Transcriptome analysis provides StMYBA1 gene that regulates potato anthocyanin biosynthesis by activating structural genes.

Anthocyanin biosynthesis is affected by light, temperature, and other environmental factors. The regulation mode of light on anthocyanin synthesis in apple, pear, tomato and other species has been reported, while not clear in potato. In this study, potato RM-210 tubers whose peel will turn purple gradually after exposure to light were selected. Transcriptome analysis was performed on RM-210 tubers during anthocyanin accumulation. The expression of StMYBA1 gene continued to increase during the anthocyanin accumulation in RM-210 tubers. Moreover, co-expression cluster analysis of differentially expressed genes showed that the expression patterns of StMYBA1 gene were highly correlated with structural genes CHS and CHI. The promoter activity of StMYBA1 was significantly higher in light conditions, and StMYBA1 could activate the promoter activity of structural genes StCHS, StCHI, and StF3H. Further gene function analysis found that overexpression of StMYBA1 gene could promote anthocyanin accumulation and structural gene expression in potato leaves. These results demonstrated that StMYBA1 gene promoted potato anthocyanin biosynthesis by activating the expression of structural genes under light conditions. These findings provide a theoretical basis and genetic resources for the regulatory mechanism of potato anthocyanin synthesis.

Comparative transcriptome analysis of purple-fleshed sweet potato and its yellow-fleshed mutant provides insight into the transcription factors involved in anthocyanin biosynthesis in tuberous root.

In various plant species, many transcription factors (TFs), such as MYB, bHLH, and WD40, have been identified as regulators of anthocyanin biosynthesis in underground organs. However, the regulatory elements of anthocyanin biosynthesis in the tuberous roots of sweet potato have not been elucidated yet. Here, we selected the purple-fleshed sweet potato cultivar "Zhezi1" (ZZ P ) and its spontaneous yellow-fleshed mutant "Xinli" (XL Y ) to investigate the regulatory mechanism of the anthocyanin biosynthesis in the tuberous roots of sweet potato. By analyzing the IbMYB1 genotype in ZZ P and XL Y , we found that the IbMYB1-2, a MYB TF involved in anthocyanin biosynthesis, was missing in the XL Y genome, which might lead to an extreme decrease in anthocyanins in XL Y . A comparative transcriptome analysis of ZZ P and XL Y was conducted to find the TFs involved in anthocyanin biosynthesis in ZZ P and XL Y . The anthocyanin structural genes were significantly enriched among the differentially expressed genes. Moreover, one MYB activator (IbMYB1), one bHLH (IbbHLH2), three WRKY activator candidates (IbWRKY21, IbWRKY24, and IbWRKY44), and two MYB repressors (IbMYB27 and IbMYBx-ZZ) were highly expressed in ZZ P accompanied with anthocyanin structural genes. We also tested the expression of these TFs in six purple- and two orange-fleshed sweet potato cultivars. Interestingly, most of these TFs were significantly positively correlated with anthocyanin contents in these cultivars. The function of the anthocyanin biosynthesis repression of IbMYB27 and IbMYBx-ZZ was verified through transient co-transformation with IbMYB1 into tobacco leaves. Further functional verification of the above TFs was conducted by Y2H, BiFC, and dual-luciferase assays. These tests showed that the MYB-bHLH-WD40/MYB-bHLH-WD40-WRKY complex activated the promoter of anthocyanin structural gene IbDFR and promoters for IbWRKY44, IbMYB27, and IbMYBx-ZZ, indicating reinforcement and feedback regulation to maintain the level of anthocyanin accumulation in the tuberous roots of purple-fleshed sweet potato. These results may provide new insights into the regulatory mechanism of anthocyanin biosynthesis and accumulation in underground organs of sweet potatoes.

Eukaryotic translation initiation factor 4E family member nCBP facilitates the accumulation of TGB-encoding viruses by recognizing the viral coat protein in potato and tobacco.

Due to their limited coding capacity, plant viruses have to depend on various host factors for successful infection of the host. Loss of function of these host factors will result in recessively inherited resistance, and therefore, these host factors are also described as susceptibility genes or recessive resistance genes. Most of the identified recessive resistance genes are members of the eukaryotic translation initiation factors 4E family (eIF4E) and its isoforms. Recently, an eIF4E-type gene, novel cap-binding protein (nCBP), was reported to be associated with the infection of several viruses encoding triple gene block proteins (TGBps) in Arabidopsis. Here, we, for the first time, report that the knockdown of nCBP in potato (StnCBP) compromises the accumulation of potato virus S (PVS) but not that of potato virus M (PVM) and potato virus X (PVX), which are three potato viruses encoding TGBps. Further assays demonstrated that StnCBP interacts with the coat proteins (CPs) of PVS and PVM but not with that of PVX, and substitution of PVS CP in the PVS infectious clone by PVM CP recovered the virus infection in StnCBP-silenced transgenic plants, suggesting that the recognition of PVS CP is crucial for StnCBP-mediated recessive resistance to PVS. Moreover, the knockdown of nCBP in Nicotiana benthamiana (NbnCBP) by virus-induced gene silencing suppressed PVX accumulation but not PVM, while NbnCBP interacted with the CPs of both PVX and PVM. Our results indicate that the nCBP orthologues in potato and tobacco have conserved function as in Arabidopsis in terms of recessive resistance against TGB-encoding viruses, and the interaction between nCBP and the CP of TGB-encoding virus is necessary but not sufficient to determine the function of nCBP as a susceptibility gene.

Physiology and Gene Expression Analysis of Potato (Solanum tuberosum L.) in Salt Stress.

The production of potato (Solanum tuberosum L.) faces a severe challenge due to the salinization of arable land worldwide. The cultivation of salt-tolerant potatoes is of great significance to ensure food security. In this study, two cultivars of 'Longshu 5' and 'Qingshu 9' were compared for physiological responses to salt stress, and then the salt tolerance of the two cultivars were assessed via principal component analysis. Furthermore, the Na+, K+, and Ca2+ flux of the cultivars under salt stress was recorded. Finally, the expression levels of ion transport-related genes and transcription factors in salt-tolerant cultivars were explored under NaCl stress. The results showed that the seven physiological indicators of salt tolerance were differed between the cultivars. Interestingly, soluble protein and sugar were early responsive to salt stress than proline in the salt-tolerance cultivar. Peroxidase and superoxide dismutase activity were significantly different in 'Longshu 5' under NaCl stress and without being significantly different in 'Qingshu9'. In addition, the salt tolerance of 'Longshu 5' was more tolerant than 'Qingshu 9' based on principal component evaluation. Meanwhile, the strong efflux of Na+, the stability of K+, and the high absorption of Ca2+ in 'Longshu 5' indicated salt adaption mechanisms in the salt-tolerant potato. In addition, we found that ion transport-related genes and transcription factors, such as StSOS1, StNHX4, StAKT1, StNAC24, and StCYP707A, played a role in the salt tolerance of 'Longshu 5'. In conclusion, the salt-tolerant potato can regulate physiological substances to adapt to salt stress, and ion transport related genes and transcription factors play a role in improving salt tolerance.

Transcriptome analysis uncovers the gene expression profile of salt-stressed potato (Solanum tuberosum L.).

Potato (Solanum tuberosum L.) is an important staple food worldwide. However, its growth has been heavily suppressed by salt stress. The molecular mechanisms of salt tolerance in potato remain unclear. It has been shown that the tetraploid potato Longshu No. 5 is a salt-tolerant genotype. Therefore, in this study we conducted research to identify salt stress response genes in Longshu No. 5 using a NaCl treatment and time-course RNA sequencing. The total number of differentially expressed genes (DEGs) in response to salt stress was 5508. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, it was found that DEGs were significantly enriched in the categories of nucleic acid binding, transporter activity, ion or molecule transport, ion binding, kinase activity and oxidative phosphorylation. Particularly, the significant differential expression of encoding ion transport signaling genes suggests that this signaling pathway plays a vital role in salt stress response in potato. Finally, the DEGs in the salt response pathway were verified by Quantitative real-time PCR (qRT-PCR). These results provide valuable information on the salt tolerance of molecular mechanisms in potatoes, and establish a basis for breeding salt-tolerant cultivars.

Cultivar diversity and organ differences of cadmium accumulation in potato (Solanum tuberosum L.) allow the potential for Cd-safe staple food production on contaminated soils.

Cadmium (Cd) is a toxic element that can accumulate in plants and poses a threat to human health through biomagnification. There are differences in Cd levels among different plants tissues. Hence, an optimal crop that possesses low Cd levels in the edible parts but high levels in the inedible parts is urgently needed to simultaneously lower soil-Cd levels in contaminated fields and to produce Cd-safe crops. In this study, we investigated the Cd levels in tubers and other tissues of potato (Solanum tuberosum L.) using different experimental approaches, and identified variations in Cd accumulation in different potato cultivars and characterized the Cd-distribution pattern in potato. Our results showed that Cd accumulation in tubers of the tested cultivars varied greatly, and that tuber-Cd levels were much lower than in the stems or leaves. Two-way ANOVA revealed that the tuber-Cd levels in potato are determined by both genotypic differences and the soil-Cd levels of the farmlands. Among the cultivars tested, one cultivar, 'Eshu10', was found to have the lowest tuber-Cd levels but had much higher Cd levels in leaf and stem tissues. Our study shows that the Cd-distribution pattern within potato plants makes it an ideal candidate for the safe production of a staple food that also has the potential to contribute to the remediation of contaminated soils.

Development of SYBR Green real-time PCR and nested RT-PCR for the detection of Potato Mop-top Virus (PMTV) and viral surveys in Progeny tubers derived from PMTV infected Potato tubers.

In this study, a new SYBR Green qPCR (qRT-PCR) and a nested RT-PCR (nRT-PCR) were developed to detect Potato mop-top virus (PMTV) in potato tuber tissues. The SYBR Green qRT-PCR and nRT-PCR assays were approximately 104- and 103- fold more sensitive than the conventional RT-PCR assay. The progeny tubers derived from PMTV-infected potato tubers were tested by conventional RT-PCR, SYBR Green qRT-PCR and nRT-PCR assays. Of the 17 samples, 9 (52.9%) were positive for PMTV by conventional RT-PCR, 11 (64.7%) were positive by nRT-PCR, and 17 (100%) were positive by SYBR Green qRT-PCR. Compared to nRT-PCR, SYBR Green qRT-PCR was showed to be more sensitive. The progeny plants exhibited foliar symptoms including chlorosis and reduction in leaf size when the PMTV-positive tubers were planted in a growth chamber at 20-22 °C. These findings suggest that PMTV has been passed on to the progeny plants and tubers.

Integrated mRNA and microRNA transcriptome analysis reveals miRNA regulation in response to PVA in potato.

Potato (Solanum tuberosum L.) is the fourth most important crop worldwide. Potato virus A (PVA) is one of the most harmful viruses infecting potatoes. However, the molecular mechanisms governing the responses to PVA infection in potato at the transcriptional and post-transcriptional levels are not well understood. In this study, we performed both mRNA and small RNA sequencing in potato leaves to identify the genes and miRNAs involved in the response to PVA infection. A total of 2,062 differentially expressed genes (DEGs) and 201 miRNAs (DEMs) were identified, respectively. Gene ontology (GO) and KEGG analysis revealed that these DEGs were involved in the transduction of pathogen signals, transcriptional reprogramming, induction of hormone signaling, activation of pathogenesis-related (PR) genes, and changes in secondary metabolism. Small RNA sequencing revealed 58 miRNA-mRNA interactions related to PVA infection. Some of the miRNAs (stu-miR482d-3p, stu-miR397-5p, etc) which target PR genes showed negative correlations between the DEMs and DEGs. Eight of the DEGs and three DEMs with their target genes were further validated by quantitative real time-PCR (qRT-PCR). Overall, this study provides a transcriptome-wide insight into the molecular basis of resistance to PVA infection in potato leaves and potenital candidate genes for improving resistance cultivars.

Molecular characterization of a Chinese isolate of potato virus A (PVA) and evidence of a genome recombination event between PVA variants at the 3'-proximal end of the genome.

Potato plants that exhibited mosaic symptoms were collected in Xiangxi, Hunan province, China. Multiplex RT-PCR screening for common viruses revealed the presence of potato virus A (PVA) in these samples. ELISA with virus-specific antibodies confirmed infection by PVA in the plants. Rod-shaped virions of ~750 nm in length and ~13 nm in width were observed by transmission electron microscopy. One virus isolate (designated PVA-Hunan) was subjected to molecular characterization. The viral genome consisted of 9,567 nucleotides, excluding the poly(A) tail, and encoded a polyprotein of 3,059 amino acids. A second characteristic potyvirus open reading frame (ORF), pretty interesting Potyviridae ORF (pipo), was located at nucleotides 2,834-3,139. The isolate shared 84% to 98% and 93% to 99% sequence identity with other PVA isolates at the nucleotide and amino acid level, respectively. Phylogenetic analysis demonstrated that, within the PVA group, PVA-Hunan clustered most closely with the Finnish isolate Her, then with isolates 143, U, Ali, M and B11. The isolate TamMV stood alone at a separate branch. However, scanning of complete genome sequences using SimPlot revealed 99%-sequence identity between PVA-Hunan and TamMV in the 3'-proximal end of the genome (~nt 9,160 to the 3'end) and a 50%-94% (average~83%) identity upstream of nt 9,160. In contrast, 98% identity between PVA-Hunan and isolates M and B11 was detected for nucleotides 1 to ~9,160, but only ~94% for the 3'-proximal region, suggesting a genome recombination event (RE) at nt 9,133. The recombination breakpoint also was identified by the Recombination Detection Program (RDP). The RE was further confirmed by analysis of the CP gene, where the apparent RE was located.

Electrochemical determination of glycoalkaloids using a carbon nanotubes-phenylboronic acid modified glassy carbon electrode.

A versatile strategy for electrochemical determination of glycoalkaloids (GAs) was developed by using a carbon nanotubes-phenylboronic acid (CNTs-PBA) modified glassy carbon electrode. PBA reacts with α-solanine and α-chaconine to form a cyclic ester, which could be utilized to detect GAs. This method allowed GA detection from 1 µM to 28 µM and the detection limit was 0.3 µM. Affinity interaction of GAs and immobilized PBA caused an essential change of the peak current. The CNT-PBA modified electrodes were sensitive for detection of GAs, and the peak current values were in quite good agreement with those measured by the sensors.

Differential pathogenicity of two different recombinant PVY(NTN) isolates in Physalis floridana is likely determined by the coat protein gene.

A previous study has identified two types of recombinant variants of Potato virus Y strain NTN (PVY(NTN)) in China and sequenced the complete genome of the variant PVY(NTN)-HN2. In this study, the complete genome of isolate PVY(NTN)-HN1 was fully sequenced and analyzed. The most striking difference between the two variants was the location of recombinant joint three (RJ3). In PVY(NTN)-HN1, like other typical European-PVY(NTN) isolates such as PVY(NTN)-Hun, the RJ3 was located at nucleotide (nt) 9183, namely the 3' proximal end of the CP gene (nt. 8571-9371), thus leading to most (the first 613 nucleotides from the 5' proximal end) of the CP gene (801 bp) with a PVYN origin and PVYN-serotype; whereas in contrast, the RJ3 in PVY(NTN)-HN2 was located at nt 8572, consequently leading to a CP gene of PVYO origin and PVYO-serotype. The varied genome composition among PVY(O), PVY(N), PVY(N:O), PVY(NTN_-HN1 and PVY(NTN)-HN2 made them useful for the investigation of possible roles of gene segment(s) in symptom formation on host plants. When Physalis floridana plants were infected with different PVY isolates, two types of symptoms were induced. PVY(N) and PVY(NTN)-HN1 induced mild symptoms (mainly mild mottling) whereas PVY(O), PVY(N:O) and PVY(NTN)-HN2 induced serve symptoms including leaf and stem necrosis, leaf-drop and stunting. These results, together with a previous study using artificial PVY chimeras, demonstrate that the CP gene, especially the 5' proximal segment (nt 8572-9183), and/or CP likely determine the pathogenicity of PVY in P. floridana.

Molecular characterization and detection of recombinant isolates of potato virus Y from China.

Although potato virus Y (PVY) is one of the most economically important pathogens of potatoes in China, few studies have been carried out to characterize the virus in that country. Using reverse transcription-polymerase chain reaction (RT-PCR)-based genotyping developed previously, two types of recombinant PVY were identified in China for the first time. One resembled the European (Eu) type of potato tuber necrosis strain (Eu-PVY(NTN)), possessing three widely recognized recombinant joints (RJs 1-3) of the common strain (PVY(O)) and the Eu- type tobacco veinal necrosis strain (Eu-PVY(N)). The other, on the other hand, appeared to have only RJ1 and RJ2. The complete genome of a representative isolate, PVY-HN2, from the second type was subsequently sequenced. Comparison of the sequence of 'HN2' with those of PVY(O) and Eu-PVY(N) not only confirmed the recombinant nature of 'HN2' but also revealed the existence of three recombinant events in the isolate, similar to that in PVY(NTN)-Hun. However, the two isolates differed significantly at RJ1 (PVY(NTN)-Hun vs. HN2, nt 2419 vs. nt 2521) and RJ3 (PVY(NTN)-Hun vs. HN2, nt 9183 vs. nt 8572) and slightly at RJ2 (PVY(NTN)-Hun vs. HN2, nt 5844 vs. nt 5867). A primer pair was developed to facilitate the detection of the alternative RJ3. Using the newly and previously designed RJ primers, all targeted RJs were detected. Interestingly, tests of the available PVY samples indicated that two were doubly infected with both types of recombinant PVY, further confirming the effectiveness of the detection. Further analysis of these samples using enzyme-linked immunosorbent assay and bioassay revealed that 'HN2' possesses a PVY(O) serotype, a PVY(N) pathotype in tobacco and a PVY(NTN) pathotype in potato.