Agave REVEILLE1 regulates the onset and release of seasonal dormancy in Populus.

Deciduous woody plants like poplar (Populus spp.) have seasonal bud dormancy. It has been challenging to simultaneously delay the onset of bud dormancy in the fall and advance bud break in the spring, as bud dormancy, and bud break were thought to be controlled by different genetic factors. Here, we demonstrate that heterologous expression of the REVEILLE1 gene (named AaRVE1) from Agave (Agave americana) not only delays the onset of bud dormancy but also accelerates bud break in poplar in field trials. AaRVE1 heterologous expression increases poplar biomass yield by 166% in the greenhouse. Furthermore, we reveal that heterologous expression of AaRVE1 increases cytokinin contents, represses multiple dormancy-related genes, and up-regulates bud break-related genes, and that AaRVE1 functions as a transcriptional repressor and regulates the activity of the DORMANCY-ASSOCIATED PROTEIN 1 (DRM1) promoter. Our findings demonstrate that AaRVE1 appears to function as a regulator of bud dormancy and bud break, which has important implications for extending the growing season of deciduous trees in frost-free temperate and subtropical regions to increase crop yield.

Agrobacterium- and a single Cas9-sgRNA transcript system-mediated high efficiency gene editing in perennial ryegrass.

Genome editing technologies provide a powerful tool for genetic improvement of perennial ryegrass, an important forage and turfgrass species worldwide. The sole publication for gene editing in perennial ryegrass used gene-gun for plant transformation and a dual promoter based CRISPR/Cas9 system for editing. However, their editing efficiency was low (5.9% or only one gene-edited plant produced). To test the suitability of the maize Ubiquitin 1 (ZmUbi1) promoter in gene editing of perennial ryegrass, we produced ZmUbi1 promoter:RUBY transgenic plants. We observed that ZmUbi1 promoter was active in callus tissue prior to shoot regeneration, suggesting that the promoter is suitable for Cas9 and sgRNA expression in perennial ryegrass for high-efficiency production of bi-allelic mutant plants. We then used the ZmUbi1 promoter for controlling Cas9 and sgRNA expression in perennial ryegrass. A ribozyme cleavage target site between the Cas9 and sgRNA sequences allowed production of functional Cas9 mRNA and sgRNA after transcription. Using Agrobacterium for genetic transformation, we observed a 29% efficiency for editing the PHYTOENE DESATURASE gene in perennial ryegrass. DNA sequencing analyses revealed that most pds plants contained bi-allelic mutations. These results demonstrate that the expression of a single Cas9 and sgRNA transcript unit controlled by the ZmUbi1 promoter provides a highly efficient system for production of bi-allelic mutants of perennial ryegrass and should also be applicable in other related grass species.

An Improved Procedure for Agrobacterium-Mediated Transformation of 'Carrizo' Citrange.

Although several protocols for genetic transformation of citrus have been published, it is highly desirable to further improve its efficiency. Here we report treatments of Agrobacterium cells and citrus explants prior to and during co-cultivation process to enhance transformation efficiency using a commercially used rootstock 'Carrizo' citrange [Citrus sinensis (L.) Osb. × Poncirius trifoliata (L.) Raf.] as a model plant. We found explants from light-grown seedlings exhibited higher transformation efficiency than those from etiolated seedlings. We pre-cultured Agrobacterium cells in a 1/10 MS, 0.5 g/L 2-(N-morpholino) ethanesulfonic acid (MES) and 100 µM acetosyringone liquid medium for 6 h at 25 °C before used to infect citrus explants. We incubated epicotyl segments in an MS liquid medium containing 13.2 µM 6-BA, 4.5 µM 2,4-D, 0.5 µM NAA for 3 h at 25 °C prior to Agrobacterium infection. In the co-cultivation medium, we added 30 µM paclobutrazol and 10 µM lipoic acid. Each of these treatments significantly increased the efficiencies of transformation up to 30.4% (treating Agrobacterium with acetosyringone), 31.8% (treating explants with cytokinin and auxin), 34.9% (paclobutrazol) and 38.6% (lipoic acid), respectively. When the three treatments were combined, we observed that the transformation efficiency was enhanced from 11.5% to 52.3%. The improvement of genetic transformation efficiency mediated by these three simple treatments may facilitate more efficient applications of transgenic and gene editing technologies for functional characterization of citrus genes and for genetic improvement of citrus cultivars.

Complete Chloroplast Genomes and Comparative Analyses of Three Ornamental Impatiens Species.

Impatiens L., the largest genus in the family Balsaminaceae with approximately 1,000 species, is a controversial genus. Due to the conflict of morphological features and insufficient genomic resources, the studies of systematic evolution and understanding of taxonomic identification are considered to be very limited. Hence, we have sequenced the complete chloroplast genomes of three ornamental species (Impatiens balsamina, I. hawkeri, and I. walleriana), and compared them with previously published wild species data. We performed a detailed comparison of a highly similar basic structure, size, GC content, gene number, order, and functional array among them. Similarly, most divergent genes were detected from previous work in the literature. The mutational regions containing highly variable nucleotide hotspots were identified and may be used as potential markers for species identification and taxonomy. Furthermore, using whole chloroplast genome data to analysis the phylogenetic relationship of the Balsaminaceae species, we found that they were all part of a single clade. The three phenotypically different ornamental species were clustered together, suggesting that they were very likely to be closely related. We achieved and characterized the plastid genome structure, identified the divergence hotspots, and determined the phylogenetic and taxonomic positions of the three cultivated species in the Impatiens genus. The results may show that the chloroplast genome can be used to solve phylogenetic problems in or between the Impatiens genus and also provide genomic resources for the study of the Balsaminaceae family's systematics and evolution.

Comparative chloroplast genome analysis of Impatiens species (Balsaminaceae) in the karst area of China: insights into genome evolution and phylogenomic implications.

BACKGROUND: Impatiens L. is a genus of complex taxonomy that belongs to the family Balsaminaceae (Ericales) and contains approximately 1000 species. The genus is well known for its economic, medicinal, ornamental, and horticultural value. However, knowledge about its germplasm identification, molecular phylogeny, and chloroplast genomics is limited, and taxonomic uncertainties still exist due to overlapping morphological features and insufficient genomic resources. RESULTS: We sequenced the chloroplast genomes of six different species (Impatiens chlorosepala, Impatiens fanjingshanica, Impatiens guizhouensis, Impatiens linearisepala, Impatiens loulanensis, and Impatiens stenosepala) in the karst area of China and compared them with those of six previously published Balsaminaceae species. We contrasted genomic features and repeat sequences, assessed sequence divergence and constructed phylogenetic relationships. Except for those of I. alpicola, I. pritzelii and I. glandulifera, the complete chloroplast genomes ranging in size from 151,366 bp (I. alpicola) to 154,189 bp (Hydrocera triflora) encoded 115 distinct genes [81 protein-coding, 30 transfer RNA (tRNA), and 4 ribosomal RNA (rRNA) genes]. Moreover, the characteristics of the long repeat sequences and simple sequence repeats (SSRs) were determined. psbK-psbI, trnT-GGU-psbD, rpl36-rps8, rpoB-trnC-GCA, trnK-UUU-rps16, trnQ-UUG, trnP-UGG-psaJ, trnT-UGU-trnL-UAA, and ycf4-cemA were identified as divergence hotspot regions and thus might be suitable for species identification and phylogenetic studies. Additionally, the phylogenetic relationships based on Maximum likelihood (ML) and Bayesian inference (BI) of the whole chloroplast genomes showed that the chloroplast genome structure of I. guizhouensis represents the ancestral state of the Balsaminaceae family. CONCLUSION: Our study provided detailed information about nucleotide diversity hotspots and the types of repeats, which can be used to develop molecular markers applicable to Balsaminaceae species. We also reconstructed and analyzed the relationships of some Impatiens species and assessed their taxonomic statuses based on the complete chloroplast genomes. Together, the findings of the current study might provide valuable genomic resources for systematic evolution of the Balsaminaceae species.

Molecular and physiological characterization of the effects of auxin-enriched rootstock on grafting.

Grafting is a highly useful technique, and its success largely depends on graft union formation. In this study, we found that root-specific expression of the auxin biosynthetic gene iaaM in tobacco, when used as rootstock, resulted in more rapid callus formation and faster graft healing. However, overexpression of the auxin-inactivating iaaL gene in rootstocks delayed graft healing. We observed increased endogenous auxin levels and auxin-responsive DR5::GUS expression in scions of WT/iaaM grafts compared with those found in WT/WT grafts, which suggested that auxin is transported upward from rootstock to scion tissues. A transcriptome analysis showed that auxin enhanced graft union formation through increases in the expression of genes involved in graft healing in both rootstock and scion tissues. We also observed that the ethylene biosynthetic gene ACS1 and the ethylene-responsive gene ERF5 were upregulated in both scions and rootstocks of the WT/iaaM grafts. Furthermore, exogenous applications of the ethylene precursor ACC to the junction of WT/WT grafts promoted graft union formation, whereas application of the ethylene biosynthesis inhibitor AVG delayed graft healing in WT/WT grafts, and the observed delay was less pronounced in the WT/iaaM grafts. These results demonstrated that elevated auxin levels in the iaaM rootstock in combination with the increased auxin levels in scions caused by upward transport/diffusion enhanced graft union formation and that ethylene was partially responsible for the effects of auxin on grafting. Our findings showed that grafting success can be enhanced by increasing the auxin levels in rootstocks using transgenic or gene-editing techniques.

Complete chloroplast genomes of Impatiens cyanantha and Impatiens monticola: Insights into genome structures, mutational hotspots, comparative and phylogenetic analysis with its congeneric species.

Impatiens L., the largest genus in the family Balsaminaceae with approximately 1000 species, is a controversial and complex genus that includes many economically important species well known for medicinal and ornamental values. However, there is limited knowledge of molecular phylogeny and chloroplast genomics, and uncertainties still exist at a taxonomic level. In this study, we have assembled four chloroplast genomics specimens of Impatiens cyanantha and Impatiens monticola, which are found at the different altitudes of Guizhou and Yunnan in China, and compared them with previously published three wild Balsaminaceae species (Impatiens piufanensis, Impatiens glandlifera, and Hydrocera triflora). The complete chloroplast genome sequences ranged from 152,236 bp (I. piufanensis) to 154,189 bp (H. triflora) and encoded 115 total distinct genes, of which 81 were protein-coding, 30 were distinct transfer RNA genes(tRNA), and 4 were ribosomal RNA genes (rRNA). A comparative analysis of I. cyanantha (Guizhou) vs. I. cyanantha (Yunnan) and I. monticola (Guizhou) vs. I. monticola (Yunnan) revealed minor changes in lengths; however, similar gene contents, gene orders, and GC contents existed among them. Interestingly, highly coding and non-coding genes, and regions matK, psbK, atpH-atpI, trnC-trnT, petN, psbM, atpE, rbcL, accD, psaL, rps3-rps19, ndhG-ndhA,rpl16, rpoB, ndhB, ndhF, ycf1, and ndhH were found, which could be suitable for identification of species and phylogenetic studies. During the comparison between I. cyanantha (Guizhou) and I. cyanantha (Yunnan), we observed that the rps4, ycf2, ndhF, ycf1, and rpoC2 genes underwent positive selection. Meanwhile, in the comparative study of I. monticola (Guizhou) vs. I. monticola (Yunnan), The accD and ycf1 genes were positively selected. Additionally, phylogenetic relationships based on maximum likelihood (ML) and Bayesian inference (BI) among whole chloroplast genomes showed that a sister relationship with I. monticola (Guizhou) and I. monticola (Yunnan) formed a clade with I.piufanensis proving their close connection. Besides, I.cyanantha (Guizhou) and I. cyanantha (Yunnan) formed a clade with I. glandlifera. Along with the findings and the results, the current study might provide valuable significant genomic resources for systematics and evolution of the genus impatiens in different altitudes of regions.

Transgenic Reduction of Cytokinin Levels in Roots Inhibits Root-Sprouting in Populus.

Root sprouts-the formation of new shoots from roots-is an important mechanism for local gene flow from poplar (Populus spp). An effective strategy to reduce root sprout formation could therefore help to ensure containment during field research and commercial deployment of poplar when grown as exotic or transgenic forms. We used a flavonoid glycosyltransferase gene promoter from Scutellaria barbata (SbUGT) to drive the expression of AtCKX2, a cytokinin oxidase from Arabidopsis that converts active to inactive cytokinins in roots of poplar. In the greenhouse, SbUGT::AtCKX2 transgenic plants exhibited a similar shoot growth habit, but had enhanced root growth and fewer root sprouts, compared to the wild-type control and transgenic events with low transgene expression in roots. Under field conditions, the transgenic trees also had similar growth habits and stem growth rates that were not statistically different from wild-type trees over 3 years. Removal of trunks generally induced high rates of root sprouting; however, in selected SbUGT::AtCKX2 transgenic poplar events there was an absence or fewer root sprouts compared to wild-type trees, consistent with the greenhouse results. Our study demonstrates that the SbUGT::AtCKX2 gene can effectively inhibit root sprouting of poplar trees under field conditions, and thus may provide a useful tool to address concerns associated with root-sprouting-mediated transgene spread.

Transcriptome Analysis Reveals Differential Gene Expression and a Possible Role of Gibberellins in a Shade-Tolerant Mutant of Perennial Ryegrass.

The molecular basis behind shade tolerance in plants is not fully understood. Previously, we have shown that a connection may exist between shade tolerance and dwarfism, however, the mechanism connecting these phenotypes is not well understood. In order to clarify this connection, we analyzed the transcriptome of a previously identified shade-tolerant mutant of perennial ryegrass (Lolium perenne L.) called shadow-1. shadow-1 mutant plants are dwarf, and are significantly tolerant to shade in a number of environments compared to wild-type controls. In this study, we treated shadow-1 and wild-type plants with 95% shade for 2 weeks and compared the transcriptomes of these shade-treated individuals with both genotypes exposed to full light. We identified 2,200 differentially expressed genes (DEGs) (1,096 up-regulated and 1,104 down-regulated) in shadow-1 mutants, compared to wild type, following exposure to shade stress. Of these DEGs, 329 were unique to shadow-1 plants kept under shade and were not found in any other comparisons that we made. We found 2,245 DEGs (1,153 up-regulated and 1,092 down-regulated) in shadow-1 plants, compared to wild-type, under light, with 485 DEGs unique to shadow-1 plants under light. We examined the expression of gibberellin (GA) biosynthesis genes and found that they were down-regulated in shadow-1 plants compared to wild type, notably gibberellin 20 oxidase (GA20ox), which was down-regulated to 3.3% (96.7% reduction) of the wild-type expression level under shade conditions. One GA response gene, lipid transfer protein 3 (LTP3), was also down-regulated to 41.5% in shadow-1 plants under shade conditions when compared to the expression level in the wild type. These data provide valuable insight into a role that GA plays in dwarfism and shade tolerance, as exemplified by shadow-1 plants, and could serve as a guide for plant breeders interested in developing new cultivars with either of these traits.

A Novel Two-Step Method for Screening Shade Tolerant Mutant Plants via Dwarfism.

When subjected to shade, plants undergo rapid shoot elongation, which often makes them more prone to disease and mechanical damage. Shade-tolerant plants can be difficult to breed; however, they offer a substantial benefit over other varieties in low-light areas. Although perennial ryegrass (Lolium perenne L.) is a popular species of turf grasses because of their good appearance and fast establishment, the plant normally does not perform well under shade conditions. It has been reported that, in turfgrass, induced dwarfism can enhance shade tolerance. Here we describe a two-step procedure for isolating shade tolerant mutants of perennial ryegrass by first screening for dominant dwarf mutants, and then screening dwarf plants for shade tolerance. The two-step screening process to isolate shade tolerant mutants can be done efficiently with limited space at early seedling stages, which enables quick and efficient isolation of shade tolerant mutants, and thus facilitates development of shade tolerant new cultivars of turfgrasses. Using the method, we isolated 136 dwarf mutants from 300,000 mutagenized seeds, with 65 being shade tolerant (0.022%). When screened directly for shade tolerance, we recovered only four mutants from a population of 150,000 (0.003%) mutagenized seeds. One shade tolerant mutant, shadow-1, was characterized in detail. In addition to dwarfism, shadow-1 and its sexual progeny displayed high degrees of tolerance to both natural and artificial shade. We showed that endogenous gibberellin (GA) content in shadow-1 was higher than wild-type controls, and shadow-1 was also partially GA insensitive. Our novel, simple and effective two-step screening method should be applicable to breeding shade tolerant cultivars of turfgrasses, ground covers, and other economically important crop plants that can be used under canopies of existing vegetation to increase productivity per unit area of land.

Isolation of prostrate turfgrass mutants via screening of dwarf phenotype and characterization of a perennial ryegrass prostrate mutant.

Prostrate turf varieties are desirable because of their increased low mowing tolerance, heat resistance, traffic resistance and ground coverage compared with upright varieties. Mutation breeding may provide a powerful tool to create prostrate varieties, but there are no simple, straightforward methods to screen for such mutants. Elucidation of the molecular basis of the major 'green revolution' traits, dwarfism and semi-dwarfism, guided us to design a simple strategy for isolating dwarf mutants of perennial ryegrass (Lolium perenne L.). We have shown that gamma-ray-mediated dominant dwarf mutants can be easily screened for at the three-leaf stage. About 10% of dwarf mutant lines also displayed a prostrate phenotype at mature stages (>10 tillers). One prostrate line, Lowboy I, has been characterized in detail. Lowboy I had significantly shorter canopy, leaf blade and internode lengths compared with wild type. Lowboy I also exhibited greater tolerance to low mowing stress than wild type. Exogenous gibberellic acid (GA) restored Lowboy I to a wild-type phenotype, indicating that the dwarf and prostrate phenotypes were both due to GA deficiency. We further showed that phenotypes of Lowboy I were dominant and stably inherited through sexual reproduction. Prostrate turfgrass mutants are difficult to screen for because the phenotype is not observed at young seedling stages, therefore our method represents a simple strategy for easily isolating prostrate mutants. Furthermore, Lowboy I may provide an outstanding germplasm for breeding novel prostrate perennial ryegrass cultivars.