Morphology and chemical composition of Taiwan oil millet (Eccoilopus formosanus) epicuticular wax.

MAIN CONCLUSION: Taiwan oil millet has two types of epicuticular wax: platelet wax composed primarily of octacosanol and filament wax constituted essentially by the singular compound of octacosanoic acid. Taiwan oil millet (TOM-Eccoilopus formosanus) is an orphan crop cultivated by the Taiwan indigenous people. It has conspicuous white powder covering its leaf sheath indicating abundant epicuticular waxes, that may contribute to its resilience. Here, we characterized the epicuticular wax secretion in TOM leaf blade and leaf sheath using various microscopy techniques, as well as gas chromatography to determine its composition. Two kinds of waxes, platelet and filaments, were secreted in both the leaf blades and sheaths. The platelet wax is secreted ubiquitously by epidermal cells, whereas the filament wax is secreted by a specific cell called epidermal cork cells. The newly developed filament waxes were markedly re-synthesized by the epidermal cork cells through papillae protrusions on the external periclinal cell wall. Ultrastructural images of cork cell revealed the presence of cortical endoplasmic reticulum (ER) tubules along the periphery of plasma membrane (PM) and ER-PM contact sites (EPCS). The predominant wax component was a C28 primary alcohol in leaf blade, and a C28 free fatty acid in the leaf sheath, pseudopetiole and midrib. The wax morphology present in distinct plant organs corresponds to the specific chemical composition: platelet wax composed of alcohols exists mainly in the leaf blade, whereas filament wax constituted mainly by the singular compound C28 free fatty acids is present abundantly in leaf sheath. Our study clarifies the filament wax composition in relation to a previous study in sorghum. Both platelet and filament waxes comprise a protection barrier for TOM.

A drought-responsive rice amidohydrolase is the elusive plant guanine deaminase with the potential to modulate the epigenome.

Drought stress in plants causes differential expression of numerous genes. One of these differentially expressed genes in rice is a specific amidohydrolase. We characterized this amidohydrolase gene on the rice chromosome 12 as the first plant guanine deaminase (OsGDA1). The biochemical activity of GDA is known from tea and coffee plants where its catalytic product, xanthine, is the precursor for theine and caffeine. However, no plant gene that is coding for GDA is known so far. Recombinant OsGDA1 converted guanine to xanthine in vitro. Measurement of guanine and xanthine contents in the OsGDA1 knockout (KO) line and in the wild type Tainung 67 rice plants also suggested GDA activity in vivo. The content of cellular xanthine is important because of its catabolic products allantoin, ureides, and urea which play roles in water and nitrogen stress tolerance among others. The identification of OsGDA1 fills a critical gap in the S-adenosyl-methionine (SAM) to xanthine pathway. SAM is converted to S-adenosyl-homocysteine (SAH) and finally to xanthine. SAH is a potent inhibitor of DNA methyltransferases, the reduction of which leads to increased DNA methylation and gene silencing in Arabidopsis. We report that the OsGDA1 KO line exhibited a decrease in SAM, SAH and adenosine and an increase in rice genome methylation. The OsGDA1 protein phylogeny combined with mutational protein destabilization analysis suggested artificial selection for null mutants, which could affect genome methylation as in the KO line. Limited information on genes that may affect epigenetics indirectly requires deeper insights into such a role and effect of purine catabolism and related genetic networks.

Rice Big Grain 1 promotes cell division to enhance organ development, stress tolerance and grain yield.

Grain/seed yield and plant stress tolerance are two major traits that determine the yield potential of many crops. In cereals, grain size is one of the key factors affecting grain yield. Here, we identify and characterize a newly discovered gene Rice Big Grain 1 (RBG1) that regulates grain and organ development, as well as abiotic stress tolerance. Ectopic expression of RBG1 leads to significant increases in the size of not only grains but also other major organs such as roots, shoots and panicles. Increased grain size is primarily due to elevated cell numbers rather than cell enlargement. RBG1 is preferentially expressed in meristematic and proliferating tissues. Ectopic expression of RBG1 promotes cell division, and RBG1 co-localizes with microtubules known to be involved in cell division, which may account for the increase in organ size. Ectopic expression of RBG1 also increases auxin accumulation and sensitivity, which facilitates root development, particularly crown roots. Moreover, overexpression of RBG1 up-regulated a large number of heat-shock proteins, leading to enhanced tolerance to heat, osmotic and salt stresses, as well as rapid recovery from water-deficit stress. Ectopic expression of RBG1 regulated by a specific constitutive promoter, GOS2, enhanced harvest index and grain yield in rice. Taken together, we have discovered that RBG1 regulates two distinct and important traits in rice, namely grain yield and stress tolerance, via its effects on cell division, auxin and stress protein induction.

MicroRNA-mediated regulation of gene expression in the response of rice plants to fungal elicitors.

MicroRNAs (miRNAs) are small non-coding RNAs that have important regulatory functions in plant growth, development, and response to abiotic stress. Increasing evidence also supports that plant miRNAs contribute to immune responses to pathogens. Here, we used deep sequencing of small RNA libraries for global identification of rice miRNAs that are regulated by fungal elicitors. We also describe 9 previously uncharacterized miRNAs in rice. Combined small RNA and degradome analyses revealed regulatory networks enriched in elicitor-regulated miRNAs supported by the identification of their corresponding target genes. Specifically, we identified an important number of miRNA/target gene pairs involved in small RNA pathways, including miRNA, heterochromatic and trans-acting siRNA pathways. We present evidence for miRNA/target gene pairs implicated in hormone signaling and cross-talk among hormone pathways having great potential in regulating rice immunity. Furthermore, we describe miRNA-mediated regulation of Conserved-Peptide upstream Open Reading Frame (CPuORF)-containing genes in rice, which suggests the existence of a novel regulatory network that integrates miRNA and CPuORF functions in plants. The knowledge gained in this study will help in understanding the underlying regulatory mechanisms of miRNAs in rice immunity and develop appropriate strategies for rice protection.

Transcriptomes of the anther sporophyte: availability and uses.

An anther includes sporophytic tissues of three outer cell layers and an innermost layer, the tapetum, which encloses a locule where the gametophytic microspores mature to become pollen. The sporophytic tissues also comprise some vascular cells and specialized cells of the stomium aligning the long anther axis for anther dehiscence. Studies of the anther sporophytic cells, especially the tapetum, have recently expanded from the use of microscopy to molecular biology and transcriptomes. The available sequencing technologies, plus the use of laser microdissection and in silico subtraction, have produced high-quality anther sporophyte transcriptomes of rice, Arabidopsis and maize. These transcriptomes have been used for research discoveries and have potential for future discoveries in diverse areas, including developmental gene activity networking and changes in enzyme and metabolic domains, prediction of protein functions by quantity, secretion, antisense transcript regulation, small RNAs and promoters for generating male sterility. We anticipate that these studies with rice and other transcriptomes will expand to encompass other plants, whose genomes will be sequenced soon, with ever-advancing sequencing technologies. In comprehensive gene activity profiling of the anther sporophyte, studies involving transcriptomes will spearhead investigation of the downstream gene activity with proteomics and metabolomics.

Studies of rice Hd1 haplotypes worldwide reveal adaptation of flowering time to different environments.

Rice domestication/adaptation is a good model for studies of the development and spread of this important crop. Mutations that caused morphological and physiological change, followed by human selection/expansion, finally led to the improvement of phenotypes suitable for different kinds of environments. We used the sequence information for Heading date 1 (Hd1) gene to reveal the association between sequence changes and flowering phenotypes of rice in different regions. Seven loss-of-function hd1 haplotypes had been reported. By data-mining the genome sequencing information in the public domain, we discovered 3 other types. These loss-of-function allele haplotypes are present in subtropical and tropical regions, which indicates human selection. Some of these haplotypes are present locally. However, types 7 and 13 are present in more than one-third of the world's rice accessions, including landraces and modern varieties. In the present study, phylogenetic, allele network and selection pressure analyses revealed that these two haplotypes might have occurred early in Southeastern Asia and then were introgressed in many local landraces in nearby regions. We also demonstrate that these haplotypes are present in weedy rice populations, which again indicates that these alleles were present in rice cultivation for long time. In comparing the wild rice sequence information, these loss-of-function haplotypes occurred in agro but were not from wild rice.

A northern Chinese origin of Austronesian agriculture: new evidence on traditional Formosan cereals.

BACKGROUND: Genetic data for traditional Taiwanese (Formosan) agriculture is essential for tracing the origins on the East Asian mainland of the Austronesian language family, whose homeland is generally placed in Taiwan. Three main models for the origins of the Taiwanese Neolithic have been proposed: origins in coastal north China (Shandong); in coastal central China (Yangtze Valley), and in coastal south China. A combination of linguistic and agricultural evidence helps resolve this controversial issue. RESULTS: We report on botanically informed linguistic fieldwork of the agricultural vocabulary of Formosan aborigines, which converges with earlier findings in archaeology, genetics and historical linguistics to assign a lesser role for rice than was earlier thought, and a more important one for the millets. We next present the results of an investigation of domestication genes in a collection of traditional rice landraces maintained by the Formosan aborigines over a hundred years ago. The genes controlling awn length, shattering, caryopsis color, plant and panicle shapes contain the same mutated sequences as modern rice varieties everywhere else in the world, arguing against an independent domestication in south China or Taiwan. Early and traditional Formosan agriculture was based on foxtail millet, broomcorn millet and rice. We trace this suite of cereals to northeastern China in the period 6000-5000 BCE and argue, following earlier proposals, that the precursors of the Austronesians, expanded south along the coast from Shandong after c. 5000 BCE to reach northwest Taiwan in the second half of the 4th millennium BCE. This expansion introduced to Taiwan a mixed farming, fishing and intertidal foraging subsistence strategy; domesticated foxtail millet, broomcorn millet and japonica rice; a belief in the sacredness of foxtail millet; ritual ablation of the upper incisors in adolescents of both sexes; domesticated dogs; and a technological package including inter alia houses, nautical technology, and loom weaving. CONCLUSION: We suggest that the pre-Austronesians expanded south along the coast from that region after c. 5000 BCE to reach northwest Taiwan in the second half of the 4th millennium BCE.

Broomcorn and foxtail millet were cultivated in Taiwan about 5000 years ago.

BACKGROUND: Archaeobotanical remains of millet were found at the Nan-kuan-li East site in Tainan Science Park, southern Taiwan. This site, dated around 5000-4300 BP, is characterized by remains of the Tapenkeng culture, the earliest Neolithic culture found so far in Taiwan. A large number of millet-like carbonized and charred seeds with varied sizes and shapes were unearthed from the site by the flotation method. Since no millet grain was ever found archaeologically in Taiwan previously, this discovery is of great importance and significance. This paper is in an attempt to further analyze these plant remains for a clearer understanding of the agricultural practice of the ancient inhabitants of the Nan-kuan-li East site. RESULT: We used light and scanning electron microscopy to examine the morphological features of some modern domesticated and unearthed seeds to compare and identify the archaeobotanical remains by three criteria: caryopsis shape, embryo notch, and morphology of lemma and palea. We also developed a new methodology for distinguishing the excavated foxtail and broomcorn millet seeds. CONCLUSION: Two domesticated millet, including broomcorn millet (Panicum miliaceum) and foxtail millet (Setaria italica), as well as one wild millet species, yellow foxtail (Setaria glauca), were identified in the unearthed seeds. Together with the millet remains, rice was also cultivated in the area. Archaeological evidence shows that millet and rice farming may have been important food sources for people living about 5000 years ago in southern Taiwan.

Genome-Wide Analysis of Polycistronic MicroRNAs in Cultivated and Wild Rice.

MicroRNAs (miRNAs) are small noncoding RNAs that direct posttranscriptional gene silencing in eukaryotes. They are frequently clustered in the genomes of animals and can be independently transcribed or simultaneously transcribed into single polycistronic transcripts. Only a few miRNA clusters have been described in plants, and most of them are generated from independent transcriptional units. Here, we used a combination of bioinformatic tools and experimental analyses to discover new polycistronic miRNAs in rice. A genome-wide analysis of clustering patterns of MIRNA loci in the rice genome was carried out using a criterion of 3 kb as the maximal distance between two miRNAs. This analysis revealed 28 loci with the ability to form the typical hairpin structure of miRNA precursors in which 2 or more mature miRNAs mapped along the same structure. RT-PCR provided evidence for the polycistronic nature of seven miRNA precursors containing homologous or nonhomologous miRNA species. Polycistronic miRNAs and candidate polycistronic miRNAs are located across different rice chromosomes, except chromosome 12, and resided in both duplicated and nonduplicated chromosomal regions. Finally, most polycistronic and candidate polycistronic miRNAs showed a pattern of conservation in the genome of rice species with an AA genome. The diversity in the organization of MIR genes that are transcribed as polycistrons suggests a versatile mechanism for the control of gene expression in different biological processes and supports additional levels of complexity in miRNA functioning in plants.

Lack of Genotype and Phenotype Correlation in a Rice T-DNA Tagged Line Is Likely Caused by Introgression in the Seed Source.

Rice (Oryza sativa) is one of the most important crops in the world. Several rice insertional mutant libraries are publicly available for systematic analysis of gene functions. However, the tagging efficiency of these mutant resources-the relationship between genotype and phenotype-is very low. We used whole-genome sequencing to analyze a T-DNA-tagged transformant from the Taiwan Rice Insertional Mutants (TRIM) resource. The phenomics records for M0028590, one of the TRIM lines, revealed three phenotypes-wild type, large grains, and tillering dwarf-in the 12 T1 plants. Using the sequencing data for 7 plants from three generations of this specific line, we demonstrate that introgression from an indica rice variety might occur in one generation before the seed was used for callus generation and transformation of this line. In addition, the large-grain trait came from the GS3 gene of the introgressed region and the tillering dwarf phenotype came from a single nucleotide change in the D17 gene that occurred during the callus induction to regeneration of the transformant. As well, another regenerant showed completely heterozygous single-nucleotide polymorphisms across the whole genome. In addition to the known sequence changes such as T-DNA integration, single nucleotide polymorphism, insertion, deletion, chromosome rearrangement and doubling, spontaneous outcrossing occurred in the rice field may also explain some mutated traits in a tagged mutant population. Thus, the co-segregation of an integration event and the phenotype should be checked when using these mutant populations.

Transcriptome profiling of leaf elongation zone under drought in contrasting rice cultivars.

Inhibition of leaf elongation and expansion is one of the earliest responses of rice to water deficit. Despite this sensitivity, a great deal of genetic variation exists in the extant of leaf elongation rate (LER) reduction in response to declining soil moisture. We analyzed global gene expression in the leaf elongation zone under drought in two rice cultivars with disparate LER sensitivities to water stress. We found little overlap in gene regulation between the two varieties under moderate drought; however, the transcriptional response to severe drought was more conserved. In response to moderate drought, we found several genes related to secondary cell wall deposition that were down regulated in Moroberekan, an LER tolerant variety, but up-regulated in LER sensitive variety IR64.

Analyses of advanced rice anther transcriptomes reveal global tapetum secretory functions and potential proteins for lipid exine formation.

The anthers in flowers perform important functions in sexual reproduction. Several recent studies used microarrays to study anther transcriptomes to explore genes controlling anther development. To analyze the secretion and other functions of the tapetum, we produced transcriptomes of anthers of rice (Oryza sativa subsp. japonica) at six progressive developmental stages and pollen with sequencing-by-synthesis technology. The transcriptomes included at least 18,000 unique transcripts, about 25% of which had antisense transcripts. In silico anther-minus-pollen subtraction produced transcripts largely unique to the tapetum; these transcripts include all the reported tapetum-specific transcripts of orthologs in other species. The differential developmental profiles of the transcripts and their antisense transcripts signify extensive regulation of gene expression in the anther, especially the tapetum, during development. The transcriptomes were used to dissect two major cell/biochemical functions of the tapetum. First, we categorized and charted the developmental profiles of all transcripts encoding secretory proteins present in the cellular exterior; these transcripts represent about 12% and 30% of the those transcripts having more than 100 and 1,000 transcripts per million, respectively. Second, we successfully selected from hundreds of transcripts several transcripts encoding potential proteins for lipid exine synthesis during early anther development. These proteins include cytochrome P450, acyltransferases, and lipid transfer proteins in our hypothesized mechanism of exine synthesis in and export from the tapetum. Putative functioning of these proteins in exine formation is consistent with proteins and metabolites detected in the anther locule fluid obtained by micropipetting.

Oil bodies and oleosins in Physcomitrella possess characteristics representative of early trends in evolution.

Searches of sequenced genomes of diverse organisms revealed that the moss Physcomitrella patens is the most primitive organism possessing oleosin genes. Microscopy examination of Physcomitrella revealed that oil bodies (OBs) were abundant in the photosynthetic vegetative gametophyte and the reproductive spore. Chromatography illustrated the neutral lipids in OBs isolated from the gametophyte to be largely steryl esters and triacylglycerols, and SDS-PAGE showed the major proteins to be oleosins. Reverse transcription-PCR revealed the expression of all three oleosin genes to be tissue specific. This tissue specificity was greatly altered via alternative splicing, a control mechanism of oleosin gene expression unknown in higher plants. During the production of sex organs at the tips of gametophyte branches, the number of OBs in the top gametophyte tissue decreased concomitant with increases in the number of peroxisomes and level of transcripts encoding the glyoxylate cycle enzymes; thus, the OBs are food reserves for gluconeogenesis. In spores during germination, peroxisomes adjacent to OBs, along with transcripts encoding the glyoxylate cycle enzymes, appeared; thus, the spore OBs are food reserves for gluconeogenesis and equivalent to seed OBs. The one-cell-layer gametophyte could be observed easily with confocal microscopy for the subcellular OBs and other structures. Transient expression of various gene constructs transformed into gametophyte cells revealed that all OBs were linked to the endoplasmic reticulum (ER), that oleosins were synthesized in extended regions of the ER, and that two different oleosins were colocated in all OBs.