Tea plant (
Camellia sinensis
) has very long history of cultivation and abundant germplasm resources in China. Purple bud is a characteristic variety, which has attracted the attention of breeding ...researchers because it accumulated a large number of anthocyanins naturally. In many species, R2R3-MYB transcription factors (TFs) were proved to be involved in the regulation of anthocyanin biosynthesis. Research on anthocyanin metabolism has been relatively clear in some species, but that needs to be further elucidated in tea plants. In this research, an R2R3-MYB transcription factor
CsMYB113
related to the anthocyanin accumulation regulation was identified from tea plants. Spatial and temporal expression analysis revealed differential expression of
CsMYB113
among different tissues and organs, with highest expression occurring in the roots. Subcellular localization assays showed that CsMYB113 localized in the nucleus. Ectopic expression of
CsMYB113
increased pigmentation and anthocyanin contents by the upregulation of the expression levels of genes in anthocyanin biosynthesis pathway among different tissues of
Arabidopsis
. Moreover, transient overexpression of
35S::CsMYB113
in tea plant increased the anthocyanin contents in the leaves. Our results indicated that
CsMYB113
plays important role in the anthocyanin biosynthesis regulation in tea plants. It will also provide useful candidate gene for the modification of anthocyanin metabolism by genetic engineering in plants.
BACKGROUND: Hydroponic growth systems are a convenient platform for studying whole plant physiology. However, we found through trialling systems as they are described in the literature that our ...experiments were frequently confounded by factors that affected plant growth, including algal contamination and hypoxia. We also found the way in which the plants were grown made them poorly amenable to a number of common physiological assays. RESULTS: The drivers for the development of this hydroponic system were: 1) the exclusion of light from the growth solution; 2) to simplify the handling of individual plants, and 3) the growth of the plant to allow easy implementation of multiple assays. These aims were all met by the use of pierced lids of black microcentrifuge tubes. Seed was germinated on a lid filled with an agar-containing germination media immersed in the same solution. Following germination, the liquid growth media was exchanged with the experimental solution, and after 14-21 days seedlings were transferred to larger tanks with aerated solution where they remained until experimentation. We provide details of the protocol including composition of the basal growth solution, and separate solutions with altered calcium, magnesium, potassium or sodium supply whilst maintaining the activity of the majority of other ions. We demonstrate the adaptability of this system for: gas exchange measurement on single leaves and whole plants; qRT-PCR to probe the transcriptional response of roots or shoots to altered nutrient composition in the growth solution (we demonstrate this using high and low calcium supply); producing highly competent mesophyll protoplasts; and, accelerating the screening of Arabidopsis transformants. This system is also ideal for manipulating plants for micropipette techniques such as electrophysiology or SiCSA. CONCLUSIONS: We present an optimised plant hydroponic culture system that can be quickly and cheaply constructed, and produces plants with similar growth kinetics to soil-grown plants, but with the advantage of being a versatile platform for a myriad of physiological and molecular biological measurements on all plant tissues at all developmental stages. We present ‘tips and tricks’ for the easy adoption of this hydroponic culture system.
In a previous study, we reported a transient transformation system using repeated screening for hygromycin B (Hyg) resistance in the basidiomycete
Ceriporiopsis subvermispora
. In the present study, ...by combining this technique with CRISPR/Cas9, we demonstrated successful marker-free genome editing in
Pleurotus ostreatus
, which is one of the most economically important cultivated mushrooms as well as a model white-rot fungus. At first, transformant selection mediated by the transient expression of marker genes was demonstrated using a plasmid harboring the Hyg resistance gene (
hph
) in
P. ostreatus
. Then, genome editing of
fcy1
, which confers 5-fluorocytosine (5-FC) resistance to the host cell, was performed by the transient expression of Cas9, gRNA, and
hph
and strains with 5-FC resistance and Hyg sensitivity were isolated. Additionally, genome editing of
fcy1
in these strains was confirmed by Sanger sequencing. To our knowledge, this is the first report of marker-free genome editing through the transient expression of Cas9, gRNA, and
hph
in agaricomycetes, which opens the door for repeated genome editing in these fungi.
Genetic transformation has always been an important method for studying medical plant secondary metabolic regulation, among which stable transformation has a good reproducibility. However, it was ...time-consuming to obtain a stable transformed hairy root or transgenic plants, which was difficult to satisfy the great demand of researches on medical plant secondary metabolism–related genes. Moreover,
Agrobacterium tumefaciens
–mediated transient transformation has been extensively applied in studies of functional genes because of its simpleness, low cost, and short period. However, presently, researches on medical plant functional genes commonly used stable genetic transformation and some high-cost and high-difficulty transient transformation methods, such as gene gun and protoplast transformation. Thus, in this study, we selected the seedlings of
Nicotiana benthamiana
,
Salvia miltiorrhiza
, and
Prunella vulgaris
as the experimental material, with the methods of
Agrobacterium tumefaciens
injection, fast
Agrobacterium
-mediated seedling transformation (FAST), and FAST and mechanical damage. The results demonstrated that the injection transient transformation system of pCAMBIA1301 vector mediated by
A
.
tumefaciens
and the transient transformation of seedling system were not established in
S
.
miltiorrhiza
. In addition, the instantaneous transformation system of
N
.
benthamiana
and
P
.
vulgaris
seedlings was basically set up by FAST method. Besides, using the method of FAST and mechanical damage, the transient genetic transformation system of
P
.
vulgaris
seedlings was established for the first time.
A
.
tumefaciens
–mediated transient transformation of seedlings with pEAQ vectors provided an effective way and reference for the further study of functional genes of the medicinal plants
N
.
benthamiana
and
P
.
vulgaris
.
Avocado,
Persea americana
Mill, is one of the most traded tropical fruits in the international market. To date, stable and transient transformation has only been achieved for of zygotic embryos and ...not of adult plant tissue, which limits functional genomics research. We provide the first transient
Agrobacterium
-mediated transformation methodology in avocado leaves that overcomes the recalcitrance to transformation of this species. We investigated the effect of
Agrobacterium
strain, leaf stage, wounding pre-treatment, the phytohormone jasmonic acid, and vacuum infiltration on transient transformation of avocado leaves. Using the
Agrobacterium
strain LBA4404 and the
RUBY
reporter a transformation frequency of up to 27% was obtained for avocado detached leaves. The transformation efficiency depended on the age of the leaf, with an intermediate stage of leaf development showing the highest efficiency of transient reporter gene expression. Microwounding pre-treatment facilitates agroinfiltration and coupled with leaf age are the primary factors influencing competence for transient transformation. Jasmonic acid did not significantly affect transient transformation in the absence of microwounding. However, microwounding and 250 µM of jasmonic acid acted synergistically to significantly enhance transient expression. Using this methodology with localized vacuum agroinfiltration, transient transformation of attached avocado leaves was achieved. This method unlocks the use of
Agrobacterium
-mediated transient transformation as a tool for explore gene function and metabolic pathways in both, detached and attached avocado leaves.
Key message
Leaf age, wounding and jasmonic acid counter the recalcitrant nature of avocado, unlocking the use of
Agrobacterium
mediated transient transformation as a tool for functional genetics in this plant. In addition, localized vacuum infiltration bypasses plant size constraints for
in-planta
transient transformation.
Camellia oleifera (C. oleifera) is a woody edible oil crop of great economic importance. Because of the lack of modern biotechnology research, C. oleifera faces huge challenges in both breeding and ...basic research. The protoplast and transient transformation system plays an important role in biological breeding, plant regeneration and somatic cell fusion. The objective of this present study was to develop a highly efficient protocol for isolating and purifying mesophyll protoplasts and transient transformation of C. oleifera. Several critical factors for mesophyll protoplast isolation from C. oleifera, including starting material (leaf age), pretreatment, enzymatic treatment (type of enzyme, concentration and digestion time), osmotic pressure and purification were optimized. Then the factors affecting the transient transformation rate of mesophyll protoplasts such as PEG molecular weights, PEG4000 concentration, plasmid concentration and incubation time were explored.
The in vitro grown seedlings of C. oleifera 'Huashuo' were treated in the dark for 24 h, then the 1st to 2nd true leaves were picked and vacuumed at - 0.07 MPa for 20 min. The maximum yield (3.5 × 10
/g·FW) and viability (90.9%) of protoplast were reached when the 1st to 2nd true leaves were digested in the enzymatic solution containing1.5% (w/v) Cellulase R-10, 0.5% (w/v) Macerozyme R-10 and 0.25% (w/v) Snailase and 0.4 M mannitol for 10 h. Moreover, the protoplast isolation method was also applicable to the other two cultivars, the protoplast yield for 'TXP14' and 'DP47' was 1.1 × 10
/g·FW and 2.6 × 10
/g·FW, the protoplast viability for 'TXP14' and 'DP47' was 90.0% and 88.2%. The purification effect was the best when using W buffer as a cleaning agent by centrifugal precipitation. The maximum transfection efficiency (70.6%) was obtained with the incubation of the protoplasts with 15 µg plasmid and 40% PEG4000 for 20 min.
In summary, a simple and efficient system for isolation and transient transformation of C. oleifera mesophyll protoplast is proposed, which is of great significance in various aspects of C. oleifera research, including the study of somatic cell fusion, genome editing, protein function, signal transduction, transcriptional regulation and multi-omics analyses.
Grape (
Vitis
L.), a highly valued fruit crop, poses significant challenges in genetic transformation and functional characterization of genes. Therefore, there is an urgent need for the development ...of a rapid and effective method for grape transformation and gene function identification. Here, we introduce a streamlined
Agrobacterium
-mediated transient transformation system for grape calli. Optimal conditions were established with a leaf-derived callus induction medium; chiefly B5 medium supplemented with 0.05 mg/L NAA, 0.5 mg/L 2,4-D, and 2.0 mg/L KT; and a callus proliferation medium (B5 medium supplemented with 0.5 mg/L NAA and 2.0 mg/L 6-BA), respectively. Notably, GUS enzyme activity peaked (352.96 ± 33.95 mol 4-MU/mg/min) by sonication with
Agrobacterium tumefaciens
EHA105 and 100 μM AS for 4 min, followed by vacuum infection for 5 min, and co-culture at 25 °C in the dark for 1 day using callus as explants at an optical density (OD
600
) of 0.8.
VaCIPK18
gene was transiently transformed into calli, and transcripts of the gene (endogenous and exogenous) were detected at higher levels than in non-transformed calli (endogenous). Moreover, after 10 days of treatment at 4 °C or −4 °C, the callus net weight of transformed callus was significantly higher than that of the untransformed callus, indicating that the
VaCIPK18-
overexpressing grape callus could improve cold tolerance. Overall, we establish a simple but effective transient transformation approach for grape callus, which could serve as a useful tool for the rapid assessment of gene function in this important crop.
Gene expression profiles are powerful tools for investigating mechanisms of plant stress tolerance.
(birch) is a widely distributed tree, but its drought-tolerance mechanism has been little studied. ...Using RNA-Seq, we identified 2917 birch genes involved in its response to drought stress. These drought-responsive genes include the late embryogenesis abundant (LEA) family, heat shock protein (HSP) family, water shortage-related and ROS-scavenging proteins, and many transcription factors (TFs). Among the drought-induced TFs, the ethylene responsive factor (ERF) and myeloblastosis oncogene (MYB) families were the most abundant. BpERF2 and BpMYB102, which were strongly induced by drought and had high transcription levels, were selected to study their regulatory networks. BpERF2 and BpMYB102 both played roles in enhancing drought tolerance in birch. Chromatin immunoprecipitation combined with qRT-PCR indicated that BpERF2 regulated genes such as those in the
and
families, while BpMYB102 regulated genes such as
(
) and
(
). Multiple genes were regulated by both BpERF2 and BpMYB102. We further characterized the function of some of these genes, and the genes that encode Root Primordium Defective 1 (RPD1), PRP1, 4CL10, LEA1, SOD5, and HSPs were found to be involved in drought tolerance. Therefore, our results suggest that BpERF2 and BpMYB102 serve as transcription factors that regulate a series of drought-tolerance genes in
to improve drought tolerance.
As one of the most economically important fruit crops in the world, the grapevine (
Vitis vinifera
) suffers significant yield losses from various pathogens including powdery mildew caused by
...Erysiphe necator
. In contrast, several wild Chinese grapevines, including
Vitis pseudoreticulata
accession Baihe-35-1, are highly resistant to powdery mildew pathogens. Here, we identified a grapevine gene
CSN5
(
COP9 signalosome complex subunit 5
), designated
VvCSN5
, that was differentially expressed between the resistant ‘Baihe-35-1’ and susceptible ‘Thompson Seedless’ during powdery mildew isolate
Erysiphe necator
NAFU1 infection. Moreover, transient silencing of
VvCSN5
in ‘Thompson Seedless’ leaves enhanced resistance to
En
NAFU1. This resistance manifested in cell wall callose deposition at attempted infection sites and hypersensitive response-like cell death of penetrated epidermal cells. Several defense-related marker genes (
VvPR1
,
VvPR3
,
VvPAD4
, and
VvRBOHD
) had higher basal expression levels in
VvCSN5
-silenced leaves. In addition, we found the structure and activity of
CSN5
promoters in ‘Thompson Seedless’ and ‘Baihe-35-1’ were different, which may have been behind their different resistances to powdery mildew infection. Taken together, these results implied that grapevine CSN5 plays an important role in the response to powdery mildew infection.
Key message
Transient silencing of
VvCSN5
in
Vitis vinifera
leaves enhances powdery mildew resistance. In addition, the
CSN5
promoters in susceptible and resistant grapevines are different.
Summary
In recent years, lipid droplets have emerged as dynamic organelles rather than inactive storage sites for triacylglycerol. The number of proteins known to be associated with lipid droplets ...has increased, but remains small in comparison with those found with other organelles. Also the mechanisms of how lipid droplets are recognized and bound by proteins need deeper investigation. Here, we present a fast, simple and inexpensive approach to assay proteins for their association with lipid droplets in vivo that can help to screen protein candidates or mutated variants of proteins for their association in an efficient manner. For this, a system to transiently transform Nicotiana tabacum pollen grains was used because these naturally contain lipid droplets. We designed vectors for fast cloning of genes as fusions with either mVenus or mCherry. This allowed us to assay colocalization with lipid droplets stained with Nile Red and Bodipy 505/515, respectively. We successfully tested our system not only for proteins from Arabidopsis thaliana, but also for proteins from the moss Physcomitrella patens and the alga Chlamydomonas reinhardtii. The small size of the vector used allows easy exchange of codons by site‐directed mutagenesis. We used this to show that two proline residues in the proline knot of a caleosin are not essential for the binding of lipid droplets. We also demonstrated that peroxisomes are not associated with the lipid droplets in tobacco pollen tubes, which reduces the risk of false interpretation of microscopic data in our system.
Significance Statement
Only a few lipid droplet‐associated proteins are known, and their binding mechanisms are mostly unclear. Here we show that transient expression of constructs in tobacco pollen tubes, which naturally contain lipid droplets, can be used to test protein localization to lipid droplets.
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