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Aldehyde dehydrogenases (ALDHs) play a central role in detoxification processes of aldehydes generated in plants when exposed to the stressed conditions. In order to identify genes required for the stresses responses in the grass crop Zea mays, an ALDH (ZmALDH22A1) gene was isolated and characterized. ZmALDH22A1 belongs to the family ALDH22 that is currently known only in plants. The ZmALDH22A1 encodes a protein of 593 amino acids that shares high identity with the orthologs from Saccharum officinarum (95%), Oryza sativa (89%), Triticum aestivum (87%) and Arabidopsis thaliana (77%), respectively. Real-time PCR analysis indicates that ZmALDH22A1 is expressed differentially in different tissues. Various elevated levels of ZmALDH22A1 expression have been detected when the seedling roots exposed to abiotic stresses including dehydration, high salinity and abscisic acid (ABA). Tomato stable transformation of construct expressing the ZmALDH22A1 signal peptide fused with yellow fluorescent protein (YFP) driven by the CaMV35S-promoter reveals that the fusion protein is targeted to plastid. Transgenic tobacco plants overexpressing ZmALDH22A1 shows elevated stresses tolerance. Stresses tolerance in transgenic plants is accompanied by a reduction of malondialdehyde (MDA) derived from cellular lipid peroxidation.

Fruits are a major source of nutrition in human diets, providing carbohydrates, fiber, vitamins and phytonutrients. Carotenoids are a principal class of compounds found in many fruits, providing nutritional benefits both as precursors to essential vitamins and as antioxidants. Molecular characterization revealed that the tomato high pigment mutant genes (hp1 and hp2) encode UV-DAMAGED DNA BINDING PROTEIN-1 (DDB1) and DE-ETIOLATED-1 (DET1) homologs, respectively, and both are essential components of the recently identified CUL4-based E3 ligase complex. Here we have isolated a tomato CUL4 homolog and performed yeast two-hybrid assays to suggest possible association of tomato DDB1 with CUL4 and DET1. Real-time RT-PCR analysis indicated that both HP1 and CUL4 are expressed constitutively. Abscisic acid is implicated in plastid division control and its application substantially enhances HP1/DDB1 mRNA accumulation. Transformation of constructs expressing CUL4-YFP and DDB1-YFP fusion proteins driven by the CaMV 35S promoter reveals that both CUL4 and DDB1 are targeted to tomato plastids and nuclei simultaneously. Using fruit-specific promoters combined with RNAi technology, we show that downregulated DDB1 expression in transgenic fruits results in a significant increase in the number of plastids and corresponding enhanced pigment accumulation. CUL4-RNAi repression lines provide insight regarding CUL4 function during tomato development, and reveal that this tomato cullin is important in the regulation of plastid number and pigmentation, which in turn have a direct impact on fruit nutrient quality.

Various abilities to synthesize and accumulate glycine betaine (GB) are crucial for angiosperms to develop salt and drought tolerances. In higher plants, GB is synthesized by a two-step oxidation of choline via an intermediate form of betaine aldehyde, and catalyzed by choline monooxygenase and betaine aldehyde dehydrogenase (BADH). In this study, numerous truncated and/or recombinant transcripts of two BADH homologs resulting from an unusual posttranscriptional processing were detected in rice (Oryza sativa) and other cereal crops, including maize (Zea mays), wheat (Triticum aestivum), and barley (Hordeum vulgare). The observed events took place at the 5′ exonic region, and led to the insertion of exogenous gene sequences and a variety of deletions that resulted in the removal of translation initiation codon, loss of functional domain, and frame-shifts with premature termination by introducing stop codon. By contrast, the BADH transcripts from dicotyledonous species, such as spinach (Spinacia oleracea), Arabidopsis (Arabidopsis thaliana), and tomato (Solanum lycopersicum), had correctly processed mRNA. This suggests the differentiation of posttranscriptional processing in BADH genes potentially contributes to the variation of GB-synthesizing capacities among various plant species. In addition, comprehensive sequence analyses demonstrated that extensive sequence similarities (named as short, direct repeats) are of paired presence surrounding the junctions of both the deletion and/or insertion sites in the unusual BADH transcripts. The site selection for the deletion/insertion was altered in response to the stress conditions. This indicates that the sequence elements of short, direct repeats are probably required for the recognition of the deletion/insertion sites.

CULLIN4-RING ubiquitin ligases (CRL4s) as well as their targets are fundamental regulators functioning in many key developmental and stress responses in eukaryotes. In tomato (Solanum lycopersicum), molecular cloning has revealed that the underlying genes of natural spontaneous mutations high pigment 1 (hp1), high pigment 2 (hp2) and uniform ripening (u) encode UV-DAMAGED DNA BINDING PROTEIN 1 (DDB1), DE-ETIOLATED 1 (DET1) and GOLDEN 2-LIKE (GLK2), respectively. However, the molecular basis of the opposite actions of tomato GLK2 vs CUL4-DDB1-DET1 complex on regulating plastid level and fruit quality remains unknown. Here, we provide molecular evidence showing that the tomato GLK2 protein is a substrate of the CUL4-DDB1-DET1 ubiquitin ligase complex for the proteasome degradation. SlGLK2 is degraded by the ubiquitin-proteasome system, which is mainly determined by two lysine residues (K11 and K253). SlGLK2 associates with the CUL4-DDB1-DET1 E3 complex in plant cells. Genetically impairing CUL4, DDB1 or DET1 results in a retardation of SlGLK2 degradation by the 26S proteasome. These findings are relevant to the potential of nutrient accumulation in tomato fruit by mediating the plastid level and contribute to a deeper understanding of an important regulatory loop, linking protein turnover to gene regulation.

The kiwifruit (Actinidia chinensis) is an economically and nutritionally important fruit crop with remarkably high vitamin C content. Here we report the draft genome sequence of a heterozygous kiwifruit, assembled from ~140-fold next-generation sequencing data. The assembled genome has a total length of 616.1 Mb and contains 39,040 genes. Comparative genomic analysis reveals that the kiwifruit has undergone an ancient hexaploidization event (γ) shared by core eudicots and two more recent whole-genome duplication events. Both recent duplication events occurred after the divergence of kiwifruit from tomato and potato and have contributed to the neofunctionalization of genes involved in regulating important kiwifruit characteristics, such as fruit vitamin C, flavonoid and carotenoid metabolism. As the first sequenced species in the Ericales, the kiwifruit genome sequence provides a valuable resource not only for biological discovery and crop improvement but also for evolutionary and comparative genomics analysis, particularly in the asterid lineage., The kiwifruit is an economically and nutritionally important fruit crop with high vitamin C content. Here, the authors report the draft genome sequence of a heterozygous kiwifruit and through comparative genomic analysis provide valuable insight into kiwifruit evolution.

Stomatal movement plays a key role in plant development and response to drought and salt stress by regulating gas exchange and water loss. A number of genes have been demonstrated to be involved in the regulation of this process. Using inverse genetics approach, we characterized the function of a rice (Oryza sativa L.) vacuolar H(+)-ATPase subunit A (OsVHA-A) gene in stomatal conductance regulation and physiological response to salt and osmotic stress. OsVHA-A was constitutively expressed in different rice tissues, and the fusion protein of GFP-OsVHA-A was exclusively targeted to tonoplast when transiently expressed in the onion epidermal cells. Heterologous expression of OsVHA-A was able to rescue the yeast mutant vma1Δ (lacking subunit A activity) phenotype, suggesting that it partially restores the activity of V-ATPase. Meanwhile, RNAi-directed knockdown of OsVHA-A led to a reduction of vacuolar H(+)-ATPase activity and an enhancement of plasma membrane H(+)-ATPase activity, thereby increasing the concentrations of extracellular H(+) and intracellular K(+) and Na(+) under stress conditions. Knockdown of OsVHA-A also resulted in the upregulation of PAM3 (plasma membrane H(+)-ATPase 3) and downregulation of CAM1 (calmodulin 1), CAM3 (calmodulin 3) and YDA1 (YODA, a MAPKK gene). Altered level of the ion concentration and the gene expression by knockdown of OsVHA-A probably resulted in expanded aperture of stomatal pores and increased stomatal density. In addition, OsVHA-A RNAi plants displayed significant growth inhibition under salt and osmotic stress conditions. Taken together, our results suggest that OsVHA-A takes part in regulating stomatal density and opening via interfering with pH value and ionic equilibrium in guard cells and thereby affects the growth of rice plants.

The phenotype of tomato high pigment-1 (hp1) mutant is characterized by overproduction of pigments including chlorophyll and carotenoids during fruit development and ripening. Although the increased plastid compartment size has been thought to largely attribute to the enhanced pigmentation, the molecular aspects of how the HP1/DDB1 gene manipulates plastid biogenesis and development are largely unknown. In the present study, we compared transcriptome profiles of immature fruit pericarp tissue between tomato cv. Ailsa Craig (WT) and its isogenic hp1 mutant. Over 20 million sequence reads, representing > 1.6 Gb sequence data per sample, were generated and assembled into 21,972 and 22,167 gene models in WT and hp1, respectively, accounting for over 60 % official gene models in both samples. Subsequent analyses revealed that 8,322 and 7,989 alternative splicing events, 8833 or 8510 extended 5′-UTRs, 8,263 or 8,939 extended 3′-UTRs, and 1,136 and 1,133 novel transcripts, exist in WT and hp1, respectively. Significant differences in expression level of 880 genes were detected between the WT and hp1, many of which are involved in signaling transduction, transcription regulation and biotic and abiotic stresses response. Distinctly, RNA-seq datasets, quantitative RT-PCR analyses demonstrate that, in hp1 mutant pericarp tissue at early developmental stage, an apparent expression alteration was found in several regulators directly involved in plastid division and development. These results provide a useful reference for a more accurate and more detailed characterization of the molecular process in the development and pigmentation of tomato fruits.

Alternative splicing can produce transcripts that encode proteins with altered functions. The transcripts of the ABSCISIC ACID INSENSITIVE3 (ABI3)/VIVIPAROUS1 (VP1) gene, which is an important component in abscisic acid (ABA) signaling, are subjected to alternative splicing in both monocotyledons and dicotyledons. We identified two alternatively spliced tomato (Solanum lycopersicum) SlABI3 transcripts, SlABI3-F and SlABI3-T, which encode the nucleus-localized full-length and truncated proteins, respectively. The tissue-specific accumulation of SlABI3-F and SlABI3-T was determined, particularly in seeds at different developmental stages and in response to phytohormonal and abiotic stress. Ectopic over-expression of SlABI3-F and SlABI3-T resulted in the induction of seed-specific genes SlSOM, SlEM1 and SlEM6 in vegetative tissues. However, over-expression of SlABI3-F, but not SlABI3-T, activated expression of the downstream gene SlABI5 and conferred hypersensitivity to exogenous ABA during seed germination and primary root growth. In addition, the SlABI3-F protein interacted with SlABI5 much stronger than SlABI3-T did in the yeast two-hybrid assay. These results suggest that SlABI3-F and SlABI3-T have similar and distinct functionality in the ABA signaling, dependent on which tissue/organ they accumulate in.

Glycine betaine (GB) is a compatible quaternary amine that enables plants to tolerate abiotic stresses, including salt, drought and cold. In plants, GB is synthesized through two-step of successive oxidations from choline, catalyzed by choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), respectively. Rice is considered as a typical non-GB accumulating species, although the entire genome sequencing revealed rice contains orthologs of both CMO and BADH. Several studies unraveled that rice has a functional BADH gene, but whether rice CMO gene (OsCMO) is functional or a pseudogene remains to be elucidated. In the present study, we report the functional characterization of rice CMO gene. The OsCMO gene was isolated from rice cv. Nipponbare (Oryza sativa L. ssp. japonica) using RT-PCR. Northern blot demonstrated the transcription of OsCMO is enhanced by salt stress. Transgenic tobacco plants overexpressing OsCMO results in increased GB content and elevated tolerance to salt stress. Immunoblotting analysis demonstrates that a functional OsCMO protein with correct size was present in transgenic tobacco but rarely accumulated in wild-type rice plants. Surprisingly, a large amount of truncated proteins derived from OsCMO was induced in the rice seedlings in response to salt stresses. This suggests that it is the lack of a functional OsCMO protein that presumably results in non-GB accumulation in the tested rice plant. Key message Expression and transgenic studies demonstrate OsCMO is transcriptionally induced in response to salt stress and functions in increasing glycinebetaine accumulation and enhancing tolerance to salt stress. Immunoblotting analysis suggests that no accumulation of glycinebetaine in the Japonica rice plant presumably results from lack of a functional OsCMO protein.

The WRKY transcription factors have been implicated in multiple biological processes in plants, especially in regulating defense against biotic and abiotic stresses. However, little information is available about the WRKYs in tomato (Solanum lycopersicum). The recent release of the whole-genome sequence of tomato allowed us to perform a genome-wide investigation for tomato WRKY proteins, and to compare these positively identified proteins with their orthologs in model plants, such as Arabidopsis and rice. In the present study, based on the recently released tomato whole-genome sequences, we identified 81 SlWRKY genes that were classified into three main groups, with the second group further divided into five subgroups. Depending on WRKY domains’ sequences derived from tomato, Arabidopsis and rice, construction of a phylogenetic tree demonstrated distinct clustering and unique gene expansion of WRKY genes among the three species. Genome mapping analysis revealed that tomato WRKY genes were enriched on several chromosomes, especially on chromosome 5, and 16 % of the family members were tandemly duplicated genes. The tomato WRKYs from each group were shown to share similar motif compositions. Furthermore, tomato WRKY genes showed distinct temporal and spatial expression patterns in different developmental processes and in response to various biotic and abiotic stresses. The expression of 18 selected tomato WRKY genes in response to drought and salt stresses and Pseudomonas syringae invasion, respectively, was validated by quantitative RT-PCR. Our results will provide a platform for functional identification and molecular breeding study of WRKY genes in tomato and probably other Solanaceae plants.

Glycine betaine (GB), a quaternary ammonium solute, plays a crucial role in developing osmotic tolerance. Rice contains a choline monooxygenase (CMO) and two betaine aldehyde dehydrogenase homologues that are required for GB synthesis, but usually no GB is accumulated in rice (Oryza sativa). To elucidate the molecular processes that underlie the GB deficiency in rice, an experiment involving rice and spinach (Spinacia oleracea) was conducted to analyze the products transcribed from CMO genes. Reverse transcription-polymerase chain reaction (RT-PCR) was used to obtain CMO transcripts and a sequencing approach was employed to analyze the structural composition of various CMO transcripts. The results showed that most rice CMO transcripts were processed incorrectly, retaining introns or deleted of coding sequences; the unusual deletion events occurred at sequence elements of the short-direct repeats. In conclusion, the production of incorrect CMO transcripts results in a deficiency of the full-length CMO protein and probably reduces GB accumulation considerably in rice plants. Sequence comparison results also implied that the unusual deletion-site selection might be mediated by the short-direct repeats in response to stress conditions.

Epigenetic modification generally refers to phenotypic changes by a mechanism other than changes in DNA sequence and plays a significant role in developmental processes. In this study, we found that overexpression of one alternatively spliced tomato DDB1 transcript, DDB1(F) that is prevalently present in all tested tissues, resulted in reduction of organ size. Transgenic plants constitutively expressing the DDB1(F) from a strong cauliflower mosaic virus (CaMV) 35S promoter displayed moderately reduced size in vegetative organs (leaves and stems) and radically decreased size in reproductive organs (flowers, seeds and fruits), in which several genes encoding negative regulators for cell division were upregulated. Significantly, reduction of organ size conferred by overexpression of DDB1(F) transgene appears not to segregate in the subsequent generations, suggesting the phenotypic alternations are manipulated in an epigenetic manner and can be transmitted over generations. This notion was further substantiated by analysis of DNA methylation level at the SlWEE1 gene (encoding a negative regulator of cell division), revealing a correlation between less methylation in the promoter region and elevated expression level of this gene. Thus, our results suggest DDB1 plays an important role in regulation of the epigenetic state of genes involved in organogenesis, despite the underlying mechanism remains to be elucidated.

Background Aromatic rice is popular worldwide because of its characteristic fragrance. Genetic studies and physical fine mapping reveal that a candidate gene (fgr/OsBADH2) homologous to betaine aldehyde dehydrogenase is responsible for aroma metabolism in fragrant rice varieties, but the direct evidence demonstrating the functions of OsBADH2 is lacking. To elucidate the physiological roles of OsBADH2, sequencing approach and RNA interference (RNAi) technique were employed to analyze allelic variation and functions of OsBADH2 gene in aroma production. Semi-quantitative, real-time reverse transcription-polymerase chain reaction (RT-PCR), as well as gas chromatography-mass spectrometry (GC-MS) were conducted to determine the expression levels of OsBADH2 and the fragrant compound in wild type and transgenic OsBADH2-RNAi repression lines, respectively. Results The results showed that multiple mutations identical to fgr allele occur in the 13 fragrant rice accessions across China; OsBADH2 is expressed constitutively, with less expression abundance in mature roots; the disrupted OsBADH2 by RNA interference leads to significantly increased 2-acetyl-1-pyrroline production. Conclusion We have found that the altered expression levels of OsBADH2 gene influence aroma accumulation, and the prevalent aromatic allele probably has a single evolutionary origin.