Efficacy and safety of dacomitinib in treatment-naïve patients with advanced NSCLC harboring uncommon EGFR mutation: an ambispective cohort study.

BACKGROUND: About 10% of non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations are harbored as uncommon mutations. This study aimed to explore the efficacy and safety of dacomitinib, a second-generation EGFR tyrosine kinase inhibitor (EGFR-TKIs), in treating uncommon EGFR-mutated advanced NSCLC. METHODS: Treatment-naïve advanced NSCLC patients treated with dacomitinib at Hunan Cancer Hospital with uncommon EGFR mutations were evaluated. The primary endpoint was progression-free survival (PFS). Secondary end points included overall survival (OS), objective response rate (ORR), disease control rate (DCR) and safety. RESULT: Between December 2019 and December 2021, a total of 16 patients was included. Median PFS was 14.0 (95% CI 4.32-23.7) months, and median OS was not reached. ORR was 68.8% (95% CI 41.3 to 89.0%) and DCR was 93.8% (95%CI 69.8 to 99.8%), including three achieving complete remission (CR) and eight achieving partial remission (PR). Median PFS for patients with brain metastasis was 9.0 (95%CI 6.9 to 11.1) months. Intracranial ORR was 100%, including 2 CR and 4 PR. Major treatment-related adverse events (TRAEs) included rash (87.5%), paronychia (62.5%), oral ulcers (50.0%), and diarrhea (50.0%), none of which were ≥ grade 3 TRAEs. CONCLUSIONS: Dacomitinib showed good activity and manageable toxicity in NSCLC patients with uncommon EGFR mutations.

Patients' adherence-related beliefs about inhaled steroids: application of the Chinese version of the Beliefs about Medicines Questionnaire-specific in patients with asthma.

Objective: The main objective of the present study is to evaluate the psychometric properties of the Chinese version of Beliefs about Medicines Questionnaire-specific among asthma patients; and to assess the association between patients' belief and adherence to inhaled corticosteroid therapy. Methods: A cross-sectional survey was carried out in the asthma clinic of Zhongshan Hospital, to Fudan University (Shanghai, China) between April 2016 and March 2018. The Beliefs about Medicines Questionnaire-specific was translated into Chinese according to international guidelines. Internal consistency, test-retest reliability, and confirmatory factor analysis were calculated to validate the Beliefs about Medicines Questionnaire-specific. The relationship between the adherence and the belief subscale were assessed using Kruskal-Wallis test. Results: Two hundred and seventeen patients were recruited in this study. The Beliefs about Medicines Questionnaire-specific was deemed reliable based on the results of Cronbach's alpha coefficient and test-retest intraclass correlation coefficient (ICC, ICC= 0.759). Confirmatory factor analysis showed acceptable model fit for the two-factor model. Patients' compliance was closely related to their belief about inhaled corticosteroid. The adherence rates were highest for the accepting groups, and lowest for the skeptical groups. Higher adherence was significantly associated with higher necessity-concerns differential (p = .001) and lower concern (p = .004). Conclusions: The Chinese version of the Beliefs about Medicines Questionnaire-specific can be used as a reliable tool by the clinicians to identify beliefs and behaviors of individual to improve adherence in Chinese patients.

miR-155, miR-96 and miR-99a as potential diagnostic and prognostic tools for the clinical management of hepatocellular carcinoma.

Increasing evidence has demonstrated that circulating microRNAs (miRNAs) can be utilized as potential biomarkers for the diagnosis of cancer, as well as a prognostic tool for the management of the disease. Therefore, the present study aimed to evaluate the predictive ability of miRNA (miR)-155, miR-96 and miR-99a for the diagnosis and prognosis of hepatocellular carcinoma (HCC). Tissues were collected from 30 patients with HCC and their matched adjacent normal liver tissues, as well as from serum samples from 30 patients with HCC and 30 healthy controls. Reverse transcription-quantitative PCR was used to measure the expression levels of miR-155, miR-96 and miR-99a. The expression levels of miR-155 and miR-96 were upregulated in the tissues and serum of patients with HCC, whereas miR-99a expression levels were decreased. Receiver operating characteristics (ROC) curve analysis revealed that circulating miR-155, miR-96, miR-99a and a combination of these three miRNAs could serve as diagnostic biomarkers for HCC with areas under the curve (AUC) of 0.84, 0.824, 0.799 and 0.931, respectively. Serum α-fetoprotein (AFP) was detected using electrochemiluminescence immunoassay analyzer. The addition of AFP with the combination of these three miRNAs offered a higher accuracy of HCC diagnosis (AUC, 0.979; sensitivity, 90.0%; specificity, 100.0%). In addition, elevated expression levels of miR-155 and miR-96 were associated with poor survival time of patients with HCC. The panel of miR-155, miR-96, miR-99a and AFP had a higher sensitivity and specificity for the diagnosis of HCC when compared with a single marker. Furthermore, the present data suggested that miR-155 and miR-96 may be potential prognostic markers for the clinical management of patients with HCC.

Attitudes Toward Bile Extraction From Living Bears: Survey of Citizens and Students in Beijing.

Bear bile is a traditional Chinese medicine that has been used for millennia. Several arguments support and oppose the use of bear farming in terms of conservation and nonhuman animal welfare. This study involved designing a questionnaire and surveying a random sample of general citizens and college students in Beijing to elicit their attitudes on bile extraction from living bears. Older people and people with lower education levels used more bear bile medicines. In total, 29.47% (n = 204) of citizens and 23.14% (n = 81) of students surveyed used bear bile medicine since 1990. Students were less willing to use bear bile medicines than citizens (p < .05). The level the respondents agreed with the blue side (against the extraction of bile from living bears; anti for short) was significantly higher than that for the red side (support the extraction of bile from living bears; pro for short; p < .05). Additionally, college students had a more distinct attitude toward the opposing views, which indicates they were more inclined to oppose bile extraction from living bears.

Sirolimus versus tacrolimus in kidney transplant recipients receiving mycophenolate mofetil and steroids: focus on acute rejection, patient and graft survival.

The study aims to conduct a meta-analysis of randomized controlled trials to compare the efficacy of tacrolimus (TAC)/mycophenolate mofetil (MMF)/corticosteroids (CSs) with sirolimus (SRL)/MMF/CSs in renal transplant recipients: Research 2 databases, PubMed, and Web of Science, selecting relevant articles. Data were selected for acute rejection and patient and graft survival. Statistical value relative risk (RR) and 95% confidence intervals (CIs) were recorded. Six randomized controlled trials involving 885 patients were included. There was a significant difference in acute rejection (P = 0.001, RR = 1.69, 95% CI, 1.23-2.34). Two groups, patient survival (P = 0.96, RR = 1.02, 95% CI, 0.54-1.91) and graft survival (P = 0.09, RR = 1.56, 95% CI, 0.93-2.60), had no statistical difference. Acute rejection by those taking SRL/MMF/CSs is worse than those taking TAC/MMF/CSs. Patient and graft survival in TAC/MMF/CSs is similar to that in SRL/MMF/CSs.

Phosphoproteome and proteome analyses reveal low-phosphate mediated plasticity of root developmental and metabolic regulation in maize (Zea mays L.).

Phosphate (Pi) deficiency has become a significant challenge to worldwide agriculture due to the depletion of accessible rock phosphate that is the major source of cheap Pi fertilizers. Previous research has identified a number of diverse adaptive responses to Pi starvation in the roots of higher plants. In this study, we found that accelerated axile root elongation of Pi-deprived maize plants resulted from enhanced cell proliferation. Comparative phosphoproteome and proteome profiles of maize axile roots were conducted in four stages in response to Pi deficiency by multiplex staining of high-resolution two dimensional gel separated proteins. Pro-Q DPS stained gels revealed that 6% of phosphoprotein spots displayed changes in phosphorylation state following low-Pi treatment. These proteins were involved in a large number of metabolic and cellular pathways including carbon metabolism and signal transduction. Changes in protein abundance of a number of enzymes indicated that low-Pi induced a number of carbon flux modifications in metabolic processes including sucrose breakdown and other downstream sugar metabolic pathways. A few key metabolic enzymes, including sucrose synthase (EC 2.4.1.13) and malate dehydrogenase (EC 1.1.1.37), and several signaling components involved in protein kinase or phosphatase cascades, auxin signaling and 14-3-3 proteins displayed low-Pi responsive changes in phosphorylation state or protein abundance. A variety of key enzymes and signaling components identified as potential targets for phosphorylation provide novel clues for comprehensive understanding of Pi regulation in plants. Protein phosphorylation, coordinating with changes in protein abundance, is required for maize root metabolic regulation and developmental acclimation to Pi starvation.

Validation of Chinese version of the Morisky medication adherence scale in patients with epilepsy.

PURPOSE: This study aimed to validate a Chinese version of the Morisky Medication Adherence Scale (MMAS-8) in patients with epilepsy. The relationships between adherence, seizure frequency, and adverse effects were assessed using this method. METHODS: Data from patients diagnosed with epilepsy at the Department of Neurology of Huashan Hospital were collected between January and June 2013. To validate the MMAS-8, internal consistency, test-retest reliability, and factor analysis were calculated. Relationships between adherence, seizure frequency, and adverse effects were assessed using Pearson's correlation. RESULTS: One hundred and eleven patients were recruited. The MMAS-8 had moderate internal consistency (Cronbach's α=0.556) and good test-retest reliability (intraclass correlation coefficient=0.729). The MMAS-8 adherence rate was 79.2%. MMAS-8 adherence was negatively correlated with seizure frequency and adverse effects (r = -0.708, p<0.001; r = -0.484, p<0.001). CONCLUSION: The MMAS-8 scale can be used as a tool to assess medication adherence in Chinese patients with epilepsy. Better seizure control and lower rates of adverse effects were significantly correlated with higher adherence scores.

Identification and comparative analysis of low phosphate tolerance-associated microRNAs in two maize genotypes.

Low phosphate (Pi) availability is a major constraint on maize growth and productivity. MicroRNAs (miRNAs) are known to play critical roles in plant responses to various environmental conditions. The identification of low Pi tolerance-associated miRNAs will accelerate the development of Pi starvation tolerant maize plants. However, miRNAs associated with low Pi tolerance have not been identified. In this study, we compared deep sequencing small RNA reads from two maize genotypes, the wild type, Qi319, and the low Pi tolerant mutant, 99038, under normal and low Pi conditions. Six known miRNA families and seven novel miRNAs were found differently expressed by the two genotypes. All these miRNAs were confirmed by sequencing a second batch of small RNA libraries constructed in the same way as those used in the first sequencing. The expression profiles of some of these miRNAs were further confirmed by real-time PCR. The predicted target genes of the low Pi tolerance-associated miRNAs were involved in root development or stress responses. Expression levels of some of target genes were significantly different between Qi319 and 99038. These findings suggested that miRNAs may play important roles in low Pi tolerance in maize and may be a key factor in determining the level of low Pi tolerance in different maize genotypes. This study provides an approach for identifying low Pi tolerance-associated miRNAs and can help in the selection and manipulation of high performing maize genotypes under low Pi conditions.

Overexpression of the phosphatidylinositol synthase gene (ZmPIS) conferring drought stress tolerance by altering membrane lipid composition and increasing ABA synthesis in maize.

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.

Overexpression of Thellungiella halophila H+-pyrophosphatase gene improves low phosphate tolerance in 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.

Overexpression of the phosphatidylinositol synthase gene from Zea mays in tobacco plants alters the membrane lipids composition and improves drought stress tolerance.

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.

Heterologous expression of ApGSMT2 and ApDMT2 genes from Aphanothece halophytica enhanced drought tolerance in transgenic tobacco.

The glycine-methylation biosynthetic pathway of glycinebetaine (GB) has been investigated, but only a few studies on GB accumulation in transgenic higher plants have utilized this pathway. In this study, two methyltransferase genes named ApGSMT2 and ApDMT2, encoding proteins catalyzing GB biosynthesis from glycine, were cloned from a relative strain of Aphanothece halophytica. The potential roles of ApGSMT2 and ApDMT2 in GB synthesis were first examined in transgenic Escherichia coli, which had increased levels of GB and improved salt tolerance. Then ApGSMT2 and ApDMT2 were transferred into tobacco. Compared with transgenic tobacco expressing betA, transgenic tobacco co-expressing ApGSMT2 and ApDMT2 accumulated more GB and exhibited enhanced drought resistance with better germination performance, higher relative water content, less cell membrane damage and better photosynthetic capacity under drought stress. We concluded that the ApGSMT2 and ApDMT2 genes cloned in this study will be very useful for engineering GB-accumulating transgenic plants with enhanced drought resistance.

Generation of marker-free transgenic maize with improved salt tolerance using the FLP/FRT recombination system.

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.

Identification and characterization of two members of the FtsH gene family in maize (Zea mays L.).

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.

The transgene pyramiding tobacco with betaine synthesis and heterologous expression of AtNHX1 is more tolerant to salt stress than either of the tobacco lines with betaine synthesis or AtNHX1.

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.

Cloning and expression analysis of some genes involved in the phosphoinositide and phospholipid signaling pathways from maize (Zea mays L.).

Previous studies have indicated the phosphoinositide and phospholipid signaling pathways play a key role in plant growth, development and responses to environmental stresses. However, little is known about the phosphoinositide and phospholipid signaling pathways in maize (Zea mays L.). To better understand the function of genes involved in the phosphoinositide and phospholipid signaling pathways in maize, the cDNA sequences of ZmPIS2, ZmPLC2, ZmDGK1, ZmDGK2 and ZmDGK3 were obtained by RACE (rapid amplification of cDNA ends) or in silico cloning combined with PCR. RT-PCR analysis of cDNA from five tissues (roots, stems, leaves, tassels, and ears) indicated that the expression patterns of the five cDNAs we isolated as well as ZmPIS, ZmPLC, ZmPLD varied in different tissues. To determine the effects of different environmental conditions such as cold, drought and various phytohormones (abscisic acid, indole-3-acetic acid and gibberellic acid) on gene expression, we analyzed expression by Real-Time (RT-PCR), and found that the different isoforms of these gene families involved in the phosphoinositide and phospholipid signaling pathways have specific expression patterns. Our results suggested that these genes may be involved in the responses to environmental stresses, but have different functions. The isolation and analysis of expression patterns of genes involved in the phosphoinositide and phospholipid signaling pathways provides a good basis for further research of the phosphoinositide and phospholipid signaling pathways in maize and is a novel supplement to our comprehension of these pathways in plants.

Influence of water stress on endogenous hormone contents and cell damage of maize seedlings.

Phytohormones play critical roles in regulating plant responses to stress. We investigated the effects of water stress induced by adding 12% (w/v) polyethylene glycol to the root medium on the levels of abscisic acid (ABA), indole-3-acid (IAA), zeatin (ZT), and gibberellin(3) (GA(3)) in maize leaves. The results suggested that water stress had significant effects on the four hormone levels. There was a transient increase in the IAA content during the initial stage of adaptation to water stress in maize leaves, but it dropped sharply thereafter in response to water stress. ABA content increased dramatically in maize leaves after 24 h of exposure to water stress, and then the high levels of ABA were maintained to the end. The contents of ZT and GA(3) rapidly declined in maize leaves subjected to water stress. The effects of water stress on chlorophyll content, electrolyte leakage and malondialdehyde levels in maize leaves were also studied. The variation of cell damage was negatively correlated with ZT and GA(3) levels in maize leaves under water stress. Thus, we explored the roles of ZT and GA(3) on the growth of maize seedlings under water stress by exogenous application. It is possible that both ZT and GA(3) were effective in protecting maize seedlings from water stress, which would be of great importance for the improvement of drought tolerance in maize by genetic manipulation.

Comparative proteome analyses of phosphorus responses in maize (Zea mays L.) roots of wild-type and a low-P-tolerant mutant reveal root characteristics associated with phosphorus efficiency.

SUMMARY: Low phosphorus (P) availability is a major limitation for plant growth. To better understand the molecular mechanism of P efficiency in maize, comparative proteome analyses were performed on the roots of the low-P-tolerant mutant 99038 and wild-type Qi-319 grown under P-sufficient (+P) or P-deficient (-P) conditions. Over 10% of proteins detected on two-dimensional electrophoresis (2-DE) gels showed expression that was altered twofold or more between the genotypes under +P or -P conditions. We identified 73 (+P) and 95 (-P) differentially expressed proteins in response to phosphate (Pi) starvation. These proteins were involved in a large number of cellular and metabolic processes, with an obvious functional skew toward carbon metabolism and regulation of cell proliferation. Further analysis of proteome data, physiological measurements and cell morphological observations showed that, compared to the wild-type, the low-P-tolerant mutant could accumulate and secrete more citrate under Pi starvation, which facilitates solubilization of soil Pi and enhances Pi absorption. The proportion of sucrose in the total soluble sugars of the low-P-tolerant mutant was significantly higher, and cell proliferation in root meristem was accelerated. This resulted in better developed roots and more advantageous root morphology for Pi uptake. These results indicate that differences in citrate secretion, sugar metabolism and root-cell proliferation are the main reasons for higher tolerance to low-P conditions in the mutant compared to the wild-type. Thus, the mutant displayed specialized P-efficient root systems with a higher capacity for mobilization of external Pi and increased cell division in the root meristem under Pi starvation.

Enhanced expression of phospholipase C 1 (ZmPLC1) improves drought tolerance in transgenic maize.

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.

Heterologous expression of vacuolar H(+)-PPase enhances the electrochemical gradient across the vacuolar membrane and improves tobacco cell salt tolerance.

The vacuolar H(+)-translocating inorganic pyrophosphatase (H(+)-PPase) uses pyrophosphate as substrate to generate the proton electrochemical gradient across the vacuolar membrane to acidify vacuoles in plant cells. The heterologous expression of H(+)-PPase genes (TsVP from Thellungiella halophila and AVP1 from Arabidopsis thaliana) improved the salt tolerance of tobacco plants. Under salt stress, the transgenic seedlings showed much better growth and greater fresh weight than wild-type plants, and their protoplasts had a normal appearance and greater vigor. The cytoplasmic and vacuolar pH in transgenic and wild-type cells were measured with a pH-sensitive fluorescence indicator. The results showed that heterologous expression of H(+)-PPase produced an enhanced proton electrochemical gradient across the vacuolar membrane, which accelerated the sequestration of sodium ions into the vacuole. More Na(+) accumulated in the vacuoles of transgenic cells under salt (NaCl) stress, revealed by staining with the fluorescent indicator Sodium Green. It was concluded that the tonoplast-resident H(+)-PPase plays important roles in the maintenance of the proton gradient across the vacuolar membrane and the compartmentation of Na(+) within vacuoles, and heterologous expression of this protein enhanced the electrochemical gradient across the vacuolar membrane, thereby improving the salt tolerance of tobacco cells.