Shudong Liu

De novo assembly and transcriptome analysis of two contrary tillering mutants to learn the mechanisms of tillers outgrowth in switchgrass (Panicum virgatum L.)

Tillering is an important trait in monocotyledon plants. The switchgrass (Panicum virgatum), studied usually as a source of biomass for energy production, can produce hundreds of tillers in its lifetime. Studying the tillering of switchgrass also provides information for other monocot crops. High-tillering and low-tillering mutants were produced by ethyl methanesulfonate mutagenesis. Alteration of tillering ability resulted from different tiller buds outgrowth in the two mutants. We sequenced the tiller buds transcriptomes of high-tillering and low-tillering plants using next-generation sequencing technology, and generated 34 G data in total. In the de novo assembly results, 133,828 unigenes were detected with an average length of 1,238 bp, and 5,290 unigenes were differentially expressed between the two mutants, including 3,225 up-regulated genes and 2,065 down-regulated genes. Differentially expressed gene analysis with functional annotations was performed to identify candidate genes involved in tiller bud outgrowth processes using Gene Ontology classification, Cluster of Orthologous Groups of proteins, and Kyoto Encyclopedia of Genes and Genomes pathway analysis. This is the first study to explore the tillering transcriptome in two types of tillering mutants by de novo sequencing.

PvTB1, a Teosinte Branched1 Gene Homolog, Negatively Regulates Tillering in Switchgrass

Switchgrass belongs to the family Poaceae and genus Panicum, and is a highly versatile grass used for soil and water conservation, livestock production, and biomass production for energy conversion. Tillering plays an important role in determining the morphology of the aboveground parts and the final biomass yield of switchgrass. In this study, we first cloned and identified PvTB1, a teosinte branched1 (TB1) gene homolog in switchgrass, based on its sequence similarity with the TB1 gene, which is involved in lateral branching in maize. Similar to other TB1 genes, the PvTB1 gene encoded putative transcription factors containing a basic helix-loop-helix type of DNA-binding motif called the TCP domain. Tiller emergence and development were obviously inhibited by overexpression of PvTB1 in transgenic plants, and the mutated phenotypes could be rescued using 6-benzylaminopurine. Overexpression or suppression of PvTB1 through a transgenic approach resulted in changes in tiller number, stem height, stem diameter, and biomass yield. Taken together, our results suggest that PvTB1 negatively regulates tillering in switchgrass, presumably via its expression in axillary buds.

Characterization of a male sterile mutant from progeny of a transgenic plant containing a leaf senescence-inhibition gene in wheat

A male sterile plant of wheat (Triticum aestivum L.) segregated from progenies of a transgenic family containing the leaf senescence-inhibition gene PSAG12-IPT in the genetic background of ‘Xinong 1376’, a well adapted winter wheat cultivar. The male sterile plant (named TR1376A) showed no phenotypic changes, except for florets and male organs, compared to its male fertile sibling plants (named TR1376B). The glumes and florets of male sterile TR1376A plants widely opened whereas those of the fertile counterpart TR1376B were closed or opened only briefly at flowing. Anthers of TR1376A were slender and indehiscent, and failed to release pollen. Compared to TR1376B, TR1376A anthers contained greatly reduced amounts of pollen, which was inviable or weakly viable. Ultra-structure studies indicated that cells in the endothecium and middle layers of the anther wall were dissolved or poorly developed in the sterile anthers of TR1376A. Molecular studies showed that the male sterility of TR1376A was caused by a sequence deletion or mutation that occurred in the promoter region of the transgene. F1 hybrids of TR1376A and TR1376B gave 1:1 segregation of male fertility to sterility, indicating that the male sterility of TR1376A was heritable and controlled by a single dominant gene (named Ms1376). To date, only a few dominant nuclear male sterility genes have been characterized and one of them (Ms2) has been successfully used to improve wheat cultivars through recurrent breeding strategies. The discovery of the Ms1376 gene provides another dominant male sterile source for establishing recurrent breeding systems in wheat.

Proteomic analysis of melatonin-mediated osmotic tolerance by improving energy metabolism and autophagy in wheat (Triticum aestivum L.)

Main conclusionMelatonin-mediated osmotic tolerance was attributed to increased antioxidant capacity, energy metabolism, osmoregulation and autophagy in wheat (Triticum aestivum L.).Melatonin is known to play multiple roles in plant abiotic stress tolerance. However, its role in wheat has been rarely investigated. In this study, 25% polyethylene glycol 6000 (PEG 6000) was used to simulate osmotic stress, and wheat seeds and seedlings were treated with different concentrations of melatonin under PEG stress. Isobaric tag for relative and absolute quantification (iTRAQ)-based proteomic techniques were used to identify the differentially accumulated proteins from melatonin-treated and non-treated seedlings. Seeding priming with melatonin significantly increased the germination rate, coleoptile length, and primary root number of wheat under PEG stress, as well as the fresh weight, dry weight, and water content of wheat seedlings. Under PEG stress, melatonin significantly improved reactive oxygen species homeostasis, as revealed by lower H2O2 and O2· content; and the expression of antioxidant enzymes at the transcription and translation levels was increased. Melatonin maintained seedling growth by improving photosynthetic rates and instantaneous and intrinsic water use efficiencies, as well as carbon fixation and starch synthesis at the protein level. Melatonin treatment significantly affected the expression of glycolytic proteins, including fructose-1,6-bisphosphate aldolase, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and enolase, and remarkably increased the expression of the nicotinamide adenine dinucleotide transporter and nicotinamide adenine dinucleotide binding protein, thereby indirectly modulating electron transport in the respiratory chain. This indicated that melatonin improved energy production in PEG-stressed seedlings. Further, melatonin played a regulatory role in autophagy, protease expression, and ubiquitin-mediated protein degradation by significantly upregulating rab-related protein, fused signal recognition particle receptor, aspartyl protease, serine protease, ubiquitin-fold modifier 1, and ubiquitin at the mRNA or protein level. These findings suggested that melatonin might activate a metabolic cascade related to autophagy under PEG stress in wheat seedlings.

Identification and functional characterization of a MAX2 ortholog from switchgrass ( Panicum virgatum L.)

Switchgrass (Panicum virgatum L.) is a sustainable cellulosic energy crop with high biomass yield on marginal soils. Tillering, an important agronomic characteristic related to biomass production in gramineous plants, is regulated by complex interacting factors, such as plant hormones. Strigolactones (SLs) comprise a novel class of plant hormones that inhibit shoot branching. The MORE AXILLARY GROWTH2 (MAX2)/DWARF 3 (D3)/RAMOSUS (RMS4) genes encode proteins involved in the SL signaling pathway in various plants. The switchgrass tetraploid genome likely contains two high-similarity MAX2 homologs, one of which is 6 bp longer than the other. The longest is named PvMAX2 and is the ortholog of MAX2 in Arabidopsis, D3 in rice, and RMS4 in petunia. PvMAX2 is expressed ubiquitously in switchgrass tissues, with higher expression levels observed in the stem and shoot. PvMAX2 gene expression is upregulated by GR24, a synthetic SL analog. Ectopic expression of PvMAX2 in the Arabidopsis max2 mutant rescued the dwarf and bushy phenotypes and small leaf size in the mutant, suggesting that functions of AtMAX2 in Arabidopsis are conserved in PvMAX2. Ectopic PvMAX2 expression also restored the wild-type primary root and hypocotyl length phenotypes and restored the response to GR24. These results indicate that PvMAX2 may play an important role in switchgrass tillering through the SL pathway.

Transcriptional and physiological data reveal the dehydration memory behavior in switchgrass ( Panicum virgatum L.)

BackgroundSwitchgrass (Panicum virgatum L.) is a model biofuel plant because of its high biomass, cellulose-richness, easy degradation to ethanol, and the availability of extensive genomic information. However, a little is currently known about the molecular responses of switchgrass plants to dehydration stress, especially multiple dehydration stresses.ResultsStudies on the transcriptional profiles of 35-day-old tissue culture plants revealed 741 dehydration memory genes. Gene Ontology and pathway analysis showed that these genes were enriched in phenylpropanoid biosynthesis, starch and sucrose metabolism, and plant hormone signal transduction. Further analysis of specific pathways combined with physiological data suggested that switchgrass improved its dehydration resistance by changing various aspects of its responses to secondary dehydration stress (D2), including the regulation of abscisic acid (ABA) and jasmonic acid (JA) biosynthesis and signal transduction, the biosynthesis of osmolytes (l-proline, stachyose and trehalose), energy metabolism (i.e., metabolic process relating to photosynthetic systems, glycolysis, and the TCA cycle), and lignin biosynthesis. The transcriptional data and chemical substance assays showed that ABA was significantly accumulated during both primary (D1) and secondary (D2) dehydration stresses, whereas JA accumulated during D1 but became significantly less abundant during D2. This suggests the existence of a complicated signaling network of plant hormones in response to repeated dehydration stresses. A homology analysis focusing on switchgrass, maize, and Arabidopsis revealed the conservation and species-specific distribution of dehydration memory genes.ConclusionsThe molecular responses of switchgrass plants to successive dehydration stresses have been systematically characterized, revealing a previously unknown transcriptional memory behavior. These results provide new insights into the mechanisms of dehydration stress responses in plants. The genes and pathways identified in this study will be useful for the genetic improvement of switchgrass and other crops.

Long non-coding RNAs of switchgrass (Panicum virgatum L.) in multiple dehydration stresses

BackgroundLong non-coding RNAs (lncRNAs) play important roles in plant growth and stress responses. Studies of lncRNAs in non-model plants are quite limited, especially those investigating multiple dehydration stresses. In this study, we identified novel lncRNAs and analyzed their functions in dehydration stress memory in switchgrass, an excellent biofuel feedstock and soil-conserving plant in the Gramineae family.ResultsWe analyzed genome-wide transcriptional profiles of leaves of 5-week-old switchgrass plantlets grown via tissue culture after primary and secondary dehydration stresses (D1 and D2) and identified 16,551 novel lncRNAs, including 4554 annotated lncRNAs (targeting 3574 genes), and 11,997 unknown lncRNAs. Gene ontology and pathway enrichment analysis of annotated genes showed that the differentially expressed lncRNAs were related to abscisic acid (ABA) and ethylene (ETH) biosynthesis and signal transduction, and to starch and sucrose metabolism. The upregulated lncRNAs and genes were related to ABA synthesis and its signal transduction, and to trehalose synthesis. Meanwhile, lncRNAs and genes related to ETH biosynthesis and signal transduction were suppressed. LncRNAs and genes involved in ABA metabolism were verified using quantitative real-time PCR, and the endogenous ABA content was determined via high performance liquid chromatography mass spectrometry (HPLC-MS). These results showed that ABA accumulated significantly during dehydration stress, especially in D2. Furthermore, we identified 307 dehydration stress memory lncRNAs, and the ratios of different memory types in switchgrass were similar to those in Arabidopsis and maize.ConclusionsThe molecular responses of switchgrass lncRNAs to multiple dehydration stresses were researched systematically, revealing novel information about their transcriptional regulatory behavior. This study provides new insights into the response mechanism to dehydration stress in plants. The lncRNAs and pathways identified in this study provide valuable information for genetic modification of switchgrass and other crops.

The PIN1 family gene PvPIN1 is involved in auxin-dependent root emergence and tillering in switchgrass

Abstract Switchgrass (Panicum virgatum L.; family Poaceae) is a warm-season C4 perennial grass. Tillering plays an important role in determining the morphology of aboveground parts and the final biomass yield of switchgrass. Auxin distribution in plants can affect a variety of important growth and developmental processes, including the regulation of shoot and root branching, plant resistance and biological yield. Auxin transport and gradients in plants are mediated by influx and efflux carriers. PvPIN1, a switchgrass PIN1-like gene that is involved in regulating polar transport, is a putative auxin efflux carrier. Neighbor-joining analysis using sequences deposited in NCBI databases showed that the PvPIN1gene belongs to the PIN1 family and is evolutionarily closer to the Oryza sativa japonica group. Tiller emergence and development was significantly promoted in plants subjected toPvPIN1 RNA interference (RNAi), which yielded a phenotype similar to that of wild-type plants treated with the auxin transport inhibitor TIBA (2,3,5-triiodobenzoic acid). A transgenic approach that inducedPvPIN1 gene overexpression or suppression altered tiller number and the shoot/root ratio. These data suggest that PvPIN1plays an important role in auxin-dependent adventitious root emergence and tillering.