Water deficit triggers phospholipase D activity in the resurrection plant Craterostigma plantagineum

Abstract

Phospholipids play an important role in many signaling pathways in animal cells. Signaling cascades are triggered by the activation of phospholipid cleaving enzymes such as phospholipases C, D (PLD), and A(2). Their activities result in the formation of second messengers and amplification of the initial signal. In this study, we provide experimental evidence that PLD is involved in the early events of dehydration in the resurrection plant Craterostigma plantagineum. The enzymatic activity of the PLD protein was activated within minutes after the onset of dehydration, and although it was not inducible by abscisic acid, PLD activity did increase in response to mastoparan, which suggests a role for heterotrimeric G proteins in PLD regulation. Two cDNA clones encoding PLDs, CpPLD-1 and CpPLD-2, were isolated. The CpPLD-1 transcript was constitutively expressed, whereas CpPLD-2 was induced by dehydration and abscisic acid. Immunological studies revealed changes in the subcellular localization of the PLD protein in response to dehydration. Taken together, the data on enzymatic activity as well as transcript and protein distributions allowed us to propose a role for PLD in the events leading to desiccation tolerance in C. plantagineum.

Figure 1.

Dehydration Stimulates PLD Activity. (A) Leaf discs from C. plantagineum were prelabeled with ³²Pi for 16 hr, removed from the labeling buffer, and dried for the times indicated. To measure PLD activity, we again incubated them in buffer that included <0.75% n-butanol (ButOH). After 5 min of vacuum infiltration, the discs were incubated for 25 min at room temperature. (B) Autoradiography of a TLC plate with the separated phospholipids extracted from one leaf disc, showing the extent of PtdButOH and PtdOH formation in response to dehydration. The positions of PtdButOH and PtdOH are indicated. C, control disc. (C) Quantification of PtdButOH and PtdOH by using a PhosphorImager. The amount of each phospholipid is expressed as fold stimulation relative to that in the control. The horizontal lines in the graph indicate the amount of PtdButOH (top) and PtdOH (bottom) measured in the control discs. The values are means of two independent experiments. The error bars indicate the standard deviations. (D) Leaf discs of C. plantagineum were treated as described in (A) but were sampled after different durations of dehydration. (E) Autoradiography of the TLC plate after separation of the phospholipids (cf. [B]). (F) Quantification of PtdButOH and PtdOH by using a PhosphorImager (cf. [C]).

Figure 2.

PLD Stimulation Is Mediated by Heterotrimeric G Proteins. (A) Leaf discs of C. plantagineum were prelabeled with ³²Pi for 16 hr and incubated in the presence of 25 μM mastoparan and 0.75% n-butanol (ButOH) for 30 min at room temperature. Control discs were incubated in the absence of mastoparan. (B) Autoradiography of a TLC plate after separation of the phospholipids from mastoparan-treated discs (25 μM mastoparan) and control leaf discs not treated with mastoparan (− mastoparan) (cf. Figure 1B). (C) Quantification of PtdButOH and PtdOH by using a PhosphorImager. The values are means of two independent experiments. The horizontal line in the graphs indicates the amount of PtdButOH and PtdOH measured in the control experiments (no mastoparan added). The error bars indicate the standard deviations (cf. Figure 1C).

Figure 3.

ABA Does Not Stimulate PLD Activity. (A) Leaf discs were prelabeled with ³²Pi for 16 hr and treated with 100 μM ABA or 0.1% ethanol (control) for the times indicated. To measure PLD activity, they were incubated in the presence of 0.75% n-butanol (ButOH). After 5 min of vacuum infiltration, the discs were incubated for 25 min at room temperature. (B) Formation of PtdButOH and PtdOH in response to ABA. After separation of phospholipids by TLC, the TLC plate was exposed to a PhosphorImager screen; the amount of each phospholipid was quantified and is expressed as fold stimulation relative to the amount in the control. The values are means of four different experiments. The error bars indicate the standard deviations. (C) Comparison of PLD activity in response to dehydration and ABA. The values for PtdButOH (top) and PtdOH (bottom), shown in Figures 1C and 3B, are compared. They demonstrate that the activation of PLD by dehydration is independent of ABA. The horizontal lines in the graphs indicate the amount of PtdButOH and PtdOH measured in the control discs. Error bars indicate the standard deviation.

Figure 4.

Amino Acid Sequence Comparison of CpPLD-1 and CpPLD-2. In the aligned amino acid sequences deduced from the cDNA clones CpPLD-1 and CpPLD-2, vertical lines denote identical amino acids present in both sequences. Invariant amino acid residues found in 11 other plant α-class PLD proteins are indicated by black boxes. The C2 (CalB) domain, which is involved in Ca²⁺/phospholipid binding, is indicated by a thick black line. The duplicated HKD catalytic domains are indicated by open bars. The region of CpPLD-1 used for antiserum production is indicated by an arrowhead. The GenBank accession numbers for CpPLD-1 and CpPLD-2 are AJ133001 (CpPLD-1) and AJ133000 (CpPLD-2).

Figure 5.

DNA Gel Blot Analyses. C. plantagineum DNA (10 μg per lane) was digested with the indicated restriction enzymes. Identical membranes were probed at high stringency with the gene-specific fragments of CpPLD-1 and CpPLD-2.

Figure 6.

Expression Pattern of CpPLD-1 and CpPLD-2 Transcripts. (A) RNA gel blot loaded with 3 μg of poly(A)⁺ RNA per lane extracted from leaves harvested from dried plants at the times indicated. The membrane was probed with the gene-specific fragments of CpPLD-1 and CpPLD-2. The same membrane was used for control hybridizations with the LEA-type cDNA pcC27-45 and an rDNA gene from wheat. (B) RNA gel blot loaded with 3 μg of poly(A)⁺ RNA per lane from roots harvested from dried plants at the times indicated. The membrane was probed with the gene-specific fragments of CpPLD-1 and CpPLD-2. (C) RNA gel blot loaded with 40 μg of total RNA per lane from detached leaves, which were dried for the times indicated, and hybridized with the same probes as described in (A). (D) RNA gel blot loaded with 40 μg of total RNA per lane from detached leaves, which were incubated in 100 μM ABA for the times indicated, and hybridized with the same probes as described in (A). (E) RNA gel blot with 40 μg of total RNA per lane from leaf discs, which were dried for the times indicated, and hybridized with the same probes as described in (A). Control discs were left in buffer for 4 hr.

Figure 7.

Protein Gel Blot Analyses with the Anti–CpPLD-1 Antiserum. (A) One hundred micrograms of total and soluble proteins per lane from leaves and roots harvested from plants treated as indicated was separated by 10% SDS-PAGE and blotted onto a polyvinyl difluoride membrane. Immunodetection was performed by using the polyclonal anti–CpPLD-1 antiserum. The membrane with the separated soluble proteins was reprobed with a polyclonal anti–pcC27-45 antiserum. (B) Fifty micrograms of the soluble protein extracts described in (A) was separated by one-dimensional isoelectric focusing (pH 3.5 to 9.5) and blotted onto a polyvinyl difluoride membrane. Immunodetection was performed as described in (A). The pH values of 1-cm gel slices were determined and are marked by horizontal lines.