Functional Analysis Of Alkali Stress Responsive Genes TaARF9 And TaNTL5

Soil salinity usually occurs with alkalinity, causing serious negative impact on plants’ growth and agricultural production. Salt stress in the soil often bring about osmotic stress and ion toxicity to plants, while alkali stress would additionally affect the soil structure seriously, decrease plants’ability to take up ions, negatively impact on cellular ion homeostasis, damage the cell membrane, as well as cause harm to the root and photosynthesis system, thus becoming more destructive to plants growth and development. So far, much attention has been paid on the regulatory pathways of plants’responses to salinity, but few studies were focused on alkalinity stress tolerance mechanisms. Considering the great threaten alkalinity has been to plants, more in-depth studies should be given to analyze plants’resistance mechanisms for alkali stress.Previously in our lab, a serious of wheat introgression lines were generated by asymmetric somatic hybridization technologies, with JN177 and tall wheatgrass as parents, and the introgression line——SR4 was cultivated from them which displayed strong tolerance towards saline-alkalinity. Experiments in both the field and the lab confirmed its strong resistance to alkalinity. Thus, the alkali-treated transcriptome in the root of SR4 was sequenced using the second generation high-throughput sequencing technologies. Two genes were selected for further studies as they were both up-regulated under alkali stress. One of them is an auxin response factor TaARF9, and the other is a membrane-bound transcriptional factor TaNTL5 which belongs to the NAC superfamily.1. Functional Analysis of a Wheat Auxin Response Factor TaARF9We cloned the ORF of TaARF9 with a length of 1.9 kb from SR4. TaARF9 is an auxin response factor which displayed different expression patterns under various abiotic treatment. TaARF9 was localized in the nucleus, and its transcriptional activation activity was verified in vitro via the yeast system. Agrobacterium-mediated transformation method was applied to generate TaARF9 overexpression (OE) lines in wild type Arabidopsis (Col-0), and at last, two TaARF9 homozygous OE lines in Arabidopsis were generated. In the soil, TaARF9 OE lines displayed strong inhibition in growth and development. It also growed faster, resulting in a shorter life cycle. Similarly, the growth of TaARF9 OE lines was also seriously inhibited on the culture medium under the control condition, with both the leaf area and main root length significantly reduced when compared with the control. However, under alkali and oxidative stress, TaARF9 OE lines developed significantly larger leaf areas than the control, indicating strong tolerance for alkali and oxidative stress. DAB and NBT staining implied that OE lines had accumulated a higher level of ROS, while activities of ROS-scavenging enzymes were also significantly increased. In addition, when exogenous NAA applied, OE lines were always promoted in relative growth than the wild type. We speculate that ROS signaling as well as auxin signaling might play a role in TaARF9-mediated tolerance for alkalinity.2. Functional Identification of a Wheat Transcriptional Factor TaNTL5We cloned the ORF of TaNTL5 with a length of 1.9 kb from SR4. TaNTL5 is a member of the NAC superfamily which had a transmembrane domain in the C-terminal. TaNTL5 responded differently under various abiotic treatment. However, the transcriptional activation activity of full length TaNTL5 was not detectable in the yeast system. Agrobacterium-mediated transformation method was applied to generate TaNTL5 overexpression (OE) lines in wild type Arabidopsis (Col-0), and finally, two TaNTL5 homozygous OE lines in Arabidopsis were generated. On the culture medium under the control condition, the leaf area of TaNTL5 OE lines was significantly reduced when compared with the wild type, while no significant difference was observed between their main root length. When treated with alkali and ABA, TaARF9 OE lines showed significantly larger leaf area than the control, indicating strong tolerance to both alkalinity and ABA in Arabidopsis leaves. At the same time, OE lines was more sensitive to salt stress than the control. Marker genes analysis on the ABA signaling pathway and salt-related genes implied that TaNTL5 up-regulated ABA2, and we propose that ABA signaling might play an important role in mediating plants’ resistance to alkalinity. However, TaNTL5 might regulate some components negatively in the downstream of ABA-independent pathway, which might be correlated with the sensitivity towards salt in OE lines.