Temperature stress differentially modulates transcription in meiotic anthers of heat-tolerant and heat-sensitive tomato plants

Abstract

Background: Fluctuations in temperature occur naturally during plant growth and reproduction. However, in the hot summers this variation may become stressful and damaging for the molecular mechanisms involved in proper cell growth, impairing thus plant development and particularly fruit-set in many crop plants. Tolerance to such a stress can be achieved by constitutive gene expression or by rapid changes in gene expression, which ultimately leads to protection against thermal damage. We have used cDNA-AFLP and microarray analyses to compare the early response of the tomato meiotic anther transcriptome to moderate heat stress conditions (32°C) in a heat-tolerant and a heat-sensitive tomato genotype. In the light of the expected global temperature increases, elucidating such protective mechanisms and identifying candidate tolerance genes can be used to improve breeding strategies for crop tolerance to heat stress.

Results: The cDNA-AFLP analysis shows that 30 h of moderate heat stress (MHS) alter the expression of approximately 1% of the studied transcript-derived fragments in a heat-sensitive genotype. The major effect is gene down-regulation after the first 2 h of stress. The microarray analysis subsequently applied to elucidate early responses of a heat-tolerant and a heat-sensitive tomato genotype, also shows about 1% of the genes having significant changes in expression after the 2 h of stress. The tolerant genotype not only reacts with moderate transcriptomic changes but also exhibits constitutively higher expression levels of genes involved in protection and thermotolerance.

Conclusion: In contrast to the heat-sensitive genotype, the heat-tolerant genotype exhibits moderate transcriptional changes under moderate heat stress. Moreover, the heat-tolerant genotype also shows a different constitutive gene expression profile compared to the heat-sensitive genotype, indicating genetic differences in adaptation to increased temperatures. In the heat-tolerant genotype, the majority of changes in gene expression is represented by up-regulation, while in the heat-sensitive genotype there is a general trend to down-regulate gene expression upon MHS. The putative functions associated with the genes identified by cDNA-AFLP or microarray indicate the involvement of heat shock, metabolism, antioxidant and development pathways. Based on the observed differences in response to MHS and on literature sources, we identified a number of candidate transcripts involved in heat-tolerance.

Figure 1

cDNA-AFLP gene expression profiling. Example of cDNA-AFLP profiling. Bands of interest are shown with circles. Panels I through VI represent individual cDNA-AFLP primer combinations. TDFs are displayed as bands over 5 different timpoints (1-5) under standard conditions (ST) and under MHS. Various different forms of differentially displayed fragments are shown (A through F).

Figure 2

Validation of the cDNA-AFLP profiling with q-PCR. Four TDFs (Panel A) corresponding to TC211882, TC191544, TC197647 and TC213983 were verified with quantitative q-PCR in the genotype Money Maker (MM; Panel B) at time points 0 h, 2 h, 6 h, 16 h and 30 h under standard growth and MHS conditions. To further analyze the expression of the same genes in the heat-sensitive (FR) and heat-tolerant (HS1) genotypes, q-PCRs were also carried out on RNA from FR and HS1, for the time points 0 h, 2 h and 6 h of MHS (Panel C). The bars represent expression values derived from the delta CT values and error bars are given as standard deviation.

Figure 3

Tomato phenotypes under heat stress. Comparison of flower and anther development under control and MHS conditions (two weeks) in the tolerant genotype HS1 and the sensitive FR. Panels a, d, g, j: whole flowers; panels b, e, h and k: isolated anther cones; panels c, f, i and l: germinating pollen. Size bars represent 10 and 3 mm respectively.

Figure 4

O2PLS analysis of the components of variation in gene expression in the microarray experiment. The main components of the changes in gene expression in response to MHS were determined using an O2PLS analysis with SIMCA P+. The plots of predictive component (T2, T3 and T4) versus orthogonal component 1 (T1) are presented. HS1-0a, b and c, HS1-2a, b and c, HS1-6a, b and c, FR-0a, b and c, FR-2a, b and c and FR-6a, b and c represent the three biological replicates (a, b and c) for each experimental time-point (0, 2 and 6) for each genotype (HS1 and FR). Each component clearly discriminates between the two genotypes.

Figure 5

Euclidian clustering of significantly differentially expressed genes in the heat-tolerant genotype (HS1) and the heat-sensitive genotype (FR). Heat maps of gene expression are shown with high expression shown in shades of red and low expression shown in blue. A dendogram of the expression profiles is shown to the left in HS1 (Heat Set 1) and the right in FR (Falcorosso). On either side of the heat maps, the TC number of the relevant probe is shown and the functional classification of the relevant gene. TCs common to both genotypes are indicated with lines linking the heat maps.

Figure 6

Pie charts of the probable functional categorisation of the transcripts found to be significantly differentially expressed in the cDNA-AFLP and microarray experiment. The two charts reflect the general (SAM multiclass) and in depth (SAM Paired) moderate temperature stress response in HS1, the heat-tolerant, and FR, the heat-sensitive genotype. The functional classes are colour coded.

Figure 7

Validation of the microarray profiling with q-PCR. Candidate genes in heat-sensitive and heat-tolerant genotypes. Five transcripts corresponding to TC170030, TC171292, TC190555, TC185448 and TC170322 were verified with q-PCR in two pairs of contrasting genotypes: HS1 (heat-tolerant): FR(heat-sensitive), and Saladette (heat-tolerant): Pull (heat-sensitive), subjected to the same stress conditions at the same time points of 0 h, 2 h and 6 h of MHS.

Figure 8

Temperature regime for moderate heat stress (MHS) used in the cDNA-ALFP profiling (top) and in microarray experiment (bottom). Black lines represent the day/night temperatures prior to application of the MHS (acclimation). Sampling times are shown as hours after experimental onset below the temperature line. Red lines indicate the heat stress period with sampling times given as hours after onset of the MHS.