Differential activity of F-box genes and E3 ligases distinguishes sexual versus apomictic germline specification in Boechera.

Germline specification is the first step during sexual and apomictic plant reproduction, and takes place in the nucellus of the ovule, a specialized domain of the reproductive flower tissues. In each case, a sporophytic cell is determined to form the sexual megaspore mother cell (MMC) or an apomictic initial cell (AIC). These differ in their developmental fates: while the MMC undergoes meiosis, the AIC modifies or omits meiosis to form the female gametophyte. Despite great interest in these distinct developmental processes, little is known about their gene regulatory basis. To elucidate the gene regulatory networks underlying germline specification, we conducted tissue-specific transcriptional profiling using laser-assisted microdissection and RNA sequencing to compare the transcriptomes of nucellar tissues between different sexual and apomictic Boechera accessions representing four species and two ploidy levels. This allowed us to distinguish between expression differences caused by genetic background or reproductive mode. Statistical data analysis revealed 45 genes that were significantly differentially expressed, and which potentially play a role for determination of the reproductive mode. Based on annotations, these included F-box genes and E3 ligases that most likely relate to genes previously described as regulators important for germline development. Our findings provide novel insights into the transcriptional basis of sexual and apomictic reproduction.

Temporal and spatial changes in gene expression, metabolite accumulation and phytohormone content in rice seedlings grown under drought stress conditions.

In order to analyze the molecular mechanisms underlying the responses of plants to different levels of drought stress, we developed a soil matric potential (SMP)-based irrigation system that precisely controls soil moisture. Using this system, rice seedlings were grown under three different drought levels, denoted Md1, Md2 and Md3, with SMP values set to -9.8, -31.0 and -309.9 kPa, respectively. Although the Md1 treatment did not alter the visible phenotype, the Md2 treatment caused stomatal closure and shoot growth retardation (SGR). The Md3 treatment markedly induced SGR, without inhibition of photosynthesis. More severe drought (Sds) treatment, under which irrigation was terminated, resulted in the wilting of leaves and inhibition of photosynthesis. Metabolome analysis revealed the accumulation of primary sugars under Md3 and Sds and of most amino acids under Sds. The starch content was increased under Md3 and decreased under Sds. Transcriptome data showed that the expression profiles of associated genes supported the observed changes in photosynthesis and metabolites, suggesting that the time lag from SGR to inhibition of photosynthesis might lead to the accumulation of photosynthates under Md3, which can be used as osmolytes under Sds. To gain further insight into the observed SGR, transcriptome and hormonome analyses were performed in specific tissues. The results showed specific decreases in indole-3-acetic acid (IAA) and cytokinin levels in Md2-, Md3- and Sds-treated shoot bases, though the expression levels of hormone metabolism-related genes were not reflected in IAA and cytokinin contents. These observations suggest that drought stress affects the distribution or degradation of cytokinin and IAA molecules.

New insight into the genetic basis of oil content based on noninvasive three-dimensional phenotyping and tissue-specific transcriptome in Brassica napus.

BACKGROUND: Increasing seed oil content is the most important breeding goal in Brassica napus, and phenotyping is crucial to dissect its genetic basis in crops. To date, QTL mapping for oil content has been based on whole seeds, and the lipid distribution is far from uniform in different tissues of seeds in B. napus. In this case, the phenotype based on whole seeds was unable to sufficiently reveal the complex genetic characteristics of seed oil content. RESULTS: Here, the three-dimensional (3D) distribution of lipid was determined for B. napus seeds by magnetic resonance imaging (MRI) and 3D quantitative analysis, and ten novel oil content-related traits were obtained by subdividing the seeds. Based on a high-density genetic linkage map, 35 QTLs were identified for 4 tissues, the outer cotyledon (OC), inner cotyledon (IC), radicle (R) and seed coat (SC), which explained up to 13.76% of the phenotypic variation. Notably, 14 tissue-specific QTLs were reported for the first time, 7 of which were novel. Moreover, haplotype analysis showed that the favorable alleles for different seed tissues exhibited cumulative effects on oil content. Furthermore, tissue-specific transcriptomes revealed that more active energy and pyruvate metabolism influenced carbon flow in the IC, OC and R than in the SC at the early and middle seed development stages, thus affecting the distribution difference in oil content. Combining tissue-specific QTL mapping and transcriptomics, 86 important candidate genes associated with lipid metabolism were identified that underlie 19 unique QTLs, including the fatty acid synthesis rate-limiting enzyme-related gene CAC2, in the QTLs for OC and IC. CONCLUSIONS: The present study provides further insight into the genetic basis of seed oil content at the tissue-specific level.

Identification of Conserved Gene-Regulatory Networks that Integrate Environmental Sensing and Growth in the Root Cambium.

Cambium drives the lateral growth of stems and roots, contributing to diverse plant growth forms. The root crop is one of the outstanding examples of the cambium-driven growth. To understand its molecular basis, we used radish to generate a compendium of root-tissue- and stage-specific transcriptomes from two contrasting inbred lines during root growth. Expression patterns of key cambium regulators and hormone signaling components were validated. Clustering and gene ontology (GO) enrichment analyses of radish datasets followed by a comparative analysis against the newly established Arabidopsis early cambium data revealed evolutionary conserved stress-response transcription factors that may intimately control the cambium. Indeed, an in vivo network consisting of selected stress-response and cambium regulators indicated ERF-1 as a potential key checkpoint of cambial activities, explaining how cambium-driven growth is altered in response to environmental changes. The findings here provide valuable information about dynamic gene expression changes during cambium-driven root growth and have implications with regard to future engineering schemes, leading to better crop yields.

Wood Transcriptome Profiling Identifies Critical Pathway Genes of Secondary Wall Biosynthesis and Novel Regulators for Vascular Cambium Development in Populus.

Wood, the most abundant biomass on Earth, is composed of secondary xylem differentiated from vascular cambium. However, the underlying molecular mechanisms of wood formation remain largely unclear. To gain insight into wood formation, we performed a series of wood-forming tissue-specific transcriptome analyses from a hybrid poplar (Populus alba × P. glandulosa, clone BH) using RNA-seq. Together with shoot apex and leaf tissue, cambium and xylem tissues were isolated from vertical stem segments representing a gradient of secondary growth developmental stages (i.e., immature, intermediate, and mature stem). In a comparative transcriptome analysis of the 'developing xylem' and 'leaf' tissue, we could identify critical players catalyzing each biosynthetic step of secondary wall components (e.g., cellulose, xylan, and lignin). Several candidate genes involved in the initiation of vascular cambium formation were found via a co-expression network analysis using abundantly expressed genes in the 'intermediate stem-derived cambium' tissue. We found that transgenic Arabidopsis plants overexpressing the PtrHAM4-1, a GRAS family transcription factor, resulted in a significant increase of vascular cambium development. This phenotype was successfully reproduced in the transgenic poplars overexpressing the PtrHAM4-1. Taken together, our results may serve as a springboard for further research to unravel the molecular mechanism of wood formation, one of the most important biological processes on this planet.

Tissue-Specific Transcriptome and Hormonal Regulation of Pollinated and Parthenocarpic Fig (Ficus carica L.) Fruit Suggest that Fruit Ripening Is Coordinated by the Reproductive Part of the Syconium.

In the unconventional climacteric fig (Ficus carica) fruit, pollinated and parthenocarpic fruit of the same genotype exhibit different ripening characteristics. Integrative comparative analyses of tissue-specific transcript and of hormone levels during fruit repining from pollinated vs. parthenocarpic fig fruit were employed to unravel the similarities and differences in their regulatory processes during fruit repining. Assembling tissue-specific transcripts into 147,000 transcripts with 53,000 annotated genes provided new insights into the spatial distribution of many classes of regulatory and structural genes, including those related to color, taste and aroma, storage, protein degradation, seeds and embryos, chlorophyll, and hormones. Comparison of the pollinated and parthenocarpic tissues during fruit ripening showed differential gene expression, especially in the fruit inflorescence. The distinct physiological green phase II and ripening phase III differed significantly in their gene-transcript patterns in both pulp and inflorescence tissues. Gas chromatographic analysis of whole fruits enabled the first determination of ripening-related hormone levels from pollinated and non-pollinated figs. Ethylene and auxin both increased during fruit ripening, irrespective of pollination, whereas no production of active gibberellins or cytokinins was found in parthenocarpic or pollinated ripening fruit. Tissue-specific transcriptome revealed apparent different metabolic gene patterns for ethylene, auxin and ABA in pollinated vs. parthenocarpic fruit, mostly in the fruit inflorescence. Our results demonstrate that the production of abscisic acid (ABA), non-active ABA-GE conjugate and non-active indoleacetic acid (IAA)-Asp conjugate in pollinated fruits is much higher than in parthenocarpic fruits. We suggest that fruit ripening is coordinated by the reproductive part of the syconium and the differences in ABA production between pollinated and parthenocarpic fig fruit might be the key to their different ripening characteristics.

Tissue-specific transcriptomics in the field cricket Teleogryllus oceanicus.

Field crickets (family Gryllidae) frequently are used in studies of behavioral genetics, sexual selection, and sexual conflict, but there have been no studies of transcriptomic differences among different tissue types. We evaluated transcriptome variation among testis, accessory gland, and the remaining whole-body preparations from males of the field cricket, Teleogryllus oceanicus. Non-normalized cDNA libraries from each tissue were sequenced on the Roche 454 platform, and a master assembly was constructed using testis, accessory gland, and whole-body preparations. A total of 940,200 reads were assembled into 41,962 contigs, to which 36,856 singletons (reads not assembled into a contig) were added to provide a total of 78,818 sequences used in annotation analysis. A total of 59,072 sequences (75%) were unique to one of the three tissues. Testis tissue had the greatest proportion of tissue-specific sequences (62.6%), followed by general body (56.43%) and accessory gland tissue (44.16%). We tested the hypothesis that tissues expressing gene products expected to evolve rapidly as a result of sexual selection--testis and accessory gland--would yield a smaller proportion of BLASTx matches to homologous genes in the model organism Drosophila melanogaster compared with whole-body tissue. Uniquely expressed sequences in both testis and accessory gland showed a significantly lower rate of matching to annotated D. melanogaster genes compared with those from general body tissue. These results correspond with empirical evidence that genes expressed in testis and accessory gland tissue are rapidly evolving targets of selection.