Cloning, Characterization and Transformation of Methyltransferase 2a Gene (Zmet2a) in Maize (Zea mays L.)

DNA methylation is an important epigenetic regulatory mechanism,it regulates gene expression by recruiting proteins involved in gene repression or by inhibiting the binding of transcription factor(s)to DNA.In this study,a novel methyltransferase 2a gene(Zmet2a)was cloned in maize and identified by polymerase chain reaction-base(PCR-base)using a bioinformatics strategy.The Zmet2a cDNA sequence is 2739 bp long and translates to 912 amino acid peptides.The Zmet2a protein revealed that it contains BAH and CHROMO structural domains,is a non-transmembrane protein that is hydrophilically unstable,and has no signal peptide structure.Meanwhile,we verified the biological roles of Zmet2a using transgenic Arabidopsis overexpressing Zmet2a and Zmet2a-knockout maize.Transgenic Zmet2a Arabidopsis thaliana showed highly significant advancement inflowering time,and Zmet2a-knockout maize showed advancement inflowering time,with significant changes in several traits.Altogether,these report the role of Zmet2a in the regulation offlowering time,which will lay a foundation for revealing the biological function and epigenetic regulation mechanism of Zmet2a in the growth,development andflowering of maize.

Activity of Acetolactate Synthase from Maize (Zea mays L.) as Influenced by Chlorsulfuron and Tribenuron-methyl

Study on relative sensitivity of maize (Zea mays L.) Nongda108 and Nongda3138 to sulfony-lurea herbicide chlorsulfuron and tribenuron-methyl using maize taproot length by sand bioassy indicated that, Nongda3138 had higher tolerance to chlorsulfuron and tribenuron-methyl than Nongda108 did. Chlorsulfuron had stronger growth inhibition to maize Nongda108 and Nongda3138 than tribenuron-methyl did. Study on target enzyme of sulfonylurea herbicide acetolactate synthase (ALS) showed that, chlorsulfuron and tribenuron-methyl inhibited ALS in vitro strongly, and non-competitively. In the same concentration of inhibitors, chlorsulfuron had stronger ALS activity inhibition than tribenuron-methyl did. Lower level of chlorsulfuron and tribenuron-methyl has no ALS activity inhibition in vivo, the ALS inhibition only occurred in the condition of high concentration of chlorsulfuron and tribenuron-methyl in vivo.

Top-grain filling characteristics at an early stage of maize(Zea mays L.) with different nitrogen use efficiencies

Maize genotypes vary significantly in their nitrogen use efficiencies（NUEs）.Better understanding of early grain filling characteristics of maize is important,especially for maize with different NUEs.The objectives of this research were（i）to investigate the difference in apical kernel development of maize with different NUEs,（ii）to determine the reaction of apical kernel development to N application levels,and（iii）to evaluate the relationship between apical kernel development and grain yield（GY）for different genotypes of maize.Three maize hybrid varieties with different NUEs were cultivated in a field with different levels of N fertilizer arranged during two growing seasons.Kernel fresh weight（KFW）,volume（KV）and dry weight（KDW）of apical kernel were evaluated at an early grain filling stage.Ear characteristics,GY and its components were determined at maturity stage.Apical kernel of the high N and high efficiency（HN-HE）type（under low N,the yield is lower,and under higher N,the yield is higher）developed better under high N（N210 and N240,pure N of 210 and 240 kg ha^–1）than at low N（N120 and N140,pure N of 120 and 140 kg ha^–1）.The low N and high efficiency（LN-HE）type（under low N,the yield is higher,while under higher N,the yield is not significantly higher）developed better under low N than at high N.The double high efficiency（D-HE）type（for both low and high N,the yield is higher）performed well under both high and low N.Apical kernel reacted differently to the N supply.Apical kernel developed well at an early grain filling stage and resulted in a higher kernel number（KN）,kernel weight（KW）and GY with better ear characteristics at maturity.

QTL analysis of the developmental changes in cell wall components and forage digestibility in maize(Zea mays L.)

Cell wall architecture plays a key role in stalk strength and forage digestibility.Lignin,cellulose,and hemicellulose are the three main components of plant cell walls,and they can impact stalk quality by affecting the structure and strength of the cell wall.To explore cell wall development during secondary cell wall lignification in maize stalks,conventional and conditional genetic mapping were used to identify the dynamic quantitative trait loci(QTLs)of the cell wall components and digestibility traits during five growth stages after silking.Acid detergent lignin(ADL),cellulose(CEL),acid detergent fiber(ADF),neutral detergent fiber(NDF),and in vitro dry matter digestibility(IVDMD)were evaluated in a maize recombinant inbred line(RIL)population.ADL,CEL,ADF,and NDF gradually increased from 10 to 40 days after silking(DAS),and then they decreased.IVDMD initially decreased until 40 DAS,and then it increased slightly.Seventytwo QTLs were identified for the five traits,and each accounted for 3.48–24.04%of the phenotypic variation.Six QTL hotspots were found,and they were localized in the 1.08,2.04,2.07,7.03,8.05,and 9.03 bins of the maize genome.Within the interval of the pleiotropic QTL identified in bin 1.08 of the maize genome,six genes associated with cell wall component biosynthesis were identified as potential candidate genes for stalk strength as well as cell wall-related traits.In addition,26 conditional QTLs were detected in the five stages for all of the investigated traits.Twenty-two of the 26 conditional QTLs were found at 30 DAS conditioned using the values of 20 DAS,and at 50 DAS conditioned using the values of 40 DAS.These results indicated that cell wall-related traits are regulated by many genes,which are specifically expressed at different stages after silking.Simultaneous improvements in both forage digestibility and lodging resistance could be achieved by pyramiding multiple beneficial QTL alleles identified in this study.

Screening Methods for Waterlogging Tolerance at Maize (Zea mays L.) Seedling Stage

Waterlogging strongly affects agronomic performance of maize （Zea mays L.）. In order to investigate the suitable selection criteria of waterflooding tolerant genotypes, and identify the most susceptible stage and the best continuous treatment time to waterlogging, 20 common maize inbred lines were subjected to successive artificial waterflooding at seedling stage, and waterlogging tolerance coefficient （WTC） was used to screen waterflooding tolerant genotypes. In addition, peroxidase （POD） activities and malondialdehyde （MDA） contents were measured for 6 of 20 lines. The results showed that the second leaf stage （V2） was the most susceptible stage, and 6 d after waterflooding was the best continuous treatment time. Dry weight （DW） of both shoots and roots of all lines were significantly reduced at 6 d time-point of waterlogging, compared to control. POD activities and MDA contents were negatively and significantly correlated, and the correlation coefficient was -0.9686 （P 〈 0.0001）. According to the results, WTC of shoot DW can be used for practical screening as a suitable index, which is significantly different from control and waterlogged plants happened 6 d earlier. Furthermore, leaf chlorosis, MDA content and POD activities could also be used as reference index for material screening. The implications of the results for waterlogging-tolerant material screening and waterlogging-tolerant breeding have been discussed in maize.

Influence of Zinc Nutrition on Growth and Yield Behaviour of Maize (<i>Zea mays</i>L.) Hybrids

A field experiment was conducted during spring 2011 at Agronomic Research Area, University of Agriculture, Faisalabad, Pakistan to evaluate the comparative efficacy of Zn uptake and grain yield in three maize hybrids namely Pioneer-32F 10, Monsanto-6525 and Hycorn-8288 through the application of Zn in the form of ZnSO4. The ZnSO4 treatments comprised;soil application at the time of sowing @ 12 kg&middotha-1 (Zn1), foliar application at vegetative stage (9 leaf stage) @ 1% ZnSO4 solution (Zn2) and foliar application at reproductive stage (anthesis) @ 1% ZnSO4 solution (Zn3) and one treatment was kept as a control, where zinc was not applied (Zn0). The experimental results showed substantial difference in all physiological and yield parameters except plant height and stem diameter. Statistically maximum grain yield (8.76 t&middotha-1) was obtained with foliar spray of ZnSO4 at 9 leaf stage (Zn2) in case of Monsanto-6525. As regard to quality parameters, Pioneer-32F 10 and Hycorn-8288 accumulated more zinc contents in grains but Monsanto-6525 attained more zinc concentration in straw. Foliar spray of ZnSO4 at 9 leaf stage produced 19.42% more zinc contents in grains as compared to other ZnSO4 treatments. Foliar spray of ZnSO4 at 9 leaf stage in Monsanto-6525 hybrid produced higher grain yield.

Establishment and Optimization of the Regeneration System of Mature Embryos of Maize (Zea mays L.)

A reliable system was developed for regeneration from mature embryos derived from callus of four maize inbred lines （Liao 7980, Dan 9818, Dan 340, and Dan 5026）. The protocol was mainly based on a series of experiments involving the composition of culture medium. We found that 9 pM 2,4-dichlorophenoxyacetic acid in MS medium was optimum for the induction of callus. The induction frequency of primary calli was over 85% for four inbred lines tested. The addition of L- proline （12 mM） in subculture medium significantly promoted the formation of embryogenic callus but it did not significantly enhance growth rate of callus. Efficient shoot regeneration was obtained on regeneration medium containing 2.22 μM 6- benzylaminopurine in combinations with 4.64 μM Kinetin. Regenerated shoots were rooted on half-strength MS medium containing 2.85 μM indole-3-butyric acid. This plant regeneration system provides a foundation for genetic transformation of maize.

Breeding for Drought Tolerance in Maize (Zea mays L.)

Drought, like many other environmental stresses, has adverse effects on crop yield including maize (Zea mays L.). Low water availability is one of the major causes for maize yield reductions affecting the majority of the farmed regions around the world. Therefore, the development of drought-tolerant lines becomes increasingly more important. In maize, a major effect of water stress is a delay in silking, resulting in an increase in the anthesis-silking interval, which is an important cause of yield failures. Diverse strategies are used by breeding programs to improve drought tolerance. Conventional breeding has improved the drought tolerance of temperate maize hybrids and the use of managed drought environments, accurate phenotyping, and the identification and deployment of secondary traits has been effective in improving the drought tolerance of tropical maize populations and hybrids as well. The contribution of molecular biology will be potential to identify key genes involved in metabolic pathways related to the stress response. Functional genomics, reverse and forward genetics, and comparative genomics are all being deployed with a view to achieving these goals. However, a multidisciplinary approach, which ties together breeding, physiology and molecular genetics, can bring a synergistic understanding to the response of maize to water deficit and improve the breeding efficiency.

Genomic and transcriptomic insights into cytochrome P450 monooxygenase genes involved in nicosulfuron tolerance in maize(Zea mays L.)

Postemergence application of nicosulfuron for weed control in maize fields can cause great damage to certain maize inbred lines and hybrids. Two maize genotypes, tolerant inbred(HBR) and sensitive inbred(HBS), were found to significantly differ in their phenotypic responses to nicosulfuron, with the EC50(50% effective concentration) values differed statistically(763.6 and 5.9 g a.i. ha–1, respectively). Pre-treatment with malathion, a known cytochrome P450 inhibitor, increased nicosulfuron injury in both HBR and HBS. Our results support the hypothesis that nicosulfuron selectivity in maize is associated with cytochrome P450 metabolism. Further analysis of the maize genome resulted in the identification of 314 full length cytochrome P450 monooxygenase(CYP) genes. These genes were classified into 2 types, 10 clans and 44 families. The CYP71 clan was represented by all A-type genes(168) belonging to 17 families. Nine clans possessed 27 families containing 146 non-A-type genes. The consensus sequences of the heme-binding regions of A-type and non-A-type CYP proteins are ‘PFGXGRRXCPG’ and ‘FXXGPRXCXG’, respectively. Illumina transcriptome sequence results showed that there were 53 differentially expressed CYP genes on the basis of high variation in expression between HBS and HBR, nicosulfuron-treated and untreated samples. These genes may contribute to nicosulfuron tolerance in maize. A hierarchical clustering analysis obtained four main clusters named C1 to C4 in which 4, 15, 21, and 13 CYP genes were found in each respective cluster. The expression patterns of some CYP genes were confirmed by RT-q PCR analysis. The research will improve our understanding of the function of maize cytochrome P450 in herbicide metabolism.

Mapping of QTLs Associated with Seed Vigor to Artificial Aging Using Two RIL Populations in Maize (<i>Zea mays</i>L.)

Improvement in seed vigor under adverse condition is an important object in maize breeding nowadays. Because the higher sowing quality of seeds is necessary for the development of the agriculture production and better able to resist all kinds of adversity in the seeds storage. So it is helpful for long-term preservation of germplasm resource. In our study, two connected recombinant inbred line (RIL) populations, which derived from the crosses Yu82 × Shen137 and Yu537A × Shen137 respectively, were evaluated for four related traits of seed vigor under three aging treatments. Meta-analysis was used to integrate genetic maps and detected QTL across two populations. In total, 74 QTL and 20 meta-QTL (mQTL) were detected. All QTLs with contributions (R2) over 10% were consistently detected in at least one of aging treatments and integrated in mQTL. Four key mQTLs (mQTL2-2, mQTL5-3, mQTL6 and mQTL8) with R2 of some initial QTLs > 10% included 5-9 initial QTLs associated with 2-4 traits. Therefore, the chromosome regions for four mQTLs with high QTL co-localization might be hot spots of the important QTLs for the associated traits. Twenty-two key candidate genes regulating four related traits of seed vigor mapped in 14 corresponding mQTLs. In particular, At5g67360, 45238345/At1g70730/At1g09640 and 298201206 were mapped within the important mQTL5-3, mQTL6 and mQTL8 regions, respectively. Fine mapping or construction of single chromosome segment lines for genetic regions of the three mQTLs is worth further study and could be put to use molecular marker-assisted breeding and pyramiding QTLs in maize.

Genetic dissection of husk number and length across multiple environments and fine-mapping of a major-effect QTL for husk number in maize(Zea mays L.)

Husk number(HN)and husk length(HL)influence the mechanical harvesting of maize grain.We investigated the genetic basis of HN and HL using a population of 204 recombinant inbred lines phenotypically evaluated in five environments.The two husk traits showed broad phenotypic variation and high heritability.Nine stable quantitative trait loci(QTL)were identified by single-environment mapping,comprising four QTL for HN and five for HL,and three QTL explained>10%of the phenotypic variation.Joint mapping revealed 22 additive QTL and 46 epistatic QTL.Both additive and epistatic(additive×additive)effects as well as a few large-effect QTL and some minor-effect QTL appeared to contribute to the genetic architecture of HN and HL.The QTL for HN located on chromosome 7,q HN7,which accounted for^20%of phenotypic variation,was detected in all five environments.q HN7 was fine-mapped to a 721.1 kb physical region based on the maize B73 Ref Gen_v3 genome assembly.Within this interval,four genes associated with plant growth and development were selected as candidate genes.The results will be useful for improvement of maize husk traits by molecular breeding and provide a basis for the cloning of q HN7.

Influence of Soil Moisture and Air Temperature on the Stability of Cytoplasmic Male Sterility (CMS) in Maize (Zea mays L.)

Cytoplasmic male sterility (CMS) is a maternally inherited trait that suppresses the production of viable pollen. CMS is a useful biological tool for confinement strategies to facilitate coexistence of genetically modified (GM) and non-GM crops in case where it is required. The trait is reversible and can be restored to fertility in the presence of nuclear restorer genes (Rf genes) and by environmental impacts. The aim of this study was to investigate the influence of the level of irrigation on the stability of CMS maize hybrids under defined greenhouse conditions. Additionally the combination of irrigation and air temperature was studied. Three CMS maize hybrids were grown with different levels of irrigation and in different temperature regimes. Tassel characteristics, pollen production and fertility were assessed. The CMS stability was high in hot air temperatures and decreased in lower temperatures. The level of irrigation had no major effect on the level of sterility. The extent of these phenomena was depending on the genotype of CMS maize and should be known before using CMS for coexistence purposes.

Uptake and accumulation of copper by roots and shoots of maize( Zea mays L.)

The effects of different concentrations of copper sulfate on root and shoot growth of maize( Zea mays L.) and the uptake and accumulation of Cu2+ by its roots and shoots were investigated in the present study. The concentrations of copper sulfate (CuSO4 (.) 5H(2)O) used were in the range of 10(-5) -10(-3)mol/L. Root growth decreased progressively with increasing concentration of Cu2+ in solution. The seedlings exposed to 10(-3) mol/L Cu2+ exhibited substantial growth reduction, yielding only 68% of the root length of the control. The shoot growth of the seedlings grown at 10(-5) -10(-4) mol/L Cu2+ were more or less the same as the control seedlings. The leaves treated with 10(-3) mol/L Cu2+ were obviously inhibited in shoot growth. The fresh and dry weights both in roots and shots decreased progressively with increasing Cu2+ concentration. This fits well with the above mentioned effects of copper sulfate on root growth. Zea mays has considerable ability to remove Cu from solutions and accumulate it. The Cu content in roots of Z. mays increased with increasing solution concentration of Cu2+. The amount of Cu in roots of plants treated with 10(-3), 10(-4) and 10(-5) mol/L Cu2+ were 10, 8 and 1.5 fold, respectively, greater than that of roots of control plane. However, the plants transported and concentrated only a small amount of Cu in their shoots.

Efficient Somatic Embryogenesis and Plant Regeneration Through Callus Initiation From Seedling-derived Leaf Materials of Maize (Zea mays L.)

While being one of the world's most important crops,maize ( Zea mays L.) is still difficult to regenerate in tissue culture which severely limits its improvement by genetic engineering.Currently,immature zygotic embryos provide the predominantly used material for regeneration and transformation.However,the procedures involved are often laborious,time-consuming and season-dependent.Here,we further improved an efficient tissue culture and plant regeneration system that uses maize leaf segments of young seedlings as an alternative explant source.Embryogenic calli were evaluated by morphology,proliferation and regeneration capacity.All these indicated that seedling-derived leaf materials have the potential to replace immature embryos for tissue culture and regeneration.

Study on the Photosynthetic Characteristics in Spring Maize (Zea mays L.)of Different Quality Type

common maize synthetic rate（ Photosynthetic characteristics were probed by sweet maize, waxy maize, high starch maize and The results revealed that leaf area index （LAI）, chlorophyll a content, chlorophyll b content,photo-PR） showed single peak curve at the whole growth stage. The stages of peak were different according to different varieties. NEAUS4 had the lowest peak and while SIDAN 19 had the highest among all stages. Ratio of chlorophyll a to b was low at seedling stage, reached the peak atjointing stage and then declined. SIDAN 19 had the lower level at the last stages.

Maize cryptochromes 1a1 and 1a2 promote seedling photomorphogenesis and shade resistance in Zea mays and Arabidopsis

Maize growth and development are regulated by light quality,intensity and photoperiod.Cryptochromes are blue/ultraviolet-A light receptors involved in stem elongation,shade avoidance,and photoperiodic flowering.To investigate the function of cryptochrome 1(CRY1) in maize,where it is encoded by Zm CRY1,we obtained two Zm CRY1a genes(Zm CRY1a1 and Zm CRY1a2),both of which share the highest similarity with other gramineous plants,in particular rice CRY1a by phylogenetic analysis.In Arabidopsis,overexpression of Zm CRY1a genes promoted seedling de-etiolation under blue and white light,resulting in dwarfing of mature plants.In seedlings of the maize inbred line Zong 31(Zm CRY1aOE),overexpression of Zm CRY1a genes caused a reduction in the mesocotyl and first leaf sheath lengths due to down-regulation of genes influencing cell elongation.In mature transgenic maize plants,plant height,ear height,and internode length decreased in response to overexpression of Zm CRY1a genes.Expression of Zm CRY1a were insensitive to low blue light(LBL)-induced shade avoidance syndrome(SAS) in Arabidopsis and maize.This prompted us to investigate the regulatory role of the gibberellin and auxin metabolic pathways in the response of Zm CRY1a genes to LBL treatment.We confirmed a link between Zm CRY1a expression and hormonal influence on the growth and development of maize under LBL-induced SAS.These results reveal that Zm CRY1a has a relatively conservative function in regulating maize photomorphogenesis and may guide new strategies for breeding high density-tolerant maize cultivars.

The Role of Nitrogen and Sulfur Interaction in Maize Quality(Zea mays L.)

Two hybrids of maize with different responses to sulfur were used in the pool experiment. The effects of nitrogen and sulfur on the grain quality of maize were evaluated. The results indicated that grain quality changed with the nutrition supply. The contents of proteins, amino acids, soluble sugar, crude fat, oil, N, P, K, S and microelements in the grain were improved due to nitrogen and sulfur fertilizer addition. But the effects of nitrogen and sulfur were not the same. Nitrogen increased starch content of the grain, but S decreased the content. Both N and S enhanced the proportion of amylopectin in starch. Sulfur nutrition significantly improved the grain quality of maize when a large amount of nitrogen was used together. Both hybrids had similar response to N and S treatments.

Comparative proteomic analysis provides new insights into ear leaf senescence of summer maize(Zea mays L.) under fi eld condition

As the most important organ in plant photosynthesis, the leaf plays an important role in plant growth and development. Leaf senescence is associated with fundamental changes in the proteome. To research the molecular mechanisms of leaf senescence, protein expression in senescing maize ear leaves grown under field conditions was analyzed using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight mass spectrometry（MALDI-TOF/TOF MS）. A total of 60 senescence-associated proteins were identified. The identified proteins are involved in many biological processes, especially energy, metabolism and protein synthesis. Several of the identified proteins have not been previously reported as senescence-associated, including glycine-rich RNA-binding protein.

Soil tillage methods by years interaction for dry matter of plant yield of maize (Zea mays L.)using additive main effects and multiplicative interaction model

The objective of this study was to assess soil tillage methods by years interaction for dry matter of plant yield of maize(Zea mays L.) grown in West Poland by the additive main effects and multiplicative interaction model. The study comprised four soil tillage methods, analysed in 12 years through field trials arranged in a randomized complete block design, with four replicates. Dry matter of plant yield of the tested soil tillage methods varied from 86.7 dt ha(for no-plough tillage in 2005) to 246.4 dt ha(for complete conventional tillage in 2012), with an average of 146.6 dt ha. In the variance analysis, 49.07% of the total dry matter of plant yield variation was explained by years, 12.69% by differences between soil tillage methods, and 10.53% by soil tillage methods by years interaction. Dry matter of plant yield is highly influenced by soil tillage methods by years factors.

Proteome Changes in Maize Embryo(Zea mays L)Induced by Ion Beam Implantation Treatment

Low energy ion beam implantation was applied to the maize （Zea mays L） embryo proteome using two-dimensional gel electrophoresis. Protein profile analysis detected more than Ii00 protein spots, 72 of which were determined to be expressed differently in the treated and control （not exposed to ion beam implantation） embryos. Of the 72 protein spots, 53 were up- regulated in the control and 19 were more abundantly expressed in the ion beam-treated embryos. The spots of up- or down-regulated proteins were identified by matrix assisted laser desorption /ionization-time of flight mass spectrometry （MALDI-TOF-MS）. Among the identified proteins, ii were up-regulated in the treated embryos. Four of these up-regulated proteins were antioxidant molecules, three were related to stress response, two to sugar metabolism and two were associated with heat shock response. Of the five proteins up-regulated in the control embryos, three were functionally related to carbohydrate metabolism; the functions of the remaining two proteins were unknown. The data collected during this study indicate that treatment of maize embryos with low energy ion beam implantation induces changes in stress tolerance enzymes/proteins, possibly as a result of alterations in metabolism.