Longitudinal residual strain and stress-strain relationship in rat small intestine

BACKGROUND: To obtain a more detailed description of the stress-free state of the intestinal wall, longitudinal residual strain measurements are needed. Furthermore, data on longitudinal stress-strain relations in visceral organs are scarce. The present study aims to investigate the longitudinal residual strain and the longitudinal stress-strain relationship in the rat small intestine. METHODS: The longitudinal zero-stress state was obtained by cutting tissue strips parallel to the longitudinal axis of the intestine. The longitudinal residual stress was characterized by a bending angle (unit: degrees per unit length and positive when bending outwards). Residual strain was computed from the change in dimensions between the zero-stress state and the no-load state. Longitudinal stresses and strains were computed from stretch experiments in the distal ileum at luminal pressures ranging from 0–4 cmH(2)O. RESULTS: Large morphometric variations were found between the duodenum and ileum with the largest wall thickness and wall area in the duodenum and the largest inner circumference and luminal area in the distal ileum (p < 0.001). The bending angle did not differ between the duodenum and ileum (p > 0.5). The longitudinal residual strain was tensile at the serosal surface and compressive at the mucosal surface. Hence, the neutral axis was approximately in the mid-wall. The longitudinal residual strain and the bending angle was not uniform around the intestinal circumference and had the highest values on the mesenteric sides (p < 0.001). The stress-strain curves fitted well to the mono-exponential function with determination coefficients above 0.96. The α constant increased with the pressure, indicating the intestinal wall became stiffer in longitudinal direction when pressurized. CONCLUSION: Large longitudinal residual strains reside in the small intestine and showed circumferential variation. This indicates that the tissue is not uniform and cannot be treated as a homogenous material. The longitudinal stiffness of the intestinal wall increased with luminal pressure. Longitudinal residual strains must be taken into account in studies of gastrointestinal biomechanical properties

Identification and functional characterization of the soybean GmaPPO12 promoter conferring Phytophthora sojae induced expression.

Identification of pathogen-inducible promoters largely lags behind cloning of the genes for disease resistance. Here, we cloned the soybean GmaPPO12 gene and found that it was rapidly and strongly induced by Phytophthorasojae infection. Computational analysis revealed that its promoter contained many known cis-elements, including several defense related transcriptional factor-binding boxes. We showed that the promoter could mediate induction of GUS expression upon infection in both transient expression assays in Nicotianabenthamiana and stable transgenic soybean hairy roots. Importantly, we demonstrated that pathogen-induced expression of the GmaPPO12 promoter was higher than that of the soybean GmaPR1a promoter. A progressive 5' and 3' deletion analysis revealed two fragments that were essential for promoter activity. Thus, the cloned promoter could be used in transgenic plants to enhance resistance to phytophthora pathogens, and the identified fragment could serve as a candidate to produce synthetic pathogen-induced promoters

Induction of the <i>GmaPPO12 and GmaPR1a</i> promoters upon <i>Phytophthoracapsici</i> infection in <i>Nicotiana</i><i>benthamiana</i> leaves.

<div><p>A. Scheme of the pathogen-induced promoter GUS fusion constructs. The studied promoters were inserted between the <i>Kpn</i>I and <i>Bgl</i>II sites in pMDC162 upstream of the -46 35S promoter of the <i>Cauliflower mosaic virus</i> (CaMV 35S).</p> <p>B. Enzymatic assay of GUS activity in expanded 6-week-old <i>N</i><i>. benthamiana</i> leaves. The leaves were infiltrated with <i>Agrobacterium</i> harboring <i>GmaPPO12</i> or <i>GmaPR1a</i> promoter: GUS constructs, and then submerged in <i>P</i><i>. capsici</i> zoospores 3 days after agroinfiltration. GUS activity levels were measured by fluorimetric assays in protein extracts from treated leaves at 0 hpi, 0.5 hpi and 2 hpi. Each column represents the fold change of <i>P</i><i>. capsici</i> induction at 0.5 hpi and 2 hpi to 0 hpi and normalized to 35S min. Labels are as follows: <i>GmaPR1a</i> promoter; <i>GmaPPO12</i> promoter; 35S minimal promoter; 35S full-length promoter fragments. Experiments were in triplicate and error bars show the standard error.</p> <p>C. Histochemical GUS staining of <i>GmaPPO12</i> and <i>GmaPR1a</i>: GUS constructs in <i>N</i><i>. benthamiana</i> leaves. The leaves were treated as in B. GUS activity increased dramatically at <i>GmaPPO12</i> and <i>GmaPR1a</i> construct agroinfiltrated regions at 0.5 hpi and 2 hpi. No visible GUS activity was noted at 35S min agroinfiltrated regions. GUS activity was relatively strong at 35S (CaMV 35S full-length promoter fragments) agroinfiltrated regions.</p> <p>D. GUS reporter gene transcript levels at different time points post inoculation measured by quantitative RT-PCR. Total RNA was extracted from transiently expressing <i>N</i><i>. benthamiana</i> leaves. The leaves were treated as in B. Real-time RT-PCR analysis employed primers specific for GUS and the <i>N</i><i>. benthamiana</i> actin gene is <i>EF1a</i>. Transcript levels represent the GUS mRNA levels compared with actin mRNA levels normalized to the roots treated at 0 hpi. Experiments were done in triplicate with error bars showing the standard error.</p></div

C-terminal deletion analysis of <i>GmaPPO12</i> promoter activity in transiently expressing <i>Nicotiana</i><i>benthamiana</i> leaves.

<div><p>A. Enzymatic assay of C-terminal deletion constructs in <i>Nicotiana</i> transiently expressing leaves. The expanded 6-week-old <i>N</i><i>. benthamiana</i> leaves were infiltrated with <i>Agrobacterium</i> harboring constructs and inoculated with <i>Phytophthoracapsici</i> zoospores 3 days after infiltration. Enzymatic assay at 0 hpi and 2 hpi. Each column represents the fold change in <i>P</i><i>. capsici</i> induction at 2 hpi compared to 0 hpi and normalized to 35S min. Labels are as follows: <i>GmaPPO12</i> promoter; C-terminal deletion mutants: CT1 (-1500 to -413), CT2 (-1500 to -746), CT3 (-1500 to -993), CT4 (-1500 to -1175), CT5 (-1500 to -1326); <i>GmaPR1a</i> promoter. Experiments were performed in triplicate. Means that have different letters at the top are significantly different (p < 0.05 Duncan’s multiple range tests), lines designated with the same letter exhibit no significant difference of the GUS enzyme activity. Error bars show the standard error.</p> <p>B. Histochemical GUS staining of different constructs in <i>Nicotiana</i> transiently expressing leaves at 0 hpi and 2 hpi. Leaves were treated as in A. No visibly induced GUS activity was detected at agroinfiltrated regions, including 35S min; GUS activity was relatively weak at agroinfiltrated regions, including CT4, CT5; GUS activity increased dramatically at agroinfiltrated regions, including CT1, CT2, CT3, M1, <i>GmaPPO12, GmaPR1a</i>; GUS activity was continuously high at agroinfiltrated regions, including 35S (CaMV 35S full-length promoter fragments).</p></div

Induced expression of <i>GmaPPO12</i> and <i>GmaPR1a</i> in soybean roots.

<div><p>A. Expression patterns of soybean <i>GmaPPO12</i> and <i>GmaPR1a</i>. Quantitative RT-PCR analysis was carried out to detect <i>GmaPPO12</i> and <i>GmaPR1a</i> transcript abundance in 7-day-old soybean roots submerged in suspensions containing 10⁵ zoospores of <i>P</i><i>. sojae</i> per milliliter at 0 hpi, 0.5 hpi and 2 hpi (hours post inoculation). The soybean <i>ACT20</i> gene was used as a reference gene with designed primers shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067670#pone.0067670.s001" target="_blank">Table S1</a>. Transcript levels represent <i>GmaPPO12</i> or <i>GmaPR1a</i> mRNA levels compared with reference mRNA levels and then normalized to the treated roots at 0 hpi. Biological triplicates were averaged. A significant difference compared using Student’s t-test (** for p < 0.01). Bars indicate the standard error.</p> <p>B. Effects of the tested hormones on accumulation of soybean <i>GmaPPO12</i> gene transcripts. Quantitative RT-PCR analysis was carried out to detect <i>GmaPPO12</i> transcript abundance in 7-day-old soybean roots. The roots were submerged in ddH₂O (control) or JA (100 µM), SA (100 µM), MeJA (100 µM), GA₃ (100 µM), ethephon (100 µM) for 3 h. The soybean <i>ACT20</i> gene was used as a reference gene. Each column represents the relative transcript expression to the soybean reference gene then normalized to the roots treated with ddH₂O. Biological triplicates were averaged. Means with different letters at the top are significantly different (p < 0.05 Duncan’s multiple range tests), lines designated with the same letter exhibit no significant difference in response to the tested hormones. Bars indicate the standard error.</p></div

The microarray data of relative expression levels of <i>GmaPPO12</i> (A) and <i>GmaPR1a</i> (B).

<p>Relative expression levels of <i>GmaPPO12</i> (A) and <i>GmaPR1a</i> (B) in three different soybean cultivars (V710370, Williams and Sloan) were collected from the published data and calculated for relative fold change compared to these in mock samples. The error bar indicates the fold change in three independent experiments.</p

Validation of <i>GmaPPO12 and GmaPR1a</i> promoter function in soybean hairy roots.

<div><p>A. Enzymatic assay of GUS activity in multiple soybean hairy roots. The roots were inoculated with 10⁵<i>P</i><i>. sojae</i> zoospores, and analyzed at 0 hpi 0.5 hpi and 2 hpi. Each column represents the fold change in <i>P</i><i>. sojae</i> induction at 2 hpi compared to 0 hpi and normalized to 35S min. Experiments were in triplicate, and error bars show the standard error.</p> <p>B. Histochemical staining of GUS activity in transgenic soybean hairy roots. Roots were treated as in A. No visible GUS activity in 35S min transgenic soybean hairy roots; GUS activity was relatively strong in 35S (CaMV 35S full-length promoter fragments) transgenic soybean hairy roots; GUS activity increased dramatically in <i>GmaPPO12</i> transgenic soybean hairy roots at 0.5 hpi and 2 hpi.</p> <p>C. <i>P</i><i>. sojae</i>-induced expression analysis of the <i>GmaPPO12</i> promoter by quantitative RT-PCR. The expression levels of the <i>GUS</i> reporter gene in the multiple soybean hairy roots. Roots were treated as in A. The soybean <i>ACT20</i> gene was used as a reference gene. Transcript levels represent the <i>GUS</i> mRNA levels compared with soybean reference mRNA levels and then normalized to the hairy roots treated at 0 hpi. Experiments were done in triplicate with error bars showing the standard error.</p></div

Functional analysis of two fragments in the promoter.

<div><p>A. Enzymatic assay of GUS activity in transient <i>Nicotiana</i><i>benthamiana</i> leaves. Expanded 6-week-old <i>N</i><i>. benthamiana</i> leaves were infiltrated with <i>Agrobacterium</i> harboring the deletion constructs M1 (-1012 to -413) and M1-113 (-525 to -413). Leaves were inoculated with <i>P</i><i>. capsici</i> zoospores 3 day after infiltration and assayed at 0 hpi and 2 hpi. Each column represents the fold change from the <i>P</i><i>. capsici</i> induction at 2 hpi compared to 0 hpi and normalized to 35S min. Labels are as follows: M1 (-1012 to -413) and M1-113 (-525 to -413). Experiments were in triplicate, and error bars show the standard error.</p> <p>B. Histochemical GUS staining of different constructs in <i>Nicotiana</i> transiently expressing leaves at 0 hpi and 2 hpi. Leaves were treated as in A. No visible induced GUS activity was detected at the injection region in 35S min. GUS activity was relatively weak at the injection region in M1-113. GUS activity was increased dramatically at the injection region of <i>GmaPPO12</i>. GUS activity was continuously high at agroinfiltrated regions for 35S (CaMV 35S full-length promoter fragments). Experiments were done in triplicate with error bars showing the standard error.</p></div