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Phosphatidylinositol (PtdIns) synthase is a key enzyme in the phospholipid pathway and catalyses the formation of PtdIns. PtdIns is not only a structural component of cell membranes, but also the precursor of the phospholipid signal molecules that regulate plant response to environment stresses. Here, we obtained transgenic maize constitutively overexpressing or underexpressing PIS from maize (ZmPIS) under the control of a maize ubiquitin promoter. Transgenic plants were confirmed by PCR, Southern blotting analysis and real-time RT-PCR assay. The electrospray ionization tandem mass spectrometry (ESI-MS/MS)-based lipid profiling analysis showed that, under drought stress conditions, the overexpression of ZmPIS in maize resulted in significantly elevated levels of most phospholipids and galactolipids in leaves compared with those in wild type (WT). At the same time, the expression of some genes involved in the phospholipid metabolism pathway and the abscisic acid (ABA) biosynthesis pathway including ZmPLC, ZmPLD, ZmDGK1, ZmDGK3, ZmPIP5K9, ZmABA1, ZmNCED, ZmAAO1, ZmAAO2 and ZmSCA1 was markedly up-regulated in the overexpression lines after drought stress. Consistent with these results, the drought stress tolerance of the ZmPIS sense transgenic plants was enhanced significantly at the pre-flowering stages compared with WT maize plants. These results imply that ZmPIS regulates the plant response to drought stress through altering membrane lipid composition and increasing ABA synthesis in maize.

The human CD14, a high affinity receptor for lipopolysaccharides (LPS), is involved in the innate immunity system and the inflammatory response. There is increasing interest in using recombinant approaches to produce purified CD14 protein for therapeutic uses. Plants provide ideal expression systems for the production of recombinant proteins, but the levels of expression of recombinant proteins produced in planta are still not high. To improve expression levels of CD14 the 22-kDa alpha-zein signal peptide (ZSP) from maize was fused to the human CD14 cDNA so that recombinant CD14 could stably accumulate in plant cells. The human CD14 gene and the modified human CD14 cDNA with the 22-kDa ZSP were respectively transformed into tobacco to produce transgenic plants. Western blot analysis confirmed human CD14 accumulation in the transgenic tobacco. The concentration of the recombinant protein in the tobacco leaves was measured by ELISA, and the results suggested that fusion with the 22-kDa alpha-ZSP effectively increased the accumulation of the recombinant protein (rCD14). The concentration of rCD14 in some of the transgenic lines was 19.54 μg g−1 tobacco leaf (fw), which was about 0.6 % of the total soluble protein. The rCD14 protein showed natural LPS-binding bioactivity by using U937 cells mensuration. Our results suggested that the maize 22-kDa alpha-zein signal peptide could be used to increase the accumulation of recombinant protein in tobacco leaves so that proteins can be produced in abundant biomass.

Drought is a major agricultural menace reducing crop productivity and limiting the successful realization of land potential throughout the world. Therefore, breeding common wheat with improved drought-tolerance via genetic manipulation is of great importance. We have introduced the betA gene encoding choline dehydrogenase from Escherichia coli into common wheat (Triticum aestivum L.) by Agrobacterium-mediated transformation. Various levels of expression of the betA gene were confirmed by RT-PCR among the transgenic lines and different levels of glycine betaine accumulation were detected in these lines. Several wheat transgenic lines with different betA expression levels in the T3 generation and wild-type (WT) were selected to test their performance under drought stress conditions. Water deficit in plants caused a reduction in photosynthesis and activity of the PSII complex and resulted in increased accumulation of osmolytes. Drought stress also led to lower membrane stability along with much higher activities of superoxide dismutase and peroxidase in all wheat lines. However, wheat lines that were transgenic for the betA gene were less injured and exhibited greater root length and growth compared with the WT. It was concluded that the amount of injury to the wheat plants was negatively correlated with the level of accumulation of glycine betaine, and the glycine betaine acted as an important osmoprotectant in transgenic plants to improve root growth, and enhance the resistance of transgenic plants to drought stress.

The DREB/CBF transcription factors play important roles during low temperature, drought, and high-salt stress in higher plants. In this paper, we transferred TsCBF1 gene from a dicotyledonous halophyte Thellungiella halophila into the monocotyledonous crop maize (Zea mays L.). PCR, Southern blot, and RT-PCR analysis indicated that the TsCBF1 gene had been integrated into the genome of transgenic plants and was expressed in their progeny. After 14 days of drought stress treatments, transgenic plants showed improved drought tolerance with higher Relative Water Content (RWC), higher solute accumulation, and less cell damage compared with wild-type (WT) plants. Most importantly, they showed shorter anthesis-silking interval (ASI) and produced much higher grain yield than WT under drought stress. The results indicate that the TsCBF1 gene conferred enhanced drought tolerance to maize plants and may have utility for improving tolerance to other abiotic stresses as well.

A betA gene encoding choline dehydrogenase from Escherichia coli (E. coli) was transformed into wheat (Triticum aestivum L.) via Agrobacterium-mediated transformation. PCR amplification and Southern blotting confirmed the existence of transgene in transformed plants and their progeny. Levels of expression of the betA gene varied among the different transgenic lines based on RT-PCR analysis. Under salt stress conditions, transgenic lines L2 and L3 had higher levels of glycine betaine and chlorophyll, lower Na+/K+ ratios and solute potential, and less cell membrane damage. These lines also retained moderately higher photosynthesis rates and more vigorous growth than the wild-type line at 200 mM NaCl. In a field trial in a high salt field, transgenic lines L2 and L3 had higher germination rates, more tillers and higher grain yields in comparison to the wild-type plants. This suggested that the transgenic plants were more tolerant to salt stress and have potential for breeding salt-tolerant wheat.

The possible release of selectable marker genes from genetically modified transgenic plants, or of gut microbes, to the environment, has raised worldwide public concerns. In this study, we showed the generation of marker-free transgenic maize plants constitutively expressing AtNHX1, a Na(+)/H(+) antiporter gene from Arabidopsis that conferred salt tolerance on plants, using the FLP/FRT site-specific recombination system. Transgenic plant expressing a modified FLP recombinase gene was crossed with transgenic plant harboring AtNHX1 and mutant als, a selectable marker gene flanked by two directed FRT sites. The sexual crossing led to precise and complete excision of the FRT-surrounded als marker gene in the F1 progenies. Further salt tolerance examinations indicated that marker-free AtNHX1 transgenic plants accumulated more Na(+) and K(+), and produced greater biomass and yields than did the wild-type plants when grown in high saline fields. These results demonstrate the feasibility of using this FLP/FRT-based marker elimination system to generate marker-free transgenic important cereal crops with improved salt tolerance.

Previous studies have shown that the overexpression of betA (encoding choline dehydrogenase from Escherichia coli) or AtNHX1 (a vacuolar Na(+)/H(+) antiport from Arabidopsis thaliana) gene can improve the salt tolerance of transgenic plants. However, little is known about the effects of the transgene pyramiding of betA and AtNHX1. Here, betA + AtNHX1 transgene pyramiding tobacco was produced by sexual crossing, and the salt tolerance was evaluated at the cellular and plant levels. In NaCl stress, the Na(+) concentration in vacuoles and vacuolar membrane potential of transgene pyramiding cells were similar to those of AtNHX1-transgenics, and much higher than those of betA-transgenics when detected using fluorescent dye staining; transgene pyramiding cells showed a higher protoplast viability and comparable mitochondrial activity as compared with single transgenics; and transgene pyramiding plants showed comparable Na(+) content in leaves as compared with AtNHX1-transgenics and remarkably higher than betA-transgenics; and transgene pyramiding lines exhibited higher percentage of seed germination, better seedling growth and higher fresh weight than lines that had betA or AtNHX1 alone. Based on the integrative analysis of salt tolerance, the consistency between the cellular level and the whole plant level was confirmed and the transgene pyramiding plants exhibited improved salt tolerance, but compared with the plants with betA or AtNHX1 alone, the differences were relatively small. Other mechanisms involved in salt tolerance should be considered to further enhance transgene pyramiding plants salt tolerance.

The tolerance to drought stress of the homozygous transgenic cotton (Gossypium hirsutum L.) plants with enhanced glycinebetaine (GB) accumulation was investigated at three development stages. Among the five transgenic lines investigated, lines 1, 3, 4, and 5 accumulated significantly higher levels of GB than the wild-type (WT) plants either before or after drought stress, and the transgenic plants were more tolerant to drought stress than the wild-type counterparts from young seedlings to flowering plants. Under drought stress conditions, transgenic lines 1, 3, 4, and 5 had higher relative water content, increased photosynthesis, better osmotic adjustment (OA), a lower percentage of ion leakage, and less lipid membrane peroxidation than WT plants. The GB levels in transgenic plants were positively correlated with drought tolerance under water stress. The results suggested that GB may not only protect the integrity of the cell membrane from drought stress damage, but also be involved in OA in transgenic cotton plants. Most importantly, the seedcotton yield of transgenic line 4 was significantly greater than that of WT plants after drought stress, which is of great value in cotton production.

Sucrose: sucrose 1-fructosyltransferase (1-SST) cDNA from Lactuca sativa, coding the enzyme responsible for lower degree polymers fructan biosynthesis, was cloned by RT-PCR and RACE methods. The 1-SST cDNA under the control of CaMV 35S promoter was introduced into tobacco by Agrobacterium-mediated leaf disc transformation protocol. Fructan synthesis in vitro and carbohydrate analysis showed that sense transgenic tobacco plant displayed sucrose: sucrose 1-fructosyltransferse activity. After freezing stress, significant increases in electrolyte leakage and malondialdehyde were found in the wild type and anti-sense transgenic plants, while no apparent differences were observed in sense transgenic plants. Meanwhile, water soluble carbohydrate, fructan and fructose of sense transgenic plants remarkably increased, compared with those of wild type and anti-sense plants. No significant difference was detected in superoxide dismutase activity between transgenic and wild type plants. The above results demonstrated that the expression of 1-SST gene improved the freezing resistance of transgenic tobacco plants.

Small bud tips of 1–3 mm in length were taken from multiple shoot clumps that derived from immature inflorescence cultures of beet as recipient for the Agrobacterium-mediated transformation and transgenic plants were obtained from eight genotypes. The optimal genetic transformation protocol was established as followed: the buds were immersed in Agrobacterium suspension of OD600 =0.3–0.5 for 5–10 min, with vacuum infiltration (0.3–0.5 × 105 Pa) or supplemented with 0.01% Silwet L-77, co-cultured for 2–4 days and followed by 10-day culture on medium containing 100 mg l−1 cefotaxime, then the buds were selected on medium containing 10 mg l−1 hygromycin B for three consecutive generations. The percentage of hygromycin-resistant buds after three selections varied from 13.3 to 30.6% with genotypes. The results of PCR and further Southern blotting of genomic DNA of hygromycin-resistant buds or plants showed that the exogenous hpt and AtNHX1 gene had been integrated into the genomes of some transformed buds or plants. The transgenic buds or plants with AtNHX1 gene encoding Na+/H+ antiport on the vacuole membrane of Arabidopsis showed improved salt-tolerance than the controls. AtNHX1gene inherited in some transgenic lines as Mendelian segregation. This result revealed that it was feasible to improve salt-tolerance of beets by the introduction of AtNHX1 gene into cultured buds.

The inheritance and stability of the acetolactate synthase (als) transgene were compared in transgenic maize plants, generated using the pollen-tube pathway, particle bombardment, or Agrobacterium-mediated methods of transformation. Progeny populations generated by successive selfing or backcrossing of primary transformants were analyzed over three generations, using PCR and herbicide screening, to examine segregation and als activity, respectively, and transgenic homozygous plants were selected. The pollen-tube method resulted in a higher rate of primary normal transgenic plants and a less-stable transmission of the als locus than did the other two methods. When transferred by the particle bombardment and Agrobacterium-mediated methods, the als gene was in a much higher proportion of Mendelian transmission than transferred by the pollen-tube method. Compared to the Agrobacterium-mediated transformation, the particle bombardment method tends to create multiple copies and insert sites of the als gene in maize genome, which delaying the homogenization of the als locus with advancing generations. Agrobacterium-mediated transformation resulted in a greater proportion of stable, low copy number (in general 1–2) transgenic events, facilitating the stable inheritance of the als gene, and producing multiple desirable transgenic plants.

Phosphatidylinositol (PtdIns) is an important lipid because it serves as a key membrane constituent and is the precursor of the inositol-containing lipids that are found in all plants and animals. It is synthesized from cytidine-diphosphodiacylglycerol (CDP-DG) and myo-inositol by PtdIns synthase (PIS). We have previously reported that two putative PIS genes from maize (Zea mays L.), ZmPIS and ZmPIS2, are transcriptionally up-regulated in response to drought (Sui et al., Gene, 426:47-56, 2008). In this work, we report on the characterization of ZmPIS in vitro and in vivo. The ZmPIS gene successfully complemented the yeast pis mutant BY4743, and the determination of PIS activity in the yeast strain further confirmed the enzymatic function of ZmPIS. An ESI-MS/MS-based lipid profiling approach was used to identify and quantify the lipid species in transgenic and wild-type tobacco plants before and after drought treatment. The results show that the overexpression of ZmPIS significantly increases lipid levels in tobacco leaves under drought stress compared to those of wild-type tobacco, which correlated well with the increased drought tolerance of the transgenic plants. Further analysis showed that, under drought stress conditions, ZmPIS overexpressors were found to exhibit increased membrane integrity, thereby enabling the retention of more solutes and water compared with the wild-type and the vector control transgenic lines. Our findings give us new insights into the role of the ZmPIS gene in the response of maize to drought/osmotic stress and the mechanisms by which plants adapt to drought stress.

Two full-length cDNAs, designated as ZmFtsH2A and ZmFtsH2B, were isolated from maize (Zea mays L.) by suppression subtractive hybridization coupled with in silico cloning approach. The predicted proteins of ZmFtsH2A and ZmFtsH2B both consisted of 677 amino acid residues and displayed high similarity to FtsH2 protease of Arabidopsis thaliana. DNA gel blotting analysis indicated that AtFtsH2-like genes exist as two copies in maize genome. The genomic sequences of ZmFtsH2A and ZmFtsH2B were cloned and the main difference was that the first intron of ZmFtsH2B was much longer than that of ZmFtsH2A. RT-PCR analysis revealed that both genes were constitutively expressed in all examined tissues and the expression level of ZmFtsH2B transcripts was higher than that of ZmFtsH2A. The responses of the two genes in maize seedlings to PEG, cold, high salt, and ABA treatments were compared, and the results showed that ZmFtsH2B transcription in leaves was markedly up-regulated by water deficit stress and ABA treatments while ZmFtsH2A constitutively expressed both in leaves and roots under all tested stressful conditions. Drought tolerance of transgenic tobaccos overexpressing ZmFtsH2A and ZmFtsH2B weren't improved compared to wild-type controls, which indicated that two genes might not be directly involved in plant drought tolerance or the number of functional FtsH heterocomplex might not be increased in this condition. Our current study provides fundamental information for the further investigation of the maize FtsH proteins.

Phosphatidylinositol-specific phospholipase C (PI-PLC) plays an important role in a variety of physiological processes in plants, including drought tolerance. It has been reported that the ZmPLC1 gene cloned from maize (Zea mays L.) encoded a PI-PLC and up-regulated the expression in maize roots under dehydration conditions (Zhai SM, Sui ZH, Yang AF, Zhang JR in Biotechnol Lett 27:799-804, 2005). In this paper, transgenic maize expressing ZmPLC1 transgenes in sense or antisense orientation were generated by Agrobacterium-mediated transformation and confirmed by Southern blot analysis. High-level expression of the transgene was confirmed by real-time RT-PCR and PI-PLC activity assay. The tolerance to drought stress (DS) of the homogenous transgenic maize plants was investigated at two developmental stages. The results demonstrated that, under DS conditions, the sense transgenic plants had higher relative water content, better osmotic adjustment, increased photosynthesis rates, lower percentage of ion leakage and less lipid membrane peroxidation, higher grain yield than the WT; whereas those expressing the antisense transgene exhibited inferior characters compared with the WT. It was concluded that enhanced expression of sense ZmPLC1 improved the drought tolerance of maize.

Low phosphate availability is a major constraint on plant growth and agricultural productivity. Engineering a crop with enhanced low phosphate tolerance by transgenic technique could be one way of alleviating agricultural losses due to phosphate deficiency. In this study, we reported that transgenic maize plants that overexpressed the Thellungiella halophila vacuolar H(+)-pyrophosphatase gene (TsVP) were more tolerant to phosphate deficit stress than the wild type. Under phosphate sufficient conditions, transgenic plants showed more vigorous root growth than the wild type. When phosphate deficit stress was imposed, they also developed more robust root systems than the wild type, this advantage facilitated phosphate uptake, which meant that transgenic plants accumulated more phosphorus. So the growth and development in the transgenic maize plants were not damaged as much as in the wild type plants under phosphate limitation. Overexpression of TsVP increased the expression of genes involved in auxin transport, which indicated that the development of larger root systems in transgenic plants might be due in part to enhanced auxin transport which controls developmental events in plants. Moreover, transgenic plants showed less reproductive development retardation and a higher grain yield per plant than the wild type plants when grown in a low phosphate soil. The phenotypes of transgenic maize plants suggested that the overexpression of TsVP led to larger root systems that allowed transgenic maize plants to take up more phosphate, which led to less injury and better performance than the wild type under phosphate deficiency conditions. This study describes a feasible strategy for improving low phosphate tolerance in maize and reducing agricultural losses caused by phosphate deficit stress.