Characterization of the CMS genetic regulation through comparative complete mitochondrial genome sequencing in Nicotiana tabacum.

Mitochondrial genomes (mitogenomes) of flowering plants vary greatly in structure and size, which can lead to frequent gene mutation, rearrangement, or recombination, then result in the cytoplasmic male sterile (CMS) mutants. In tobacco (Nicotiana tabacum), suaCMS lines are widely used in heterosis breeding; however, the related genetic regulations are not very clear. In this study, the cytological observation indicated that the pollen abortion of tobacco suaCMS(HD) occurred at the very early stage of the stamen primordia differentiation. In this study, the complete mitochondrial genomes of suaCMS(HD) and its maintainer HD were sequenced using the PacBio and Illumina Hiseq technology. The total length of the assembled mitogenomes of suaCMS(HD) and HD was 494,317 bp and 430,694 bp, respectively. Comparative analysis indicated that the expanded 64 K bases in suaCMS(HD) were mainly located in noncoding regions, and 23 and 21 big syntenic blocks (>5000 bp) were found in suaCMS(HD) and HD with a series of repeats. Electron transport chain-related genes were highly conserved in two mitogenomes, except five genes (ATP4, ATP6, COX2, CcmFC, and SDH3) with substantial substitutions. Three suaCMS(HD)-specific genes, orf261, orf291, and orf433, were screened. Sequence analysis and RT-PCR verification showed that they were unique to suaCMS(HD). Further gene location analysis and protein property prediction indicated that all the three genes were likely candidates for suaCMS(HD). This study provides new insight into understanding the suaCMS mechanism and is useful for improving tobacco breeding.

Integration of metabolomics and transcriptomics analyses investigates the accumulation of secondary metabolites in maturing seed plumule of sacred lotus (Nelumbo nucifera).

Lotus seed plumule (LP) is rich in a variety of antioxidant and anti-inflammatory secondary metabolites, making it a traditional food and medicine widely used in China. Physiological and histological evidences indicated that LP mainly accumulated metabolites in 15-24 days after pollination (DAP) during their development. To systematically investigate the dynamic accumulation of major secondary metabolites, the UPLC-HRMS-based widely targeted metabolomics analyses were performed on maturing LP at 15, 18, 21, and 24 DAP. In total, 767 metabolites were identified, including many secondary metabolites, e.g., 27 % flavonoids and 8 % alkaloids. Among them, 591 were identified as differentially accumulated metabolites (DAMs). The majority of secondary metabolites showed great accumulation after 18 DAP even at the late stage of LP maturation, such as hesperidin, neohesperidin, orobol, serotonin, and lotus special O-nornuciferine, endowing mature LP with effective pharmaceutical properties. The paralleled transcriptomic analysis identified 11,019 differentially expressed genes (DEGs). Based on the comprehensive data, several systematical metabolic regulation maps were established for different secondary metabolites, and 18 DAP was found as a switching point for LP maturing from active primary metabolism to massive secondary metabolites deposition. This study provides valuable information for understanding the mechanism of secondary metabolite accumulation in maturing LP and facilitates its pharmaceutical application.

Quantitative ubiquitylomics approach for characterizing the dynamic change and extensive modulation of ubiquitylation in rice seed germination.

During seed germination, cells embark on extensive post-transcriptional and post-translational modifications (PTM), providing a perfect platform to study these events in embryo rebooting from relative quiescenct to highly active state. PR-619, a deubiquitylase inhibitor, delayed the rice seed germination and resulted in the accumulation of ubiquitylated proteins, which indicated the protein ubiquitylation is involved in this process. Using the K-Ɛ-GG antibody enrichment method integrated with high-resolution mass spectrometry, a list of 2576 lysine ubiquitylated (Kub) sites in 1171 proteins was compiled for rice embryos at 0, 12 and 24 h after imbibition (HAI). Of these, the abundance of 1419 Kub sites in 777 proteins changed significantly. Most of them substantially increased within the first 12 HAI, which is similar to the dynamic state previously observed for protein phosphorylation, implying that the first 12 HAI are essential for subsequent switch during rice seed germination. We also quantitatively analyzed the embryo proteome in these samples. Generally, a specific protein's abundance in the ubiquitylome was uncorrelated to that in the proteome. The differentially ubiquitinated proteins were greatly enriched in the categories of protein processing, DNA and RNA processing/regulation related, signaling, and transport. The DiGly footprint of the Kub sites was significantly reduced on K48, a linkage typically associated with proteasome-mediated degradation. These observations suggest ubiquitylation may modulate the protein function more than providing 26S degradation signals in the early stage of rice seed germination. Revealing this comprehensive ubiquitylome greatly increases our understanding of this critical PTM during seed germination.

Differential Molecular Responses of Rapeseed Cotyledons to Light and Dark Reveal Metabolic Adaptations toward Autotrophy Establishment.

Photosynthesis competent autotrophy is established during the postgerminative stage of plant growth. Among the multiple factors, light plays a decisive role in the switch from heterotrophic to autotrophic growth. Under dark conditions, the rapeseed hypocotyl extends quickly with an apical hook, and the cotyledon is yellow and folded, and maintains high levels of the isocitrate lyase (ICL). By contrast, in the light, the hypocotyl extends slowly, the cotyledon unfolds and turns green, the ICL content changes in parallel with cotyledon greening. To reveal metabolic adaptations during the establishment of postgerminative autotrophy in rapeseed, we conducted comparative proteomic and metabolomic analyses of the cotyledons of seedlings grown under light versus dark conditions. Under both conditions, the increase in proteases, fatty acid ß-oxidation and glyoxylate-cycle related proteins was accompanied by rapid degradation of the stored proteins and lipids with an accumulation of the amino acids. While light condition partially retarded these conversions. Light significantly induced the expression of chlorophyll-binding and photorespiration related proteins, resulting in an increase in reducing-sugars. However, the levels of some chlorophyllide conversion, Calvin-cycle and photorespiration related proteins also accumulated in dark grown cotyledons, implying that the transition from heterotrophy to autotrophy is programmed in the seed rather than induced by light. Various anti-stress systems, e.g., redox related proteins, salicylic acid, proline and chaperones, were employed to decrease oxidative stress, which was mainly derived from lipid oxidation or photorespiration, under both conditions. This study provides a comprehensive understanding of the differential molecular responses of rapeseed cotyledons to light and dark conditions, which will facilitate further study on the complex mechanism underlying the transition from heterotrophy to autotrophy.