A SmelAAT Acyltransferase Variant Causes a Major Difference in Eggplant (Solanum melongena L.) Peel Anthocyanin Composition

Eggplant berries are rich in anthocyanins like delphinidin-3-rutinoside (D3R) and nasunin (NAS), which are accumulated at high amounts in the peel. NAS is derived by D3R through acylation and glycosylation steps. The presence of D3R or NAS is usually associated with black-purple or lilac fruit coloration of the most cultivated varieties, respectively. Building on QTL mapping position, a candidate gene approach was used to investigate the involvement of a BAHD anthocyanin acyltransferase (SmelAAT) in determining anthocyanin type. The cDNA sequence comparison revealed the presence of a single-base deletion in D3R-type line ‘305E40’ (305E40_aat) with respect to the NAS-type reference line ‘67/3’. This is predicted to cause a frame shift mutation, leading to a loss of SmelAAT function and, thus, D3R retention. RT-qPCR analyses confirmed SmelAAT and 305E40_aat expression during berry maturation. In D3R-type lines, ‘305E40’ and ‘DR2’ overexpressing the functional SmelAAT allele from ‘67/3’, the transcript levels of the transgene, correlated with the accumulation of NAS in fruit peel. Furthermore, it was also found a higher expression of the transcript for glucosyltransferase Smel5GT1, putatively involved with SmelAAT in the last steps of anthocyanin decoration. Finally, an indel marker matching with anthocyanin type in the ‘305E40’ × ’67/3’ segregating population was developed and validated in a wide number of accessions, proving its usefulness for breeding purposes.

A critical role of PvFtsH2 in the degradation of photodamaged D1 protein in common bean

Light is required for initiating chloroplast biogenesis and photosynthesis; however, the photosystem II reaction center (PSII RC) can be photodamaged. In this study, we characterized pvsl1, a seedling-lethal mutant of Phaseolus vulgaris. This mutant showed lethality when exposed to sunlight irradiation and a yellow-green leaf phenotype when grown in a growth chamber under low-light conditions. We developed 124 insertion/deletion (INDEL) markers based on resequencing data of Dalong1 and PI60234, two local Chinese common bean cultivars, for genetic mapping. We identified Phvul.002G190900, which encodes the PvFtsH2 protein, as the candidate gene for this pvsl1 mutation through fine-mapping and functional analysis. A single-base deletion occurred in the coding region of Phvul.002G190900 in the pvsl1 mutant, resulting in a frameshift mutation and a truncated protein lacking the Zn2+ metalloprotease domain. Suppressed expression of Phvul.002G190900 at the transcriptional level was detected, while no change in the subcellular localization signal was observed. The seedlings of pvsl1 exhibited hypersensitivity to photoinhibition stress. In the pvsl1 mutant, abnormal accumulation of the D1 protein indicated a failure to rapidly degrade damaged D1 protein in the PSII RC. The results of this study demonstrated that PvFtsH2 is critically required for survival and maintaining photosynthetic activity by degrading photodamaged PSII RC D1 protein in common bean.

A High-Resolution Melting Analysis with an Unlabeled Probe for CRISPR/Cas9-Induced ZBED6 Knockout Pigs Detection

Background The zinc finger BED-type containing 6 knockout (ZBED6-KO) pigs were created to improve economic traits by increasing the expression of insulin-like growth factor 2. They were generated by CRISPR/CRISPR-associated protein 9 (Cas9) technology and a single-base deletion of ZBED6 was found. An efficient and rapid method was needed to detect this type of pig. Objective This study aimed to develop a high-resolution melting (HRM) method to detect ZBED6-KO pigs. Methods An unlabeled probe and two primers were designed to develop HRM method. The limit of detection, specificity and accuracy of established method were tested by the constructed plasmid and DNA extracts of tissue specimens. Results The limit of detection by established method was 102 copies/µL. The HRM method with an unlabeled probe showed good specificity and high accuracy. Conclusions The established HRM analysis with an unlabeled probe showed it to be a highly effective, rapid and reliable to distinguish ZBED6-KO pigs with wild-type pigs. Highlights It is the first time that HRM analysis with an unlabeled probe has been used in the detection of genome editing pigs by the CRISPR/Cas9 technology.

Mutation of IDR1 enhances drought tolerance by reducing ROS production and activating ROS scavenging in rice

To discover new mutant alleles conferring enhanced tolerance to drought stress, we screened a mutagenized rice population (cv. IAPAR9) and identified a mutant, named idr1-1 (for increased drought resistance 1-1), with obviously increased drought tolerance under upland field conditions. The idr1-1 mutant possessed a significantly enhanced ability to tolerate high-drought stress in different trials. Map-based cloning revealed that the gene LOC_Os05g26890 (corresponding to D1 or RGA1 gene), residing in the mapping region of IDR1 locus, carried a single-base deletion in the idr1-1 mutant, which caused a frameshift and premature translation termination. Complementation tests indicated that such a mutation was indeed responsible for the elevated drought tolerance in idr1-1 mutant. IDR1 protein was localized in nucleus and to plasma membrane or cell periphery. Further investigations indicated that the significantly increased drought tolerance in idr1-1 mutant stemmed from a range of physiological and morphological changes occurring in such a mutant, including greater leaf potentials, increased proline contents, heightened leaf thickness, and upregulation of antioxidant-synthesizing and drought-induced genes, etc., under drought-stressed conditions. Especially, ROS production from NADPH oxidases and chloroplasts might be remarkably impaired, while ROS-scavenging ability appeared to be markedly enhanced as a result of significantly elevated expression of a dozen ROS-scavenging enzyme genes in idr1-1 mutant under drought-stressed conditions. Besides, IDR1 physically interacted with TUD1, and idr1-1 mutant showed impaired EBR responsiveness. Altogether, these results suggest that mutation of IDR1 leads to alterations of multiple layers of regulations, which ultimately confers obviously enhanced drought tolerance to the idr1-1 mutant.One-sentence summaryMutation of IDR1 significantly enhances drought tolerance in an upland cultivar IAPAR9 by decreasing apoplastic and chloroplastic ROS production and increasing ROS-scavenging ability

Heterotrimeric PCNA Increases the Activity and Fidelity of Dbh, a Y-family Translesion DNA Polymerase Prone to Creating Single-Base Deletion Mutations

Dbh is a Y-family translesion DNA polymerase from Sulfolobus acidocaldarius, an archaeal species that grows in harsh environmental conditions. Biochemically, Dbh displays a distinctive mutational profile, creating single-base deletion mutations at extraordinarily high frequencies (up to 50%) in specific repeat sequences. In cells, however, Dbh does not appear to contribute significantly to spontaneous frameshifts in these same sequence contexts. This suggests that either the error-prone DNA synthesis activity of Dbh is reduced in vivo and/or Dbh is restricted from replicating these sequences. Here, we test the hypothesis that the propensity for Dbh to make single base deletion mutations is reduced through interaction with the S. acidocaldarius heterotrimeric sliding clamp processivity factor, PCNA-123. We first confirm that Dbh physically interacts with PCNA-123, with the interaction requiring both the PCNA-1 subunit and the C-terminal 10 amino acids of Dbh, which contain a predicted PCNA-interaction peptide (PIP) motif. This interaction stimulates the polymerase activity of Dbh, even on short, linear primer-template DNA by increasing the rate of nucleotide incorporation. This stimulation requires an intact PCNA-123 heterotrimer and a DNA duplex length of at least 18 basepairs, the minimal length predicted from structural data to bind to both the polymerase and the clamp. Finally, we find that PCNA-123 increases the fidelity of Dbh on a single-base deletion hotspot sequence 3-fold by promoting an increase in the rate of correct, but not incorrect, nucleotide addition and propose that PCNA-123 induces Dbh to adopt a more active conformation that is less prone to creating deletions during DNA synthesis.HighlightsPCNA increases the fidelity of Dbh polymerase on a deletion-hotspot sequence.The interaction stimulates incorporation of the correct, but not incorrect, nucleotide.A minimal duplex length of 18 bp is required for PCNA to stimulate polymerase activity.Structural modeling suggests that PCNA induces a conformational change in Dbh.

Mutations in the Rice OsCHR4 Gene, Encoding a CHD3 Family Chromatin Remodeler, Induce Narrow and Rolled Leaves with Increased Cuticular Wax

Leaf blade width, curvature, and cuticular wax are important agronomic traits of rice. Here, we report the rice Oschr4-5 mutant characterized by pleiotropic phenotypes, including narrow and rolled leaves, enhanced cuticular wax deposition and reduced plant height and tiller number. The reduced leaf width is caused by a reduced number of longitudinal veins and increased auxin content. The cuticular wax content was significantly higher in the Oschr4-5 mutant, resulting in reduced water loss rate and enhanced drought tolerance. Molecular characterization reveals that a single-base deletion results in a frame-shift mutation from the second chromodomain of OsCHR4, a CHD3 (chromodomain helicase DNA-binding) family chromatin remodeler, in the Oschr4-5 mutant. Expressions of seven wax biosynthesis genes (GL1-4, WSL4, OsCER7, LACS2, LACS7, ROC4 and BDG) and four auxin biosynthesis genes (YUC2, YUC3, YUC5 and YUC6) was up-regulated in the Oschr4-5 mutant. Chromatin immunoprecipitation assays revealed that the transcriptionally active histone modification H3K4me3 was increased, whereas the repressive H3K27me3 was reduced in the upregulated genes in the Oschr4-5 mutant. Therefore, OsCHR4 regulates leaf morphogenesis and cuticle wax formation by epigenetic modulation of auxin and wax biosynthetic genes expression.