The complex world of plant protease inhibitors: Insights into a Kunitz-type cysteine protease inhibitor of <i>Arabidopsis thaliana</i>

<p>Plants have evolved an intricate regulatory network of proteases and corresponding protease inhibitors (PI), which operate in various biological pathways and serve diverse spatiotemporal functions during the sedentary life of a plant. Intricacy of the regulatory network can be anticipated from the observation that, depending on the developmental stage and environmental cue(s), either a single PI or multiple PIs regulate the activity of a given protease. On the other hand, the same PI often interacts with different targets at different places, necessitating another level of fine control to be added in planta. Here, it is reported on how the activity of a papain-like cysteine protease dubbed RD21 (RESPONSIVE TO DESICCATION 21) is differentially regulated by serpin and Kunitz PIs over plant development and how this mechanism contributes to defenses against herbivorous arthropods and microbial pests.</p

Characterization of MT1 (128/Xyl, cf. Fig. S1e) transformants.

<p>(A) PCR analysis of 13 randomly chosen doubled-haploid T₁ seedlings of primary transformant MT1 B4. Lanes: 1 = 1 kb ladder; 2 =  plasmid DNA; 3 =  wild type wheat DNA; 4–16 = T₁ seeds. (B) Southern blot analysis of homozygous MT1 transformants from T₁ seedlings with the xylanase gene. Lanes: C1, C2, C4 respectively showing 2, 4 and 8 copies of 837 bp probe; M =  DNA ladder; lanes 1–10 = T₁ doubled-haploid seedlings of 6 different T₀ transformants (1 and 2 = MT1-1, 3 and 4 = MT1-2, 5 and 6 = MT1-3, 7 = MT1B5, 8 and 9 = MT1-6, and 10 = MT1-7); lanes 11–13 = T₁ doubled-haploid seedlings of MT1-B4; lanes 14–15 =  wild type DNA. (C) Zymogram assay for identification of transgenic wheat grains synthesizing recombinant 1,4-β-xylanase. Transgenic wheat grains (T₂ of MT1-B4) secrete the enzyme into the medium containing oat-spelt xylan that is stainable with Congo Red. De-polymerization of the xylan by the enzyme results in an unstained yellow ring around the seed. Wild type wheat grains lack the yellow ring (arrows).</p

Developmental pathways of pre-treated wheat microspores in culture, as determined by time-lapse tracking.

<p>According to the type of development, microspores are grouped into three classes: type I (A–H), type II (I–O) and type III (P–R). Microspores were stained with FM 4–64 (Molecular Probes Cat. # T-3166) to confirm their viability in culture. All pictures were taken at a 25× magnification and 6× optical zoom except for G, O and H, where the former two pictures were taken at the same magnification but at 3× optical zoom and the latter picture was taken at 10× magnification and 1.7× optical zoom.</p

Electron micrographs of wheat microspores after pretreatment using transmission electron microscopy.

<p>Figures a-c show differences in thickness of intine, number of cytoplasmic organelles and amount of starch accumulated in amyloplasts. (A) represents type I developmental pathway, (B) represents type II developmental pathway, and (C) represents type III developmental pathway. am =  amyloplast, ex =  exine wall, in =  intine layer, mt =  mitochondria, gl =  Golgi apparatus, p =  proplastid, rer =  rough endoplasmic reticulum, st =  starch.</p

List of barley genes differentially regulated by <i>P. indica</i> and involved in ethylene synthesis or signaling.

1<p>Gene expression data was published in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035502#pone.0035502-Schfer2" target="_blank">[27]</a>.</p

Colonization of barley and <i>Arabidopsis</i> by <i>P. indica</i> in response to ACC and MCP.

<p>(<b>A</b>) Two-day-old barley seedlings or (<b>B</b>) two-week-old <i>Arabidopsis</i> seedlings were inoculated with <i>P. indica</i> and subsequently treated with 500 ppt 1-methylcyclopropene (MCP) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035502#s4" target="_blank">Materials and Methods</a>. Barley was also treated with 100 µM 1-aminocyclopropane-1-carboxylic acid (ACC). MCP inhibited <i>P. indica</i> colonization at 3 or 7 dai in <i>Arabidopsis</i> or barley, respectively. The values are normalized to colonization in mock-treated roots (set to one). The data are based on three independent biological experiments. Student's <i>t</i>-test indicates a significant difference in <i>P. indica</i>-colonization of MCP-treated roots (* P<0.05).</p

Suppression of chitin-induced oxidative burst by <i>P. indica</i>.

<p>Chitin (1 µM <i>N</i>-acetylchitooctaose) was applied to barley root segments of seedlings harvested at 3 days after <i>P. indica</i>- or <i>Rhizoctonia solani</i> inoculation or mock-treatment, respectively. Values are given as relative light units (RLU) over time as means with standard errors of two biological experiments with three independent measurements per treatment and experiment. GP, barley cv. Golden Promise.</p

ACC content in barley roots during <i>P. indica</i> colonization.

<p>Free (<b>A</b>) and malonylated (<b>B</b>) 1-aminocyclopropane-1-carboxylic acid (ACC) contents were determined in <i>P. indica</i> and mock-treated roots at 1, 3, and 7 days after treatments. At 1 dai, the complete roots were harvested and forwarded to ACC measurements. At 3 and 7 dai, the upper two centimeters (basal part) and the remaining part of the roots (apical part) were analyzed separately. Absolute values are given in nmol • g FW⁻¹ for mock-treated and <i>P. indica</i>-colonized roots. (<b>A</b>) Free ACC levels were significantly enhanced at 3 and 7 dai in the apical zone and 7 dai in the basal part as indicated by Students <i>t</i>-test (* P<0.05, ** P<0.01, *** P<0.001). (<b>B</b>) Malonylated ACC was not significantly altered during <i>P. indica</i> colonization at any timepoint or in any tissue. Data show the mean content of four biological experiments (with at least two technical repetitions per experiment) and bars indicate standard errors.</p

GUS accumulation in roots of <i>ACS1</i>::<i>GUS</i> reporter plants colonized by <i>P. indica</i>.

<p><i>Arabidopsis</i> line <i>ACS1</i>::<i>GUS</i> was harvested at 7 dai and, after GUS and WGA-AF 488 staining, analyzed cytologically. (<b>A</b>, <b>B</b>) <i>P. indica</i> colonization at the base of lateral roots (arrows) or primordia (asterisks) of line <i>ACS1</i>::<i>GUS</i> was associated with enhanced GUS accumulation. <i>P. indica</i> (arrowsheads in A) was visualized by staining with WGA-AF 488. (<b>C</b>) In mock-treated <i>ACS1</i>::<i>GUS</i>, GUS staining was weakly detectable e.g. at the lateral root base. Bars = 60 µm.</p

Colonization of ethylene synthesis and signaling mutants by <i>P. indica</i>.

<p>(<b>A</b>) Three-week-old plants were inoculated with <i>P. indica</i> and fungal biomass was determined in <i>ein2-1</i>, <i>etr1-3, eto1-1, ctr1-1</i>, and <i>35S</i>::<i>ERF1</i> by qRT-PCR at 3 and 14 dai. (<b>B</b>) Three-week-old <i>35S::ERF1</i> plants were injured with foreceps and inoculated with <i>P. indica</i> at 1 day after wounding. Fungal biomass was determined by qRT-PCR at 3 and 7 dai. All values were related to Col-0 (set to one). The data are based on at least three independent experiments. Students <i>t</i>-test indicated significant difference in <i>P. indica</i>-colonization (* P<0.05, ** P<0.001).</p