The first two whole mitochondrial genomes for the genus Dactylis species: assembly and comparative genomics analysis.

BACKGROUND: Orchardgrass (Dactylis glomerata L.), a perennial forage, has the advantages of rich leaves, high yield, and good quality and is one of the most significant forage for grassland animal husbandry and ecological management in southwest China. Mitochondrial (mt) genome is one of the major genetic systems in plants. Studying the mt genome of the genus Dactylis could provide more genetic information in addition to the nuclear genome project of the genus. RESULTS: In this study, we sequenced and assembled two mitochondrial genomes of Dactylis species of D. glomerata (597, 281 bp) and D. aschersoniana (613, 769 bp), based on a combination of PacBio and Illumina. The gene content in the mitochondrial genome of D. aschersoniana is almost identical to the mitochondrial genome of D. glomerata, which contains 22-23 protein-coding genes (PCGs), 8 ribosomal RNAs (rRNAs) and 30 transfer RNAs (tRNAs), while D. glomerata lacks the gene encoding the Ribosomal protein (rps1) and D. aschersoniana contains one pseudo gene (atp8). Twenty-three introns were found among eight of the 30 protein-coding genes, and introns of three genes (nad 1, nad2, and nad5) were trans-spliced in Dactylis aschersoniana. Further, our mitochondrial genome characteristics investigation of the genus Dactylis included codon usage, sequences repeats, RNA editing and selective pressure. The results showed that a large number of short repetitive sequences existed in the mitochondrial genome of D. aschersoniana, the size variation of two mitochondrial genomes is due largely to the presence of a large number of short repetitive sequences. We also identified 52-53 large fragments that were transferred from the chloroplast genome to the mitochondrial genome, and found that the similarity was more than 70%. ML and BI methods used in phylogenetic analysis revealed that the evolutionary status of the genus Dactylis. CONCLUSIONS: Thus, this study reveals the significant rearrangements in the mt genomes of Pooideae species. The sequenced Dactylis mt genome can provide more genetic information and improve our evolutionary understanding of the mt genomes of gramineous plants.

DNAJ protein gene expansion mechanism in Panicoideae and PgDNAJ functional identification in pearl millet.

KEY MESSAGE: Analyze the evolutionary pattern of DNAJ protein genes in the Panicoideae, including pearl millet, to identify and characterize the biological function of PgDNAJ genes in pearl millet. Global warming has become a major factor threatening food security and human development. It is urgent to analyze the heat-tolerant mechanism of plants and cultivate crops that are adapted to high temperature conditions. The Panicoideae are the second largest subfamily of the Poaceae, widely distributed in warm temperate and tropical regions. Many of these species have been reported to have strong adaptability to high temperature stress, such as pearl millet, foxtail millet and sorghum. The evolutionary differences in DNAJ protein genes among 12 Panicoideae species and 10 other species were identified and analyzed. Among them, 79% of Panicoideae DNAJ protein genes were associated with retrotransposon insertion. Analysis of the DNAJ protein pan-gene family in six pearl millet accessions revealed that the non-core genes contained significantly more TEs than the core genes. By identifying and analyzing the distribution and types of TEs near the DNAJ protein genes, it was found that the insertion of Copia and Gypsy retrotransposons provided the source of expansion for the DNAJ protein genes in the Panicoideae. Based on the analysis of the evolutionary pattern of DNAJ protein genes in Panicoideae, the PgDNAJ was obtained from pearl millet through identification. PgDNAJ reduces the accumulation of reactive oxygen species caused by high temperature by activating ascorbate peroxidase (APX), thereby improving the heat resistance of plants. In summary, these data provide new ideas for mining potential heat-tolerant genes in Panicoideae, and help to improve the heat tolerance of other crops.

Milletdb: a multi-omics database to accelerate the research of functional genomics and molecular breeding of millets.

Millets are a class of nutrient-rich coarse cereals with high resistance to abiotic stress; thus, they guarantee food security for people living in areas with extreme climatic conditions and provide stress-related genetic resources for other crops. However, no platform is available to provide a comprehensive and systematic multi-omics analysis for millets, which seriously hinders the mining of stress-related genes and the molecular breeding of millets. Here, a free, web-accessible, user-friendly millets multi-omics database platform (Milletdb, http://milletdb.novogene.com) has been developed. The Milletdb contains six millets and their one related species genomes, graph-based pan-genomics of pearl millet, and stress-related multi-omics data, which enable Milletdb to be the most complete millets multi-omics database available. We stored GWAS (genome-wide association study) results of 20 yield-related trait data obtained under three environmental conditions [field (no stress), early drought and late drought] for 2 years in the database, allowing users to identify stress-related genes that support yield improvement. Milletdb can simplify the functional genomics analysis of millets by providing users with 20 different tools (e.g., 'Gene mapping', 'Co-expression', 'KEGG/GO Enrichment' analysis, etc.). On the Milletdb platform, a gene PMA1G03779.1 was identified through 'GWAS', which has the potential to modulate yield and respond to different environmental stresses. Using the tools provided by Milletdb, we found that the stress-related PLATZs TFs (transcription factors) family expands in 87.5% of millet accessions and contributes to vegetative growth and abiotic stress responses. Milletdb can effectively serve researchers in the mining of key genes, genome editing and molecular breeding of millets.

The NAC factor LpNAL delays leaf senescence by repressing two chlorophyll catabolic genes in perennial ryegrass.

Expression of chlorophyll (Chl) catabolic genes during leaf senescence is tightly controlled at the transcriptional level. Here, we identified a NAC family transcription factor, LpNAL, involved in regulating Chl catabolic genes via the yeast one-hybrid system based on truncated promoter analysis of STAYGREEN (LpSGR) in perennial ryegrass (Lolium perenne L.). LpNAL was found to be a transcriptional repressor, directly repressing LpSGR as well as the Chl b reductase gene, NONYELLOWING COLORING1. Perennial ryegrass plants over-expressing LpNAL exhibited delayed leaf senescence or stay-green phenotypes, whereas knocking down LpNAL using RNA interference accelerated leaf senescence. Comparative transcriptome analysis of leaves at 30 d after emergence in wild-type, LpNAL-overexpression, and knock-down transgenic plants revealed that LpNAL-regulated stay-green phenotypes possess altered light reactions of photosynthesis, antioxidant metabolism, ABA and ethylene synthesis and signaling, and Chl catabolism. Collectively, the transcriptional repressor LpNAL targets both Chl a and Chl b catabolic genes and acts as a brake to fine-tune the rate of Chl degradation during leaf senescence in perennial ryegrass.

STRONG STAYGREEN inhibits DNA binding of PvNAP transcription factors during leaf senescence in switchgrass.

Fine tuning the progression of leaf senescence is important for plant fitness in nature, while the "staygreen" phenotype with delayed leaf senescence has been considered a valuable agronomic trait in crop genetic improvement. In this study, a switchgrass (Panicum virgatum L.) CCCH-type Zinc finger gene, Strong Staygreen (PvSSG), was characterized as a suppressor of leaf senescence as overexpression or suppression of the gene led to delayed or accelerated leaf senescence, respectively. Transcriptomic analysis marked that chlorophyll (Chl) catabolic pathway genes were involved in the PvSSG-regulated leaf senescence. PvSSG was identified as a nucleus-localized protein with no transcriptional activity. By yeast two-hybrid screening, we identified its interacting proteins, including a pair of paralogous transcription factors, PvNAP1/2 (NAC-LIKE, ACTIVATED BY AP3/PI). Overexpression of PvNAPs led to precocious leaf senescence at least partially by directly targeting and transactivating Chl catabolic genes to promote Chl degradation. PvSSG, through protein-protein interaction, repressed the DNA-binding efficiency of PvNAPs and alleviated its transactivating effect on downstream genes, thereby functioning as a "brake" in the progression of leaf senescence. Moreover, overexpression of PvSSG resulted in up to 47% higher biomass yield and improved biomass feedstock quality, reiterating the importance of leaf senescence regulation in the genetic improvement of switchgrass and other feedstock crops.

NOL-mediated functional stay-green traits in perennial ryegrass (Lolium perenne L.) involving multifaceted molecular factors and metabolic pathways regulating leaf senescence.

Loss of chlorophyll (Chl) is a hallmark of leaf senescence, which may be regulated by Chl catabolic genes, including NON-YELLOW COLORING 1 (NYC1)-like (NOL). The objective of this study was to determine molecular factors and metabolic pathways underlying NOL regulation of leaf senescence in perennial grass species. LpNOL was cloned from perennial ryegrass (Lolium perenne L.) and found to be highly expressed in senescent leaves. Transient overexpression of LpNOL accelerated leaf senescence and Chl b degradation in Nicotiana benthamiana. LpNOL RNA interference (NOLi) in perennial ryegrass not only significantly blocked Chl degradation in senescent leaves, but also delayed initiation and progression of leaf senescence. This study found that NOL, in addition to functioning as a Chl b reductase, could enact the functional stay-green phenotype in perennial grass species, as manifested by increased photosynthetic activities in NOLi plants. Comparative transcriptomic analysis revealed that NOL-mediated functional stay-green in perennial ryegrass was mainly achieved through the modulation of Chl catabolism, light harvesting for photosynthesis, photorespiration, cytochrome respiration, carbohydrate catabolism, oxidative detoxification, and abscisic acid biosynthesis and signaling pathways.

Knock down of NON-YELLOW COLOURING 1-like gene or chlorophyllin application enhanced chlorophyll accumulation with antioxidant roles in suppressing heat-induced leaf senescence in perennial ryegrass.

Loss of chlorophyll and oxidative damage co-occur during heat-induced leaf senescence. This study aimed to determine the functions of a chlorophyll catabolic gene, NON-YELLOW COLOURING 1 (NYC1)-like (NOL), in regulating heat-induced leaf senescence and to characterize antioxidant roles of a chlorophyll derivative, sodium copper chlorophyllin (SCC), in suppressing heat-induced leaf senescence. In two separate experiments, one by comparing NOL RNAi transgenic and wild-type plants, and the other by analysing the effects of SCC treatment, perennial ryegrass (Lolium perenne) was exposed to heat stress (38/35 °C, day/night) or optimal temperature (25/20 °C). Results showed that both knock down of LpNOL and application of SCC suppressed heat-induced leaf senescence, as manifested by increased chlorophyll content, reduced electrolyte leakage, down-regulation of chlorophyll-catabolic genes and senescence-related genes, as well as enhanced antioxidant capacity in the peroxidase pathway for H2O2 scavenging. Ex vivo SCC incubation protected membranes from H2O2 damage in mesophyll protoplasts of perennial ryegrass. The suppression of leaf senescence by knocking down NOL or chlorophyllin application was associated with enhanced chlorophyll accumulation playing antioxidant roles in protecting leaves from heat-induced oxidative damage.

Knockdown of STAYGREEN in Perennial Ryegrass (Lolium perenne L.) Leads to Transcriptomic Alterations Related to Suppressed Leaf Senescence and Improved Forage Quality.

Chl breakdown is a hallmark of leaf senescence. Protein degradation is tightly associated with accelerated Chl catabolism during leaf senescence. Therefore, blocking or reducing Chl breakdown and thereby improving Chl and leaf protein contents is desirable for agronomic improvement in perennial forage grasses. Perennial ryegrass (Lolium perenne L.) is one principle cool-season forage grass in temperate areas throughout the world. In this study, the perennial ryegrass STAY-GREEN gene (LpSGR) was cloned and characterized. LpSGR was highly expressed in developmentally or dark-induced senescent leaves. LpSGR was subcellularly localized in chloroplast and interacted with the other Chl catabolic enzymes. RNA interference (RNAi) of LpSGR in perennial ryegrass blocked the degradation of Chl, resulting in increased Chl content and photochemical efficiency in senescent leaves. The RNAi transgenic plants had significantly improved forage quality, with up to 46.1% increased protein content in the harvested biomass. Transcriptome comparison revealed that suppression of LpSGR led to multiple alterations in metabolic pathways in locations inside the chloroplast. Most transcription factors of senescence-associated hormonal signaling pathways (e.g. ABA, ethylene and jasmonic acid) had decreased expression levels in the RNAi plants. These results provided a foundation for the further study on the regulatory mechanism of LpSGR in perennial ryegrass for the purpose of forage improvement with delayed leaf senescence and higher forage quality.

Transcriptome analysis of Cd-treated switchgrass root revealed novel transcripts and the importance of HSF/HSP network in switchgrass Cd tolerance.

KEY MESSAGE: Transcriptome analysis of Cd-treated switchgrass roots not only revealed novel switchgrass transcripts and gene structures but also highlighted the indispensable role of HSF/HSP network in switchgrass Cd tolerance. Switchgrass (Panicum virgatum L.), a C4 perennial tall grass, can be used for revegetation of Cd-contaminated soil. In the present study, a comparative transcriptome analysis of Cd-treated switchgrass roots was conducted. The result revealed a total of 462 novel transcripts and refined gene structures of 2337 transcripts. KEGG pathway and Gene Ontology analyses of the differentially expressed genes (DEGs) suggested that activation of redox homeostasis and oxidation-related metabolic processes were the primary response to Cd stress in switchgrass roots. In particular, 21 out of 23 differentially expressed shock transcription factor genes (HSFs), and 22 out of 23 differentially expressed heat shock protein genes (HSPs) had increased expression levels after Cd treatment. Furthermore, over-expressing one HSP-encoding gene in Arabidopsis significantly improved plant Cd tolerance. The result highlighted the activation of the redox homeostasis and the involvement of the HSF/HSP network in re-establishing normal protein conformation and thus cellular homeostasis in switchgrass upon Cd stress. These DEGs, especially those of the HSF/HSP network, could be used as candidate genes for further functional studies toward improved plant Cd tolerance in switchgrass and related species.

Switchgrass PvDREB1C plays opposite roles in plant cold and salt tolerance in transgenic tobacco.

BACKGROUND: The C-repeat-binding factors/DRE-binding factors (CBF/DREBs) comprise a key transcription factor family involved in plant stress tolerance. Yet, there is limited information about switchgrass DREB genes and their functional roles. RESULTS: In this study, four cold-inducible PvDREB1s were identified from switchgrass (Panicum virgatum), among which PvDREB1C was the one responded to cold stress later than the other three PvDREB1s. Yet, ectopic overexpression of PvDREB1C led to significantly compromised, instead of improved cold tolerance in transgenic tobacco. On the other hand, PvDREB1C was transcriptionally down-regulated in response to salt stress, but overexpression of PvDREB1C improved plant salt tolerance in transgenic tobacco. The improved salt tolerance was associated with increased K+/Na+ ratio and Ca2+ content, higher cellular osmotic potential, and activation of stress-related functional genes in the leaves of transgenic plants under salt stress. CONCLUSIONS: The current results implied that PvDREB1C played opposite roles in plant cold and salt tolerance. Although DREB1s were known as positive stress regulators, particular attentions shall be paid to their potential negative regulatory role(s).

Comprehensive analysis of CCCH-type zinc finger family genes facilitates functional gene discovery and reflects recent allopolyploidization event in tetraploid switchgrass.

BACKGROUND: In recent years, dozens of Arabidopsis and rice CCCH-type zinc finger genes have been functionally studied, many of which confer important traits, such as abiotic and biotic stress tolerance, delayed leaf senescence and improved plant architecture. Switchgrass (Panicum virgatum) is an important bioenergy crop. Identification of agronomically important genes and/or loci is an important step for switchgrass molecular breeding. Annotating switchgrass CCCH genes using translational genomics methods will help further the goal of understanding switchgrass genetics and creating improved varieties. RESULTS: Taking advantage of the publicly-available switchgrass genomic and transcriptomic databases, we carried out a comprehensive analysis of switchgrass CCCH genes (PvC3Hs). A total of 103 PvC3Hs were identified and divided into 21 clades according to phylogenetic analysis. Genes in the same clade shared similar gene structure and conserved motifs. Chromosomal location analysis showed that most of the duplicated PvC3H gene pairs are in homeologous chromosomes. Evolution analysis of 19 selected PvC3H pairs showed that 42.1% of them were under diversifying selection. Expression atlas of the 103 PvC3Hs in 21 different organs, tissues and developmental stages revealed genes with higher expression levels in lignified cells, vascular cells, or reproductive tissues/organs, suggesting the potential function of these genes in development. We also found that eight PvC3Hs in Clade-XIV were orthologous to ABA- or stress- responsive CCCH genes in Arabidopsis and rice with functions annotated. Promoter and qRT-PCR analyses of Clade-XIV PvC3Hs showed that these eight genes were all responsive to ABA and various stresses. CONCLUSIONS: Genome-wide analysis of PvC3Hs confirmed the recent allopolyploidization event of tetraploid switchgrass from two closely-related diploid progenitors. The short time window after the polyploidization event allowed the existence of a large number of PvC3H genes with a high positive selection pressure onto them. The homeologous pairs of PvC3Hs may contribute to the heterosis of switchgrass and its wide adaptation in different ecological niches. Phylogenetic and gene expression analyses provide informative clues for discovering PvC3H genes in some functional categories. Particularly, eight PvC3Hs in Clade-XIV were found involved in stress responses. This information provides a foundation for functional studies of these genes in the future.

Proteomic and physiological responses of contrasting two different heat-resistant orchardgrass genotypes to heat stress.

As an important forage crop worldwide, the growth and productivity of orchardgrass are greatly impacted by high temperatures. However, little information is known about how orchardgrass proteomic changes under heat conditions. Therefore, the present study investigated the proteomics and physiological changes in 667 [AKZ-NRGR667 (heat-tolerant)] and 7602 [PI237602 (heat-sensitive)] under heat stress (40/35 °C). In addition, the responses of translational regulating of heat stress in orchardgrass were analyzed through proteomic changes using the tandem mass tags (TMT) technique. Together, 410 differentially expressed proteins (DEPs) were identified from two orchardgrass genotypes under heat at 24 h. Proteomics analyses indicated that proteins related to substance metabolism, photosynthesis, and heat shock proteins (HSPs) were differentially expressed under heat stress and control conditions. Moreover, a large proportion of HSPs were expressed in the heat-tolerant genotype as compared to the heat-sensitive genotype. In conclusion, genotype 667 has higher adaptability and repairing capability due to stronger heat tolerance capacity that can make it more suited to sustaining its survival and growth than genotype 7602. These findings can provide the basis for genetic improvements in orchardgrass and other crops facing high-temperature stress or heat environment that may lead to heat resistance or tolerance.

CCCH protein-PvCCCH69 acted as a repressor for leaf senescence through suppressing ABA-signaling pathway.

CCCH is a subfamily of zinc finger proteins involved in plant growth, development, and stresses response. The function of CCCH in regulating leaf senescence, especially its roles in abscisic acid (ABA)-mediated leaf senescence is largely unknown. The objective of this study was to determine functions and mechanisms of CCCH gene in regulating leaf senescence in switchgrass (Panicum virgatum). A CCCH gene, PvCCCH69 (PvC3H69), was cloned from switchgrass. Overexpressing PvC3H69 in rice suppressed both natural senescence with leaf aging and dark-induced leaf senescence. Endogenous ABA content, ABA biosynthesis genes (NCED3, NCED5, and AAO3), and ABA signaling-related genes (SnRKs, ABI5, and ABF2/3/4) exhibited significantly lower levels in senescencing leaves of PvC3H69-OE plants than those in WT plants. PvC3H69-suppression of leaf senescence was associated with transcriptional upregulation of genes mainly involved in the light-dependent process of photosynthesis, including light-harvesting complex proteins, PSI proteins, and PSII proteins and downregulation of ABA biosynthesis and signaling genes and senescence-associated genes. PvC3H69 could act as a repressor for leaf senescence via upregulating photosynthetic proteins and repressing ABA synthesis and ABA signaling pathways.

Improved cold tolerance in switchgrass by a novel CCCH-type zinc finger transcription factor gene, PvC3H72, associated with ICE1-CBF-COR regulon and ABA-responsive genes.

BACKGROUND: Switchgrass (Panicum virgatum) is a warm-season perennial grass. Improving its cold tolerance is important for its sustainable production in cooler regions. Through genome-wide bioinformatic analysis of switchgrass Zinc finger-CCCH genes (PvC3Hs), we found that several PvC3Hs, including PvC3H72, might play regulatory roles in plant cold tolerance. The objectives of this study were to characterize PvC3H72 using reverse genetics approach and to understand its functional role in cold signal transduction and cold tolerance in switchgrass. RESULTS: PvC3H72 is an intronless gene encoding a transcriptional activation factor. The expression of PvC3H72 was rapidly and highly induced by cold stress. Transgenic switchgrass with over-expressed PvC3H72 driven under maize ubiquitin promoter showed significantly improved chilling tolerance at 4 °C as demonstrated by less electrolyte leakage and higher relative water content than wild-type (WT) plants, as well as significantly higher survival rate after freezing treatment at - 5 °C. Improved cold tolerance of PvC3H72 transgenic lines was associated with significantly up-regulated expression of ICE1-CBF-COR regulon and ABA-responsive genes during cold treatment. CONCLUSIONS: PvC3H72 was the first characterized switchgrass cold-tolerance gene and also the only Znf-CCCH family gene known as a transcription factor in plant cold tolerance. PvC3H72 was an added signaling component in plant cold tolerance associated with regulation of ICE1-CBF-COR regulon and ABA-responsive genes. Knowledge gained in this study not only added another acting component into plant cold-tolerance mechanism, but also be of high value for genetic improvement of cold tolerance in switchgrass as well as other warm-season grasses.

An efficient protocol for perennial ryegrass mesophyll protoplast isolation and transformation, and its application on interaction study between LpNOL and LpNYC1.

BACKGROUND: Perennial ryegrass (Lolium perenne L.) is an important temperate grass used for turf and forage purposes. With the increasing accumulation of genomic and transcriptomic data of perennial ryegrass, an efficient protoplast and transient gene expression protocol is highly desirable for in vivo gene functional studies in its homologous system. RESULTS: In this report, a highly efficient protoplast isolation (5.6 × 107 protoplasts per gram of leaf material) and transient expression (plasmid transformation efficiency at 55.2%) was developed and the detailed protocol presented. Using this protocol, the subcellular locations of two ryegrass proteins were visualized in chloroplasts and nuclei, respectively, and protein-protein interaction between two chlorophyll catabolic enzymes (LpNOL and LpNYC1) was recorded in its homologous system for the first time. CONCLUSION: This efficient protoplast isolation and transformation protocol is sufficient for studies on protein subcellular localization and protein-protein interaction, and shall be suitable for many other molecular biology applications where the mesophyll protoplast system is desirable in perennial ryegrass.