Loss of CRWN Nuclear Proteins Induces Cell Death and Salicylic Acid Defense Signaling

Abstract

Defects in the nuclear lamina of animal cell nuclei have dramatic effects on nuclear structure and gene expression as well as diverse physiological manifestations. We report that deficiencies in CROWDED NUCLEI (CRWN), which are candidate nuclear lamina proteins in Arabidopsis (Arabidopsis thaliana), trigger widespread changes in transcript levels and whole-plant phenotypes, including dwarfing and spontaneous cell death lesions. These phenotypes are caused in part by ectopic induction of plant defense responses via the salicylic acid pathway. Loss of CRWN proteins induces the expression of the salicylic acid biosynthetic gene ISOCHORISMATE SYNTHASE1, which leads to spontaneous defense responses in crwn1 crwn2 and crwn1 crwn4 mutants, which are deficient in two of the four CRWN paralogs. The symptoms of ectopic defense response, including pathogenesis marker gene expression and cell death, increase in older crwn double mutants. These age-dependent effects are postulated to reflect an increase in nuclear dysfunction or damage over time, a phenomenon reminiscent of aging effects seen in animal nuclei and in some human laminopathy patients.

Figure 1.

The patterns of up-regulated genes in crwn mutants demonstrate both synergistic and antagonistic relationships among crwn mutations. The sizes of each circle in the Venn diagrams are proportional to the number of genes up-regulated at least 2-fold in our RNA-seq data (q < 0.01). A, Up-regulated genes in three crwn single mutants, crwn1, crwn2, and crwn4, showing shared transcriptomic alterations. B, Up-regulated genes in the double mutant crwn1 crwn2 and corresponding single mutants, crwn1 and crwn2, showing the synergistic effects of the crwn1 and crwn2 mutations. C, Up-regulated genes in the double mutant crwn1 crwn4 and corresponding single mutants, crwn1 and crwn4, showing the antagonistic effects of these mutations. D, Up-regulated genes in two double mutants, crwn1 crwn2 and crwn1 crwn4, and one single mutant, crwn4, illustrating overlapping profiles. Note that PR1 and PR2 fell in the overlap between crwn1 crwn2 and crwn1 crwn4 mutants (this overlap contains 119 genes), and PR5 was found only in the sector corresponding to the crwn1 crwn2 mutant (region with 565 genes).

Figure 2.

crwn1 crwn2 and crwn1 crwn4 mutants exhibit spontaneous defense responses. A, DC3000 growth in rosette leaves of 22-d-old plants. The two double mutants have elevated resistance against DC3000. Log₁₀ values of colony-forming units (cfu) from each mutant were calculated 2 and 4 d after infection (DAI). Error bars indicate sd (n = 4). Each mutant was compared with the wild type (WT) using Student’s t test (**, P < 0.01 and ***, P < 0.001). B, RT-qPCR data showing expression of three PR genes in 27-d-old plants. Transcript levels of the marker genes are significantly higher in the two double mutants, consistent with their DC3000 resistance phenotype. Error bars indicate se (n = 3). Student’s t tests were performed based on delta-C_t values (*, P < 0.05; **, P < 0.01; and ***, P < 0.001).

Figure 3.

crwn double mutants exhibit cell death and PR gene expression, which are exacerbated with aging. A, Trypan Blue staining showed that crwn1 crwn2 and crwn1 crwn4 mutants form spontaneous lesions. As plants matured, cell death was more apparent and more frequent. B, PR1 expression in leaves from crwn1 crwn2 mutants of different ages. Expression of PR1 is higher in chronologically older leaves of crwn1 crwn2 mutants, consistent with our observation that older plants develop more lesions. PR1 expression at each stage was normalized to transcript levels in wild-type samples for the housekeeping gene ROC1. Error bars indicate se (n = 3). Student’s t tests were performed based on delta-C_t values (*, P < 0.05 and ***, P < 0.001). C, The sid2-1 mutation suppressed cell death in 22-d-old crwn1 crwn2 mutants; however, 30-d-old plants still exhibited some lesion formation. Bars = 100 mm.

Figure 4.

SA levels in crwn1 crwn2 and crwn1 crwn4 mutants are significantly higher than in wild-type (WT) plants or crwn single mutants. A, HPLC/MS detection of free SA levels from 27-d-old plants. Peak intensities of free SA per fresh sample weight were normalized to the spiked internal control d4-SA. Each mutant was compared with the wild type using Student’s t test, and error bars indicate sd (*, P < 0.05; n = 3). B, HPLC/MS detection of glucosylated SA (SA-Glc, indicating either SA glucoside or salicylate Glc ester) levels from each mutant. The detection method, units, and statistical analysis were identical to those described for A (*, P < 0.05 and **, P < 0.01). C, Overlap of misregulated genes in our RNA-seq data from crwn1 crwn2 mutants and those in microarray data from 1 mm SA-treated wild-type plants (Zhou et al., 2015). For SA-treated wild-type microarray data, genes at least 2-fold increased or decreased with adjusted P < 0.05 were used. For the RNA-seq of crwn1 crwn2 mutants, the criteria described in Figure 1 were used. The significance of the overlap of both up-regulated and down-regulated gene comparisons was assessed by the hypergeometric test; the resulting P values in both cases were far less than 0.001. D, RT-qPCR data showing expression of the SID2 gene in 27-d-old plants. Error bars indicate se (n = 3). Student’s t tests were performed based on delta-C_t values (*, P < 0.05 and **, P < 0.01).

Figure 5.

Two models to explain how crwn mutations affect disease signaling and other phenotypes. A, The first model posits that crwn mutations lead to transcriptional changes via the disruption in the direct action of CRWN proteins or epigenetic mechanisms (e.g. through changes in chromatin modification or three-dimensional genomic configuration). In this first model, overexpression of SID2 and subsequent SA accumulation lead to pathogen defense phenotypes and an LMM syndrome. B, In the alternative model, cell damage and/or death due to nuclear dysfunction is the primary effect of crwn mutations. Downstream effects of age-dependent cell damage/death are amplified via an SA-mediated pathway, but an SA-independent pathway also mediates developmental defects.