In planta regulation of the Arabidopsis Ca(2+)/H(+) antiporter CAX1

Vacuolar localized Ca(2+)/H(+) exchangers such as Arabidopsis thaliana cation exchanger 1 (CAX1) play important roles in Ca(2+) homeostasis. When expressed in yeast, CAX1 is regulated via an N-terminal autoinhibitory domain. In yeast expression assays, a 36 amino acid N-terminal truncation of CAX1, termed sCAX1, and variants with specific mutations in this N-terminus, show CAX1-mediated Ca(2+)/H(+) antiport activity. Furthermore, transgenic plants expressing sCAX1 display increased Ca(2+) accumulation and heightened activity of vacuolar Ca(2+)/H(+) antiport. Here the properties of N-terminal CAX1 variants in plants and yeast expression systems are compared and contrasted to determine if autoinhibition of CAX1 is occurring in planta. Initially, using ionome analysis, it has been demonstrated that only yeast cells expressing activated CAX1 transporters have altered total calcium content and fluctuations in zinc and nickel. Tobacco plants expressing activated CAX1 variants displayed hypersensitivity to ion imbalances, increased calcium accumulation, heightened concentrations of other mineral nutrients such as potassium, magnesium and manganese, and increased activity of tonoplast-enriched Ca(2+)/H(+) transport. Despite high in planta gene expression, CAX1 and N-terminal variants of CAX1 which were not active in yeast, displayed none of the aforementioned phenotypes. Although several plant transporters appear to contain N-terminal autoinhibitory domains, this work is the first to document clearly N-terminal-dependent regulation of a Ca(2+) transporter in transgenic plants. Engineering the autoinhibitory domain thus provides a strategy to enhance transport function to affect agronomic traits.     

Ectopic expression of a maize calreticulin mitigates calcium deficiency-like disorders in sCAX1-expressing tobacco and tomato

Deregulated expression of an Arabidopsis H⁺/Ca²⁺ antiporter (sCAX1) in agricultural crops increases total calcium (Ca²⁺) but may result in yield losses due to Ca²⁺ deficiency-like symptoms. Here we demonstrate that co-expression of a maize calreticulin (CRT, a Ca²⁺ binding protein located at endoplasmic reticulum) in sCAX1-expressing tobacco and tomato plants mitigated these adverse effects while maintaining enhanced Ca²⁺ content. Co-expression of CRT and sCAX1 could alleviate the hypersensitivity to ion imbalance in tobacco plants. Furthermore, blossom-end rot (BER) in tomato may be linked to changes in CAX activity and enhanced CRT expression mitigated BER in sCAX1 expressing lines. These findings suggest that co-expressing Ca²⁺ transporters and binding proteins at different intracellular compartments can alter the content and distribution of Ca²⁺ within the plant matrix.     

Ca2+‐regulated and diurnal rhythm‐regulated Na+/Ca2+ exchanger AtNCL affects flowering time and auxin signalling in Arabidopsis

Calcium (Ca²⁺) is vital for plant growth, development, hormone response and adaptation to environmental stresses, yet the mechanisms regulating plant cytosolic Ca²⁺ homeostasis are not fully understood. Here, we characterize an Arabidopsis Ca²⁺‐regulated Na⁺/Ca²⁺ exchanger AtNCL that regulates Ca²⁺ and multiple physiological processes. AtNCL was localized to the tonoplast in yeast and plant cells. AtNCL appeared to mediate sodium (Na⁺) vacuolar sequestration and meanwhile Ca²⁺ release. The EF‐hand domains within AtNCL regulated Ca²⁺ binding and transport of Ca²⁺ and Na⁺. Plants with diminished AtNCL expression were more tolerant to high CaCl₂ but more sensitive to both NaCl and auxin; heightened expression of AtNCL rendered plants more sensitive to CaCl₂ but tolerant to NaCl. AtNCL expression appeared to be regulated by the diurnal rhythm and suppressed by auxin. DR5::GUS expression and root responses to auxin were altered in AtNCL mutants. The auxin‐induced suppression of AtNCL was attenuated in SLR/IAA14 and ARF6/8 mutants. The mutants with altered AtNCL expression also altered flowering time and FT and CO expression; FT may mediate AtNCL‐regulated flowering time change. Therefore, AtNCL is a vacuolar Ca²⁺‐regulated Na⁺/Ca²⁺ exchanger that regulates auxin responses and flowering time.     

Expression of an Arabidopsis Ca/H antiporter CAX1 variant in petunia enhances cadmium tolerance and accumulation

Phytoremediation is a cost-effective and minimally invasive technology to cleanse soils contaminated with heavy metals. However, few plant species are suitable for phytoremediation of metals such as cadmium (Cd). Genetic engineering offers a powerful tool to generate plants that can hyperaccumulate Cd. An Arabidopsis CAX1 mutant (CAXcd), which confers enhanced Cd transport in yeast, was ectopically expressed in petunia to evaluate whether the CAXcd expression would enhance Cd tolerance and accumulation in planta. The CAXcd-expressing petunia plants showed significantly greater Cd tolerance and accumulation than the controls. After being treated with either 50 or 100 μM CdCl₂ for 6 weeks, the CAXcd-expressing plants showed more vigorous growth compared with controls, and the transgenic plants accumulated significantly more Cd (up to 2.5-fold) than controls. Moreover, the accumulation of Cd did not affect the development and morphology of the CAXcd-expressing petunia plants until the flowering and ultimately the maturing of seeds. Therefore, petunia has the potential to serve as a model species for developing herbaceous, ornamental plants for phytoremediation.     

Extremely Flexible Transparent Conducting Electrodes for Organic Devices

Extremely flexible transparent conducting electrodes are developed using a combination of metal‐embedding architecture into plastic substrate and ultrathin transparent electrodes, which leads to highly transparent (optical transmittance ≈93% at a wavelength of 550 nm), highly conducting (sheet resistance ≈13 Ω □^?1), and extremely flexible (bending radius ≈ 200 μm) electrodes. The electrodes are used to fabricate flexible organic solar cells and organic light‐emitting diodes that exhibit performance similar or superior to that of devices fabricated on glass substrates. Moreover, the flexible devices do not show degradation in their performance even after being folded with a radius of ≈200 μm.     

Role of pectin methylesterases in cellular calcium distribution and blossom-end rot development in tomato fruit

Blossom-end rot (BER) in tomato fruit (Solanum lycopersicum) is believed to be a calcium (Ca(2+) ) deficiency disorder, but the mechanisms involved in its development are poorly understood. Our hypothesis is that high expression of pectin methylesterases (PMEs) increases Ca(2+) bound to the cell wall, subsequently decreasing Ca(2+) available for other cellular functions and thereby increasing fruit susceptibility to BER. The objectives of this study were to evaluate the effect of PME expression, and amount of esterified pectins and Ca(2+) bound to the cell wall on BER development in tomato fruit. Wild-type and PME-silenced tomato plants were grown in a greenhouse. At full bloom, flowers were pollinated and Ca(2+) was no longer provided to the plants to induce BER. Our results show that suppressing expression of PMEs in tomato fruit reduced the amount of Ca(2+) bound to the cell wall, and also reduced fruit susceptibility to BER. Both the wild-type and PME-silenced fruit had similar total tissue, cytosolic and vacuolar Ca(2+) concentrations, but wild-type fruit had lower water-soluble apoplastic Ca(2+) content and higher membrane leakage, one of the first symptoms of BER. Our results suggest that apoplastic water-soluble Ca(2+) concentration influences fruit susceptibility to Ca(2+) deficiency disorders.     

Improved watermelon quality using bottle gourd rootstock expressing a Ca2+/H+ antiporter

Bottle gourd (Lagenaria siceraria Standl.) has been commonly used as a source of rootstock for watermelon. To improve its performance as a rootstock without adverse effects on the scion, the bottle gourd was genetically engineered using a modified Arabidopsis Ca²⁺/H⁺ exchanger sCAX2B. This transporter provides enhanced Ca²⁺ substrate specificity and decreased Mn²⁺ transport capability. Our previous work demonstrated that sCAX2B bottle gourds were more robust and nutrient dense than controls. Here, several cucurbit crops were test-grafted onto the transgenic bottle gourd to determine its effect on the scions. The grafted watermelons and melons onto the transgenic rootstocks appeared to show more robust growth than the controls 35 days after greenhouse transplanting. Watermelon fruits with the watermelon/transgenic bottle gourd (scion/rootstock) combination demonstrated higher osmotic pressure and more soluble solids than controls. These results suggest that sCAX2B expression in the bottle gourd rootstock facilitates improved watermelon quality through the translocation of nutrients and/or water toward enhancing the biomass of scion.     

Increased calcium levels and prolonged shelf life in tomatoes expressing Arabidopsis H+/Ca2+ transporters

Here we demonstrate that fruit from tomato (Lycopersicon esculentum) plants expressing Arabidopsis (Arabidopsis thaliana) H(+)/cation exchangers (CAX) have more calcium (Ca2+) and prolonged shelf life when compared to controls. Previously, using the prototypical CAX1, it has been demonstrated that, in yeast (Saccharomyces cerevisiae) cells, CAX transporters are activated when the N-terminal autoinhibitory region is deleted, to give an N-terminally truncated CAX (sCAX), or altered through specific manipulations. To continue to understand the diversity of CAX function, we used yeast assays to characterize the putative transport properties of CAX4 and N-terminal variants of CAX4. CAX4 variants can suppress the Ca2+ hypersensitive yeast phenotypes and also appear to be more specific Ca2+ transporters than sCAX1. We then compared the phenotypes of sCAX1- and CAX4-expressing tomato lines. The sCAX1-expressing tomato lines demonstrate increased vacuolar H(+)/Ca2+ transport, when measured in root tissue, elevated fruit Ca2+ level, and prolonged shelf life but have severe alterations in plant development and morphology, including increased incidence of blossom-end rot. The CAX4-expressing plants demonstrate more modest increases in Ca2+ levels and shelf life but no deleterious effects on plant growth. These findings suggest that CAX expression may fortify plants with Ca2+ and may serve as an alternative to the application of CaCl2 used to extend the shelf life of numerous agriculturally important commodities. However, judicious regulation of CAX transport is required to assure optimal plant growth.