In order to better understand the role of antioxidant enzymes in plant stress protection mechanisms, transgenic tobacco ( Nicotiana tabacum cv. Xanthi) plants were developed that overexpress both superoxide dismutase (SOD) and ascorbate peroxidase (APX) in chloroplasts. These plants were evaluated for protection against methyl viologen (MV, paraquat)-mediated oxidative damage both in leaf discs and whole plants. Transgenic plants that express either chloroplast-targeted CuZnSOD (C) or MnSOD (M) and APX (A) were developed (referred to as CA plants and AM plants, respectively). These plant lines were crossed to produce plants that express all three transgenes (CMA plants and AMC plants). These plants had higher total APX and SOD activities than non-transgenic (NT) plants and exhibit novel APX and SOD isoenzymes not detected in NT plants. As expected, transgenic plants that expressed single SODs showed levels of protection from MV that were only slightly improved compared to NT plants. The expression of either SOD isoform along with APX led to increased protection while expression of both SODs and APX provided the highest levels of protection against membrane damage in leaf discs and visual symptoms in whole plants.

Calmodulin (CaM) influences many cellular processes by interacting with various proteins. Here, we isolated AtBAG6, an Arabidopsis CaM-binding protein that contains a central BCL-2-associated athanogene (BAG) domain. In yeast and plants, overexpression of AtBAG6 induced cell death phenotypes consistent with programmed cell death (PCD). Recombinant AtBAG6 had higher affinity for CaM in the absence of free Ca 2 þ than in its presence. An IQ motif (IQXXXRGXXXR, where X denotes any amino-acid) was required for Ca 2 þ -independent CaM complex formation and single amino-acid changes within this motif abrogated both AtBAG6-activated CaM-binding and cell death in yeast and plants. A 134-amino-acid stretch, encompassing both the IQ motif and BAG domain, was sufficient to induce cell death. Agents generating oxygen radicals, which are known to be involved in plant PCD, specifically induced the AtBAG6 transcript. Collectively, these results suggest that AtBAG6 is a stress-upregulated CaM-binding protein involved in plant PCD.

Reverse transcription quantitative real-time PCR (RT-qPCR) has become one of the most widely used methods for gene expression analysis, but its successful application depends on the stability of suitable reference genes used for data normalization. In plant studies, the choice and optimal number of reference genes must be experimentally determined for the specific conditions, plant species, and cultivars. In this study, ten candidate reference genes of sweetpotato (Ipomoea batatas) were isolated and the stability of their expression was analyzed using two algorithms, geNorm and NormFinder. The samples consisted of tissues from four sweetpotato cultivars subjected to four different environmental stress treatments, i.e., cold, drought, salt and oxidative stress. The results showed that, for sweetpotato, individual reference genes or combinations thereof should be selected for use in data normalization depending on the experimental conditions and the particular cultivar. In general, the genes ARF, UBI, COX, GAP and RPL were validated as the most suitable reference gene set for every cultivar across total tested samples. Interestingly, the genes ACT and TUB, although widely used, were not the most suitable reference genes in different sweetpotato sample sets. Taken together, these results provide guidelines for reference gene(s) selection under different experimental conditions. In addition, they serve as a foundation for the more accurate and widespread use of RT-qPCR in various sweetpotato cultivars.