Overexpression of microRNA319 impacts leaf morphogenesis and leads to enhanced cold tolerance in rice (Oryza sativa L.)

MicroRNA319 (miR319) family is one of the conserved microRNA (miRNA) families among diverse plant species. It has been reported that miR319 regulates plant development in dicotyledons, but little is known at present about its functions in monocotyledons. In rice (Oryza sativa L.), the MIR319 gene family comprises two members, Osa‐MIR319a and Osa‐MIR319b. Here, we report an expression pattern analysis and a functional characterization of the two Osa‐MIR319 genes in rice. We found that overexpressing Osa‐MIR319a and Osa‐MIR319b in rice both resulted in wider leaf blades. Leaves of osa‐miR319 overexpression transgenic plants showed an increased number of longitudinal small veins, which probably accounted for the increased leaf blade width. In addition, we observed that overexpressing osa‐miR319 led to enhanced cold tolerance (4 °C) after chilling acclimation (12 °C) in transgenic rice seedlings. Notably, under both 4 and 12 °C low temperatures, Osa‐MIR319a and Osa‐MIR319b were down‐regulated while the expression of miR319‐targeted genes was induced. Furthermore, genetically down‐regulating the expression of either of the two miR319‐targeted genes, OsPCF5 and OsPCF8, in RNA interference (RNAi) plants also resulted in enhanced cold tolerance after chilling acclimation. Our findings in this study demonstrate that miR319 plays important roles in leaf morphogenesis and cold tolerance in rice.     

Silicon‐mediated changes in polyamines participate in silicon‐induced salt tolerance in Sorghum bicolor L.

Silicon (Si) is generally considered a beneficial element for the growth of higher plants, especially under stress conditions, but the mechanisms remain unclear. Here, we tested the hypothesis that Si improves salt tolerance through mediating important metabolism processes rather than acting as a mere mechanical barrier. Seedlings of sorghum (Sorghum bicolor L.) growing in hydroponic culture were treated with NaCl (100 mm ) combined with or without Si (0.83 mm ). The result showed that supplemental Si enhanced sorghum salt tolerance by decreasing Na⁺ accumulation. Simultaneously, polyamine (PA) levels were increased and ethylene precursor (1‐aminocyclopropane‐1‐carboxylic acid: ACC) concentrations were decreased. Several key PA synthesis genes were up‐regulated by Si under salt stress. To further confirm the role of PA in Si‐mediated salt tolerance, seedlings were exposed to spermidine (Spd) or a PA synthesis inhibitor (dicyclohexylammonium sulphate, DCHA) combined with salt and Si. Exogenous Spd showed similar effects as Si under salt stress whereas exogenous DCHA eliminated Si‐enhanced salt tolerance and the beneficial effect of Si in decreasing Na⁺ accumulation. These results indicate that PAs and ACC are involved in Si‐induced salt tolerance in sorghum and provide evidence that Si plays an active role in mediating salt tolerance.     

Transgenic creeping bentgrass overexpressing Osa‐miR393a exhibits altered plant development and improved multiple stress tolerance

MicroRNA393 (miR393) has been implicated in plant growth, development and multiple stress responses in annual species such as Arabidopsis and rice. However, the role of miR393 in perennial grasses remains unexplored. Creeping bentgrass (Agrostis stolonifera L.) is an environmentally and economically important C3 cool‐season perennial turfgrass. Understanding how miR393 functions in this representative turf species would allow the development of novel strategies in genetically engineering grass species for improved abiotic stress tolerance. We have generated and characterized transgenic creeping bentgrass plants overexpressing rice pri‐miR393a (Osa‐miR393a). We found that Osa‐miR393a transgenics had fewer, but longer tillers, enhanced drought stress tolerance associated with reduced stomata density and denser cuticles, improved salt stress tolerance associated with increased uptake of potassium and enhanced heat stress tolerance associated with induced expression of small heat‐shock protein in comparison with wild‐type controls. We also identified two targets of miR393, AsAFB2 and AsTIR1, whose expression is repressed in transgenics. Taken together, our results revealed the distinctive roles of miR393/target module in plant development and stress responses between creeping bentgrass and other annual species, suggesting that miR393 would be a promising candidate for generating superior crop cultivars with enhanced multiple stress tolerance, thus contributing to agricultural productivity.     

Regulation of miR319 during cold stress in sugarcane

MicroRNAs (miRNAs) are part of a novel mechanism of gene regulation that is active in plants under abiotic stress conditions. In the present study, 12 miRNAs were analysed to identify miRNAs differentially expressed in sugarcane subjected to cold stress (4 °C). The expression of miRNAs assayed by stem–loop RT‐PCR showed that miR319 is up‐regulated in sugarcane plantlets exposed to 4 °C for 24 h. The induction of miR319 expression during cold stress was observed in both roots and shoots. Sugarcane miR319 was also regulated by treatment with abscisic acid. Putative targets of this miRNA were identified and their expression levels were decreased in sugarcane plantlets exposed to cold. The cleavage sites of two targets were mapped using a 5′ RACE PCR assay confirming the regulation of these genes by miR319. When sugarcane cultivars contrasting in cold tolerance were subjected to 4 °C, we observed up‐regulation of miR319 and down‐regulation of the targets in both varieties; however, the changes in expression were delayed in the cold‐tolerant cultivar. These results suggest that differences in timing and levels of the expression of miR319 and its targets could be tested as markers for selection of cold‐tolerant sugarcane cultivars.     

Ectopic expression of wheat expansin gene TaEXPA2 improved the salt tolerance of transgenic tobacco by regulating Na+/K+ and antioxidant competence

High salinity is one of the most serious environmental stresses that limit crop growth. Expansins are cell wall proteins that regulate plant development and abiotic stress tolerance by mediating cell wall expansion. We studied the function of a wheat expansin gene, TaEXPA2, in salt stress tolerance by overexpressing it in tobacco. Overexpression of TaEXPA2 enhanced the salt stress tolerance of transgenic tobacco plants as indicated by the presence of higher germination rates, longer root length, more lateral roots, higher survival rates and more green leaves under salt stress than in the wild type (WT). Further, when leaf disks of WT plants were incubated in cell wall protein extracts from the transgenic tobacco plants, their chlorophyll content was higher under salt stress, and this improvement from TaEXPA2 overexpression in transgenic tobacco was inhibited by TaEXPA2 protein antibody. The water status of transgenic tobacco plants was improved, perhaps by the accumulation of osmolytes such as proline and soluble sugar. TaEXPA2‐overexpressing tobacco lines exhibited lower Na⁺ but higher K⁺ accumulation than WT plants. Antioxidant competence increased in the transgenic plants because of the increased activity of antioxidant enzymes. TaEXPA2 protein abundance in wheat was induced by NaCl, and ABA signaling was involved. Gene expression regulation was involved in the enhanced salt stress tolerance of the TaEXPA2 transgenic plants. Our results suggest that TaEXPA2 overexpression confers salt stress tolerance on the transgenic plants, and this is associated with improved water status, Na⁺/K⁺ homeostasis, and antioxidant competence. ABA signaling participates in TaEXPA2‐regulated salt stress tolerance.     

Solar UV‐B radiation and ethylene play a key role in modulating effective defenses against Anticarsia gemmatalis larvae in field‐grown soybean

Solar UV‐B radiation has been reported to enhance plant defenses against herbivore insects in many species. However, the mechanism and traits involved in the UV‐B mediated increment of plant resistance are unknown in crops species, such as soybean. Here, we studied defense‐related responses in undamaged and Anticarsia gemmatalis larvae‐damaged leaves of two soybean cultivars grown under attenuated or full solar UV‐B radiation. We determined changes in jasmonates, ethylene (ET), salicylic acid, trypsin protease inhibitor activity, flavonoids, and mRNA expression of genes related with defenses. ET emission induced by Anticarsia gemmatalis damage was synergistically increased in plants grown under solar UV‐B radiation and was positively correlated with malonyl genistin concentration, trypsin proteinase inhibitor activity and expression of IFS2, and the pathogenesis protein PR2, while was negatively correlated with leaf consumption. The precursor of ET, aminocyclopropane‐carboxylic acid, applied exogenously to soybean was sufficient to strongly induce leaf isoflavonoids. Our results showed that in field‐grown soybean isoflavonoids were regulated by both herbivory and solar UV‐B inducible ET, whereas flavonols were regulated by solar UV‐B radiation only and not by herbivory or ET. Our study suggests that, although ET can modulate UV‐B‐mediated priming of inducible plant defenses, some plant defenses, such as isoflavonoids, are regulated by ET alone.     

Activation of ethylene‐related genes in response to aphid feeding on resistant and susceptible melon and tomato plants

The simple gaseous compound ethylene (ET) has long been recognized as a common component of plant responses to insect feeding and pathogen attack. However, it is presently uncertain whether it plays a role in host–plant resistance to piercing–sucking insects such as aphids. In these experiments, we investigated the expression of key ET‐associated genes in resistant and susceptible interactions in two model systems: the tomato‐Mi‐Macrosiphum euphorbiae (Thomas) (Hemiptera: Aphididae: Macrosiphini) system and the melon‐virus aphid transmission gene (Vat)‐Aphis gossypii Glover (Hemiptera: Aphididiae: Aphidini) system. We examined expression patterns of genes associated with ET synthesis, perception, signal transduction, and downstream response. When compared with control plants, plants infested with aphids showed marked differences in gene expression. In particular, ET signaling pathway genes and downstream response genes were highly upregulated in the resistant interaction between A. gossypii and Vat⁺, indicating ET may play a role in Vat‐mediated host–plant resistance. A key integrator between the ET and jasmonic acid pathways (Cm‐ERF1) showed the strongest response.     

The miR156‐SPL9‐DFR pathway coordinates the relationship between development and abiotic stress tolerance in plants

Young organisms have relatively strong resistance to diseases and adverse conditions. When confronted with adversity, the process of development is delayed in plants. This phenomenon is thought to result from the rebalancing of energy, which helps plants to coordinate the relationship between development and stress tolerance; however, the molecular mechanism underlying this phenomenon remains mysterious. In this study, we found that miR156 integrates environmental signals to ensure timely flowering, thus enabling the completion of breeding. Under stress conditions, miR156 is induced to maintain the plant in the juvenile state for a relatively long period of time, whereas under favorable conditions, miR156 is suppressed to accelerate the developmental transition. Blocking the miR156 signaling pathway in Arabidopsis thaliana with 35S::MIM156 (via target mimicry) increased the sensitivity of the plant to stress treatment, whereas overexpression of miR156 increased stress tolerance. In fact, this mechanism is also conserved in Oryza sativa (rice). We also identified downstream genes of miR156, i.e. SQUAMOSA PROMOTER BINDING PROTEIN‐LIKE 9 (SPL9) and DIHYDROFLAVONOL‐4‐REDUCTASE (DFR), which take part in this process by influencing the metabolism of anthocyanin. Our results uncover a molecular mechanism for plant adaptation to the environment through the miR156‐SPLs‐DFR pathway, which coordinates development and abiotic stress tolerance.