Plant Science & Landscape Architecture Research Works

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A Comparison of Irrigation-Water Containment Methods and Management Strategies Between Two Ornamental Production Systems to Minimize Water Security Threats
(MDPI, 2019-12-03) Ristvey, Andrew G.; Belayneh, Bruk E.; Lea-Cox, John D.
Water security in ornamental plant production systems is vital for maintaining profitability. Expensive, complicated, or potentially dangerous treatment systems, together with skilled labor, is often necessary to ensure water quality and plant health. Two contrasting commercial ornamental crop production systems in a mesic region are compared, providing insight into the various strategies employed using irrigation-water containment and treatment systems. The first is a greenhouse/outdoor container operation which grows annual ornamental plants throughout the year using irrigation booms, drip emitters, and/or ebb and flow systems depending on the crop, container size, and/or stage of growth. The operation contains and recycles 50–75% of applied water through a system of underground cisterns, using a recycling reservoir and a newly constructed 0.25 ha slow-sand filtration (SSF) unit. Groundwater provides additional water when needed. Water quantity is not a problem in this operation, but disease and water quality issues, including agrochemicals, are of potential concern. The second is a perennial-plant nursery which propagates cuttings and produces field-grown trees and containerized plants. It has a series of containment/recycling reservoirs that capture rainwater and irrigation return water, together with wells of limited output. Water quantity is a more important issue for this nursery, but poor water quality has had some negative economic effects. Irrigation return water is filtered and sanitized with chlorine gas before being applied to plants via overhead and micro-irrigation systems. The agrochemical paclobutrazol was monitored for one year in the first operation and plant pathogens were qualified and quantified over two seasons for both production systems. The two operations employ very different water treatment systems based on their access to water, growing methods, land topography, and capital investment. Each operation has experienced different water quantity and quality vulnerabilities, and has addressed these threats using a variety of technologies and management techniques to reduce their impacts.
Acclimation and Compensating Metabolite Responses to UV-B Radiation in Natural and Transgenic Populus spp. Defective in Lignin Biosynthesis
(MDPI, 2022-08-20) Wong, Tiffany M.; Sullivan, Joe H.; Eisenstein, Edward
Plants have evolved to protect leaf mesophyll tissue from damage caused by UV-B radiation by producing an array of UV-absorbing secondary metabolites. Flavonoids (phenolic glycosides) and sinapate esters (hydroxycinnamates) have been implicated as UV-B protective compounds because of the accumulation in the leaf epidermis and the strong absorption in the wavelengths corresponding to UV. Environmental adaptations by plants also generate a suite of responses for protection against damage caused by UV-B radiation, with plants from high elevations or low latitudes generally displaying greater adaptation or tolerance to UV-B radiation. In an effort to explore the relationships between plant lignin levels and composition, the origin of growth elevation, and the hierarchical synthesis of UV-screening compounds, a collection of natural variants as well as transgenic Populus spp. were examined for sensitivity or acclimation to UV-B radiation under greenhouse and laboratory conditions. Noninvasive, ecophysiological measurements using epidermal transmittance and chlorophyll fluorescence as well as metabolite measurements using UPLC-MS generally revealed that the synthesis of anthocyanins, flavonoids, and lignin precursors are increased in Populus upon moderate to high UV-B treatment. However, poplar plants with genetic modifications that affect lignin biosynthesis, or natural variants with altered lignin levels and compositions, displayed complex changes in phenylpropanoid metabolites. A balance between elevated metabolic precursors to protective phenylpropanoids and increased biosynthesis of these anthocyanins, flavonoids, and lignin is proposed to play a role in the acclimation of Populus to UV-B radiation and may provide a useful tool in engineering plants as improved bioenergy feedstocks.
Apple skin patterning is associated with differential expression of MYB10
(2011-05-20) Telias, Adriana; Lin-Wang, Kui; Stevenson, David E; Cooney, Janine M; Hellens, Roger P; Allan, Andrew C; Hoover, Emily E; Bradeen, James M
Background: Some apple (Malus × domestica Borkh.) varieties have attractive striping patterns, a quality attribute that is important for determining apple fruit market acceptance. Most apple cultivars (e.g. ‘Royal Gala’) produce fruit with a defined fruit pigment pattern, but in the case of ‘Honeycrisp’ apple, trees can produce fruits of two different kinds: striped and blushed. The causes of this phenomenon are unknown. Results: Here we show that striped areas of ‘Honeycrisp’ and ‘Royal Gala’ are due to sectorial increases in anthocyanin concentration. Transcript levels of the major biosynthetic genes and MYB10, a transcription factor that upregulates apple anthocyanin production, correlated with increased anthocyanin concentration in stripes. However, nucleotide changes in the promoter and coding sequence of MYB10 do not correlate with skin pattern in ‘Honeycrisp’ and other cultivars differing in peel pigmentation patterns. A survey of methylation levels throughout the coding region of MYB10 and a 2.5 Kb region 5’ of the ATG translation start site indicated that an area 900 bp long, starting 1400 bp upstream of the translation start site, is highly methylated. Cytosine methylation was present in all three contexts, with higher methylation levels observed for CHH and CHG (where H is A, C or T) than for CG. Comparisons of methylation levels of the MYB10 promoter in ‘Honeycrisp’ red and green stripes indicated that they correlate with peel phenotypes, with an enrichment of methylation observed in green stripes. Conclusions: Differences in anthocyanin levels between red and green stripes can be explained by differential transcript accumulation of MYB10. Different levels of MYB10 transcript in red versus green stripes are inversely associated with methylation levels in the promoter region. Although observed methylation differences are modest, trends are consistent across years and differences are statistically significant. Methylation may be associated with the presence of a TRIM retrotransposon within the promoter region, but the presence of the TRIM element alone cannot explain the phenotypic variability observed in ‘Honeycrisp’. We suggest that methylation in the MYB10 promoter is more variable in ‘Honeycrisp’ than in ‘Royal Gala’, leading to more variable color patterns in the peel of this cultivar.
Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis
(Springer Nature, 2019-01-31) Malzahn, Aimee A.; Tang, Xu; Lee, Keunsub; Ren, Qiurong; Sretenovic, Simon; Zhang, Yingxiao; Chen, Hongqiao; Kang, Minjeong; Bao, Yu; Zheng, Xuelian; Deng, Kejun; Zhang, Tao; Salcedo, Valeria; Wang, Kan; Zhang, Yong; Qi, Yiping
CRISPR-Cas12a (formerly Cpf1) is an RNA-guided endonuclease with distinct features that have expanded genome editing capabilities. Cas12a-mediated genome editing is temperature sensitive in plants, but a lack of a comprehensive understanding on Cas12a temperature sensitivity in plant cells has hampered effective application of Cas12a nucleases in plant genome editing. We compared AsCas12a, FnCas12a, and LbCas12a for their editing efficiencies and non-homologous end joining (NHEJ) repair profiles at four different temperatures in rice. We found that AsCas12a is more sensitive to temperature and that it requires a temperature of over 28 °C for high activity. Each Cas12a nuclease exhibited distinct indel mutation profiles which were not affected by temperatures. For the first time, we successfully applied AsCas12a for generating rice mutants with high frequencies up to 93% among T0 lines. We next pursued editing in the dicot model plant Arabidopsis, for which Cas12a-based genome editing has not been previously demonstrated. While LbCas12a barely showed any editing activity at 22 °C, its editing activity was rescued by growing the transgenic plants at 29 °C. With an early high-temperature treatment regime, we successfully achieved germline editing at the two target genes, GL2 and TT4, in Arabidopsis transgenic lines. We then used high-temperature treatment to improve Cas12a-mediated genome editing in maize. By growing LbCas12a T0 maize lines at 28 °C, we obtained Cas12a-edited mutants at frequencies up to 100% in the T1 generation. Finally, we demonstrated DNA binding of Cas12a was not abolished at lower temperatures by using a dCas12a-SRDX-based transcriptional repression system in Arabidopsis. Our study demonstrates the use of high-temperature regimes to achieve high editing efficiencies with Cas12a systems in rice, Arabidopsis, and maize and sheds light on the mechanism of temperature sensitivity for Cas12a in plants.
Auxin regulates adventitious root formation in tomato cuttings
(Springer Nature, 2019-10-21) Guan, Ling; Tayengwa, Reuben; Cheng, Zongming (Max); Peer, Wendy Ann; Murphy, Angus S.; Zhao, Mizhen
Adventitious root (AR) formation is a critical developmental process in cutting propagation for the horticultural industry. While auxin has been shown to regulate this process, the exact mechanism and details preceding AR formation remain unclear. Even though AR and lateral root (LR) formation share common developmental processes, there are exist some differences that need to be closely examined at the cytological level. Tomato stem cuttings, which readily form adventitious roots, represent the perfect system to study the influence of auxin on AR formation and to compare AR and LR organogenesis.
Bacterial community diversity and variation in spray water sources and the tomato fruit surface
(2011-04-21) Telias, Adriana; White, James R; Pahl, Donna M; Ottesen, Andrea R; Walsh, Christopher S
Background: Tomato (Solanum lycopersicum) consumption has been one of the most common causes of produceassociated salmonellosis in the United States. Contamination may originate from animal waste, insects, soil or water. Current guidelines for fresh tomato production recommend the use of potable water for applications coming in direct contact with the fruit, but due to high demand, water from other sources is frequently used. We sought to describe the overall bacterial diversity on the surface of tomato fruit and the effect of two different water sources (ground and surface water) when used for direct crop applications by generating a 454- pyrosequencing 16S rRNA dataset of these different environments. This study represents the first in depth characterization of bacterial communities in the tomato fruit surface and the water sources commonly used in commercial vegetable production. Results: The two water sources tested had a significantly different bacterial composition. Proteobacteria was predominant in groundwater samples, whereas in the significantly more diverse surface water, abundant phyla also included Firmicutes, Actinobacteria and Verrucomicrobia. The fruit surface bacterial communities on tomatoes sprayed with both water sources could not be differentiated using various statistical methods. Both fruit surface environments had a high representation of Gammaproteobacteria, and within this class the genera Pantoea and Enterobacter were the most abundant. Conclusions: Despite the major differences observed in the bacterial composition of ground and surface water, the season long use of these very different water sources did not have a significant impact on the bacterial composition of the tomato fruit surface. This study has provided the first next-generation sequencing database describing the bacterial communities living in the fruit surface of a tomato crop under two different spray water regimes, and therefore represents an important step forward towards the development of science-based metrics for Good Agricultural Practices.
Benefits of gene flow are mediated by individual variability in self-compatibility in small isolated populations of an endemic plant species
(John Wiley & Sons Ltd., 2016-09-05) Frye, Christopher T.; Neel, Maile C.
Many rare and endemic species experience increased rates of self-fertilization and mating among close relatives as a consequence of existing in small populations within isolated habitat patches. Variability in self-compatibility among individuals within populations may reflect adaptation to local demography and genetic architecture, inbreeding, or drift. We use experimental hand-pollinations under natural field conditions to assess the effects of gene flow in 21 populations of the central Appalachian endemic Trifolium virginicum that varied in population size and degree of isolation. We quantified the effects of distance from pollen source on pollination success and fruit set. Rates of self-compatibility varied dramatically among maternal plants, ranging from 0% to 100%. This variation was unrelated to population size or degree of isolation. Nearly continuous variation in the success of selfing and near-cross-matings via hand pollination suggests that T. virginicum expresses pseudo-self- fertility, whereby plants carrying the same S-allele mate successfully by altering the self-incompatibility reaction. However, outcrossing among populations produced significantly higher fruit set than within populations, an indication of drift load. These results are consistent with strong selection acting to break down self-incompatibility in these small populations and/or early-acting inbreeding depression expressed upon selfing.
Can City Lifestyle be a Catalyst for Smart Suburban Change?
(National Center for Smart Growth Research, 2004) Chang, Shenglin
The research investigates how Asian and Latino immigrants’ prior urban experiences can inform the future planning and growth of suburban communities in Maryland. The investigation of Maryland immigrants’ various built environment (dwelling, landscape, neighborhood, transportation mode) preferences will result in a set of ecologically appropriate and culturally sensitive design guidelines that will help shape the future of the rapidly growing suburban communities.
Comparative genome analyses reveal sequence features reflecting distinct modes of host-adaptation between dicot and monocot powdery mildew
(Springer Nature, 2018-09-25) Wu, Ying; Ma, Xianfeng; Pan, Zhiyong; Kale, Shiv D.; Song, Yi; King, Harlan; Zhang, Qiong; Presley, Christian; Deng, Xiuxin; Wei, Cheng-I; Xiao, Shunyuan
Powdery mildew (PM) is one of the most important and widespread plant diseases caused by biotrophic fungi. Notably, while monocot (grass) PM fungi exhibit high-level of host-specialization, many dicot PM fungi display a broad host range. To understand such distinct modes of host-adaptation, we sequenced the genomes of four dicot PM biotypes belonging to Golovinomyces cichoracearum or Oidium neolycopersici. We compared genomes of the four dicot PM together with those of Blumeria graminis f.sp. hordei (both DH14 and RACE1 isolates), B. graminis f.sp. tritici, and Erysiphe necator infectious on barley, wheat and grapevine, respectively. We found that despite having a similar gene number (6620–6961), the PM genomes vary from 120 to 222 Mb in size. This high-level of genome size variation is indicative of highly differential transposon activities in the PM genomes. While the total number of genes in any given PM genome is only about half of that in the genomes of closely related ascomycete fungi, most (~ 93%) of the ascomycete core genes (ACGs) can be found in the PM genomes. Yet, 186 ACGs were found absent in at least two of the eight PM genomes, of which 35 are missing in some dicot PM biotypes, but present in the three monocot PM genomes, indicating remarkable, independent and perhaps ongoing gene loss in different PM lineages. Consistent with this, we found that only 4192 (3819 singleton) genes are shared by all the eight PM genomes, the remaining genes are lineage- or biotype-specific. Strikingly, whereas the three monocot PM genomes possess up to 661 genes encoding candidate secreted effector proteins (CSEPs) with families containing up to 38 members, all the five dicot PM fungi have only 116–175 genes encoding CSEPs with limited gene amplification. Compared to monocot (grass) PM fungi, dicot PM fungi have a much smaller effectorome. This is consistent with their contrasting modes of host-adaption: while the monocot PM fungi show a high-level of host specialization, which may reflect an advanced host-pathogen arms race, the dicot PM fungi tend to practice polyphagy, which might have lessened selective pressure for escalating an with a particular host.
CRISPR enables directed evolution in plants
(Springer Nature, 2019-04-30) Zhang, Yingxiao; Qi, Yiping
A proof-of-concept study has demonstrated the application of CRISPR-Cas9 for directed evolution in rice, engineering crops for desired traits.
CRISPR-enabled investigation of fitness costs associated with the E198A mutation in β-tubulin of Colletotrichum siamense
(Frontiers, 2023-11-02) Cosseboom, Scott D.; Agarwal, Chiti; Hu, Mengjun
Introduction: Understanding fitness costs associated with fungicide resistance is critical to improve resistance management strategies. E198A in b-tubulin confers resistance to the fungicide thiophanate-methyl and has been widely reported in several plant pathogens including Colletotrichum siamense. Method: To better understand potential fitness costs associated with the resistance, a ribonucleoprotein (RNP) complex mediated CRISPR/Cas9 system was used to create a point mutation (E198A) through homology directed repair (HDR) in each of the sensitive (E198) C. siamense isolates collected from strawberries, raspberries, and peaches. The RNP complex was delivered into fungal protoplasts using polyethylene glycol-mediated (PEG) transfection. Results: The transformation efficiency, the proportion of transformants of sensitive parental isolates containing the E198A mutation, averaged 72%. No off-target mutations were observed when sequences similar to the b-tubulin target region with a maximum of four mismatch sites were analyzed, suggesting that the CRISPR/Cas9 system used in this study was highly specific for genome editing in C. siamense. Of the 41 comparisons of fitness between mutant and wild type isolates through in vitro and detached fruit assays, mutant isolates appeared to be as fit (24 of 41 comparisons), if not more fit than wild-type isolates (10 of 41 comparisons). Discussion: The use of CRISPR/Cas9 to evaluate fitness costs associated with point mutations in this study represents a novel and useful method, since wild-type and mutant isolates were genetically identical except for the target mutation.
Directed plant cell-wall accumulation of iron: embedding co-catalyst for efficient biomass conversion
(Springer Nature, 2016-10-21) Lin, Chien-Yuan; Jakes, Joseph E.; Donohoe, Bryon S.; Ciesielski, Peter N.; Yang, Haibing; Gleber, Sophie-Charlotte; Vogt, Stefan; Ding, Shi-You; Peer, Wendy A.; Murphy, Angus S.; McCann, Maureen C.; Himmel, Michael E.; Tucker, Melvin P.; Wei, Hui
Plant lignocellulosic biomass is an abundant, renewable feedstock for the production of biobased fuels and chemicals. Previously, we showed that iron can act as a co-catalyst to improve the deconstruction of lignocellulosic biomass. However, directly adding iron catalysts into biomass prior to pretreatment is diffusion limited, and increases the cost of biorefinery operations. Recently, we developed a new strategy for expressing iron-storage protein ferritin intracellularly to accumulate iron as a catalyst for the downstream deconstruction of lignocellulosic biomass. In this study, we extend this approach by fusing the heterologous ferritin gene with a signal peptide for secretion into Arabidopsis cell walls (referred to here as FerEX). The transgenic Arabidopsis plants. FerEX. accumulated iron under both normal and iron-fertilized growth conditions; under the latter (iron-fertilized) condition, FerEX transgenic plants showed an increase in plant height and dry weight by 12 and 18 %, respectively, compared with the empty vector control plants. The SDS- and native-PAGE separation of cell-wall protein extracts followed by Western blot analyses confirmed the extracellular expression of ferritin in FerEX plants. Meanwhile, Perls' Prussian blue staining and X-ray fluorescence microscopy (XFM) maps revealed iron depositions in both the secondary and compound middle lamellae cell-wall layers, as well as in some of the corner compound middle lamella in FerEX. Remarkably, their harvested biomasses showed enhanced pretreatability and digestibility, releasing, respectively, 21 % more glucose and 34 % more xylose than the empty vector control plants. These values are significantly higher than those of our recently obtained ferritin intracellularly expressed plants. This study demonstrated that extracellular expression of ferritin in Arabidopsis can produce plants with increased growth and iron accumulation, and reduced thermal and enzymatic recalcitrance. The results are attributed to the intimate colocation of the iron co-catalyst and the cellulose and hemicellulose within the plant cell-wall region, supporting the genetic modification strategy for incorporating conversion catalysts into energy crops prior to harvesting or processing at the biorefinery.
DNA binding specificity of ATAF2, a NAC domain transcription factor targeted for degradation by Tobacco mosaic virus
(2012-08-31) Wang, Xiao; Culver, James N
Background: Control of the host transcriptome represents a key battleground in the interaction of plants and pathogens. Specifically, plants have evolved complex defense systems that induce profound transcriptional changes in response to pathogen attack while pathogens have evolved mechanisms to subvert or disable these defenses. Several NAC transcription factors such as ATAF2 have been linked to plant defense responses, including those targeting viruses. The replication protein of Tobacco mosaic virus (TMV) has been shown to interact with and target the degradation of ATAF2. These findings suggest that the transcriptional targets of ATAF2 are involved in defense against TMV. Results: To detect potential ATAF2 transcriptional targets, a genomic pull-down assay was utilized to identify ATAF2 promoter binding sequences. Subsequent mobility shift and DNA footprinting assays identified a 30-bp ATAF2 binding sequence. An in vivo GUS reporter system confirmed the function of the identified 30-bp binding sequence as an ATAF2 specific transcriptional activator in planta. Gel filtration studies of purified ATAF2 protein and mutagenesis studies of the 30-bp binding sequence indicate ATAF2 functions as a dimer. Computational analysis of interacting promoter sequences identified a corresponding 25-bp A/T-rich consensus sequence with repeating [GC] AAA motifs. Upon ATAF2 induction real-time qRT-PCR studies confirmed the accumulation of select gene transcripts whose promoters contain this consensus sequence. Conclusion: We report the identification of a cis-regulatory binding sequence for ATAF2. Different from other known NAC protein binding sequences, the A/T-rich ATAF2 binding motif represents a novel binding sequence for NAC family proteins. Combined this information represents a unique tool for the identification of ATAF2 target genes.
Elucidating the evolutionary history and expression patterns of nucleoside phosphorylase paralogs (vegetative storage proteins) in Populusand the plant kingdom
(Springer Nature, 2013-08-19) Pettengill, Emily A; Pettengill, James B; Coleman, Gary D
Nucleoside phosphorylases (NPs) have been extensively investigated in human and bacterial systems for their role in metabolic nucleotide salvaging and links to oncogenesis. In plants, NP-like proteins have not been comprehensively studied, likely because there is no evidence of a metabolic function in nucleoside salvage. However, in the forest trees genus Populus a family of NP-like proteins function as an important ecophysiological adaptation for inter- and intra-seasonal nitrogen storage and cycling. We conducted phylogenetic analyses to determine the distribution and evolution of NP-like proteins in plants. These analyses revealed two major clusters of NP-like proteins in plants. Group I proteins were encoded by genes across a wide range of plant taxa while proteins encoded by Group II genes were dominated by species belonging to the order Malpighiales and included the Populus Bark Storage Protein (BSP) and WIN4-like proteins. Additionally, we evaluated the NP-like genes in Populus by examining the transcript abundance of the 13 NP-like genes found in the Populus genome in various tissues of plants exposed to long-day (LD) and short-day (SD) photoperiods. We found that all 13 of the Populus NP-like genes belonging to either Group I or II are expressed in various tissues in both LD and SD conditions. Tests of natural selection and expression evolution analysis of the Populus genes suggests that divergence in gene expression may have occurred recently during the evolution of Populus, which supports the adaptive maintenance models. Lastly, in silico analysis of cis-regulatory elements in the promoters of the 13 NP-like genes in Populus revealed common regulatory elements known to be involved in light regulation, stress/pathogenesis and phytohormone responses. In Populus, the evolution of the NP-like protein and gene family has been shaped by duplication events and natural selection. Expression data suggest that previously uncharacterized NP-like proteins may function in nutrient sensing and/or signaling. These proteins are members of Group I NP-like proteins, which are widely distributed in many plant taxa. We conclude that NP-like proteins may function in plants, although this function is undefined.
Evaluation of qPCR reference genes in two genotypes of Populus for use in photoperiod and low-temperature studies
(2012-07-23) Pettengill, Emily A; Parmentier-Line, Cecile; Coleman, Gary D
Background: Quantitative PCR (qPCR) is a widely used technique for gene expression analysis. A common normalization method for accurate qPCR data analysis involves stable reference genes to determine relative gene expression. Despite extensive research in the forest tree species Populus, there is not a resource for reference genes that meet the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) standards for qPCR techniques and analysis. Since Populus is a woody perennial species, studies of seasonal changes in gene expression are important towards advancing knowledge of this important developmental and physiological trait. The objective of this study was to evaluate reference gene expression stability in various tissues and growth conditions in two important Populus genotypes (P. trichocarpa “Nisqually 1” and P. tremula x P. alba 717 1-B4) following MIQE guidelines. Results: We evaluated gene expression stability in shoot tips, young leaves, mature leaves and bark tissues from P. trichocarpa and P. tremula. x P. alba grown under long-day (LD), short-day (SD) or SD plus low-temperatures conditions. Gene expression data were analyzed for stable reference genes among 18S rRNA, ACT2, CDC2, CYC063, TIP4-like, UBQ7, PT1 and ANT using two software packages, geNorm PLUS and BestKeeper. GeNorm PLUS ranked TIP4-like and PT1 among the most stable genes in most genotype/tissue combinations while BestKeeper ranked CDC2 and ACT2 among the most stable genes. Conclusions: This is the first comprehensive evaluation of reference genes in two important Populus genotypes and the only study in Populus that meets MIQE standards. Both analysis programs identified stable reference genes in both genotypes and all tissues grown under different photoperiods. This set of reference genes was found to be suitable for either genotype considered here and may potentially be suitable for other Populus species and genotypes. These results provide a valuable resource for the Populus research community.
Expanding the targeting scope of FokI-dCas nuclease systems with SpRY and Mb2Cas12a
(Wiley, 2022-04-04) Cheng, Yanhao; Sretenovic, Simon; Zhang, Yingxiao; Pan, Changtian; Huang, Ji; Qi, Yiping
CRISPR-Cas9 and Cas12a are widely used sequence-specific nucleases (SSNs) for genome editing. The nuclease domains of Cas proteins can induce DNA double strand breaks upon RNA guided DNA targeting. Zinc finger nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) have been popular SSNs prior to CRISPR. Both ZFNs and TALENs are based on reconstitution of two monomers with each consisting of a DNA binding domain and a FokI nuclease domain. Inspired by the configuration of ZFNs and TALENs, dimeric FokI-dCas9 systems were previously demonstrated in human cells. Such configuration, based on a pair of guide RNAs (gRNAs), offers great improvement on targeting specificity. To expand the targeting scope of dimeric FokI-dCas systems, the PAM (protospacer adjacent motif)-less SpRY Cas9 variant and the PAM-relaxed Mb2Cas12a system were explored. Rice cells showed that FokI-dSpRY had more robust editing efficiency than a paired SpRY nickase system. Furthermore, a dimeric FokI-dMb2Cas12a system was developed that displayed comparable editing activity to Mb2Cas12a nuclease in rice cells. Finally, a single-chain FokI-FokI-dMb2Cas12a system was developed that cuts DNA outside its targeting sequence, which could be useful for many versatile applications. Together, this work greatly expanded the FokI based CRISPR-Cas systems for genome editing.
Fruit Morphology Measurements of Jujube Cultivar ‘Lingwu Changzao’ (Ziziphus jujuba Mill. cv. Lingwuchangzao) during Fruit Development
(MDPI, 2021-02-06) Ma, Yaping; Zhang, Dapeng; Wang, Zhuangji; Song, Lihua; Cao, Bing
‘Lingwu Changzao’ (Ziziphus jujuba Mill. cv. Lingwuchangzao), a cultivar of Ziziphus in the Rhamnaceae family, is a traditional jujube cultivar in Ningxia, China. For ‘Lingwu Changzao’, morphological traits are prominent in characterizing fruit yield, quality, and consumer acceptance. However, morphological measurements for ‘Lingwu Changzao’ cultivation are limited. Therefore, the objective of this study is to measure the growing patterns of selected morphological traits during ‘Lingwu Changzao’ fruit development. Eight morphological traits, including four fruit traits (fruit length, diameter, weight, and flesh (mesocarp) thickness), three stone traits (stone length, diameter, and weight), and fruit firmness (also known as fruit hardness), were measured over a 3-mo (months) period, covering a completed fruit development period. Results indicate that the growing patterns of fruit traits coincide with double ‘S’ growth curves, which mainly present the growth of ‘Lingwu Changzao’ fruit. Increases of stone traits terminated in the early fruit growth period, while fruit traits continuously increased till the end of the 3-mo period. That implies a high fruit-stone ratio, i.e., a desirable quality attribute for ‘Lingwu Changzao’ as fresh-eating fruits. The results presented in this study can serve as one part of the standard dataset for jujube fruit cultivation in China, and it can also support decisions in plant breeding and field managements for ‘Lingwu Changzao’.
Genome editing is revolutionizing biology
(Springer Nature, 2017-07-14) Qi, Yiping
High throughput sequencing reveals novel and abiotic stress-regulated microRNAs in the inflorescences of rice
(Springer Nature, 2012-08-03) Barrera-Figueroaroa, Blanca E; Gao, Lei; Wu, Zhigang; Zhou, Xuefeng; Zhu, Jianhua; Jin, Hailing; Liu, Renyi; Zhu, Jian-Kang
MicroRNAs (miRNAs) are small RNA molecules that play important regulatory roles in plant development and stress responses. Identification of stress-regulated miRNAs is crucial for understanding how plants respond to environmental stimuli. Abiotic stresses are one of the major factors that limit crop growth and yield. Whereas abiotic stress-regulated miRNAs have been identified in vegetative tissues in several plants, they are not well studied in reproductive tissues such as inflorescences. We used Illumina deep sequencing technology to sequence four small RNA libraries that were constructed from the inflorescences of rice plants that were grown under control condition and drought, cold, or salt stress. We identified 227 miRNAs that belong to 127 families, including 70 miRNAs that are not present in the miRBase. We validated 62 miRNAs (including 10 novel miRNAs) using published small RNA expression data in DCL1, DCL3, and RDR2 RNAi lines and confirmed 210 targets from 86 miRNAs using published degradome data. By comparing the expression levels of miRNAs, we identified 18, 15, and 10 miRNAs that were regulated by drought, cold and salt stress conditions, respectively. In addition, we identified 80 candidate miRNAs that originated from transposable elements or repeats, especially miniature inverted-repeat elements (MITEs). We discovered novel miRNAs and stress-regulated miRNAs that may play critical roles in stress response in rice inflorescences. Transposable elements or repeats, especially MITEs, are rich sources for miRNA origination.
A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 (Cas12a) nucleases in rice
(Springer Nature, 2018-07-04) Tang, Xu; Liu, Guanqing; Zhou, Jianping; Ren, Qiurong; You, Qi; Tian, Li; Xin, Xuhui; Zhong, Zhaohui; Liu, Binglin; Zheng, Xuelian; Zhang, Dengwei; Malzahn, Aimee; Gong, Zhiyun; Qi, Yiping; Zhang, Tao; Zhang, Yong
Targeting specificity has been a barrier to applying genome editing systems in functional genomics, precise medicine and plant breeding. In plants, only limited studies have used whole-genome sequencing (WGS) to test off-target effects of Cas9. The cause of numerous discovered mutations is still controversial. Furthermore, WGS-based off-target analysis of Cpf1 (Cas12a) has not been reported in any higher organism to date. We conduct a WGS analysis of 34 plants edited by Cas9 and 15 plants edited by Cpf1 in T0 and T1 generations along with 20 diverse control plants in rice. The sequencing depths range from 45× to 105× with read mapping rates above 96%. Our results clearly show that most mutations in edited plants are created by the tissue culture process, which causes approximately 102 to 148 single nucleotide variations (SNVs) and approximately 32 to 83 insertions/deletions (indels) per plant. Among 12 Cas9 single guide RNAs (sgRNAs) and three Cpf1 CRISPR RNAs (crRNAs) assessed by WGS, only one Cas9 sgRNA resulted in off-target mutations in T0 lines at sites predicted by computer programs. Moreover, we cannot find evidence for bona fide off-target mutations due to continued expression of Cas9 or Cpf1 with guide RNAs in T1 generation. Our comprehensive and rigorous analysis of WGS data across multiple sample types suggests both Cas9 and Cpf1 nucleases are very specific in generating targeted DNA modifications and off-targeting can be avoided by designing guide RNAs with high specificity.