Xiangli Niu

Draft genome of the kiwifruit Actinidia chinensis

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.

SlNAC1, a stress‐related transcription factor, is fine‐tuned on both the transcriptional and the post‐translational level

The plant-specific NAC (NAM, ATAF1,2, CUC2) transcription factors play significant roles in diverse physiological processes. In this study, we determined the regulation of a stress-related tomato (Solanum lycopersicum) NAC1 (SlNAC1) transcription factor at both the transcriptional and the post-translational level. The SlNAC1 protein was found to be stable in the presence of proteasome-specific inhibitor MG132 or MG115 and ubiquitinated in plant cells, suggesting that the SlNAC1 is subject to the ubiquitin-proteasome system-mediated degradation. Deletion analysis identified a short segment of 10 amino acids (aa261-270) that was required for ubiquitin-proteasome system-mediated degradation, among which two leucine residues (L268 and L269) were critical for the protein instability of SlNAC1. Fusion of the degron (SlNAC1(191-270) ) containing these 10 amino acids to green fluorescent protein was found to be sufficient to trigger the degradation of the fusion protein. In addition, the SlNAC1 gene is strongly upregulated during Pseudomonas infection, while repression of the NAC1 ortholog in Nicotiana benthamiana resulted in enhanced susceptibility to Pseudomonas bacteria. These results suggest that rapid upregulation of the NAC1 gene resulting in more protein production is likely one of the strategies plants use to defend themselves against pathogen infection.

RNAi-Directed Downregulation of Vacuolar H+-ATPase Subunit A Results in Enhanced Stomatal Aperture and Density in Rice

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.

A Role of Tomato UV-Damaged DNA Binding Protein 1 (DDB1) in Organ Size Control via an Epigenetic Manner

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, DDB1F that is prevalently present in all tested tissues, resulted in reduction of organ size. Transgenic plants constitutively expressing the DDB1F 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 DDB1F 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.

Comprehensive Transcriptome Profiling Reveals Long Noncoding RNA Expression and Alternative Splicing Regulation during Fruit Development and Ripening in Kiwifruit (Actinidia chinensis)

Genomic and transcriptomic data on kiwifruit (Actinidia chinensis) in public databases are very limited despite its nutritional and economic value. Previously, we have constructed and sequenced nine fruit RNA-Seq libraries of A. chinensis “Hongyang” at immature, mature, and postharvest ripening stages of fruit development, and generated over 66.2 million paired-end and 24.4 million single-end reads. From this dataset, here we have identified 7051 long noncoding RNAs (lncRNAs), 29,327 alternative splicing (AS) events and 2980 novel protein-coding genes that were not annotated in the draft genome of “Hongyang.” AS events were demonstrated in genes involved in the synthesis of nutritional metabolites in fruit, such as ascorbic acids, carotenoids, anthocyanins, and chlorophylls, and also in genes in the ethylene signaling pathway, which plays an indispensable role in fruit ripening. Additionally, transcriptome profiles and the contents of sugars, organic and main amino acids were compared between immature, mature, and postharvest ripening stages in kiwifruits. A total of 5931 differentially expressed genes were identified, including those associated with the metabolism of sugar, organic acid, and main amino acids. The data generated in this study provide a foundation for further studies of fruit development and ripening in kiwifruit, and identify candidate genes and regulatory elements that could serve as targets for improving important agronomic traits through marker assisted breeding and biotechnology.

Functional characterization of two alternatively spliced transcripts of tomato ABSCISIC ACID INSENSITIVE3 ( ABI3 ) gene

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 (Solanumlycopersicum) 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.

Down-regulation of a LBD-like gene, OsIG1, leads to occurrence of unusual double ovules and developmental abnormalities of various floral organs and megagametophyte in rice

Highlight This study demonstrates that OsIG1 plays an essential role in the regulation of empty-glume identity, floral organ number control and female gametophyte development in rice.

Ubiquitin‐conjugated degradation of golden 2‐like transcription factor is mediated by CUL4‐DDB1‐based E3 ligase complex in tomato

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.

Overexpression of a glyoxalase gene, OsGly I, improves abiotic stress tolerance and grain yield in rice (Oryza sativa L.)

Glyoxalase I (Gly I) is a component of the glyoxalase system which is involved in the detoxification of methylglyoxal, a byproduct of glycolysis. In the present study, a gene of rice (Oryza sativa L., cv. Nipponbare) encoding Gly I was cloned and characterized. The quantitative real-time PCR analysis indicated that rice Gly I (OsGly I) was ubiquitously expressed in root, stem, leaf, leaf sheath and spikelet with varying abundance. OsGly I was markedly upregulated in response to NaCl, ZnCl2 and mannitol in rice seedlings. For further functional investigation, OsGly I was overexpressed in rice using Agrobacterium-mediated transformation. Transgenic rice lines exhibited increased glyoxalase enzyme activity, decreased methylglyoxal level and improved tolerance to NaCl, ZnCl2 and mannitol compared to wild-type plants. Enhancement of stress tolerance in transgenic lines was associated with reduction of malondialdehyde content which was derived from cellular lipid peroxidation. In addition, the OsGly I-overexpression transgenic plants performed higher seed setting rate and yield. Collectively, these results indicate the potential of bioengineering the Gly I gene in crops.

Roles of UV-damaged DNA binding protein 1 (DDB1) in epigenetically modifying multiple traits of agronomic importance in tomato

Epigenetic regulation participates broadly in many fundamentally cellular and physiological processes. In this study, we found that DDB1, a protein originally identified as a factor involved in DNA repairing, plays important roles in regulating organ size, growth habit and photosynthesis in tomato via an epigenetic manner. We generated transgenic tomato plants overexpressing an alternatively spliced DDB1 transcript (DDB1F, prevalently present in tomato tissues) and found the primary transformants displayed small-fruited “cherry tomato” in companion with strikingly enhanced shoot branching and biomass, dark-green leaves with elevated chlorophyll accumulation, and increased soluble solids in fruits. Significantly, these phenotypic alterations did not segregate with the DDB1F transgene in subsequent generations, suggesting that the effect of DDB1F on multiple agronomic traits is implemented via an epigenetic manner and is inheritable over generations. We speculate that DDB1, as a core subunit in the recently identified CUL4-based E3 ligase complex, mediates the 26S proteasome-dependent degradation of a large number of proteins, some of which might be required for perpetuating epigenetic marks on chromatins.