Transcriptome changes in Arabidopsis expressing Vitreoscilla hemoglobin under hypoxic and NO donor treatments.

Hypoxia, a naturally occurring phenomenon, results in tremendous losses in the growth of plants and in their productivity. On the physiological level oxygen deprivation results in distinct metabolic rearrangements, is accompanied by dehydration, and oxidative and nitrosative stresses. Six to seven core hypoxia-responsive genes, which are always induced under oxygen deprivation, have been shown to include hemoglobin 1 (Hb1) across a wide range of plant species. However, the function of this induced Hb1 is not completely understood .The importance of elevated Hb expression has been attributed mainly to its nitric oxide (NO) scavenging properties, and hence, to the elimination of this free-radical and signaling species. NO is known to accumulate under hypoxia and its level is in reciprocal relationship with the Hb expression levels. However, evidence has accumulated on Hbs as essential for normal plant development and they take part in the regulation of e.g. flowering, germination and lateral and adventitious root emergence. The physiological role of induced hemoglobin (Hb) expression and its role in NO scavenging were assessed by introducing bacterial (Vitreoscilla stercoraria) hemoglobin (VHb) to the cytoplasm and mitochondria of Arabidopsis thaliana. Heterologous expression was chosen to avoid interference with developmental functions of endogenous Hbs. Sixteen day old transformants were subjected to hypoxia or to NO donor treatment (DETA, 500 µM) for 2h and 24h and global changes in gene expression were assessed using a microarray approach. Both hypoxic and NO-treatment experiments were aimed also to reveal novel functions for Hb under abiotic stress conditions, exemplified by oxygen deprivation and NO/oxidative stresses and during normal development of Arabidopsis transformants on the background of normally expressed endogenous Hbs Hypoxia, a naturally occurring phenomenon, results in tremendous losses in the growth of plants and in their productivity. On the physiological level oxygen deprivation results in distinct metabolic rearrangements, is accompanied by dehydration, and oxidative and nitrosative stresses. Six to seven core hypoxia-responsive genes, which are always induced under oxygen deprivation, have been shown to include hemoglobin 1 (Hb1) across a wide range of plant species. However, the function of this induced Hb1 is not completely understood .The importance of elevated Hb expression has been attributed mainly to its nitric oxide (NO) scavenging properties, and hence, to the elimination of this free-radical and signaling species. NO is known to accumulate under hypoxia and its level is in reciprocal relationship with the Hb expression levels. However, evidence has accumulated on Hbs as essential for normal plant development and they take part in the regulation of e.g. flowering, germination and lateral and adventitious root emergence. The physiological role of induced hemoglobin (Hb) expression and its role in NO scavenging were assessed by introducing bacterial (Vitreoscilla stercoraria) hemoglobin (VHb) to the cytoplasm and mitochondria of Arabidopsis thaliana. Heterologous expression was chosen to avoid interference with developmental functions of endogenous Hbs. Sixteen day old transformants were subjected to hypoxia or to NO donor treatment (DETA, 500 µM) for 2h and 24h and global changes in gene expression were assessed using a microarray approach. Both hypoxic and NO-treatment experiments were aimed also to reveal novel functions for Hb under abiotic stress conditions, exemplified by oxygen deprivation and NO/oxidative stresses and during normal development of Arabidopsis transformants on the background of normally expressed endogenous Hbs Three lines of Arabidopsis thaliana (Landsberg) were used: wild type WT; line C13, expressing Vitreoscilla hemoglobin (VHb) in the cytoplasm; line Mt62 – expressing VHb in mitochondria. Analysis used total RNA isolated from the shoots of 16 days old control plants (at 0h = 16 day-old plants, start of experiment, and after 2h and 24h) and from the experimental plants after hypoxic treatment ( 4% O2 for 2h and 24h) and DETA treatment ( 500 µm, 2h and 24 h incubation after the onset of NO release). Treatments were performed separately. Indirect comparisons were made across multiple arrays with raw data pulled from different channels for data analysis and comparison to the control data. Each experimental variant had four biological replicates randomly labelled with three different dyes: Cy3, HyPer5 and Alexa Fluor 488 (See 'Arrays layout' tab). Each microarray field was used to hybridize three different samples labelled with three different dyes. For each experimental variant there are four raw data files = 4 replicates. Name of the raw data file includes: First 13 digits - Agilent slide number; AxFy - array x, field y; Hy5, Cy3, Al - dyes Hyper 5, Cy3, Alexa 488; WT - wild type, C13 – line expressing VHb in the cytosol; Mt62 – line expressing VHb in mitochondria; c – control; H- hypoxic treatment; DETA - treatment with NO donor DETA; 0h, 2h, 24h - duration of treatment, hours. Gene expression data normalization was performed by R language and BioConductor Packages. First the data was normalized using quantile normalization and in-house modified version of COMBAT software. We selected significantly regulated probes from the normalized data using Empirical Bayes T-test. Next, probe-level scores were summarized for each gene by taking average of scores of probes targeting the same gene. In the matrix table LOG FCs which have a T score p value > 0.01, are disregarded and marked as ‘null’. GPR files: Agilent-015059 Arabidopsis 3 Oligo Microarray 4x44K G2519F, 7 slides AxFy = array x, field y; Hy5, Cy3, Al = dyes Hyper 5, Cy3, Alexa 488; WT, C13, Mt62 = wild type, cytoplasmic VHb line 1:3 , mitochondrial VHb line 6:2; Ctrl = control; H = hypoxic treatment; DETA = NO donor DETA (500 µM) treatment; 0h, 2h, 24h, = duration of the treatment, hours;