Ivr2, a candidate gene for a QTL of vacuolar invertase activity in maize leaves. Gene-specific expression under water stress

Water shortage produced an early and large stimulation of acid- soluble invertase activity in adult maize leaves whereas cell wall invertase activity remained constant. This response was closely related to the mRNA level for only one of the invertase gene (Ivr2), encoding a vacuolar isoform. In parallel, four quantitative trait loci (QTLs) were detected for invertase activity under control and nine under stressful conditions. One QTL in control and one in stressed plants was located near to the lvr2 gene on chromosome 5. Other QTLs for invertase activity were found close to carbohydrate QTLs; some of them formed stress clusters.     

玉米雄穗分枝数与主轴长的QTL鉴定

在包含103个SSR标记的连锁图谱基础上, 运用复合区间作图法检测玉米组合(N87-1×9526 )F3家系在正常与干旱胁迫环境下的雄穗分枝数与主轴长性状QTL。雄穗分枝数在正常环境下被检测到2个QTL座位, 分别位于第5和7连锁群上; 在胁迫环境下被检测到4个QTL座位分别位于 2、5、7和10连锁群上, 其中位于第5和7连锁群上的QTL不仅具有一致性而且与本作图群体中曾检测到的耐旱相关性状QTL存在连锁。雄穗主轴长在正常环境下被检测到2个QTL位于第2和第6连锁群上, 在干旱胁迫环境下被检测到了3个QTL分别于第2、4和10连锁群上, 其中位于第2染色体上的QTL是两种环境下所共同检测到的QTL。分析QTL的遗传作用方式表明, 雄穗分枝数以部分加性效应为主, 而雄主轴长全部表现为显性和超显性。     

玉米子粒中锌及铁铜锰含量的遗传及QLT分析

分别利用三交组合DH8×登海40和DH86×沈137创建F1代DH群体A和群体B,比较2个不同遗传背景下DH群体子粒中锌及铁铜锰含量的变化,并对2个供试群体2年间的试验结果进行了QTL分析。发现玉米子粒中锌及铁铜锰的含量在不同个体间、不同年份间受环境影响比较大,且表现不稳定;群体内呈现连续性数量性状变化,服从于正态分布。对群体A2年子粒中锌及铁铜锰含量进行QTL定位,结果2007年检测到了5个与这些性状有关的QTL,可解释的遗传变异范围为9.41%~43.67%;2008年检测到9个QTL,可解释的遗传变异范围为11.21%~42.96%。2年间末检测到相同的QTL位点。对群体B进行QTL定位,2年间检测到18个QTL位点,分布于除第5染色体以外的其余9条染色体上。其中,2007年检测到12个QTL,2008年获得6个QTL,相同的QTL为2个,控制锌含量的1个位点位于第3染色体的p-umc1399-p-bnlg1605区段,控制铜含量的1个位点位于第2染色体的bnlg1746区段。     

A genome-wide survey of maize lipid-related genes: candidate genes mining,digital gene expression profiling and co-location with QTL for maize kernel oil

Lipids play an important role in plants due to their abundance and their extensive participation in many metabolic processes. Genes involved in lipid metabolism have been extensively studied in Arabidopsis and other plant species. In this study, a total of 1003 maize lipid-related genes were cloned and annotated, including 42 genes with experimental validation, 732 genes with full-length cDNA and protein sequences in public databases and 229 newly cloned genes. Ninety-seven maize lipid-related genes with tissue-preferential expression were discovered by in silico gene expression profiling based on 1984483 maize Expressed Sequence Tags collected from 182 cDNA libraries. Meanwhile, 70 QTL clusters for maize kernel oil were identified, covering 34.5% of the maize genome. Fifty-nine (84%) QTL clusters co-located with at least one lipid-related gene, and the total number of these genes amounted to 147. Interestingly, thirteen genes with kernel-preferential expression profiles fell within QTL clusters for maize kernel oil content. All the maize lipid-related genes identified here may provide good targets for maize kernel oil QTL cloning and thus help us to better understand the molecular mechanism of maize kernel oil accumulation.     

Toward positional cloning of Vgt1, a QTL controlling the transition from the vegetative to the reproductive phase in maize

Vgt1 (Vegetative to generative transition 1) is a quantitative trait locus (QTL) for flowering time in maize (Zea mays L.). Vgt1 was initially mapped in a ca. 5-cM interval on chromosome bin 8.05, using a set of near-isogenic lines (NILs) in the genetic background of the late dent line N28, with the earliness allele introgressed from the early variety Gaspé Flint. A new large mapping population was produced by crossing N28 and one early NIL with a ca. 6-cM long Gaspé Flint introgression at the Vgt1 region. Using PCR-based assays at markers flanking Vgt1, 69 segmental NILs homozygous for independent crossovers near the QTL were developed. When the NILs were tested in replicated field trials for days to pollen shed (DPS) and plant node number (ND), the QTL followed a Mendelian segregation. Using bulk segregant analysis and AFLP profiling, 17 AFLP markers linked to the QTL region were identified. Statistical analysis indicated a substantial coincidence of the effects of Vgt1 on both DPS and ND. Vgt1 was mapped at ca. 0.3 cM from an AFLP marker. As compared to DPS, the higher heritability of ND allowed for a more accurate assessment of the effects of Vgt1. The feasibility of the positional cloning of Vgt1 is discussed.     

An allele of ZmPORB2 encoding a protochlorophyllide oxidoreductase promotes tocopherol accumulation in both leaves and kernels of maize

Phytol is one of the key precursors for tocopherol synthesis in plants, however, the underlying mechanisms concerning the accumulation of tocopherol remain poorly understood. In this study, qVE5, a major QTL affecting tocopherol accumulation in maize kernels was identified via a positional cloning approach. qVE5 encodes a protochlorophyllide oxidoreductase (ZmPORB2), which localizes to the chloroplast. Overexpression of ZmPORB2 increased tocopherol content in both leaves and kernels. Candidate gene association analysis identified a 5/8‐bp insertion/deletion (InDel058) in the 5′ untranslated region (UTR) as the causal polymorphism in affecting ZmPORB2 expression and being highly associated with tocopherol content. We showed that higher expression of ZmPORB2 correlated with more chlorophyll metabolites in the leaf following pollination. RNA‐sequencing and metabolic analysis in near isogenic lines (NILs) support that ZmPORB2 participates in chlorophyll metabolism enabling the production of phytol, an important precursor of tocopherol. We also found that the tocopherol content in the kernel is mainly determined by the maternal genotype, a fact that was further confirmed by in vitro culture experiments. Finally, a PCR‐based marker based on Indel058 was developed in order to facilitate the high tocopherol (vitamin E) maize breeding.     

Mapping QTLs with epistatic effects and QTL×environment interactions for plant height using a doubled haploid population in cultivated wheat

Quantitative trait loci (QTLs) for plant height in wheat (Triticum aestivum L.) were studied using a set of 168 doubled haploid (DH) lines, which were derived from the cross Huapei 3/Yumai 57. A genetic linkage map was constructed using 283 SSR and 22 EST-SSR markers. The DH population and the parents were evaluated for wheat plant height in 2005 and 2006 in Tai’an and 2006 in Suzhou. QTL analyses were performed using the software of QTLNetwork version 2.0 based on the mixed linear model. Four additive QTLs and five pairs of epistatic effects were detected, which were distributed on chromosomes 3A, 4B, 4D, 5A, 6A, 7B, and 7D. Among them, three additive QTLs and three pairs of epistatic QTLs showed QTL×environment interactions (QEs). Two major QTLs, Qph4B and Qph4D, which accounted for 14.51% and 20.22% of the phenotypic variation, were located similar to the reported locations of the dwarfing genes Rht1 and Rht2, respectively. The Qph3A-2 with additive effect was not reported in previous linkage mapping studies. The total QTL effects detected for the plant height explained 85.04% of the phenotypic variation, with additive effects 46.07%, epistatic effects 19.89%, and QEs 19.09%. The results showed that both additive effects and epistatic effects were important genetic bases of wheat plant height, which were subjected to environmental modifications, and caused dramatic changes in phenotypic effects. The information obtained in this study will be useful for manipulating the QTLs for wheat plant height by molecular marker-assisted selection (MAS).     

Coconut malformation: An emerging disease caused by Fusarium proliferatum in southern Iran

Symptoms of vegetative malformation were observed on coconut palms (Cocos nucifera L.) in the Qeshm Island, Bandar Abbas and Minab, in Hormozgan province, southern Iran. The symptoms included misshapen and dwarfed leaves with shortened, thickened and tightened leaflets in wavy and zigzag form. The aim of this study was to identify the causal pathogen of coconut palm malformation and complete Koch's postulates for putative pathogen. Small pieces of surface‐disinfested malformed vegetative tissues of coconut palms were cultured on potato dextrose agar (PDA) medium. Fusarium isolates were permanently obtained from the symptomatic tissues. Sequence data from the internal transcribed spacer region (ITS1–5.8S‐ITS2) and translation elongation factor 1 alpha (TEF‐1α) gene were used for molecular identification of the isolates. BLAST search of the sequences showed 99%–100% identity to several Fusarium proliferatum strains in the GenBank, FUSARIUM‐ID and Fusarium MLST databases. A phylogeny inferred using individual sequence data from ITS region and TEF‐1α gene placed our isolates together with the other F. proliferatum sequences retrieved from the GenBank. Pathogenicity tests were carried out using one‐year‐old healthy coconut palm seedlings and conidial suspensions (10⁶ conidia/ml) of the F. proliferatum isolates. The first visible symptoms appeared on newly produced leaves of the inoculated seedlings during the 16th week after inoculation, wherease no disease symptoms were observed on the control plants until the end of the experiment. Reisolation from symptomatic tissues of the inoculated seedlings yielded isolates of F. proliferatum with morphological and molecular characteristics identical to those of the isolates used for inoculations. This is the first report of coconut palm malformation caused by F. proliferatum worldwide.     

Genotypic and phenotypic characterization of a large,diverse population of maize near‐isogenic lines

Genome‐wide association (GWA) studies can identify quantitative trait loci (QTL) putatively underlying traits of interest, and nested association mapping (NAM) can further assess allelic series. Near‐isogenic lines (NILs) can be used to characterize, dissect and validate QTL, but the development of NILs is costly. Previous studies have utilized limited numbers of NILs and introgression donors. We characterized a panel of 1270 maize NILs derived from crosses between 18 diverse inbred lines and the recurrent inbred parent B73, referred to as the nested NILs (nNILs). The nNILs were phenotyped for flowering time, height and resistance to three foliar diseases, and genotyped with genotyping‐by‐sequencing. Across traits, broad‐sense heritability (0.4–0.8) was relatively high. The 896 genotyped nNILs contain 2638 introgressions, which span the entire genome with substantial overlap within and among allele donors. GWA with the whole panel identified 29 QTL for height and disease resistance with allelic variation across donors. To date, this is the largest and most diverse publicly available panel of maize NILs to be phenotypically and genotypically characterized. The nNILs are a valuable resource for the maize community, providing an extensive collection of introgressions from the founders of the maize NAM population in a B73 background combined with data on six agronomically important traits and from genotyping‐by‐sequencing. We demonstrate that the nNILs can be used for QTL mapping and allelic testing. The majority of nNILs had four or fewer introgressions, and could readily be used for future fine mapping studies.     

Probing formation of cargo/importin‐α transport complexes in plant cells using a pathogen effector

Importin‐αs are essential adapter proteins that recruit cytoplasmic proteins destined for active nuclear import to the nuclear transport machinery. Cargo proteins interact with the importin‐α armadillo repeat domain via nuclear localization sequences (NLSs), short amino acids motifs enriched in Lys and Arg residues. Plant genomes typically encode several importin‐α paralogs that can have both specific and partially redundant functions. Although some cargos are preferentially imported by a distinct importin‐α it remains unknown how this specificity is generated and to what extent cargos compete for binding to nuclear transport receptors. Here we report that the effector protein HaRxL106 from the oomycete pathogen Hyaloperonospora arabidopsidis co‐opts the host cell's nuclear import machinery. We use HaRxL106 as a probe to determine redundant and specific functions of importin‐α paralogs from Arabidopsis thaliana. A crystal structure of the importin‐α3/MOS6 armadillo repeat domain suggests that five of the six Arabidopsis importin‐αs expressed in rosette leaves have an almost identical NLS‐binding site. Comparison of the importin‐α binding affinities of HaRxL106 and other cargos in vitro and in plant cells suggests that relatively small affinity differences in vitro affect the rate of transport complex formation in vivo. Our results suggest that cargo affinity for importin‐α, sequence variation at the importin‐α NLS‐binding sites and tissue‐specific expression levels of importin‐αs determine formation of cargo/importin‐α transport complexes in plant cells.     

A TLC bioautographic method for the detection of α‐ and β‐glucosidase inhibitors in plant extracts

Introduction – Bioautographic assays using TLC play an important role in the search for active compounds from plants. A TLC assay has previously been established for the detection of β‐glucosidase inhibitors but not for α‐glucosidase. Nonetheless, α‐glucosidase inhibition is an important target for therapeutic agents against of type 2 diabetes and anti‐viral infections. Objective – To develop a TLC bioautographic method to detect α‐ and β‐glucosidase inhibitors in plant extracts. Methodology – The enzymes α‐ and β‐d ‐glucosidase were dissolved in sodium acetate buffer. After migration of the samples, the TLC plate was sprayed with enzyme solution and incubated at room temperature for 60 min in the case of α‐d ‐glucosidase, and 37°C for 20 min in the case of β‐d ‐glucosidase. For detection of the active enzyme, solutions of 2‐naphthyl‐α‐D‐glucopyranoside or 2‐naphthyl‐β‐D‐glucopyranoside and Fast Blue Salt were mixed at a ratio of 1 : 1 (for α‐d ‐glucosidase) or 1 : 4 (for β‐d ‐glucosidase) and sprayed onto the plate to give a purple background colouration after 2–5 min. Results – Enzyme inhibitors were visualised as white spots on the TLC plates. Conduritol B epoxide inhibited α‐d ‐glucosidase and β‐d ‐glucosidase down to 0.1 µg. Methanol extracts of Tussilago farfara and Urtica dioica after migration on TLC gave enzymatic inhibition when applied in amounts of 100 µg for α‐glucosidase and 50 µg for β‐glucosidase. Conclusion – The screening test was able to detect inhibition of α‐ and β‐glucosidases by pure reference substances and by compounds present in complex matrices, such as plant extracts. Copyright © 2009 John Wiley & Sons, Ltd.     

The truncated NLR protein TIR‐NBS13 is a MOS6/IMPORTIN‐α3 interaction partner required for plant immunity

Importin‐α proteins mediate the translocation of nuclear localization signal (NLS)‐containing proteins from the cytoplasm into the nucleus through nuclear pore complexes (NPCs). Genetically, Arabidopsis IMPORTIN‐α3/MOS6 (MODIFIER OF SNC1, 6) is required for basal plant immunity and constitutive disease resistance activated in the autoimmune mutant snc1 (suppressor of npr1‐1, constitutive 1), suggesting that MOS6 plays a role in the nuclear import of proteins involved in plant defense signaling. Here, we sought to identify and characterize defense‐regulatory cargo proteins and interaction partners of MOS6. We conducted both in silico database analyses and affinity purification of functional epitope‐tagged MOS6 from pathogen‐challenged stable transgenic plants coupled with mass spectrometry. We show that among the 13 candidate MOS6 interactors we selected for further functional characterization, the TIR‐NBS‐type protein TN13 is required for resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 lacking the type‐III effector proteins AvrPto and AvrPtoB. When expressed transiently in N. benthamiana leaves, TN13 co‐immunoprecipitates with MOS6, but not with its closest homolog IMPORTIN‐α6, and localizes to the endoplasmic reticulum (ER), consistent with a predicted N‐terminal transmembrane domain in TN13. Our work uncovered the truncated NLR protein TN13 as a component of plant innate immunity that selectively binds to MOS6/IMPORTIN‐α3 in planta. We speculate that the release of TN13 from the ER membrane in response to pathogen stimulus, and its subsequent nuclear translocation, is important for plant defense signal transduction.     

A novel Fe(II)/α‐ketoglutarate‐dependent dioxygenase from Burkholderia ambifaria has β‐hydroxylating activity of N‐succinyl l‐leucine

An Fe(II)/α‐ketoglutarate‐dependent dioxygenase, SadA, was obtained from Burkholderia ambifaria AMMD and heterologously expressed in Escherichia coli. Purified recombinant SadA had catalytic activity towards several N‐substituted l‐amino acids, which was especially strong with N‐succinyl l‐leucine. With the NMR and LC‐MS analysis, SadA converted N‐succinyl l‐leucine into N‐succinyl l‐threo‐β‐hydroxyleucine with >99% diastereoselectivity. SadA is the first enzyme catalysing β‐hydroxylation of aliphatic amino acid‐related substances and a potent biocatalyst for the preparation of optically active β‐hydroxy amino acids.     

The genetic and functional analysis of flavor in commercial tomato: the FLORAL4 gene underlies a QTL for floral aroma volatiles in tomato fruit

Tomato (Solanum lycopersicum L.) has become a popular model for genetic studies of fruit flavor in the last two decades. In this article we present a study of tomato fruit flavor, including an analysis of the genetic, metabolic and sensorial variation of a collection of contemporary commercial glasshouse tomato cultivars, followed by a validation of the associations found by quantitative trait locus (QTL) analysis of representative biparental segregating populations. This led to the identification of the major sensorial and chemical components determining fruit flavor variation and detection of the underlying QTLs. The high representation of QTL haplotypes in the breeders’ germplasm suggests that there is great potential for applying these QTLs in current breeding programs aimed at improving tomato flavor. A QTL on chromosome 4 was found to affect the levels of the phenylalanine‐derived volatiles (PHEVs) 2‐phenylethanol, phenylacetaldehyde and 1‐nitro‐2‐phenylethane. Fruits of near‐isogenic lines contrasting for this locus and in the composition of PHEVs significantly differed in the perception of fruity and rose‐hip‐like aroma. The PHEV locus was fine mapped, which allowed for the identification of FLORAL4 as a candidate gene for PHEV regulation. Using a gene‐editing‐based (CRISPR‐CAS9) reverse‐genetics approach, FLORAL4 was demonstrated to be the key factor in this QTL affecting PHEV accumulation in tomato fruit.     

Longer lifespan in male mice treated with a weakly estrogenic agonist,an antioxidant,an α‐glucosidase inhibitor or a Nrf2‐inducer

The National Institute on Aging Interventions Testing Program (ITP) evaluates agents hypothesized to increase healthy lifespan in genetically heterogeneous mice. Each compound is tested in parallel at three sites, and all results are published. We report the effects of lifelong treatment of mice with four agents not previously tested: Protandim, fish oil, ursodeoxycholic acid (UDCA) and metformin – the latter with and without rapamycin, and two drugs previously examined: 17‐α‐estradiol and nordihydroguaiaretic acid (NDGA), at doses greater and less than used previously. 17‐α‐estradiol at a threefold higher dose robustly extended both median and maximal lifespan, but still only in males. The male‐specific extension of median lifespan by NDGA was replicated at the original dose, and using doses threefold lower and higher. The effects of NDGA were dose dependent and male specific but without an effect on maximal lifespan. Protandim, a mixture of botanical extracts that activate Nrf2, extended median lifespan in males only. Metformin alone, at a dose of 0.1% in the diet, did not significantly extend lifespan. Metformin (0.1%) combined with rapamycin (14 ppm) robustly extended lifespan, suggestive of an added benefit, based on historical comparison with earlier studies of rapamycin given alone. The α‐glucosidase inhibitor, acarbose, at a concentration previously tested (1000 ppm), significantly increased median longevity in males and 90th percentile lifespan in both sexes, even when treatment was started at 16 months. Neither fish oil nor UDCA extended lifespan. These results underscore the reproducibility of ITP longevity studies and illustrate the importance of identifying optimal doses in lifespan studies.