Transcriptomic and proteomic approach to identify differentially expressed genes and proteins in Arabidopsis thaliana mutants lacking chloroplastic 1 and cytosolic FBPases reveals several levels of metabolic regulation

Abstract

BackgroundDuring the photosynthesis, two isoforms of the fructose-1,6-bisphosphatase (FBPase), the chloroplastidial (cFBP1) and the cytosolic (cyFBP), catalyse the first irreversible step during the conversion of triose phosphates (TP) to starch or sucrose, respectively. Deficiency in cyFBP and cFBP1 isoforms provokes an imbalance of the starch/sucrose ratio, causing a dramatic effect on plant development when the plastidial enzyme is lacking.ResultsWe study the correlation between the transcriptome and proteome profile in rosettes and roots when cFBP1 or cyFBP genes are disrupted in Arabidopsis thaliana knock-out mutants. By using a 70-mer oligonucleotide microarray representing the genome of Arabidopsis we were able to identify 1067 and 1243 genes whose expressions are altered in the rosettes and roots of the cfbp1 mutant respectively; whilst in rosettes and roots of cyfbp mutant 1068 and 1079 genes are being up- or down-regulated respectively. Quantitative real-time PCR validated 100% of a set of 14 selected genes differentially expressed according to our microarray analysis. Two-dimensional (2-D) gel electrophoresis-based proteomic analysis revealed quantitative differences in 36 and 26 proteins regulated in rosettes and roots of cfbp1, respectively, whereas the 18 and 48 others were regulated in rosettes and roots of cyfbp mutant, respectively. The genes differentially expressed and the proteins more or less abundant revealed changes in protein metabolism, RNA regulation, cell signalling and organization, carbon metabolism, redox regulation, and transport together with biotic and abiotic stress. Notably, a significant set (25%) of the proteins identified were also found to be regulated at a transcriptional level.ConclusionThis transcriptomic and proteomic analysis is the first comprehensive and comparative study of the gene/protein re-adjustment that occurs in photosynthetic and non-photosynthetic organs of Arabidopsis mutants lacking FBPase isoforms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0945-7) contains supplementary material, which is available to authorized users.

Transcriptional Regulation of the Glucose-6-Phosphate/Phosphate Translocator 2 Is Related to Carbon Exchange Across the Chloroplast Envelope

“…Plants lacking the stromal FBP aldolase have higher cyclic electron flow consistent with this hypothesis (Gotoh et al, 2010) although it is not known if they have increased expression of GPT2. In another study using a chloroplast FBPase mutant, elevated GPT2 transcript was seen but only in the roots (Soto-Suárez et al, 2016). The reasons for the spatial difference in GPT2 expression observed between our study and that one are unclear.…”

Secondary Wall Regulating NACs Differentially Bind at the Promoter at a CELLULOSE SYNTHASE A4 Cis-eQTL

“…While transcript analysis is a powerful tool and can provide invaluable data on genome regulation, stress response, and more, mRNA accumulation alone is not enough to draw definitive conclusions about protein expression. Indeed, the global correlation between the transcriptome and proteome in both prokaryotes and eukaryotes has been found to be weak, at best (Gygi et al, 1999; Washburn et al, 2003; Soto-Suárez et al, 2016). Posttranscriptional regulation mechanisms, half-life, and localization and interactions all may play a role in protein expression levels, causing them to differ from transcript abundance.…”

Proteomic Analyses of Thioredoxins f and m Arabidopsis thaliana Mutants Indicate Specific Functions for These Proteins in Plants

“…The final pellet was suspended in 600 μL of protein solubilization buffer (9 M urea, 4% 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS), 0.5% TritonX100, and 100 mM dithiothreitol (DTT)). Protein content was quantified by the method of Bradford [19], using bovine serum albumin (BSA) as standard. Three technical replicates of the quantified protein were performed per sample [20].…”

“…IEF, 2-D electrophoresis, gel staining, image capture, protein spot digestion and MALDI (Matriz-Assisted Laser Desorption/Ionizacion)-TOF (Time of flight) were analyzed in the Universidad of Córdoba UCO-SCAI proteomics facility (Córdoba, Spain), a member of Carlos III Networked Proteomics Platform, ProteoRed-ISCIII. The methodology of Soto et al was followed [19] as below. Isoelectrofocusing (IEF) was carried out on Precast 17 cm IPG pH 5–8 linear gradient (Bio-Rad, Hercules, CA, USA) strips.…”