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Nitrogen recycling and redistribution are important for the environmental stress response

Nitrogen recycling and redistribution are important for the environmental stress response of plants. activating stress-response genes genome-wide (Watanabe et al., 2014b). Despite several genes of ureide metabolism being associated with stress, the involvement of the remaining components, especially those downstream of allantoate degradation, is largely unknown. More importantly, the regulation mechanisms of stress induction are not well understood. (to hydrolyze ureidoglycolate into glyoxylate, carbon dioxide, and two molecules of ammonia (Werner et al., 2010). Here, we report the LT-responsive expression of the rice gene (is investigated. The results obtained here indicate that C-repeat-binding factors/DRE-binding proteins 1 (CBFs/DREB1s) play a critical role in the LT-responsive expression of L. ssp. (was PCR-amplified from rice genomic DNA using gene-specific primers. To identify potential functional elements, the full-length sequence of Pwas analyzed with the PLACE1 and Plant-PAN2 software packages as previously described (Luo et al., 2013). Promoter-Chimeric Vector Construction and Generation of Transgenic Rice Plants The 5 deletions of Pat positions C1227, C717, C522, C420, and C137 were generated by PCR amplification using different forward primers and a single downstream primer. A coding sequence. The corresponding plasmids 1229582-33-5 supplier were designated as Paccording to the position at the 5 end. Site-specific mutation was performed using the Quick Change Site-Directed Mutagenesis Kit (Transgene, China). The pEASY-T plasmid containing the Pfragment was used as the PCR template. The obtained mutated construct was cut by coding sequence. The obtained construct was named Pand used as a control. A 103-bp fragment that was located in the region from C522 to C420 of Pwas obtained by PCR using sequence-specific primers with a to obtain the recombinant plasmid Pwas also fused to Pas a positive control (construct Pstrain EHA105. The rice transformation constructs that were used contained the gene under the control of the 35S promoter to enable hygromycin-based plant selection. Embryonic calli from the mature rice seeds (L. ssp. gene under temperature stress, 10-days-after-germination (DAG) seedlings on agar plates were placed in a growth chamber at constant temperatures of 4 or 42C under a light/dark cycle of 16 h/8 h. The seedlings were incubated in 1/2 MS solution containing 250 mM NaCl for salt treatment and 100 M ABA for ABA treatment. For drought stress, the seedlings were dried 1229582-33-5 supplier at 40% relative humidity. Then, the samples were harvested at 0, 4, 8, 12, and 24 h and frozen in liquid nitrogen for RNA extraction. To analyze the response of Pto LT stress at different temperatures, 10-DAG seedlings on agar plates were placed in growth chambers at 4, 10, and 15C. The control seedlings were grown under the same conditions but at 30C. The samples were harvested at 0, 4, 8, 12, and 24 h. Mature plants at 60 DAG were treated for 24 h at 4C, after which the roots, stems and leaves were collected. To analyze the response to LT stress, transgenic plants of truncation and mutation constructs were Serpine1 treated for 24 h at 4C as above. RNA Isolation and qRT-PCR Analysis The total RNA was extracted from rice using the RNAprep Pure Plant Kit (TIANGEN, China) in accordance with the manufacturers instructions. To amplify the corresponding genes, cDNAs were synthesized with random primers using the FastQuant RT Kit (TIANGEN, China) as the template for the qRT-PCR. Real-time 1229582-33-5 supplier quantitative PCR was performed using an ABI PRISM 7500 real-time PCR system (Applied Biosystems, USA) with SYBR Green (TIANGEN, China). The real-time PCR conditions were 95C for 10 min, followed by 40 cycles of 15 s at.