Comparative functional genomics analysis of cytochrome P450 gene superfamily in wheat and maize.

BACKGROUND: The cytochrome P450s (CYP450s) as the largest enzyme family of plant metabolism participate in various physiological processes, whereas no study has demonstrated interest in comprehensive comparison of the genes in wheat and maize. Genome-wide survey, characterization and comparison of wheat and maize CYP450 gene superfamily are useful for genetic manipulation of the Gramineae crops. RESULTS: In total, 1285 and 263 full-length CYP450s were identified in wheat and maize, respectively. According to standard nomenclature, wheat CYP450s (TaCYP450s) were categorized into 45 families, while maize CYP450s (ZmCYP450s) into 43 families. A comprehensive analysis of wheat and maize CYP450s, involved in functional domains, conserved motifs, phylogeny, gene structures, chromosome locations and duplicated events was performed. The result showed that each family/subfamily in both species exhibited characteristic features, suggesting their phylogenetic relationship and the potential divergence in their functions. Functional divergence analysis at the amino acid level of representative clans CYP51, CYP74 and CYP97 in wheat, maize and rice identified some critical amino acid sites that are responsible for functional divergence of a gene family. Expression profiles of Ta-, ZmCYP450s were investigated using RNA-seq data, which contribute to infer the potential functions of the genes during development and stress responses. We found in both species CYP450s had preferential expression in specific tissues, and many tissue-specific genes were identified. Under water-deficit condition, 82 and 39 significantly differentially expressed CYP450s were respectively detected in wheat and maize. These genes may have some roles in protecting plants against drought damage. Thereinto, fourteen CYP450s were selected to validate their expression level through qRT-PCR. To further elucidating molecular mechanisms of CYP450 action, gene co-expression network was constructed. In total, 477 TaCYP450s were distributed in 22 co-expression modules, and some co-expressed genes that likely take part in the same biochemical pathway were identified. For instance, the expression of TaCYP74A98_4D was highly correlated with TaLOX9, TaLOX36, TaLOX39, TaLOX44 and TaOPR8, and all of them may be involved in jasmonate (JA) biosynthesis. TaCYP73A201_3A showed coexpression with TaPAL1.25, TaCCoAOMT1.2, TaCOMT.1, TaCCR1.6 and TaLAC5, which probably act in the wheat stem and/or root lignin synthesis pathway. CONCLUSION: Our study first established systematic information about evolutionary relationship, expression pattern and function characterization of CYP450s in wheat and maize.

Comparative functional genomics analysis of bHLH gene family in rice, maize and wheat.

BACKGROUND: The basic helix-loop-helix transcription factors play important roles in diverse cellular and molecular processes. Comparative functional genomics can provide powerful approaches to draw inferences about gene function and evolution among species. The comprehensive comparison of bHLH gene family in different gramineous plants has not yet been reported. RESULTS: In this study, a total of 183, 231 and 571 bHLHs were identified in rice, maize and wheat genomes respectively, and 1154 bHLH genes from the three species and Arabidopsis were classified into 36 subfamilies. Of the identified genes, 110 OsbHLHs, 188 ZmbHLHs and 209 TabHLHs with relatively high mRNA abundances were detected in one or more tissues during development, and some of them exhibited tissue-specific expression such as TabHLH454-459, ZmbHLH099-101 and OsbHLH037 in root, TabHLH559-562, - 046, - 047 and ZmbHLH010, - 072, - 226 in leaf, TabHLH216-221, - 333, - 335, - 340 and OsbHLH005, - 141 in inflorescence, TabHLH081, ZmbHLH139 and OsbHLH144 in seed. Forty five, twenty nine and thirty one differentially expressed bHLHs were respectively detected in wheat, maize and rice under drought stresses using RNA-seq technology. Among them, the expressions of TabHLH046, - 047, ZmbHLH097, - 098, OsbHLH006 and - 185 were strongly induced, whereas TabHLH303, - 562, ZmbHLH155, - 154, OsbHLH152 and - 113 showed significant down-regulation. Twenty two TabHLHs were induced after stripe rust infection at 24 h and nine of them were suppressed at 72 hpi, whereas 28 and 6 TabHLHs exhibited obviously down- and up-regulation after powdery mildew attack respectively. Forty one ZmbHLHs were differentially expressed in response to F. verticillioides infection. Twenty two co-expression modules were identified by the WGCNA, some of which were associated with particular tissue types. And GO enrichment analysis for the modules showed that some TabHLHs were involved in the control of several biological processes, such as tapetal PCD, lipid metabolism, iron absorption, stress responses and signal regulation. CONCLUSION: The present study identifies the bHLH family in rice, maize and wheat genomes, and detailedly discusses the evolutionary relationships, expression and function of bHLHs. This study provides some novel and detail information about bHLHs, and may facilitate understanding the molecular basis of the plant growth, development and stress physiology.

Global identification, structural analysis and expression characterization of cytochrome P450 monooxygenase superfamily in rice.

BACKGROUND: The cytochrome P450 monooxygenases (CYP450, CYP, P450) catalyze numerous monooxygenation/hydroxylation reactions in biochemical pathways. Although CYP superfamily has been systematically studied in a few species, the genome-scale research about it in rice has not been done. RESULTS: In this study, a total of 355 CYPs encoded by 326 genes were identified in japonica genome. The OsCYP genes are classified into 10 clans including 45 families according to phylogenetic analysis. More than half of the genes are distributed in 53 tandem duplicated gene clusters. Intron-exon structure of OsCYPs exhibits highly conserved and specificity within a family, and divergences of duplicate genes in gene structure result in non-functionalization, neo-functionalization or sub-functionalization. Selection pressure analysis showed that rice CYPs are under purifying selection. The microarray data analysis shows that some genes are tissue-specific expression, such as OsCYP710A5 and OsCYP71X14 in endosperm, OsCYP99A3 and OsCYP78A16 in root and OsCYP93G2 and OsCYP97D7 in leaf. Analysis of RNA-seq data derived from rice leaf developmental gradient indicates that some OsCYPs exhibit zone-specific expression patterns. OsCYP87C2, OsCYP96B5, OsCYP96B8 and OsCYP84A5 were specifically expressed in leaf base and transitional zone. The transcripts of lineages II and IV-1 members were highly abundant in maturing zone. Eighty three OsCYPs are differentially expressed in response to drought stress, of which OsCYP51G3, OsCYP709C9, OsCYP709C5, OsCYP81A6, OsCYP72A18 and OsCYP704A5 are strongly induced and OsCYP78A16, OsCYP89C9 and OsCYP704A5 are down-regulated significantly, and some of the results were validated by qPCR. And 23 up-regulated and 17 down-regulated genes are specific to Osbhlh148 mutation under drought stress. Compared to those in wild type, the changes in transcript levels of several genes are slight in the mutant, such as OsCYP51G3, OsCYP94C2, OsCYP709C9 and OsCYP709C5. CONCLUSION: The whole-genomic analysis of rice P450 superfamily provides a clue to understanding biological function of OsCYPs in development regulation and drought stress response, and is helpful to rice molecular breeding.

Global evolutionary history and spatio-temporal dynamics of dengue virus type 2.

DENV-2 spread throughout the tropical and subtropical regions globally, which is implicated in deadly outbreaks of DHF and DSS. Since dengue cases have grown dramatically in recent years, about half of the world's population is now at risk. Our timescale analysis indicated that the most recent common ancestor existed about 100 years ago. The rate of nucleotide substitution was estimated to be 8.94 × 10-4 subs/site/year. Selection pressure analysis showed that two sites 160 and 403 were under positive selection, while E gene is mainly shaped by stronger purifying selection. BSP analysis showed that estimating effective population size from samples of sequences has undergone three obvious increases, additionally, Caribbean and Puerto Rico maintained higher levels of genetic diversity relative to other 6 representative geographical populations using GMRF method. The phylogeographic analysis indicated that two major transmission routes are from South America to Caribbean and East&SouthAsia to Puerto Rico. The trunk reconstruction confirmed that the viral evolution spanned 50 years occurred primarily in Southeast Asia and East&South Asia. In addition, phylogeographic association-trait analysis indicated that the viral phenotypes are highly correlated with phylogeny in Nicaragua and Puerto Rico (P < 0.05).

Genome-scale phylodynamics and evolution analysis of global H7N7 influenza viruses.

Previous studies lacked of comprehensive analysis about the evolutionary history and phylogeography of global H7N7 viruses. In this study, it is essential to undertake a genome-scale analysis to investigate the evolutionary processes in a global perspective. There was local phylogenetic divergence among eight trees based on individual segments of 132 strains. We detected four reassortments between four distinct groups of viruses divided by HA gene, suggesting intrasubtype reassortment could accelerate the emergence of highly pathogenic virus. The molecular clock estimated that H7N7 virus evolved at a slower evolutionary rate ranged from 1.03E-03 to 2.81E-03subs/site/year. And we also showed that all gene segments of the virus were under strong purifying selection. A total of 11 positively selected sites were detected by at least two out of three methods. We reconstructed the population dynamics of global H7N7 viruses spanning over a century, revealing that temporal trends of the effective population size were consistent with the major epidemics previously reported. Our study adopt a Bayesian phylogeographic approach to investigate the geographic spread of H7N7 viruses, which combined with temporal and spatial information of all sequences. We have confirmed several migration events between different geographic locations supported by higher values of Bayes factor. The diffusion patterns of H7N7 viruses reveal that the virus is more likely to evolve to expand their host ranges even cross the species.

Non-specific lipid transfer proteins in maize.

BACKGROUND: In plant, non-specific lipid transfer proteins (nsLTPs) are small, basic proteins that have been reported to be involved in numerous biological processes such as transfer of phospholipids, reproductive development, pathogen defence and abiotic stress response. To date, only a tiny fraction of plant nsLTPs have been functionally identified, and even fewer have been identified in maize [Zea mays (Zm)]. RESULTS: In this study, we carried out a genome-wide analysis of nsLTP gene family in maize and identified 63 nsLTP genes, which can be divided into five types (1, 2, C, D and G). Similar intron/exon structural patterns were observed in the same type, strongly supporting their close evolutionary relationship. Gene duplication analysis indicated that both tandem and segmental duplication contribute to the diversification of this gene family. Additionally, the three-dimensional structures of representative nsLTPs were studied with homology modeling to understand their molecular functions. Gene ontology analysis was performed to obtain clues about biological function of the maize nsLTPs (ZmLTPs). The analyses of putative upstream regulatory elements showed both shared and distinct transcriptional regulation motifs of ZmLTPs, further indicating that ZmLTPs may play roles in diverse biological processes. The dynamic expression patterns of ZmLTPs family across the different developmental stages showed that several of them exhibit tissue-specific expression, indicative of their important roles in maize life cycle. Furthermore, we focused on the roles of maize nsLTPs in biotic and abiotic stress responses. Our analyses demonstrated that some ZmLTPs exhibited a delayed expression pattern after the infection of Ustilago maydis and differentially expressed under drought, salt and cold stresses, and these may be a great help for further studies to improve the stress resistance and tolerance in maize breeding. CONCLUSIONS: Our results provide new insights into the phylogenetic relationships and characteristic functions of maize nsLTPs and will be useful in studies aimed at revealing the global regulatory network in maize development and stress responses, thereby contributing to the maize molecular breeding with enhanced quality traits.

Maize protein phosphatase gene family: identification and molecular characterization.

BACKGROUND: Protein phosphatases (PPs) play critical roles in various cellular processes through the reversible protein phosphorylation that dictates many signal transduction pathways among organisms. Recently, PPs in Arabidopsis and rice have been identified, while the whole complement of PPs in maize is yet to be reported. RESULTS: In this study, we have identified 159 PP-encoding genes in the maize genome. Phylogenetic analyses categorized the ZmPP gene family into 3 classes (PP2C, PTP, and PP2A) with considerable conservation among classes. Similar intron/exon structural patterns were observed in the same classes. Moreover, detailed gene structures and duplicative events were then researched. The expression profiles of ZmPPs under different developmental stages and abiotic stresses (including salt, drought, and cold) were analyzed using microarray and RNA-seq data. A total of 152 members were detected in 18 different tissues representing distinct stages of maize plant developments. Under salt stress, one gene was significantly up-expressed in seed root (SR) and one gene was down-expressed in primary root (PR) and crown root (CR), respectively. As for drought stress condition, 13 genes were found to be differentially expressed in leaf, out of which 10 were up-regulated and 3 exhibited down-regulation. Additionally, 13 up-regulated and 3 down-regulated genes were found in cold-tolerant line ETH-DH7. Furthermore, real-time PCR was used to confirm the expression patterns of ZmPPs. CONCLUSIONS: Our results provide new insights into the phylogenetic relationships and characteristic functions of maize PPs and will be useful in studies aimed at revealing the global regulatory network in maize abiotic stress responses, thereby contributing to the maize molecular breeding with enhanced quality traits.

Genetic dynamic analysis of the influenza A H5N1 NS1 gene in China.

The direct precursors of the A/Goose/Guangdong/1/1996 (GS/GD) virus lineage and its reassortants have been established geographically and ecologically. To investigate the variation and evolutionary dynamics of H5N1 viruses, whole-genome viral sequences (n = 164) were retrieved from the NCBI Influenza Virus Resource. Here, we present phylogenetic evidence for intrasubtype reassortments among H5N1 viruses isolated from China during 1996-2012. On the basis of phylogenetic analysis, we identified four major groups and further classified the reassortant viruses into three subgroups. Putative mosaic structures were mostly found in the viral ribonucleoprotein (vRNP) complexes and 91.0% (10/11) mosaics were obtained from terrestrial birds. Sequence variability and selection pressure analyses revealed that both surface glycoproteins (HA and NA) and nonstructural protein 1 (NS1) have higher dN/dS ratio and variability than other internal proteins. Furthermore, we detected 47 positively selected sites in genomic segments with the exception of PB2 and M1 genes. Hemagglutinin (HA) and neuraminidase (NA) are considered highly variable due to host immune pressure, however, it is not known what drives NS1 variability. Therefore, we performed a thorough analysis of the genetic variation and selective pressure of NS1 protein (462 available NS1 sequences). We found that most of positively selected sites and variable amino acids were located in the C-terminal effector domain (ED) of NS1. In addition, we focused on the NS1-RNA and NS1-protein interactions that were involved in viral replication mechanisms and host immune response. Transcriptomic analysis of H5N1-infected monkey lungs showed that certain PI3K-related genes were up-regulated.

Genome-scale evolution and phylodynamics of H5N1 influenza virus in China during 1996-2012.

Highly pathogenic avian influenza A (HPAI) H5N1 is endemic in China. It is expanding its borders over time and the associated economic and health consequences are critically important. To determine the evolutionary history and phylodynamics of HPAI H5N1, a comprehensive analysis of the demographic, adaptive and spatial dynamics of H5N1 viruses in China over almost two decades based on whole genome sequences was performed. We divided HPAI H5N1 isolates into five and seven distinct groups for HA and NA respectively, and several regionally dominant subgroups were found as well. We detected five reassortants, and our analysis suggested that the intrasubtype reassortment may resulted in enhanced virulence. Seven and eight positively selected sites were detected in HA and NA genes respectively, and the relatively higher dN/dS ratio and nucleotide substitution rate were observed in NS gene. Our BSP analysis about the effective population size increase and decrease showed similar temporal patterns to those in the epidemiological studies. Here we show that the time to most recent common ancestor (tMRCA) of earlier reassortments was dated back to 1970s. And our phylogeographic analysis indicated that the geographic spread of HPAI H5N1 accelerated the viral evolution and expanded their host ranges, prompting potential cross-species transmission.

Argonaute protein as a linker to command center of physiological processes.

MicroRNAs (miRNAs) post-transcriptionally regulate gene expression by binding to target mRNAs with perfect or imperfect complementarity, recruiting an Argonaute (AGO) protein complex that usually results in degradation or translational repression of the target mRNA. AGO proteins function as the Slicer enzyme in miRNA and small interfering RNA (siRNA) pathways involved in human physiological and pathophysiological processes, such as antiviral responses and disease formation. Although the past decade has witnessed rapid advancement in studies of AGO protein functions, to further elucidate the molecular mechanism of AGO proteins in cellular function and biochemical process is really a challenging area for researchers. In order to understand the molecular causes underlying the pathological processes, we mainly focus on five fundamental problems of AGO proteins, including evolution, functional domain, subcellular location, post-translational modification and protein-protein interactions. Our discussion highlight their roles in early diagnosis, disease prevention, drug target identification, drug response, etc.

Genome-wide analysis of bZIP-encoding genes in maize.

In plants, basic leucine zipper (bZIP) proteins regulate numerous biological processes such as seed maturation, flower and vascular development, stress signalling and pathogen defence. We have carried out a genome-wide identification and analysis of 125 bZIP genes that exist in the maize genome, encoding 170 distinct bZIP proteins. This family can be divided into 11 groups according to the phylogenetic relationship among the maize bZIP proteins and those in Arabidopsis and rice. Six kinds of intron patterns (a-f) within the basic and hinge regions are defined. The additional conserved motifs have been identified and present the group specificity. Detailed three-dimensional structure analysis has been done to display the sequence conservation and potential distribution of the bZIP domain. Further, we predict the DNA-binding pattern and the dimerization property on the basis of the characteristic features in the basic and hinge regions and the leucine zipper, respectively, which supports our classification greatly and helps to classify 26 distinct subfamilies. The chromosome distribution and the genetic analysis reveal that 58 ZmbZIP genes are located in the segmental duplicate regions in the maize genome, suggesting that the segment chromosomal duplications contribute greatly to the expansion of the maize bZIP family. Across the 60 different developmental stages of 11 organs, three apparent clusters formed represent three kinds of different expression patterns among the ZmbZIP gene family in maize development. A similar but slightly different expression pattern of bZIPs in two inbred lines displays that 22 detected ZmbZIP genes might be involved in drought stress. Thirteen pairs and 143 pairs of ZmbZIP genes show strongly negative and positive correlations in the four distinct fungal infections, respectively, based on the expression profile and Pearson's correlation coefficient analysis.

Multiple-strategy analyses of ZmWRKY subgroups and functional exploration of ZmWRKY genes in pathogen responses.

The WRKY transcription factor family plays crucial roles in biotic responses, such as fungi, bacteria, viruses and nematode infections and insect attacks. In this article, multiple-strategy analyses of the three subgroups were performed in order to gain structural and evolutionary proofs of the overall WRKY family and unravel the functions possessed by each group or subgroup. Thus we analyzed the similarity of WRKY factors between maize and Arabidopsis based on homology modelling. The gene structure and motif analyses of Group II demonstrated that specific motifs existing in the given subgroups may contribute to the functional diversification of WRKY proteins and the two types of conserved intron splice sites suggest their evolutionary conservation. The evolutionary divergence time estimation of Group III proteins indicated that the divergence of Group III occurred during the Neogene period. Further, we focused on the roles of maize WRKYs in pathogen responses based on publicly available microarray experiments. The result suggested that some ZmWRKYs are expressed specifically under the infection of certain fungus, among which some are up-regulated and some are down-regulated, indicating their positive or negative roles in pathogen response. Also, some genes remain unchanged upon fungal infection. Pearson correlation coefficient (PCC) analysis was performed using 62 fungal infection experiments to calculate the correlation between each pair of genes. A PCC value higher than 0.6 was regarded as strong correlation - in these circumstances, ninety pairs of genes showed a strong positive correlation, while fifteen pairs of genes displayed a strong negative correlation. These correlated genes form a co-regulatory network and help us investigate the existence of interactions between WRKY proteins.

Structural evolution and functional diversification analyses of argonaute protein.

Argonaute (AGO) proteins are highly specialized small-RNA-binding modules and small RNAs are anchored to their specific binding pockets guiding AGO proteins to target mRNA molecules for silencing or destruction. The 135 full-length AGO protein sequences derived from 36 species covering prokaryote, archaea, and eukaryote are chosen for structural and functional analyses. The results show that bacteria and archaeal AGO proteins are clustered in the same clade and there exist multiple AGO proteins in most eukaryotic species, demonstrating that the increase of AGO gene copy number and horizontal gene transfer (HGT) have been the main evolutionary driving forces for adaptability and biodiversity. And the emergence of PAZ domain in AGO proteins is the unique evolutionary event. The analysis of middle domain (MID)-nucleotide contaction shows that either the position of sulfate I bond in Nc_QDE2 or the site of phosphate I bond in Hs_AGO2 represents the 5'-nucleotide binding site of miRNA. Also, H334, T335, and Y336 of Hs_AGO1 can form hydrogen bonds with 3'-overhanging ends of miRNAs and the same situation exists in Hs_AGO2, Hs_AGO3, Hs_AGO4, Dm_AGO1, and Ce_Alg1. Some PIWI domains containing conserved DDH motif have no slicer activity, and post-translational modifications may be associated with the endonucleolytic activities of AGOs. With the numbers of AGO genes increasing and fewer crystal structures available, the evolutionary and functional analyses of AGO proteins can help clarify the molecular mechanism of function diversification in response to environmental changes, and solve major issues including host defense mechanism against virus infection and molecular basis of disease.

[Molecular mechanism for dynamic regulation of endogenous ABA signal level].

The process from stress signal perception and the trigger of ABA biosynthesis to dynamic regulation of ABA level is an important stress signaling pathway in cells. Compared to the downstream events in ABA signal transduction, the researches in this field are relatively lagged. Expression of synthase genes, such as ZEP in roots and rate-limiting enzyme genes NCED, AtRGS1 and ABA2, can be activated in response to stresses. However, the expression of genes encoding degradative enzymes, including 7'-, 8'-, 9'-hydroxylase and glucosyltransferase, negatively regulates ABA accumulation. Meanwhile, the expressions of the synthases, such as ZEP and NCED3, are induced by increasing endogenous ABA contents. Additionally, the analyses of gene expression and source-sink dynamics indicates that sustained supply from root-sourced ABA is required for the maintenance of leaf ABA dynamic pool. It is notable that miRNAs should be involved in ABA signal origin and ABA level dynamic adjustment. Further dynamic analysis of ABA metabolism revealed that endogenous ABA signal levels are synergistically controlled by the expressions of synthases and degradative enzymes.

Evolution and adaptation of hemagglutinin gene of human H5N1 influenza virus.

The H5N1 HPAI virus has brought heavy loss to poultry industry. Although, there exists limited human-to-human transmission, it poses potential serious risks to public health. HA is responsible for receptor-binding and membrane-fusion and contains the host receptor-binding sites and major epitopes for neutralizing antibodies. To investigate molecular adaption of HPAI H5N1 viruses, we performed a phylogenetic analysis of HA sequences with 240 HPAI virus strains isolated from human. The topology of the tree reveals overall clustering of strains in four major clusters based on geographic location, and shows antigenic diversity of HA of human H5N1 isolates co-circulating in Asia, Africa, and Europe. The four clusters possess distinct features within the cleavage site and glycosylation sites, respectively. We identified six sites apparently evolving under positive selection, five of which persist in the population. Three positively selected sites are found to be located either within or flanking the receptor-binding sites, suggesting that selection at these sites may increase the affinity to human-type receptor. Furthermore, some sites are also associated with glycosylation and antigenic changes. In addition, two sites are found to be selected differentially in the two clusters. The analyses provide us deep insight into the adaptive evolution of human H5N1 viruses, show us several candidate mutations that could cause a pandemic, and suggest that efficiency measures should be taken to deal with potential risks.

Molecular phylogenetic and expression analysis of the complete WRKY transcription factor family in maize.

The WRKY transcription factors function in plant growth and development, and response to the biotic and abiotic stresses. Although many studies have focused on the functional identification of the WRKY transcription factors, much less is known about molecular phylogenetic and global expression analysis of the complete WRKY family in maize. In this study, we identified 136 WRKY proteins coded by 119 genes in the B73 inbred line from the complete genome and named them in an orderly manner. Then, a comprehensive phylogenetic analysis of five species was performed to explore the origin and evolutionary patterns of these WRKY genes, and the result showed that gene duplication is the major driving force for the origin of new groups and subgroups and functional divergence during evolution. Chromosomal location analysis of maize WRKY genes indicated that 20 gene clusters are distributed unevenly in the genome. Microarray-based expression analysis has revealed that 131 WRKY transcripts encoded by 116 genes may participate in the regulation of maize growth and development. Among them, 102 transcripts are stably expressed with a coefficient of variation (CV) value of <15%. The remaining 29 transcripts produced by 25 WRKY genes with the CV value of >15% are further analysed to discover new organ- or tissue-specific genes. In addition, microarray analyses of transcriptional responses to drought stress and fungal infection showed that maize WRKY proteins are involved in stress responses. All these results contribute to a deep probing into the roles of WRKY transcription factors in maize growth and development and stress tolerance.

[Establishment of high efficiency genetic transformation system of maize mediated by Agrobacterium tumefaciens].

In order to establish high-frequency regeneration and high-efficiency genetic transformation system in maize, the significance of the 11 factors influencing maize embryonic callus induction and 9 factors affecting embryonic callus differentiation was researched by orthogonal experiment. The results showed that genotype had highly significant impact on induction of embryonic callus. The concentration of 6-BA, AgNO3, 2,4-D, ABA, and medium are the significant factors. The Multi-comparison showed that ABA 2 mg/L has a significant influence. Among the callus differentiation factors, the genotype and 6-BA concentration showed a strong main effect, the concentrations of NAA, medium, KT and 2,4-D had significant impacts on callus differentiation. Southern blotting analysis demonstrated that the resistant callus rate under the selection pressure of 25 mg/L hygromycin was a reliable indicator for system optimization in resistance screening. The concentration of acetosyringone (AS) showed sensitive differences among genotypes. The highest transformation rate was found with the optimized combination of 24-25 degrees C for co-culture temperature, 0.7 ODx15 min for Agrobacterium tumefa-ciens concentration and incubation-time, and pH 5.5-6.2. By this optimized combination, the survival rate of resistant calli as an index for the stable transformation rates of inbred lines Huangzao 4 and Zong 31 by introducing GUS gene into maize inbred lines was as high as 48.6% and 46.2%, respectively.

Dynamic analysis of ABA accumulation in relation to the rate of ABA catabolism in maize tissues under water deficit.

The plant hormone abscisic acid (ABA) accumulates in plant tissues which experience water deficit (stress ABA). This study analysed its accumulation as a function of both synthesis and catabolism in maize tissues. By following the disappearance of the stress ABA when ABA synthesis was blocked by nordihydroguaiaretic acid (NDGA), the rate of the catabolism of stress ABA was determined. When compared with the catabolic rate of baseline (non-stress) ABA, stress ABA showed a catabolic rate >11 times higher. With such an elevated catabolic rate, it is proposed that the xanthophyll precursor pool may not be able to sustain the ABA accumulation, and such a proposition has been substantiated by further experiments where fluridone is used to limit the availability of upstream ABA precursors. When fluridone was used, stress ABA accumulation could only be sustained for a few hours, i.e. approximately 5 h for leaf and 1 h for root tissues. In detached roots, stress ABA accumulation could not be sustained even if fluridone was not used, suggesting that stress ABA accumulation in root systems requires the continuous import of ABA precursors from the shoots. Such an assumption was substantiated by the observation that defoliation or shading significantly reduced ABA accumulation in intact roots. The present study suggests that ABA catabolism is rapid enough to play an important role in the regulation of ABA accumulation.