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General translational repression is a key process that reduces energy consumption under hypoxia. Here, we show that plant stress-activated general control nonderepressible 2 (GCN2) was activated to regulate the reduction in polysome loading during submergence in Arabidopsis . GCN2 signaling was activated by ethylene under submergence. GCN2 activity was reduced in etr1-1 , but not in ein2-5 or eil1ein3 , under submergence, suggesting that GCN2 activity is regulated by a noncanonical ethylene signaling pathway. Polysome loading was not reduced in ein2-5 under submergence, implying that ethylene modulates translation via both EIN2 and GCN2. Transcriptomic analysis demonstrated that EIN2 and GCN2 regulate not only general translational repression but also translational enhancement of specific mRNAs under submergence. Together, these results demonstrate that during submergence, entrapped ethylene triggers GCN2 and EIN2 to regulate translation dynamics and ensure the translation of stress response proteins.

Phalaenopsis aphrodite, an epiphytic orchid native to Taiwan and Philippines, possesses many desirable horticultural characteristics permitting its use as parents in breeding commercial orchid hybrids. In order to facilitate the development of genomics/omics breeding tools for orchids, our effort was geared to making P. aphrodite gene annotations and transcriptome resources more accessible to the orchid research community. In this chapter, we review our major progress in P. aphrodite genomics and functional genomics, focusing on the genome sequencing and annotation, whole transcriptome analysis of flower development, analysis of orchid miRNA, and the integrative functional genomics database for the orchid family.

Background and Aims Orchidaceae is a large plant family, and its extraordinary adaptations may have guaranteed its evolutionary success. Flavonoids are a group of secondary metabolites that mediate plant acclimation to challenge environments. Chalcone synthase (CHS) catalyses the initial step in the flavonoid biosynthetic pathway. This is the first chromosome-level investigation of the CHS gene family in Phalaenopsis aphrodite and was conducted to elucidate if divergence of this gene family is associated with chromosome evolution. Methods Complete CHS genes were identified from our whole-genome sequencing data sets and their gene expression profiles were obtained from our transcriptomic data sets. Fluorescence in situ hybridization (FISH) was conducted to position five CHS genes to high-resolution pachytene chromosomes. Key Results The five Phalaenopsis CHS genes can be classified into three groups, PaCHS1, PaCHS2 and the tandemly arrayed three-gene cluster, which diverged earlier than those of the orchid genera and species. Additionally, pachytene chromosome-based FISH mapping showed that the three groups of CHS genes are localized on three distinct chromosomes. Moreover, an expression analysis of RNA sequencing revealed that the five CHS genes had highly differentiated expression patterns and its expression pattern-based clustering showed high correlations between sequence divergences and chromosomal localizations of the CHS gene family in P. aphrodite. Conclusions Based on their phylogenetic relationships, expression clustering analysis and chromosomal distributions of the five paralogous PaCHS genes, we proposed that expansion of this gene family in P. aphrodite occurred through segmental duplications, followed by tandem duplications. These findings provide information for further studies of CHS functions and regulations, and shed light on the divergence of an important gene family in orchids.

Summary The Orchidaceae is a diverse and ecologically important plant family. Approximately 69% of all orchid species are epiphytes, which provide diverse microhabitats for many small animals and fungi in the canopy of tropical rainforests. Moreover, many orchids are of economic importance as food flavourings or ornamental plants. Phalaenopsis aphrodite, an epiphytic orchid, is a major breeding parent of many commercial orchid hybrids. We provide a high‐quality chromosome‐scale assembly of the P. aphrodite genome. The total length of all scaffolds is 1025.1 Mb, with N50 scaffold size of 19.7 Mb. A total of 28 902 protein‐coding genes were identified. We constructed an orchid genetic linkage map, and then anchored and ordered the genomic scaffolds along the linkage groups. We also established a high‐resolution pachytene karyotype of P. aphrodite and completed the assignment of linkage groups to the 19 chromosomes using fluorescence in situ hybridization. We identified an expansion in the epiphytic orchid lineage of FRS5‐like subclade associated with adaptations to the life in the canopy. Phylogenetic analysis further provides new insights into the orchid lineage‐specific duplications of MADS‐box genes, which might have contributed to the variation in labellum and pollinium morphology and its accessory structure. To our knowledge, this is the first orchid genome to be integrated with a SNP‐based genetic linkage map and validated by physical mapping. The genome and genetic map not only offer unprecedented resources for increasing breeding efficiency in horticultural orchids but also provide an important foundation for future studies in adaptation genomics of epiphytes.

The rice SUB1A-1 gene, which encodes a group VII ethylene response factor (ERFVII), plays a pivotal role in rice survival under flooding stress, as well as other abiotic stresses. In Arabidopsis , five ERFVII factors play roles in regulating hypoxic responses. A characteristic feature of Arabidopsis ERFVIIs is a destabilizing N terminus, which functions as an N-degron that targets them for degradation via the oxygen-dependent N-end rule pathway of proteolysis, but permits their stabilization during hypoxia for hypoxia-responsive signaling. Despite having the canonical N-degron sequence, SUB1A-1 is not under N-end rule regulation, suggesting a distinct hypoxia signaling pathway in rice during submergence. Herein we show that two other rice ERFVIIs gene, ERF66 and ERF67 , are directly transcriptionally up-regulated by SUB1A-1 under submergence. In contrast to SUB1A-1, ERF66 and ERF67 are substrates of the N-end rule pathway that are stabilized under hypoxia and may be responsible for triggering a stronger transcriptional response to promote submergence survival. In support of this, overexpression of ERF66 or ERF67 leads to activation of anaerobic survival genes and enhanced submergence tolerance. Furthermore, by using structural and protein-interaction analyses, we show that the C terminus of SUB1A-1 prevents its degradation via the N-end rule and directly interacts with the SUB1A-1 N terminus, which may explain the enhanced stability of SUB1A-1 despite bearing an N-degron sequence. In summary, our results suggest that SUB1A-1 , ERF66 , and ERF67 form a regulatory cascade involving transcriptional and N-end rule control, which allows rice to distinguish flooding from other SUB1A-1–regulated stresses.

Orchidaceae, the orchid family, encompasses more than 25,000 species and five subfamilies. Due to their beautiful and exotic flowers, distinct biological and ecological features, orchids have aroused wide interest among both researchers and the general public. We constructed the Orchidstra database, a resource for orchid transcriptome assembly and gene annotations. The Orchistra database has been under active development since 2013. To accommodate the increasing amount of orchid transcriptome data and house more comprehensive information, Orchidstra 2.0 has been built with a new database system to store the annotations of 510,947 protein-coding genes and 161,826 noncoding transcripts, covering 18 orchid species belonging to 12 genera in five subfamilies of Orchidaceae. We have improved the N50 size of protein-coding genes, provided new functional annotations (including protein-coding gene annotations, protein domain/family information, pathways analysis, Gene Ontology term assignments, orthologous genes across orchid species, cross-links to the database of model species, and miRNA information), and improved the user interface with better website performance. We also provide new database functionalities for database searching and sequence retrieval. Moreover, the Orchidstra 2.0 database incorporates detailed RNA-Seq gene expression data from various tissues and developmental stages in different orchid species. The database will be useful for gene prediction and gene family studies, and for exploring gene expression in orchid species. The Orchidstra 2.0 database is freely accessible at http://orchidstra2.abrc.sinica.edu.tw.

Calcium ions are a well-known essential component for pollen germination and tube elongation. Several calcium-dependent protein kinases (CDPKs) are expressed predominantly in mature pollen grains and play a critical role in pollen. However, none of their interacting proteins or downstream substrates has been identified. Using yeast two-hybrid screening, we isolated OsCPK25/26-interacting protein 30 (OIP30), which is also predominantly expressed in pollen. OIP30 encodes a RuvB-like DNA helicase 2 (RuvBL2) that is well conserved in eukaryotic species from yeast to human. Yeast and Drosophila defective in RuvBL2 are non-viable. The interaction between OsCPK26 and OIP30 was confirmed by far-Western blot and pull-down experiments. OIP30 was phosphorylated in a calcium-dependent manner by OsCPK26 but not OsCPK2, which is highly similar to OsCPK26 in sequence and expression profile. OIP30 unwound partial duplex DNA with a 3' to 5' directionality by ATP hydrolysis. Concurrently, the ATPase activity of OIP30 depended on single-stranded DNA. OsCPK26 phosphorylated OIP30 and enhanced both its helicase and ATPase activity about 3-fold. OIP30 may be the potential downstream substrate for OsCPK25/26 in pollen. This report characterizes a RuvBL in plants and links its activities with its upstream regulator.

Previously we developed genomic resources for orchids, including transcriptomic analyses using next-generation sequencing techniques and construction of a web-based orchid genomic database. Here, we report a modified molecular model of flower development in the Orchidaceae based on functional analysis of gene expression profiles in Phalaenopsis aphrodite (a moth orchid) that revealed novel roles for the transcription factors involved in floral organ pattern formation. Phalaenopsis orchid floral organ-specific genes were identified by microarray analysis. Several critical transcription factors including AP3, PI, AP1 and AGL6, displayed distinct spatial distribution patterns. Phylogenetic analysis of orchid MADS box genes was conducted to infer the evolutionary relationship among floral organ-specific genes. The results suggest that gene duplication MADS box genes in orchid may have resulted in their gaining novel functions during evolution. Based on these analyses, a modified model of orchid flowering was proposed. Comparison of the expression profiles of flowers of a peloric mutant and wild-type Phalaenopsis orchid further identified genes associated with lip morphology and peloric effects. Large scale investigation of gene expression profiles revealed that homeotic genes from the ABCDE model of flower development classes A and B in the Phalaenopsis orchid have novel functions due to evolutionary diversification, and display differential expression patterns.

Being one of the largest families in the angiosperms, Orchidaceae display a great biodiversity resulting from adaptation to diverse habitats. Genomic information on orchids is rather limited, despite their unique and interesting biological features, thus impeding advanced molecular research. Here we report a strategy to integrate sequence outputs of the moth orchid, Phalaenopsis aphrodite, from two high-throughput sequencing platform technologies, Roche 454 and Illumina/Solexa, in order to maximize assembly efficiency. Tissues collected for cDNA library preparation included a wide range of vegetative and reproductive tissues. We also designed an effective workflow for annotation and functional analysis. After assembly and trimming processes, 233,823 unique sequences were obtained. Among them, 42,590 contigs averaging 875 bp in length were annotated to protein-coding genes, of which 7,263 coding genes were found to be nearly full length. The sequence accuracy of the assembled contigs was validated to be as high as 99.9%. Genes with tissue-specific expression were also categorized by profiling analysis with RNA-Seq. Gene products targeted to specific subcellular localizations were identified by their annotations. We concluded that, with proper assembly to combine outputs of next-generation sequencing platforms, transcriptome information can be enriched in gene discovery, functional annotation and expression profiling of a non-model organism.

A specialized orchid database, named Orchidstra (URL: http://orchidstra.abrc.sinica.edu.tw), has been constructed to collect, annotate and share genomic information for orchid functional genomics studies. The Orchidaceae is a large family of Angiosperms that exhibits extraordinary biodiversity in terms of both the number of species and their distribution worldwide. Orchids exhibit many unique biological features; however, investigation of these traits is currently constrained due to the limited availability of genomic information. Transcriptome information for five orchid species and one commercial hybrid has been included in the Orchidstra database. Altogether, these comprise >380,000 non-redundant orchid transcript sequences, of which >110,000 are protein-coding genes. Sequences from the transcriptome shotgun assembly (TSA) were obtained either from output reads from next-generation sequencing technologies assembled into contigs, or from conventional cDNA library approaches. An annotation pipeline using Gene Ontology, KEGG and Pfam was built to assign gene descriptions and functional annotation to protein-coding genes. Deep sequencing of small RNA was also performed for Phalaenopsis aphrodite to search for microRNAs (miRNAs), extending the information archived for this species to miRNA annotation, precursors and putative target genes. The P. aphrodite transcriptome information was further used to design probes for an oligonucleotide microarray, and expression profiling analysis was carried out. The intensities of hybridized probes derived from microarray assays of various tissues were incorporated into the database as part of the functional evidence. In the future, the content of the Orchidstra database will be expanded with transcriptome data and genomic information from more orchid species.

Orchids display unique phenotypes, functional characteristics and ecological adaptations that are not found in model plants. In this study, we aimed to characterize the microRNA (miRNA) transcriptome and identify species- and tissue-specific miRNAs in Phalaenopsis aphrodite. After data filtering and cleanup, a total of 59,387,374 reads, representing 1,649,996 unique reads, were obtained from four P. aphrodite small RNA libraries. A systematic bioinformatics analysis pipeline was developed that can be used for miRNA and precursor mining, and target gene prediction in non-model plants. A total of 3,251 unique reads for 181 known plant miRNAs (belonging to 88 miRNA families), 23 new miRNAs and 91 precursors were identified. All the miRNA star sequences (miRNA*), the complementary strands of miRNA that from miRNA/miRNA* duplexes, of the predicted new miRNAs were detected in our small RNA libraries, providing additional evidence for their existence as new miRNAs in P. aphrodite. Furthermore, 240 potential miRNA-targets that appear to be involved in many different biological activities and molecular functions, especially transcription factors, were identified, suggesting that miRNAs can impact multiple processes in P. aphrodite. We also verified the cleavage sites for six targets using RNA ligase-mediated rapid amplification of 5′ ends assay. The results provide valuable information about the composition, expression and function of miRNA in P. aphrodite, and will aid functional genomics studies of orchids. Electronic supplementary material The online version of this article (doi:10.1007/s11103-013-0150-0) contains supplementary material, which is available to authorized users.