Key message
N-cre
and
C-cre
added in separate lines reassemble functional Cre in F1 progeny to excise unnecessary DNA, including
cre
DNA, thereby eliminating generations needed to cross in and out
...cre
.
Crop improvement via transgenesis can benefit through efficient DNA integration strategies. As new traits are developed, new transgenes can be stacked by
in planta
site-specific integration near previous transgenes, thereby facilitating their introgression to field cultivars as a single segregation locus. However, as each round of integration often requires use of selectable markers, it is more convenient to reuse the selection scheme. The Cre recombinase can be used to delete away previously used selection genes, and other DNA no longer needed after transformation, but the constitutive production of this DNA scanning protein can also affect plant growth. We had previously described in
Arabidopsis
a split Cre protein fragment complement scheme to reassemble a functional Cre recombinase. As our goal for developing this system was to deploy its use in major crop plants, here we show that Cre protein fragment complementation works in rice with precise recombination structures confirmed by DNA sequencing. As each N-terminal and C-terminal fragment is also flanked by
lox
recombination sites, they can also self-excise to avoid the need to segregate away the
cre
DNA. Options to form F1 hybrids homozygous for one transgene, or hemizygous for two different transgenes at the same chromosome location, are discussed.
Cadmium (Cd) is a toxic heavy metal that can accumulate in crop plants. We reported previously the engineering of a low cadmium-accumulating line (2B) of rice through overexpression of a truncated ...OsO3L2 gene. As expression of this transgene was highest in plant roots, amplicon and metatranscriptome sequencing were used to investigate the possibility that its expression affects root associated microbes. Based on amplicon sequencing of bacterial 16S rRNA, but less so from fungal ITS, the OTUs (operational taxonomic units) showed less diversity in soil tightly (rhizoplane) than loosely (rhizosphere) associated with plant roots. Significantly changed OTUs caused by the low-Cd accumulating plant 2B, Cd treatment or both were found, and 10 of the 13 OTUs (77%) that were enriched in Cd treated 2B samples over the wild type counterpart have been previously described as involved in tolerance to Cd or other heavy metals. Metatranscriptome sequencing of rhizosphere microbiome found that bacteria accounted for 70–75% of the microbial RNA. Photosynthesis-antenna proteins and nitrogen metabolism pathways were most active in soil microbes treated with Cd and grown with plant 2B. Correspondingly, the relative abundance of Cyanobacteria was enriched to < 1% of Cd treated rhizosphere bacteria, yet accounted for up to 13% of Cd treated 2B rhizospheric transcripts. These enriched microbes by transgene and Cd are worthy candidates for future application on reducing crop uptake of Cd.
•Overexpression of a cadmium-reducing gene reshaped rice root microbiome.•Cadmium treatment contributed to shifts of the rice root microbiome.•Microbial taxa involved in low Cd accumulation of transgenic rice were identified.•Cyanobacteria could play a role in rice low Cd accumulation trait formation.
Survival of a species depends on reproductive fitness and a plant's floral transition is controlled by developmental and environmental signals. In Arabidopsis, the floral integrators SOC1 (SUPPRESSOR ...OF OVEREXPRESSION OF CONSTANS 1) and FT (FLOWERING LOCUS T) sense various pathway signals to activate floral meristem identity genes. At high stress intensity, greater nuclear accumulation of the zinc-finger transcription factor OXS2 (OXIDATIVE STRESS 2) activates an early-flowering stress-escape response. Curiously, accumulation of OXS2 in the cytoplasm can delay flowering, prompting the hypothesis that in absence of stress, OXS2 helps to maintain vegetative growth. While the mechanism of stress-escape was identified as the OXS2-mediated transcription of SOC1, how cytoplasmic OXS2 delays flowering was unknown. Here, we report that OXS2 can interact indirectly with florigen FT and transcription factor FD (FLOWERING LOCUS D), the two proteins known to induce floral transition. By using 14-3-3Ω as a bridge linker, OXS2 can alter the subcellular distribution of FT. This lead to a speculation on how cytoplasmic OXS2 is able to prevent early flowering, by keeping FT from the nucleus.
•OXS2 regulates flowering time under stress.•14-3-3Ω links OXS2 to florigen FT as bridge protein.•Interaction between OXS2 and FT appears in cytoplasm and nucleus.•OXS2 increases cytoplasmic accumulation of FT in the absence of stress.
Abscisic acid (ABA) and the AP2/ERF (APETALA2/ETHYLENE-RESPONSIVE FACTOR)-type transcription factor called ABA INSENSITIVE 4 (ABI4) play pivotal roles in plant growth responses to environmental ...stress. An analysis of seedling development in Arabidopsis ABA hypersensitive mutants suggested that OXS3 (OXIDATIVE STRESS 3), OXS3b, O3L3 (OXS3 LIKE 3), O3L4, and O3L6 were negative regulators of ABI4 expression. We therefore characterized the roles of the OXS3 family members in ABA signaling. All the above five OXS3 proteins were found to interact with AFP1 (ABI FIVE BINDING PROTEIN 1) in yeast two hybrid assays. Seven OXS3 family members including two other members O3L1 and O3L5 were found to interact with histone H2A.X, although OXS3b, O3L3, and O3L5 showed weaker interactions. ChIP-qPCR analysis showed that the absence of some of these OXS3 family proteins was associated with increased occupancy of histone γ-H2A.X at the ABI4 promoter, which also corresponded with de-repression of ABI4 expression. Repression of ABI4 expression, however, required both AFP1 and OXS3, OXS3b or O3L6. We conclude that in the absence of stress, OXS3 family proteins regulate γ-H2A.X deposition at the ABI4 promoter and that together with AFP1, OXS3 family proteins function to prevent ABA-induced growth arrest by co-repressing ABI4 through decreased promoter occupancy of histone γ-H2A.X.
As plants encounter various environmental stresses, judicial allocation of resources to stress response is crucial for plant fitness. The plant OXS2 (OXIDATIVE STRESS 2) family has been reported to ...play important roles in growth regulation and stress response. Here, we report that the maize OXS2 family member ZmOXS2a when expressed in Arabidopsis retards growth including delayed flowering, but improves heat tolerance. ZmOXS2a can be found in the cytoplasm, nucleus and PBs/P bodies (mRNA processing bodies), but heat treatment induces higher accumulation in the PBs. Deletion of ARR (arginine rich region) and TZF (tandem zinc finger) domains for high-affinity RNA-binding reduced PBs accumulation of ZmOXS2a; and unlike ZmOXS2a, expression of this deletion mutant gene affected neither Arabidopsis growth nor heat tolerance. This suggests that ZmOXS2a might be involved in RNA degradation, which would also account for the larger amount of down-regulated genes found in ZmOXS2a expressing lines. Furthermore, 240 of 890 down-regulated genes contain ARE (AU-rich elements) in the mRNA 3′UTR that might be potential targets of ZmOXS2a. Expression of ZmOXS2a also disturbs the response to ABA (abscisic acid) and cytokinin, as GO (gene ontology) analysis shows that 50 and 15 DEGs (differentially expressed genes) are enriched in the GO term for ABA and cytokinin responses, respectively. ZmOXS2a expression lines are more sensitive to ABA, but less sensitive to cytokinin. It is likely that ZmOXS2a promotes the degradation of the mRNA of down-regulated genes containing ARE, which consequently perturbs the hormone pathways that affect stress response-related plant growth.
•Constitutive expression of ZmOXS2a in Arabidopsis retards growth but improves heat tolerance.•Deletion of ARR and TZF domains reduces localization to the PBs and restores wild type phenotype.•Constitutive expression of ZmOXS2a in Arabidopsis down-regulates 890 genes and 27% of them contain ARE.•Transgenic lines are more sensitive to ABA but less sensitive to cytokinin.
Abstract
As millions of seeds are produced from a breeding line, the long-term stability of transgene expression is vital for commercial-scale production of seeds with transgenic traits. Transgenes ...can be silenced by epigenetic mechanisms, but reactivation of expression can occur as a result of treatment with chromatin modification inhibitors such as 5-azacytidine, from stress such as heat or UV-B, or in mutants that have acquired a defect in gene silencing. Previously, we targeted a gfp reporter gene into the tobacco (Nicotiana tabacum) genome by site-specific recombination but still found some silenced lines among independent integration events. One such line also had a second random copy and both copies showed DNA hypermethylation. To test whether removing the second copy would reactivate gfp expression, two T1 plants were backcrossed to the wild type. Whereas the silenced status was maintained in the progenies from one backcross, spontaneous partial reactivation of gfp expression was found among progenies from a second backcross. However, this reactivation did not correlate with loss of the second random copy or with a significant change in the pattern or amount of DNA hypermethylation. This finding supports the suggestion that gene reactivation does not necessarily involve loss of DNA homology or methylation.
A site-specific silenced gfp transgene associated with a second gfp copy and DNA hypermethylation can be reactivated independent of a change in DNA methylation or transgene copy number.
Key message
Five soybean target lines with recombinase sites at suitable genomic positions were obtained and tested for site-specific gene stacking.
For introgression of new transgenic traits to ...field cultivars, adding new DNA to an existing transgene locus would reduce the number of segregating loci to reassemble back into a breeding line. We described previously an
in planta
transgene stacking system using the Bxb1 integrase to direct new DNA into a genomic target, but for this system to operate, the target locus must have a preexisting recombination site for Bxb1-mediated integration. Here, we describe 5 soybean target lines from the screening of 118
Agrobacterium
-mediated transgenic plants that were positive for
gus
expression. Each of the 5 target lines has a single copy of the transgenic DNA with precise DNA sequences of the recombinase recognition sites, located at least 1 kb away from the nearest coding region, not close to the centromere, and showed good expression of the reporter gene. We tested Bxb1 integrase-mediated integration of a
gfp
-containing plasmid into each of these lines and showed precise site-specific integration in bombarded calluses. For plant regeneration, we used embryonic axes of mature soybean seeds to conduct a new set of biolistic transformation with a
DsRed
-containing plasmid. Three integration events were regenerated into whole plants, demonstrating the principle that target lines can serve as foundation lines for the stacking of DNA to predefined locations in the soybean genome.
Transgene integration typically takes place in an easy-to-transform laboratory variety before the transformation event is introgressed through backcrosses to elite cultivars. As new traits are added ...to existing transgenic lines, site-specific integration can stack new transgenes into a previously created transgenic locus.
site-specific integration minimizes the number of segregating loci to assemble into a breeding line, but cannot break genetic linkage between the transgenic locus and nearby undesirable traits. In this study, we describe an additional feature of an
gene-stacking scheme, in which the Cre (control of recombination) recombinase not only deletes transgenic DNA no longer needed after transformation but also mediates recombination between homologous or non-homologous chromosomes. Although the target site must first be introgressed through conventional breeding, subsequent transgenes inserted into the same locus would be able to use Cre-mediated translocation to expedite a linkage drag-free introgression to field cultivars.
Dear Editor,
Cadmium （Cd） is a toxic heavy metal and carcinogen, and its en- try into the food chain has serious effects on food security and public health （Uraguchi et al., 2011）. As rice is a ...staple food crop for half of the world＇s population, Cd in rice grain has become a major source of dietary Cd for this part of the world.
Abstract
Histone replacement in chromatin-remodeling plays an important role in eukaryotic gene expression. New histone variants replacing their canonical counterparts often lead to a change in ...transcription, including responses to stresses caused by temperature, drought, salinity, and heavy metals. In this study, we describe a chromatin-remodeling process triggered by eviction of Rad3/Tel1-phosphorylated H2Aα, in which a heterologous plant protein AtOXS3 can subsequently bind fission yeast HA2.Z and Swc2, a component of the SWR1 complex, to facilitate replacement of H2Aα with H2A.Z. The histone replacement increases occupancy of the oxidative stress-responsive transcription factor Pap1 at the promoters of at least three drug-resistant genes, which enhances their transcription and hence primes the cell for higher stress tolerance.
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