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Water availability influences all aspects of plant growth and development; however, most studies of plant responses to drought have focused on vegetative organs, notably roots and leaves. Far less is known about the molecular bases of drought acclimation responses in fruits, which are complex organs with distinct tissue types. To obtain a more comprehensive picture of the molecular mechanisms governing fruit development under drought, we profiled the transcriptomes of a spectrum of fruit tissues from tomato (Solanum lycopersicum), spanning early growth through ripening and collected from plants grown under varying intensities of water stress. In addition, we compared transcriptional changes in fruit with those in leaves to highlight different and conserved transcriptome signatures in vegetative and reproductive organs. We observed extensive and diverse genetic reprogramming in different fruit tissues and leaves, each associated with a unique response to drought acclimation. These included major transcriptional shifts in the placenta of growing fruit and in the seeds of ripe fruit related to cell growth and epigenetic regulation, respectively. Changes in metabolic and hormonal pathways, such as those related to starch, carotenoids, jasmonic acid, and ethylene metabolism, were associated with distinct fruit tissues and developmental stages. Gene coexpression network analysis provided further insights into the tissue-specific regulation of distinct responses to water stress. Our data highlight the spatiotemporal specificity of drought responses in tomato fruit and indicate known and unrevealed molecular regulatory mechanisms involved in drought acclimation, during both vegetative and reproductive stages of development., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Fruit softening is a key component of the irreversible ripening program, contributing to the palatability necessary for frugivore-mediated seed dispersal. The underlying textural changes are complex and result from cell wall remodeling and changes in both cell adhesion and turgor. While a number of transcription factors (TFs) that regulate ripening have been identified, these affect most canonical ripening-related physiological processes. Here, we show that a tomato fruit ripening-specific LATERAL ORGAN BOUNDRIES ( LOB ) TF, SlLOB1 , up-regulates a suite of cell wall-associated genes during late maturation and ripening of locule and pericarp tissues. SlLOB1 repression in transgenic fruit impedes softening, while overexpression throughout the plant under the direction of the 35s promoter confers precocious induction of cell wall gene expression and premature softening. Transcript and protein levels of the wall-loosening protein EXPANSIN1 ( EXP1 ) are strongly suppressed in Sl LOB1 RNA interference lines, while EXP1 is induced in Sl LOB1 -overexpressing transgenic leaves and fruit. In contrast to the role of ethylene and previously characterized ripening TFs, which are comprehensive facilitators of ripening phenomena including softening, Sl LOB1 participates in a regulatory subcircuit predominant to cell wall dynamics and softening., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Softening is a hallmark of ripening in fleshy fruits, and has both desirable and undesirable implications for texture and postharvest stability. Accordingly, the timing and extent of pre-harvest ripening and associated textural changes following harvest are key targets for improving fruit quality through breeding. Previously, we identified a large effect locus associated with harvest date and firmness in apple ( Malus domestica ) using genome-wide association studies (GWAS). Here, we present additional evidence that polymorphisms in or around a transcription factor gene, NAC18.1 , may cause variation in these traits. First, we confirmed our previous findings with new phenotype and genotype data from ∼800 apple accessions. In this population, we compared a genetic marker within NAC18.1 to markers targeting three other firmness-related genes currently used by breeders ( ACS1 , ACO1 , and PG1 ), and found that the NAC18.1 marker was the strongest predictor of both firmness at harvest and firmness after 3 months of cold storage. By sequencing NAC18.1 across 18 accessions, we revealed two predominant haplotypes containing the single nucleotide polymorphism (SNP) previously identified using GWAS, as well as dozens of additional SNPs and indels in both the coding and promoter sequences. NAC18.1 encodes a protein that is orthogolous to the NON-RIPENING (NOR) transcription factor, a regulator of ripening in tomato ( Solanum lycopersicum ). We introduced both NAC18.1 transgene haplotypes into the tomato nor mutant and showed that both haplotypes complement the nor ripening deficiency. Taken together, these results indicate that polymorphisms in NAC18.1 may underlie substantial variation in apple firmness through modulation of a conserved ripening program., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Migicovsky, Yeats, Watts, Song, Forney, Burgher-MacLellan, Somers, Gong, Zhang, Vrebalov, van Velzen, Giovannoni, Rose and Myles.)

Solanum pimpinellifolium (SP) is the wild progenitor of cultivated tomato. Because of its remarkable stress tolerance and intense flavor, SP has been used as an important germplasm donor in modern tomato breeding. Here, we present a high-quality chromosome-scale genome sequence of SP LA2093. Genome comparison identifies more than 92,000 structural variants (SVs) between LA2093 and the modern cultivar, Heinz 1706. Genotyping these SVs in ~600 representative tomato accessions identifies alleles under selection during tomato domestication, improvement and modern breeding, and discovers numerous SVs overlapping genes known to regulate important breeding traits such as fruit weight and lycopene content. Expression quantitative trait locus (eQTL) analysis detects hotspots harboring master regulators controlling important fruit quality traits, including cuticular wax accumulation and flavonoid biosynthesis, and SVs contributing to these complex regulatory networks. The LA2093 genome sequence and the identified SVs provide rich resources for future research and biodiversity-based breeding.

Background: MicroRNAs (miRNAs) are short non-coding RNAs that can influence gene expression via diverse mechanisms. Tomato is a fruit widely consumed for its flavor, culinary attributes, and high nutritional quality. Tomato fruit are climacteric and fleshy, and their ripening is regulated by endogenous and exogenous signals operating through a coordinated genetic network. Much research has been conducted on mechanisms of tomato fruit ripening, but the roles of miRNA-regulated repression/expression of specific regulatory genes are not well documented., Results: In this study, we demonstrate that miR172 specifically targets four SlAP2 transcription factor genes in tomato. Among them, SlAP2a was repressed by the overexpression of SlmiR172, manifesting in altered flower morphology, development and accelerated ripening. miR172 over-expression lines specifically repressed SlAP2a, enhancing ethylene biosynthesis, fruit color and additional ripening characteristics. Most previously described ripening-regulatory genes, including RIN-MADS, NR, TAGL1 and LeHB-1 were not influenced by miR172 while CNR showed altered expression., Conclusions: Tomato fruit ripening is directly influenced by miR172 targeting of the APETALA2 transcription factor, SlAP2a, with minimal influence over additional known ripening-regulatory genes. miR172a-guided SlAP2a expression provides insight into another layer of genetic control of ripening and a target for modifying the quality and nutritional value of tomato and possibly other fleshy fruit crops.

Carotenoids are critically important to plants and humans. The ORANGE (OR) gene is a key regulator for carotenoid accumulation, but its physiological roles in crops remain elusive. In this study, we generated transgenic tomato ectopically overexpressing the Arabidopsis wild-type OR (AtOR WT ) and a 'golden SNP'-containing OR (AtOR H is ). We found that AtOR H is initiated chromoplast formation in very young fruit and stimulated carotenoid accumulation at all fruit developmental stages, uncoupled from other ripening activities. The elevated levels of carotenoids in the AtOR lines were distributed in the same subplastidial fractions as in wild-type tomato, indicating an adaptive response of plastids to sequester the increased carotenoids. Microscopic analysis revealed that the plastid sizes were increased in both AtOR WT and AtOR H is lines at early fruit developmental stages. Moreover, AtOR overexpression promoted early flowering, fruit set and seed production. Ethylene production and the expression of ripening-associated genes were also significantly increased in the AtOR transgenic fruit at ripening stages. RNA-Seq transcriptomic profiling highlighted the primary effects of OR overexpression on the genes in the processes related to RNA, protein and signalling in tomato fruit. Taken together, these results expand our understanding of OR in mediating carotenoid accumulation in plants and suggest additional roles of OR in affecting plastid size as well as flower and fruit development, thus making OR a target gene not only for nutritional biofortification of agricultural products but also for alteration of horticultural traits., (© 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Background: While the role of ethylene in fruit ripening has been widely studied, the contributions of additional plant hormones are less clear. Here we examined the interactions between the transcription factor MaMADS2-box which plays a major role in banana fruit ripening and hormonal regulation. Specifically, we used MaMADS2 repressed lines in transcriptome and hormonal analyses throughout ripening and assessed hormone and gene expression perturbations as compared to wild-type (WT) control fruit., Results: Our analyses revealed major differences in hormones levels and in expression of hormone synthesis and signaling genes mediated by MaMADS2 especially in preclimacteric pulp. Genes encoding ethylene biosynthesis enzymes had lower expression in the pulp of the repressed lines, consistent with reduced ethylene production. Generally, the expression of other hormone (auxin, gibberellins, abscisic acid, jasmonic acid and salicylic acid) response pathway genes were down regulated in the WT pulp prior to ripening, but remained high in MaMADS2 repressed lines. Hormone levels of abscisic acid were also higher, however, active gibberellin levels were lower and auxin levels were similar with MaMADS2 repression as compared to WT. Although abscisic level was higher in MaMADS2 repression, exogenous abscisic acid shortened the time to ethylene production and increased MaMADS2 mRNA accumulation in WT. Exogenous ethylene did not influence abscisic acid level. CRE - a cytokinin receptor, increased its expression during maturation in WT and was lower especially at prebreaker in the repressed line and zeatin level was lower at mature green of the repressed line in comparison to WT., Conclusions: In addition to previously reported effects of MaMADS2 on ethylene, this transcription factor also influences other plant hormones, particularly at the pre-climacteric stage. The cytokinin pathway may play a previously unanticipated role via MaMADS2 in banana ripening. Finally, abscisic acid enhances MaMADS2 expression to promote ripening, but the transcription factor in turn auto inhibits ABA synthesis and signaling. Together, these results demonstrate a complex interaction of plant hormones and banana fruit ripening mediated by MaMADS2.

Main Conclusion: Systemic responses to an arbuscular mycorrhizal fungus reveal opposite phenological patterns in two tomato ripening mutants depending whether ethylene or light reception is involved. The availability of tomato ripening mutants has revealed many aspects of the genetics behind fleshy fruit ripening, plant hormones and light signal reception. Since previous analyses revealed that arbuscular mycorrhizal symbiosis influences tomato berry ripening, we wanted to test the hypothesis that an interplay might occur between root symbiosis and fruit ripening. With this aim, we screened seven tomato mutants affected in the ripening process for their responsiveness to the arbuscular mycorrhizal fungus Funneliformis mosseae. Following their phenological responses we selected two mutants for a deeper analysis: Green ripe (Gr), deficient in fruit ethylene perception and high-pigment-1 (hp-1), displaying enhanced light signal perception throughout the plant. We investigated the putative interactions between ripening processes, mycorrhizal establishment and systemic effects using biochemical and gene expression tools. Our experiments showed that both mutants, notwithstanding a normal mycorrhizal phenotype at root level, exhibit altered arbuscule functionality. Furthermore, in contrast to wild type, mycorrhization did not lead to a higher phosphate concentration in berries of both mutants. These results suggest that the mutations considered interfere with arbuscular mycorrhiza inducing systemic changes in plant phenology and fruits metabolism. We hypothesize a cross talk mechanism between AM and ripening processes that involves genes related to ethylene and light signaling.

Genetic solutions to postharvest crop loss can reduce cost and energy inputs while increasing food security, especially for banana (Musa acuminata), which is a significant component of worldwide food commerce. We have functionally characterized two banana E class (SEPALLATA3 [SEP3]) MADS box genes, MaMADS1 and MaMADS2, homologous to the tomato (Solanum lycopersicum) RIN-MADS ripening gene. Transgenic banana plants repressing either gene (via antisense or RNA interference [RNAi]) were created and exhibited specific ripening delay and extended shelf-life phenotypes, including delayed color development and softening. The delay in fruit ripening is associated with a delay in climacteric respiration and reduced synthesis of the ripening hormone ethylene; in the most severe repressed lines, no ethylene was produced and ripening was most delayed. Unlike tomato rin mutants, banana fruits of all transgenic repression lines responded to exogenous ethylene by ripening normally, likely due to incomplete transgene repression and/or compensation by other MADS box genes. Our results show that, although MADS box ripening gene necessity is conserved across diverse taxa (monocots to dicots), unlike tomato, banana ripening requires at least two necessary members of the SEPALLATA MADS box gene group, and either can serve as a target for ripening control. The utility of such genes as tools for ripening control is especially relevant in important parthenocarpic crops such as the vegetatively propagated and widely consumed Cavendish banana, where breeding options for trait improvement are severely limited., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

A previous EST study identified a MADS box transcription factor coding sequence, AcMADS1, that is strongly induced during non-climacteric pineapple fruit ripening. Phylogenetic analyses place the AcMADS1 protein in the same superclade as LeMADS-RIN, a master regulator of fruit ripening upstream of ethylene in climacteric tomato. LeMADS-RIN has been proposed to be a global ripening regulator shared among climacteric and non-climacteric species, although few functional homologs of LeMADS-RIN have been identified in non-climacteric species. AcMADS1 shares 67 % protein sequence similarity and a similar expression pattern in ripening fruits as LeMADS-RIN. However, in this study AcMADS1 was not able to complement the tomato rin mutant phenotype, indicating AcMADS1 may not be a functionally conserved homolog of LeMADS-RIN or has sufficiently diverged to be unable to act in the context of the tomato network of interacting proteins. The AcMADS1 promoter directed strong expression of the GUS reporter gene to fruits and developing floral organs in tomato and Arabidopsis thaliana, suggesting AcMADS1 may play a role in flower development as well as fruitlet ripening. The AcMADS1 promoter provides a useful molecular tool for directing transgene expression, particularly where up-regulation in developing flowers and fruits is desirable.

Solanum pennellii is a wild tomato species endemic to Andean regions in South America, where it has evolved to thrive in arid habitats. Because of its extreme stress tolerance and unusual morphology, it is an important donor of germplasm for the cultivated tomato Solanum lycopersicum. Introgression lines (ILs) in which large genomic regions of S. lycopersicum are replaced with the corresponding segments from S. pennellii can show remarkably superior agronomic performance. Here we describe a high-quality genome assembly of the parents of the IL population. By anchoring the S. pennellii genome to the genetic map, we define candidate genes for stress tolerance and provide evidence that transposable elements had a role in the evolution of these traits. Our work paves a path toward further tomato improvement and for deciphering the mechanisms underlying the myriad other agronomic traits that can be improved with S. pennellii germplasm.

Ripening of tomato fruits is triggered by the plant hormone ethylene, but its effect is restricted by an unknown developmental cue to mature fruits containing viable seeds. To determine whether this cue involves epigenetic remodeling, we expose tomatoes to the methyltransferase inhibitor 5-azacytidine and find that they ripen prematurely. We performed whole-genome bisulfite sequencing on fruit in four stages of development, from immature to ripe. We identified 52,095 differentially methylated regions (representing 1% of the genome) in the 90% of the genome covered by our analysis. Furthermore, binding sites for RIN, one of the main ripening transcription factors, are frequently localized in the demethylated regions of the promoters of numerous ripening genes, and binding occurs in concert with demethylation. Our data show that the epigenome is not static during development and may have been selected to ensure the fidelity of developmental processes such as ripening. Crop-improvement strategies could benefit by taking into account not only DNA sequence variation among plant lines, but also the information encoded in the epigenome.

Nicotiana benthamiana is a widely used model plant species for the study of fundamental questions in molecular plant-microbe interactions and other areas of plant biology. This popularity derives from its well-characterized susceptibility to diverse pathogens and, especially, its amenability to virus-induced gene silencing and transient protein expression methods. Here, we report the generation of a 63-fold coverage draft genome sequence of N. benthamiana and its availability on the Sol Genomics Network for both BLAST searches and for downloading to local servers. The estimated genome size of N. benthamiana is 3 Gb (gigabases). The current assembly consists of approximately 141,000 scaffolds, spanning 2.6 Gb with 50% of the genome sequence contained within scaffolds >89 kilobases. Of the approximately 16,000 N. benthamiana unigenes available in GenBank, >90% are represented in the assembly. The usefulness of the sequence was demonstrated by the retrieval of N. benthamiana orthologs for 24 immunity-associated genes from other species including Ago2, Ago7, Bak1, Bik1, Crt1, Fls2, Pto, Prf, Rar1, and mitogen-activated protein kinases. The sequence will also be useful for comparative genomics in the Solanaceae family as shown here by the discovery of microsynteny between N. benthamiana and tomato in the region encompassing the Pto and Prf genes.

Fruit ripening is a complex developmental process responsible for the transformation of the seed-containing organ into a tissue attractive to seed dispersers and agricultural consumers. The coordinated regulation of the different biochemical pathways necessary to achieve this change receives considerable research attention. The MADS-box transcription factor RIPENING INHIBITOR (RIN) is an essential regulator of tomato (Solanum lycopersicum) fruit ripening but the exact mechanism by which it influences the expression of ripening-related genes remains unclear. Using a chromatin immunoprecipitation approach, we provide evidence that RIN interacts with the promoters of genes involved in the major pathways associated with observed and well-studied ripening phenotypes and phenomena, including the transcriptional control network involved in overall ripening regulation, ethylene biosynthesis, ethylene perception, downstream ethylene response, cell wall metabolism, and carotenoid biosynthesis. Furthermore, in the cases of ethylene and carotenoid biosynthesis, RIN interacts with the promoters of genes encoding rate-limiting activities. We also show that RIN recruitment to target loci is dependent on a normally functioning allele at the ripening-specific transcription factor COLORLESS NONRIPENING gene locus, further clarifying the relationship between these two ripening regulators.

Climacteric and non-climacteric fruits have traditionally been viewed as representing two distinct programmes of ripening associated with differential respiration and ethylene hormone effects. In climacteric fruits, such as tomato and banana, the ripening process is marked by increased respiration and is induced and co-ordinated by ethylene, while in non-climacteric fruits, such as strawberry and grape, it is controlled by an ethylene-independent process with little change in respiration rate. The two contrasting mechanisms, however, both lead to texture, colour, and flavour changes that probably reflect some common programmes of regulatory control. It has been shown that a SEPALLATA(SEP)4-like gene is necessary for normal ripening in tomato. It has been demonstrated here that silencing a fruit-related SEP1/2-like (FaMADS9) gene in strawberry leads to the inhibition of normal development and ripening in the petal, achene, and receptacle tissues. In addition, analysis of transcriptome profiles reveals pleiotropic effects of FaMADS9 on fruit development and ripening-related gene expression. It is concluded that SEP genes play a central role in the developmental regulation of ripening in both climacteric and non-climacteric fruits. These findings provide important information to extend the molecular control of ripening in a non-climacteric fruit beyond the limited genetic and cultural options currently available.

The transition of fleshy fruit maturation to ripening is regulated by exogenous and endogenous signals that coordinate the transition of the fruit to a final state of attractiveness to seed dispersing organisms. Tomato is a model for biology and genetics regulating specific ripening pathways including ethylene, carotenoids and cell wall metabolism in addition to upstream signaling and transcriptional regulators. Ripening-associated transcription factors described to date including the RIN-MADS, CLEAR NON-RIPENING, TAGL1 and LeHB-1 genes all encode positive regulators of ripening phenomena. Here we describe an APETALA2 transcription factor (SlAP2a) identified through transcriptional profiling of fruit maturation that is induced during, and which negatively regulates, tomato fruit ripening. RNAi repression of SlAP2a results in fruits that over-produce ethylene, ripen early and modify carotenoid accumulation profiles by altering carotenoid pathway flux. These results suggest that SlAP2a functions during normal tomato fruit ripening as a modulator of ripening activity and acts to balance the activities of positive ripening regulators., (© 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd.)

With the aim of determining the genetic basis of metabolic regulation in tomato fruit, we constructed a detailed physical map of genomic regions spanning previously described metabolic quantitative trait loci of a Solanum pennellii introgression line population. Two genomic libraries from S. pennellii were screened with 104 colocated markers from five selected genomic regions, and a total of 614 bacterial artificial chromosome (BAC)/cosmids were identified as seed clones. Integration of sequence data with the genetic and physical maps of Solanum lycopersicum facilitated the anchoring of 374 of these BAC/cosmid clones. The analysis of this information resulted in a genome-wide map of a nondomesticated plant species and covers 10% of the physical distance of the selected regions corresponding to approximately 1% of the wild tomato genome. Comparative analyses revealed that S. pennellii and domesticated tomato genomes can be considered as largely colinear. A total of 1,238,705 bp from both BAC/cosmid ends and nine large insert clones were sequenced, annotated, and functionally categorized. The sequence data allowed the evaluation of the level of polymorphism between the wild and cultivated tomato species. An exhaustive microsynteny analysis allowed us to estimate the divergence date of S. pennellii and S. lycopersicum at 2.7 million years ago. The combined results serve as a reference for comparative studies both at the macrosyntenic and microsyntenic levels. They also provide a valuable tool for fine-mapping of quantitative trait loci in tomato. Furthermore, they will contribute to a deeper understanding of the regulatory factors underpinning metabolism and hence defining crop chemical composition.

Six MaMADS-box genes have been cloned from the banana fruit cultivar Grand Nain. The similarity of these genes to tomato LeRIN is low and neither MaMADS2 nor MaMADS1 complement the tomato rin mutation. Nevertheless, the expression patterns, specifically in fruit and the induction during ripening and in response to ethylene and 1-MCP, suggest that some of these genes may participate in ripening. MaMADS1, 2, and 3, are highly expressed in fruit only, while the others are expressed in fruit as well as in other organs. Moreover, the suites of MaMADS-box genes and their temporal expression differ in peel and pulp during ripening. In the pulp, the increase in MaMADS2, 3, 4, and 5 expression preceded an increase in ethylene production, but coincides with the CO(2) peak. However, MaMADS1 expression in pulp coincided with ethylene production, but a massive increase in its expression occurred late during ripening, together with a second wave in the expression of MaMADS2, 3, and 4. In the peel, on the other hand, an increase in expression of MaMADS1, 3, and to a lesser degree also of MaMADS4 and 2 coincided with an increase in ethylene production. Except MaMADS3, which was induced by ethylene in pulp and peel, only MaMADS4, and 5 in pulp and MaMADS1 in peel were induced by ethylene. 1-MCP applied at the onset of the increase in ethylene production, increased the levels of MaMADS4 and MaMADS1 in pulp, while it decreased MaMADS1, 3, 4, and 5 in peel, suggesting that MaMADS4 and MaMADS1 are negatively controlled by ethylene at the onset of ethylene production only in pulp. Only MaMADS2 is neither induced by ethylene nor by 1-MCP, and it is expressed mainly in pulp. Our results suggest that two independent ripening programs are employed in pulp and peel which involve the activation of mainly MaMADS2, 4, and 5 and later on also MaMADS1 in pulp, and mainly MaMADS1, and 3 in peel. Hence, our results are consistent with MaMADS2, a SEP3 homologue, acting in the pulp upstream of the increase in ethylene production similarly to LeMADS-RIN.

The maturation and ripening of fleshy fruits is a developmental program that synchronizes seed maturation with metabolism, rendering fruit tissues desirable to seed dispersing organisms. Through RNA interference repression, we show that Tomato AGAMOUS-LIKE1 (TAGL1), the tomato (Solanum lycopersicum) ortholog of the duplicated SHATTERPROOF (SHP) MADS box genes of Arabidopsis thaliana, is necessary for fruit ripening. Tomato plants with reduced TAGL1 mRNA produced yellow-orange fruit with reduced carotenoids and thin pericarps. These fruit are also decreased in ethylene, indicating a comprehensive inhibition of maturation mediated through reduced ACC Synthase 2 expression. Furthermore, ectopic expression of TAGL1 in tomato resulted in expansion of sepals and accumulation of lycopene, supporting the role of TAGL1 in ripening. In Arabidopsis, the duplicate SHP1 and SHP2 MADS box genes regulate the development of separation layers essential for pod shatter. Expression of TAGL1 in Arabidopsis failed to completely rescue the shp1 shp2 mutant phenotypes, indicating that TAGL1 has evolved distinct molecular functions compared with its Arabidopsis counterparts. These analyses demonstrate that TAGL1 plays an important role in regulating both fleshy fruit expansion and the ripening process that together are necessary to promote seed dispersal of fleshy fruit. From this broad perspective, SHP1/2 and TAGL1, while distinct in molecular function, regulate similar activities via their necessity for seed dispersal in Arabidopsis and tomato, respectively.

Comparative genomics is a powerful tool for gaining insight into genomic function and evolution. However, in plants, sequence data that would enable detailed comparisons of both coding and noncoding regions have been limited in availability. Here we report the generation and analysis of sequences for an unduplicated conserved syntenic segment (CSS) in the genomes of five members of the agriculturally important plant family Solanaceae. This CSS includes a 105-kb region of tomato chromosome 2 and orthologous regions of the potato, eggplant, pepper, and petunia genomes. With a total neutral divergence of 0.73-0.78 substitutions/site, these sequences are similar enough that most noncoding regions can be aligned, yet divergent enough to be informative about evolutionary dynamics and selective pressures. The CSS contains 17 distinct genes with generally conserved order and orientation, but with numerous small-scale differences between species. Our analysis indicates that the last common ancestor of these species lived approximately 27-36 million years ago, that more than one-third of short genomic segments (5-15 bp) are under selection, and that more than two-thirds of selected bases fall in noncoding regions. In addition, we identify genes under positive selection and analyze hundreds of conserved noncoding elements. This analysis provides a window into 30 million years of plant evolution in the absence of polyploidization.

We report the cloning of Style2.1, the major quantitative trait locus responsible for a key floral attribute (style length) associated with the evolution of self-pollination in cultivated tomatoes. The gene encodes a putative transcription factor that regulates cell elongation in developing styles. The transition from cross-pollination to self-pollination was accompanied, not by a change in the STYLE2.1 protein, but rather by a mutation in the Style2.1 promoter that results in a down-regulation of Style2.1 expression during flower development.

As part of a larger project to sequence the Populus genome and generate genomic resources for this emerging model tree, we constructed a physical map of the Populus genome, representing one of the few such maps of an undomesticated, highly heterozygous plant species. The physical map, consisting of 2802 contigs, was constructed from fingerprinted bacterial artificial chromosome (BAC) clones. The map represents approximately 9.4-fold coverage of the Populus genome, which has been estimated from the genome sequence assembly to be 485 +/- 10 Mb in size. BAC ends were sequenced to assist long-range assembly of whole-genome shotgun sequence scaffolds and to anchor the physical map to the genome sequence. Simple sequence repeat-based markers were derived from the end sequences and used to initiate integration of the BAC and genetic maps. A total of 2411 physical map contigs, representing 97% of all clones assigned to contigs, were aligned to the sequence assembly (JGI Populus trichocarpa, version 1.0). These alignments represent a total coverage of 384 Mb (79%) of the entire poplar sequence assembly and 295 Mb (96%) of linkage group sequence assemblies. A striking result of the physical map contig alignments to the sequence assembly was the co-localization of multiple contigs across numerous regions of the 19 linkage groups. Targeted sequencing of BAC clones and genetic analysis in a small number of representative regions showed that these co-aligning contigs represent distinct haplotypes in the heterozygous individual sequenced, and revealed the nature of these haplotype sequence differences.

Loss of self-incompatibility (SI) in Arabidopsis thaliana was accompanied by inactivation of genes required for SI, including S-LOCUS RECEPTOR KINASE (SRK) and S-LOCUS CYSTEINE-RICH PROTEIN (SCR), coadapted genes that constitute the SI specificity-determining S haplotype. Arabidopsis accessions are polymorphic for PsiSRK and PsiSCR, but it is unknown if the species harbors structurally different S haplotypes, either representing relics of ancestral functional and structurally heteromorphic S haplotypes or resulting from decay concomitant with or subsequent to the switch to self-fertility. We cloned and sequenced the S haplotype from C24, in which self-fertility is due solely to S locus inactivation, and show that this haplotype was produced by interhaplotypic recombination. The highly divergent organization and sequence of the C24 and Columbia-0 (Col-0) S haplotypes demonstrate that the A. thaliana S locus underwent extensive structural remodeling in conjunction with a relaxation of selective pressures that once preserved the integrity and linkage of coadapted SRK and SCR alleles. Additional evidence for this process was obtained by assaying 70 accessions for the presence of C24- or Col-0-specific sequences. Furthermore, analysis of SRK and SCR polymorphisms in these accessions argues against the occurrence of a selective sweep of a particular allele of SCR, as previously proposed.

Despite recent progress in our understanding of carotenogenesis in plants, the mechanisms that govern overall carotenoid accumulation remain largely unknown. The Orange (Or) gene mutation in cauliflower (Brassica oleracea var botrytis) confers the accumulation of high levels of beta-carotene in various tissues normally devoid of carotenoids. Using positional cloning, we isolated the gene representing Or and verified it by functional complementation in wild-type cauliflower. Or encodes a plastid-associated protein containing a DnaJ Cys-rich domain. The Or gene mutation is due to the insertion of a long terminal repeat retrotransposon in the Or allele. Or appears to be plant specific and is highly conserved among divergent plant species. Analyses of the gene, the gene product, and the cytological effects of the Or transgene suggest that the functional role of Or is associated with a cellular process that triggers the differentiation of proplastids or other noncolored plastids into chromoplasts for carotenoid accumulation. Moreover, we demonstrate that Or can be used as a novel genetic tool to induce carotenoid accumulation in a major staple food crop. We show here that controlling the formation of chromoplasts is an important mechanism by which carotenoid accumulation is regulated in plants.

Jasmonic acid (JA) is a lipid-derived signal that regulates plant defense responses to biotic stress. Here, we report the characterization of a JA-deficient mutant of tomato (Lycopersicon esculentum) that lacks local and systemic expression of defensive proteinase inhibitors (PIs) in response to wounding. Map-based cloning studies demonstrated that this phenotype results from loss of function of an acyl-CoA oxidase (ACX1A) that catalyzes the first step in the peroxisomal beta-oxidation stage of JA biosynthesis. Recombinant ACX1A exhibited a preference for C12 and C14 straight-chain acyl-CoAs and also was active in the metabolism of C18 cyclopentanoid-CoA precursors of JA. The overall growth, development, and reproduction of acx1 plants were similar to wild-type plants. However, the mutant was compromised in its defense against tobacco hornworm (Manduca sexta) attack. Grafting experiments showed that loss of ACX1A function disrupts the production of the transmissible signal for wound-induced PI expression but does not affect the recognition of this signal in undamaged responding leaves. We conclude that ACX1A is essential for the beta-oxidation stage of JA biosynthesis and that JA or its derivatives is required both for antiherbivore resistance and the production of the systemic wound signal. These findings support a role for peroxisomes in the production of lipid-based signaling molecules that promote systemic defense responses.

Fruit constitutes a major component of human diets, providing fiber, vitamins, and phytonutrients. Carotenoids are a major class of compounds found in many fruits, providing nutritional benefits as precursors to essential vitamins and as antioxidants. Although recent gene isolation efforts and metabolic engineering have primarily targeted genes involved in carotenoid biosynthesis, factors that regulate flux through the carotenoid pathway remain largely unknown. Characterization of the tomato high-pigment mutations (hp1 and hp2) suggests the manipulation of light signal transduction machinery may be an effective approach toward practical manipulation of plant carotenoids. We demonstrate here that hp1 alleles represent mutations in a tomato UV-DAMAGED DNA-BINDING PROTEIN 1 (DDB1) homolog. We further demonstrate that two tomato light signal transduction genes, LeHY5 and LeCOP1LIKE, are positive and negative regulators of fruit pigmentation, respectively. Down-regulated LeHY5 plants exhibit defects in light responses, including inhibited seedling photomorphogenesis, loss of thylakoid organization, and reduced carotenoid accumulation. In contrast, repression of LeCOP1LIKE expression results in plants with exaggerated photomorphogenesis, dark green leaves, and elevated fruit carotenoid levels. These results suggest genes encoding components of light signal transduction machinery also influence fruit pigmentation and represent genetic tools for manipulation of fruit quality and nutritional value.

We constructed and characterized a bacterial artificial chromosome (BAC) library for Epichloë festucae, a genetically tractable fungal plant mutualist. The 6144 clone library with an average insert size of 87kb represents at least 18-fold coverage of the 29 Mb genome. We used the library to assemble a 110kb contig spanning the putative ornithine decarboxylase (odc) ortholog and subsequently expanded it to 228kb with a single walking step in each direction. Furthermore, we evaluated conservation of microsynteny between E. festucae and some model filamentous fungi by comparing sequence available from a 43kb region at the end of one BAC to publicly available fungal genome sequences. Orthologs to the 13 contiguous open reading frames (ORFs) identified in E. festucae are syntenic in Neurospora crassa and Magnaporthe grisea occurring in small sets of two, three or four colinear ORFs. This library is a valuable resource for research into traits important for the development and maintenance of a plant-fungus mutualistic symbiosis.

The completed rice genome sequence will accelerate progress on the identification and functional classification of biologically important genes and serve as an invaluable resource for the comparative analysis of grass genomes. In this study, methods were developed for sequence-based alignment of sorghum and rice chromosomes and for refining the sorghum genetic/physical map based on the rice genome sequence. A framework of 135 BAC contigs spanning approximately 33 Mbp was anchored to sorghum chromosome 3. A limited number of sequences were collected from 118 of the BACs and subjected to BLASTX analysis to identify putative genes and BLASTN analysis to identify sequence matches to the rice genome. Extensive conservation of gene content and order between sorghum chromosome 3 and the homeologous rice chromosome 1 was observed. One large-scale rearrangement was detected involving the inversion of an approximately 59 cM block of the short arm of sorghum chromosome 3. Several small-scale changes in gene collinearity were detected, indicating that single genes and/or small clusters of genes have moved since the divergence of sorghum and rice. Additionally, the alignment of the sorghum physical map to the rice genome sequence allowed sequence-assisted assembly of an approximately 1.6 Mbp sorghum BAC contig. This streamlined approach to high-resolution genome alignment and map building will yield important information about the relationships between rice and sorghum genes and genomic segments and ultimately enhance our understanding of cereal genome structure and evolution.

Development, maturation and ripening of fruits has received considerable experimental attention, primarily due to the uniqueness of such processes to plant species and the importance of fruit as a significant aspect of human dietary intake and nutrition. Molecular and genetic analysis of fruit development, and especially ripening of fleshy fruits, has resulted in significant gains in knowledge over recent years, especially with respect to understanding ethylene biosynthesis and response, cell wall metabolism and, to a lesser extent, environmental cues which impact ripening. Tomato has proved to be an excellent model system for the analysis of fruit ripening and development, in part due to the availability of well characterized ripening mutants. Especially interesting are the non-allelic ripening-inhibitor (rin) and non-ripening (nor) mutations which result in non-ripening fruit. Fruit from both mutants are deficient in climacteric respiration and the associated burst in ethylene biosynthesis. Exogenous ethylene does not restore ripening yet does induce expression of ethylene-regulated ripening genes, suggesting both mutations block necessary aspects of ripening outside the realm of ethylene's influence. Both mutations therefore represent genes upstream of ethylene control and additional non-ethylene mediated aspects of ripening. Both genes have recently been isolated through positional cloning strategies and it was shown that ripening is regulated, in part, by a MADS-box transcription factor at the rin locus. Recent development of tools for tomato genomics summarized here have further expanded the potential of the tomato system for the elucidation of genetic regulatory components impacting fruit development, ripening and nutritional quality.

Tomato plants harboring the ripening-inhibitor (rin) mutation yield fruits that fail to ripen. Additionally, rin plants display enlarged sepals and loss of inflorescence determinacy. Positional cloning of the rin locus revealed two tandem MADS-box genes (LeMADS-RIN and LeMADS-MC), whose expression patterns suggested roles in fruit ripening and sepal development, respectively. The rin mutation alters expression of both genes. Gene repression and mutant complementation demonstrate that LeMADS-RIN regulates ripening, whereas LeMADS-MC affects sepal development and inflorescence determinacy. LeMADS-RIN demonstrates an agriculturally important function of plant MADS-box genes and provides molecular insight into nonhormonal (developmental) regulation of ripening.

As a starting point for a phylogenetic study of self-incompatibility (SI) in crucifers and to elucidate the genetic basis of transitions between outcrossing and self-fertilizing mating systems in this family, we investigated the SI system of Arabidopsis lyrata. A. lyrata is an outcrossing close relative of the self-fertile A. thaliana and is thought to have diverged from A. thaliana approximately 5 million years ago and from Brassica spp 15 to 20 million years ago. Analysis of two S (sterility) locus haplotypes demonstrates that the A. lyrata S locus contains tightly linked orthologs of the S locus receptor kinase (SRK) gene and the S locus cysteine-rich protein (SCR) gene, which are the determinants of SI specificity in stigma and pollen, respectively, but lacks an S locus glycoprotein gene. As described previously in Brassica, the S haplotypes of A. lyrata differ by the rearranged order of their genes and by their variable physical sizes. Comparative mapping of the A. lyrata and Brassica S loci indicates that the S locus of crucifers is a dynamic locus that has undergone several duplication events since the Arabidopsis--Brassica split and was translocated as a unit between two distant chromosomal locations during diversification of the two taxa. Furthermore, comparative analysis of the S locus region of A. lyrata and its homeolog in self-fertile A. thaliana identified orthologs of the SRK and SCR genes and demonstrated that self-compatibility in this species is associated with inactivation of SI specificity genes.

We have constructed a Bacterial Artificial Chromosome (BAC) library that provides 5.5-fold redundant coverage of the chicken genome. The library was made by cloning partial HindIII-digested high-molecular-weight (HMW) DNA of a female White Leghorn chicken into the HindIII site of the vector pECBAC1. Several modifications of standard protocols were necessary to clone efficiently large partial HindIII DNA fragments. The library consists of 49,920 clones arranged in 130 384-well plates. An average insert size of 134 kb was estimated from the analysis of 152 randomly selected BAC clones. The average number of NotI restriction sites per clone was 0.77. After individual growth, DNA was isolated of the pooled clones of each 384-well plate, and subsequently DNA of each plate was isolated from the individual row and column pools. Screening of the Wageningen chicken BAC library was performed by two-dimensional PCR with 125 microsatellite markers. For 124 markers at least one BAC clone was obtained. FISH experiments of 108 BAC clones revealed chimerism in less than 1%. The number of different BAC clones per marker present in the BAC library was examined for 35 markers which resulted in a total of 167 different BAC clones. Per marker the number of BAC clones varied from 1 to 11, with an average of 4.77. The chicken BAC library constitutes an invaluable tool for positional cloning and for comparative mapping studies.

A genetic analysis was performed to study the frequency of recombination for intervals across the Brassica S locus region. No recombination was observed between the S locus glycoprotein gene and the S receptor kinase gene in the segregating populations that we analyzed. However, a number of recombination breakpoints in regions flanking these genes were identified, allowing the construction of an integrated genetic and physical map of the genomic region encompassing one S haplotype. We identified, based on the pollination phenotype of plants homozygous for recombinant S haplotypes, a 50-kb region that encompasses all specificity functions in the S haplotype that we analyzed. Mechanisms that might operate to preserve the tight linkage of self-incompatibility specificity genes within the S locus complex are discussed in light of the relatively uniform recombination frequencies that we observed across the S locus region and of the structural heteromorphisms that characterize different S haplotypes.

In this paper we describe a novel, dominant pleiotropic tomato (Lycopersicon esculentum)-ripening mutation, Cnr (colorless nonripening). This mutant occurred spontaneously in a commercial population. Cnr has a phenotype that is quite distinct from that of the other pleiotropic tomato-ripening mutants and is characterized by fruit that show greatly reduced ethylene production, an inhibition of softening, a yellow skin, and a nonpigmented pericarp. The ripening-related biosynthesis of carotenoid pigments was abolished in the pericarp tissue. The pericarp also showed a significant reduction in cell-to-cell adhesion, with cell separation occurring when blocks of tissue were incubated in water alone. The mutant phenotype was not reversed by exposure to exogenous ethylene. Crosses with other mutant lines and the use of a restriction fragment length polymorphism marker demonstrated that Cnr was not allelic with the pleiotropic ripening mutants nor, alc, rin, Nr, Gr, and Nr-2. The gene has been mapped to the top of chromosome 2, also indicating that it is distinct from the other pleiotropic ripening mutants. We undertook the molecular characterization of Cnr by examining the expression of a panel of ripening-related genes in the presence and absence of exogenous ethylene. The pattern of gene expression in Cnr was related to, but differed from, that of several of the other well-characterized mutants. We discuss here the possible relationships among nor, Cnr, and rin in a putative ripening signal cascade.