Calcium‐ and potassium‐permeable plasma membrane transporters are activated by copper in Arabidopsis root tips: linking copper transport with cytosolic hydroxyl radical production

Transition metals such as copper can interact with ascorbate or hydrogen peroxide to form highly reactive hydroxyl radicals (OH^?), with numerous implications to membrane transport activity and cell metabolism. So far, such interaction was described for extracellular (apoplastic) space but not cytosol. Here, a range of advanced electrophysiological and imaging techniques were applied to Arabidopsis thaliana plants differing in their copper‐transport activity: Col‐0, high‐affinity copper transporter COPT1‐overexpressing (C1^OE) seedlings, and T‐DNA COPT1 insertion mutant (copt1). Low Cu concentrations (10 µm ) stimulated a dose‐dependent Gd³⁺ and verapamil sensitive net Ca²⁺ influx in the root apex but not in mature zone. C1^OE also showed a fivefold higher Cu‐induced K⁺ efflux at the root tip level compared with Col‐0, and a reduction in basal peroxide accumulation at the root tip after copper exposure. Copper caused membrane disruptions of the root apex in C1^OE seedlings but not in copt1 plants; this damage was prevented by pretreatment with Gd³⁺. Our results suggest that copper transport into cytosol in root apex results in hydroxyl radical generation at the cytosolic side, with a consequent regulation of plasma membrane OH^?‐sensitive Ca²⁺ and K⁺ transport systems.     

Heteromeric p97/p97R155C Complexes Induce Dominant Negative Changes in Wild-Type and Autophagy 9-Deficient Dictyostelium strains

Heterozygous mutations in the human VCP (p97) gene cause autosomal-dominant IBMPFD (inclusion body myopathy with early onset Paget’s disease of bone and frontotemporal dementia), ALS14 (amyotrophic lateral sclerosis with or without frontotemporal dementia) and HSP (hereditary spastic paraplegia). Most prevalent is the R155C point mutation. We studied the function of p97 in the social amoeba Dictyostelium discoideum and have generated strains that ectopically express wild-type (p97) or mutant p97 (p97^R155C) fused to RFP in AX2 wild-type and autophagy 9 knock-out (ATG9^KO) cells. Native gel electrophoresis showed that both p97 and p97^R155C assemble into hexamers. Co-immunoprecipitation studies revealed that endogenous p97 and p97^R155C-RFP form heteromers. The mutant strains displayed changes in cell growth, phototaxis, development, proteasomal activity, ubiquitinylated proteins, and ATG8(LC3) indicating mis-regulation of multiple essential cellular processes. Additionally, immunofluorescence analysis revealed an increase of protein aggregates in ATG9^KO/p97^R155C-RFP and ATG9^KO cells. They were positive for ubiquitin in both strains, however, solely immunoreactive for p97 in the ATG9^KO mutant. A major finding is that the expression of p97^R155C-RFP in the ATG9^KO strain partially or fully rescued the pleiotropic phenotype. We also observed dose-dependent effects of p97 on several cellular processes. Based on findings in the single versus the double mutants we propose a novel mode of p97 interaction with the core autophagy protein ATG9 which is based on mutual inhibition.     

The overexpression of OsNAC9 alters the root architecture of rice plants enhancing drought resistance and grain yield under field conditions

Drought conditions limit agricultural production by preventing crops from reaching their genetically predetermined maximum yields. Here, we present the results of field evaluations of rice overexpressing OsNAC9, a member of the rice NAC domain family. Root‐specific (RCc3) and constitutive (GOS2) promoters were used to overexpress OsNAC9 and produced the transgenic RCc3:OsNAC9 and GOS2:OsNAC9 plants. Field evaluations over two cultivating seasons showed that grain yields of the RCc3:OsNAC9 and the GOS2:OsNAC9 plants were increased by 13%–18% and 13%–32% under normal conditions, respectively. Under drought conditions, RCc3:OsNAC9 plants showed an increased grain yield of 28%–72%, whilst the GOS2:OsNAC9 plants remained unchanged. Both transgenic lines exhibited altered root architecture involving an enlarged stele and aerenchyma. The aerenchyma of RCc3:OsNAC9 roots was enlarged to a greater extent than those of GOS2:OsNAC9 and non‐transgenic (NT) roots, suggesting the importance of this phenotype for enhanced drought resistance. Microarray experiments identified 40 up‐regulated genes by more than threefold (P < 0.01) in the roots of both transgenic lines. These included 9‐cis‐epoxycarotenoid dioxygenase, an ABA biosynthesis gene, calcium‐transporting ATPase, a component of the Ca²⁺ signalling pathway involved in cortical cell death and aerenchyma formation, cinnamoyl CoA reductase 1, a gene involved in lignin biosynthesis, and wall‐associated kinases¸ genes involved in cell elongation and morphogenesis. Interestingly, O‐methyltransferase, a gene necessary for barrier formation, was specifically up‐regulated only in the RCc3:OsNAC9 roots. Such up‐regulated genes that are commonly and specifically up‐regulated in OsNAC9 transgenic roots may account for the altered root architecture conferring increased drought resistance phenotype.     

Bicarbonate blocks iron translocation from cotyledons inducing iron stress responses in Citrus roots

The effect of bicarbonate ion (HCO₃⁻) on the mobilization of iron (Fe) reserves from cotyledons to roots during early growth of citrus seedlings and its influence on the components of the iron acquisition system were studied. Monoembryonic seeds of Citrus limon (L.) were germinated “in vitro” on two iron-deprived media, supplemented or not with 10 mM HCO₃⁻ (−Fe+Bic and −Fe, respectively). After 21 d of culture, Fe concentration in seedling organs was measured, as well as gene expression and enzymatic activities. Finally, the effect of Fe resupply on the above responses was tested in the presence and absence of HCO₃⁻ (+Fe+Bic or +Fe, respectively). −Fe+Bic seedlings exhibited lower Fe concentration in shoots and roots than −Fe ones but higher in cotyledons, associated to a significative inhibition of NRAMP3 expression. HCO₃⁻ upregulated Strategy I related genes (FRO1, FRO2, HA1 and IRT1) and FC-R and H⁺-ATPase activities in roots of Fe-starved seedlings. PEPC1 expression and PEPCase activity were also increased. When −Fe+Bic pre-treated seedlings were transferred to Fe-containing media for 15 d, Fe content in shoots and roots increased, although to a lower extent in the +Fe+Bic medium. Consequently, the above-described root responses became markedly repressed, however, this effect was less pronounced in +Fe+Bic seedlings. In conclusion, it appears that HCO₃⁻ prevents Fe translocation from cotyledons to shoot and root, therefore reducing their Fe levels. This triggers Fe-stress responses in the root, enhancing the expression of genes related with Fe uptake and the corresponding enzymatic activities.     

Subcellular localization of rice histone deacetylases in organelles

Histone deacetylases (HDACs) are known to function in the nucleus. Here, we report on the organellar localization of three rice HDACs, OsSIR2b, OsHDAC6, and OsHDAC10. The 35S:OsSIR2b-GFP and 35S:OsHDAC10-GFP constructs were introduced into tobacco BY2 cells. Co-localization analysis of the green fluorescent protein and MitoTracker fluorescent signals in the transformed BY2 cells indicated that OsSIR2b and OsHDAC10 are localized in the mitochondria. Transgenic Arabidopsis lines harboring 35S:OsHDAC6-GFP and 35S:OsHDAC10-GFP constructs were similarly analyzed, revealing that OsHDAC6-GFP is localized exclusively in chloroplasts, whereas OsHDAC10-GFP is localized in both mitochondria and chloroplasts. The presence of OsHDAC6-GFP and OsHDAC10-GFP in chloroplasts was verified by immunodetection.     

Cadmium effect on vacuolar H+-ATPase gene expression in the roots of barley seedlings of different age

Expression of two genes (HvVHA E and HvVHA c) of vacuolar H⁺-ATPase was studied in the cells of barley (Hordeum vulgare L., cv. Zazerskii 85) roots in seedlings of different age in the presence of cadmium (100 μM). Three-day-old seedlings were kept for four days on cadmium solution, which caused suppression of root growth accompanied by an increase in HvVHA E gene expression. In this case, the content of reduced glutathione (GSH) decreased. When seven-day-old seedlings were placed on cadmium solution and kept there for four days, the content of metal in the root was higher, but its growth was not suppressed and expression of both studied genes markedly increased. The content of GSH also apparently increased, ensuring the maintenance of high enzyme activity. A comparison of gene expression encoding two subunits of vacuolar H⁺-ATPase and cadmium resistance of the seedlings of different age showed that the enzyme participates in the mechanism of the improvement of barley tolerance to this metal.     

Effects of potassium deficiency on root morphology,ultrastructure and antioxidant enzyme system in sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis> [L.] Lam.) during early growth

Potassium (K⁺) deficiency is an important abiotic stress which has severe influence on the growth and development of sweet potato. To investigate the difference on root morphology, ultrastructure and antioxidant enzyme system at early growth stage, two representative sweet potato cultivars (Ipomoea batatas [L.] Lam.) with different K⁺ deficiency tolerance capacities were hydroponically cultivated under normal K⁺ (control) and K⁺ deficiency (?K) treatments. The results showed that after 14 days of treatment, the root length, surface area, volume, and average diameter of Ningzishu 1 (sensitive to K⁺ deficiency) were significantly decreased under ?K treatment, by comparison, those corresponding decreases of Xushu 32 (tolerant to K⁺ deficiency) were lower and not significant (except for root length). In addition, the proportion of fine roots (diameter <0.5 mm) and thick root (d > 1.0 mm) of Xushu 32 seedlings increased significantly under condition of K⁺ deficiency. Through transmission electron microscopy observations, it can be found that Xushu 32 seedlings showed a more complete root cellular structure and slighter oxidative damage than Ningzishu 1 under ?K treatment. Moreover, the hydrogen peroxide content and malondialdehyde content in the root of Xushu 32 seedlings was significantly lower than that of Ningzishu 1, and the variations of superoxide dismutase, peroxidase, and catalase activities were pronouncedly less than those of Ningzishu 1. These differences between the two cultivars indicated that the stronger root morphology and nutrients absorption ability, and the better root cellular structure and physiological role of Xushu 32 seedlings could alleviate the damage of K⁺ deficiency stress. Thus, it might be a potential mechanism for cultivar tolerance to low K⁺ in sweet potato.