Long-distance mobile mRNA CAX3 modulates iron uptake and zinc compartmentalization.

Iron deficiency in plants can lead to excessive absorption of zinc; however, important details of this mechanism have yet to be elucidated. Here, we report that MdCAX3 mRNA is transported from the leaf to the root, and that MdCAX3 is then activated by MdCXIP1. Suppression of MdCAX3 expression leads to an increase in the root apoplastic pH, which is associated with the iron deficiency response. Notably, overexpression of MdCAX3 does not affect the apoplastic pH in a MdCXIP1 loss-of-function Malus baccata (Mb) mutant that has a deletion in the MdCXIP1 promoter. This deletion in Mb weakens MdCXIP1 expression. Co-expression of MdCAX3 and MdCXIP1 in Mb causes a decrease in the root apoplastic pH. Furthermore, suppressing MdCAX3 in Malus significantly reduces zinc vacuole compartmentalization. We also show that MdCAX3 activated by MdCXIP1 is not only involved in iron uptake, but also in regulating zinc detoxification by compartmentalizing zinc in vacuoles to avoid iron starvation-induced zinc toxicity. Thus, mobile MdCAX3 mRNA is involved in the regulation of iron and zinc homeostasis in response to iron starvation.

CAX3 (cation/proton exchanger) mediates a Cd tolerance by decreasing ROS through Ca elevation in Arabidopsis.

KEY MESSAGE: Over-expression of CAX3 encoding a cation/proton exchanger enhances Cd tolerance by decreasing ROS (Reactive Oxygen Species) through activating anti-oxidative enzymes via elevation of Ca level in Arabidopsis CAXs (cation/proton exchangers) are involved in the sequestration of cations such as Mn, Li, and Cd, as well as Ca, from cytosol into the vacuole using proton gradients. In addition, it has been reported that CAX1, 2 and 4 are involved in Cd tolerance. Interestingly, it has been reported that CAX3 expressions were enhanced by Cd in Cd-tolerant transgenic plants expressing Hb1 (hemoglobin 1) or UBC1 (Ub-conjugating enzyme 1). Therefore, to investigate whether CAX3 plays a role in increasing Cd tolerance, CAX3 of Arabidopsis and tobacco were over-expressed in Arabidopsis thaliana. Compared to control plants, both transgenic plants displayed an increase in Cd tolerance, no change in Cd accumulation, and enhanced Ca levels. In support of these, AtCAX3-Arabidopsis showed no change in expressions of Cd transporters, but reduced expressions of Ca exporters and lower rate of Ca efflux. By contrast, atcax3 knockout Arabidopsis exhibited a reduced Cd tolerance, while the Cd level was not altered. The expression of Δ90-AtCAX3 (deletion of autoinhibitory domain) increased Cd and Ca tolerance in yeast, while AtCAX3 expression did not. Interestingly, less accumulation of ROS (H2O2 and O2-) was observed in CAX3-expressing transgenic plants and was accompanied with higher antioxidant enzyme activities (SOD, CAT, GR). Taken together, CAX3 over-expression may enhance Cd tolerance by decreasing Cd-induced ROS production by activating antioxidant enzymes and by intervening the positive feedback circuit between ROS generation and Cd-induced spikes of cytoplasmic Ca.

[CAX3 Gene is Involved in Flax Response to High Soil Acidity and Aluminum Exposure].

Understanding the molecular mechanisms of plant response to unfavorable conditions is necessary for the effective selection of tolerant genotypes. Earlier, using high-throughput transcriptome sequencing of flax plants after exposure to aluminum ions (Al^(3+)) and high soil acidity, we detected stress-induced alteration in the expression of several genes, including CAX3, which encodes Ca^(2+)/H^(+)-exchanger involved in calcium ion transport. Here we describe CAX3 mRNA levels in flax cultivars either tolerant (Hermes and TMP1919) or sensitive (Lira and Orshanskiy) to Al^(3+). Stress-induced increased expression of CAX3 was detected only in aluminum-tolerant flax cultivars. The product of CAX3 gene may participate in flax response to high soil acidity and high Al^(3+) concentration through Ca^(2+)-mediated intracellular regulation.

Ectopic overexpression of Eleusine coracana CAX3 confers tolerance to metal and ion stress in yeast and Arabidopsis.

Ionic and metal toxicity in plants is still a global problem for the environment, agricultural productivity and ultimately poses human health threats when these metal ions accumulate in edible organs of plants. Metal and ion transport from cytosol to the vacuole is considered an important component of metal and ion tolerance and a plant's potential utility in phytoremediation. Finger millet (Eleusine coracana) is an orphan crop but has prominent nutritional value in comparison to other cereals. Previous transcriptomic studies suggested that one of the calcium/proton exchanger (EcCAX3) is strongly upregulated during different developmental stages of spikes development in plant. This finding led us to speculate that high calcium accumulation in the grain might be because of CAX3 function. Moreover, phylogenetic analysis shows that EcCAX3 is more closely related to foxtail millet, sorghum and rice CAX3 protein. To decipher the functional role of EcCAX3, we have adopted complementation of yeast triple mutant K677 (Δpmc1Δvcx1Δcnb1), which has defective calcium transport machinery. Furthermore, metal tolerance assay shows that EcCAX3 expression conferred tolerance to different metal stresses in yeast. The gain-of-function study suggests that EcCAX3 overexpressing Arabidopsis plants shows better tolerance to higher concentration of different metal ions as compared to wild type Col-0 plants. EcCAX3-overexpression transgenic lines exhibits abundance of metal transporters and cation exchanger transporter transcripts under metal stress conditions. Furthermore, EcCAX3-overexpression lines have higher accumulation of macro- and micro-elements under different metal stress. Overall, this finding highlights the functional role of EcCAX3 in the regulation of metal and ion homeostasis and this could be potentially utilized to engineer metal fortification and generation of stress tolerant crops in near future.

Melatonin antagonizes ABA action to promote seed germination by regulating Ca2+ efflux and H2O2 accumulation.

Seed germination is a vital stage in the plant life-cycle that greatly contributes to plant establishment. Melatonin has been shown to promote seed germination under various environmental stresses; however, the mechanism remains largely underexplored. Here, we reported that melatonin antagonized abscisic acid (ABA) to promote seed germination by regulating ABA and gibberellic acid (GA3) balance. Transcriptomic analysis revealed that such a role of melatonin was associated with Ca2+ and redox signaling. Melatonin pretreatment induced Ca2+ efflux accompanied by an up-regulation of vacuolar H+/Ca2+ antiporter 3 (CAX3). AtCAX3 deletion in Arabidopsis exhibited reduced Ca2+ efflux. Inhibition of Ca2+ efflux in the seeds of melon and Arabidopsis mutant AtCAX3 compromised melatonin-induced germination under ABA stress. Melatonin increased H2O2 accumulation, and H2O2 pretreatment decreased ABA/GA3 ratio and promoted seed germination under ABA stress. However, complete inhibition of H2O2 accumulation abolished melatonin-induced ABA and GA3 balance and seed germination. Our study reveals a novel regulatory mechanism in which melatonin counteracts ABA to induce seed germination that essentially involves CAX3-mediated Ca2+ efflux and H2O2 accumulation, which, in turn, regulate ABA and GA3 balance by promoting ABA catabolism and/or GA3 biosynthesis.