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Protein lipid modification of cysteine residues, referred to as S-palmitoylation or S-acylation, is an important secondary and reversible modification that regulates membrane association, trafficking, and function of target proteins. This enzymatic reaction is mediated by protein S-acyl transferases (PATs). Here, the phylogeny, genomic organization, protein topology, expression, and localization pattern of the 24 PAT family members from Arabidopsis (Arabidopsis thaliana) is described. Most PATs are expressed at ubiquitous levels and tissues throughout the development, while few genes are expressed especially during flower development preferentially in pollen and stamen. The proteins display large sequence and structural variations but exhibit a common protein topology that is preserved in PATs from various organisms. Arabidopsis PAT proteins display a complex targeting pattern and were detected at the endoplasmic reticulum, Golgi, endosomal compartments, and the vacuolar membrane. However, most proteins were targeted to the plasma membrane. This large concentration of plant PAT activity to the plasma membrane suggests that the plant cellular S-acylation machinery is functionally different compared with that of yeast (Saccharomyces cerevisiae) and mammalians.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Fruit firmness in tomato (Solanum lycopersicum) is determined by a number of factors including cell wall structure, turgor, and cuticle properties. Firmness is a complex polygenic trait involving the coregulation of many genes and has proved especially challenging to unravel. In this study, a quantitative trait locus (QTL) for fruit firmness was mapped to tomato chromosome 2 using the Zamir Solanum pennellii interspecific introgression lines (ILs) and fine-mapped in a population consisting of 7,500 F2 and F3 lines from IL 2-3 and IL 2-4. This firmness QTL contained five distinct subpeaks, Fir(s.p.)QTL2.1 to Fir(s.p.)QTL2.5, and an effect on a distal region of IL 2-4 that was nonoverlapping with IL 2-3. All these effects were located within an 8.6-Mb region. Using genetic markers, each subpeak within this combinatorial locus was mapped to a physical location within the genome, and an ethylene response factor (ERF) underlying Fir(s.p.)QTL2.2 and a region containing three pectin methylesterase (PME) genes underlying Fir(s.p.)QTL2.5 were nominated as QTL candidate genes. Statistical models used to explain the observed variability between lines indicated that these candidates and the nonoverlapping portion of IL 2-4 were sufficient to account for the majority of the fruit firmness effects. Quantitative reverse transcription-polymerase chain reaction was used to quantify the expression of each candidate gene. ERF showed increased expression associated with soft fruit texture in the mapping population. In contrast, PME expression was tightly linked with firm fruit texture. Analysis of a range of recombinant lines revealed evidence for an epistatic interaction that was associated with this combinatorial locus.

The inheritance of flower color in pea (Pisum sativum) has been studied for more than a century, but many of the genes corresponding to these classical loci remain unidentified. Anthocyanins are the main flower pigments in pea. These are generated via the flavonoid biosynthetic pathway, which has been studied in detail and is well conserved among higher plants. A previous proposal that the Clariroseus (B) gene of pea controls hydroxylation at the 5′ position of the B ring of flavonoid precursors of the anthocyanins suggested to us that the gene encoding flavonoid 3′,5′-hydroxylase (F3′5′H), the enzyme that hydroxylates the 5′ position of the B ring, was a good candidate for B. In order to test this hypothesis, we examined mutants generated by fast neutron bombardment. We found allelic pink-flowered b mutant lines that carried a variety of lesions in an F3′5′H gene, including complete gene deletions. The b mutants lacked glycosylated delphinidin and petunidin, the major pigments present in the progenitor purple-flowered wild-type pea. These results, combined with the finding that the F3′5′H gene cosegregates with b in a genetic mapping population, strongly support our hypothesis that the B gene of pea corresponds to a F3′5′H gene. The molecular characterization of genes involved in pigmentation in pea provides valuable anchor markers for comparative legume genomics and will help to identify differences in anthocyanin biosynthesis that lead to variation in pigmentation among legume species.

The pentatricopeptide repeat (PPR) gene family represents one of the largest gene families in higher plants. Accumulating data suggest that PPR proteins play a central and broad role in modulating the expression of organellar genes in plants. Here we report a rice (Oryza sativa) mutant named young seedling albino (ysa) derived from the rice thermo/photoperiod-sensitive genic male-sterile line Pei'ai64S, which is a leading male-sterile line for commercial two-line hybrid rice production. The ysa mutant develops albino leaves before the three-leaf stage, but the mutant gradually turns green and recovers to normal green at the six-leaf stage. Further investigation showed that the change in leaf color in ysa mutant is associated with changes in chlorophyll content and chloroplast development. Map-based cloning revealed that YSA encodes a PPR protein with 16 tandem PPR motifs. YSA is highly expressed in young leaves and stems, and its expression level is regulated by light. We showed that the ysa mutation has no apparent negative effects on several important agronomic traits, such as fertility, stigma extrusion rate, selfed seed-setting rate, hybrid seed-setting rate, and yield heterosis under normal growth conditions. We further demonstrated that ysa can be used as an early marker for efficient identification and elimination of false hybrids in commercial hybrid rice production, resulting in yield increases by up to approximately 537 kg ha−1.

Membrane transporters play a central role in many cellular processes that rely on the movement of ions and organic molecules between the environment and the cell, and between cellular compartments. Transporters have been well characterized in plants and green algae, but little is known about transporters or their evolutionary histories in the red algae. Here we examined 482 expressed sequence tag contigs that encode putative membrane transporters in the economically important red seaweed Porphyra (Bangiophyceae, Rhodophyta). These contigs are part of a comprehensive transcriptome dataset from Porphyra umbilicalis and Porphyra purpurea. Using phylogenomics, we identified 30 trees that support the expected monophyly of red and green algae/plants (i.e. the Plantae hypothesis) and 19 expressed sequence tag contigs that show evidence of endosymbiotic/horizontal gene transfer involving stramenopiles. The majority (77%) of analyzed contigs encode transporters with unresolved phylogenies, demonstrating the difficulty in resolving the evolutionary history of genes. We observed molecular features of many sodium-coupled transport systems in marine algae, and the potential for coregulation of Porphyra transporter genes that are associated with fatty acid biosynthesis and intracellular lipid trafficking. Although both the tissue-specific and subcellular locations of the encoded proteins require further investigation, our study provides red algal gene candidates associated with transport functions and novel insights into the biology and evolution of these transporters.

Improvement of strawberry (Fragaria × ananassa) fruit flavor is an important goal in breeding programs. To investigate genetic factors controlling this complex trait, a strawberry mapping population derived from genotype ‘1392’, selected for its superior flavor, and ‘232’ was profiled for volatile compounds over 4 years by headspace solid phase microextraction coupled to gas chromatography and mass spectrometry. More than 300 volatile compounds were detected, of which 87 were identified by comparison of mass spectrum and retention time to those of pure standards. Parental line ‘1392’ displayed higher volatile levels than ‘232’, and these and many other compounds with similar levels in both parents segregated in the progeny. Cluster analysis grouped the volatiles into distinct chemically related families and revealed a complex metabolic network underlying volatile production in strawberry fruit. Quantitative trait loci (QTL) detection was carried out over 3 years based on a double pseudo-testcross strategy. Seventy QTLs covering 48 different volatiles were detected, with several of them being stable over time and mapped as major QTLs. Loci controlling γ-decalactone and mesifurane content were mapped as qualitative traits. Using a candidate gene approach we have assigned genes that are likely responsible for several of the QTLs. As a proof of concept we show that one homoeolog of the O-methyltransferase gene (FaOMT) is the locus responsible for the natural variation of mesifurane content. Sequence analysis identified 30 bp in the promoter of this FaOMT homoeolog containing putative binding sites for basic/helix-loop-helix, MYB, and BZIP transcription factors. This polymorphism fully cosegregates with both the presence of mesifurane and the high expression of FaOMT during ripening.

MicroRNAs (miRNAs) and small interfering RNAs are important regulators of plant development and seed formation, yet their population and abundance in the oil crop Brassica napus are still not well understood, especially at different developmental stages and among cultivars with varied seed oil contents. Here, we systematically analyzed the small RNA expression profiles of Brassica napus seeds at early embryonic developmental stages in high-oil-content and low-oil-content B. napus cultivars, both cultured in two environments. A total of 50 conserved miRNAs and 9 new miRNAs were identified, together with some new miRNA targets. Expression analysis revealed some miRNAs with varied expression levels in different seed oil content cultivars or at different embryonic developmental stages. A large number of 23-nucleotide small RNAs with specific nucleotide composition preferences were also identified, which may present new classes of functional small RNAs.

The maize (Zea mays) kernel plays a critical role in feeding humans and livestock around the world and in a wide array of industrial applications. An understanding of the regulation of kernel starch, protein, and oil is needed in order to manipulate composition to meet future needs. We conducted joint-linkage quantitative trait locus mapping and genome-wide association studies (GWAS) for kernel starch, protein, and oil in the maize nested association mapping population, composed of 25 recombinant inbred line families derived from diverse inbred lines. Joint-linkage mapping revealed that the genetic architecture of kernel composition traits is controlled by 21–26 quantitative trait loci. Numerous GWAS associations were detected, including several oil and starch associations in acyl-CoA:diacylglycerol acyltransferase1-2, a gene that regulates oil composition and quantity. Results from nested association mapping were verified in a 282 inbred association panel using both GWAS and candidate gene association approaches. We identified many beneficial alleles that will be useful for improving kernel starch, protein, and oil content.

The LOSS OF APOMEIOSIS (LOA) locus is one of two dominant loci known to control apomixis in the eudicot Hieracium praealtum. LOA stimulates the differentiation of somatic aposporous initial cells after the initiation of meiosis in ovules. Aposporous initial cells undergo nuclear proliferation close to sexual megaspores, forming unreduced aposporous embryo sacs, and the sexual program ceases. LOA-linked genetic markers were used to isolate 1.2 Mb of LOA-associated DNAs from H. praealtum. Physical mapping defined the genomic region essential for LOA function between two markers, flanking 400 kb of identified sequence and central unknown sequences. Cytogenetic and sequence analyses revealed that the LOA locus is located on a single chromosome near the tip of the long arm and surrounded by extensive, abundant complex repeat and transposon sequences. Chromosomal features and LOA-linked markers are conserved in aposporous Hieracium caespitosum and Hieracium piloselloides but absent in sexual Hieracium pilosella. Their absence in apomictic Hieracium aurantiacum suggests that meiotic avoidance may have evolved independently in aposporous subgenus Pilosella species. The structure of the hemizygous chromosomal region containing the LOA locus in the three Hieracium subgenus Pilosella species resembles that of the hemizygous apospory-specific genomic regions in monocot Pennisetum squamulatum and Cenchrus ciliaris. Analyses of partial DNA sequences at these loci show no obvious conservation, indicating that they are unlikely to share a common ancestral origin. This suggests convergent evolution of repeat-rich hemizygous chromosomal regions containing apospory loci in these monocot and eudicot species, which may be required for the function and maintenance of the trait.

The phosphatidyl ethanolamine-binding protein (PEBP) gene family is present in all eukaryote kingdoms, with three subfamilies identified in angiosperms (FLOWERING LOCUS T [FT], MOTHER OF FT AND TFL1 [MFT], and TERMINAL FLOWER1 [TFL1] like). In angiosperms, PEBP genes have been shown to function both as promoters and suppressors of flowering and to control plant architecture. In this study, we focus on previously uncharacterized PEBP genes from gymnosperms. Extensive database searches suggest that gymnosperms possess only two types of PEBP genes, MFT-like and a group that occupies an intermediate phylogenetic position between the FT-like and TFL1-like (FT/TFL1-like). Overexpression of Picea abies PEBP genes in Arabidopsis (Arabidopsis thaliana) suggests that the FT/TFL1-like genes (PaFTL1 and PaFTL2) code for proteins with a TFL1-like function. However, PaFTL1 and PaFTL2 also show highly divergent expression patterns. While the expression of PaFTL2 is correlated with annual growth rhythm and mainly confined to needles and vegetative and reproductive buds, the expression of PaFTL1 is largely restricted to microsporophylls of male cones. The P. abies MFT-like genes (PaMFT1 and PaMFT2) show a predominant expression during embryo development, a pattern that is also found for many MFT-like genes from angiosperms. P. abies PEBP gene expression is primarily detected in tissues undergoing physiological changes related to growth arrest and dormancy. A first duplication event resulting in two families of plant PEBP genes (MFT-like and FT/TFL1-like) seems to coincide with the evolution of seed plants, in which independent control of bud and seed dormancy was required, and the second duplication resulting in the FT-like and TFL1-like clades probably coincided with the evolution of angiosperms.

It has been well established that DNA cytosine methylation plays essential regulatory roles in imprinting gene expression in endosperm, and hence normal embryonic development, in the model plant Arabidopsis (Arabidopsis thaliana). Nonetheless, the developmental role of this epigenetic marker in cereal crops remains largely unexplored. Here, we report for sorghum (Sorghum bicolor) differences in relative cytosine methylation levels and patterns at 5′-CCGG sites in seven tissues (endosperm, embryo, leaf, root, young inflorescence, anther, and ovary), and characterize a set of tissue-specific differentially methylated regions (TDMRs). We found that the most enriched TDMRs in sorghum are specific for the endosperm and are generated concomitantly but imbalanced by decrease versus increase in cytosine methylation at multiple 5′-CCGG sites across the genome. This leads to more extensive demethylation in the endosperm than in other tissues, where TDMRs are mainly tissue nonspecific rather than specific to a particular tissue. Accordingly, relative to endosperm, the other six tissues showed grossly similar levels though distinct patterns of cytosine methylation, presumably as a result of a similar extent of concomitant decrease versus increase in cytosine methylation that occurred at variable genomic loci. All four tested TDMRs were validated by bisulfite genomic sequencing. Diverse sequences were found to underlie the TDMRs, including those encoding various known-function or predicted proteins, transposable elements, and those bearing homology to putative imprinted genes in maize (Zea mays). We further found that the expression pattern of at least some genic TDMRs was correlated with its tissue-specific methylation state, implicating a developmental role of DNA methylation in regulating tissue-specific or -preferential gene expression in sorghum.

Grass plants develop unique floral patterns that determine grain production. However, the molecular mechanism underlying the specification of floral organ identities and meristem determinacy, including the interaction among floral homeotic genes, remains largely unknown in grasses. Here, we report the interactions of rice (Oryza sativa) floral homeotic genes, OsMADS3 (a C-class gene), OsMADS13 (a D-class gene), and DROOPING LEAF (DL), in specifying floral organ identities and floral meristem determinacy. The interaction among these genes was revealed through the analysis of double mutants. osmads13-3 osmads3-4 displayed a loss of floral meristem determinacy and generated abundant carpelloid structures containing severe defective ovules in the flower center, which were not detectable in the single mutant. In addition, in situ hybridization and yeast two-hybrid analyses revealed that OsMADS13 and OsMADS3 did not regulate each other’s transcription or interact at the protein level. This indicates that OsMADS3 plays a synergistic role with OsMADS13 in both ovule development and floral meristem termination. Strikingly, osmads3-4 dl-sup6 displayed a severe loss of floral meristem determinacy and produced supernumerary whorls of lodicule-like organs at the forth whorl, suggesting that OsMADS3 and DL synergistically terminate the floral meristem. Furthermore, the defects of osmads13-3 dl-sup6 flowers appeared identical to those of dl-sup6, and the OsMADS13 expression was undetectable in dl-sup6 flowers. These observations suggest that DL and OsMADS13 may function in the same pathway specifying the identity of carpel/ovule and floral meristem. Collectively, we propose a model to illustrate the role of OsMADS3, DL, and OsMADS13 in the specification of flower organ identity and meristem determinacy in rice.

Phenotypic diversity within cultivated tomato (Solanum lycopersicum) is particularly evident for fruit shape and size. Four genes that control tomato fruit shape have been cloned. SUN and OVATE control elongated shape whereas FASCIATED (FAS) and LOCULE NUMBER (LC) control fruit locule number and flat shape. We investigated the distribution of the fruit shape alleles in the tomato germplasm and evaluated their contribution to morphology in a diverse collection of 368 predominantly tomato and tomato var. cerasiforme accessions. Fruits were visually classified into eight shape categories that were supported by objective measurements obtained from image analysis using the Tomato Analyzer software. The allele distribution of SUN, OVATE, LC, and FAS in all accessions was strongly associated with fruit shape classification. We also genotyped 116 representative accessions with additional 25 markers distributed evenly across the genome. Through a model-based clustering we demonstrated that shape categories, germplasm classes, and the shape genes were nonrandomly distributed among five genetic clusters (P < 0.001), implying that selection for fruit shape genes was critical to subpopulation differentiation within cultivated tomato. Our data suggested that the LC, FAS, and SUN mutations arose in the same ancestral population while the OVATE mutation arose in a separate lineage. Furthermore, LC, OVATE, and FAS mutations may have arisen prior to domestication or early during the selection of cultivated tomato whereas the SUN mutation appeared to be a postdomestication event arising in Europe.

The three most important agronomic traits of rice (Oryza sativa), yield, plant height, and flowering time, are controlled by many quantitative trait loci (QTLs). In this study, a newly identified QTL, DTH8 (QTL for days to heading on chromosome 8), was found to regulate these three traits in rice. Map-based cloning reveals that DTH8 encodes a putative HAP3 subunit of the CCAAT-box-binding transcription factor and the complementary experiment increased significantly days to heading, plant height, and number of grains per panicle in CSSL61 (a chromosome segment substitution line that carries the nonfunctional DTH8 allele) with the Asominori functional DTH8 allele under long-day conditions. DTH8 is expressed in most tissues and its protein is localized to the nucleus exclusively. The quantitative real-time PCR assay revealed that DTH8 could down-regulate the transcriptions of Ehd1 (for Early heading date1) and Hd3a (for Heading date3a; a rice ortholog of FLOWERING LOCUS T) under long-day conditions. Ehd1 and Hd3a can also be down-regulated by the photoperiodic flowering genes Ghd7 and Hd1 (a rice ortholog of CONSTANS). Meanwhile, the transcription of DTH8 has been proved to be independent of Ghd7 and Hd1, and the natural mutation of this gene caused weak photoperiod sensitivity and shorter plant height. Taken together, these data indicate that DTH8 probably plays an important role in the signal network of photoperiodic flowering as a novel suppressor as well as in the regulation of plant height and yield potential.

Transposons are major components of all eukaryotic genomes. Although traditionally regarded as causes of detrimental mutations, recent evidence suggests that transposons may play a role in host gene diversification and evolution. For example, host gene transduction by retroelements has been suggested to be both common and to have the potential to create new chimeric genes by the shuffling of existing sequences. We have previously shown that the maize (Zea mays subsp. mays) retrotransposon Bs1 has transduced sequences from three different host genes. Here, we provide evidence that these transduction events led to the generation of a chimeric new gene that is both transcribed and translated. Expression of Bs1 is tightly controlled and occurs during a narrow developmental window in early ear development. Although all Bs1-associated transduction events took place before Zea speciation, a full uninterrupted open reading frame encoding the BS1 protein may have arisen in domesticated maize or in the diverse populations of its progenitor Z. mays subsp. parviglumis. We discuss potential functions based on domain conservation and evidence for functional constraints between the transduced sequences and their host gene counterparts.

Knowledge of phosphorylation events and their regulation is crucial to understand the functional biology of plants. Here, we report a large-scale phosphoproteome analysis in the model monocot rice (Oryza sativa japonica ‘Nipponbare’), an economically important crop. Using unfractionated whole-cell lysates of rice cells, we identified 6,919 phosphopeptides from 3,393 proteins. To investigate the conservation of phosphoproteomes between plant species, we developed a novel phosphorylation-site evaluation method and performed a comparative analysis of rice and Arabidopsis (Arabidopsis thaliana). The ratio of tyrosine phosphorylation in the phosphoresidues of rice was equivalent to those in Arabidopsis and human. Furthermore, despite the phylogenetic distance and the use of different cell types, more than 50% of the phosphoproteins identified in rice and Arabidopsis, which possessed ortholog(s), had an orthologous phosphoprotein in the other species. Moreover, nearly half of the phosphorylated orthologous pairs were phosphorylated at equivalent sites. Further comparative analyses against the Medicago phosphoproteome also showed similar results. These data provide direct evidence for conserved regulatory mechanisms based on phosphorylation in plants. We also assessed the phosphorylation sites on nucleotide-binding leucine-rich repeat proteins and identified novel conserved phosphorylation sites that may regulate this class of proteins.

Carotenoid pigments in fruits are indicative of the ripening process and potential nutritional value. Papaya (Carica papaya) fruit flesh color is caused by the accumulation of lycopene or beta-carotenoids in chromoplasts. It is a distinct feature affecting nutritional composition, fruit quality, shelf life, and consumer preference. To uncover the molecular basis of papaya flesh color, we took map-based cloning and candidate gene approaches using integrated genetic and physical maps. A DNA marker tightly linked to flesh color colocalized on a contig of the physical map with a cDNA probe of the tomato (Solanum lycopersicum) chromoplast-specific lycopene beta-cyclase, CYC-b. Candidate gene sequences were obtained from amplified fragments and verified by sequencing two bacterial artificial chromosomes containing the two alleles. Sequence comparison revealed a 2-bp insertion in the coding region of the recessive red flesh allele resulting in a frame-shift mutation and a premature stop codon. A color complementation test in bacteria confirmed that the papaya CpCYC-b is the gene controlling fruit flesh color. Sequence analysis of wild and cultivated papaya accessions showed the presence of this frame-shift mutation in all red flesh accessions examined. Evaluation of DNA markers near CpCYC-b revealed a recombination hot spot, showing that CpCYC-b is located in a gene-rich region with a recombination rate at 3.7 kb per centimorgan, more than 100-fold higher than the genome average at 400 kb per centimorgan. Conserved microsynteny of the CpCYC-b region is indicated by colinearity of two to four genes between papaya, Arabidopsis (Arabidopsis thaliana), grape (Vitis vinifera), and tomato. Our results enhanced our understanding of papaya flesh color inheritance and generated new tools for papaya improvement.

Phosphate homeostasis was studied in a monocotyledonous model plant through the characterization of the PHO1 gene family in rice (Oryza sativa). Bioinformatics and phylogenetic analysis showed that the rice genome has three PHO1 homologs, which cluster with the Arabidopsis (Arabidopsis thaliana) AtPHO1 and AtPHO1;H1, the only two genes known to be involved in root-to-shoot transfer of phosphate. In contrast to the Arabidopsis PHO1 gene family, all three rice PHO1 genes have a cis-natural antisense transcript located at the 5 ′ end of the genes. Strand-specific quantitative reverse transcription-PCR analyses revealed distinct patterns of expression for sense and antisense transcripts for all three genes, both at the level of tissue expression and in response to nutrient stress. The most abundantly expressed gene was OsPHO1;2 in the roots, for both sense and antisense transcripts. However, while the OsPHO1;2 sense transcript was relatively stable under various nutrient deficiencies, the antisense transcript was highly induced by inorganic phosphate (Pi) deficiency. Characterization of Ospho1;1 and Ospho1;2 insertion mutants revealed that only Ospho1;2 mutants had defects in Pi homeostasis, namely strong reduction in Pi transfer from root to shoot, which was accompanied by low-shoot and high-root Pi. Our data identify OsPHO1;2 as playing a key role in the transfer of Pi from roots to shoots in rice, and indicate that this gene could be regulated by its cis-natural antisense transcripts. Furthermore, phylogenetic analysis of PHO1 homologs in monocotyledons and dicotyledons revealed the emergence of a distinct clade of PHO1 genes in dicotyledons, which include members having roles other than long-distance Pi transport.

We analyzed 6,749 lines tagged by the gene trap vector pGA2707. This resulted in the isolation of 3,793 genomic sequences flanking the T-DNA. Among the insertions, 1,846 T-DNAs were integrated into genic regions, and 1,864 were located in intergenic regions. Frequencies were also higher at the beginning and end of the coding regions and upstream near the ATG start codon. The overall GC content at the insertion sites was close to that measured from the entire rice (Oryza sativa) genome. Functional classification of these 1,846 tagged genes showed a distribution similar to that observed for all the genes in the rice chromosomes. This indicates that T-DNA insertion is not biased toward a particular class of genes. There were 764, 327, and 346 T-DNA insertions in chromosomes 1, 4 and 10, respectively. Insertions were not evenly distributed; frequencies were higher at the ends of the chromosomes and lower near the centromere. At certain sites, the frequency was higher than in the surrounding regions. This sequence database will be valuable in identifying knockout mutants for elucidating gene function in rice. This resource is available to the scientific community at http://www.postech.ac.kr/life/pfg/risd.

To investigate the relationship between T-DNA integration and double-stranded break (DSB) repair in Arabidopsis, we studied 67 T-DNA/plant DNA junctions and 13 T-DNA/T-DNA junctions derived from transgenic plants. Three different types of T-DNA-associated joining could be distinguished. A minority of T-DNA/plant DNA junctions were joined by a simple ligation-like mechanism, resulting in a junction without microhomology or filler DNA insertions. For about one-half of all analyzed junctions, joining of the two ends occurred without insertion of filler sequences. For these junctions, microhomology was strikingly combined with deletions of the T-DNA ends. For the remaining plant DNA/T-DNA junctions, up to 51-bp-long filler sequences were present between plant DNA and T-DNA contiguous sequences. These filler segments are built from several short sequence motifs, identical to sequence blocks that occur in the T-DNA ends and/or the plant DNA close to the integration site. Mutual microhomologies among the sequence motifs that constitute a filler segment were frequently observed. When T-DNA integration and DSB repair were compared, the most conspicuous difference was the frequency and the structural organization of the filler insertions. In Arabidopsis, no filler insertions were found at DSB repair junctions. In maize (Zea mays) and tobacco (Nicotiana tabacum), DSB repair-associated filler was normally composed of simple, uninterrupted sequence blocks. Thus, although DSB repair and T-DNA integration are probably closely related, both mechanisms have some exclusive and specific characteristics.

We have identified an R2R3-type MYB factor, GMYB10, from Gerbera hybrida (Asteraceae) that shares high sequence homology to and is phylogenetically grouped together with the previously characterized regulators of anthocyanin pigmentation in petunia (Petunia hybrida) and Arabidopsis. GMYB10 is able to induce anthocyanin pigmentation in transgenic tobacco (Nicotiana tabacum), especially in vegetative parts and anthers. In G. hybrida, GMYB10 is involved in activation of anthocyanin biosynthesis in leaves, floral stems, and flowers. In flowers, its expression is restricted to petal epidermal cell layers in correlation with the anthocyanin accumulation pattern. We have shown, using yeast (Saccharomyces cerevisiae) two-hybrid assay, that GMYB10 interacts with the previously isolated bHLH factor GMYC1. Particle bombardment analysis was used to show that GMYB10 is required for activation of a late anthocyanin biosynthetic gene promoter, PGDFR2. cis-Analysis of the target PGDFR2 revealed a sequence element with a key role in activation by GMYB10/GMYC1. This element shares high homology with the anthocyanin regulatory elements characterized in maize (Zea mays) anthocyanin promoters, suggesting that the regulatory mechanisms involved in activation of anthocyanin biosynthesis have been conserved for over 125 million years not only at the level of transcriptional regulators but also at the level of the biosynthetic gene promoters.

The use of a nonlethal selection scheme, most often using the aadA gene that confers resistance to spectinomycin and streptomycin, has been considered critical for recovery of plastid transformation events. In this study, the plastid-lethal markers, glyphosate or phosphinothricin herbicides, were used to develop a selection scheme for plastids that circumvents the need for integration of an antibiotic resistance marker. The effect of selective agents on tobacco (Nicotiana tabacum) mesophyll chloroplasts was first examined by transmission electron microscopy. We found that at concentrations typically used for selection of nuclear transformants, herbicides caused rapid disintegration of plastid membranes, whereas antibiotics had no apparent effect. To overcome this apparent herbicide lethality to plastids, a “transformation segregation” scheme was developed that used two independent transformation vectors for a cotransformation approach and two different selective agents in a phased selection scheme. One transformation vector carried an antibiotic resistance (aadA) marker used for early nonlethal selection, and the other transformation vector carried the herbicide (CP4 or bar) resistance marker for use in a subsequent lethal selection phase. Because the two markers were carried on separate plasmids and were targeted to different locations on the plastid genome, we reasoned that segregation of the two markers in some transplastomic lines could occur. We report here a plastid cotransformation frequency of 50% to 64%, with a high frequency (20%) of these giving rise to transformation segregants containing exclusively the initially nonselected herbicide resistance marker. Our studies indicate a high degree of persistence of unselected transforming DNA, providing useful insights into plastid chromosome dynamics.

Ubiquitin-mediated proteolysis by the proteasome is a critical regulatory mechanism controlling many biological processes. In particular, SKP1, cullin/CDC53, F-box protein (SCF) complexes play important roles in selecting substrates for proteolysis by facilitating the ligation of ubiquitin to specific proteins. In plants, SCF complexes have been found to regulate auxin responses and jasmonate signaling and may be involved in several other processes, such as flower development, circadian clock, and gibberellin signaling. Although 21 Skp1-related genes, called Arabidopsis-SKP1-like (ASK), have been uncovered in the Arabidopsis genome, ASK1 is the only gene that has been analyzed genetically. As a first step toward understanding their functions, we tested for expression of 20 ASK genes using reverse transcription-polymerase chain reaction experiments. Also, we examined the expression patterns of 11 ASK genes by in situ hybridizations. The ASK genes exhibit a spectrum of expression levels and patterns, with a large subset showing expression in the flower and/or fruit. In addition, the ASK genes that have similar sequences tend to have similar expression patterns. On the basis of the expression results, we selectively suppressed the expression of a few ASK genes using RNA interference. Compared with the ask1 mutant, the strong ASK1 RNA interference (RNAi) line exhibited similar or enhanced phenotypes in both vegetative and floral development, whereas ASK11 RNAi plants had normal vegetative growth but mild defects in flower development. The diverse expression patterns and distinct defects observed in RNAi plants suggest that the ASK gene family may collectively perform a range of functions and may regulate different developmental and physiological processes.

Dormancy is a state of metabolic arrest that facilitates the survival of organisms during environmental conditions incompatible with their regular course of life. Many organisms have deep dormant stages to promote an extended life span (increased longevity). In contrast, plants have seed dormancy and seed longevity described as two traits. Seed dormancy is defined as a temporary failure of a viable seed to germinate in conditions that favor germination, whereas seed longevity is defined as seed viability after dry storage (storability). In plants, the association of seed longevity with seed dormancy has not been studied in detail. This is surprising given the ecological, agronomical, and economic importance of seed longevity. We studied seed longevity to reveal its genetic regulators and its association with seed dormancy in Arabidopsis (Arabidopsis thaliana). Integrated quantitative trait locus analyses for seed longevity, in six recombinant inbred line populations, revealed five loci: Germination Ability After Storage1 (GAAS1) to GAAS5. GAAS loci colocated with seed dormancy loci, Delay Of Germination (DOG), earlier identified in the same six recombinant inbred line populations. Both GAAS loci and their colocation with DOG loci were validated by near isogenic lines. A negative correlation was observed, deep seed dormancy correlating with low seed longevity and vice versa. Detailed analysis on the collocating GAAS5 and DOG1 quantitative trait loci revealed that the DOG1-Cape Verde Islands allele both reduces seed longevity and increases seed dormancy. To our knowledge, this study is the first to report a negative correlation between seed longevity and seed dormancy.

To identify the genetic factors underlying the regulation of fruit vitamin C (L-ascorbic acid [AsA]) concentrations, quantitative trait loci (QTL) studies were carried out in an F1 progeny derived from a cross between the apple (Malus × domestica) cultivars Telamon and Braeburn over three years. QTL were identified for AsA, glutathione, total antioxidant activity in both flesh and skin tissues, and various quality traits, including flesh browning. Four regions on chromosomes 10, 11, 16, and 17 contained stable fruit AsA-QTL clusters. Mapping of AsA metabolic genes identified colocations between orthologs of GDP-L-galactose phosphorylase (GGP), dehydroascorbate reductase (DHAR), and nucleobase-ascorbate transporter within these QTL clusters. Of particular interest are the three paralogs of MdGGP, which all colocated within AsA-QTL clusters. Allelic variants of MdGGP1 and MdGGP3 derived from the cultivar Braeburn parent were also consistently associated with higher fruit total AsA concentrations both within the mapping population (up to 10-fold) and across a range of commercial apple germplasm (up to 6-fold). Striking differences in the expression of the cv Braeburn MdGGP1 allele between fruit from high- and low-AsA genotypes clearly indicate a key role for MdGGP1 in the regulation of fruit AsA concentrations, and this MdGGP allele-specific single-nucleotide polymorphism marker represents an excellent candidate for directed breeding for enhanced fruit AsA concentrations. Interestingly, colocations were also found between MdDHAR3-3 and a stable QTL for browning in the cv Telamon parent, highlighting links between the redox status of the AsA pool and susceptibility to flesh browning.

Chloroplasts and other members of the plastid organelle family contain a small genome of bacterial ancestry. Young chloroplasts contain hundreds of genome copies, but the functional significance of this high genome copy number has been unclear. We describe molecular phenotypes associated with mutations in a nuclear gene in maize (Zea mays), white2 (w2), encoding a predicted organellar DNA polymerase. Weak and strong mutant alleles cause a moderate (approximately 5-fold) and severe (approximately 100-fold) decrease in plastid DNA copy number, respectively, as assayed by quantitative PCR and Southern-blot hybridization of leaf DNA. Both alleles condition a decrease in most chloroplast RNAs, with the magnitude of the RNA deficiencies roughly paralleling that of the DNA deficiency. However, some RNAs are more sensitive to a decrease in genome copy number than others. The rpoB messenger RNA (mRNA) exhibited a unique response, accumulating to dramatically elevated levels in response to a moderate reduction in plastid DNA. Subunits of photosynthetic enzyme complexes were reduced more severely than were plastid mRNAs, possibly because of impaired translation resulting from limiting ribosomal RNA, transfer RNA, and ribosomal protein mRNA. These results indicate that chloroplast genome copy number is a limiting factor for the expression of a subset of chloroplast genes in maize. Whereas in Arabidopsis (Arabidopsis thaliana) a pair of orthologous genes function redundantly to catalyze DNA replication in both mitochondria and chloroplasts, the w2 gene is responsible for virtually all chloroplast DNA replication in maize. Mitochondrial DNA copy number was reduced approximately 2-fold in mutants harboring strong w2 alleles, suggesting that w2 also contributes to mitochondrial DNA replication.

Stilbenes are a small family of phenylpropanoids produced in a number of unrelated plant species, including grapevine (Vitis vinifera). In addition to their participation in defense mechanisms in plants, stilbenes, such as resveratrol, display important pharmacological properties and are postulated to be involved in the health benefits associated with a moderate consumption of red wine. Stilbene synthases (STSs), which catalyze the biosynthesis of the stilbene backbone, seem to have evolved from chalcone synthases (CHSs) several times independently in stilbene-producing plants. STS genes usually form small families of two to five closely related paralogs. By contrast, the sequence of grapevine reference genome (cv PN40024) has revealed an unusually large STS gene family. Here, we combine molecular evolution and structural and functional analyses to investigate further the high number of STS genes in grapevine. Our reannotation of the STS and CHS gene families yielded 48 STS genes, including at least 32 potentially functional ones. Functional characterization of nine genes representing most of the STS gene family diversity clearly indicated that these genes do encode for proteins with STS activity. Evolutionary analysis of the STS gene family revealed that both STS and CHS evolution are dominated by purifying selection, with no evidence for strong selection for new functions among STS genes. However, we found a few sites under different selection pressures in CHS and STS sequences, whose potential functional consequences are discussed using a structural model of a typical STS from grapevine that we developed.

Dicer-Like1 (DCL1), an RNaseIII endonuclease, and Hyponastic Leaves1 (HYL1), a double-stranded RNA-binding protein, are core components of the plant microRNA (miRNA) biogenesis machinery. hyl1 null mutants accumulate low levels of miRNAs and display pleiotropic developmental phenotypes. We report the identification of five new hyl1 suppressor mutants, all of which are alleles of DCL1. These new alleles affect either the helicase or the RNaseIIIa domains of DCL1, highlighting the critical functions of these domains. Biochemical analysis of the DCL1 suppressor variants reveals that they process the primary transcript (pri-miRNA) more efficiently than wild-type DCL1, with both higher K cat and lower K m values. The DCL1 variants largely rescue wild-type miRNA accumulation levels in vivo, but do not rescue the MIRNA processing precision defects of the hyl1 null mutant. In vitro, the helicase domain confers ATP dependence on DCL1-catalyzed MIRNA processing, attenuates DCL1 cleavage activity, and is required for precise MIRNA processing of some substrates.

The proper use of resistance genes (R genes) requires a comprehensive understanding of their genomics and evolution. We analyzed genes encoding nucleotide-binding sites and leucine-rich repeats in the genomes of rice (Oryza sativa), maize (Zea mays), sorghum (Sorghum bicolor), and Brachypodium distachyon. Frequent deletions and translocations of R genes generated prevalent presence/absence polymorphism between different accessions/species. The deletions were caused by unequal crossover, homologous repair, nonhomologous repair, or other unknown mechanisms. R gene loci identified from different genomes were mapped onto the chromosomes of rice cv Nipponbare using comparative genomics, resulting in an integrated map of 495 R loci. Sequence analysis of R genes from the partially sequenced genomes of an African rice cultivar and 10 wild accessions suggested that there are many additional R gene lineages in the AA genome of Oryza. The R genes with chimeric structures (termed type I R genes) are diverse in different rice accessions but only account for 5.8% of all R genes in the Nipponbare genome. In contrast, the vast majority of R genes in the rice genome are type II R genes, which are highly conserved in different accessions. Surprisingly, pseudogene-causing mutations in some type II lineages are often conserved, indicating that their conservations were not due to their functions. Functional R genes cloned from rice so far have more type II R genes than type I R genes, but type I R genes are predicted to contribute considerable diversity in wild species. Type I R genes tend to reduce the microsynteny of their flanking regions significantly more than type II R genes, and their flanking regions have slightly but significantly lower G/C content than those of type II R genes.

Plant mitochondria have very active DNA recombination activities that are responsible for its plastic structures and that should be involved in the repair of double-strand breaks in the mitochondrial genome. Little is still known on plant mitochondrial DNA repair, but repair by recombination is believed to be a major determinant in the rapid evolution of plant mitochondrial genomes. In flowering plants, mitochondria possess at least two eubacteria-type RecA proteins that should be core components of the mitochondrial repair mechanisms. We have performed functional analyses of the two Arabidopsis (Arabidopsis thaliana) mitochondrial RecAs (RECA2 and RECA3) to assess their potential roles in recombination-dependent repair. Heterologous expression in Escherichia coli revealed that RECA2 and RECA3 have overlapping as well as specific activities that allow them to partially complement bacterial repair pathways. RECA2 and RECA3 have similar patterns of expression, and mutants of either display the same molecular phenotypes of increased recombination between intermediate-size repeats, thus suggesting that they act in the same recombination pathways. However, RECA2 is essential past the seedling stage and should have additional important functions. Treatment of plants with several DNA-damaging drugs further showed that RECA3 is required for different recombination-dependent repair pathways that significantly contribute to plant fitness under stress. Replication repair of double-strand breaks results in the accumulation of crossovers that increase the heteroplasmic state of the mitochondrial DNA. It was shown that these are transmitted to the plant progeny, enhancing the potential for mitochondrial genome evolution.

The S5 locus regulates spikelet fertility of indica/japonica hybrid rice (Oryza sativa). There are three alleles at the S5 locus, including an indica allele (S5i), a japonica allele (S5j), and a wide-compatibility allele (S5n). This study analyzed the molecular basis for S5-dependent reproductive isolation and compatibility of indica/japonica rice hybrids. Three S5 alleles were expressed at extremely low levels, and only in the ovary. S5n was more similar to S5i in both RNA and protein expression profiles. The S5 locus was not essential for embryo sac development, although deleterious interactions between S5i and S5j resulted in reduced rates of spikelet fertility. The yeast two-hybrid system was used to test direct interactions between S5-encoded proteins. The results indicated that the S5i- and S5j-encoded eukaryotic aspartyl proteases formed both homodimers and heterodimers, whereas the S5n-encoded aspartyl protease was incapable of dimerization. Site-directed mutagenesis revealed that a single amino acid difference between S5i- and S5j-encoded aspartyl proteases (phenylalanine/leucine at residue 273) was primarily responsible for embryo sac abortion. The S5 locus may have promoted the subspeciation of indica and japonica, but it also enables gene flow between them.

Recent advances in -omics technologies such as transcriptomics, metabolomics, and proteomics along with genotypic profiling have permitted dissection of the genetics of complex traits represented by molecular phenotypes in nonmodel species. To identify the genetic factors underlying variation in primary metabolism in potato (Solanum tuberosum), we have profiled primary metabolite content in a diploid potato mapping population, derived from crosses between S. tuberosum and wild relatives, using gas chromatography-time of flight-mass spectrometry. In total, 139 polar metabolites were detected, of which we identified metabolite quantitative trait loci for approximately 72% of the detected compounds. In order to obtain an insight into the relationships between metabolic traits and classical phenotypic traits, we also analyzed statistical associations between them. The combined analysis of genetic information through quantitative trait locus coincidence and the application of statistical learning methods provide information on putative indicators associated with the alterations in metabolic networks that affect complex phenotypic traits.

Trehalose is a nonreducing sugar used as a reserve carbohydrate and stress protectant in a variety of organisms. While higher plants typically do not accumulate high levels of trehalose, they encode large families of putative trehalose biosynthesis genes. Trehalose biosynthesis in plants involves a two-step reaction in which trehalose-6-phosphate (T6P) is synthesized from UDP-glucose and glucose-6-phosphate (catalyzed by T6P synthase [TPS]), and subsequently dephosphorylated to produce the disaccharide trehalose (catalyzed by T6P phosphatase [TPP]). In Arabidopsis (Arabidopsis thaliana), 11 genes encode proteins with both TPS- and TPP-like domains but only one of these (AtTPS1) appears to be an active (TPS) enzyme. In addition, plants contain a large family of smaller proteins with a conserved TPP domain. Here, we present an in-depth analysis of the 10 TPP genes and gene products in Arabidopsis (TPPA-TPPJ). Collinearity analysis revealed that all of these genes originate from whole-genome duplication events. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that all encode active TPP enzymes with an essential role for some conserved residues in the catalytic domain. These results suggest that the TPP genes function in the regulation of T6P levels, with T6P emerging as a novel key regulator of growth and development in higher plants. Extensive gene expression analyses using a complete set of promoter-β-glucuronidase/green fluorescent protein reporter lines further uncovered cell- and tissue-specific expression patterns, conferring spatiotemporal control of trehalose metabolism. Consistently, phenotypic characterization of knockdown and overexpression lines of a single TPP, AtTPPG, points to unique properties of individual TPPs in Arabidopsis, and underlines the intimate connection between trehalose metabolism and abscisic acid signaling.

The cross between Arabidopsis thaliana and the closely related species Arabidopsis arenosa results in postzygotic hybrid incompatibility, manifested as seed death. Ecotypes of A. thaliana were tested for their ability to produce live seed when crossed to A. arenosa. The identified genetic variation was used to map quantitative trait loci (QTLs) encoded by the A. thaliana genome that affect the frequency of postzygotic lethality and the phenotypes of surviving seeds. Seven QTLs affecting the A. thaliana component of this hybrid incompatibility were identified by crossing a Columbia × C24 recombinant inbred line population to diploid A. arenosa pollen donors. Additional epistatic loci were identified based on their pairwise interaction with one or several of these QTLs. Epistatic interactions were detected for all seven QTLs. The two largest additive QTLs were subjected to fine-mapping, indicating the action of at least two genes in each. The topology of this network reveals a large set of minor-effect loci from the maternal genome controlling hybrid growth and viability at different developmental stages. Our study establishes a framework that will enable the identification and characterization of genes and pathways in A. thaliana responsible for hybrid lethality in the A. thaliana × A. arenosa interspecific cross.

The nuclear genome of eukaryotes contains large amounts of cytoplasmic organelle DNA (nuclear integrants of organelle DNA [norgs]). The recent sequencing of many mitochondrial and chloroplast genomes has enabled investigation of the potential role of norgs in endosymbiotic evolution. In this article, we describe a new polymerase chain reaction-based method that allows the identification and evolutionary study of recent and older norgs in a range of eukaryotes. We tested this method in the genus Nicotiana and obtained sequences from seven nuclear integrants of plastid DNA (nupts) totaling 25 kb in length. These nupts were estimated to have been transferred 0.033 to 5.81 million years ago. The spectrum of mutations present in the potential protein-coding sequences compared with the noncoding sequences of each nupt revealed that nupts evolve in a nuclear-specific manner and are under neutral evolution. Indels were more frequent in noncoding regions than in potential coding sequences of former chloroplastic DNA, most probably due to the presence of a higher number of homopolymeric sequences. Unexpectedly, some potential protein-coding sequences within the nupts still contained intact open reading frames for up to 5.81 million years. These results suggest that chloroplast genes transferred to the nucleus have in some cases several millions of years to acquire nuclear regulatory elements and become functional. The different factors influencing this time frame and the potential role of nupts in endosymbiotic gene transfer are discussed.

Plant mitochondrial genomes have features that distinguish them radically from their animal counterparts: a high rate of rearrangement, of uptake and loss of DNA sequences, and an extremely low point mutation rate. Perhaps the most unique structural feature of plant mitochondrial DNAs is the presence of large repeated sequences involved in intramolecular and intermolecular recombination. In addition, rare recombination events can occur across shorter repeats, creating rearrangements that result in aberrant phenotypes, including pollen abortion, which is known as cytoplasmic male sterility (CMS). Using next-generation sequencing, we pyrosequenced two rice (Oryza sativa) mitochondrial genomes that belong to the indica subspecies. One genome is normal, while the other carries the wild abortive-CMS. We find that numerous rearrangements in the rice mitochondrial genome occur even between close cytotypes during rice evolution. Unlike maize (Zea mays), a closely related species also belonging to the grass family, integration of plastid sequences did not play a role in the sequence divergence between rice cytotypes. This study also uncovered an excellent candidate for the wild abortive-CMS-encoding gene; like most of the CMS-associated open reading frames that are known in other species, this candidate was created via a rearrangement, is chimeric in structure, possesses predicted transmembrane domains, and coopted the promoter of a genuine mitochondrial gene. Our data give new insights into rice mitochondrial evolution, correcting previous reports.

Cysteine proteases (CPs) accumulate to high concentration in many fruit, where they are believed to play a role in fungal and insect defense. The fruit of Actinidia species (kiwifruit) exhibit a range of CP activities (e.g. the Actinidia chinensis variety YellowA shows less than 2% of the activity of Actinidia deliciosa variety Hayward). A major quantitative trait locus for CP activity was mapped to linkage group 16 in a segregating population of A. chinensis. This quantitative trait locus colocated with the gene encoding actinidin, the major acidic CP in ripe Hayward fruit encoded by the ACT1A-1 allele. Sequence analysis indicated that the ACT1A locus in the segregating A. chinensis population contained one functional allele (A-2) and three nonfunctional alleles (a-3, a-4, and a-5) each containing a unique frameshift mutation. YellowA kiwifruit contained two further alleles: a-6, which was nonfunctional because of a large insertion, and a-7, which produced an inactive enzyme. Site-directed mutagenesis of the act1a-7 protein revealed a residue that restored CP activity. Expression of the functional ACT1A-1 cDNA in transgenic plants complemented the natural YellowA mutations and partially restored CP activity in fruit. Two consequences of the increase in CP activity were enhanced degradation of gelatin-based jellies in vitro and an increase in the processing of a class IV chitinase in planta. These results provide new insight into key residues required for CP activity and the in vivo protein targets of actinidin.

Postpollination nonrandom mating among compatible mates is a widespread phenomenon in plants and is genetically undefined. In this study, we used the recombinant inbred line (RIL) population between Landsberg erecta and Columbia (Col) accessions of Arabidopsis (Arabidopsis thaliana) to define the genetic architecture underlying both female- and male-mediated nonrandom mating traits. To map the genetic loci responsible for male-mediated nonrandom mating, we performed mixed pollinations with Col and RIL pollen on Col pistils. To map the genetic loci responsible for female-mediated nonrandom mating, we performed mixed pollinations with Col and Landsberg erecta pollen on RIL pistils. With these data, we performed composite interval mapping to identify two quantitative trait loci (QTLs) that control male-mediated nonrandom mating. We detected epistatic interactions between these two loci. We also explored female- and male-mediated traits involved in seed yield in mixed pollinations. We detected three female QTLs and one male QTL involved in directing seed number per fruit. To our knowledge, the results of these experiments represent the first time the female and male components of seed yield and nonrandom mating have been separately mapped.

Extensive natural variation has been described for the timing of flowering initiation in many annual plants, including the model wild species Arabidopsis (Arabidopsis thaliana), which is presumed to be involved in adaptation to different climates. However, the environmental factors that might shape this genetic variation, as well as the molecular bases of climatic adaptation by modifications of flowering time, remain mostly unknown. To approach both goals, we characterized the flowering behavior in relation to vernalization of 182 Arabidopsis wild genotypes collected in a native region spanning a broad climatic range. Phenotype-environment association analyses identified strong altitudinal clines (0–2600 m) in seven out of nine flowering-related traits. Altitudinal clines were dissected in terms of minimum winter temperature and precipitation, indicating that these are the main climatic factors that might act as selective pressures on flowering traits. In addition, we used an association analysis approach with four candidate genes, FRIGIDA (FRI), FLOWERING LOCUS C (FLC), PHYTOCHROME C (PHYC), and CRYPTOCHROME2, to decipher the genetic bases of this variation. Eleven different loss-of-function FRI alleles of low frequency accounted for up to 16% of the variation for most traits. Furthermore, an FLC allelic series of six novel putative loss- and change-of-function alleles, with low to moderate frequency, revealed that a broader FLC functional diversification might contribute to flowering variation. Finally, environment-genotype association analyses showed that the spatial patterns of FRI, FLC, and PHYC polymorphisms are significantly associated with winter temperatures and spring and winter precipi- tations, respectively. These results support that allelic variation in these genes is involved in climatic adaptation

Germination represents a rapid transition from dormancy to a high level of metabolic activity. In-depth transcriptomic profiling at 10 time points in Arabidopsis (Arabidopsis thaliana), including fresh seed, ripened seed, during stratification, germination, and postgermination per se, revealed specific temporal expression patterns that to our knowledge have not previously been identified. Over 10,000 transcripts were differentially expressed during cold stratification, with subequal numbers up-regulated as down-regulated, revealing an active period in preparing seeds for germination, where transcription and RNA degradation both play important roles in regulating the molecular sequence of events. A previously unidentified transient expression pattern was observed for a group of genes, whereby a significant rise in expression was observed at the end of stratification and significantly lower expression was observed 6 h later. These genes were further defined as germination specific, as they were most highly expressed at this time in germination, in comparison with all developmental tissues in the AtGenExpress data set. Functional analysis of these genes using genetic inactivation revealed that they displayed a significant enrichment for embryo-defective or -arrested phenotype. This group was enriched in genes encoding mitochondrial and nuclear RNA-processing proteins, including more than 45% of all pentatricopeptide domain-containing proteins expressed during germination. The presence of mitochondrial DNA replication factors and RNA-processing functions in this germination-specific subset represents the earliest events in organelle biogenesis, preceding any changes associated with energy metabolism. Green fluorescent protein analysis also confirmed organellar localization for 65 proteins, largely showing germination-specific expression. These results suggest that mitochondrial biogenesis involves a two-step process to produce energetically active organelles: an initial phase at the end of stratification involving mitochondrial DNA synthesis and RNA processing, and a later phase for building the better-known energetic functions. This also suggests that signals with a mitochondrial origin and retrograde signals may be crucial for successful germination.

Compounds of the terpenoid class play numerous roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of cultivated tomato (Solanum lycopersicum) contains 44 terpene synthase (TPS) genes, including 29 that are functional or potentially functional. Of these 29 TPS genes, 26 were expressed in at least some organs or tissues of the plant. The enzymatic functions of eight of the TPS proteins were previously reported, and here we report the specific in vitro catalytic activity of 10 additional tomato terpene synthases. Many of the tomato TPS genes are found in clusters, notably on chromosomes 1, 2, 6, 8, and 10. All TPS family clades previously identified in angiosperms are also present in tomato. The largest clade of functional TPS genes found in tomato, with 12 members, is the TPS-a clade, and it appears to encode only sesquiterpene synthases, one of which is localized to the mitochondria, while the rest are likely cytosolic. A few additional sesquiterpene synthases are encoded by TPS-b clade genes. Some of the tomato sesquiterpene synthases use z,z-farnesyl diphosphate in vitro as well, or more efficiently than, the e,e-farnesyl diphosphate substrate. Genes encoding monoterpene synthases are also prevalent, and they fall into three clades: TPS-b, TPS-g, and TPS-e/f. With the exception of two enzymes involved in the synthesis of ent-kaurene, the precursor of gibberellins, no other tomato TPS genes could be demonstrated to encode diterpene synthases so far.

The S locus, a single polymorphic locus, is responsible for self-incompatibility (SI) in the Brassicaceae family and many related plant families. Despite its importance, our knowledge of S-locus evolution is largely restricted to the causal genes encoding the S-locus receptor kinase (SRK) receptor and S-locus cysteine-rich protein (SCR) ligand of the SI system. Here, we present high-quality sequences of the genomic region of six S-locus haplotypes: Arabidopsis (Arabidopsis thaliana; one haplotype), Arabidopsis lyrata (four haplotypes), and Capsella rubella (one haplotype). We compared these with reference S-locus haplotypes of the self-compatible Arabidopsis and its SI congener A. lyrata. We subsequently reconstructed the likely genomic organization of the S locus in the most recent common ancestor of Arabidopsis and Capsella. As previously reported, the two SI-determining genes, SCR and SRK, showed a pattern of coevolution. In addition, consistent with previous studies, we found that duplication, gene conversion, and positive selection have been important factors in the evolution of these two genes and appear to contribute to the generation of new recognition specificities. Intriguingly, the inactive pseudo-S-locus haplotype in the self-compatible species C. rubella is likely to be an old S-locus haplotype that only very recently became fixed when C. rubella split off from its SI ancestor, Capsella grandiflora.

Plants, like animals, use several lines of defense against pathogen attack. Prominent among genes that confer disease resistance are those encoding nucleotide-binding site-leucine-rich repeat (NB-LRR) proteins. Likely due to selection pressures caused by pathogens, NB-LRR genes are the most variable gene family in plants, but there appear to be species-specific limits to the number of NB-LRR genes in a genome. Allelic diversity within an individual is also increased by obligatory outcrossing, which leads to genome-wide heterozygosity. In this study, we compared the NB-LRR gene complement of the selfer Arabidopsis thaliana and its outcrossing close relative Arabidopsis lyrata. We then complemented and contrasted the interspecific patterns with studies of NB-LRR diversity within A. thaliana. Three important insights are as follows: (1) that both species have similar numbers of NB-LRR genes; (2) that loci with single NB-LRR genes are less variable than tandem arrays; and (3) that presence-absence polymorphisms within A. thaliana are not strongly correlated with the presence or absence of orthologs in A. lyrata. Although A. thaliana individuals are mostly homozygous and thus potentially less likely to suffer from aberrant interaction of NB-LRR proteins with newly introduced alleles, the number of NB-LRR genes is similar to that in A. lyrata. In intraspecific and interspecific comparisons, NB-LRR genes are also more variable than receptor-like protein genes. Finally, in contrast to Drosophila, there is a clearly positive relationship between interspecific divergence and intraspecific polymorphisms.

The cactus Opuntia ficus-indica is a constitutive Crassulacean acid metabolism (CAM) species. Current knowledge of CAM metabolism suggests that the enzyme phosphoenolpyruvate carboxylase kinase (PPCK) is circadian regulated at the transcriptional level, whereas phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), NADP-malic enzyme (NADP-ME), and pyruvate phosphate dikinase (PPDK) are posttranslationally controlled. As little transcriptomic data are available from obligate CAM plants, we created an expressed sequence tag database derived from different organs and developmental stages. Sequences were assembled, compared with sequences in the National Center for Biotechnology Information nonredundant database for identification of putative orthologs, and mapped using Kyoto Encyclopedia of Genes and Genomes Orthology and Gene Ontology. We identified genes involved in circadian regulation and CAM metabolism for transcriptomic analysis in plants grown in long days. We identified stable reference genes for quantitative polymerase chain reaction and found that OfiSAND, like its counterpart in Arabidopsis (Arabidopsis thaliana), and OfiTUB are generally appropriate standards for use in the quantification of gene expression in O. ficus-indica. Three kinds of expression profiles were found: transcripts of OfiPPCK oscillated with a 24-h periodicity; transcripts of the light-active OfiNADP-ME and OfiPPDK genes adapted to 12-h cycles, while transcript accumulation patterns of OfiPEPC and OfiMDH were arrhythmic. Expression of the circadian clock gene OfiTOC1, similar to Arabidopsis, oscillated with a 24-h periodicity, peaking at night. Expression of OfiCCA1 and OfiPRR9, unlike in Arabidopsis, adapted best to a 12-h rhythm, suggesting that circadian clock gene interactions differ from those of Arabidopsis. Our results indicate that the evolution of CAM metabolism could be the result of modified circadian regulation at both the transcriptional and posttranscriptional levels.

DNA polymerases play a central role in the process of DNA replication. Yet, the proteins in charge of the replication of plant organelle DNA have not been unambiguously identified. There are however many indications that a family of proteins homologous to bacterial DNA polymerase I (PolI) is implicated in organelle DNA replication. Here, we have isolated mutant lines of the PolIA and PolIB genes of Arabidopsis (Arabidopsis thaliana) to test this hypothesis. We find that mutation of both genes is lethal, thus confirming an essential and redundant role for these two proteins. However, the mutation of a single gene is sufficient to cause a reduction in the levels of DNA in both mitochondria and plastids. We also demonstrate that polIb, but not polIa mutant lines, are hypersensitive to ciprofloxacin, a small molecule that specifically induces DNA double-strand breaks in plant organelles, suggesting a function for PolIB in DNA repair. In agreement with this result, a cross between polIb and a plastid Whirly mutant line yielded plants with high levels of DNA rearrangements and severe growth defects, indicating impairments in plastid DNA repair pathways. Taken together, this work provides further evidences for the involvement of the plant PolI-like genes in organelle DNA replication and suggests an additional role for PolIB in DNA repair.

Mutagenized populations have become indispensable resources for introducing variation and studying gene function in plant genomics research. In this study, fast neutron (FN) radiation was used to induce deletion mutations in the soybean (Glycine max) genome. Approximately 120,000 soybean seeds were exposed to FN radiation doses of up to 32 Gray units to develop over 23,000 independent M2 lines. Here, we demonstrate the utility of this population for phenotypic screening and associated genomic characterization of striking and agronomically important traits. Plant variation was cataloged for seed composition, maturity, morphology, pigmentation, and nodulation traits. Mutants that showed significant increases or decreases in seed protein and oil content across multiple generations and environments were identified. The application of comparative genomic hybridization (CGH) to lesion-induced mutants for deletion mapping was validated on a midoleate x-ray mutant, M23, with a known FAD2-1A (for fatty acid desaturase) gene deletion. Using CGH, a subset of mutants was characterized, revealing deletion regions and candidate genes associated with phenotypes of interest. Exome resequencing and sequencing of PCR products confirmed FN-induced deletions detected by CGH. Beyond characterization of soybean FN mutants, this study demonstrates the utility of CGH, exome sequence capture, and next-generation sequencing approaches for analyses of mutant plant genomes. We present this FN mutant soybean population as a valuable public resource for future genetic screens and functional genomics research.

The likelihood of duplicate gene retention following polyploidy varies by functional properties (e.g. gene ontologies or protein family domains), but little is known about the effects of whole-genome duplication on gene networks related by a common physiological process. Here, we examined the effects of both polyploid and nonpolyploid duplications on genes encoding the major functional groups of photosynthesis (photosystem I, photosystem II, the light-harvesting complex, and the Calvin cycle) in the cultivated soybean (Glycine max), which has experienced two rounds of whole-genome duplication. Photosystem gene families exhibit retention patterns consistent with dosage sensitivity (preferential retention of polyploid duplicates and elimination of nonpolyploid duplicates), whereas Calvin cycle and light-harvesting complex gene families do not. We observed similar patterns in barrel medic (Medicago truncatula), which shared the older genome duplication with soybean but has evolved independently for approximately 50 million years, and in Arabidopsis (Arabidopsis thaliana), which experienced two nested polyploidy events independent from the legume duplications. In both soybean and Arabidopsis, Calvin cycle gene duplicates exhibit a greater capacity for functional differentiation than do duplicates within the photosystems, which likely explains the greater retention of ancient, nonpolyploid duplicates and larger average gene family size for the Calvin cycle relative to the photosystems.

The genetic mechanism involved in a transition from the black-colored seed hull of the ancestral wild rice (Oryza rufipogon and Oryza nivara) to the straw-white seed hull of cultivated rice (Oryza sativa) during grain ripening remains unknown. We report that the black hull of O. rufipogon was controlled by the Black hull4 (Bh4) gene, which was fine-mapped to an 8.8-kb region on rice chromosome 4 using a cross between O. rufipogon W1943 (black hull) and O. sativa indica cv Guangluai 4 (straw-white hull). Bh4 encodes an amino acid transporter. A 22-bp deletion within exon 3 of the bh4 variant disrupted the Bh4 function, leading to the straw-white hull in cultivated rice. Transgenic study indicated that Bh4 could restore the black pigment on hulls in cv Guangluai 4 and Kasalath. Bh4 sequence alignment of all taxa with the outgroup Oryza barthii showed that the wild rice maintained comparable levels of nucleotide diversity that were about 70 times higher than those in the cultivated rice. The results from the maximum likelihood Hudson-Kreitman-Aguade test suggested that the significant reduction in nucleotide diversity in rice cultivars could be caused by artificial selection. We propose that the straw-white hull was selected as an important visual phenotype of nonshattered grains during rice domestication.

To expand the repertoire of Arabidopsis (Arabidopsis thaliana) mutation-reporter transgenes, we constructed six mutant alleles in the same codon of the β-glucuronidase-encoding GUS transgene. Each allele reverts to GUS + only via a particular one of the six transition/transversion pathways. AcV5 epitope tags, fused carboxyl terminal to the inactive GUS– proteins, enabled semiquantitative immunoassays in plant protein extracts. Spontaneous G:C→T:A transversions, previously not measured using reporter transgenes, were quite frequent. This may reflect mispairing of adenine with 8-oxoguanine in DNA attacked by endogenous oxyradicals. Spontaneous G:C→A:T was modest and other reversions were relatively low, as reported previously. Frequencies of ultraviolet C-induced TT→TC and TC→TT reversions were both high. With increased transgene copy number, spontaneous G:C→T:A reversions increased but ultraviolet C-induced reversions decreased. Frequencies of some reversion events were reduced among T4 versus T3 generation plants. Based on these and other analyses of sources of experimental variation, we propose guidelines for the employment of these lines to study genotoxic stress in planta.

L'article original est publié par The American Society of Plant Biologists; WRINKLED1 (WRI1), a key regulator of seed oil biosynthesis in Arabidopsis (Arabidopsis thaliana), was duplicated during the genome amplification of the cereal ancestor genome 90 million years ago. Both maize (Zea mays) coorthologs ZmWri1a and ZmWri1b show a strong transcriptional induction during the early filling stage of the embryo and complement the reduced fatty acid content of Arabidopsis wri1-4 seeds, suggesting conservation of molecular function. Overexpression of ZmWri1a not only increases the fatty acid content of the mature maize grain but also the content of certain amino acids, of several compounds involved in amino acid biosynthesis, and of two intermediates of the tricarboxylic acid cycle. Transcriptomic experiments identified 18 putative target genes of this transcription factor, 12 of which contain in their upstream regions an AW box, the cis-element bound by AtWRI1. In addition to functions related to late glycolysis and fatty acid biosynthesis in plastids, the target genes also have functions related to coenzyme A biosynthesis in mitochondria and the production of glycerol backbones for triacylglycerol biosynthesis in the cytoplasm. Interestingly, the higher seed oil content in ZmWri1a overexpression lines is not accompanied by a reduction in starch, thus opening possibilities for the use of the transgenic maize lines in breeding programs.