which to study their development and functions. The first experimental chapter comprises a survey of the Brassicaceae for the presence of phi thickenings in roots and their induction by water stress. This body of work has been published under the title “Developmental biology and induction of phi thickenings by abiotic stress in roots of the Brassicaceae” (Aleamotuʻa et al., 2018). By analysing seedlings grown on nutrient agar plates in the presence or absence of added salt, a considerable variation within the Brassicaceae was documented in not only the presence of phi thickenings within roots, but also responses to salt stress. However, induction also occurred under sucrose-induced water stress in conditions that did not inhibit root elongation, thus demonstrating that root growth inhibition and phi thickening induction are not directly linked. More importantly, strong variations were found in the ability of two species, Brassica oleracea and B. napus, to form phi thickenings. Using confocal microscopy and 3D reconstructions, it was shown that phi thickenings form a continuous, lignified ring around the inner cortex of some Brassica roots, immediately outside the endodermis, and more intriguingly, that these thickenings often extend to form a delicate, reticulate network of secondary wall material along the inner face of these cells adjacent to the endodermal layer. These observations reveal the diverse nature of phi thickening development in the Brassicaceae, and that variations can occur in phi thickening responses to abiotic stress not just within a family but even within a single species. The second experimental aim was to characterise the developmental biology of phi thickening deposition. This work has been published under the title, “Phi thickenings in Brassica oleracea roots are induced by osmotic stress and mechanical effects, both involving jasmonic acid” (Aleamotu’a et al., 2022). In this chapter, a simple system for rapid induction of phi thickenings in primary roots of Brassica was used. Four-day old seedlings were transferred from control agar plates to new plates containing increasing levels of different osmotica and phi thickening development was shown to be directly proportional to osmotic stress. Thickening induction occurred within a narrow region of the differentiation zone, with cellulose deposition and lignification starting after twelve and fifteen hours respectively. However, osmoprotectants such as glycine betaine not only failed to induce phi thickenings, but inhibited induction when tested in combination with thickening-inducing osmotica. Evidence also suggests existence of an independent biomechanical pathway regulating phi thickening induction, with root elongation rates and substrate texture also important for thickening induction. Intriguingly, for the first time, phi thickening development was found to be controlled by stress-related plant hormones, most notably jasmonic acid (JA) and to a lesser extent abscisic acid (ABA). Collectively, these findings not only provide the first understanding of the developmental pathways controlling phi thickening induction, but provide an experimental approach through which the functions of these enigmatic structures might be clarified. In Chapter 2, wide variations in the ability of different Brassica cultivars to induce phi thickenings was reported. In the third experimental chapter, this genetic variation was exploited, in conjunction with genetic resources available within Australia that exist because of the economic importance of this family, notably B. napus (canola), to Australian agriculture. Two different genetic studies were completed in two different Brassica species, namely; (i) a Genome Wide Association Study (GWAS) using 225 inbred lines of a B. napus BnASSYST diversity panel, and (ii) Quantitative Trait Locus (QTL) association mapping of an F2 population (239 lines) derived from B. rapa BC80000 x BC79995 parental cross. For the breeding population, the two parental lines showed markedly different phi thickening induction responses and the population was assessed by phenotyping phi thickening induction in six day-old seedlings grown in agar plates in response to the inclusion of 1 μM JA. These screens aimed to identify major QTL associated with phi thickening induction, and to subsequently pinpoint candidate genes controlling phi thickening development and responses to stresses, with the use of the multiple species providing an independent validation of the identified QTL. Commercial software successfully identified genetic loci and major QTL linked to phi thickening development in the GWAS and F2 mapping populations. Numerous candidate genes linked to QTL were identified, with some having Arabidopsis thaliana homologues annotated to be involved in secondary cell wall deposition and biogenesis, or in various abiotic stresses responses induced by JA. Looking specifically at the B. rapa BC80000 x BC79995 parental cross, the distribution of two separate phenotypes (phi thickening induction, and a distinct phenotype associated with the formation of the reticulate network) in the F2 progeny was consistent with the presence of multiple genetic differences in both JA signalling occurring late in the phi thickening developmental pathway. To validate candidate genes near the major locus for phi thickenings mapped to chromosome A07 and a major locus for reticulate network mapped to chromosome A06 of the B. rapa genome, RT-qPCR was undertaken to assess whether expression of selected candidate genes correlated with phi thickening development. This study has provided the first developmental and genetic insights into phi thickenings as a particular aspect of root biology. Whilst the data generated does not directly demonstrate the functions of phi thickenings, the experimental tools that was developed will allow potential roles to be tested in future projects. Collectively, the findings generated from this research project may prove valuable for plant breeders working in Brassica seeking to develop more resilient cultivars as phi thickenings is likely to be an important trait in plant root stress adaptation. Additionally, the work may lead to a further understanding of JA signalling within plant roots, a subject of considerably importance because of the roles played by JA in biotic stress responses.
Subject: phi thickenings; jasmonic acid; Brassica root development; secondary cell structure; root abiotic stress
Identifier: http://hdl.handle.net/1959.13/1513629
Identifier: uon:56750
Language: eng

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