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

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

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

List of QTLs detected on chromosome 3A in the glasshouse study conducted at Pullman, WA.

<p>HD = heading date, PH = plant height, TKW = 1000-kernal weight, KPS = kernels per spike, SB = shoot biomass, TB = total biomass, SWPS = seed weight per spike.</p>*<p>QTLs detected in field data analysis.</p

Cytogenetic-ladder map of wheat chromosome 3A showing locations of genes and/or QTLs influencing a number of agronomically important traits.

<p>Markers showing connection between genetic and cytogenetic maps are highlighted in blue on the cytogenetic map. (A) Consensus cytogenetic map of chromosome 3A developed by integrating information for additional markers, genes (underlined) and QTLs on the reference map <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070526#pone.0070526-Dilbirligi1" target="_blank">[11]</a>. (B) Integrated genetic linkage map developed by incorporating SSR, STM and DArT markers on the RFLP skeleton map. The map was used to demarcate locations of consistent QTLs detected in the present study and to depict locations of QTLs detected for a number of agronomical traits in the published literature. (C) List of traits and symbols used to demarcate locations of QTLs published elsewhere. YLD = yield; HD = heading date; GW = grain weight; Yr/Sr/Lr = yellow, stem and leaf rust resistance; FHB = <i>Fusarium</i> head blight resistance; LGSA = leaf glutamine synthetase activity; KPS = kernels per spike; GPC = grain protein content; PH = plant height; GL = grain length; LW = leaf waxiness; DSF = domestication syndrome factor; APT = adult plant type; SWPS = seed weight per spike; LV/BS/DS = loaf volume, bread score and dough score; MGFR = mean grain filling rate; LFW = leaf fresh weight; TE = transpiration efficiency; FN/PHS/GC = falling number, preharvest sprouting tolerance and grain color; GL&GW = grain length and grain width; GVWT = grain volume weight; PGMS = percent greenness at maximum senescence; UTEB = Phosphorus utilization efficiencies based on biomass yield; UTEG = Phosphorus utilization efficiencies based on grain yield; ABAR = ABA responsiveness. (D) List of traits mapped during the present study (see M&M for details). Traits mapped using data recorded in glasshouse are marked with a star.</p

List of consistent QTLs^* detected on chromosome 3A using data recorded over fourteen different environment years.

**<p>For years 2005 and 2008 data was recorded at Lincoln and Pullman, respectively; SPSM = spike/square meter, PH = plant height, HD = heading date, TKW = 1000-kernel weight, GY = grain yield, KPSM = kernels/square meter, GVWT = grain volume weight, and KPS = kernels per spike.</p>*<p>For the purpose of identifying consistent QTLs, the QTLs detected in overlapping marker intervals with one common marker flanking the region were treated as the same QTL. Among these QTLs the marker interval showing the highest LOD score and R² value was documented in the table (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070526#pone.0070526.s007" target="_blank">table S4</a> for complete list of QTLs).</p

Microsatellite markers classified according to repeat element type.

<p>Number of simple sequence repeats (SSRs) or microsatellites falling in each category is listed and the range of alleles detected by SSRs in these categories and their average PIC (polymorphic information content) values are shown.</p

List of two-rowed spring barley varieties/breeding lines used in the study.

<p>List of two-rowed spring barley varieties/breeding lines used in the study.</p

Genetic linkage map of chromosome 6H showing the respective locations of 61 microsatellite markers used in the present study (left).

<p>Various alleles detected from six barley genotypes used for crossing with the Bob <i>AHAS</i> mutant are indicated by colored boxes (middle), where each color represents a unique allele and the white color represents the ‘Bob’-type allele. The total number of polymorphic markers identified per genotype pair with the Bob mutant is shown below. The PIC value calculated for each marker was plotted against its location on the genetic linkage map (right) to indicate the level of nucleotide diversity observed using 13 barley genotypes, and its distribution along the entire length of chromosome 6H.</p