Knockout of Arabidopsis accelerated-cell-death11 encoding a sphingosine transfer protein causes activation of programmed cell death and defense

Abstract

We describe the lethal, recessive accelerated-cell-death11 Arabidopsis mutant (acd11). Cell death in acd11 exhibits characteristics of animal apoptosis monitored by flow cytometry, and acd11 constitutively expresses defense-related genes that accompany the hypersensitive response normally triggered by avirulent pathogens. Global transcriptional changes during programmed cell death (PCD) and defense activation in acd11 were monitored by cDNA microarray hybridization. The PCD and defense pathways activated in acd11 are salicylic acid (SA) dependent, but do not require intact jasmonic acid or ethylene signaling pathways. Light is required for PCD execution in acd11, as application of an SA-analog to SA-deficient acd11 induced death in the light, but not in the dark. Epistatic analysis showed that the SA-dependent pathways require two regulators of SA-mediated resistance responses, PAD4 and EDS1. Furthermore, acd11 PR1 gene expression, but not cell death, depends on the SA signal tranducer NPR1, suggesting that the npr1-1 mutation uncouples resistance responses and cell death in acd11. The acd11 phenotype is caused by deletion of the ACD11 gene encoding a protein homologous to a mammalian glycolipid transfer protein (GLTP). In contrast to GLTP, ACD11 accelerates the transfer of sphingosine, but not of glycosphingolipids, between membranes in vitro.

Figure 1

Phenotype of acd11 mutant. (A) Ler and acd11. (B) Cross sections of Ler and acd11 rosette leaves. Bar, 25 μm. (C) Trypan blue staining of Ler and acd11 rosette leaves. Bar, 25 mm.

Figure 2

ROS and apoptosis measurements. (A) NBT staining of Ler and acd11 seedlings. (B) Flow cytometry of 7-AAD stained protoplasts isolated from Ler and acd11 rosette leaves showing number of cells (events) vs. 7-AAD intensity. Bars indicate fluorescence intensity intervals, which define apoptotic cells. (Inset) Side vs. forward scatter (cell size). Each experiment was repeated three times. (C) As in B with diphenylene iodonium (DPI), staurosporine (STA), okadaic acid (OKA), or N(6)-benzyladenine (BA).

Figure 3

Accumulation of defense-related markers. (A) Autofluorescence (top) and callose staining (bottom) of Ler and acd11 rosette leaves. Bar, 100 μM. (B,C) RNA gel blots of 10 μg total RNA hybridized with radiolabeled probes as indicated. The SAG12 probe detected ∼1 pg of the 1200-bp fragment used as a probe template spotted onto a filter. Equal loading of RNA was checked on all blots by hybridization with EF-1α. A representative blot is shown.

Figure 4

Epistatic analyses. (A) Phenotypes of mock- (left) and BTH- (middle) treated acd11/nahG, acd11/pad4-2, and acd11/eds1-2 plants 6 d after treatment. Trypan blue staining of BTH-treated leaves is also shown (right). (B) Accumulation of PR1 and SAG13 mRNA in mock- and BTH-treated Ler, acd11/nahG, acd11/pad4-2 and acd11/eds1-2 plants 6 d after treatment. At longer exposures, weak PR1 and SAG13 signals are detected in BTH-treated acd11/eds1-2. (C) Phenotypes of acd11/npr1-1, acd11 and npr1-1 mutants. (D) Accumulation of PR1 and SAG13 mRNA in acd11/npr1-1, acd11 and npr1-1 mutants. (E) Accumulation of PR1 and SAG13 in acd11/ein2-1, acd11/jar1-1 and in single mutants. (F) Representative mock- or BTH-treated leaves of nahG and acd11/nahG after 4 d in light or dark.

Figure 5

ACD11 is homologous to glycolipid transfer proteins and accelerates the transfer of sphingosine between membranes in vitro. (A) Alignment of ACD11 protein homologs of the same length as ACD11 ordered according to their similarity to ACD11. (Os) Oryza sativa; (At) A. thaliana; (Mm) Mus musculus; (Ce) C. elegans; (Bt) Bos taurus; (Pa) P. anserina. GI numbers corresponding to the proteins are OSJNBa0018H01.3, 13702819; T19P19.60, 7487218; MGC7473, 13277382; K08E4.2, 7505546; GLTP, 6959680; Het-C2, 2133321. (B) Sphingosine transfer activity as a function of recombinant ACD11 input. (C) ACD11 and GLTP sphingolipid transfer specificity. The background transfer levels are subtracted from transfer in the presence of ACD11 or GLTP.