Heterodimerization of Arabidopsis calcium/proton exchangers contributes to regulation of guard cell dynamics and plant defense responses.

Arabidopsis thaliana cation exchangers (CAX1 and CAX3) are closely related tonoplast-localized calcium/proton (Ca2+/H+) antiporters that contribute to cellular Ca2+ homeostasis. CAX1 and CAX3 were previously shown to interact in yeast; however, the function of this complex in plants has remained elusive. Here, we demonstrate that expression of CAX1 and CAX3 occurs in guard cells. Additionally, CAX1 and CAX3 are co-expressed in mesophyll tissue in response to wounding or flg22 treatment, due to the induction of CAX3 expression. Having shown that the transporters can be co-expressed in the same cells, we demonstrate that CAX1 and CAX3 can form homomeric and heteromeric complexes in plants. Consistent with the formation of a functional CAX1-CAX3 complex, CAX1 and CAX3 integrated into the yeast genome suppressed a Ca2+-hypersensitive phenotype of mutants defective in vacuolar Ca2+ transport, and demonstrated enzyme kinetics different from those of either CAX protein expressed by itself. We demonstrate that the interactions between CAX proteins contribute to the functioning of stomata, because stomata were more closed in cax1-1, cax3-1, and cax1-1/cax3-1 loss-of-function mutants due to an inability to buffer Ca2+ effectively. We hypothesize that the formation of CAX1-CAX3 complexes may occur in the mesophyll to affect intracellular Ca2+ signaling during defense responses

A calmodulin-like protein regulates plasmodesmal closure during bacterial immune responses

Plants sense microbial signatures via activation of pattern recognition receptors (PPRs), which trigger a range of cellular defences. One response is the closure of plasmodesmata, which reduces symplastic connectivity and the capacity for direct molecular exchange between host cells. Plasmodesmal flux is regulated by a variety of environmental cues but the downstream signalling pathways are poorly defined, especially the way in which calcium regulates plasmodesmal closure. Here, we identify that closure of plasmodesmata in response to bacterial flagellin, but not fungal chitin, is mediated by a plasmodesmal-localized Ca2+ -binding protein Calmodulin-like 41 (CML41). CML41 is transcriptionally upregulated by flg22 and facilitates rapid callose deposition at plasmodesmata following flg22 treatment. CML41 acts independently of other defence responses triggered by flg22 perception and reduces bacterial infection. We propose that CML41 enables Ca2+ -signalling specificity during bacterial pathogen attack and is required for a complete defence response against Pseudomonas syringae.Bo Xu, Cecilia Cheval, Anuphon Laohavisit, Bradleigh Hocking, David Chiasson, Tjelvar S. G. Olsson, Ken Shirasu, Christine Faulkner and Matthew Gilliha

The Arabidopsis Ca²⁺ / H⁺ exchangers, AtCAX1 and AtCAX3, are shown by co-localisation, interaction and complementation to participate in plant Ca²⁺ homeostasis.

This research was performed over 10 months as part of a Masters in Biotechnology (Plant Biotechnology). The literature review was previously assessed but has been modified somewhat to address small changes in the research focus of the project. Although the research manuscript contained herein will provide the first draft of a future publication in Plant Cell and Physiology Journal, due to time constraints, additional data relevant to that publication may be collected. Additional data that has been collected which was not conclusive or integral to the focus of the research manuscript is provided within the appendices. The research manuscript details investigations into the interactions of Arabidopsis calcium transporters, while the appendices contain additional information pertaining to protoplast transformations, plasmid construction and plant media recipes.Thesis (M.Bio (PB)) - University of Adelaide, School of Agriculture, Food and Wine, 200

The role of calcium in the cell wall of grape berries

Calcium has defined roles in plant signalling, water relations and cell wall interactions. Calcium nutrition impacts fruit quality by facilitating developmental and stress response signalling, stabilising membranes, and modifying cell wall properties through cross-linking of de-esterified pectins. The importance of calcium in fruit development and ripening is reviewed, experimental work probing the relationship between calcium nutrition and fruit development in grape berries is undertaken. Relationships between calcium uptake and pectin modification were investigated in a survey of red, white, and table grape varieties collected from two sites varying in calcium levels. Grapes harvested at the Barossa site showed higher calcium concentrations within apoplastic fluid, skin and mesocarp tissues than those from Waite. Chenin Blanc had higher apoplastic calcium content than other varieties. Fluorescent immuno-labelling revealed de-esterified pectin localisation in the middle lamella of all varieties with punctillate staining patterns observed in Grenache and Thompson Seedless. A negative correlation between apoplastic pH and apoplastic calcium concentration was observed. Shiraz was the only variety to demonstrate any significant difference between sites in apoplastic pH and apoplastic calcium activity. Effects of low and high calcium supply in grapevines were investigated. Low calcium grown Shiraz showed early berry softening and onset of berry weight loss. High calcium grown Shiraz showed delayed and asynchronous fruit development. Berry hydration assays indicated that early onset of berry weight loss in low calcium grown berries was a result of higher post-veraison berry transpiration. High calcium grown berries demonstrated lower berry water uptake rate pre-veraison, and lower berry transpiration rates throughout development. Whole vine physiology was assessed in Chenin Blanc; high calcium grown vines demonstrated reduced transpiration and net assimilation rates compared to basal and low calcium grown vines. An image analysis macro was developed for quantification of cell vitality (with fluorescein diacetate; FDA) and pectin de-esterification (with propidium iodide; PI) staining patterns. Chenin Blanc maintained higher PI staining in skin tissue than Shiraz throughout development; higher magnification imaging revealed this staining to be localised to the epidermis and peripheral vasculature of Chenin Blanc berries. Transmission electron microscopy demonstrated cuticle localisation of de-esterified pectin in Chenin Blanc and Shiraz berries, particularly of low calcium grown berries; low levels of calcium-pectin crosslinkages and high rates of berry transpiration result in increased movement of de-esterified pectin from the epidermis into the cuticle. Shiraz cuticle de-esterified pectin levels increased throughout development, indicating pectin solubilisation. Chenin Blanc showed strong de-esterified pectin labelling in epidermal and hypodermal cell walls, consistent with patterns visualised using PI staining. Low calcium grown Chenin Blanc berries showed a higher Botrytis infection rate than basal or high calcium grown berries. Differences in calcium accumulation and pectin modification contribute to varietal diversity in ripening physiology. Berries supplied with low calcium are early softening and susceptible to shrivel and Botrytis infection, whereas high calcium supply results in changes in vine physiology, including delayed and asynchronous berry development.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2015

Heterodimerization of Arabidopsis calcium/proton exchangers contributes to regulation of guard cell dynamics and plant defense responses

Arabidopsis thaliana cation exchangers (CAX1 and CAX3) are closely related tonoplast-localized calcium/proton (Ca2+/H+) antiporters that contribute to cellular Ca2+ homeostasis. CAX1 and CAX3 were previously shown to interact in yeast; however, the function of this complex in plants has remained elusive. Here, we demonstrate that expression of CAX1 and CAX3 occurs in guard cells. Additionally, CAX1 and CAX3 are co-expressed in mesophyll tissue in response to wounding or flg22 treatment, due to the induction of CAX3 expression. Having shown that the transporters can be co-expressed in the same cells, we demonstrate that CAX1 and CAX3 can form homomeric and heteromeric complexes in plants. Consistent with the formation of a functional CAX1-CAX3 complex, CAX1 and CAX3 integrated into the yeast genome suppressed a Ca2+-hypersensitive phenotype of mutants defective in vacuolar Ca2+ transport, and demonstrated enzyme kinetics different from those of either CAX protein expressed by itself. We demonstrate that the interactions between CAX proteins contribute to the functioning of stomata, because stomata were more closed in cax1-1, cax3-1, and cax1-1/cax3-1 loss-of-function mutants due to an inability to buffer Ca2+ effectively. We hypothesize that the formation of CAX1-CAX3 complexes may occur in the mesophyll to affect intracellular Ca2+ signaling during defense responses