Targeted mutagenesis of the vacuolar H+ translocating pyrophosphatase gene reduces grain chalkiness in rice.

Grain chalkiness is a major concern in rice production because it impacts milling yield and cooking quality, eventually reducing market value of the rice. A gene encoding vacuolar H+ translocating pyrophosphatase (V-PPase) is a major quantitative trait locus in indica rice, controlling grain chalkiness. Higher transcriptional activity of this gene is associated with increased chalk content. However, whether the suppression of V-PPase could reduce chalkiness is not clear. Furthermore, natural variation in the chalkiness of japonica rice has not been linked with V-PPase. Here, we describe promoter targeting of the japonica V-PPase allele that led to reduced grain chalkiness and the development of more translucent grains. Disruption of a putative GATA element by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 suppressed V-PPase activity, reduced grain chalkiness and impacted post-germination growth that could be rescued by the exogenous supply of sucrose. The mature grains of the targeted lines showed a much lower percentage of large or medium chalk. Interestingly, the targeted lines developed a significantly lower chalk under heat stress, a major inducer of grain chalk. Metabolomic analysis showed that pathways related to starch and sugar metabolism were affected in the developing grains of the targeted lines that correlated with higher inorganic pyrophosphate and starch contents and upregulation of starch biosynthesis genes. In summary, we show a biotechnology approach of reducing grain chalkiness in rice by downregulating the transcriptional activity of V-PPase that presumably leads to altered metabolic rates, including starch biosynthesis, resulting in more compact packing of starch granules and formation of translucent rice grains.

Effect of Interfacial Schemes on the Optical and Structural Properties of InAs/GaSb Type-II Superlattices.

Incorporating an intentional strain compensating InSb interface (IF) layer in InAs/GaSb type-II superlattices (T2SLs) enhances device performance. But there is a lack of studies that correlate this approach's optical and structural quality, so the mechanisms by which this improvement is achieved remain unclear. One critical issue in increasing the performance of InAs/GaSb T2SLs arises from the lattice mismatch between InAs and GaSb, leading to interfacial strain in the structure. Not only that but also, since each side of the InAs/GaSb heterosystem does not have common atoms, there is a possibility of atomic intermixing at the IFs. To address such issues, an intentional InSb interfacial layer is commonly introduced at the InAs-on-GaSb and GaSb-on-InAs IFs to compensate for the strain and the chemical mismatches. In this report, we investigate InAs/GaSb T2SLs with (Sample A) and without (Sample B) InSb IF layers emitting in the mid-wavelength infrared (MWIR) through photoluminescence (PL) and band structure simulations. The PL studies indicate that the maximum PL intensity of Sample A is 1.6 times stronger than that of Sample B. This could be attributed to the effect of migration-enhanced epitaxy (MEE) growth mode. Band structure simulations understand the impact of atomic intermixing and segregation at T2SL IFs on the bandgap energy and PL intensity. It is observed that atomic intermixing at the IFs changes the bandgap energy and significantly affects the wave function overlap and the optical property of the samples. Transmission electron microscopy (TEM) measurements reveal that the T2SL IFs in Sample A are very rough compared to sharp IFs in Sample B, indicating a high possibility of atomic intermixing and segregation. Based on these results, it is believed that high-quality heterostructure could be achieved by controlling the IFs to enhance its structural and compositional homogeneities and the optical properties of the T2SLs.

Phenotypic and transcriptomic analysis reveals early stress responses in transgenic rice expressing Arabidopsis DREB1a.

Overexpression of Arabidopsis dehydration response element binding 1a (DREB1a) is a well-known approach for developing salinity, cold and/or drought stress tolerance. However, understanding of the genetic mechanisms associated with DREB1a expression in rice is generally limited. In this study, DREB1a-associated early responses were investigated in a transgenic rice line harboring cold-inducible DREB1a at a gene stacked locus. Although the function of other genes in the stacked locus was not relevant to stress tolerance, this study demonstrates DREB1a can be co-localized with other genes for multigenic trait enhancement. As expected, the transgenic lines displayed improved tolerance to salinity stress and water withholding as compared with non-transgenic controls. RNA sequencing and transcriptome analysis showed upregulation of complex transcriptional networks and metabolic reprogramming as DREB1a expression led to the upregulation of multiple transcription factor gene families, suppression of photosynthesis, and induction of secondary metabolism. In addition to the detection of previously described mechanisms such as production of protective molecules, potentially novel pathways were also revealed. These include jasmonate, auxin, and ethylene signaling, induction of JAZ and WRKY regulons, trehalose synthesis, and polyamine catabolism. These genes regulate various stress responses and ensure timely attenuation of the stress signal. Furthermore, genes associated with heat stress response were downregulated in DREB1a expressing lines, suggesting antagonism between heat and dehydration stress response pathways. In summary, through a complex transcriptional network, multiple stress signaling pathways are induced by DREB1a that presumably lead to early perception and prompt response toward stress tolerance as well as attenuation of the stress signal to prevent deleterious effects of the runoff response.

Targeting TOR and SnRK1 Genes in Rice with CRISPR/Cas9.

Genome targeting with CRISPR/Cas9 is a popular method for introducing mutations and creating knock-out effects. However, limited information is currently available on the mutagenesis of essential genes. This study investigated the efficiency of CRISPR/Cas9 in targeting rice essential genes: the singleton TARGET OF RAPAMYCIN (OsTOR) and the three paralogs of the Sucrose non-fermenting-1 (SNF1)-related kinase 1 (OsSnRK1α), OsSnRK1αA, OsSnRK1αB and OsSnRK1αC. Strong activity of constitutively expressed CRISPR/Cas9 was effective in creating mutations in OsTOR and OsSnRK1α genes, but inducible CRISPR/Cas9 failed to generate detectable mutations. The rate of OsTOR mutagenesis was relatively lower and only the kinase domain of OsTOR could be targeted, while mutations in the HEAT region were unrecoverable. OsSnRK1α paralogs could be targeted at higher rates; however, sterility or early senescence was observed in >50% of the primary mutants. Additionally, OsSnRK1αB and OsSnRK1αC, which bear high sequence homologies, could be targeted simultaneously to generate double-mutants. Further, although limited types of mutations were found in the surviving mutants, the recovered lines displayed loss-of-function or knockdown tor or snrk1 phenotypes. Overall, our data show that mutations in these essential genes can be created by CRISPR/Cas9 to facilitate investigations on their roles in plant development and environmental response in rice.

Field-Evolved ΔG210-ppo2 from Palmer Amaranth Confers Pre-emergence Tolerance to PPO-Inhibitors in Rice and Arabidopsis.

Resistance to protoporphyrinogen IX oxidase (PPO)-inhibitors in Amaranthus palmeri and Amaranthus tuberculatus is mainly contributed by mutations in the PPO enzyme, which renders herbicide molecules ineffective. The deletion of glycine210 (ΔG210) is the most predominant PPO mutation. ΔG210-ppo2 is overexpressed in rice (Oryza sativa c. 'Nipponbare') and Arabidopsis thaliana (Col-0). A foliar assay was conducted on transgenic T1 rice plants with 2× dose of fomesafen (780 g ha−1), showing less injury than the non-transgenic (WT) plants. A soil-based assay conducted with T2 rice seeds confirmed tolerance to fomesafen applied pre-emergence. In agar medium, root growth of WT rice seedlings was inhibited >90% at 5 µM fomesafen, while root growth of T2 seedlings was inhibited by 50% at 45 µM fomesafen. The presence and expression of the transgene were confirmed in the T2 rice survivors of soil-applied fomesafen. A soil-based assay was also conducted with transgenic A. thaliana expressing ΔG210-ppo2 which confirmed tolerance to the pre-emergence application of fomesafen and saflufenacil. The expression of A. palmeri ΔG210-ppo2 successfully conferred tolerance to soil-applied fomesafen in rice and Arabidopsis. This mutant also confers cross-tolerance to saflufenacil in Arabidopsis. This trait could be introduced into high-value crops that lack chemical options for weed management.

Multigene Transformation Through Cre-lox Mediated Site-Specific Integration in Rice.

Plant transformation with multiple genes is a major challenge, rendering multi-trait engineering extremely difficult in crop plants. One of the hurdles in multigene transformation is the uncontrolled integration process that leads to low quality transgenic lines that are unsuitable for practical application. Recombinase-mediated site-specific integration has been tested and validated for developing high quality transgenic lines expressing one, two, or multiple genes. Of the numerous recombinase systems tested, Cre-lox and FLP-FRT show high efficiency in plants. Recently, Cre-lox system was successfully used to stack a set of 3 constitutive, 1 heat-induced, and 1 cold-induced gene. A number of transgenic lines were obtained through a relatively small effort, and the resulting transgenic lines all expressed the genes properly as determined by their promoter-specificity. Here, a method of Cre-lox mediated stacking of a multigene construct is described using rice as a model crop.

Suppression of ERECTA Signaling Impacts Agronomic Performance of Soybean (Glycine max (L) Merril) in the Greenhouse.

The ERECTA (ER) family of genes, encoding leucine-rich repeat receptor-like kinase (RLK), influences complex morphological and physiological aspects of plants. Modulation of ER signaling leads to abiotic stress tolerance in diverse plant species. However, whether the gain in stress tolerance is accompanied with desirable agronomic performance is not clearly known. In this study, soybean plants potentially suppressed in ER signaling were evaluated for the phenotypic performance and drought response in the greenhouse. These plants expressed a dominant-negative Arabidopsis thaliana ER (AtER) called ΔKinase to suppress ER signaling, which has previously been linked with the tolerance to water deficit, a major limiting factor for plant growth and development, directly compromising agricultural production. With the aim to select agronomically superior plants as stress-tolerant lines, transgenic soybean plants were subjected to phenotypic selection and subsequently to water stress analysis. This study found a strong inverse correlation of ΔKinase expression with the agronomic performance of soybean plants, indicating detrimental effects of expressing ΔKinase that presumably led to the suppression of ER signaling. Two lines were identified that showed favorable agronomic traits and expression of ΔKinase gene, although at lower levels compared with the rest of the transgenic lines. The drought stress analysis on the progenies of these lines, however, showed that these plants were more susceptible to water-deficit stress as compared with the non-transgenic controls. The selected transgenic plants showed greater stomata density and conductance, which potentially led to higher biomass, and consequently more water demand and greater susceptibility to the periods of water withholding.

SNF1-related protein kinase 1: the many-faced signaling hub regulating developmental plasticity in plants.

The Snf1-related protein kinase 1 (SnRK1) is the plant homolog of the heterotrimeric AMP-activated protein kinase/sucrose non-fermenting 1 (AMPK/Snf1), which works as a major regulator of growth under nutrient-limiting conditions in eukaryotes. Along with its conserved role as a master regulator of sugar starvation responses, SnRK1 is involved in controlling the developmental plasticity and resilience under diverse environmental conditions in plants. In this review, through mining and analyzing the interactome and phosphoproteome data of SnRK1, we are highlighting its role in fundamental cellular processes such as gene regulation, protein synthesis, primary metabolism, protein trafficking, nutrient homeostasis, and autophagy. Along with the well-characterized molecular interaction in SnRK1 signaling, our analysis highlights several unchartered regions of SnRK1 signaling in plants such as its possible communication with chromatin remodelers, histone modifiers, and inositol phosphate signaling. We also discuss potential reciprocal interactions of SnRK1 signaling with other signaling pathways and cellular processes, which could be involved in maintaining flexibility and homeostasis under different environmental conditions. Overall, this review provides a comprehensive overview of the SnRK1 signaling network in plants and suggests many novel directions for future research.

FLP-Mediated Site-Specific Gene Integration in Rice.

Enabling precise gene integration is important for installing traits in the plants. One of the practical methods of achieving precise gene integration is by using the yeast FLP-FRT recombination system that is efficient in directing DNA integration into the "engineered" genomic sites. The critical parameters of this method include the use of the thermostable version of FLP protein and the promoter trap design to select site-specific integration clones. The resulting transgenic plants display stable expression that is transmitted to the progeny. Therefore, FLP-mediated site-specific integration method could be used for trait engineering in the crop plants or testing gene functions in the model plants.

Genotype-dependent and heat-induced grain chalkiness in rice correlates with the expression patterns of starch biosynthesis genes.

Starch biosynthesis is a complex process underlying grain chalkiness in rice in a genotype-dependent manner. Coordinated expression of starch biosynthesis genes is important for producing translucent rice grains, while disruption in this process leads to opaque or chalky grains. To better understand the dynamics of starch biosynthesis genes in grain chalkiness, six rice genotypes showing variable chalk levels were subjected to gene expression analysis during reproductive stages. In the chalky genotypes, peak expression of the large subunit genes of ADP-glucose pyrophosphorylase (AGPase), encoding the first key step in starch biosynthesis, occurred in the stages before grain filling commenced, creating a gap with the upregulation of starch synthase genes, granule bound starch synthase I (GBSSI) and starch synthase IIA (SSIIA). Whereas, in low-chalk genotypes, AGPase large subunit genes expressed at later stages, generally following the expression patterns of GBSSI and SSIIA. However, heat treatment altered the expression in a genotype-dependent manner that was accompanied by transformed grain morphology and increased chalkiness. The suppression of AGPase subunit genes during early grain filling stages was observed in the chalky genotypes or upon heat treatment, which could result in a limited pool of ADP-Glucose for synthesizing amylose and amylopectin, the major components of the starch. This suboptimal starch biosynthesis process could subsequently lead to inefficient grain filling and air pockets that contribute to chalkiness. In summary, this study suggests a mechanism of grain chalkiness based on the expression patterns of the starch biosynthesis genes in rice.

Recombinase-mediated integration of a multigene cassette in rice leads to stable expression and inheritance of the stacked locus.

Efficient methods for multigene transformation are important for developing novel crop varieties. Methods based on random integrations of multiple genes have been successfully used for metabolic engineering in plants. However, efficiency of co-integration and co-expression of the genes could present a bottleneck. Recombinase-mediated integration into the engineered target sites is arguably a more efficient method of targeted integration that leads to the generation of stable transgenic lines at a high rate. This method has the potential to streamline multigene transformation for metabolic engineering and trait stacking in plants. Therefore, empirical testing of transgene(s) stability from the multigene site-specific integration locus is needed. Here, the recombinase technology based on Cre-lox recombination was evaluated for developing multigenic lines harboring constitutively-expressed and inducible genes. Targeted integration of a five genes cassette in the rice genome generated a precise full-length integration of the cassette at a high rate, and the resulting multigenic lines expressed each gene reliably as defined by their promoter activity. The stable constitutive or inducible expression was faithfully transmitted to the progeny, indicating inheritance-stability of the multigene locus. Co-localization of two distinctly inducible genes by heat or cold with the strongly constitutive genes did not appear to interfere with each other's expression pattern. In summary, high rate of co-integration and co-expression of the multigene cassette installed by the recombinase technology in rice shows that this approach is appropriate for multigene transformation and introduction of co-segregating traits. SIGNIFICANCE STATEMENT: Recombinase-mediated site-specific integration approach was found to be highly efficacious in multigene transformation of rice showing proper regulation of each gene driven by constitutive or inducible promoter. This approach holds promise for streamlining gene stacking in crops and expressing complex multigenic traits.

Modification of soybean growth and abiotic stress tolerance by expression of truncated ERECTA protein from Arabidopsis thaliana.

ERECTA gene family encodes leucine-rich repeat receptor-like kinases that control major aspects of plant development such as elongation of aboveground organs, leaf initiation, development of flowers, and epidermis differentiation. To clarify the importance of ERECTA signaling for the development of soybean (Glycine max), we expressed the dominant-negative ERECTA gene from Arabidopsis thaliana that is truncated in the kinase domain (AtΔKinase). Expression of AtΔKinase in soybean resulted in the short stature, reduced number of leaves, reduced leaf surface area and enhanced branching in the transgenic plants. The transgenic AtΔKinase soybean plants exhibited increased tolerance to water deficit stress due to the reduction of total leaf area and reduced transpiration compared to the wild-type plants. Production of seeds in AtΔKinase lines was higher compared to wild type at regular conditions of cultivation and after exposure to drought stress. Transgenic seedlings expressing AtΔKinase were also able to withstand salt stress better than the wild-type. Established results demonstrated the significance of native soybean genes (GmER and GmERL) in development and stress response of soybean, and suggested that the truncated ERECTA gene of Arabidopsis thaliana can be used to manipulate the growth and stress response of different crop species.

Heat-shock-inducible CRISPR/Cas9 system generates heritable mutations in rice.

Transient expression of CRISPR/Cas9 is an effective approach for limiting its activities and improving its precision in genome editing. Here, we describe the heat-shock-inducible CRISPR/Cas9 for controlled genome editing, and demonstrate its efficiency in the model crop, rice. Using the soybean heat-shock protein gene promoter and the rice U3 promoter to express Cas9 and sgRNA, respectively, we developed the heat-shock (HS)-inducible CRISPR/Cas9 system, and tested its efficacy in targeted mutagenesis. Two loci were targeted in rice, and the presence of targeted mutations was determined before and after the HS treatment. Only a low rate of targeted mutagenesis was detected before HS (~16%), but an increased rate of mutagenesis was observed after the HS treatment among the transgenic lines (50-63%). Analysis of regenerated plants harboring HS-CRISPR/Cas9 revealed that targeted mutagenesis was suppressed in the plants but induced by HS, which was detectable by Sanger sequencing after a few weeks of HS treatments. Most importantly, the HS-induced mutations were transmitted to the progeny at a high rate, generating monoallelic and biallelic mutations that independently segregated from the Cas9 gene. Additionally, off-target mutations were either undetectable or found at a lower rate in HS-CRISPR/Cas9 lines as compared to the constitutive-overexpression CRISPR/Cas9 lines. Taken together, this work shows that HS-CRISPR/Cas9 is a controlled and reasonably efficient platform for genome editing, and therefore, a promising tool for limiting genome-wide off-target effects and improving the precision of genome editing.

Utility of I-SceI and CCR5-ZFN nucleases in excising selectable marker genes from transgenic plants.

OBJECTIVES: Removal of selection marker genes from transgenic plants is highly desirable for their regulatory approval and public acceptance. This study evaluated the use of two nucleases, the yeast homing endonuclease, I-SceI, and the designed zinc finger nuclease, CCR5-ZFN, in excising marker genes from plants using rice and Arabidopsis as the models. RESULTS: In an in vitro culture assay, both nucleases were effective in precisely excising the DNA fragments marked by the nuclease target sites. However, rice cultures were found to be refractory to transformation with the I-SceI and CCR5-ZFN overexpressing constructs. The inducible I-SceI expression was also problematic in rice as the progeny of the transgenic lines expressing the heat-inducible I-SceI did not inherit the functional gene. On the other hand, heat-inducible I-SceI expression in Arabidopsis was effective in creating somatic excisions in transgenic plants but ineffective in generating heritable excisions. The inducible expression of CCR5-ZFN in rice, although transmitted stably to the progeny, appeared ineffective in creating detectable excisions. Therefore, toxicity of these nucleases in plant cells poses major bottleneck in their application in plant biotechnology, which could be avoided by expressing them transiently in cultures in vitro.

Dual-targeting by CRISPR/Cas9 leads to efficient point mutagenesis but only rare targeted deletions in the rice genome.

The present study investigated the efficiency of CRISPR/Cas9 in creating genomic deletions as the basis of its application in removing selection marker genes or the intergenic regions. Three loci, representing a transgene and two rice genes, were targeted at two sites each, in separate experiments, and the deletion of the defined fragments was investigated by PCR and sequencing. Genomic deletions were found at a low rate among the transformed callus lines that could be isolated, cultured, and regenerated into plants harboring the deletion. However, randomly regenerated plants showed mixed genomic effects, and generally did not harbor heritable genomic deletions. To determine whether point mutations occurred at each targeted site, a total of 114 plants consisting of primary transgenic lines and their progeny were analyzed. Ninety-three plants showed targeting, 60 of which were targeted at both sites. The presence of point mutations at both sites was correlated with the guide RNA efficiency. In summary, genomic deletions through dual-targeting by the paired-guide RNAs were generally observed in callus, while de novo point mutations at one or both sites occurred at high rates in transgenic plants and their progeny, generating a variety of insertion-deletions or single-nucleotide variations. In this study, point mutations were exceedingly favored over genomic deletions; therefore, for the recovery of plant lines harboring targeted deletions, identifying early transformed clones harboring the deletions, and isolating them for plant regeneration is recommended.

Generation of a selectable marker free, highly expressed single copy locus as landing pad for transgene stacking in sugarcane.

KEY MESSAGE: A selectable marker free, highly expressed single copy locus flanked by insulators was created as landing pad for transgene stacking in sugarcane. These events displayed superior transgene expression compared to single-copy transgenic lines lacking insulators. Excision of the selectable marker gene from transgenic sugarcane lines was supported by FLPe/FRT site-specific recombination. Sugarcane, a tropical C4 grass in the genus Saccharum (Poaceae), accounts for nearly 80% of sugar produced worldwide and is also an important feedstock for biofuel production. Generating transgenic sugarcane with predictable and stable transgene expression is critical for crop improvement. In this study, we generated a highly expressed single copy locus as landing pad for transgene stacking. Transgenic sugarcane lines with stable integration of a single copy nptII expression cassette flanked by insulators supported higher transgene expression along with reduced line to line variation when compared to single copy events without insulators by NPTII ELISA analysis. Subsequently, the nptII selectable marker gene was efficiently excised from the sugarcane genome by the FLPe/FRT site-specific recombination system to create selectable marker free plants. This study provides valuable resources for future gene stacking using site-specific recombination or genome editing tools.

Gene Stacking in Plants Through the Application of Site-Specific Recombination and Nuclease Activity.

Biotechnology methods for inserting genes one by one or as a block of fragment into plant genomes are needed to introduce valuable traits into crop varieties. Insertion of multiple genes into a single site, called as molecular stacking, is important to allow co-inheritance of the genes into the progeny. Generally, two approaches are available for creating gene stacks: nuclease-induced targeted gene integration into native sites and recombinase-mediated gene integration into the engineered sites. The recombinase application is attractive as several recombinases show high efficiency and precision in plant genomes. This chapter describes a gene stacking method based on the use of Cre-lox site-specific recombination system to integrate genes into the engineered sites and nucleases to delete selection genes leading to stacking of traits into a single genomic site. High efficiency and precision, and undetectable off-target effects of Cre-lox in a number of plant species, make it an attractive tool for complex applications such as gene stacking.

Association analysis of salt tolerance in cowpea (Vigna unguiculata (L.) Walp) at germination and seedling stages.

KEY MESSAGE: This is the first report on association analysis of salt tolerance and identification of SNP markers associated with salt tolerance in cowpea. Cowpea (Vigna unguiculata (L.) Walp) is one of the most important cultivated legumes in Africa. The worldwide annual production in cowpea dry seed is 5.4 million metric tons. However, cowpea is unfavorably affected by salinity stress at germination and seedling stages, which is exacerbated by the effects of climate change. The lack of knowledge on the genetic underlying salt tolerance in cowpea limits the establishment of a breeding strategy for developing salt-tolerant cowpea cultivars. The objectives of this study were to conduct association mapping for salt tolerance at germination and seedling stages and to identify SNP markers associated with salt tolerance in cowpea. We analyzed the salt tolerance index of 116 and 155 cowpea accessions at germination and seedling stages, respectively. A total of 1049 SNPs postulated from genotyping-by-sequencing were used for association analysis. Population structure was inferred using Structure 2.3.4; K optimal was determined using Structure Harvester. TASSEL 5, GAPIT, and FarmCPU involving three models such as single marker regression, general linear model, and mixed linear model were used for the association study. Substantial variation in salt tolerance index for germination rate, plant height reduction, fresh and dry shoot biomass reduction, foliar leaf injury, and inhibition of the first trifoliate leaf was observed. The cowpea accessions were structured into two subpopulations. Three SNPs, Scaffold87490_622, Scaffold87490_630, and C35017374_128 were highly associated with salt tolerance at germination stage. Seven SNPs, Scaffold93827_270, Scaffold68489_600, Scaffold87490_633, Scaffold87490_640, Scaffold82042_3387, C35069468_1916, and Scaffold93942_1089 were found to be associated with salt tolerance at seedling stage. The SNP markers were consistent across the three models and could be used as a tool to select salt-tolerant lines for breeding improved cowpea tolerance to salinity.

Effect of gene order in DNA constructs on gene expression upon integration into plant genome.

Several plant biotechnology applications are based on the expression of multiple genes located on a single transformation vector. The principles of stable expression of foreign genes in plant cells include integration of full-length gene fragments consisting of promoter and transcription terminator sequences, and avoiding converging orientation of the gene transcriptional direction. Therefore, investigators usually generate constructs in which genes are assembled in the same orientation. However, no specific information is available on the effect of the order in which genes should be assembled in the construct to support optimum expression of each gene upon integration in the genome. While many factors, including genomic position and the integration structure, could affect gene expression, the investigators judiciously design DNA constructs to avoid glitches. However, the gene order in a multigene assembly remains an open question. This study addressed the effect of gene order in the DNA construct on gene expression in rice using a simple design of two genes placed in two possible orders with respect to the genomic context. Transgenic rice lines containing green fluorescent protein (GFP) and ß-glucuronidase (GUS) genes in two distinct orders were developed by Cre-lox-mediated site-specific integration. Gene expression analysis of transgenic lines showed that both genes were expressed at similar levels in either orientation, and different transgenic lines expressed each gene within 1-2× range. Thus, no significant effect of the gene order on gene expression was found in the transformed rice lines containing precise site-specific integrations and stable gene expression in plant cells could be obtained with altered gene orders. Therefore, gene orientation and integration structures are more important factors governing gene expression than gene orders in the genomic context.