Liming Lu

De novo characterization of the alligator weed ( Alternanthera philoxeroides ) transcriptome illuminates gene expression under potassium deprivation

As one of the three macronutrients, potassium participates in many physiological processes in plant life cycle. Recently, potassium-dependent transcriptome analysis has been reported in Arabidopsis, rice and soybean. Alligator weed is well known, particularly for its strong ability to accumulate potassium. However, the molecular mechanism that underlies potassium starvation responses has not yet been described. In this study, we used Illumina (Solexa) sequencing technology to analyse the root transcriptome information of alligator weed under low potassium stress. Further analysis suggested that 9253 differentially expressed genes (DEGs) were upregulated, and 2138 DEGs were downregulated after seven days of potassium deficiency. These factors included 121 transcription factors, 108 kinases, 136 transporters and 178 genes that were related to stress. Twelve transcription factors were randomly selected for further analysis. The expression level of each transcription factor was confirmed by quantitative RT-PCR, and the results of this secondary analysis were consistent with the results of Solexa sequencing. Enrichment analysis indicated that 10,993 DEGs were assigned to 54 gene ontology terms and 123 KEGG pathways. Approximately 24% of DEGs belong to the metabolic, ribosome and biosynthesis of secondary metabolite KEGG pathways. Our results provide a comprehensive analysis of the gene regulatory network of alligator weed under low potassium stress, and afford a valuable resource for genetic and genomic research on plant potassium deficiency.

Transcriptome analysis reveals dynamic changes in the gene expression of tobacco seedlings under low potassium stress

Potassium plays a key role in plant development and reproduction. In agricultural practice, potassium deficiency is common worldwide, and leads to crop growth inhibition and output reduction. In this study, we analysed the transcriptome of tobacco seedlings under low potassium stress. Tobacco seedlings with or without decreased potassium treatment were harvested after 0 (control), 6, 12, or 24 h and were submitted for microarray analysis. The results showed that up to 3790 genes were upregulated or downregulated more than 2-fold as a result of the decreased potassium treatment. Gene ontology analysis revealed significantly differentially expressed genes that were categorized as cation binding, transcription regulation, metabolic processes, transporter activity and enzyme regulation. Some potassium, nitrogen and phosphorus transporters; transcription factors; and plant signal molecules, such as CPKs were also significantly differentially expressed under potassium deficiency. Our results indicate that the expression profiles of a large number of genes involved in various plant physiological processes are significantly altered in response to potassium deficiency, which can result in physiological and morphological changes in tobacco plants.

Ectopic Expression of AtCIPK23 Enhances Tolerance Against Low-K+ Stress in Transgenic Potato

Potato (Solanum tuberosum L.) has a relatively high requirement for potassium (K+). In the face of the declining of potato yield and quality as a result of reduction of soil K+ content, it is necessary to determine how to improve the tolerance of potatoes to low-K+ stresses. The protein kinase AtCIPK23 is essential for K+ uptake, and its overexpression significantly enhances K+ uptake and tolerance to low-K+ in Arabidopsis. In this research, AtCIPK23 was transformed into an E3 potato cultivar successfully. In low-K+ conditions, the transgenic potato lines had lower levels of leaf chlorosis, higher K+ uptake rate, increased dry weight and K+ content. The non-transgenic lines displayed reduced stature while the transgenic lines exhibited sustained growth. These results indicate that ectopic expression of AtCIPK23 increases low-K+ tolerance in potato. This study demonstrated the potential use of the transgenic approach to increase potato production in low-K+ fields.ResumenLa papa (Solanum tuberosum L.), tiene un requerimiento relativamente alto de potasio (K+). Encarando la reducción en rendimientos y de la calidad de la papa como resultado de una reducción en el contenido de K+ en el suelo, es necesario determinar cómo mejorar la tolerancia de la papa al agobio por bajo K+. La proteína kinasa AtCIPK23 es esencial para la absorción de K+, y su sobreexpresión aumenta significativamente la absorción de K+ y tolerancia a bajo K+ en Arabidopsis. En esta investigación, se incorporó AtCIPK23 a una variedad E3 con éxito. Bajo condiciones de bajo K+, las líneas trangénicas de papa tuvieron niveles más bajos de clorosis foliar, un nivel más alto de absorción de K+, aumento en peso seco y del contenido de K+. Las líneas no trangénicas expresaron altura reducida mientras que las trangénicas mostraron crecimiento sostenido. Estos resultados indican que la expresión ectópica de AtCIPK23 aumenta la tolerancia al bajo K+ en papa. Este estudio demostró el uso potencial del enfoque trangénico para aumentar la producción de papa en campos de bajo K+.

Identifying the Genes Regulated by AtWRKY6 Using Comparative Transcript and Proteomic Analysis under Phosphorus Deficiency

Phosphorus (P) is an important mineral nutrient for plant growth and development. Overexpressing AtWRKY6 (35S:WRKY6-9) was more sensitive and wrky6 (wrky6-1) was more resistant under low Pi conditions. To better understand the function of AtWRKY6 under low phosphate stress conditions, we applied two-dimensional gel electrophoresis (2-DE) to analyse differentially expressed proteins in the shoots and roots between wild type, 35S:WRKY6-9 and wrky6-1 after phosphorus deficiency treatment for three days. The results showed 88 differentially abundant protein spots, which were identified between the shoots and roots of 35S:WRKY6-9 and wrky6-1 plants. In addition, 59 differentially expressed proteins were identified in the leaves and roots of 35S:WRKY6-9 plants. After analysis, 9 genes with W-box elements in their promoter sequences were identified in the leaves, while 6 genes with W-box elements in their promoter sequences were identified in the roots. A total of 8 genes were identified as potential target genes according to the quantitative PCR (QPCR) and two dimension difference gel electrophoresis, (2D-DIGE) results, including ATP synthase, gln synthetase, nitrilase, 14-3-3 protein, carbonic anhydrases 2, and tryptophan synthase. These results provide important information concerning the AtWRKY6 regulation network and reveal potential vital target genes of AtWRKY6 under low phosphorus stress. two dimension difference gel electrophoresis, 2D-DIGE

Identification of low potassium stress-responsive proteins in tobacco (Nicotiana tabacum) seedling roots using an iTRAQ-based analysis.

Potassium is one of the three main mineral nutrients, and is vital for leaf growth and the quality of tobacco (Nicotiana tabacum) plants. In recent years, the isobaric tags for relative and absolute quantitation (iTRAQ) method has been one of the most popular techniques for quantitative proteomic analysis. In this study, we used iTRAQ to compare protein abundances in the roots of control and low potassium-treated tobacco seedlings, and found that 108 proteins were differentially expressed between the two treatments. Of these, 34 were upregulated and 74 were downregulated, and 39 (36%) were in the chloroplasts. Kyoto Encyclopedia of Genes and Genomes pathway enrichment results suggested that metabolic pathways were the dominant pathways (10 upregulated and 14 downregulated proteins). Ten proteins involved in the pyruvate metabolism pathway increased their expression levels, and 17 upregulated proteins were enriched in the ribosomes category. To evaluate correlations between protein and gene transcript abundances, the expression patterns of 12 randomly chosen genes were examined. A quantitative real-time polymerase chain reaction revealed that the 12 genes were induced after low potassium treatment for 3, 6, 12, and 24 h. Our results demonstrate that low potassium levels affect protein profiles in tobacco roots.

Variation of genomic DNA methylation in the nitrate reductase gene of sibling tobacco (Nicotiana tabacum) cultivars.

To better understand genomic DNA methylation in sibling plant cultivars, methylation-sensitive amplification polymorphism analysis was used to investigate two sibling tobacco cultivars, Yunyan85 and Yunyan87, and their two parents, K326 and Yunyan No. 2. Differences in the degree of genomic DNA methylation were found among the four tobacco cultivars. Compared with parents, the two sibling cultivars had fewer methylated sites. Twenty-nine methylation-sensitive amplification polymorphism fragments that exhibited methylation alteration in the four tobacco cultivars were recovered and sequenced. BLAST (nucleotide BLAST) searches showed that two of the 29 sequences have 99% similarity with nucleotides 1442-1694 of the nia-1 gene and the other 27 sequences contain GC, CAAT or TATA box. The nitrate reductase genes from Yunyan87, K326 and Yunyan No. 2 were found to be identical; however, the third intron of the nitrate reductase gene from Yunyan85 was different compared to the third introns of Yunyan87, K326 and Yunyan No. 2. We conclude that methylation alteration of promoter regions could be responsible for the different phenotypes in tobacco and that introns of the nitrate reductase gene can vary as a result of intra-species crossing in tobacco.

Proteome analysis of tobacco leaves reveals dynamic changes in protein expression among different cultivation areas

The leaves of tobacco plants were used to analyze differences in protein content of tobacco grown in the four main flue-cured tobacco-producing areas of Sichuan Province, China. An improved protein extraction method, isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis two-dimensional gel electrophoretic separation, was used to extract and separate total protein from tobacco leaves. Proteomic maps with relatively high resolution and repeatability were produced. At isoelectric points 4 to 7 and molecular weight ranging from 20-100 kDa, we detected 1032, 1030, 1019, and 1011 clearly visible protein spots in tobacco leaves from the four study areas. Proteome comparison between these protein spots showed that 119 spots with a greater than 2-fold change in expression quantity contributed to the variation in expression. Of which, 115 were successfully identified and annotated. According to the annotation results, these proteins participate in photosynthesis, energy metabolism, mineral nutrition, terpene metabolism, defensive reaction, and other physiological and biochemical processes. This study preliminarily explains the effects of ecological conditions on the physiological metabolism of tobacco leaves and how such effects directly or indirectly contribute to tobacco leaf quality.

Different tobacco cultivation regions lead to variations in tobacco leaf gene expression profiles involved in carbonhydrate metabolism and ion transportation

Tobacco is an important crop economically in China, and tobacco quality varies across its cultivated regions. To unveil the mechanisms that result in such varied tobacco leaf qualities, we have analyzed the tobacco leaf gene expression profiles of four areas in Sichuan province, China. Tobacco leaves in the 12 position were collected from plants in four different regions, namely Huili, Miyi, Xingwen and Jiange, and submitted for microarray analysis. Gene expression levels from Miyi group were used as control, and that from Jiange, Xingwen and Huili were compared to Miyi group respectively. A total of 5154 differentially expressed genes (DEGs) were detected, among which 2731 genes were up-regulated and 2423 genes were down-regulated. Based on gene ontology (GO) analysis, significant DEGs were classified into 15 categories (P≤0.05) based on properties pertaining to transferase activity, transmembrane transporter activity, binding, transcription regulator activity, metabolic processes, secondary metabolic processes, etc. Our results also show that these DEGs were significantly enriched in 11 pathways, including metabolic pathways, starch and sucrose metabolism, photosynthesis, etc. Our study suggests that many of the genes involved in various plant physiological and biochemical processes significantly differed across these four different regions, which may explain their differences in tobacco leaf quality.

De novo transcriptome analysis of tobacco seedlings and identification of the early response gene network under low-potassium stress

Tobacco is an economically important crop, and its potassium content can greatly affect the quality of tobacco leaves. However, the molecular mechanism involved in potassium starvation in tobacco has not been elucidated to date. In this study, Illumina (Solexa) sequencing technology was used to analyze the transcriptome of tobacco seedlings under low-potassium stress for 6, 12, and 24 h. After analysis, 107,824 assembled unigenes were categorized into 57 GO functional groups, and 31,379 unigenes (29.08%) were clustered into 25 COG categories. A total of 9945 genes were classified into 233 KEGG pathways, and 15,209 SSRs were found among the 107,824 unigenes. Between the two samples, 1034 genes were differentially expressed. Twelve randomly selected gene expression levels were analyzed by quantitative RT-PCR, and the results were highly consistent with those obtained by Solexa sequencing. Our results provide a comprehensive analysis of the gene-regulatory network of tobacco seedlings under low-potassium stress.