Genome-wide identification,systematic analysis and characterization of <Emphasis Type="Italic">SRO</Emphasis> family genes in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

SIMILAR TO RCD ONE (SRO) is a small plant-specific gene family, which play essential roles in plant growth and development as well as in abiotic stresses. However, the function of SROs in maize is still unknown. In our study, six putative SRO genes were isolated from the maize genome. A systematic analysis was performed to characterize the ZmSRO gene family. The ZmSRO gene family was divided into two groups according to the motif and intron/exon analysis. Phylogenetic analysis of them with other plants showed that the clades of SROs along with the divergence of monocot and dicot and ZmSROs were more closely with OsSROs. Many abiotic stress response and hormone-induced cis-regulatory elements were identified from the promoter region of ZmSROs. Furthermore, RNA-seq analysis indicated that SRO genes were widely expressed in different tissues and development stages in maize, and the expression divergence was also obviously observed. Analyses of expression in response to PEG6000 and NaCl treatment, in addition to exogenous application of ABA and GA hormones showed that the majority of the members display stress-induced expression patterns. Taken together, our results provide valuable reference for further functional analysis of the SRO gene family in maize, especially in abiotic stress responses.     

Artificial chromosome formation in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

We report on the construction of maize minichromosomes using shuttle vectors harboring native centromeric segments, origins of replication, selectable marker genes, and telomeric repeats. These vectors were introduced into scutellar cells of maize immature embryos by microprojectile bombardment. Several independent transformation events were identified containing minichromosomes in addition to the normal diploid complement of 20 maize chromosomes. Immunostaining indicated that the minichromosomes recruited centromeric protein C, which is a specific component of the centromere/kinetochore complex. Minichromosomes were estimated to be 15–30 Mb in size based on cytological measurements. Fluorescent in situ hybridization (FISH) showed that minichromosomes contain the centromeric, telomeric, and exogenous unique marker sequences interspersed with maize retrotransposons. Minichromosomes were detected for at least a year in actively dividing callus cultures, providing evidence for their stability through numerous cell cycles. Plants were regenerated and minichromosomes were detected in root tips, providing confirmation of their normal replication and transmission during mitosis and through organogenesis. Assembly of maize artificial chromosomes may provide a tool to study centromere function and a foundation for developing new high capacity vectors for plant functional genomics and breeding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Evgueni V. Ananiev, deceased Evgueni V. Ananiev and Chengcang Wu contributed equally to this work. Novel materials described in this publication may be available for noncommercial research purposes on acceptance and signing of a material transfer agreement. In some cases, such materials may contain or be derived from materials obtained from a third party. In such cases, the distribution of material will be subject to the requisite permission from any third-party owners, licensors, or controllers of all or parts of the material. Obtaining any permission will be the sole responsibility of the requestor.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Analysis of synonymous codon usage in <Emphasis Type="Italic">Zea mays</Emphasis>

It is important and meaningful to understand the codon usage pattern and the factors that shape codon usage of maize. In this study, trends in synonymous codon usage in maize have been firstly examined through the multivariate statistical analysis on 7402 cDNA sequences. The results showed that the genes positions on the primary axis were strongly negatively correlated with GC3s, GC content of individual gene and gene expression level assessed by the codon adaptation index (CAI) values, which indicated that nucleotide composition and gene expression level were the main factors in shaping the codon usage of maize, and the variation in codon usage among genes may be due to mutational bias at the DNA level and natural selection acting at the level of mRNA translation. At the same time, CDS length and the hydrophobicity of each protein were, respectively, significantly correlated with the genes locations on the primary axis, GC3s and CAI values. We infer that genes length and the hydrophobicity of the encoded protein may play minor role in shaping codon usage bias. Additional 28 codons ending with a G or C base have been defined as “optimal codons”, which may provide useful information for maize gene-transformation and gene prediction.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cloning,characterization, and transformation of the phosphoethanolamine <Emphasis Type="Italic">N</Emphasis>-methyltransferase gene (<Emphasis Type="Italic">ZmPEAMT1</Emphasis>) in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

N-methylation of phosphoethanolamine, the committing step in choline (Cho) biosynthesis in plants, is catalyzed by S-adenosyl-l-methionine: phosphoethanolamine N-methyltransferase (PEAMT, EC 2.1.1.103). Herein we report the cloning and characterization of the novel maize phosphoethanolamine N-methyltransferase gene (ZmPEAMT1) using a combination of bioinformatics and a PCR-based allele mining strategy. The cDNA sequence of ZmPEAMT1 gene is 1,806 bp in length and translates a 495 amino acids peptide. The upstream promoter sequence of ZmPEAMT1 were obtained by TAIL-PCR, and contained four kinds of putative cis-acting regulatory elements, including stress-responsive elements, phytohormone-responsive elements, pollen developmental special activation elements, and light-induced signal transduction elements, as well as several other structural features in common with the promoter of rice and Arabidopsis homologies. RT-PCR analysis showed that expression of ZmPEAMT1 was induced by salt stress and suppressed by high temperature. Over-expression of ZmPEAMT1 enhanced the salt tolerance, root length, and silique number in transgenic Arabidopsis. These data indicated that ZmPEAMT1 maybe involved in maize root development and stress resistance, and maybe having a potential application in maize genetic engineering. Note: Nucleotide sequence data are available in GenBank under the following accession numbers: maize (Zea mays, ZmPEAMT1, AY626156; ZmPEAMT2, AY103779); rice (Oryza sativa, OsPEAMT1/Os01g50030, NM_192178; OsPEAMT2/Os05g47540, XM_475841); wheat (Triticum aestivum, TaPEAMT, AY065971); Arabidopsis (Arabidopsis thaliana, AtNMT1/At3g18000, AY091683; AtNMT2/At1g48600, NM_202264; AtNMT3/At1g73600, NM_106018); oilseed rape (Brassica napus, BnPEAMT, AY319479), tomato (Lycopersicon esculentum, AF328858), spinach (Spinacia oleracea, AF237633).     

Fine mapping of a quantitative resistance gene for gray leaf spot of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) derived from teosinte (<Emphasis Type="Italic">Z. mays</Emphasis> ssp. <Emphasis Type="Italic">parviglumis</Emphasis>)

Key message

In this study we mapped the QTL Qgls8 for gray leaf spot (GLS) resistance in maize to a ~130 kb region on chromosome 8 including five predicted genes.

Abstract

In previous work, using near isogenic line (NIL) populations in which segments of the teosinte (Zea mays ssp. parviglumis) genome had been introgressed into the background of the maize line B73, we had identified a QTL on chromosome 8, here called Qgls8, for gray leaf spot (GLS) resistance. We identified alternate teosinte alleles at this QTL, one conferring increased GLS resistance and one increased susceptibility relative to the B73 allele. Using segregating populations derived from NIL parents carrying these contrasting alleles, we were able to delimit the QTL region to a ~130 kb (based on the B73 genome) which encompassed five predicted genes.

     

Improvement of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in Hi-II maize (<Emphasis Type="Italic">Zea mays</Emphasis>) using standard binary vectors

High-frequency transformation of maize (Zea mays L.) using standard binary vectors is advantageous for functional genomics and other genetic engineering studies. Recent advances in Agrobacterium tumefaciens-mediated transformation of maize have made it possible for the public to transform maize using standard binary vectors without a need of the superbinary vector. While maize Hi-II has been a preferred maize genotype to use in various maize transformation efforts, there is still potential and need in further improving its transformation frequency. Here we report the enhanced Agrobacterium-mediated transformation of immature zygotic embryos of maize Hi-II using standard binary vectors. This improved transformation process employs low-salt media in combined use with antioxidant l-cysteine alone or l-cysteine and dithiothreitol (DTT) during the Agrobacterium infection stage. Three levels of N6 medium salts, 10, 50, and 100%, were tested. Both 10 and 50% salts were found to enhance the T-DNA transfer in Hi-II. Addition of DTT to the cocultivation medium also improves the T-DNA transformation. About 12% overall and the highest average of 18% transformation frequencies were achieved from a large number of experiments using immature embryos grown in various seasons. The enhanced transformation protocol established here will be advantageous for maize genetic engineering studies including transformation-based functional genomics.     

Induction of two cyclotide-like genes <Emphasis Type="Italic">Zmcyc1</Emphasis> and <Emphasis Type="Italic">Zmcyc5</Emphasis> by abiotic and biotic stresses in <Emphasis Type="Italic">Zea mays</Emphasis>

Cyclotides are small plant disulfide-rich and cyclic proteins with a diverse range of biological activities. Cyclotide-like genes show key sequence features of cyclotides and are present in the Poaceae. In this study the cDNA of the nine cyclotide-like genes were cloned and sequenced using 3′RACE from Zea mays. The gene expression of two of these genes (Zmcyc1 and Zmcyc5) were analyzed by real-time PCR in response to biotic (Fusarium graminearum, Ustilago maydis and Rhopalosiphum maydis) and abiotic (mechanical wounding, water deficit and salinity) stresses, as well as in response to salicylic acid and methyl jasmonate elicitors to mimic biotic stresses. All isolated genes showed significant similarity to other cyclotide-like genes and were classified in two separate clusters. Both Zmcyc1 and Zmcyc5 were expressed in all studied tissues with the highest expression in leaves and lowest expression in roots. Wounding, methyl jasmonate and salicylic acid significantly induced the expression of Zmcyc1 and Zmcyc5 genes, but the higher expression was observed for Zmcyc1 as compared with Zmcyc5. Expression levels of these two genes were also induced in inoculated leaves with F. graminearum, U. maydis and also in response to insect infestation. In addition, the 1000-base-pairs (bp) upstream of the promoter of Zmcyc1 and Zmcyc5 genes were identified and analyzed using the PlantCARE database and consequently a large number of similar biotic and abiotic cis-regulatory elements were identified for these two genes.     

Responses of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) near isogenic lines carrying <Emphasis Type="Italic">Wsm1</Emphasis>, <Emphasis Type="Italic">Wsm2,</Emphasis> and <Emphasis Type="Italic">Wsm3</Emphasis> to three viruses in the Potyviridae

Genes on chromosomes six (Wsm1), three (Wsm2) and ten (Wsm3) in the maize (Zea mays L.) inbred line Pa405 control resistance to Wheat streak mosaic virus (WSMV), and the same or closely linked genes control resistance to Maize dwarf mosaic virus (MDMV) and Sugarcane mosaic virus (SCMV). Near isogenic lines (NIL) carrying one or two of the genes were developed by introgressing regions of the respective chromosomes into the susceptible line Oh28 and tested for their responses to WSMV, MDMV, and SCMV in the field and greenhouse. F₁ progeny from NIL × Oh28 were also tested. Wsm1, or closely linked genes, provided resistance to all three viruses, as determined by symptom incidence and severity. Wsm2 and Wsm3 provided resistance to WSMV. Wsm2 and/or Wsm3 provided no resistance to MDMV, but significantly increased resistance in plants with one Wsm1 allele. NIL carrying Wsm1, Wsm2, or Wsm3 had similar SCMV resistance in the field, but NIL with Wsm2 and Wsm3 were not resistant in the greenhouse. Addition of Wsm2 to Wsm1 increased SCMV resistance in the field. For all viruses, symptom incidence was higher in the greenhouse than in the field, and relative disease severity was higher in the greenhouse for WSMV and MDMV. An Italian MDMV isolate and the Ohio SCMV infected the Wsm1 NIL, while the Ohio MDMV and Seehausen SCMV isolates did not. Our results indicate that the three genes, or closely linked loci, provide virus resistance. Resistance conferred by the three genes is influenced by interactions among the genes, the virus species, the virus isolate, and the environment.     

Effect of high temperature on photosynthesis and transpiration of sweet corn (<Emphasis Type="Italic">Zea mays</Emphasis> L. var. <Emphasis Type="Italic">rugosa</Emphasis>)

Four temperature treatments were studied in the climate controlled growth chambers of the Georgia Envirotron: 25/20, 30/25, 35/30, and 40/35 °C during 14/10 h light/dark cycle. For the first growth stage (V3-5), the highest net photosynthetic rate (P _N) of sweet corn was found for the lowest temperature of 28–34 μmol m⁻² s⁻¹ while the P _N for the highest temperature treatment was 50–60 % lower. We detected a gradual decline of about 1 P _N unit per 1 °C increase in temperature. Maximum transpiration rate (E) fluctuated between 0.36 and 0.54 mm h⁻¹ (≈5.0–6.5 mm d⁻¹) for the high temperature treatment and the minimum E fluctuated between 0.25 and 0.36 mm h⁻¹ (≈3.5–5.0 mm d⁻¹) for the low temperature treatment. Cumulative CO₂ fixation of the 40/35 °C treatment was 33.7 g m⁻² d⁻¹ and it increased by about 50 % as temperature declined. The corresponding water use efficiency (WUE) decreased from 14 to 5 g(CO₂) kg⁻¹(H₂O) for the lowest and highest temperature treatments, respectively. Three main factors affected WUE, P _N, and E of Zea: the high temperature which reduced P _N, vapor pressure deficit (VPD) that was directly related to E but did not affect P _N, and quasi stem conductance (QC) that was directly related to P _N but did not affect E. As a result, WUE of the 25/20 °C temperature treatment was almost three times larger than that of 40/35 °C temperature treatment.     

Cadmium uptake,localization and detoxification in <Emphasis Type="Italic">Zea mays</Emphasis>

Cadmium uptake, translocation and localization in maize roots and shoots at the tissue and cellular level were investigated. Metal accumulation in plant organs as well as symptoms of Cd toxicity were closely correlated with an increase in Cd concentration applied (5 – 300 M). Most of the metal taken up was retained in roots, mainly inside the cells of endodermis, pericycle and central cylinder parenchyma. Accumulation of phytochelatins and related peptides also depended on Cd concentration in the nutrient solution.     

Genetic dissection of the maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) MAMP response

Key message

Loci associated with variation in maize responses to two microbe-associated molecular patterns (MAMPs) were identified. MAMP responses were correlated. No relationship between MAMP responses and quantitative disease resistance was identified.

Abstract

Microbe-associated molecular patterns (MAMPs) are highly conserved molecules commonly found in microbes which can be recognized by plant pattern recognition receptors. Recognition triggers a suite of responses including production of reactive oxygen species (ROS) and nitric oxide (NO) and expression changes of defense-related genes. In this study, we used two well-studied MAMPs (flg22 and chitooctaose) to challenge different maize lines to determine whether there was variation in the level of responses to these MAMPs, to dissect the genetic basis underlying that variation and to understand the relationship between MAMP response and quantitative disease resistance (QDR). Naturally occurring quantitative variation in ROS, NO production, and defense genes expression levels triggered by MAMPs was observed. A major quantitative traits locus (QTL) associated with variation in the ROS production response to both flg22 and chitooctaose was identified on chromosome 2 in a recombinant inbred line (RIL) population derived from the maize inbred lines B73 and CML228. Minor QTL associated with variation in the flg22 ROS response was identified on chromosomes 1 and 4. Comparison of these results with data previously obtained for variation in QDR and the defense response in the same RIL population did not provide any evidence for a common genetic basis controlling variation in these traits.

     

Genetic analysis and molecular mapping of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) stalk rot resistant gene<Emphasis Type="Italic"> Rfg</Emphasis>1

One single pathogen Fusarium graminearum Schw. was inoculated to maize inbred lines 1,145 (Resistant) and Y331 (Susceptive), and their progenies of F₁, F₂ and BC₁F₁ populations. Field statistical data revealed that all of the F₁ individuals were resistant to the disease and that the ratio of resistant plants to susceptive plants was 3:1 in the F₂ population, and 1:1 in the BC₁F₁ population. The results revealed that a single dominant gene controls the resistance to F. graminearum Schw.. The resistant gene to F. graminearum Schw. was denominated as Rfg1 according to the standard principle of the nomenclature of the plant disease resistant genes. RAPD (randomly amplified polymorphic DNA) combined with BSA (bulked segregant analysis) analysis was carried out in the developed F₂ and BC₁F₁ populations, respectively. Three RAPD products screened from the RAPD analysis with 820 Operon 10-mer primers showed the linkage relation with the resistant gene Rfg1. The three RAPD amplification products (OPD-20₁₀₀₀, OPA-04₁₁₀₀ and OPY-04₉₀₀) were cloned and their copy numbers were determined. The results indicated that only OPY-04₉₀₀ was a single-copy sequence. Then, OPY-04₉₀₀ was used as a probe to map the Rfg1 gene with a RIL F₇ mapping population provided by Henry Nguyen, which was developed from the cross S3×Mo17. Rfg1 was primarily mapped on chromosome 6 between the two linked markers OPY-04₉₀₀ and umc21 (Bin 6.04–6.05). In order to confirm the primary mapping result, 25 SSR (simple sequence repeat) markers and six RFLP (restriction fragment length polymorphism) markers in the Rfg1 gene-encompassing region were selected, and their linkage relation with Rfg1 was analyzed in our F₂ population. Results indicated that SSR marker mmc0241 and RFLP marker bnl3.03 are flanking the Rfg1 gene with a genetic distance of 3.0 cM and 2.0 cM, respectively. This is the first time to name and to map a single resistant gene of maize stalk rot through a single pathogen inoculation and molecular marker analysis.Communicated by H.F. Linskens     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.