Efficient and genotype independent maize transformation using pollen transfected by DNA-coated magnetic nanoparticles.

Current gene delivery methods for maize are limited to specific genotypes and depend on time-consuming and labor-intensive tissue culture techniques. Here, we report a new method to transfect maize that is culture-free and genotype independent. To enhance efficiency of DNA entry and maintain high pollen viability of 32%-55%, transfection was performed at cool temperature using pollen pretreated to open the germination aperture (40%-55%). Magnetic nanoparticles (MNPs) coated with DNA encoding either red fluorescent protein (RFP), ß-glucuronidase gene (GUS), enhanced green fluorescent protein (EGFP) or bialaphos resistance (bar) was delivered into pollen grains, and female florets of maize inbred lines were pollinated. Red fluorescence was detected in 22% transfected pollen grains, and GUS stained 55% embryos at 18 d after pollination. Green fluorescence was detected in both silk filaments and immature kernels. The T1 generation of six inbred lines showed considerable EGFP or GUS transcripts (29%-74%) quantitated by polymerase chain reaction, and 5%-16% of the T1 seedlings showed immunologically active EGFP or GUS protein. Moreover, 1.41% of the bar transfected T1 plants were glufosinate resistant, and heritable bar gene was integrated into the maize genome effectively as verified by DNA hybridization. These results demonstrate that exogenous DNA could be delivered efficiently into elite maize inbred lines recalcitrant to tissue culture-mediated transformation and expressed normally through our genotype-independent pollen transfection system.

Nucleotide variation in the NCED3 region of Arabidopsis thaliana and its association study with abscisic acid content under drought stress.

Drought tolerance is a comprehensive quantitative trait that is being understood further at the molecular genetic level. Abscisic acid (ABA) is the main drought-induced hormone that regulates the expression of many genes related to drought responses. 9-cis-epoxycarotenoid dioxygenase (NCED3) is thought to be a key enzyme in ABA biosynthesis. In this paper, we measured the ABA content increase under drought stress, and sequenced and compared the sequence of AtNCED3 among 22 Arabidopsis thaliana accessions. The results showed that the fold of ABA content increase under drought stress was highly variable among these accessions. High density single nucleotide polymorphism (SNP) and insertion/deletion (indel) were found in the AtNCED3 region, on average one SNP per 87.4 bp and one indel per 502 bp. Nucleotide diversity was significantly lower in the coding region than that in non-coding regions. The results of an association study with anova analysis suggested that the 274th site (P<-->S) and the 327th site (P<-->R) amino acid variations might be the cause of ABA content increase of 163av accession under drought stress.

Size dependent magnetization and high-vacuum annealing enhanced ferromagnetism in Zn(1-x)Co(x)O nanowires.

Diameter controllable ZnO nanowires have been fabricated by thermal evaporation (vapor transport) with various sizes of gold nanoparticles as catalysts. Diluted magnetic semiconductor (DMS) Zn(1-x)Co(x)O nanowires were then made by high energy Co ion implantation. The as-implanted and the argon-annealed Zn(1-x)Co(x)O nanowires displayed weak ferromagnetism while the high-vacuum annealed nanowires exhibited strong ferromagnetic ordering at room temperature. Size dependent behavior has been observed in the magnetic field and temperature dependences of magnetization. The shrinkage of the nanowire diameter reduced the spontaneous magnetization as well as the hysteresis loops. Field cooled and zero-field cooled magnetization and coercivity measurements were performed between 2 and 300 K to study the evolution of magnetism from the weak to the strong ferromagnetic states. In particular, superparamagnetic features were observed and shown to be intrinsic characteristics of the DMS Zn(1-x)Co(x)O nanowires. The room-temperature spontaneous magnetization of individual Zn(1-x)Co(x)O nanowires was also established by using magnetic force microscope measurements.

[Localization of microtubule-associated protein AtMAP65-1 in guard cells of arabidopsis].

AtMAP65-1 gene was cloned from Arabidopsis genome. A plant expression vector containing AtMAP65-1-GFP fusion gene driven by 35S promoter was constructed and introduced into Arabidopsis by Agrobacterium infection. Under confocal laser scanning microscope (CLSM), it was found that in the light-induced opening guard cells AtMAP65-1 was distributed radiately from dorsal wall to ventral wall in a bundling way. Treated with microtubule specific inhibitor-Vinblastine (10 micromol/L) for 90 min, the organization of AtMAP65-1 in guard cell was disrupted dramatically. Following the depolymerization of cortical microtubule and the decreasing of stomatal aperture, the fluorescence of AtMAP65-1 became to be fragmented into dotted spots. The organization of AtMAP65-1 was broken up completely when the treatment was up to 120 min. In the dark-induced closing guard cells, AtMAP65-1 was turned into fragments and was randomly dispersed throughout the cells. That is, AtMAP65-1 is co-localized with cortical microtubules. The results suggest that AtMAP65-1 binds cortical microtubules, and it may regulate the arrangement of cortical microtubules, which helps us gain more insights into the role of AtMAP65-1 in stomatal movement.

[Nucleotide polymorphism in the drought induced transcription factor CBF4 region of Arabidopsis thaliana and its molecular evolution analyses].

Intraspecific nucleotide polymorphism in the drought induced transcription factor CBF4 region of Arabidopsis thaliana was analyzed with 17 core accessions growing in different ecoclimate. High density of single nucleotide polymorphism (SNP) and insertion/deletion (Indel) were found, on average 1 SNP per 35.8 bp and 1 Indel per 143 bp. Nucleotide polymorphism in non-coding region was three times higher than that in coding region. In coding region of CBF4, SNP frequency is one SNP per 96.4 bp, one nonsynonymous mutation was detected from 25 av, 203 av and 244 av accessions, which is the 205th site amino acid variation: gly <--> val caused by the 1034th site (corresponding to 19,696 site nucleotide of GenBank No. AB015478 as 1) nucleotide variation: G <--> T. Statistical result of nucleotide diversity showed that linkage disequilibrium (LD) existed in large-scale region of CBF4 and recombination event was also detected in 5' non-coding region. Identical to the results of other genes of Arabidopsis, different regions of the gene were seemingly under different selective pressures. Balancing selection resulted in high nucleotide diversity in 3' non-coding region, and the neutral mutation hypothesis can explain the DNA polymorphism in coding region, whereas, nature positive selection in the population affected nucleotide variation in 5' non-coding region of gene.

[ATHK1 gene regulates signal transduction of osmotic stress in Arabidopsis thaliana].

Differences in physiology and gene expression between ATHK1 knock-out mutant caused by T-DNA insertion and wild type (WT) of WS accession of Arabidopsis thaliana were analysed. Water loss ratio of detached leaf of ATHK1-mutant was obviously higher than that of WT. After being treated with 30% PEG-6000, ion leakage ratio of cell membrane in wild type leaves was 50% higher than that before PEG treatment, while in mutant leaves it increased 80%. The wilted phenotype of ATHK1-mutant after PEG treatment for 48 h was higher than that of WT. All these results showed that ATHK1-mutant was more sensitive to osmotic stress compared to WT and ATHK1 involved in osmotic stress adaptation. Differential-Display Reverse Transcription-PCR (DDRT-PCR) analysis was carried out to investigate the difference of gene expression between ATHK1-mutant and WT. Nine differential cDNA fragments involved in stress adaptation were identified, including the MAPKKK18 and serine/threonine protein kinase genes. These fragments were up-regulated by PEG treatment in WT, but not in ATHK1-mutant. These results indicate that ATHK1 plays an important role up-stream from MAPK in the osmotic stress signal transduction pathway. ATHK1 may be working as a plant osmosensor.

Purification and identification of a 42-kilodalton abscisic acid-specific-binding protein from epidermis of broad bean leaves.

Purification of abscisic acid (ABA)-binding proteins is considered to constitute a major step toward isolating ABA receptors. We report here that an ABA-binding protein was for the first time, to our knowledge, purified from the epidermis of broad bean (Vicia faba) leaves via affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, and isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis two-dimensional electrophoresis of the purified ABA-binding protein all identified a single protein band with a molecular mass of 42 kD and an isoelectric point 4.86. The Scatchard plot for the purified protein showed a linear function with a maximum binding activity of 0.87 mol mol(-1) protein and an equilibrium dissociation constant of 21 nM, indicating that the purified protein may be a monomeric one, possessing one binding site. The ABA-binding protein was enriched more than 300-fold with a yield of 14%. (-)ABA and trans-ABA were substantially incapable of displacing (3)H-(+/-)ABA bound to the ABA-binding protein, and (+/-)ABA was less effective than (+)ABA in the competition. These findings allow establishment of the stereospecificity of the 42-kD protein and suggest its ABA receptor nature. Pretreatment of the guard cell protoplasts of broad bean leaves with the monoclonal antibody raised against the 42-kD protein significantly decreased the ABA specific-induced phospholipase D activity in a dose-dependent manner. This physiological significance provides more clear evidence for the potential ABA-receptor nature of the 42-kD protein.

Identification of the Photoaffinity-labeled Abscisic Acid Binding Proteins from Maize Root Microsome.

ABA binding protein (ABA-BP) was solubilized with 0.2% (W/V) Triton X-100 from the microsomal membrane of maize root, with a specific binding activity of 0.56 pmol ABA/g FW. When ABA was cross-linked to its binding sites by irradiating the microsomal proteins with light of 330 nm after incubation with low levels of H(3)-(-/+)ABA, it was found that this kind of photoaffinity labeling was specific to ABA-BP. On PAGE and SDS-PAGE three major protein bands with molecular weights of 72 kD, 47 kD and 37 kD were found with high radioactivity.