Fig uEffect of Runx and

Among these senescence-associated NAC TFs, the role of ORE1/ANAC092 has been widely studied. It can be induced by darkness, salinity, ABA, and JA (Kim et al., 2009). Additionally, it could enhance the expression of SINA1, SAG29/SWEET15, and BFN1, further accelerating senescence and developmental PCD (programmed cell death) ( Balazadeh et al., 2010uanduMatallana-Ramirez et al., 2013). Furthermore, ANAC059/ORS1 is considered as a duplication of the ANAC092/NAC2/ORE1 ( Balazadeh et al., 2011). Similarly to ORE1, it triggers expression of senescence-associated genes through a regulatory network.
In cotton, seventy-seven NAC genes in Gossypium hirsutum L. and 145 NAC genes in G raimondii Ulbr. have been reported to respond to many stresses ( Meng et al., 2009, Shah et al., 2013, Shang et al., 2013uanduShah et al., 2014). However, few studies were focused on the relationship between leaf senescence and the function of single NAC TFs in cotton.
In this study, we performed functional analyses of an up-regulated gene during leaf senescence, G. hirsutum L. NAC12 (GhNAC12), one of the 103 NAC unigenes (partly published) cloned by our lab ( Shah et al., 2013). Overexpression of GhNAC12 in Arabidopsis led to early senescence. And it exhibited a differential expression pattern between two types of upland cotton varieties. All of our results suggested that it is a candidate early-aging gene in cotton. Therefore, our data provide valuable information for understanding the molecular mechanism of early aging and for selecting early-maturing but non-early-aging image varieties.
2. Materials and methods
2.1. Plant materials, growth conditions, and transformation
Arabidopsis thaliana ecotype Col-0 was the parent strain for the transgenic lines used in this study. Surface-sterilized seeds were plated on 1/2 MS medium (2.2ug.Lu1 MS salts, 1% sucrose, and 10ug.Lu1 agar; pHu5.7a5.8) and were imbibed for 4udays at 4uC to improve germination uniformity. The plants were grown in a greenhouse at 22uC under a 16uh light/8uh dark Phos-tag  (PAR of 100a150uμEumu2.su1) and a relative humidity of approximately 80%.
The CCRI 10 seeds were germinated in potting soil at 25uC in a culture room with a 16uh light/8uh dark cycle. The cotyledons were sampled every 10udays beginning at 15udays after spreading out.
To detect the variety-specific expression of GhNAC12, two cotton varieties (CCRI 10 and Liao 4086) were planted in the experimental field in the Institute of Cotton Research of CAAS (ICR of CAAS, Anyang, Henan Province). All plants were topped 100udays after seeding, and the top leaf after topping was sampled every 5udays from the top day onward.
For neutrality tests, 27 upland cotton cultivars were also planted in the experimental field in the ICR of CAAS. All of the race latifolium cultivar samples were provided by the National Wild Cotton Nursery (Sanya, China). Young leaves were taken as samples.
All samples were quickly frozen in liquid nitrogen and stored at u80uC for RNA extraction.
To obtain GhNAC12-overexpressing transgenic plants, the complete coding sequence of GhNAC12 was digested with Xba I and Sac I, and the digested fragment was cloned into the pBI121 vector containing a 35S promoter. The constructed overexpression plasmid was transferred into Agrobacterium tumefaciens (strain LBA4404) and used to transform Arabidopsis plants via the floral dip method ( Clough and Bent, 1998). Positive transgenic lines were screened on 1/2 MS medium with kanamycin (Solarbio, Beijing, China; 50uμg.mlu1) and confirmed through PCR.
2.2. RNA extraction, qRT-PCR, RT-PCR, and DNA preparation
Total RNA was isolated from plant tissues with the RNAprep Pure Plant Kit (Tiangen, China). Potential genomic DNA was removed using RNase-free DNase I (Tiangen, China). First-strand cDNA samples were generated from 2uμg of RNA via reverse transcription using the SuperScript III First-Strand Synthesis System for qRT-PCR (Invitrogen, USA) and were used as templates for RT-PCR-based gene expression analyses. qRT-PCR was performed using the UltraSYBR Mixture (with ROX) (CWBIO, China), and the obtained data were analyzed with GraphPad Prism5. Marker genes of senesce
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