Nitric oxide content analysis in the aerial parts and in the roots of ARG-26 and ARG-38 seedlings grown on 1/2 MS medium, 1/2 MS medium with 4×NH4NO3, and 1/2 MS medium lacking nitrogen.

<p>Compared to non-transgenic control plants, the nitric oxide content decreased in the aerial parts and in the roots of ARG-26 and ARG-38 seedlings on all of the various 1/2 MS media compositions. no differences were observed for the aerial parts of the plants. (A) the nitric oxide content in seedlings of transgenic cotton grown on 1/2 MS medium. (B) the nitric oxide content in transgenic cotton seedlings grown on 1/2 MS medium with 4× NH₄NO₃. (C) the nitric oxide content in transgenic cotton seedlings grown on 1/2 MS medium lacking NH₄NO3 and KNO3. ‘FW’ refers to fresh leaf weight. ‘WT’ refers to non-transgenic control plants of wild type cotton. *: P<0.05.</p

Arginase enzyme activity in ARG-26 and ARG-38 transgenic cotton leaves.

<p>Compared with arginase activity of non-transgenic control plants, expression of OsARG increase the total arginase activity in ARG-26 and ARG-38. ‘WT’ refers to non-transgenic control plants of wild type cotton. **: P<0.01.</p

Expression of the Rice Arginase Gene <i>OsARG</i> in Cotton Influences the Morphology and Nitrogen Transition of Seedlings

<div><p>Arginase is the only enzyme capable of producing urea in plants. This enzyme also contributes to many important biological functions during plant growth and development, such as seed development, root development and plant nitrogen using. The unique rice arginase gene <i>OsARG</i> is known to affect nitrogen use efficiency and is also associated with higher yields in rice. In this study, we transformed <i>OsARG</i> into upland cotton R18 by <i>Agrobacterium</i>-mediated genetic transformation and analyzed the function of <i>OsARG</i> in transgenic cotton. Two independent <i>OsARG</i> expression transgenic cotton lines, ARG-26 and ARG-38, were obtained via transformation. Southern blot analysis indicated that two copies and one copy of the <i>OsARG</i> gene were integrated into the ARG-26 and ARG-38 genomes, respectively. Enzyme activity and RNA transcription analysis revealed that the <i>OsARG</i> gene is highly expressed in cotton. The nitric oxide content and the morphology of ARG-26 and ARG-38 seedlings were both affected by expression of the <i>OsARG</i> gene. Field experiments indicated that the polyamine and nitrogen content increased by more than two-fold in the T3 generation plants of the transgenic cotton lines ARG-26-2, ARG-26-7, ARG-38-8, and ARG-38-11, as compared with the control plants. After harvesting cotton fibers grown in field conditions, we analyzed the quality of fiber and found that the fiber length was increased in the transgenic lines. The average cotton fiber length for all of the transgenic cotton lines was two millimeters longer than the fibers of the control plants; the average cotton fiber lengths were 31.94 mm, 32.00 mm, 32.68 mm and 32.84 mm in the ARG-26ARG-26-2, ARG-26-7, ARG-38-8 and ARG-38-11 lines, respectively, but the average fiber length of the control plants was 29.36mm. Our results indicate that the <i>OsARG</i> gene could potentially be used to improve cotton fiber length traits.</p></div

PCR identification of the <i>OsARG</i> gene in transgenic cotton.

<p>The under labels indicate the following samples: M, marker. 1, positive control. 2, negative control. 3–6, PCR analysis of four independent cotton plants containing ARG-26 genomic DNA. 7–10, PCR analysis of four independent cotton plants containing the ARG-38 genome DNA.</p

Southern blot analysis of <i>OsARG</i> gene copy number in the genomes of transgenic cotton plants.

<p>The probe was labeled with the radioactive isotope [α-³²P] dCTP. (A) Two copies of <i>OsARG</i> were integrated into the ARG-26 genome. (B) One copy of <i>OsARG</i> was integrated into the ARG-38 genome. The under labels indicate the following samples: M, marker. 1, positive control. 2, negative control. 3, genomic DNA digested using <i>Eco</i>RI. 4, genomic DNA digested using <i>Hind</i>III.</p

Quantitative PCR analysis of OsArg expression in the leaves of ARG-26 and ARG-38 transgenic cotton.

<p>The <i>OsARG</i> gene was detected in ARG-26 and ARG-38 leaves but not in non-transgenic control leaves. ‘WT’ refers to non-transgenic control plants of wild type cotton. **: P<0.01</p

Map of the plant expression vector.

<p>Map of the plant expression vector.</p

Concentrations of amino acids in the leaves of control and transgenic cotton plants.

<p>Values are the means ± SD (μmol/g DW). ‘Control’ refers to non-transgenic control plants.</p><p>‘*’ indicates that the amino acid content of the transgenic line was significantly decreased compared with that of control.</p><p>‘◆’ indicates that the amino acid content of the transgenic line was significantly increased compared with that of control.</p><p>‘Control’ refers to non-transgenic wild type plants. P ≤ 0.05.</p><p>Concentrations of amino acids in the leaves of control and transgenic cotton plants.</p

Comparison of the polyamine content in transgenic and control cotton plants grown under field conditions.

<p>The polyamine content here refers to the content of putrescine, spermine, and spermidine. All data in this figure are displayed as the mean values for putrescine, spermine, and spermidine content in the WT, ARG-26-2, ARG-26-7, ARG-38-8, and ARG-38-11 plants. The spermine and spermidine content in transgenic cotton plants were both imilar to those of the WT plants. The putrescine content in transgenic plants was two-fold greater than that in WT plants. ‘FW’ refers to fresh leaf weight. ‘WT’ refers to non-transgenic control plants of wild type cotton. **: P<0.01.</p

Fiber length analysis in transgenic cotton and control plants.

<p>ARG-26-2 and ARG-26-7 were bred from the ARG-26 line. ARG-38-8 and ARG-38-11 were bred from theARG-38 line. ‘Control’ refers to non-transgenic wild type plants.</p><p>Fiber length analysis in transgenic cotton and control plants.</p