TOND1 confers tolerance to nitrogen deficiency in rice

Nitrogen (N), the most important mineral nutrient for plants, is critical to agricultural production systems. N deficiency severely affects rice growth and decreases rice yields. However, excessive use of N fertilizer has caused severe pollution to agricultural and ecological environments. The necessity of breeding of crops that require lower input of N fertilizer has been recognized. Here we identified a major quantitative trait locus on chromosome 12, Tolerance Of Nitrogen Deficiency 1 (TOND1), that confers tolerance to N deficiency in the indica cultivar Teqing. Sequence verification of 75 indica and 75 japonica cultivars from 18 countries and regions demonstrated that only 27.3% of cultivars (41 indica cultivars) contain TOND1, whereas 72.7% of cultivars, including the remaining 34 indica cultivars and all 75 japonica cultivars, do not harbor the TOND1 allele. Over‐expression of TOND1 increased the tolerance to N deficiency in the TOND1‐deficient rice cultivars. The identification of TOND1 provides a molecular basis for breeding rice varieties with improved grain yield despite decreased input of N fertilizers.     

Long noncoding RNA AK088388 regulates autophagy through miR-30a to affect cardiomyocyte injury

Finding ways to reduce myocardial ischemia/reperfusion injury in the process of myocardial infarction has been an area of intense study in the field of heart disease. Recent studies have shown that long noncoding RNA (lncRNA) and autophagy play important roles in cardiovascular diseases. In our study, software analysis and dual-luciferase reporter assays have shown that miR-30a has binding sites on both AK088388 and Beclin-1. Continuing experiments found that miR-30a expression is downregulated, while the expressions of AK088388, Beclin-1, and LC3-II are upregulated in hypoxia/reoxygenation (H/R) cardiomyocytes; miR-30a inhibits the expression of AK088388, Beclin-1, and LC3-II in H/R cardiomyocytes, while AK088388 promotes the expression of Beclin-1 and LC3-II and inhibits miR-30a expression. AK088388 small interfering RNA and miR-30a mimics can promote the viability of H/R cardiomyocytes, reduce lactate dehydrogenase release, and reduce apoptosis. Mutations of the miR-30a binding site in AK088388 could not block the effects of miR-30a mentioned above. Therefore, AK088388 can competitively bind to miR-30a, promoting the expression of Beclin-1 and LC3-II, autophagy, and eventually cell damage. This finding provides new evidence for understanding the role of lncRNA in myocardial ischemia/reperfusion injury.     

miR‐18a promotes Mycobacterial survival in macrophages via inhibiting autophagy by down‐regulation of ATM

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of leading causes of global deaths. This study aimed to explore the role of miR‐18a in RAW264.7 cells response to Mtb infection. Exosomes derived from Mtb‐infected cells were isolated and further validated by size, transmission electron microscopy and Western blot. RT‐PCR was utilized to measure miR‐18a expression. Cell viability and ultrastructure were examined by CFU counting, CCK‐8 and electron microscope, respectively. Potential target genes of miR‐18a were predicted with bioinformatics and further confirmed using RT‐PCR, Western blot and laser confocal microscope analysis, respectively. LC3, AMPK and mTOR were measured using Western blot. We found that miR‐18a was induced both in Mtb‐infected RAW264.7 cells and its derived exosomes compared with the controls. In addition, up‐regulation of miR‐18a promoted intracellular Mtb survival, attenuated cell viability and reduced LC3‐II level, while its down‐regulation had the opposite effect. miR‐18a overexpression suppressed level of ATM, one possible target of miR‐18a, while its underexpression enhanced ATM. We also found that inhibition of ATM induced LC3‐II decrease in Mtb‐infected cells and could reverse the increase of LC3‐II caused by inhibition of miR‐18a. Moreover, down‐regulation of miR‐18a increased p‐AMPK level while reduction of ATM could reverse the change. Taken together, our results suggest that miR‐18a is up‐regulated in macrophages response to Mtb infection, and it promotes intracellular Mtb survival through repressing autophagic process by down‐regulation of ATM pathway. This provides new thought for TB pathogenesis, diagnosis and treatment.     

Overexpression of GmAAP6a enhances tolerance to low nitrogen and improves seed nitrogen status by optimizing amino acid partitioning in soybean

Amino acid transport via phloem is one of the major source‐to‐sink nitrogen translocation pathways in most plant species. Amino acid permeases (AAPs) play essential roles in amino acid transport between plant cells and subsequent phloem or seed loading. In this study, a soybean AAP gene, annotated as GmAAP6a, was cloned and demonstrated to be significantly induced by nitrogen starvation. Histochemical staining of GmAAP6a:GmAAP6a‐GUS transgenic soybean revealed that GmAAP6a is predominantly expressed in phloem and xylem parenchyma cells. Growth and transport studies using toxic amino acid analogs or single amino acids as a sole nitrogen source suggest that GmAAP6a can selectively absorb and transport neutral and acidic amino acids. Overexpression of GmAAP6a in Arabidopsis and soybean resulted in elevated tolerance to nitrogen limitation. Furthermore, the source‐to‐sink transfer of amino acids in the transgenic soybean was markedly improved under low nitrogen conditions. At the vegetative stage, GmAAP6a‐overexpressing soybean showed significantly increased nitrogen export from source cotyledons and simultaneously enhanced nitrogen import into sink primary leaves. At the reproductive stage, nitrogen import into seeds was greatly enhanced under both sufficient and limited nitrogen conditions. Collectively, our results imply that overexpression of GmAAP6a enhances nitrogen stress tolerance and source‐to‐sink transport and improves seed quality in soybean. Co‐expression of GmAAP6a with genes specialized in source nitrogen recycling and seed loading may represent an interesting application potential in breeding.     

The Arabidopsis tt19-4 mutant differentially accumulates proanthocyanidin and anthocyanin through a 3' amino acid substitution in glutathione S-transferase

The Arabidopsis transparent testa (tt) mutant tt19-4 shows reduced seed coat colour, but stains darkly with DMACA and accumulates anthocyanins in aerial tissues. Positional cloning showed that tt19-4 was allelic to tt19-1 and has a G-to-T mutation in a conserved 3'-domain in the TT19-4 gene. Soluble and unextractable seed proanthocyanidins and hydrolysis of unextractable proanthocyanidin differ between wild-type Col-4 and both mutants. However, seed quercetins, unextractable proanthocyanidin hydrolysis, and seedling anthocyanin content, and flavonoid gene expression differ between tt19-1 and tt19-4. Transformation of tt19-1 with a TT19-4 cDNA results in vegetative anthocyanins, whereas TT19-4 cDNA cannot complement the proanthocyanidin and pale seed coat phenotype of tt19-1. Both recombinant TT19 and TT19-4 enzymes are functional GSTs and are localized in the cytosol, but TT19 did not function with wide range of flavonoids and natural products to produce conjugation products. We suggest that the dark seed coat of Arabidopsis is related to soluble proanthocyanidin content and that quercetin holds the key to the function of TT19. In addition, TT19 appears to have a 5' GSH-binding domain influencing both anthocyanin and proanthocyanidin accumulation and a 3' domain affecting proanthocyanidin accumulation by a single amino acid substitution.     

Influence of spermine and nitrogen deficiency on growth and secondary metabolites accumulation in Castilleja tenuiflora Benth. cultured in a RITA® temporary immersion system

The effect of exogenous spermine (SPM) on Castilleja tenuiflora shoots developing under nitrogen deficiency (ND) stress was evaluated. Shoots cultivated in a temporary immersion system were subjected to four experimental treatments: (1) control; (2) exogenous SPM; (3) ND; and (4) ND+SPM. Shoots were longer in the ND+SPM treatment (6.3 ± 0.5 cm) than in the ND treatment (4.2 ± 0.5 cm). The total chlorophyll content was similar in the control and SPM treatments (0.41 µg mg^?1 FM) and the highest values of total phenolic content were detected at 21 days in the ND+SPM treatment (84.1 ± 0.05 GAE g^?1 DM). In the ND+SPM treatment, the phenylalanine ammonia lyase activity increased earlier than in ND treatment, and reached its maximum at day 21 (3.9 ± 0.2 µmol E‐CIN h^?1 mg^?1 protein). Compared with the control, the ND and ND+SPM treatments resulted in increased secondary metabolites contents in both root and aerial parts. The strongest effect was in the roots, where the SPM and ND+SPM treatments both resulted in increased quercetin content (4.3‐fold that in the control). Our results showed that SPM partially counteract the damage caused by ND and results in increased contents of valuable bioactive compounds.