ACTCAT, a novel cis-acting element for proline- and hypoosmolarity-responsive expression of the ProDH gene encoding proline dehydrogenase in Arabidopsis

Abstract

Proline (Pro) is one of the most widely distributed osmolytes in water-stressed plants. We previously isolated from Arabidopsis a gene encoding Pro dehydrogenase (ProDH), a mitochondrial enzyme involved in the first step of the conversion of Pro to glutamic acid. The ProDH gene in Arabidopsis is up-regulated by rehydration after dehydration but is down-regulated by dehydration. ProDH is also induced by L-Pro and hypoosmolarity. The induction of ProDH expression under rehydration seems to be caused by both accumulated Pro and hypoosmolarity. We analyzed a DNA region that is located 5' to the transcription start site (a promoter region) of ProDH to identify cis-acting elements involved in L-Pro-induced and hypoosmolarity-induced expression in transgenic tobacco (Nicotiana tabacum) and Arabidopsis plants. We found that a 9-bp sequence, ACTCATCCT, in the ProDH promoter is necessary for the efficient expression of ProDH in response to L-Pro and hypoosmolarity. Moreover, ACTCAT is a core cis-acting element, which we have called Pro- or hypoosmolarity-responsive element (PRE), that is necessary for L-Pro-responsive and hypoosmolarity-responsive expression of ProDH. Microarray and RNA gel-blot analyses showed that 21 L-Pro-inducible genes have the PRE sequences in their promoter regions. These results indicate that the PRE sequence play an important role in the L-Pro-responsive gene expression.

Figure 1

Nucleotide sequence of the promoter region of ProDH. Underlines show the putative TATA box (TATAA), G-box-like sequence (ACGTG), MYB recognition site (PyAACNPu), MYC recognition site (CANNTG), and as-1 sequence (TGACG). ▪, The direct repeat sequence ACTCATCCT. ▴, Start points of 5′-deleted derivatives. The nucleotide sequence was analyzed with the Genetyx software system (Software Development, Tokyo).

Figure 2

5′-Deletion analysis of the ProDH promoter for the l-Pro- and hypoosmolarity-responsive induction of the LUC reporter gene in transgenic tobacco and Arabidopsis plants. A, Schematics of the 5′-terminal deletions of the ProDH promoter fused to the LUC reporter gene. Arrows indicate 9-bp direct repeat sequences. B, LUC activity in transgenic tobacco plants containing 5′-terminal deletions of the ProDH promoter fused to the LUC gene. T2 seedlings of tobacco were incubated in distilled water (DW) or in 0.09 m l-Pro for 24 h. LUC activity was measured in three plants of seven independent transformant lines for each construct. Multiplication factors of induction of LUC activity (ratio of after treatment to before treatment) by DW and l-Pro treatments are shown at the right. Bars indicate se. C, LUC activity in transgenic Arabidopsis plants containing 5′-terminal deletions of the ProDH promoter fused to LUC. T2 seedlings of Arabidopsis were incubated in DW or 0.09 m l-Pro for 24 h. LUC activity was measured in seven independent transformant lines for each construct. We measured three plants for each line and showed average values.

Figure 3

3′-Deletion analysis of the ProDH promoter for the l-Pro- and hypoosmolarity-responsive induction of the LUC reporter gene in transgenic tobacco and Arabidopsis plants. A, Schematics of the 3′-terminal deletions of promoters fused to the LUC reporter gene. Arrows indicate 9-bp direct repeat sequences. B, LUC activity in transgenic tobacco plants containing 3′-terminal deletions of the ProDH promoter fused to LUC. T1 leaves of tobacco were incubated in DW or in 0.09 m l-Pro for 24 h. LUC activity was measured in 20 independently obtained transgenic plants for each construct. Multiplication factors of induction of LUC activity by DW and l-Pro treatments are shown on the right. Bars indicate se. C, LUC activity in transgenic Arabidopsis plants containing 3′-terminal deletions of the ProDH promoter fused to the LUC reporter gene. T2 seedlings of Arabidopsis were incubated in DW or 0.09 m l-Pro for 24 h. LUC activity was measured in seven independent transformant lines for each construct. We measured three plants for each line and showed average values.

Figure 4

Base substitution analysis of the 70-bp region from positions −730 to −661 of the ProDH promoter for the l-Pro- and hypoosmolarity-responsive induction of LUC in transgenic tobacco plants. A, Upper strand sequences of the 70-bp fragment of the ProDH promoter and its mutated sequences (M1–M8). Each fragment containing each mutation was ligated to the −42 ProDH minimal TATA promoter-LUC construct. Dashes indicate the sequence of the 3D6 construct. B, Effect of base substitutions in the direct repeat sequence for l-Pro- and hypoosmolarity-responsive expression of ProDH. T1 leaves of tobacco were incubated in DW or 0.09 m l-Pro for 24 h. LUC activity was measured in 12 to 26 leaves of independent transformant lines for each construct and is shown as average values. Bars indicate se.

Figure 5

GUS activity and histochemical analysis in transgenic Arabidopsis plants containing the 1.4-kb ProDH promoter or the 3D5 fragment fused to the GUS reporter gene after treatment with DW or l-Pro. A, GUS activity in transgenic Arabidopsis containing the 1.4-kb promoter-GUS fusion gene before and after treatment with DW or 0.09 m l-Pro for 24 h. GUS activity was measured in two independent transformant lines for each construct. We measured five plants for each line and showed average values. B, GUS activity in transgenic Arabidopsis containing the 3D5 fragment fused to the GUS reporter gene instead of the LUC reporter gene before and after treatment with DW or 0.09 m l-Pro for 24 h. GUS activity was measured in three independent transformant lines for each construct. We measured five plants for each line and showed average values. C through E, Control plants containing the 1.4-kb promoter-GUS fusion gene grown for 2 weeks and stained for 4 h. F through H, 3D5 constructs containing the GUS reporter gene instead of the LUC reporter gene grown for 14 d and stained overnight. C and F, Before treatment; D and G, after treatment with DW for 24 h; E and H, after treatment with 0.09 m l-Pro for 24 h.

Figure 6

RNA gel-blot analysis in transgenic Arabidopsis plants containing the 1.4-kb ProDH promoter, 3D5, 3D6, M3, M4, M5, or M6 fragments fused to the GUS reporter gene after treatment with DW, GM, 0.09 m l-Pro, or 0.09 m d-Pro for 2 h or dehydration for 2 h. The 35S-GUS construct contains the CaMV 35S-promoter and the GUS reporter gene.

Figure 7

RNA gel-blot analysis of l-Pro-inducible genes verified by using the full-length cDNA microarray. RNA samples from Arabidopsis plants transferred from GM plates to GM solution (GM) or GM plates to 0.09 m l-Pro solution (l-Pro) for 1, 2, 5, 10, and 24 h and untreated plants (control) were hybridized with PCR-amplified or sfiI-digested DNA fragments as probes.

Figure 8

A model for the induction of ProDH under rehydration. ProDH expression is up-regulated by rehydration after dehydration, through l-Pro accumulation and hypoosmolarity. The transcription factor (TF) may be activated by Pro signaling or hypoosmolarity and bind to the ACTCAT sequence. The expression of ProDH may then be induced.