Ai-Qin Wang

Transformation of sugarcane with ACC oxidase antisense gene

The present study is an attempt to validate the function of sugarcane ACO by construction of plant expression vectors for sugarcane ACO sense and anti-sense genes and their effective transformation. Plant expression vectors for sugarcane ACO anti-sense gene was constructed and then transformed into sugarcane variety ROC22 mediated by Agrobacterium tumefaciens. The transgenic shoots were screened under the selective pressure of 40 mg/l G418 and 300 mg/l cefalexin and 19 resistant plants were obtained, two of which were further proved to be positive through detecting NPTII by PCR. The transformation rate was found 10.5%. The positive plants were found to grow slowly and were more dwarf compared to non-transgenic ones. The present work reveals the efficient genetic transformation system and development of transgenic sugarcane plant with ACO antisense gene. This work will help in understanding the ethylene and growth relationship and will also help breeders in breeding programs tailored to develop new varieties using molecular approaches.

Aluminum induces rapidly mitochondria- dependent programmed cell death in Al-sensitive peanut root tips

BackgroundAlthough many studies suggested that aluminum (Al) induced programmed cell death (PCD) in plants, the mechanism of Al-induced PCD and its effects in Al tolerance is limited. This study was to investigate the mechanism and type of Al induced PCD and the relationship between PCD and Al tolerance.ResultsIn this study, two genotypes of peanut 99-1507 (Al tolerant) and ZH2 (Al sensitive) were used to investigate Al-induced PCD. Peanut root growth inhibition induced by AlCl3 was concentration and time-dependent in two peanut varieties. AlCl3 at 100 μM could induce rapidly peanut root tip PCD involved in DNA cleavage, typical apoptotic chromatin condensation staining with DAPI, apoptosis related gene Hrs203j expression and cytochrome C (Cyt c) release from mitochondria to cytosol. Caspase3-like protease was activated by Al; it was higher in ZH2 than in 99-1507. Al increased the opening of mitochondrial permeability transition pore (MPTP), decreased inner membrane potential (ΔΨm) of mitochondria. Compared with the control, Al stress increased O2•- and H2O2 production in mitochondria. Reactive oxygen species (ROS) burst was produced at Al treatment for 4 h.ConclusionsAl-induced PCD is earlier and faster in Al-sensitive peanut cultivar than in Al-tolerant cultivar. There is a negative relationship between PCD and Al resistance. Mitochondria- dependence PCD was induced by Al and ROS was involved in this process. The mechanism can be explained by the model of acceleration of senescence under Al stress.

Molecular cloning and tissue specific expression of an elongation factor 1A gene in sugarcane stalks

Genomic reconstruction done by using the cDNA insert containing the ORF of GZ-1-alpha as the probe indicated the presence of about 5 copies of eEF1A gene per haploid sugarcane genome. Northern hybridization studies revealed the presence of eEF1A transcripts in Saccharum officinarum stalks at late growth stage. A full-length cDNA clone (GZ-1-alpha) encoding elongation factor 1-alpha has been isolated from stalks of S. officinarum L. (accession number EF581011). The 1736 bp nucleotide sequence and the deduced 447 amino acid sequence shared 65–92 % and 95–99 % identity, respectively, to the other plant elongation factor 1As. The 400 mg/L ethephon treatment significantly induced the expression of GZ-1-alpha and maintained higher level in the top internodes for a long time, but did not induce the expression in the mid and bottom internodes. The results suggest that ethephon mediates the tissue and growth stage specific expressions of eEF1A gene isolated from S. officinarum stalks.

Cloning of three members of acc synthase gene family in sugarcane

To clone the members of ACC synthase gene family in sugarcane, two degenerate oligonucleotide primers were designed, coding for two conservative acid regons in ACC synthase protein family. PCR amplification was performed on sugarcane DNA template, which produced three fragments that were 1041 bp (Sc-ACS1), 1345bp (Sc-ACS2) and 1707bp (Sc-ACS3), respectively. Analyses by using the program of BLAST on NCBI GenBank database showed that the polypeptide sequences of their coding regions highly matched with the family of ACC synthase genes from grass plants, the identity of their nucleotide sequence reached 88% to 98%. The nucleotide sequence of both Sc-ACS1 and Sc-ACS3 in opposite directons shared a homology of 98% but they were less similar to Sc-ACS2 (49%). Alignment and phylogenetic analyses of the amino acid sequence deduced from these fragments and sequences of ACC synthase from other plants in the GenBank. According to the ACC synthase protein family, the intron was excluding and the three sequences coded 326,242 and 310 amino acids, respectively, which spanned 67%, 50% and 64.6%, respectively, of the putative whole sequence in length. The putative amino acid sequence of both Sc-ACS1 and Sc-ACS3 shared significant identity to other ACC synthases (53–96%) and they were more similar to the sequences in grass plant species (80% with Zea mays Zm-ACS6, 75% withAsparagus officinaland andPhyllostachs edulis, and 73% withOryza sativa OS-ACS1) than to Sc-ACS2 (45–49%). The amino acid sequence of both Sc-ACS1 and Sc-ACS3 in opposite directions shared a homology of 96%. Based on the Northern analysis of the three ACC synthase gene members isolated it was concluded that accumulation of Sc-ACS1 RNA increased moderately in the sugarcane leaves treated with dark-growing condition, ethrel, IAA, LiCl and cold-stress, but not expressed while treated with BA and light- growing condition. The Sc-ACS2 was only expressed in the ethrel treatment, whereas Sc-ACS3 did not express in any treatment. The present study provided a new evidence of ethylene regulation in sugarcane at molecular level, as part of the regulation mechanism of ethylene to the growth of sugarcane plants.

Nitric Oxide Inhibits Al-Induced Programmed Cell Death in Root Tips of Peanut (Arachis hypogaea L.) by Affecting Physiological Properties of Antioxidants Systems and Cell Wall

It has been reported that nitric oxide (NO) is a negative regulator of aluminum (Al)-induced programmed cell death (PCD) in peanut root tips. However, the inhibiting mechanism of NO on Al-induced PCD is unclear. In order to investigate the mechanism by which NO inhibits Al-induced PCD, the effects of co-treatment Al with the exogenous NO donor or the NO-specific scavenger on peanut root tips, the physiological properties of antioxidants systems and cell wall (CW) in root tip cells of NO inhibiting Al-induced PCD were studied with two peanut cultivars. The results showed that Al exposure induced endogenous NO accumulation, and endogenous NO burst increased antioxidant enzyme activity in response to Al stress. The addition of NO donor sodium nitroprusside (SNP) relieved Al-induced root elongation inhibition, cell death and Al adsorption in CW, as well as oxidative damage and ROS accumulation. Furthermore, co-treatment with the exogenous NO donor decreased MDA content, LOX activity and pectin methylesterase (PME) activity, increased xyloglucan endotransglucosylase (XET) activity and relative expression of the xyloglucan endotransglucosylase/hydrolase (XTH-32) gene. Taken together, exogenous NO alleviated Al-induced PCD by inhibiting Al adsorption in CW, enhancing antioxidant defense and reducing peroxidation of membrane lipids, alleviating the inhibition of Al on root elongation by maintaining the extensibility of CW, decreasing PME activity, and increasing XET activity and relative XTH-32 expression of CW.

Analysis of Mitochondrial Markers of Programmed Cell Death

Mitochondria play a crucial role in programmed cell death (PCD) in plants. In most cases of mitochondria-dependent PCD, cytochrome c (Cyt c) released from mitochondria due to the opening of mitochondrial permeability transition pore (MPTP) and the activation of caspase-like proteases. Here we describe the analytic methods of mitochondrial markers of PCD including mitochondria isolation, mitochondrial membrane permeability, mitochondrial inner membrane potential, Cytc release, ATP, and mitochondrial reactive oxygen species (ROS).

Nitric oxide suppresses aluminum-induced programmed cell death in peanut ( Arachis hypoganea L.) root tips by improving mitochondrial physiological properties

Aluminum (Al) stress alters nitric oxide (NO) and induces programmed cell death (PCD) in plants. Recent study has shown that NO inhibits Al-induced PCD. However, the mechanism of NO inhibiting Al-induced PCD has not been revealed yet. Here, we investigated the behavior of mitochondria during Al-induced PCD suppressed by NO in peanut. Seedlings of peanut was grown hydroponically in a controllable growth room. The mitochondrial physiological parameters were determined spectrophotometrically. The expression of AhANT and AhHsp70 was determined by quantitative RT-PCR. Al-induced cell death rapidly in peanut root tips is mitochondria-dependent PCD. There was a significantly negative relationship between PCD and mitochondrial NO/H2O2 level. Compared with Al treatment alone, the addition of NO donor sodium nitroprusside (SNP) increased the ratio of NO/H2O2, down-regulated AhANT expression and inhibited the opening of mitochondrial permeability transition pore (MPTP), up-regulated AhHsp70 expression and increased mitochondrial inner membrane potential (ΔΨm), reduced cytochrome c (Cyt c) release from mitochondria and caspase 3-like protease activity, while the effect of NO specific scavenger cPTIO supplement was opposite. NO suppresses Al-induce PCD in peanut root tips by improving mitochondrial physiological properties.