Pan-Genome Analyses of the Genus Cohnella and Proposal of the Novel Species Cohnella silvisoli sp. nov., Isolated from Forest Soil.

Two strains, designated NL03-T5T and NL03-T5-1, were isolated from a soil sample collected from the Nanling National Forests, Guangdong Province, PR China. The two strains were Gram-stain-positive, aerobic, rod-shaped and had lophotrichous flagellation. Strain NL03-T5T could secrete extracellular mucus whereas NL03-T5-1 could not. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the two strains belong to the genus Cohnella, were most closely related to Cohnella lupini LMG 27416T (95.9% and 96.1% similarities), and both showed 94.0% similarity with Cohnella arctica NRRL B-59459T, respectively. The two strains showed 99.8% 16S rRNA gene sequence similarity between them. The draft genome size of strain NL03-T5T was 7.44 Mbp with a DNA G+C content of 49.2 mol%. The average nucleotide identities (ANI) and the digital DNA-DNA hybridization (dDDH) values between NL03-T5T and NL03-T5-1 were 99.98% and 100%, indicating the two strains were of the same species. Additionally, the ANI and dDDH values between NL03-T5T and C. lupini LMG 27416T were 76.1% and 20.4%, respectively. The major cellular fatty acids of strain NL03-T5T included anteiso-C15:0 and iso-C16:0. The major polar lipids and predominant respiratory quinone were diphosphatidylglycerol (DPG) and menaquinone-7 (MK-7). Based on phylogenetic analysis, phenotypic and chemotaxonomic characterization, genomic DNA G+C content, and ANI and dDDH values, strains NL03-T5T and NL03-T5-1 represent novel species in the genus Cohnella, for which the name Cohnella silvisoli is proposed. The type strain is NL03-T5T (=GDMCC 1.2294T = JCM 34999T). Furthermore, comparative genomics revealed that the genus Cohnella had an open pan-genome. The pan-genome of 29 Cohnella strains contained 41,356 gene families, and the number of strain-specific genes ranged from 6 to 1649. The results may explain the good adaptability of the Cohnella strains to different habitats at the genetic level.

Transcription Factor OsbZIP60-like Regulating OsP5CS1 Gene and 2-Acetyl-1-pyrroline (2-AP) Biosynthesis in Aromatic Rice.

The most important volatile in determining the aroma of fragrant rice is 2-Acetyl-1-pyrroline (2-AP); however, the transcriptional regulation mechanism of 2-AP biosynthesis in fragrant rice is still unclear. In this study, Osp5cs1 knockout mutant lines and OsP5CS1 over-expression lines were constructed by the genetic transformation of the Indica rice cultivar, i.e., 'Zhonghua11', which knocks out OsBADH2 to produce fragrance in aromatic rice. The OsP5CS1 gene was also identified as a key gene in the 2-AP biosynthesis pathway of aromatic rice. The OsP5CS1 promoter was used as bait, and the OsbZIP60-like transcription factor was screened by yeast one-hybrid assays. The OsbZIP60-like transcription factor specifically bound to the OsP5CS1 gene. The dual luciferase reporting system found that the OsbZIP60-like transcription factor promoted the transcriptional activation of OsP5CS1. Compared with the wild type, OsP5CS1 gene expression was significantly down-regulated in the Osbzip60-like mutant and resulted in a substantial reduction in 2-AP biosynthesis. Moreover, the OsP5CS1 gene expression was significantly up-regulated in OsbZIP60-like over-expressed plants, and the 2-AP concentrations were also increased, whereas the Osbzip60-like mutants were found to be sensitive to Zn deficiency. Overall, the OsbZIP60-like transcription factor promoted the 2-AP accumulation. This study provides a theoretical basis for the transcriptional regulation mechanism of 2-AP biosynthesis and explores the function of the OsbZIP transcription factor in fragrant rice.

Insights of Improved Aroma under Additional Nitrogen Application at Booting Stage in Fragrant Rice.

Plant mineral nutrition substantially affects the growth, yield and quality of rice, whereas nitrogen (N) application contributes significantly in this regard. Undoubtedly, N application improves rice aroma biosynthesis; however, the molecular mechanism underlying the regulation of grain 2-acetyl-1-pyrroline (2-AP) biosynthesis in the presence of nitrogen application at the booting stage has remained largely unexplored. The present study examined the effects of three N levels, i.e., 0 g per pot (N0), 0.43 g per pot (N1) and 0.86 g per pot (N2) on intermediates, enzymes and genes involved in 2-AP biosynthesis, as well as on the yield of two fragrant rice cultivars viz, Meixiangzhan2 and Xiangyaxiangzhan. N was additionally applied at the booting stage. The results depicted that the levels of precursor, such as proline, and the activity of enzymes involved in 2-AP biosynthesis, such as Δ1-pyrroline-5-carboxylate synthetase (P5CS) and diamine oxidase (DAO), and P5CS1 gene expression were comparatively higher under N1 than N0 in both fragrant rice cultivars. Moreover, the N2 treatment increased the grain panicle-1, filled grain percentage and grain yield of both rice cultivars, while the grain yield of Meixiangzhan2 and Xiangyaxiangzhan was increased by 15.87% and 12.09%, respectively, under N2 compared to N1 treatment. Hence, 0.43 g per pot of N showed positive performances in yield and aroma accumulation in fragrant rice and should be further employed in the practice and production for better cultivation in the rice market.

Transcriptomic Analysis Provides Insights into the Differential Effects of Aluminum on Peanut (Arachis hypogaea L.).

In acidic soils, high concentrations of aluminum ions (Al3+) in dissolved form reduce root growth and development of most crops. In addition, Al3+ is also a beneficial element in some plant species in low concentrations. However, the regulatory mechanism of the growth and development of peanut (Arachis hypogaea L.) treated with different concentrations of Al3+ has been rarely studied. In this study, peanut seedlings were treated with AlCl3.18H2O in Hoagland nutrient solution at four different concentrations of Al3+, i.e., 0 (pH 6.85), 1.25 (pH 4.03), 2.5 (pH 3.85), and 5 (pH 3.69) mmol/L, which are regarded as Al0, Al1, Al2, and Al3. The results showed that low concentrations of Al treatment (Al1) promoted peanut growth, while high concentrations of Al treatments (Al2 and Al3) significantly inhibited peanut growth. Compared with the control (Al0), transcriptome analysis showed that the differentially expressed genes (DEGs) of starch and sucrose metabolic pathways were significantly enriched at low concentrations, i.e., Al1 treatment, whereas the expression of AhERD6 (sugar transporter) was significantly up-regulated, and the soluble sugar content was significantly increased. The DEGs of the plant hormone signaling transduction pathway were significantly enriched at high concentrations of Al2 and Al3 treatments, whereas the expression of AhNCED1 (9-cis-epoxycarotenoid dioxygenase) was significantly up-regulated, and the content of ABA was significantly increased. Moreover, the expression of transcription factors (TFs) in peanut was affected by different concentrations of Al. Overall, low concentrations of Al1 promoted peanut growth by increasing soluble sugar content, while high concentrations of Al2 and Al3 inhibited the growth of peanut, induced AhNCED1 gene expression, and increased endogenous ABA content. For peanut, the exposure of Al at low concentrations not only derived an adaptive mechanism to cope with Al stress, but also acted as a stimulator to promote its growth and development.

Post-transcriptional regulation of 2-acetyl-1-pyrroline (2-AP) biosynthesis pathway, silicon, and heavy metal transporters in response to Zn in fragrant rice.

Fragrant rice (Oryza sativa L.) has a high economic and nutritional value, and the application of micronutrients regulates 2-acetyl-1-pyrroline (2-AP) production, which is responsible for aroma in fragrant rice. Alternative splicing (AS) is an important post-transcriptional regulatory mechanism to generate transcript variability and proteome diversity in plants. However, no systematic investigation of AS events in response to micronutrients (Zn) has been performed in fragrant rice. Furthermore, the post-transcriptional regulation of genes involved in 2-AP biosynthesis is also not known. In this study, a comprehensive analysis of AS events under two gradients of Zn treatment in two different fragrant rice cultivars (Meixiangzhan-2 and Xiangyaxiangzhan) was performed based on RNA-seq analysis. A total of 386 and 598 significant AS events were found in Meixiangzhan-2 treated with low and high doses of Zn, respectively. In Xiangyaxiangzhan, a total of 449 and 598 significant AS events were found in low and high doses of Zn, respectively. Go analysis indicated that these genes were highly enriched in physiological processes, metabolism, and cellular processes in both cultivars. However, genotype and dose-dependent AS events were also detected in both cultivars. By comparing differential AS (DAS) events with differentially expressed genes (DEGs), we found a weak overlap among DAS and DEGs in both fragrant rice cultivars indicating that only a few genes are post-transcriptionally regulated in response to Zn treatment. We further report that Zn differentially regulates the expression of 2-AP biosynthesis-related genes in both cultivars and Zn treatment altered the editing frequency of single nucleotide polymorphism (SNPs) in the genes involved in 2-AP biosynthesis. Finally, we showed that epigenetic modifications associated with active gene transcription are generally enriched over 2-AP biosynthesis-related genes. Similar to the 2-AP pathway, we found that heavy metal transporters (genes related to silicon, iron, Zn and other metal transport) are also regulated at transcriptional and post-transcriptional levels in response to Zn in fragrant rice. Taken together, our results provide evidence of the post-transcriptional gene regulation in fragrant rice in response to Zn treatment and highlight that the 2-AP biosynthesis pathway and heavy metal transporters may also be regulated through epigenetic modifications. These findings will serve as a cornerstone for further investigation to understand the molecular mechanisms of 2-AP biosynthesis and regulation of heavy metal transporters in fragrant rice.

Transcriptomic Analysis Provides Insights into Foliar Zinc Application Induced Upregulation in 2-Acetyl-1-pyrroline and Related Transcriptional Regulatory Mechanism in Fragrant Rice.

The involvement of zinc (Zn) in terms of aroma formation has been rarely investigated. This study shows that the regulation of 2-acetyl-1-pyrroline (2AP) biosynthesis was evaluated in two different rice cultivars under foliar Zn application. The results showed that the 2AP and Zn contents in leaves and grains were improved substantially under foliar Zn application. The 2AP content was positively related to the expression P5CS2 gene, contents of proline, 1-pyrroline, and Δ1-pyrroline-5-carboxylate (P5C), and the activity of pyrroline-5-carboxylate synthase (P5CS) under Zn application in fragrant rice. Multiple transcription factors (TFs) were differently expressed, such as bZIPs, NACs, and MYBs, to play a role under Zn treatments in fragrant rice, suggesting the crucial role of 46 differently expressed TFs in 2AP improvements in fragrant rice. Furthermore, this study showed that the optimal foliar Zn application at a concentration of 30 mg L-1 could increase the 2AP content of aromatic rice and keep the yield stable or increase the yield. TFs were involved in regulating to promote the 2AP formation in aromatic rice under the foliar Zn application. However, the relationship between 2AP biosynthesis pathway genes and TFs in fragrant rice remains to be further studied.

Transcriptome Analysis Revealed the Mechanisms Involved in Ultrasonic Seed Treatment-Induced Aluminum Tolerance in Peanut.

Ultrasonic (US) treatment is an efficient method to induce crop tolerance against heavy metal toxicity; however, US-induced aluminum (Al) tolerance in peanuts was rarely studied. This study was comprised of two treatments, namely, CK, without ultrasonic treatment, and US, an ultrasonic seed treatment, for 15 min. Both treated and non-treated treatments were applied with Al in the form of AlCl3.18H2O at 5 mmol L-1 in Hoagland solution at one leaf stage. Results depicted that plant height, main root length, and number of lateral roots increased significantly under US treatment. Transcriptome analysis revealed that plant hormone signal transduction and transcription factors (TFs) were significantly enriched in the differentially expressed genes (DEGs) in US treatment, and the plant hormones were measured, including salicylic acid (SA) and abscisic acid (ABA) contents, were substantially increased, while indole acetic acid (IAA) and jasmonic acid (JA) contents were decreased significantly in US treatment. The TFs were verified using quantitative real-time (qRT)-PCR, and it was found that multiple TFs genes were significantly upregulated in US treatment, and ALMT9 and FRDL1 genes were also significantly upregulated in US treatment. Overall, the US treatment induced the regulation of hormone content and regulated gene expression by regulating TFs to improve Al tolerance in peanuts. This study provided a theoretical rationale for US treatment to improve Al tolerance in peanuts.

Rhizobium glycinendophyticum sp. nov., isolated from roots of Glycine max (Linn. Merr.).

A Gram-stain-negative, rod-shaped and aerobic bacterium, designated CL12T, was isolated from roots of Glycine max (Linn. Merr.) collected from an experimental field in the campus of South China Agricultural University, PR China (22°58'46″S, 110°51'10″E). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CL12T belongs to the genus Rhizobium, closely related to Rhizobium wuzhouense W44T (99.3%), followed by Rhizobium rosettiformans W3T (98.0%) and Rhizobium ipomoeae Shin9-1T (97.9%). The results of analysis of sequences of four housekeeping genes (recA, atpD, rpoB and glnA) also revealed strain CL12T to be closely related to R. wuzhouense W44T with the similarities 91.0%, 95.0%, 94.2% and 90.5%, respectively. The major fatty acid of strain CL12T was Summed Feature 8 (C18:1ω7c and/or C18:1ω6c). Strain CL12T had not the nodulation genes (nodC and nodA) and nitrogenase reductase gene (nifH), and could not cause formation of nodule on soybean. The draft genome size of strain CL12T was 4.84 Mbp with a genomic DNA G + C content of 61.1 mol%. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) of strain CL12T and R. wuzhouense W44T were 27.4% and 84.7%, respectively. Based on genomic, phenotypic and phylogenetic analysis, strain CL12T is suggested to represent a new species of the genus Rhizobium, for which the name Rhizobium glycinendophyticum sp. nov. is proposed. The type strain is CL12T (=GDMCC 1.1597T = KACC 21281T).

Deminuibacter soli gen. nov., sp. nov., isolated from forest soil, and reclassification of Filimonas aurantiibacter as Arvibacter aurantiibacter comb. nov.

A novel strain, designated K23C18032701T, was isolated from a sample of forest soil collected from Dinghushan Biosphere Reserve, Guangdong Province, PR China. The strain was Gram-stain-negative, aerobic, motile and showed a shape change from a filamentous cell to coccobacilli. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the novel strain belongs to the family Chitinophagaceae, and showed the highest similarities to Arvibacter flaviflagrans JCM 31293T (95.0 %) and Filimonas aurantiibacter LMG 29039T (94.4 %). The major cellular fatty acids included iso-C15 : 0, iso-C17 : 0 3-OH and iso-C15 : 1 G. The predominant polar lipid was phosphatidylethanolamine (PE). The predominant respiratory quinone was menaquinone-7. The major polyamine was sym-homospermidine. The draft genome size of strain K23C18032701T was 5.84 Mb with a DNA G+C content of 47.2 mol%. Based on phenotypic, genotypic and phylogenetic analysis, strain K23C18032701T represents a novel species of a new genus in the family Chitinophagaceae, for which the name Deminuibacter soli is proposed. The type strain is K23C18032701T (=GDMCC 1.1403T=KCTC 62913T). We also propose the reclassification of Filimonas aurantiibacter as Arvibacter aurantiibacter comb. nov. (type strain 1458T=NRRL B-65305T=LMG 29039T).

Chitinophaga silvisoli sp. nov., isolated from forest soil.

A Gram-stain-negative, rod-shaped and aerobic bacterium, designated K20C18050901T, was isolated from forest soil collected on 11 September 2017 from Dinghushan Biosphere Reserve, Guangdong Province, PR China (23° 10' 24'' N; 112° 32' 10'' E). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain K20C18050901T belongs to the genus Chitinophaga, and showed the highest similarities to Chitinophaga sancti NBRC 15057T (98.6 %) and Chitinophaga oryziterrae JCM 16595T (96.9 %). The major fatty acids (>10 %) were iso-C15 : 0, C16 : 1ω5c, summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c) and iso-C17 : 0 3-OH. The predominant respiratory quinone was menaquinone-7. The major polar lipid was phosphatidylethanolamine. The draft genome size of strain K20C18050901T was 8.36 Mb with a DNA G+C content of 44.7 mol%. The digital DNA-DNA hybridization and average nucleotide identity values between strain K20C18050901T and C. sancti NBRC 15057T were 31.40 and 85.82 %, respectively. On the basis of phenotypic, genotypic and phylogenetic analysis, strain K20C18050901T represents a novel species of the genus Chitinophaga, for which the name Chitinophagasilvisoli sp. nov. is proposed. The type strain is K20C18050901T (=GDMCC 1.1411T=KCTC 62860T).

Molecular basis for increased 2-acetyl-1-pyrroline contents under alternate wetting and drying (AWD) conditions in fragrant rice.

Factors affecting rice aroma biosynthesis have been well documented previously, however the molecular mechanism lies behind the regulations in grain 2-acetyl-1-pyrroline (2AP) biosynthesis under alternate wetting and drying (AWD) remained largely unexplored. Present study investigated the effects of three irrigation regimes i.e., conventional irrigation (CI), alternate wetting and moderate drying (WMD), and alternate wetting and severe drying (WSD) on the yield, quality traits, intermediates, enzyme activities and genes involved in 2-acetyl-1-pyrroline biosynthesis in two fragrant rice cultivars viz, Meixiangzhan2 and Xiangyaxiangzhan. Results revealed that the levels of intermediates such as Δ1-pyrroline-5-carboxylate (P5C) and Δ1-pyrroline, and the activity of enzymes such as proline dehydrogenase (PRODH), Δ1-pyrroline-5-carboxylate synthetase (P5CS), diamine oxidase (DAO), and gene expressions of PRODH, P5CS2 and DAO were comparatively higher under AWD than CI in both aromatic rice cultivars. The levels of gamma-aminobutyric acid (GABA), betaine aldehyde dehydrogenase 2 (BADH2) and BADH2 gene were lower that together led to enhanced 2-AP contents in rice grains. Moreover, WMD irrigation improved yield and yield characters, while WSD irrigation reduced yield and quality traits of rice. Overall, up-regulation of P5C and Δ1-pyrroline and down-regulation of GABA under AWD treatments resulted in enhanced 2AP biosynthesis in both rice cultivars. Evaluation and adoption of AWD (within safe limits) at field level could be an alternative option to conventional flooded rice to get better yield and quality.

Co-expression of NCED and ALO improves vitamin C level and tolerance to drought and chilling in transgenic tobacco and stylo plants.

Abscisic acid (ABA) regulates plant adaptive responses to various environmental stresses, while L-ascorbic acid (AsA) that is also named vitamin C is an important antioxidant and involves in plant stress tolerance and the immune system in domestic animals. Transgenic tobacco (Nicotiana tabacum L.) and stylo [Stylosanthes guianensis (Aublet) Swartz], a forage legume, plants co-expressing stylo 9-cis-epoxycarotenoid dioxygenase (SgNCED1) and yeast D-arabinono-1,4-lactone oxidase (ALO) genes were generated in this study, and tolerance to drought and chilling was analysed in comparison with transgenic tobacco overexpressing SgNCED1 or ALO and the wild-type plants. Compared to the SgNCED1 or ALO transgenic plants, in which only ABA or AsA levels were increased, both ABA and AsA levels were increased in transgenic tobacco and stylo plants co-expressing SgNCED1 and ALO genes. Compared to the wild type, an enhanced drought tolerance was observed in SgNCED1 transgenic tobacco plants with induced expression of drought-responsive genes, but not in ALO plants, while an enhanced chilling tolerance was observed in ALO transgenic tobaccos with induced expression of cold-responsive genes, but not in SgNCED1 plants. Co-expression of SgNCED1 and ALO genes resulted in elevated tolerance to both drought and chilling in transgenic tobacco and stylo plants with induced expression of both drought and cold-responsive genes. Our result suggests that co-expression of SgNCED1 and ALO genes is an effective way for use in forage plant improvement for increased tolerance to drought and chilling and nutrition quality.