An efficient TILLING platform for cultivated tobacco.

Targeting-induced local lesions in genomes (TILLING) is a powerful reverse-genetics tool that enables high-throughput screening of genomic variations in plants. Although TILLING has been developed for many diploid plants, the technology has been used in very few polyploid species due to their genomic complexity. Here, we established an efficient capillary electrophoresis-based TILLING platform for allotetraploid cultivated tobacco (Nicotiana tabacum L.) using an ethyl methanesulfonate (EMS)-mutagenized population of 1,536 individuals. We optimized the procedures for endonuclease preparation, leaf tissue sampling, DNA extraction, normalization, pooling, PCR amplification, heteroduplex formation, and capillary electrophoresis. In a test screen using seven target genes with eight PCR fragments, we obtained 118 mutants. The mutation density was estimated to be approximately one mutation per 106 kb on average. Phenotypic analyses showed that mutations in two heavy metal transporter genes, HMA2S and HMA4T, led to reduced accumulation of cadmium and zinc, which was confirmed independently using CRISPR/Cas9 to generate knockout mutants. Our results demonstrate that this powerful TILLING platform (available at http://www.croptilling.org) can be used in tobacco to facilitate functional genomics applications.

Phytomelatonin receptor PMTR1-mediated signaling regulates stomatal closure in Arabidopsis thaliana.

Melatonin has been detected in plants in 1995; however, the function and signaling pathway of this putative phytohormone are largely undetermined due to a lack of knowledge about its receptor. Here, we discovered the first phytomelatonin receptor (CAND2/PMTR1) in Arabidopsis thaliana and found that melatonin governs the receptor-dependent stomatal closure. The application of melatonin induced stomatal closure through the heterotrimeric G protein α subunit-regulated H2 O2 and Ca2+ signals. The Arabidopsis mutant lines lacking AtCand2 that encodes a candidate G protein-coupled receptor were insensitive to melatonin-induced stomatal closure. Accordingly, the melatonin-induced H2 O2 production and Ca2+ influx were completely abolished in cand2. CAND2 is a membrane protein that interacts with GPA1 and the expression of AtCand2 was tightly regulated by melatonin in various organs and guard cells. CAND2 showed saturable and specific 125 I-melatonin binding, with apparent Kd (dissociation constant) of 0.73 ± 0.10 nmol/L (r2  = .99), demonstrating this protein is a phytomelatonin receptor (PMTR1). Our results suggest that the phytomelatonin regulation of stomatal closure is dependent on its receptor CAND2/PMTR1-mediated H2 O2 and Ca2+ signaling transduction cascade.

C1 metabolism and the Calvin cycle function simultaneously and independently during HCHO metabolism and detoxification in Arabidopsis thaliana treated with HCHO solutions.

Formaldehyde (HCHO) is suggested to be detoxified through one-carbon (C1) metabolism or assimilated by the Calvin cycle in plants. To further understand the function of the Calvin cycle and C1 metabolism in HCHO metabolism in plants, HCHO elimination and metabolism by Arabidopsis thaliana in HCHO solutions was investigated in this study. Results verified that Arabidopsis could completely eliminate aqueous HCHO from the HCHO solutions. Carbon-13 nuclear magnetic resonance ((13)C-NMR) analysis showed that H(13)CHO absorbed by Arabidopsis was first oxidized to H(13)COOH. Subsequently, a clear increase in [U-(13)C]Gluc peaks accompanied by a strong enhancement in peaks of [2-(13)C]Ser and [3-(13)C]Ser appeared in Arabidopsis. Pretreatment with cyclosporin A or L-carnitine, which might inhibit the transport of (13)C-enriched compounds into chloroplasts and mitochondria, caused a remarkable decline in yields of both [U-(13)C]Gluc and [3-(13)C]Ser in H(13)CHO-treated Arabidopsis. These results suggested that both the Calvin cycle and the C1 metabolism functioned simultaneously during HCHO detoxification. Moreover, both functioned more quickly under high H(13)CHO stress than low H(13)CHO stress. When a photorespiration mutant was treated in 6 mm H(13)CHO solution, formation of [U-(13)C]Gluc and [2-(13)C]Ser was completely inhibited, but generation of [3-(13)C]Ser was not significantly affected. This evidence suggested that the Calvin cycle and C1 metabolism functioned independently in Arabidopsis during HCHO metabolism.

[Physiological differences between HPS/PHI over-expressing transgenic and wild-type geraniums under formaldehyde stress revealed by FTIR analysis].

In the present study, FTIR was used to analyze changes in chemical component contents and spectra characters of 3-hexulose-6-phosphate synthase/6-phosphate-3-hexuloisomerase (HPS/PHI) over-expressing transgenic and wild-type (WT) geraniums under formaldehyde (HCHO) stress to examine if FTIR could be a new method for identification of phenotypic differences between the transgenic plants with a photosynthetic HCHO-assimilation pathway and the WT plants. The WT and transgenic geranium plants were treated with 4 mmol x L(-1) HCHO for 0, 1, 2, 3 and 4 days, respectively. The comparison of FTIR spectral characteristics at different time points between the transgenic and WT plants indicated that the contents of carbohydrate, proteins and aliphatic compounds were significantly higher than those in the WT plants after 4 days of HCHO-treatment. This may be due to installation of the photosynthetic HCHO-assimilation pathway in the transgenic geranium, which enhanced its ability to metabolize and assimilate HCHO, thus allowed more HCHO to be fixed to 6-phosphate fructose, and then entered assimilation pathways for synthesis of a variety of intracellular components. The results suggest that FTIR can be a new method to identify the phenotypic differences between transgenic plants with a photosynthetic HCHO-assimilation pathway and WT plants.

[Investigation on the difference in HCHO metabolic mechanism between Arabidopsis and tobacco using FTIR].

In the present study, the model plants, arabidopsis and tobacco, were chosen for FTIR analysis to investigate the spectrum characters and the changes in their chemical component contents in the time course of HCHO treatment, providing clues to explain the difference in HCHO metabolic mechanism between the two plants. The FTIR data showed that all the chemical components of arabidopsis and tobacco varied under HCHO stress conditions. An interested peak near 1,376 cm(-1) which was assigned as the absorption of methyl group of cellulose was specially existed in the spectrum of arabidopsis. This peak showed a mild decrease compared with other peaks at the beginning (at 1 day) of HCHO stress. This indicated that the major part of HCHO metabolic flux was introduced towards its oxidation pathway to form HCOOH and CO2 subsequently and only small amount of HCHO entered the other pathways. The CO2 was assimilated in Calvin cycle to form sugars which might be used to synthesis of cellulose later. At 7 day of HCHO treatment, the height of the peak decreased whereas the height of the other peaks still increased. This might suggest that the gene expression of some enzymes in the HCHO oxidation pathway was inhibited under HCHO stress conditions and the inhibition might not happen to the gene expression of the enzymes in other pathways. In the case of tobacco, the contents of all chemical components showed the same variation on the FTIR spectrum in the time course of HCHO treatment, which indicated that there was no much difference in HCHO metabolism flux in each pathway. At 4 day of HCHO treatment, the decrease in the height of all peaks is the result of the poor ability of HCHO metabolism of tobacco, which also demonstrated the lower HCHO tolerance of tobacco compared with arabidopsis.

[Bacterial ribulose monophosphate pathway and formaldehyde assimilation].

Ribulose monophosphate pathway (RuMP), which was originally found in methylotrophic bacteria, is now recognized as a metabolic pathway widespread in most bacteria and involved in formaldehyde assimilation and detoxification. 3-Hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) are the key enzymes of this pathway. This review describes the physiological significance of RuMP pathway derived from a variety of bacteria, the organizations and expressional regulations of HPS and PHI genes and the perspectives for applications of the two genes.