Comprehensive transcriptome analysis unravels the existence of crucial genes regulating primary metabolism during adventitious root formation in Petunia hybrida

To identify specific genes determining the initiation and formation of adventitious roots (AR), a microarray-based transcriptome analysis in the stem base of the cuttings of Petunia hybrida (line W115) was conducted. A microarray carrying 24,816 unique, non-redundant annotated sequences was hybridized to probes derived from different stages of AR formation. After exclusion of wound-responsive and root-regulated genes, 1,354 of them were identified which were significantly and specifically induced during various phases of AR formation. Based on a recent physiological model distinguishing three metabolic phases in AR formation, the present paper focuses on the response of genes related to particular metabolic pathways. Key genes involved in primary carbohydrate metabolism such as those mediating apoplastic sucrose unloading were induced at the early sink establishment phase of AR formation. Transcriptome changes also pointed to a possible role of trehalose metabolism and SnRK1 (sucrose non-fermenting 1- related protein kinase) in sugar sensing during this early step of AR formation. Symplastic sucrose unloading and nucleotide biosynthesis were the major processes induced during the later recovery and maintenance phases. Moreover, transcripts involved in peroxisomal beta-oxidation were up-regulated during different phases of AR formation. In addition to metabolic pathways, the analysis revealed the activation of cell division at the two later phases and in particular the induction of G1- specific genes in the maintenance phase. Furthermore, results point towards a specific demand for certain mineral nutrients starting in the recovery phase

Cluster analysis.

<p>Two major clusters of genes specifically induced during different phases of AR formation created via K-means clustering method by Genesis software. Three types of associated expression graphs of each cluster are shown. SEP: Sink Establishment Phase; RP: Recovery Phase; MP: Maintenance Phase.</p

Schematic illustration of metabolic pathways regulated during AR formation in petunia cuttings.

<p>In the figure, only important induced key genes in primary metabolism, cell replication machinery, signalling and membrane transport are included. Induced genes are shown in red color. Because the observed gene expression patterns in R and M phases were not significantly different and to simplify the figure, only two phases have been illustrated. (<b>A</b>) SE phase (<b>B</b>) R and M phases. NT: nitrate transporter, PT: phosphate transporter, Zn/Fe.T: zinc/iron transporter, Cyt. Inv: cytosolic invertase; CW Inv: cell wall invertase; SuT: sucrose transporter; PFK: phosphofructokinase, HXK: hexokinase, RNR: ribonucleotide reductase; PEPC: phosphoenolpyruvate carboxylase; H.A.CoA DH: 3-hydroxyacyl-CoA dehydrogenase; E.CoA hydratase: enoyl-CoA hydratase; Pro: proline; ProDH: proline dehydrogenase; P5CDH: pyrroline-5-carboxylate dehydrogenase; Met: methionine; SAMS: S-adenosylmethionine synthase; SAM: S-adenosylmethionine.</p

Negative correlation between the significance of time and replicate effects.

<p>Plot of -Log10 of the significance of the replicate effect (Y-axis) versus the -Log10 of the significance of the time effect (X-axis), indicating a much stronger time effect than undesired variation between replicates. Minimum P-value within replicates of all time points is located on the abscissa (X-axis) and minimum P-value of all ratios compared to 0hpe is located on the ordinate (Y-axis).</p

Comparison of fold change differences of seven genes specifically induced during different phases of AR formation based on microarray and Real-Time qPCR.

<p>Each value in qPCR data is represented by the mean of three independent replicates ± standard error. Sequence ID for each gene is: pectinase (GO_drpoolB-CL9530Contig1), glucose transporter (cn1111), nitrate transporter (cn8317), F-box family protein (IP_PHBS008L07u), ubiquitin-protein ligase (IP_PHBS007P04u), trehalose-p-phosphatase (cn3641) and zinc/iron transporter (cn5371).</p

cDNA library of petunia cuttings.

<p>Classification of putative proteins into different categories of metabolism during various developmental stages of adventitious root formation in petunia cuttings. Numbers of putative proteins in different categories of the KEGG superpathway are shown in parentheses. Total protein number was 607 (out of 4,700 EST‘s).</p

Fold change ratios of mineral nutrient transporters during AR formation in petunia cuttings.

<p>M values (Log2 of fold change ratios) of genes involved in mineral nutrient transport and processing during various phases of AR formation in petunia cuttings compared to the time point 0 hpe. SEP: Sink Establishment Phase; RP: Recovery Phase; MP: Maintenance Phase. Sequence ID for each gene in sequential order as illustrated in the figure: high affinity nitrate transporters (GO_drpoolB-CL2736 Contig1, GO_drs31P0006M14_R_ab1), nitrate transporters (cn1356, cn7095, GO_drpoolB-CL8921Contig1), phosphate transporter 1 (cn1272, cn8235), phosphate transporter 5 (cn8236), sulfate adenylyltransferases (GO_drpoolB-CL2256Contig1, GO_drpoolB-CL4792Contig1), sulfate transporter (cn6307), zinc/iron transporters (cn5371, GO_dr004P0016I22_F_ab1), ATZIP4 zinc transporter (IP_PHBS009K04u), ferric-chelate reductase (GO_drpoolB-CL1069Contig 1), high-affinity K+ transporter (cn7074), NRAMP metal ion transporter (GO_drpoolB-CL1711Contig1). Significant changes are indicated by asterisks.</p