Yutao Zou

Soil salinization is a worldwide and increasing problem that leads to a decrease of arable land and affects food supply. Unlike halophyte species, most crops are sensitive to salt stress, which affects their growth and yield. Therefore, to make progress on generating more salt tolerant crops, we need to uncover the underlying mechanisms of salt-induced plant responses. In this thesis, we use the model species Arabidopsis (Arabidopsis thaliana) to obtain new knowledge that we anticipate can be used to transfer to crops.

In Chapter 1, I introduce how soil salinity threatens plant growth, function and survival and emphasize the importance of maintaining Na+ and K+ ionic homeostasis during ionic stresses. I also introduce various phytohormones, including abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) that are involved in salt signaling pathways. Moreover, the current knowledge of salt-induced root directional growth and how plant cell walls monitor the outside environment -especially under salt stress- are introduced in this chapter.

In Chapter 2 the processes involved in several major responses of root growth, including the modulation of root system architecture (RSA), root directional growth, root cell wall modifications are further addressed. The developmental and molecular mechanisms that govern growth responses of root systems under salinity are reviewed in more detail in this chapter. Additionally, we discuss the recent literature on genetic variation between and within plant species in response to salinity stress, in Arabidopsis and several crop species.

Building on the observation of the salt-specific directional response named root halotropism (Galvan-Ampudia et al., 2013; Deolu-Ajayi et al., 2019), we established a rapid salt-induced tilting assay (SITA) which also reports the specific modulation of root growth direction by NaCl (but not osmotic stress) upon gravitropic challenge with high time resolution (every 20 mins) in Chapter 3. We show that the ABA signaling pathway and ethylene overproduction are involved in modulating SITA responses. Natural variation is a powerful tool to identify new genetic components underlying the diverse phenotypic range of adaptations in growth responses to salt. Combining SITA with natural variation of the HapMap population of Arabidopsis accessions, we performed genome wide association studies (GWAS) to find the genetic basis of the response. Two candidate genes that were found to be associated with the root response vector angle in SITA were identified: unknown Protein kinase (PK, AT4G37540) and Extensin Arabinose Deficient Transferase (ExAD, AT4G37550). By characterization of T-DNA insertion mutants of candidate genes, the role of the two candidate genes PK and ExAD were shown to be required for modulating root vector angle in SITA response to salt.

To continue, in Chapter 4 we further characterized the candidate gene ExAD for its function in root responses to salinity. Being a member of the arabinosyltransferases enzyme family, ExAD adds the arabinose residue to form a Hyp-Araf1-4 side-chain at the α-(1,3)-fourth specific position on extensins, which are cell wall hydroxyproline-rich glycoproteins (HRGPs) (Petersen et al., 2021; Møller et al., 2017). We show the roles of ExAD in root directional growth responses and ion accumulation in response to salt treatment. By Comprehensive Microarray Polymer Profiling (CoMPP) and dot-blot, we reveal that ExAD is involved in the alteration of polysaccharide signals under salt stress, and JIM11 is the specific anti-extensin-Hyp-Araf4 antibody to detect the function of ExAD. We also show that ExAD-dependent Hyp-Araf4 is increased when exposed to salt in Col-0, but is absent in the exad-1 mutant. Moreover, cell wall thickness increases in exad-1 root epidermal cells under salt stress in both the root elongation zone and maturation zone. Therefore, we suggest a correlation between salt-induced cell wall extensin Hyp-arabinosylation, cell wall structure and plant root directional responses to salinity.

To monitor environmental stresses, plant cell walls are crucial for governing downstream signaling pathways and triggering adequate responses. Besides cell wall structural proteins (e.g. extensins), cell wall localized receptor-like kinases are also crucial for mediating salt responses. Focusing on the cell wall localized Arabidopsis L-type LecRKs genes, in Chapter 5, we characterize the role of LecRK-IV.1 and LecRK-IV.2 in salt responses. A double mutant for these genes, named lecrk4.1/4.2, shows reduced salt-induced root directional changes in both halotropism and SITA response assays. The lecrk4.1/4.2 mutant shows enhanced lateral root density, lower response in shoot Na+/K+ ratio, and higher seedling survival under severe salt stress. Additionally, we show that LecRK-IV.1/LecRK-IV.2 regulate cell wall integrity (CWI) responses with a higher accumulation of JA and lignin in the lecrk4.1/4.2 double mutant induced by the cellulose biosynthesis inhibitor isoxaben (ISX) compared to Col-0. The role of LecRK-IV.1/LecRK-IV.2 was distinct from THESEUS1 (THE1), but they function in a THE1 dependent fashion, since both the1-1 mutant and lecrk4.1/4.2/the1-1 triple mutant showed significantly lower accumulation of JA and lignin induced by ISX compared to Col-0. Interestingly, the lecrk4.1/4.2/the1-1 triple mutant showed a reduced halotropism response in the long term, compared to the lecrk4.1/4.2 and the1-1 mutants. These data may suggest that THE1 and LecRK-IV.1/LecRK-IV.2 have different roles in responding to cellulose biosynthesis inhibition-induced CWI, but together contribute to salt-induced long-term root halotropism responses.

In Chapter 6, I further discuss the results from the previous chapters and provide future perspectives on the role of cell wall post-translational modifications of extensin and function of LecRKs in salt sensing and root responses. I proposed two hypothetical models in this chapter that summarize the role of cell wall modifications during salt stress and propose speculative new links that provide new routes to uncover plant salt signaling pathways. Together, this study suggests that well-arabinosylated versions of the cell wall structural protein extensin and several cell wall localized LecRKs are involved in modulating root growth responses and ion homeostasis under salt stress conditions, which may provide insight into how plants coordinate cell wall changes to further proceed signal transduction. This work has revealed some of the underlying mechanisms that modulate root directional growth and root shape of plants exposed to salt stress, and ultimately may contribute to improving salt stress resilience of plants.