Characterization of subunits encoded by SnRK1 and dissection of combinations among these subunits in sorghum (Sorghum bicolor L.)
Sucrose nonfermenting-related protein kinase 1 (SnRK1) is one of the critical serine/threonine protein kinases. It commonly mediates plant growth and development, cross-talks with metabolism processes and physiological responses to biotic or abiotic stresses. It plays a key role in distributing carbohydrates and sugar signal transporting. In the present study, eight SnRK1 coding genes were identified in sorghum (Sorghum bicolor L.) via sequences alignment, with three for α subunits (SnRK1α1 to SnRK1α3), three for β (SnRK1β1 to SnRK1β3), and one for both γ (SnRK1γ) and βγ (SnRK1βγ). These eight corresponding genes located on five chromosomes (Chr) of Chr1–3, Chr7, and Chr9 and presented collinearities to SnRK1s from maize and rice, exhibiting highly conserved domains within the same subunits from the three kinds of cereals. Expression results via qRT-PCR showed that different coding genes of SnRK1s in sorghum possessed similar expression patterns except for SnRK1α3 with a low expression level in grains and SnRK1β2 with a relatively high expression level in inflorescences. Results of subcellular localization in sorghum leaf protoplast showed that SnRK1α1/α2/α3/γ mainly located on organelles, while the rest four of SnRK1β1/β2/β3/βγ located on both membranes and some organelles. Besides, three combinations were discovered among eight SnRK1 subunits in sorghum through yeast two hybrid, including α1-β2-βγ, α2-β3-γ, and α3-β3-γ. These results provide informative references for the following functional dissection of SnRK1 subunits in sorghum.
The relationship between the fate of nitrogen (N) fertilizer and the N application rate in paddy fields in Northeast China is unclear, as is the fate of residual N. To clarify these issues, paddy field and 15N microplot experiments were carried out in 2017 and 2018, with N applications at five levels: 0, 75, 105, 135 and 165 kg N ha–1 (N0, N75, N105, N135 and N165, respectively). 15N-labeled urea was applied to the microplots in 2017, and the same amount of unlabeled urea was applied in 2018. Ammonia (NH3) volatilization, leaching, surface runoff, rice yield, the N contents and 15N abundances of both plants and soil were analyzed. The results indicated a linear platform model for rice yield and the application rate of N fertilizer, and the optimal rate was 135 kg N ha–1. N uptake increased with an increasing N rate, and the recovery efficiency of applied N (REN) values of the difference subtraction method were 45.23 and 56.98% on average in 2017 and 2018, respectively. The REN was the highest at the N rate of 135 kg ha–1 in 2017 and it was insignificantly affected by the N application rate in 2018, while the agronomic efficiency of applied N (AEN) and physiological efficiency of applied N (PEN) decreased significantly when excessive N was applied. N loss through NH3 volatilization, leaching and surface runoff was low in the paddy fields in Northeast China. NH3 volatilization accounted for 0.81 and 2.99% of the total N application in 2017 and 2018, respectively. On average, the leaching and surface runoff rates were 4.45% and less than 1.05%, respectively, but the apparent denitrification loss was approximately 42.63%. The residual N fertilizer in the soil layer (0–40 cm) was 18.37–31.81 kg N ha–1 in 2017, and the residual rate was 19.28–24.50%. Residual 15N from fertilizer in the soil increased significantly with increasing N fertilizer, which was mainly concentrated in the 0–10 cm soil layer, accounting for 58.45–83.54% of the total residual N, and decreased with increasing depth. While the ratio of residual N in the 0–10 cm soil layer to that in the 0–40 cm soil layer was decreased with increasing N application. Furthermore, of the residual N, approximately 5.4% was taken up on average in the following season and 50.2% was lost, but 44.4% remained in the soil. Hence, the amount of applied N fertilizer should be reduced appropriately due to the high residual N in paddy fields in Northeast China. The appropriate N fertilizer rate in the northern fields in China was determined to be 105–135 kg N ha–1 in order to achieve a balance between rice yield and high N fertilizer uptake.
Efficient and stable expression of foreign genes in cells and transgenic animals is important for gain-of-function studies and the establishment of bioreactors. Safe harbor loci in the animal genome enable consistent overexpression of foreign genes, without side effects. However, relatively few safe harbor loci are available in pigs, a fact which has impeded the development of multi-transgenic pig research. We report a strategy for efficient transgene knock-in in the endogenous collagen type I alpha 1 chain (COL1A1) gene using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. After the knock-in of a 2A peptide-green fluorescence protein (2A-GFP) transgene in the last codon of COL1A1 in multiple porcine cells, including porcine kidney epithelial (PK15), porcine embryonic fibroblast (PEF) and porcine intestinal epithelial (IPI-2I) cells, quantitative PCR (qPCR), Western blotting, RNA-seq and CCK8 assay were performed to assess the safety of COL1A1 locus. The qPCR results showed that the GFP knock-in had no effect (P=0.29, P=0.66 and P=0.20 for PK15, PEF and IPI-2I cells, respectively) on the mRNA expression of COL1A1 gene. Similarly, no significant differences (P=0.64, P=0.48 and P=0.80 for PK15, PEF and IPI-2I cells, respectively) were found between the GFP knock-in and wild type cells by Western blotting. RNA-seq results revealed that the transcriptome of GFP knock-in PEF cells had a significant positive correlation (P<2.2e–16) with that of the wild type cells, indicating that the GFP knock-in did not alter the global expression of endogenous genes. Furthermore, the CCK8 assay showed that the GFP knock-in events had no adverse effects (P24h=0.31, P48h=0.96, P72h=0.24, P96h=0.17, and P120h=0.38) on cell proliferation of PK15 cells. These results indicate that the COL1A1 locus can be used as a safe harbor for foreign genes knock-in into the pig genome and can be broadly applied to farm animal breeding and biomedical model establishment
In maize, two root epidermis-expressed ammonium transporters ZmAMT1;1a and ZmAMT1;3 play major roles in high-affinity ammonium uptake. However, the transcriptional regulation of ZmAMT1s in roots for ensuring optimal ammonium acquisition remains largely unknown. Here, using a split root system we showed that ZmAMT1;1a and ZmAMT1;3 transcript levels were induced by localized ammonium supply to nitrogen-deficient roots. This enhanced expression of ZmAMT1s correlated with increases in 15NH4+ influx rates and tissue glutamine concentrations in roots. When ammonium was supplied together with methionine sulfoximine, an inhibitor of glutamine synthase, ammonium-induced expression of ZmAMT1s disappeared, suggesting that glutamine rather than ammonium itself regulated ZmAMT1s expression. When glutamine was supplied to nitrogen-deficient roots, expression levels of ZmAMT1s were enhanced, and negative feedback regulation could subsequently occur by supply of glutamine at a high level. Thus, our results indicated an ammonium-dependent regulation of ZmAMT1s at transcript levels, and a dual role of glutamine was suggested in the regulation of ammonium uptake in maize roots.
Soybean cyst nematode (SCN) Heterodera glycines is considered as the major constraint to soybean production. GmSHMT08 at Rhg4 locus on chromosome 08, encoding a serine hydroxylmethyltransferase, is a major gene underlying resistance against H. glycines in Peking-type soybeans. However, the molecular mechanism underpinning this resistance is less well characterized, and whether GmSHMT08 could interact with proteins in H. glycines remains unclear. In this study, yeast two-hybrid screening was conducted using GmSHMT08 as a bait protein, and a fragment of a 70-kDa heat shock protein (HgHSP70) was screened from H. glycines that exhibited interaction with GmSHMT08. This interaction was verified by both GST pull-down and bimolecular fluorescence complementation assays. Our finding reveals HgHSP70 could be applied as a potential candidate gene for further exploring the mechanism on GmSHMT08-mediated resistance against SCN H. glycines.
Sugar transporters are essential for osmotic process regulation, various signaling pathways and plant growth and development. Currently, few studies are available on the function of sugar transporters in sorghum (Sorghum bicolor L.). In this study, we performed a genome-wide survey of sugar transporters in sorghum. In total, 98 sorghum sugar transporters (SSTs) were identified via BLASTP. These SSTs were classified into three families based on the phylogenetic and conserved domain analysis, including six sucrose transporters (SUTs), 23 sugars will eventually be exported transporters (SWEETs), and 69 monosaccharide transporters (MSTs). The sorghum MSTs were further divided into seven subfamilies, including 24 STPs, 23 PLTs, two VGTs, four INTs, three pGlcT/SBG1s, five TMTs, and eight ERDs. Chromosomal localization of the SST genes showed that they were randomly distributed on 10 chromosomes, and substantial clustering was evident on the specific chromosomes. Twenty-seven SST genes from the families of SWEET, ERD, STP, and PLT were found to cluster in eight tandem repeat event regions. In total, 22 SSTs comprising 11 paralogous pairs and accounting for 22.4% of all the genes were located on the duplicated blocks. The different subfamilies of SST proteins possessed the same conserved domain, but there were some differences in features of the motif and transmembrane helices (TMH). The publicly-accessible RNA-sequencing data and real-time PCR revealed that the SST genes exhibited distinctive tissue specific patterns. Functional studies showed that seven SSTs were mainly located on the cell membrane and membrane organelles, and 14 of the SSTs could transport different types of monosaccharides in yeast. These findings will help us to further elucidate their roles in the sorghum sugar transport and sugar signaling pathways.