A Key ABA Catabolic Gene, <i>OsABA8ox3</i>, Is Involved in Drought Stress Resistance in Rice

<div><p>Expressions of ABA biosynthesis genes and catabolism genes are generally co-regulated in plant development and responses to environmental stress. Up-regulation of <i>OsNCED3</i> gene, a key gene in ABA biosynthesis, has been suggested as a way to enhance plant drought resistance but little is known for the role of ABA catabolic genes during drought stress. In this study, we found that <i>OsABA8ox3</i> was the most highly expressed gene of the <i>OsABA8ox</i> family in rice leaves. Expression of <i>OsABA8ox3</i> was promptly induced by rehydration after PEG-mimic dehydration, a tendency opposite to the changes of ABA level. We therefore constructed rice <i>OsABA8ox3</i> silencing (RNA interference, RNAi) and overexpression plants. There were no obvious phenotype differences between the transgenic seedlings and wild type under normal condition. However, <i>OsABA8ox3</i> RNAi lines showed significant improvement in drought stress tolerance while the overexpression seedlings were hypersensitive to drought stress when compared with wild type in terms of plant survival rates after 10 days of unwatering. Enzyme activity analysis indicated that <i>OsABA8ox3</i> RNAi plants had higher superoxide dismutase (SOD) and catalase (CAT) activities and less malondialdehyde (MDA) content than those of wild type when the plants were exposed to dehydration treatment, indicating a better anti-oxidative stress capability and less membrane damage. DNA microarray and real-time PCR analysis under dehydration treatment revealed that expressions of a group of stress/drought-related genes, i.e. <i>LEA</i> genes, were enhanced with higher transcript levels in <i>OsABA8ox3</i> RNAi transgenic seedlings. We therefore conclude that that <i>OsABA8ox3</i> gene plays an important role in controlling ABA level and drought stress resistance in rice.</p></div

ABA sensitivity of the <i>OsABA8ox3</i> transgenic seeds during germination and post-germination growth.

<p>(A) Seed germination, (B) Post-germination root growth and (C) Root length of WT and <i>OsABA8ox3</i>-RNAi and -overexpression transgenic plants in response to exogenous ABA. Seeds treated with water and ABA (1μM and 5μM) were imbibed at 28°C to facilitate germination and post-germination growth. Error bars are standard deviations based on three replicates. ** Indicates significant difference from wild-type at P = 0.01.</p

Relative expression levels of 9 stress-related genes in the WT and <i>OsABA8ox3</i> RNAi transgenic plants under normal and PEG-treated conditions detected by qRT-PCR.

<p>Four-leaf stage seedlings were treated with 20% PEG for 2 h, and then the samples were quickly frozen with liquid nitrogen for RNA extraction. The genes include dehydration-responsive element-binding protein (Os06g03670), zinc finger protein (Os05g10670), late embryogenesis abundant proteins (Os05g46480, Os01g50910 and Os04g49980), dehydrin family proteins (Os11g26760 and Os01g50700) and heat shock protein (Os03g16920 and Os02g54140).</p

Physiological changes in the <i>OsABA8ox3</i>-RNAi and -overexpression transgenic plants.

<p>(A) ABA contents, (B) MDA contents and (C) antioxidant enzyme activities in the WT and <i>OsABA8ox3</i>-RNAi and -overexpression transgenic seedlings under normal condition and PEG treatment. Four-leaf stage seedlings were treated with 20% PEG for 2 h, then the samples were quickly frozen with liquid nitrogen. Error bars are standard deviations based on three replicates. ** Indicates significant difference from wild-type at P = 0.01.</p

Stress tolerance assays of wild type plants and the transgenic rice.

<p>(A) Relative expressions of <i>OsABA8ox3</i> gene in RNAi and overexpression lines; (B) Performance of WT control and <i>OsABA8ox3</i> RNAi-9, RNAi -27 and overexpression lines after 7 d soil drought stress and 4 d recovery. (C) Survival rate of rice seedlings after drought stress and rewatering. Error bars are standard deviations based on three replicates (n > 40). ** Indicates significant difference from wild-type at P = 0.01.</p

Comparison of Endoscopic Submuscosal Implantation vs. Surgical Intramuscular Implantation of VX2 Fragments for Establishing a Rabbit Esophageal Tumor Model for Mimicking Human Esophageal Squamous Carcinoma

<div><p>Purpose</p><p>This study was undertaken to establish a rabbit esophageal tumor model for mimicking human esophageal squamous carcinoma (ESC) by endoscopic and surgical implantation of VX2 tumors.</p><p>Methods</p><p>Fragments of a VX2 tumour were endoscopically implanted in the submucosal layer of the thoracic esophagus of 32 New Zealand white rabbits, while 34 animals received surgical implantation into the muscular layer. Then, the animals were studied endoscopically and pathologically. The safety and efficiency of the two methods and the pathological features of the animal models were analyzed.</p><p>Results</p><p>Both the endoscopic and the surgical method had a relatively high success rate of tumor implantation [93.7% (30/32) vs. 97.1% (33/34)] and tumor growth [86.7% (26/30) vs. 81.8% (27/33)], and the variation in the results was not statistically significant (<i>P</i>>0.05). Compared with those produced by the surgical method, the models produced by the endoscopic method had a higher rate of severe esophageal stricture [61.5% (16/26) vs. 29.6% (8/27)] and of intra-luminal tumor growth [73.1% (19/26) vs. 37.0% (10/27)], and had a lower rate of tumor invasion of adjacent organs [53.8% (14/26) vs. 81.5% (22/27)]; all of these results were statistically significant (P<0.05). However, the difference in the survival time and the rates of tumor regional/distant metastasis [38.5% (10/26) vs. 51.8% (14/27)] between the two methods were not statistically significant (P>0.05).</p><p>Conclusion</p><p>The endoscopic and surgical methods are both safe and effective for establishment of VX2 tumors in the rabbit esophagus. The models produced by the two methods have different pathologic features mimicking that of human ESC. We recommend the models for studies on surgical procedures and minimally invasive treatments.</p></div

Needles for endoscopic implantation.

<p>(A) Tip of the endoscopic needle (fine needle). (B) Tip of the revised needle (large needle).</p

Tumor invasion of adjacent tissues.

<p>(A) Invasion was observed into the trachea/bronchus (green arrow) and the pleura and the lung in the upper lobes (blue arrows), but invasion of the heart and the pericardium (white arrow) was not observed. The black arrows point to the esophagus. (B) Tumor invasion was observed into the trachea/bronchus (green arrows) and the heart and the pericardium (white arrow), but invasion of the pleura and the lung (blue arrows) was not observed.</p

Tumor regional/distant metastases.

<p>(A) Macroscopic view of the lymph node metastases (black arrow) and the yellow arrow points to the esophageal tumor. (B) Microscopy observation of lymph node metastases (200x). (C) Macroscopic view of the liver metastases (yellow arrows). (D) Microscopy observation of liver metastases (200x). (E) Macroscopic view of the lung metastases (black arrows). (F) Microscopy view of lung metastases (200x).</p

The tumor growth patterns.

<p>Tumor intra-luminal growth: (A) macroscopic external view of the esophageal tumor (black arrow) and (B) internal view of the esophageal tumor (black arrow) in a sacrificed animal model produced by the endoscopic method. Tumor extra-luminal growth: (C) macroscopic view of the tumor (black arrows) with esophageal congestion (blue arrow) from the external esophagus in a sacrificed animal model produced by the surgical method, and (D) macroscopic view of no tumor but esophageal congestion (blue arrows) from the internal esophagus.</p