We isolated a cDNA clone, named TLHS-1, for a low molecular weight heat-shock protein (LMW HSP) from tobacco. The nucleotide sequence determination of the clone identified an open reading frame for 159 amino acids. To the upstream of the open reading frame, a sequence of 124 nucleotides was determined. To the 3' downstream of the open reading frame, 212 nucleotides were identified which carried a poly(A) tail. A comparison of the open reading frame of TLHS-1 with the previously reported class I LMW HSPs showed a high identity which classified the TLHS-1 as a class I LMW HSP cDNA clone. Reexamination of the amino acid sequences for LMW HSPs including the TLHS-1 argued for the necessity to redraw the consensus region. Six consensus regions were proposed instead of the two consensus regions suggested previously. A RNA blot hybridization for TLHS-1 showed the typical expression pattern of a heat-shock inducible gene from 3 common tobacco cultivars. The genomic DNA blot hybridization for the TLHS-1 gene from the 3 tobacco cultivars showed identical patterns. These results implied that the expression control and structure of the TLHS-1 gene have been well conserved in the tobacco cultivars which are not bred for high temperature stress resistance.

In plants small heat shock proteins (sHsp) are abundantly expressed upon heat stress in vegetative tissue, however, sHsp expression is also developmentally induced in pollen. The developmental induction of sHsp has been related to the potential for stress-induced microspore embryogenesis. We investigated the polymorphism among sHsp and their expression during pollen development and after heat stress in tobacco. Real-time RT-PCR was used for quantification of mRNA of two known and nine newly isolated cDNAs representing cytosolic sHsp. At normal temperature most of these genes are not transcribed in vegetative tissues, however, all genes were expressed during pollen development. Low levels of mRNAs were found for sHsp-1A and -1B in early-unicellular stage, increasing four to sevenfold in mature pollen. Nine other genes are up-regulated in unicellular and down-regulated in bicellular pollen; three these genes show stage-specific expression. Western analysis revealed that cytosolic class I and II sHsp are developmentally expressed during all stages of pollen development. Different subsets of cytosolic sHsp genes are expressed in a stage-specific fashion suggesting that certain sHsp genes may play specific roles in early, others during later stages of pollen development. Heat stress results in a relatively weak and incomplete response in pollen: (i) the heat-induced levels of mRNA (excepting sHsp-2B, -3C and -6) are much lower than in leaves, (ii) several sHsp are not detected after heat stress in pollen, although, they are heat-inducibly expressed in leaves. Application of heat stress, cold, and starvation, which induce microspore embryogenesis, modify mRNA levels and the patterns of 2-D-separated sHsp, but only heat stress enhances the expression of sHsp in microspores. There is no correlation of the expression of specific sHsp with the potential for microspore embryogenesis.