CAX1 Vacuolar Antiporter Overexpression in Potato Results in Calcium Deficiency in Leaves and Tubers by Sequestering Calcium as Calcium Oxalate

Abstract

Recent studies show that overexpression of a short version of the cation exchanger 1 gene (sCAX1) can cause Ca2+ deficiency symptoms in tomato (Solanum lycopersicum L.). However, the Ca2+ deficiency in relation to the overexpression of this gene has not been investigated in potato (Solanum tuberosum L.). The objective of our study was to investigate the production of known Ca2+ deficiency symptoms in potato in relation to the overexpression of sCAX1. Plantlets of S. tuberosum cultivar ‘Atlantic’ overexpressing the sCAX1 gene were produced using Agrobacterium tumefaciens. Transgenic plants grown with normal amounts of Ca2+ under in vitro or greenhouse conditions showed known Ca2+ deficiency symptoms in potato plants such as shoot tips damage and leaf margin necrosis, as well as tuber internal defects (hollow heart). Growing the transgenic plants with higher amounts of Ca2+ in the media or soil nutrient solution mitigated these symptoms. These results support the notion that both shoot tip necrosis and hollow heart are associated with Ca2+ deficiency. There was abundance of calcium oxalate (CaC2O4) crystals present only in the transgenic plants suggesting that these plants sequester Ca2+ in the form of CaC2O4 in the vacuoles of transgenic plants, reducing Ca2+ in the other pools. Since both shoot tip necrosis and hollow heart are known to be associated with poor cell wall health, our results suggest that CAX1 is a regulator of Ca2+ in the cell wall. In support of this concept, we found reduced cell wall biomass in the transgenic plants compared with the wild type.

Fractionating of Calcium in Tuber and Leaf Tissues Explains the Calcium Deficiency Symptoms in Potato Plant Overexpressing CAX1

“…More oxalate crystals were detected at 10 mM concentration in the young and mature leaves. These observations are consistent with our recent results on potato transgenic lines overexpressing sCAX1 (Zorrilla et al, 2019).Over expression of sCAX1 is expected to increase vacuolar calcium concentration (de Freitas et al, 2011) and vacuolar calcium once stored in the vacuole is generally not redistributed. Excess storage of calcium has been shown to result in the formation of calcium oxalate crystal FIGURE 8 | Effects of exogenous calcium on the contents of water-extractable calcium (A), HCl-extractable calcium (B) and total calcium (C) in the tubers of wildtype "Atlantic" (WT Atl) and "AT010901" transgenic plants under high-calcium (10 mM) and low-calcium (1 mM) treatments for 3 weeks.…”

“…Furthermore, the wild type potato plants did not show any of these calcium deficiency symptoms (Figure 1). These results are consistent with recent results from our group for transgenic potato lines (Zorrilla et al, 2019). These symptoms occurred even though the total calcium concentration was greater in thetransgenic plants than the wild type in young leaves (Figure 3), mature leaves ( Figure 5) and tubers (Figure 8) of the transgenic plants at 1 mM calcium treatment.…”

“…Recently our laboratory has reported similar results for transgenic potato plants over-expressing sCAX1 (Zorrilla et al, 2019). In this study one of the transgenic line (AT010901) was shown to have calcium deficiency symptoms in leaf and tuber tissues at normal calcium concentration in the nutrient media while the wild type did not show ant deficiency symptoms.…”

“…Experiments were conducted at the University of Wisconsin-Madison using a transgenic line of potato cultivar`Atlantic' designated as AT010901 and the corresponding wild type. This transgenic line carried the cdc2a promoter cdc2a::sCAX1and a single copy of sCAX1 (Zorrilla et al, 2019). The cdc2a::sCAX1 construct was kindly provided by Dr Kendall Hirschi.…”

“…The transgenic line was found to sequester calcium in the form of calcium oxalate in the vacuole. Furthermore, these deficiencies in AT010901 were mitigated when plants were grown with much higher calcium concentration in the nutrient solution (Zorrilla et al, 2019). The present study was undertaken to quantify calcium in various fraction of leaf and tuber tissues of this transgenic and wild type potato clones to understand the development of these deficiency symptoms at normal calcium nutrition and its mitigation at higher calcium nutrition.…”