Comprehensive expression profile analysis of the Arabidopsis Hsp70 gene family

Abstract

We isolated cDNA clones for two nuclear-encoded, organellar members of the Arabidopsis hsp70 gene family, mtHsc70-2 (AF217458) and cpHsc70-2 (AF217459). Together with the completion of the genome sequence, the hsp70 family in Arabidopsis consists of 14 members unequally distributed among the five chromosomes. To establish detailed expression data of this gene family, a comprehensive reverse transcriptase-polymerase chain reaction analysis for 11 hsp70s was conducted including analysis of organ-specific and developmental expression and expression in response to temperature extremes. All hsp70s showed 2- to 20-fold induction by heat shock treatment except cpHsc70-1 and mtHsc70-1, which were unchanged or repressed. The expression profiles in response to low temperature treatment were more diverse than those evoked by heat shock treatment. Both mitochondrial and all cytosolic members of the family except Hsp70b were strongly induced by low temperature, whereas endoplasmic reticulum and chloroplast members were not induced or were slightly repressed. Developmentally regulated expression of the heat-inducible Hsp70 in mature dry seed and roots in the absence of temperature stress suggests prominent roles in seed maturation and root growth for this member of the hsp70 family. This reverse transcriptase-polymerase chain reaction analysis establishes the complex differential expression pattern for the hsp70s in Arabidopsis that portends specialized functions even among members localized to the same subcellular compartment.

Figure 1

Chromosomal location of hsp70 genes in Arabidopsis. Hsp70 genes were positioned on a sequence map created by the Arabidopsis Genome Initiative (AGI, http://www.Arabidopsis.org/agi.html). This map contains molecular markers as well as genetic markers.

Figure 2

Sequence alignment of chloroplast and mitochondrial hsp70 proteins. Arabidopsis plastid hsp70s (cpHsc70-1, cpHsc70-2) were aligned with chloroplast hsp70s from pea, spinach, and cucumber. Arabidopsis mitochondrial hsp70s (mtHsc70-1, mtHsc70-2) were aligned with mitochondrial hsp70s from potato, bean, pea, and spinach. Black shading indicates consensus residues common to both chloroplast and mitochondrial hsp70s. Gray shading indicates consensus residues specific in either chloroplast or mitochondrial hsp70s. Brackets indicate the beginning of the N-terminal, highly conserved ATP-binding motif. Underlined residues are C-terminal signature motifs for organelle localization.

Figure 3

Rooted Neighbor-Joining analysis using the CLUSTAL program in DNASTAR for protein sequences of the Arabidopsis hsp70 gene family. Full-length protein sequences of 12 Arabidopsis hsp70 proteins and two truncated sequences (Hsp70t-1, Hsp70t-2) were used in this analysis. The scale at the bottom represents the branch distance as the number of changes in character states between neighbors. Each shaded area represents subcellular localization; from the top, cytosol, ER, mitochondrion, and chloroplast.

Figure 4

RT-PCR optimization. Equivalent increase of duplex Hsc70-1 and 18S rRNA signal in the range of 1 to 256 ng of total RNA was tested. The 16-ng amount of total RNA was selected for subsequent experiments. *, RT-PCR band for Hsc70-1. **, RT-PCR band for 18S rRNA.

Figure 5

Response of cytosolic hsp70s to temperature extremes. Two-week-old Arabidopsis plants were subjected to 4°C for 12 and 48 h for low temperature treatment or 40°C for 30, 60, and 90 min for heat shock treatment. Control plants (C) were incubated at 20°C, simultaneously. Signal values obtained from each gene were normalized with the 18S rRNA signal value, and the resulting mean values were presented as relative units. Error bar represents sd.

Figure 6

Response of organellar hsp70s to temperature extremes. Empty space indicated by a white line in the gel pictures of cpHsc70-1 and mtHsc70-2 was cut out to achieve uniform spatial arrangement of the images.

Figure 7

Predicted cis elements in the promoters of Arabidopsis hsp70 genes. Promoter sequences for 11 Arabidopsis hsp70 genes, a cold-inducible gene (Rd29A/Cor78), and a heat-inducible gene (Hsp17.4) were analyzed. The numbers at the bottom indicate the number of nucleotides upstream to the translation initiation codon, ATG. Induction fold of each gene in response to heat and cold are indicated as solid diamonds; one diamond, less than 5-fold; two diamonds, 5- to 10-fold; and three diamonds, more than 10-fold. ●, Perfect HSE (nTTCnnGAAnnTTCn or nGAAnnTTCnnGAAn). ○, Imperfect HSE. ▴, The core sequence of CRT/DRE (CCGAC). ▪, TATA box. Only a small portion (approximately 200 bp) of the promoter region for Hsp70 was available at the time of analysis.

Figure 8

Expression of cytosolic hsp70s during seed maturation and germination. Silique samples (S1, S2) were harvested from 5- and 6-week-old plants. S1, Silique at 7 DAP; S2, silique at 14 DAP; Sd, mature dry seed. Samples were also harvested at 6, 12, 24, 48, and 96 h after imbibition.

Figure 9

Expression of organellar hsp70s during seed maturation and germination.

Figure 10

Expression of cytosolic hsp70s in different organs. Rt, Root; Lf, leaf; St, stem; Fl, flower; Si, silique at 3 DAP.

Figure 11

Expression of organellar hsp70s in different organs. Rt, Root; Lf, leaf; St, stem; Fl, flower; Si, silique at 3 DAP.