Transcriptomic and physiological analysis provide new insight into seed shattering mechanism in Pennisetum alopecuroides 'Liqiu'.

KEY MESSAGE: Through the histological, physiological, and transcriptome-level identification of the abscission zone of Pennisetum alopecuroides 'Liqiu', we explored the structure and the genes related to seed shattering, ultimately revealing the regulatory network of seed shattering in P. alopecuroides. Pennisetum alopecuroides is one of the most representative ornamental grass species of Pennisetum genus. It has unique inflorescence, elegant appearance, and strong stress tolerance. However, the shattering of seeds not only reduces the ornamental effect, but also hinders the seed production. In order to understand the potential mechanisms of seed shattering in P. alopecuroides, we conducted morphological, histological, physiological, and transcriptomic analyses on P. alopecuroides cv. 'Liqiu'. According to histological findings, the seed shattering of 'Liqiu' was determined by the abscission zone at the base of the pedicel. Correlation analysis showed that seed shattering was significantly correlated with cellulase, lignin, auxin, gibberellin, cytokinin and jasmonic acid. Through a combination of histological and physiological analyses, we observed the accumulation of cellulase and lignin during 'Liqiu' seed abscission. We used PacBio full-length transcriptome sequencing (SMRT) combined with next-generation sequencing (NGS) transcriptome technology to improve the transcriptome data of 'Liqiu'. Transcriptomics further identified many differential genes involved in cellulase, lignin and plant hormone-related pathways. This study will provide new insights into the research on the shattering mechanism of P. alopecuroides.

The first complete mitochondrial genome of Carex (C. breviculmis): a significantly expanded genome with highly structural variations.

MAIN CONCLUSION: The first complete mitochondrial genome of Carex (C. breviculmis) was sequenced and assembled, and its genomic signature was analyzed and the possible conformations of its mitochondrial genome were validated. Carex breviculmis is a very adaptable grass that is highly resistant to environmental stresses such as drought and low light. It is also admired as a landscape plant with high development prospects and scientific research value. In this study, the mitochondrial genome of C. breviculmis was assembled using Pacbio and Illumina sequencing data. We detected 267 pairs of repeats and found that three pairs of repeats could mediate the recombination of its mitochondrial genome and formed four possible conformations, of which we verified the two conformations mediated by the shortest pair of repeats using PCR amplification and Sanger sequencing. The major conformation of the C. breviculmis mitochondrial genome is a 1,414,795 bp long circular molecule with 33 annotated protein-coding genes, 15 tRNA genes, and three rRNA genes. We detected a total of 25 homologous sequences between the chloroplast and mitochondrial genomes, corresponding to 0.40% of the mitochondrial genome. Combined with the low GC content (41.24%), we conclude that the reduction in RNA editing sites in the C. breviculmis mitochondrial genome may be due to an accumulation of point mutations in C-to-T or retroprocessing events within the genome. The relatively low number of RNA editing sites in its mitochondrial genome could serve as important material for subsequent studies on the selection pressure of RNA editing in angiosperms. A maximum likelihood analysis based on 23 conserved mitochondrial genes from 28 species reflects an accurate evolutionary and taxonomic position of C. breviculmis. This research provided us with a comprehensive understanding of the mitochondrial genome of Carex and also provided important information for its molecular breeding.

PacBio single-molecule long-read sequencing shed new light on the complexity of the Carex breviculmis transcriptome.

BACKGROUND: Carex L., a grass genus commonly known as sedges, is distributed worldwide and contributes constructively to turf management, forage production, and ecological conservation. The development of next-generation sequencing (NGS) technologies has considerably improved our understanding of transcriptome complexity of Carex L. and provided a valuable genetic reference. However, the current transcriptome is not satisfactory mainly because of the enormous difficulty in obtaining full-length transcripts. RESULTS: In this study, we employed PacBio single-molecule long-read sequencing (SMRT) technology for whole-transcriptome profiling in Carex breviculmis. We generated 60,353 high-confidence non-redundant transcripts with an average length of 2302-bp. A total of 3588 alternative splicing events, and 1273 long non-coding RNAs were identified. Furthermore, 40,347 complete coding sequences were predicted, providing an informative reference transcriptome. In addition, the transcriptional regulation mechanism of C. breviculmis in response to shade stress was further explored by mapping the NGS data to the reference transcriptome constructed by SMRT sequencing. CONCLUSIONS: This study provided a full-length reference transcriptome of C. breviculmis using the SMRT sequencing method for the first time. The transcriptome atlas obtained will not only facilitate future functional genomics studies but also pave the way for further selective and genic engineering breeding projects for C. breviculmis.

MADS1, a novel MADS-box protein, is involved in the response of Nicotiana benthamiana to bacterial harpin(Xoo).

MADS-box transcription factor genes are well known for their role in floral organ and seed development. In this study, a novel MADS-box-containing gene, designated NbMADS1, was isolated from leaves of Nicotiana benthamiana. The full-length cDNA was 666 bp and encoded a putative polypeptide of 221 aa with a mass of 24.3 kDa. To assess the role of NbMADS1 in the defence response to bacterial harpin(Xoo), an elicitor of the hypersensitive response, a loss-of-function experiment was performed in N. benthamiana plants using virus-induced gene silencing. Analyses of electrolyte leakage revealed more extensive cell death in the control plants than in NbMADS1-silenced plants. The NbMADS1-silenced plants showed impaired harpin(Xoo)-induced stomatal closure, decreased harpin(Xoo)-induced production of hydrogen peroxide (H2O2) and nitric oxide (NO) in guard cells, and reduced harpin(Xoo)-induced resistance to Phytophthora nicotianae. The compromised stomatal closure observed in the NbMADS1-silenced plants was inhibited by the application of H2O2 and sodium nitroprusside (an NO donor). Taken together, these results demonstrate that the NbMADS1-H2O2-NO pathway mediates multiple harpin(Xoo)-triggered responses, including stomatal closure, hypersensitive cell death, and defence-related gene expression, suggesting that NbMADS1 plays an important role in regulating the response to harpin(Xoo) in N. benthamiana plants.

ALY proteins participate in multifaceted Nep1Mo-triggered responses in Nicotiana benthamiana and Arabidopsis thaliana.

Previously, it was found that Nep1Mo (a Nep1-like protein from Magnaporthe oryzae) could trigger a variety of plant responses, including stomatal closure, hypersensitive cell death (HCD), and defence-related gene expression, in Nicotiana benthamiana. In this study, it was found that Nep1Mo-induced cell death could be inhibited by the virus-induced gene silencing of NbALY916 in N. benthamiana. NbALY916-silenced plants showed impaired Nep1Mo-induced stomatal closure, decreased Nep1Mo-induced production of hydrogen peroxide (H2O2) and nitric oxide (NO) in guard cells, and reduced Nep1Mo-induced resistance against Phytophthora nicotianae. It also found that the deletion of AtALY4, an orthologue of NbALY916 in Arabidopsis thaliana, impaired Nep1Mo-triggered stomatal closure, HCD, and defence-related gene expression. The compromised stomatal closure observed in the NbALY916-silenced plants and AtALY4 mutants was inhibited by the application of H2O2 and sodium nitroprusside (an NO donor), and both Nep1Mo and H2O2 stimulated guard cell NO synthesis. Conversely, NO-induced stomatal closure was found not to require H2O2 synthesis; and NO treatment did not induce H2O2 production in guard cells. Taken together, these results demonstrate that the NbAlY916/AtAlY4-H2O2-NO pathway mediates multiple Nep1Mo-triggered responses, including stomatal closure, HCD, and defence-related gene expression.

Octoploids Show Enhanced Salt Tolerance through Chromosome Doubling in Switchgrass (Panicum virgatum L.).

Polyploid plants often exhibit enhanced stress tolerance. Switchgrass is a perennial rhizomatous bunchgrass that is considered ideal for cultivation in marginal lands, including sites with saline soil. In this study, we investigated the physiological responses and transcriptome changes in the octoploid and tetraploid of switchgrass (Panicum virgatum L. 'Alamo') under salt stress. We found that autoploid 8× switchgrass had enhanced salt tolerance compared with the amphidiploid 4× precursor, as indicated by physiological and phenotypic traits. Octoploids had increased salt tolerance by significant changes to the osmoregulatory and antioxidant systems. The salt-treated 8× Alamo plants showed greater potassium (K+) accumulation and an increase in the K+/Na+ ratio. Root transcriptome analysis for octoploid and tetraploid plants with or without salt stress revealed that 302 upregulated and 546 downregulated differentially expressed genes were enriched in genes involved in plant hormone signal transduction pathways and were specifically associated with the auxin, cytokinin, abscisic acid, and ethylene pathways. Weighted gene co-expression network analysis (WGCNA) detected four significant salt stress-related modules. This study explored the changes in the osmoregulatory system, inorganic ions, antioxidant enzyme system, and the root transcriptome in response to salt stress in 8× and 4× Alamo switchgrass. The results enhance knowledge of the salt tolerance of artificially induced homologous polyploid plants and provide experimental and sequencing data to aid research on the short-term adaptability and breeding of salt-tolerant biofuel plants.

Chromosome-level reference genome assembly provides insights into the evolution of Pennisetum alopecuroides.

Pennisetum alopecuroides is an important forage grass resource, which plays a vital role in ecological environment improvement. Therefore, the acquisition of P. alopecuroides genome resources is conducive to the study of the adaptability of Pennisetum species in ecological remediation and forage breeding development. Here we assembled a P. alopecuroides cv. 'Liqiu' genome at the chromosome level with a size of approximately 845.71 Mb, contig N50 of 84.83Mb, and genome integrity of 99.13% as assessed by CEGMA. A total of 833.41-Mb sequences were mounted on nine chromosomes by Hi-C technology. In total, 60.66% of the repetitive sequences and 34,312 genes were predicted. The genomic evolution analysis showed that P. alopecuroides cv. 'Liqiu' was isolated from Setaria 7.53-13.80 million years ago and from Cenchrus 5.33-8.99 million years ago, respectively. The whole-genome event analysis showed that P. alopecuroides cv. 'Liqiu' underwent two whole-genome duplication (WGD) events in the evolution process, and the duplication events occurred at a similar time to that of Oryza sativa and Setaria viridis. The completion of the genome sequencing of P. alopecuroides cv. 'Liqiu' provides data support for mining high-quality genetic resources of P. alopecuroides and provides a theoretical basis for the origin and evolutionary characteristics of Pennisetum.

The Nicotiana benthamiana mitogen-activated protein kinase cascade and WRKY transcription factor participate in Nep1(Mo)-triggered plant responses.

Many bacterial, fungal, and oomycete species secrete necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLP) that trigger programmed cell death (PCD) and innate immune responses in dicotyledonous plants. However, how NLP induce such immune responses is not understood. Here, we show that silencing of the MAPKKKα-MEK2-WIPK mitogen-activated protein kinase (MAPK) cascade through virus-induced gene silencing compromises hydrogen peroxide accumulation and PCD induced by Nep1(Mo) from Magnaporthe oryzae. WIPK interacts with NbWRKY2, a transcription factor in Nicotiana benthamiana, in vitro and in vivo, suggesting an effector pathway that mediates Nep1(Mo)-induced cell death. Unexpectedly, salicylic acid-induced protein kinase (SIPK)- and NbWRKY2-silenced plants showed impaired Nep1(Mo)-induced stomatal closure, decreased Nep1(Mo)-promoted nitric oxide (NO) production in guard cells, and a reduction in Nep1(Mo)-induced resistance against Phytophthora nicotianae. Expression studies by real-time polymerase chain reaction suggested that the MEK2-WIPK-NbWRKY2 pathway regulated Nep1(Mo)triggered NO accumulation could be partly dependent on nitrate reductase, which was implicated in NO synthesis. Taken together, these studies demonstrate that the MAPK cascade is involved in Nep1(Mo)-triggered plant responses and MAPK signaling associated with PCD exhibits shared and distinct components with that for stomatal closure.

Cadmium uptake by a hyperaccumulator and three Pennisetum grasses with associated rhizosphere effects.

Pennisetum grasses (P. purpureum Schumach. 'Purple', P. alopecuroides (L.) Spreng. 'Liren' and P. alopecuroides (L.) Spreng. 'Changsui'), and a cadmium (Cd) hyperaccumulator (Thlaspi caerulescens J.Presl & C.Presl), were grown in soil with four Cd addition levels of 0, 2, 20 and 200 mg/kg. Toxicity symptoms were not observed although growth of all plants decreased as Cd addition increased. Shoot bioconcentration factor (BCFS), the translocation factor (TF) and shoot accumulation of Cd for most plants first increased and then declined as Cd concentrations increased. In contrast, the root bioconcentration factor (BCFR) for T. caerulescens declined and root Cd accumulation for T. caerulescens and two P. alopecuroides cultivars increased consistently as Cd levels increased. P. purpureum had the largest biomass with shoot Cd accumulation similar to that of T. caerulescens, despite lower foliar Cd concentration. Although shoot Cd concentrations of two P. alopecuroides cultivars were lower than for P. purpureum, root Cd concentrations were greater. P. purpureum had Cd BCFS and TF (> 1) at 2- and 20-mg/kg Cd addition treatments, similar to T. caerulescens. P. alopecuroides cultivars had Cd BCFR (> 1) and TF (< 1) at all Cd levels. Roots did not affect rhizosphere pH. However, concentrations of acid extractable Cd in rhizosphere soil were lower than those of corresponding non-rhizosphere soil at all Cd levels for T. caerulescens and P. purpureum; T. caerulescens and P. purpureum did not affect less bioavailable Cd fractions. Concentrations of acid extractable Cd in the rhizosphere of the P. alopecuroides cultivars were not reduced at any Cd level. Differences in Cd accumulation among the three Pennisetum grasses were mainly attributable to root biomass and Cd TFs rather than rhizosphere Cd mobility.

Effects of Nitrogen Forms on the Growth and Nitrogen Accumulation in Buchloe dactyloides Seedlings.

Buffalograss [Buchloe dactyloides (Nutt.) Engelm.] has become the most widely cultivated warm-season turfgrass in northern China because of its low-maintenance requirements. Nitrogen (N) can be applied to plants in a range of formulations. However, preference of nitrogen uptake and the effects of N form on plant growth and nitrogen accumulation has not been established in buffalograss. In this study, we evaluated the effects of different inorganic nitrogen forms (NO3--N, NH4+-N, and NO3--N: NH4+-N = 1:1) on growth and nitrogen accumulation in buffalograss seedlings. Results showed that supply of three N forms significantly increased buffalograss seedlings growth, biomass, and N contents of all plant organs compared with the seedlings receiving free nitrogen. Plants achieved better growth performance when they received nitrate as the sole N source, which stimulated stolon growth and increased the biomass of ramets, spacers, and aboveground and total plant biomass, and also allocated more biomass to ramets and more N to spacers. Meanwhile, those plants supplied with the treatment +NH4NO3 displayed a significantly greater N content in the ramet, 15N abundance, and 15N accumulation amount in all organs. These data suggest NO3--N supplied either singly or in mixture increased vegetative propagation and thus facilitates buffalograss establishment. However, applications of ammonium caused detrimental effects on buffalograss seedlings growth, but +NO3- could alleviate NH4+-induced morphological disorders. Thus, recommendations to increase vegetative propagation and biomass accumulation in buffalograss seedlings should consider increasing NO3--N in a fertility program and avoiding applications of nitrogen as NH4+-N.

Seed Germination Mechanism of Carex rigescens Under Variable Temperature Determinded Using Integrated Single-Molecule Long-Read and Illumina Sequence Analysis.

The turfgrass species Carex rigescens has broad development and utilization prospects in landscaping construction. However, seed dormancy and a low germination rate have inhibited its application. Furthermore, the molecular mechanisms of seed germination in C. rigescens have not been thoroughly studied. Therefore, in the present study, PacBio full-length transcriptome sequencing combined with Illumina sequencing was employed to elucidate the germination mechanism of C. rigescens seeds under variable temperatures. In general, 156,750 full-length non-chimeric sequences, including those for 62,086 high-quality transcripts, were obtained using single-molecule long read sequencing. In total, 40,810 high-quality non-redundant, 1,675 alternative splicing, 28,393 putative coding sequences, and 1,052 long non-coding RNAs were generated. Based on the newly constructed full-length reference transcriptome, 23,147 differentially expressed genes were identified. We screened four hub genes participating in seed germination using weighted gene co-expression network analysis. Combining these results with the physiological observations, the important roles of sucrose and starch metabolic pathways in germination are further discussed. In conclusion, we report the first full-length transcriptome of C. rigescens, and investigated the physiological and transcriptional mechanisms of seed germination under variable temperatures. Our results provide valuable information for future transcriptional analyses and gene function studies of C. rigescens.

[Index system and fuzzy synthetic assessment in landscape evaluation of ornamental grasses].

A composite index system for landscape evaluation of ornamental grasses was built from the aspects of ecological constraints, resources requirement and aesthetic values, and the ornamental grass landscapes in Beijing Botanical Garden and in Nanzhongzhou public green spaces were evaluated by fuzzy synthetic assessment (FSA) method. The results showed that the scores of "very apt" for the ornamental grasses in Beijing Botanical Garden and in Nanzhongzhou public green spaces were 0.459 and 0.413, respectively, while those of "apt" for the two landscapes were 0.302 and 0.301, respectively. The evaluation results were consistent with the public feedbacks, suggesting that the composite index system and FSA method were applicable for the landscape evaluation of ornamental grasses.