Evolution and natural selection of ribosome-inactivating proteins in bacteria, fungi, and plants.

Ribosome-inactivating proteins (RIPs) are found in bacteria, fungi, and plants, with a wide range of biological resistances such as anti-fungal, anti-viral, anti-insect, and anti-tumor. They can be roughly divided into proactive defense bacterial or fungal types and passive defense plant types. We identified 1592 RIP genes in bacteria, fungi, and plants. Approximately 88 % of the 764 bacterial RIPs were Shiga or Shiga-like toxins which were exotoxins and could rapidly enter cells to possess strong biotoxicity, and about 98 % of fungal RIPs were predicted as secreted proteins. RIPs were not detected in non-seed plants such as algae, bryophytes, and ferns. However, we found RIPs in some flowering and non-flowering seed plants. The existence of plant RIPs might be related to the structure of seeds or fruits, which might be associated with whether seeds are easy to survive and spread. The evolutionary characteristics of RIPs were different between dicotyledons and monocotyledons. In addition, we also found that RIP2 genes might emerge very early and be plant-specific. Some plant RIP1 genes might evolve from RIP2 genes. This study provides new insights into the evolution of RIPs.

Dynamic Changes in Seed Germination under Low-Temperature Stress in Maize.

Low-temperature stress delays seed germination in maize. Different maize inbred lines display various low-temperature resistance, but the dynamic changes in seed germination under low-temperature stress in maize remain unknown, especially at the transcriptome level. In this study, low-temperature-resistant maize (RM) inbred line 04Qun0522-1-1 had a significantly faster germination speed than low-temperature-sensitive maize (SM) line B283-1 under low-temperature stress. Moreover, the total antioxidant capacity, superoxide dismutase, and peroxidase activities were notably higher in the RM line than in the SM line from 3 to 6 d. In contrast, the SM line showed significantly higher malondialdehyde (MDA) content than the RM line at 6 d. Gene ontology (GO) enrichment analysis showed that in 2dvs0d, both SM and RM lines displayed the downregulation of ribosome-related genes. Moreover, photosystem II and heat shock protein binding-related genes were also downregulated in the SM line. In 4dvs2d, the RM line showed a higher degree of upregulation of the ribosome and peroxidase (POD)-related genes than the SM line. In 6dvs4d, POD-related genes were continuously upregulated in both SM and RM lines, but the degree of upregulation of the genes was higher in the SM line than in the RM line. Moreover, vitamin B6-related genes were specifically upregulated in the RM line. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that in 6dvs4d, phenylpropanoid biosynthesis was the most significantly enriched pathway in both SM and RM lines. Moreover, phenylpropanoid biosynthesis was also enriched in the RM line in 4dvs2d. More than half of the differentially expressed genes (DEGs) in phenylpropanoid biosynthesis were peroxidase, and the DEGs were similar to the GO enrichment analysis. The results provide new insights into maize seed germination in response to low-temperature stress.

Maize Seed Germination Under Low-Temperature Stress Impacts Seedling Growth Under Normal Temperature by Modulating Photosynthesis and Antioxidant Metabolism.

Spring maize is usually subjected to low-temperature stress during seed germination, which retards seedling growth later even under a suitable temperature. However, the mechanism underlying maize seed germination under low-temperature stress impacting seedling growth is still ambiguous. In this study, we used one low-temperature sensitive maize (SM) and one low-temperature resistance maize (RM) to investigate the mechanism. The results showed that the SM line had higher malondialdehyde content and lower total antioxidant capacity (TAC) and germination percentage than the RM line under low-temperature stress, indicating the vulnerability of SM line to low-temperature stress. Further transcriptome analysis revealed that seed germination under low-temperature stress caused the down-regulation of photosynthesis-related gene ontology terms in two lines. Moreover, the SM line displayed down-regulation of ribosome and superoxide dismutase (SOD) related genes, whereas genes involved in SOD and vitamin B6 were up-regulated in the RM line. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that photosynthesis and antioxidant metabolism-related pathways played essential roles in response to low-temperature stress during seed germination. The photosynthetic system displayed a higher degree of damage in the SM line. Both qRT-PCR and physiological characteristics experiments showed similar results with transcriptome data. Taken together, we propose a model for maize seed germination in response to low-temperature stress.

Transcriptome analysis reveals the mechanism of internode development affecting maize stalk strength.

BACKGROUND: The stalk rind is one of the important factors affecting maize stalk strength that is closely related to stalk lodging. However, the mechanism of rind development in maize is still largely unknown. RESULTS: In this study, we analyzed the mechanical, anatomical, and biochemical properties of the third basal internode in one maize non-stiff-stalk (NSS) line and two stiff-stalk (SS) lines. Compared with the NSS line, the two SS lines had a significantly higher rind penetrometer resistance, thicker rind, and higher dry matter, hemicellulose, cellulose, and lignin weights per unit length. RNA-seq analysis was used to compare transcriptomes of the third basal internode of the two SS lines and the NSS line at the ninth leaf and tasseling stages. Gene Ontology (GO) enrichment analysis revealed that genes involved in hydrolase activity (hydrolyzing O-glycosyl compounds) and cytoskeleton organization were significantly up-regulated in the two SS lines at the ninth leaf stage and that microtubule process-related genes were significantly up-regulated in the two SS lines at the tasseling stage. Moreover, the two SS lines had enhanced expression of cell wall metabolism-related genes at the tasseling stage. CONCLUSIONS: The synthesis of cell wall polysaccharides and the cytoskeleton might play important roles in internode development. Our results can be applied for screening lodging-resistant inbred lines and breeding lodging-resistant cultivars in maize.

Proteomic analysis of wheat seeds produced under different nitrogen levels before and after germination.

The objective of this study was to investigate differentially abundant proteins (DAPs) of wheat seeds produced under two nitrogen levels (0 and 240 kg/ha) before and after germination. We selected samples at 8 and 72 h after imbibition (HAI) to identify DAPs by iTRAQ. The results showed 190 and 124 DAPs at 8 and 72 HAI, respectively. Alpha-gliadin and chlorophyll a-b binding protein showed the biggest difference in abundance before and after germination. In GO enrichment analysis, the most significantly enriched GO term was nutrient reservoir activity at 8 HAI and endopeptidase inhibitor activity at 72 HAI. Moreover, many DAPs involved in mobilization of stored nutrients and photosynthesis were mapped to KEGG pathways. Dough development time, dough stability time and seedling chlorophyll content under N240 were significantly higher than those under N0, which validated the results of proteomic analysis. These results are crucial for food nutrition and food processing.

Transcriptome analysis reveals the mechanism by which spraying diethyl aminoethyl hexanoate after anthesis regulates wheat grain filling.

BACKGROUND: Diethyl aminoethyl hexanoate (DA-6), a plant growth regulator, has many beneficial effects on agricultural production. DA-6 has been applied to many plant species, but the molecular mechanism by which spraying DA-6 after anthesis regulates wheat grain filling is still unknown. RESULTS: In this study, we used four DA-6 concentrations: C0 (0 g/L), C2 (2 g/L), C4 (4 g/L), and C6 (6 g/L). The results showed that C4 and C6 led to a significantly higher 1000-grain weight and seed protein content than C0 during two wheat growing seasons. We then subjected samples at 24 days after anthesis (at which point the grain weight increased rapidly) to transcriptome analysis. Flag leaf (L), seed (S), and stem (T) samples under C6 and C0 were used for RNA-seq. The seed samples under C6 compared with C0 (S6vsS0) presented the most differentially expressed genes (DEGs; 2164). Plant hormone signal transduction (p = 1.97 × 10- 4), protein processing in the endoplasmic reticulum (ER; p = 9.04 × 10- 11) and starch and sucrose metabolism (p = 1.90 × 10- 10) pathways were the most markedly enriched pathways in the flag leaves, stems, and seeds, respectively. DEGs involved in sucrose synthesis in the flag leaves, protein processing in ER in the stems, and starch synthesis and protein processing in ER in the seeds were significantly upregulated under C6 compared with C0. CONCLUSIONS: Overall, we propose a model for spraying DA-6 after anthesis to regulate metabolic pathways in wheat, which provides new insights into wheat in response to DA-6.

Effects of Nitrogen Level during Seed Production on Wheat Seed Vigor and Seedling Establishment at the Transcriptome Level.

Nitrogen fertilizer is a critical determinant of grain yield and seed quality in wheat. However, the mechanism of nitrogen level during seed production affecting wheat seed vigor and seedling establishment at the transcriptome level remains unknown. Here, we report that wheat seeds produced under different nitrogen levels (N0, N168, N240, and N300) showed significant differences in seed vigor and seedling establishment. In grain yield and seed vigor, N0 and N240 treatments showed the minimum and maximum, respectively. Subsequently, we used RNA-seq to analyze the transcriptomes of seeds and seedlings under N0 and N240 at the early stage of seedling establishment. Gene Ontology (GO) term enrichment analysis revealed that dioxygenase-activity-related genes were dramatically upregulated in faster growing seedlings. Among these genes, the top three involved linoleate 9S-lipoxygenase (Traes_2DL_D4BCDAA76, Traes_2DL_CE85DC5C0, and Traes_2DL_B5B62EE11). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that pathways involved in nutrient mobilization and the antioxidant system showed enhanced expression under N240. Moreover, seeds with faster growing seedlings had a higher gene expression level of α-amylase, which was consistent with α-amylase activity. Taken together, we propose a model for seedling establishment and seed vigor in response to nitrogen level during seed production.

Rapid evaluation of seed vigor by the absolute content of protein in seed within the same crop.

Seed vigor, an important index of seed quality, determines the potential for rapid and uniform emergence of plants. The objective of this study was to explore a rapid method for evaluating seed vigor. To analyze the correlation of seed traits and seedling traits related to seed vigor, we designed five experiments including nitrogen fertilizer, irrigation and seed sorting treatments in wheat. The results showed that only the absolute content of protein (ACP) in wheat seed was significantly correlated with plant dry weight in five experiments. Subsequently, another experiment including 30 wheat seed lots was used to validate the above results. Although 100-grain weight was also correlated with plant dry weight (R = 0.799, p < 0.01), the correlation coefficient was lower than that between ACP in seed and plant dry weight (R = 0.897, p < 0.01). Moreover, the results of three experiments using maize seeds was similar with above. The relative content of protein in seed detected by near-infrared spectrum combining with seed weight could realize rapid and nondestructive testing ACP in seed. Collectively, ACP in crop seed could be applied in rapid evaluation of seed vigor and could potentially be used for processing and screening high vigor seeds.

A loose endosperm structure of wheat seed produced under low nitrogen level promotes early germination by accelerating water uptake.

Water uptake is the fundamental requirement for the initiation and completion of seed germination that is a vital phase in the life cycle of seed plants. We found that seeds produced under four nitrogen levels showed significantly different germination speed. The objective of this study was to study the mechanism of rapid seed germination and explore which pathways and genes play critical roles in radicle protrusion. Anatomical data revealed that seed protein content affected endosperm structure of seeds. Moreover, scanning electron microscope maps showed that faster germinated seeds had a looser endosperm structure compared with other seeds. Subsequently, high throughout RNA-seq data were used to compare the transcriptomes of imbibed seeds with different germination speed. Gene ontology (GO) term enrichment analysis revealed that cell wall metabolism related genes significantly up-regulated in faster germinated seeds. In these genes, the top four were chitinase that had about fourfold higher expression in faster germinated seeds. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that faster germinated seeds had enhanced expression in glutathione metabolism. By combining these results, we propose a model for nitrogen fertilizer affects germination speed of wheat seed, which provide new insights into seed germination.

Universal Multiplex PCR: a novel method of simultaneous amplification of multiple DNA fragments.

BACKGROUND: Multiplex PCR has been successfully applied in many areas since it was first reported in 1988; however, it suffers from poor universality. RESULTS: A novel method called Universal Multiplex PCR (UM-PCR) was created, which simultaneously amplifies multiple target fragments from genomic DNA. The method has two steps. First, the universal adapter-F and universal adapter-R are connected to the forward primers and the reverse primers, respectively. Hairpin structures and cross dimers of five pairs of adapter-primers are detected. Second, UM-PCR amplification is implemented using a novel PCR procedure termed "Two Rounds Mode" (three and 28-32 cycles). The first round (the first three cycles) is named the "One by One Annealing Round". The second round (28-32 cycles) combines annealing with extension. In the first two cycles of the first round, primers only amplify the specific templates; there are no templates for the universal adapters. The templates of universal adapters begin to be synthesized from the second cycle of the first round, and universal adapters and primers commence full amplification from the third cycle of the first round. CONCLUSIONS: UM-PCR greatly improves the universality of multiplex PCR. UM-PCR could rapidly detect the genetic purity of maize seeds. In addition, it could be applied in other areas, such as analysis of polymorphisms, quantitative assays and identifications of species.