Metal homeostasis is critical for plant growth, development and adaptation to environmental stresses and largely governed by a variety of metal transporters. The plant ZIP (Zn-regulated transporter, Iron-regulated transporter-like Protein) family proteins belong to the integral membrane transporters responsible for uptake and allocation of essential and non-essential metals. However, whether the ZIP family members mediate metal efflux and its regulatory mechanism remains unknown. In this report, we provided evidence that OsZIP1 is a metal-detoxified transporter through preventing excess Zn, Cu and Cd accumulation in rice. OsZIP1 is abundantly expressed in roots throughout the life span and sufficiently induced by excess Zn, Cu and Cd but not by Mn and Fe at transcriptional and translational levels. Expression of OsZIP-GFP fusion in rice protoplasts and tobacco leaves shows that OsZIP1 resides in the endoplasmic reticulum (ER) and plasma membrane (PM). The yeast (Saccharomyces cerevisiae) complementation test shows that expression of OsZIP1 reduced Zn accumulation. Transgenic rice overexpressing OsZIP1 grew better under excess metal stress but accumulated less of the metals in plants. In contrast, both oszip1 mutant and RNA interference (RNAi) lines accumulated more metal in roots and contributed to metal sensitive phenotypes. These results suggest OsZIP1 is able to function as a metal exporter in rice when Zn, Cu and Cd are excess in environment. We further identified the DNA methylation of histone H3K9me2 of OsZIP1 and found that OsZIP1 locus, whose transcribed regions imbed a 242 bp sequence, is demethylated, suggesting that epigenetic modification is likely associated with OsZIP1 function under Cd stress. OsZIP1 is a transporter that is required for detoxification of excess Zn, Cu and Cd in rice.

Nitric oxide (NO) as a key signaling molecule has been involved in mediation of various biotic and abiotic stress-induced physiological responses in plants. In the present study, we investigated the effect of NO on Cassia tora L. plants exposed to aluminum (Al). Plants pre-treated for 12 h with 0.4 mM sodium nitroprusside (SNP), an NO donor, and subsequently exposed to 10 µM Al treatment for 24 h exhibited significantly greater root elongation as compared with the plants without SNP treatment. The NO-promoted root elongation was correlated with a decrease in Al accumulation in root apexes. Furthermore, oxidative stress associated with Al treatment increased lipid peroxidation and reactive oxygen species, and the activation of lipoxygenase and antioxidant enzymes was reduced by NO. Such effects were confirmed by the histochemical staining for the detection of peroxidation of lipids and loss of membrane integrity in roots. The ameliorating effect of NO was specific, because the NO scavenger cPTIO [2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylinidazoline-1-oxyl-3-oxide] completely reversed the effect of NO on root growth in the presence of Al. These results indicate that NO plays an important role in protecting the plant against Al-induced oxidative stress.

Background: MicroRNAs (miRNAs) are a class of short, endogenous non-coding small RNAs that have ability to base pair with their target mRNAs to induce their degradation in plants. miR394a/b are conserved small RNAs and its target gene LCR (LEAF CURLING RESPONSIVENESS) encodes an F-box protein (SKP1-Cullin/CDC53-F-box) but whether miR394a/b and its target gene LCR are involved in regulation of plant response to abscisic acid (ABA) and abiotic stresses is unknown. Results: Mature miR394 and precursor miR394a/b are shown to be slightly induced by ABA. By contrast, LCR expression is depressed by ABA. Analysis of LCR and its promoter (pLCR::GUS) revealed that LCR is expressed at all development stages. MIR394a/b over-expression (35S::MIR394a/b) and lcr (LCR loss of function) mutant plants are hypersensitive to salt stress, but LCR over-expressing (35S::m5LCR) plants display the salt-tolerant phenotype. Both 35S::MIR394a/b and lcr plants are highly tolerant to severe drought stress compared with wild-type, but 35S::m5LCR plants are susceptible to water deficiency. Over-expression of MIR394a/b led to ABA hypersensitivity and ABA-associated phenotypes, whereas 35S::m5LCR plants show ABA resistance phenotypes. Moreover, 35S::MIR394a/b plants accumulated higher levels of ABA-induced hydrogen peroxide and superoxide anion radicals than wild-type and 35S::m5LCR plants. Expressions of ABA- and stress-responsive genes, ABI3, ABI4, ABI5, ABF3, and ABF4 are up-regulated in MIR394a/b over-expressing plants but down-regulated in 35S::m5LCR plants. Over-expression of MIR394a in abi4-1 or abi5-1 background resulted in loss of ABA-sensitivity in 35S::MIR394a plants. Conclusions: The silencing of LCR mRNA by miR394 is essential to maintain a certain phenotype favorable for the adaptive response to abiotic stresses. The contrasting phenotypes of salt and drought responses may be mediated by a functional balance between miR394 and LCR. If the balance is perturbed in case of the abiotic stress, an identical phenotype related to the stress response occurs, resulting in either ABA sensitive or insensitive response. Thus, miR394-regulated LCR abundance may allow plants to fine-tune their responses to ABA and abiotic stress.

We adopted a deep sequencing approach developed by Solexa (Illumina Inc., San Diego, CA, USA) to investigate global expression and complexity of microRNAs (miRNAs) and their targets from Medicago truncatula. Two small RNA libraries and two degradome libraries were constructed from mercury (Hg)‐treated and Hg‐free M. truncatula seedlings. For miRNAs, each library generated 18.5–18.6 million short sequences, resulting in 10.2–10.8 million clean reads. At least 52 new miRNA candidates with ∼21 nucleotides are perfectly matched to the M. truncatula genome. Statistical analysis on transcript abundance of the new candidate miRNAs revealed that most of them were differentially regulated by the heavy metal mercury Hg(II), with 12 miRNAs being specifically induced by Hg exposure. Additionally, we identified 201 individual miRNAs representing 63 known M. truncatula miRNA families, including 12 new conserved and one non‐conserved miRNAs that have not been described before. Finally, 130 targets for 58 known (37 conserved and 21 non‐conserved) miRNA families and 37 targets for 18 new M. truncatula‐specific candidate miRNA families were identified by high‐throughput degradome sequencing approach.

Excess copper affects the growth and metabolism of plants and green algae. However, the physiological processes under Cu stress are largely unknown. In this study, we investigated Cu-induced nitric oxide (NO) generation and its relationship to proline synthesis in Chlamydomonas reinhardtii. The test alga accumulated a large amount of proline after exposure to relatively low Cu concentrations (2.5 and 5.0 μM Cu²⁺). A concomitant increase in the intracellular NO level was observed with increasing concentrations of Cu applied. Data analysis revealed that the endogenous NO generated was positively associated with the proline level in Cu-stressed algae. The involvement of NO in Cu-induced proline accumulation was confirmed by using an NO-specific donor, sodium nitroprusside (SNP), and an NO scavenger cPTIO [2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylinidazoline-1-oxyl-3-oxide]. Pre-treatment with 10 μM SNP increased the proline accumulation in Cu-treated cells by about 1.5-fold, while this effect could be blocked by addition of 10 μM cPTIO. We further investigated the effect of Cu and NO on the activity and transcript amount of Δ¹-pyrroline-5-carboxylate synthetase (P5CS, EC 2.7.2.11), the key enzyme of proline biosynthesis, and observed that application of SNP was able to stimulate the P5CS activity and up-regulate the expression of P5CS in the Cu-treated algae. These results indicate that Cu-responsive proline synthesis is closely related to NO generation in C. reinhardtii, suggesting the regulatory function of NO in proline metabolism under heavy metal stress.