Identification of differential expression genes in leaves of rice (Oryza sativa L.) in response to heat stress by cDNA-AFLP analysis

Abstract

High temperature impedes the growth and productivity of various crop species. To date, rice (Oryza sativa L.) has not been exploited to understand the molecular basis of its abnormally high level of temperature tolerance. To identify transcripts induced by heat stress, twenty-day-old rice seedlings of different rice cultivars suffering from heat stress were treated at different times, and differential gene expression analyses in leaves were performed by cDNA-AFLP and further verified by real-time RT-PCR. In aggregate, more than three thousand different fragments were indentified, and 49 fragments were selected for the sequence and differential expressed genes were classified functionally into different groups. 6 of 49 fragments were measured by real-time RT-PCR. In addition, the variations of three different polyamine contents in response to heat stress through high-performance liquid chromatography (HPLC) analysis were also performed. The results and their direct and indirect relationships to heat stress tolerance mechanism were discussed.

Figure 1

Behavior of rice seedlings (Shuanggui 1). (a) 28°C day/22°C night treatment; (b) 39°C day/30°C night treatment; (c) The comparison of plant height after 48 h at 39°C. Data from plant heights are means of three replications (50 plants per replication) ± SE. Dissimilar letters above bars differ significantly at P ≤ 0.05.

Figure 2

Relative expression of Os04g0429600, Os06g0124900, SRP14, Os02g0611200, Os02g0788800, and Os07g0620200 compared to that of actin detected by real-time quantitative RT-PCR in plants at 28/22°C or plants at 39/30°C for 24 hours and 48 hours in heat-sensitive Shuanggui 1 and heat-tolerant Huanghuazhan. Values indicate relative expression levels against those of the same genes at 28/22°C in three biological replications from cDNA prepared from leaves. Os04g0429600, cellulose synthase-like protein H1; Os06g0124900, hypothetical protein; SRP14, signal recognition particle subunit 14; Os02g0611200, S-adenosylmethionine decarboxylase proenzyme (AdoMetDC) (SamDC); Os02g0788800, amino acid/polyamine transporter II; Os07g0620200, heat shock protein DnaJ, N-terminal domain containing protein.

Figure 3

Variation of levels of free spermidine, spermine, and putrescine in rice seedlings under 39°C and 28°C treatments with HPLC analysis. Data from levels of free spermidine, spermine, and putrescine in rice seedlings are means of three replications ± SE. Dissimilar letters above bars differ significantly at P ≤ 0.05.