SUMMARYSpike mosses (Selaginellaceae) represent an ancient lineage of vascular plants in which some species have evolved desiccation tolerance (DT). A sister-group contrast to reveal the metabolic basis of DT was conducted between a desiccation-tolerant species, Selaginella lepidophylla, and a desiccation-sensitive species, Selaginella moellendorffii, at 100% relative water content (RWC) and 50% RWC using non-biased, global metabolomics profiling technology, based on GC/MS and UHLC/MS/MS 2 platforms. A total of 301 metabolites, including 170 named (56.5%) and 131 (43.5%) unnamed compounds, were characterized across both species. S. lepidophylla retained significantly higher abundances of sucrose, mono-and polysaccharides, and sugar alcohols than did S. moellendorffii. Aromatic amino acids, the well-known osmoprotectant betaine and flavonoids were also more abundant in S. lepidophylla. Notably, levels of c-glutamyl amino acid, linked with glutathione metabolism in the detoxification of reactive oxygen species, and with possible nitrogen remobilization following rehydration, were markedly higher in S. lepidophylla. Markers for lipoxygenase activity were also greater in S. lepidophylla, especially at 50% RWC. S. moellendorffii contained more than twice the number of unnamed compounds, with only a slightly greater abundance than in S. lepidophylla. In contrast, S. lepidophylla contained 14 unnamed compounds of fivefold or greater abundance than in S. moellendorffii, suggesting that these compounds might play critical roles in DT. Overall, S. lepidophylla appears poised to tolerate desiccation in a constitutive manner using a wide range of metabolites with some inducible components, whereas S. moellendorffii mounts only limited metabolic responses to dehydration stress.

Selaginella lepidophylla is one of only a few species of spike mosses (Selaginellaceae) that have evolved desiccation tolerance (DT) or the ability to 'resurrect' from an air-dried state. In order to understand the metabolic basis of DT, S. lepidophylla was subjected to a five-stage, rehydration/dehydration cycle, then analyzed using non-biased, global metabolomics profiling technology based on GC/MS and UHLC/MS/MS(2) platforms. A total of 251 metabolites including 167 named (66.5%) and 84 (33.4%) unnamed compounds were characterized. Only 42 (16.7%) and 74 (29.5%) of compounds showed significantly increased or decreased abundance, respectively, indicating that most compounds were produced constitutively, including highly abundant trehalose, sucrose, and glucose. Several glycolysis/gluconeogenesis and tricarboxylic acid (TCA) cycle intermediates showed increased abundance at 100% relative water content (RWC) and 50% RWC. Vanillate, a potent antioxidant, was also more abundant in the hydrated state. Many different sugar alcohols and sugar acids were more abundant in the hydrated state. These polyols likely decelerate the rate of water loss during the drying process as well as slow water absorption during rehydration, stabilize proteins, and scavenge reactive oxygen species (ROS). In contrast, nitrogen-rich and γ-glutamyl amino acids, citrulline, and nucleotide catabolism products (e.g. allantoin) were more abundant in the dry states, suggesting that these compounds might play important roles in nitrogen remobilization during rehydration or in ROS scavenging. UV-protective compounds such as 3-(3-hydroxyphenyl)propionate, apigenin, and naringenin, were more abundant in the dry states. Most lipids were produced constitutively, with the exception of choline phosphate, which was more abundant in dry states and likely plays a role in membrane hydration and stabilization. In contrast, several polyunsaturated fatty acids were more abundant in the hydrated states, suggesting that these compounds likely help maintain membrane fluidity during dehydration. Lastly, S. lepidophylla contained seven unnamed compounds that displayed twofold or greater abundance in dry or rehydrating states, suggesting that these compounds might play adaptive roles in DT.

14Nebraska-Lincoln, Lincoln, NE 68588 (C.Z.) 16One-sentence summary: Single-consensus guide RNA partially reduces kafirin levels in sorghum 17 (Sorghum bicolor) grain, leading to an increased proportion of non-kafirins and improved digestibility 18 and protein quality.

Introducing traits from dent corn to popcorn is challenging because it is difficult to recover adequate popping characteristics. QPM (Quality Protein Maize) is a dent corn variety carrying the opaque-2 (o2) mutation, specifying increased amounts of normally limiting essential amino acids, and modifier genes which restore the wild type vitreous kernel phenotype. In this study, we introgressed o2 and selected for endosperm modification using vitreousness and high 27-kD gamma zein content. In this way, we recovered high-lysine, fully poppable Quality Protein Popcorn (QPP). BC2F4 individuals with vitreous kernels were confirmed to be o2 mutants by both genotyping and SDS-PAGE. Amino acid profiling of BC2F4 individuals showed that they all have significantly increased lysine compared with popcorn parental lines. Principal Component Analysis of the amino acid profiles showed that all introgressions were grouped with corresponding QPM parental lines. Popping analysis of the BC2F5 individuals showed that while there is variability in popping volume between lines, some lines show equivalent popping to the popcorn parent. In this proof-of-concept study for QPP, we have shown that it is possible to rapidly recover sufficient popcorn characteristics in a modified o2 background using simple phenotypic, biochemical and genetic selection. Furthermore, this shows increased γ-zein is an acceptable substitute for α-zein for full poppability. Since we have developed multiple QPP introgressions, this gives good scope for ongoing hybrid production and future evaluation of agronomic performance and selection of elite hybrids. In a wider context, this study shows the potential for breeding beneficial traits into popcorn for agronomic improvement.

Popcorn varieties are agronomically sub-optimal and genetically limited compared to other maize subspecies. To increase genetic diversity and improve popcorn agronomics, dent germplasm has been introduced to popcorn with limited success and generally, major loss of popping. Between 2013 and 2018, 12 Quality Protein Popcorn (QPP) inbreds containing Quality Protein Maize (QPM) and popcorn germplasm were produced that maintained popping while carrying the opaque-2 allele conferring elevated kernel lysine. This is an opportune trait in the growing market for healthier snacks and a model for mining QPM traits into popcorn. We crossed QPP inbreds to explore the effects of heterosis on popcorn protein, popping quality, and plant agronomics and selected hybrids for further production. To rank and intermediately prescreen hybrids, we utilized a novel hybrid-ranking model adapted from a rank summation index while examining the inbred general combining ability and hybrid specific combining ability estimates for all traits. We observed a biological manifestation of heterosis by categorizing hybrids by pedigree that resulted in a stepwise progression of trait improvement. These results corroborated our hybrid selection and offered insight in basic heterosis research. Estimates for popcorn quality and agronomic trait covariances also suggest the synergistic introgression of highly vitreous dent maize (QPM) into popcorn, providing a likely explanation for the successfully maintained vitreous endosperm, protein quality and popping traits in line with a remodeled proteome. QPP hybrids maintained improved amino acid profiles although different popping methods variably affected popcorn's protein bound and free amino acid levels. This preliminary screening of QPP hybrids is enabling further quantitative selection for large-scale, complex trait comparison to currently marketed elite popcorn varieties.

BackgroundUnderstanding the response of resurrection angiosperms to dehydration and rehydration is critical for deciphering the mechanisms of how plants cope with the rigors of water loss from their vegetative tissues. We have focused our studies on the C4 resurrection grass, Sporobolus stapfianus Gandoger, as a member of a group of important forage grasses.MethodsWe have combined non-targeted metabolomics with transcriptomics, via a NimbleGen array platform, to develop an understanding of how gene expression and metabolite profiles can be linked to generate a more detailed mechanistic appreciation of the cellular response to both desiccation and rehydration.ResultsThe rehydration transcriptome and metabolome are primarily geared towards the rapid return of photosynthesis, energy metabolism, protein turnover, and protein synthesis during the rehydration phase. However, there are some metabolites associated with ROS protection that remain elevated during rehydration, most notably the tocopherols. The analysis of the dehydration transcriptome reveals a strong concordance between transcript abundance and the associated metabolite abundance reported earlier, but only in responses that are directly related to cellular protection during dehydration: carbohydrate metabolism and redox homeostasis. The transcriptome response also provides strong support for the involvement of cellular protection processes as exemplified by the increases in the abundance of transcripts encoding late embryogenesis abundant (LEA) proteins, anti-oxidant enzymes, early light-induced proteins (ELIP) proteins, and cell-wall modification enzymes. There is little concordance between transcript and metabolite abundance for processes such as amino acid metabolism that do not appear to contribute directly to cellular protection, but are nonetheless important for the desiccation tolerant phenotype of S. stapfianus.ConclusionsThe transcriptomes of both dehydration and rehydration offer insight into the complexity of the regulation of responses to these processes that involve complex signaling pathways and associated transcription factors. ABA appears to be important in the control of gene expression in both the latter stages of the dehydration and the early stages of rehydration. These findings add to the growing body of information detailing how plants tolerate and survive the severe cellular perturbations of dehydration, desiccation, and rehydration.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-017-1013-7) contains supplementary material, which is available to authorized users.

The moss Physcomitrella patens, a model system for basal land plants, tolerates several abiotic stresses, including dehydration. We previously reported that Physcomitrella patens survives equilibrium dehydration to −13 MPa in a closed system at 91% RH. Tolerance of desiccation to water potentials below −100 MPa was only achieved by pretreatment with exogenous abscisic acid (ABA). We report here that gametophores, but not protonemata, can survive desiccation below −100 MPa after a gradual drying regime in an open system, without exogenous ABA. In contrast, faster equilibrium drying at 90% RH for 3-5 days did not induce desiccation tolerance in either tissue. Endogenous ABA accumulated in protonemata and gametophores under both drying regimes, so did not correlate directly with desiccation tolerance. Gametophores of a Ppabi3a/b/ c triple knock out transgenic line also survived the gradual dehydration regime, despite impaired ABA signaling. Our results suggest that the initial drying rate, and not the amount of endogenous ABA, may be critical in the acquisition of desiccation tolerance. Results from this work will provide insight into ongoing studies to uncover the role of ABA in the dehydration response and the underlying mechanisms of desiccation tolerance in this bryophyte.

In this unit, we describe a high-throughput absolute quantification protocol for 16 protein-bound amino acids (PBAAs) that combines a microscale protein hydrolysis step and an absolute quantification step using multiple reaction monitoring-based liquid chromatography-tandem mass spectrometry detection. The approach facilitates analysis of a few hundred samples per week by using a 96-well-plate extraction setup and avoiding use of additives. Importantly, the method uses only ß3 mg of tissue per sample and includes 12 heavy-amino-acid internal standards to enable quantification of the absolute levels of PBAAs with high precision, accuracy, and reproducibility. The protocol described herein has been optimized for seed samples but is applicable to other plant tissues. C 2018 by John Wiley & Sons, Inc.Keywords: amino acids r LC-MS/MS r protein hydrolysis r seeds How to cite this article: Yobi, A., & Angelovici, R. (2018). A high-throughput absolute-level quantification of protein-bound amino acids in seeds.