Yoshihito Shinozaki

Proteomic Analysis of Embryonic Proteins Synthesized from Long-Lived mRNAs During Germination of Rice Seeds

Dry seeds contain translatable, long-lived mRNAs that are stored during seed maturation. Early studies using transcriptional inhibitors supported the view that protein synthesis during the initial phase of germination occurs on long-lived mRNA templates. Rice seeds were treated with the transcriptional inhibitor actinomycin D (Act D), and the embryonic proteins translated from long-lived mRNAs during germination were identified using a proteomic analysis. De novo transcription was not required for germination of rice seeds, since >80% of seeds germinated when transcription was prevented by treatment with Act D. In contrast, germination was completely inhibited in the presence of cycloheximide, an inhibitor of translation. Thus, de novo protein synthesis is necessary for germination of rice seeds. The proteomic analysis revealed that 20 proteins are up-regulated during germination, even after Act D treatment. Many of the up-regulated proteins are involved in carbohydrate metabolism and cytoskeleton formation. These results indicate that some of the germination-specific proteins involved in energy production and maintenance of cell structure in rice seeds are synthesized from long-lived mRNAs. The timing of translation of eight up-regulated proteins was clearly later than that of the other up-regulated proteins under conditions in which transcription was inhibited by Act D, suggesting that translation of long-lived mRNAs in rice seeds is regulated according to the germination phase.

Ethylene suppresses tomato (Solanum lycopersicum) fruit set through modification of gibberellin metabolism

Fruit set in angiosperms marks the transition from flowering to fruit production and a commitment to seed dispersal. Studies with Solanum lycopersicum (tomato) fruit have shown that pollination and subsequent fertilization induce the biosynthesis of several hormones, including auxin and gibberellins (GAs), which stimulate fruit set. Circumstantial evidence suggests that the gaseous hormone ethylene may also influence fruit set, but this has yet to be substantiated with molecular or mechanistic data. Here, we examined fruit set at the biochemical and genetic levels, using hormone and inhibitor treatments, and mutants that affect auxin or ethylene signaling. The expression of system-1 ethylene biosynthetic genes and the production of ethylene decreased during pollination-dependent fruit set in wild-type tomato and during pollination-independent fruit set in the auxin hypersensitive mutant iaa9-3. Blocking ethylene perception in emasculated flowers, using either the ethylene-insensitive Sletr1-1 mutation or 1-methylcyclopropene (1-MCP), resulted in elongated parthenocarpic fruit and increased cell expansion, whereas simultaneous treatment with the GA biosynthesis inhibitor paclobutrazol (PAC) inhibited parthenocarpy. Additionally, the application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) to pollinated ovaries reduced fruit set. Furthermore, Sletr1-1 parthenocarpic fruits did not exhibit increased auxin accumulation, but rather had elevated levels of bioactive GAs, most likely reflecting an increase in transcripts encoding the GA-biosynthetic enzyme SlGA20ox3, as well as a reduction in the levels of transcripts encoding the GA-inactivating enzymes SlGA2ox4 and SlGA2ox5. Taken together, our results suggest that ethylene plays a role in tomato fruit set by suppressing GA metabolism.

Genes that influence yield in tomato

Yield is the most important breeding trait of crops. For fruit-bearing plants such as Solanum lycopersicum (tomato), fruit formation directly affects yield. The final fruit size depends on the number and volume of cell layers in the pericarp of the fruit, which is determined by the degree of cell division and expansion in the fertilized ovaries. Thus, fruit yield in tomato is predominantly determined by the efficiency of fruit set and the final cell number and size of the fruits. Through domestication, tomato fruit yield has been markedly increased as a result of mutations associated with fruit size and genetic studies have identified the genes that influence the cell cycle, carpel number and fruit set. Additionally, several lines of evidence have demonstrated that plant hormones control fruit set and size through the delicate regulation of genes that trigger physiological responses associated with fruit expansion. In this review, we introduce the key genes involved in tomato breeding and describe how they affect the physiological processes that contribute to tomato yield.

Application of Digital Image Analysis System for Fine Evaluation of Varietal Differences and the Role of Ethylene in Visible Petal Senescence of Morning Glory

We detected differences in both onset and progression of visible petal senescence among morning glory cultivars by application of a digital image analysis system. The system is based on semiautomated time-lapse measurement of corolla areas. The system could also be applied to evaluate the effects of ethylene and its inhibitor on visible petal senescence. Both onset and progression of visible petal senescence were accelerated by ethylene treatment in all six cultivars tested. Treatment with aminooxyacetic acid (AOA), an ethylene biosynthesis inhibitor, prolonged time to onset of visible petal senescence in three of the six tested cultivars. In contrast, AOA treatment had no effect on duration of visible petal senescence in any tested cultivars. These data suggested differences among morning glory cultivars in the role of endogenous ethylene in controlling onset of visible petal senescence. In addition, we propose a new application of image analysis to fine quantification of time-lapse changes in the shape of plant organs.

High-resolution spatiotemporal transcriptome mapping of tomato fruit development and ripening

Tomato (Solanum lycopersicum) is an established model for studying fruit biology; however, most studies of tomato fruit growth and ripening are based on homogenized pericarp, and do not consider the internal tissues, or the expression signatures of individual cell and tissue types. We present a spatiotemporally resolved transcriptome analysis of tomato fruit ontogeny, using laser microdissection (LM) or hand dissection coupled with RNA-Seq analysis. Regulatory and structural gene networks, including families of transcription factors and hormone synthesis and signaling pathways, are defined across tissue and developmental spectra. The ripening program is revealed as comprising gradients of gene expression, initiating in internal tissues then radiating outward, and basipetally along a latitudinal axis. We also identify spatial variations in the patterns of epigenetic control superimposed on ripening gradients. Functional studies elucidate previously masked regulatory phenomena and relationships, including those associated with fruit quality traits, such as texture, color, aroma, and metabolite profiles.Cell-type transcriptome profiling greatly elucidate organismal development. Here, the authors report a spatiotemporally resolved comprehensive transcriptome analysis of tomato fruit ontogeny and suggest a new model of fruit maturation which initiates in internal tissues then radiates outwards.

The Tomato Expression Atlas

Summary: With the development of new high‐throughput DNA sequencing technologies and decreasing costs, large gene expression datasets are being generated at an accelerating rate, but can be complex to visualize. New, more interactive and intuitive tools are needed to visualize the spatiotemporal context of expression data and help elucidate gene function. Using tomato fruit as a model, we have developed the Tomato Expression Atlas to facilitate effective data analysis, allowing the simultaneous visualization of groups of genes at a cell/tissue level of resolution within an organ, enhancing hypothesis development and testing in addition to candidate gene identification. This atlas can be adapted to different types of expression data from diverse multicellular species. Availability and Implementation: The Tomato Expression Atlas is available at http://tea.solgenomics.net/. Source code is available at https://github.com/solgenomics/Tea. Contact: jr286@cornell.edu or lam87@cornell.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Expression of an AtNAP gene homolog in senescing morning glory (Ipomoea nil) petals of two cultivars with a different flower life span.

AtNAP, a NAC family transcription factor, has been shown to promote leaf senescence in Arabidopsis. We isolated an AtNAP homolog in morning glory (Ipomoea nil), designated InNAP, and investigated its expression during petal senescence. We used two cultivars, one showing a normal short flower life span (cv. Peking Tendan) and another a longer life span (cv. Violet). InNAP was highly expressed in both cultivars. Expression was high before that of the senescence marker gene InSAG12. InNAP and InSAG12 expression was high in cv. Peking Tendan before cv. Violet. The expression of both genes was therefore temporally related to the onset of the visible senescence symptoms. An inhibitor of ethylene action (silver thiosulphate, STS) delayed petal senescence in cv. Peking Tendan but had no effect in cv. Violet. STS treatment had no clear effect on the InNAP expression in petals of both cultivars, suggesting that endogenous ethylene may not be necessary for its induction. These data suggest the hypothesis that InNAP plays a role in petal senescence, independent of the role of endogenous ethylene.

Laser microdissection of tomato fruit cell and tissue types for transcriptome profiling

This protocol enables transcriptome profiling of specific cell or tissue types that are isolated from tomato using laser microdissection (LM). To prepare tissue for LM, fruit samples are first fixed in optimal cutting temperature (OCT) medium and frozen in molds. The tissue is then sectioned using a cryostat before being dissected using an LM instrument. The RNAs contained in the harvested cells are purified and subjected to two rounds of amplification to yield sufficient quantities of RNA to generate cDNA libraries. Unlike several other techniques that are used to isolate specific cell types, LM has the advantage of being readily applied to any plant species without having to generate transgenic plants. Using the protocols described here, LM-mediated cell-type transcriptomic analysis of two samples requires ∼8 d from tissue harvest to RNA sequencing (RNA-seq), whereas each additional sample, up to a total of 12 samples, requires ∼1 additional day for the LM step. RNA obtained using this method has been successfully used for deep-coverage transcriptome profiling, which is a particularly effective strategy for identifying genes that are differentially expressed between cell or tissue types.

Length of the dark period affects flower opening and the expression of circadian-clock associated genes as well as xyloglucan endotransglucosylase/hydrolase genes in petals of morning glory (Ipomoea nil).

Key messageWe isolated differentially expressed and dark-responsive genes during flower development and opening in petals of morning glory.AbstractFlower opening usually depends on petal expansion and is regulated by both genetic and environmental factors. Flower opening in morning glory (Ipomoea nil) is controlled by the dark/light regime just prior to opening. Opening was normal after 8- or 12-h dark periods but progressed very slowly after a 4-h dark period or in continuous light. Four genes (InXTH1–InXTH4) encoding xyloglucan endotransglucosylase/hydrolases (XTHs) and three genes (InEXPA1–InEXPA3) encoding alpha-expansins (EXPAs) were isolated. The expression patterns of InXTH2, InXTH3, and InXTH4 in petals were closely correlated with the rate of flower opening controlled by the length of the dark period prior to opening, but those of the EXPA genes were not. The expression pattern of InXTH1 gene was closely correlated with petal elongation. Suppression subtractive hybridization was used to isolate dark-responsive genes accompanying flower opening. The expressions of ten isolated genes were associated with the length of the dark period prior to flower opening. One gene was highly homologous to Arabidopsis PSEUDO-RESPONSE REGULATOR7, which is associated with the circadian clock and phytochrome signaling; another to Arabidopsis REVEILLE1, which affects the output of the circadian clock. Other genes were related to light responses, plant hormone effects and signal transduction. The possible roles of these genes in regulation of flower opening are discussed.

The role of ethylene in the regulation of ovary senescence and fruit set in tomato (Solanum lycopersicum)

ABSTRACT Fruit set is the developmental transition from ovary to young fruit, and generally requires pollination and fertilization. Although the mechanism for fruit set remains elusive, several lines of evidence have demonstrated that fruit set is triggered by activated metabolism of or increased sensitivity to the plant hormones auxin or gibberellins (GAs), which stimulate cell division and expansion within the ovary. Our recent study with tomato (Solanum lycopersicum) suggested that the gaseous hormone ethylene connects auxin and GA, suppressing initiation of fruit set by down-regulating GA accumulation. By contrast, reduced sensitivity to ethylene triggers accumulation of GA, but not auxin, through increasing bioactive GA biosynthesis and decreasing GA inactivation. These changes induce parthenocarpy accompanied by pollination-independent cell expansion in the ovary. Here, we provide evidence that ethylene likely promotes mRNA expression of the senescence-associated genes SlSAG12 and SlNAP in unpollinated ovaries. These results suggest that ethylene acts downstream of auxin and upstream of GA, and also suggest that ethylene promotes senescence of ovary that fail to set fruit in tomato.