Huaijun Si

Identification of Novel and Conserved MicroRNAs Related to Drought Stress in Potato by Deep Sequencing

MicroRNAs (miRNAs) are a group of small, non-coding RNAs that play important roles in plant growth, development and stress response. There have been an increasing number of investigations aimed at discovering miRNAs and analyzing their functions in model plants (such as Arabidopsis thaliana and rice). In this research, we constructed small RNA libraries from both polyethylene glycol (PEG 6,000) treated and control potato samples, and a large number of known and novel miRNAs were identified. Differential expression analysis showed that 100 of the known miRNAs were down-regulated and 99 were up-regulated as a result of PEG stress, while 119 of the novel miRNAs were up-regulated and 151 were down-regulated. Based on target prediction, annotation and expression analysis of the miRNAs and their putative target genes, 4 miRNAs were identified as regulating drought-related genes (miR811, miR814, miR835, miR4398). Their target genes were MYB transcription factor (CV431094), hydroxyproline-rich glycoprotein (TC225721), quaporin (TC223412) and WRKY transcription factor (TC199112), respectively. Relative expression trends of those miRNAs were the same as that predicted by Solexa sequencing and they showed a negative correlation with the expression of the target genes. The results provide molecular evidence for the possible involvement of miRNAs in the process of drought response and/or tolerance in the potato plant.

Identification of miR159s and their target genes and expression analysis under drought stress in potato

The MYB proteins comprise one of the largest families of plant transcription factors (TFs) and many of MYB families, which play essential roles in plant growth, development and respond to environmental stresses, and have yet been identified in plant. Previous research has shown that miR159 family members repressed the conserved plant R2R3 MYB domain TFs in model plants. In the present research, we identified three potato novel miR159 family members named as stu-miR159a, stu-miR159b and stu-miR159c based on bioinformatics analysis. Target prediction showed that they have a bite sit on the three GAMyb-like genes (StGAMyb-like1, StGAMyb-like2.1 and StGAMyb-like2.2) of potato. Those GAMyb-like genes also have been selected and cloned from potato, which belong to R2R3 MYB domain TFs. We further measured expressional levels of stu-miR159s and potato GAMyb-like genes during the different periods of drought treated samples using quantitative real-time PCR (qRT-PCR). The results showed that they had a opposite expression pattern, briefly, three stu-miR159 members showed similar expressional trends which were significantly decreased expression after experiencing 25 days of drought stress treatment, while the potato GAMyb-like family members were greatly increased. Therefore, we suggested that stu-miR159s negatively regulated the expression of potato GAMyb-like genes which responsible for drought stress. The findings can facilitate functional studies of miRNAs in plants and provide molecular evidence for involvement process of drought tolerance in potato.

Transcriptome characterization and sequencing-based identification of drought-responsive genes in potato

Abstract Potato (Solanum tubersosum L.) is relatively vulnerable to abiotic stress conditions such as drought, but the tolerance mechanisms to such stress in potato are largely unknown. To gain insight into the transcriptome dynamics that are associated with drought stress, genome-wide gene expression profile was conducted by Solexa sequencing to generate a large dataset and a comprehensive transcriptome profile for potato. Here, we report a reference for the potato transcriptome using leaf tissues under drought-stressed condition from a local potato cultivar ‘Longshu 3’. Analysis of 86,965,482 RNA-Seq reads permitted the detection and quantification of expression levels of 7,284 genes at transcriptional levels, among them, 6,754 genes were enriched in draught-treated leaves while 6,419 in control. We identified 842 drought-responsive up-regulated and 494 down-regulated candidate genes with significantly differentially expression under continued drought stress treatments. Those differently expressed genes were mostly enriched in 89 gene categories and 21 KEGG pathways. Drought-stressed leaves had increased expression of genes involved in stress response compared with control leaves. A subset of differentially expressed genes associated with drought response was examined using quantitative real-time PCR. These results provide a broad spectrum of candidate genes that are essential for understanding the molecular regulation of potato in response to abiotic stresses.

Prediction and verification of microRNAs related to proline accumulation under drought stress in potato

Proline is an important osmotic adjusting material greatly accumulated under drought stress and can help plant to adapt to osmotic stress. MicroRNAs (miRNAs) are small, endogenous RNAs that play important regulatory roles in plant development and stress response by negatively affecting gene expression at post-transcriptional level. Three genes of pyrroline-5-carboxylate synthetase (P5CS), pyrroline-5-carboxylate reductase (P5CR) and proline dehydrogenase (ProDH) are regulating proline metabolism. Until now, little is known about miRNAs regulating proline accumulation. In this work, in order to understand whether miRNAs related to mRNAs of enzymes to regulate proline enrichment under drought stress, we used mRNAs of related enzymes as the targets of miRNAs to search miRBase using BLAST and find many query miRNA sequences. After a range of filtering criteria, 11 known miRNAs classified into 6 miRNA families were predicted. The result from qRT-PCR assay showed that 10 out of 11 predicted miRNAs were successfully detected including 9 down-regulated miRNAs and one up-regulated miRNA. Based on expression and functional analysis, we identified miR172, miR396a, miR396c and miR4233 may regulate P5CS gene, and miR2673 and miR6461 may regulate P5CR and ProDH gene, respectively. The findings can help us make a good understanding of the roles of miRNAs in regulation of proline accumulation and provide molecular evidence for involvement process of drought tolerance in potato.

Transcriptomic changes during tuber dormancy release process revealed by RNA sequencing in potato.

Potato tuber dormancy release is a critical development process that allows potato to produce new plant. The first Illumina RNA sequencing to generate the expressed mRNAs at dormancy tuber (DT), dormancy release tuber (DRT) and sprouting tuber (ST) was performed. We identified 26,639 genes including 5,912 (3,450 up-regulated while 2,462 down-regulated) and 3,885 (2,141 up-regulated while 1,744 down-regulated) genes were differentially expressed from DT vs DRT and DRT vs ST. The RNA-Seq results were further verified using qRT-PCR. We found reserve mobilization events were activated before the bud emergence (DT vs DRT) and highlighted after dormancy release (DRT vs ST). Overexpressed genes related to metabolism of auxin, gibberellic acid, cytokinin and barssinosteriod were dominated in DT vs DRT, whereas overexpressed genes involved in metabolism of ethylene, jasmonate and salicylate were prominent in DRT vs ST. Various histone and cyclin isoforms associated genes involved in cell division/cycle were mainly up-regulated in DT vs DRT. Dormancy release process was also companied by stress response and redox regulation, those genes related to biotic stress, cell wall and second metabolism was preferentially overexpressed in DRT vs ST, which might accelerate dormancy breaking and sprout outgrowth. The metabolic processes activated during tuber dormancy release were also supported by plant seed models. These results represented the first comprehensive picture of a large number of genes involved in tuber dormancy release process.

Proteomic changes during tuber dormancy release process revealed by iTRAQ quantitative proteomics in potato

Given that limited information is available with regard to tuber dormancy release related proteome, we conducted proteome analysis of tuber dormancy release process at dormant tuber (DT), dormancy release tuber (DRT) and sprouting tuber (ST) using the iTRAQ technology. A total of 1,752 proteins were identified. Among them, a subset of 316 proteins was screened as significant up- (137) and down regulated (179) between DT vs DRT. A subset of 120 proteins experienced significant up- (40) or down-regulation (80) between DRT vs ST. The differentially expressed proteins were grouped into 11 functional categories. Proteins enriched in functional categories of major carbohydrate (CHO) metabolism, glycolysis, fermentation, amino acid metabolism, protein and transport were highly up-regulated, while functional categories of photosynthesis and RNA were down-regulated between DT vs DRT. Proteins enriched in functional groups of protein, cell wall, lipid metabolism, miscellaneous, and signaling were strongly up-regulated, while functional categories of photosynthesis, hormone metabolism and protein were down-regulated between DRT vs ST. Consistent with previous documented differentially expressed genes, most of differentially expressed proteins were also identified between DT and DRT, indicating the metabolism shift from growth suspension to growth activation as tubers dormancy breaking. The changes in protein profiles showed lower concordance with corresponding alterations in transcript levels, indicating possible transcriptional and posttranscriptional regulation. Furthermore, the possible mechanism of tuber dormancy release was discussed in relation to what was known in transcripts change and other plant models from carbohydrate metabolism, protein metabolism, stress response, redox regulation, transcription regulation, DNA metabolism, amino acid metabolism, development, signaling as well as hormone metabolism.

Transgenic potato plants expressing cry3A gene confer resistance to Colorado potato beetle

The Colorado potato beetle (Leptinotarsa decemlineata Say, CPB) is a fatal pest, which is a quarantine pest in China. The CPB has now invaded the Xinjiang Uygur Autonomous Region and is constantly spreading eastward in China. In this study, we developed transgenic potato plants expressing cry3A gene. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the cry3A gene expressed in leaves, stems and roots of the transgenic plants under the control of CaMV 35S promoter, while they expressed only in leaves and stems under the control of potato leaf and stem-specific promoter ST-LS1. The mortality of the larvae was higher (28% and 36%) on the transgenic plant line 35S1 on the 3rd and 4th days, and on ST3 (48%) on the 5th day after inoculation with instar larvae. Insect biomass accumulation on the foliage of the transgenic plant lines 35S1, 35S2 and ST3 was significantly lower (0.42%, 0.43% and 0.42%). Foliage consumption was lowest on transgenic lines 35S8 and ST2 among all plant foliage (7.47 mg/larvae/day and 12.46 mg/larvae/day). The different transgenic plant foliages had varied inhibition to larval growth. The survivors on the transgenic lines obviously were smaller than their original size and extremely weak. The transgenic potato plants with CPB resistance could be used to develop germplasms or varieties for controlling CPB damage and halting its spread in China.

Changes in ROS production and antioxidant capacity during tuber sprouting in potato

Potato dormancy is a complex process with an extensive release phase. This study investigated involvement of reactive oxygen species during tuber dormancy release. We found that tuber sprouting was delayed by treatment with diphenylene iodonium chloride, an NADPH oxidase inhibitor; NADPH oxidase catalyze the production of ROS. In situ ROS localization and ROS content estimation revealed that dormancy release was associated with an accumulation of superoxide anion and hydrogen peroxide in tuber buds. The antioxidant compounds and enzymes display important changes during the progression of dormancy. The application of Ca2+ induced superoxide anion production and promoted in vitro tuber bud sprouting. Among the seven homologues of NADPH oxidases in potato, the expression of StrbohA and StrbohB were detected in particular when dormancy break. In addition, the expression of key genes that function in the potato dormancy release are discussed in relation to ROS metabolism in other plants.

Development of selectable marker-free transgenic potato plants expressing cry3A against the Colorado potato beetle (Leptinotarsa decemlineata Say)

BACKGROUND Elimination of selectable marker genes (SMGs) is important for the safe assessment and commercial use of transgenic plants. The destructive and invasive Colorado potato beetle (CPB) poses a serious threat to potato production. In response to this need, selectable marker-free transgenic potato lines expressing cry3A were developed to control the damage and spread of CPB. RESULTS We simultaneously introduced cry3A and npt II genes harboured in different plasmids into the potato genome using the Agrobacterium-mediated cotransformation method. Four selectable marker-free transgenic potato (CT) lines expressing cry3A were developed by self-crossing segregation and molecular analyses, including Southern blot, western blot and enzyme-linked immunosorbent assay (ELISA) assays. CT lines were used in a resistance bioassay against CPB in the laboratory and field. In the laboratory, CT lines exhibited high resistance to CPB, and 100% mortality of first-instar larvae occurred 6 days after infestation. In the field, untransformed plant leaves were almost entirely consumed, with an average of 155 larvae present per plant 25 days after inoculation. However, CT lines showed no damage symptoms, with approximately 2.5 larvae surviving per plant. CONCLUSION We successfully eliminated SMGs from the transgenic potato lines expressing cry3A in order to decrease CPB damage, control the spread of this pest eastwards and alleviate the concern regarding the safe assessment of regulatory requirements.

Expression of the Galanthus nivalis agglutinin (GNA) gene in transgenic potato plants confers resistance to aphids

Aphids, the largest group of sap-sucking pests, cause significant yield losses in agricultural crops worldwide every year. The massive use of pesticides to combat this pest causes severe damage to the environment, putting in risk the human health. In this study, transgenic potato plants expressing Galanthus nivalis agglutinin (GNA) gene were developed using CaMV 35S and ST-LS1 promoters generating six transgenic lines (35S1-35S3 and ST1-ST3 corresponding to the first and second promoter, respectively). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the GNA gene was expressed in leaves, stems and roots of transgenic plants under the control of the CaMV 35S promoter, while it was only expressed in leaves and stems under the control of the ST-LS1 promoter. The levels of aphid mortality after 5 days of the inoculation in the assessed transgenic lines ranged from 20 to 53.3%. The range of the aphid population in transgenic plants 15 days after inoculation was between 17.0±1.43 (ST2) and 36.6±0.99 (35S3) aphids per plant, which corresponds to 24.9-53.5% of the aphid population in non-transformed plants. The results of our study suggest that GNA expressed in transgenic potato plants confers a potential tolerance to aphid attack, which appears to be an alternative against the use of pesticides in the future.