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.

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.

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.

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.

Gene stacking by recombinases.

Efficient methods of stacking genes into plant genomes are needed to expedite transfer of multigenic traits to crop varieties of diverse ecosystems. Over two decades of research has identified several DNA recombinases that carryout efficient cis and trans recombination between the recombination sites artificially introduced into the plant chromosome. The specificity and efficiency of recombinases make them extremely attractive for genome engineering. In plant biotechnology, recombinases have mostly been used for removing selectable marker genes and have rarely been extended to more complex applications. The reversibility of recombination, a property of the tyrosine family of recombinases, does not lend itself to gene stacking approaches that involve rounds of transformation for integrating genes into the engineered sites. However, recent developments in the field of recombinases have overcome these challenges and paved the way for gene stacking. Some of the key advancements include the application of unidirectional recombination systems, modification of recombination sites and transgene site modifications to allow repeated site-specific integrations into the selected site. Gene stacking is relevant to agriculturally important crops, many of which are difficult to transform; therefore, development of high-efficiency gene stacking systems will be important for its application on agronomically important crops, and their elite varieties. Recombinases, by virtue of their specificity and efficiency in plant cells, emerge as powerful tools for a variety of applications including gene stacking.

Gene stacking in plant cell using recombinases for gene integration and nucleases for marker gene deletion.

BACKGROUND: Practical approaches for multigene transformation and gene stacking are extremely important for engineering complex traits and adding new traits in transgenic crops. Trait deployment by gene stacking would greatly simplify downstream plant breeding and trait introgression into cultivars. Gene stacking into pre-determined genomic sites depends on mechanisms of targeted DNA integration and recycling of selectable marker genes. Targeted integrations into chromosomal breaks, created by nucleases, require large transformation efforts. Recombinases such as Cre-lox, on the other hand, efficiently drive site-specific integrations in plants. However, the reversibility of Cre-lox recombination, due to the incorporation of two cis-positioned lox sites, presents a major bottleneck in its application in gene stacking. Here, we describe a strategy of resolving this bottleneck through excision of one of the cis-positioned lox, embedded in the marker gene, by nuclease activity. METHODS: All transgenic lines were developed by particle bombardment of rice callus with plasmid constructs. Standard molecular approach was used for building the constructs. Transgene loci were analyzed by PCR, Southern hybridization, and DNA sequencing. RESULTS: We developed a highly efficient gene stacking method by utilizing powerful recombinases such as Cre-lox and FLP-FRT, for site-specific gene integrations, and nucleases for marker gene excisions. We generated Cre-mediated site-specific integration locus in rice and showed excision of marker gene by I-SceI at ~20 % efficiency, seamlessly connecting genes in the locus. Next, we showed ZFN could be used for marker excision, and the locus can be targeted again by recombinases. Hence, we extended the power of recombinases to gene stacking application in plants. Finally, we show that heat-inducible I-SceI is also suitable for marker excision, and therefore could serve as an important tool in streamlining this gene stacking platform. CONCLUSIONS: A practical approach for gene stacking in plant cell was developed that allows targeted gene insertions through rounds of transformation, a method needed for introducing new traits into transgenic lines for their rapid deployment in the field. By using Cre-lox, a powerful site-specific recombination system, this method greatly improves gene stacking efficiency, and through the application of nucleases develops marker-free, seamless stack of genes at pre-determined chromosomal sites.

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.

Site-specific methylation in gene coding region underlies transcriptional silencing of the Phytochrome A epiallele in Arabidopsis thaliana.

DNA methylation in cytosine residues plays an important role in regulating gene expression. Densely methylated transgenes are often silenced. In contrast, several eukaryotic genomes express moderately methylated genes. These methylations are found in the CG context within the coding region (gene body). The role of gene body methylation in gene expression, however, is not clear. The Arabidopsis Phytochrome A epiallele, phyA', carries hypermethylation in several CG sites resident to the coding region. As a result, phyA' is transcriptionally silenced and confers strong mutant phenotype. Mutations in chromatin modification factors and RNAi genes failed to revert the mutant phenotype, suggesting the involvement of a distinct epigenetic mechanism associated with phyA' silencing. Using the forward genetics approach, a suppressor line, termed as suppressor of p hyA' silencing 1 (sps1), was isolated. Genetic and molecular analysis revealed that sps1 mutation reactivates the phyA' locus without altering its methylation density. However, hypomethylation at a specific CG site in exon 1 was consistently associated with the release of phyA' silencing. While gene underlying sps1 mutation is yet to be identified, microarray analysis suggested that its targets are the expressed genes or euchromatic loci in Arabidopsis genome. By identifying the association of phyA' silencing with the methylation of a specific CG site in exon 1, the present work shows that site-specific methylation confers greater effect on transcription than the methylation density within gene-body. Further, as the identified site (exon 1) is not critical for the promoter activity, transcription elongation rather than transcription initiation is likely to be affected by this site-specific CG methylation.

Marker-free site-specific gene integration in rice based on the use of two recombination systems.

Transgene integration mediated by heterologous site-specific recombination (SSR) systems into the dedicated genomic sites has been demonstrated in a few different plant species. This approach of plant transformation generates a precise site-specific integration (SSI) structure consisting of a single copy of the transgene construct. As a result, stable transgene expression correlated with promoter strength and gene copy number is observed among independent transgenic lines and faithfully transmitted through subsequent generations. Site-specific integration approaches use selectable marker genes, removal of which is necessary for the implementation of this approach as a biotechnology application. As SSR systems are also excellent tools for excising marker genes from transgene locus, a molecular strategy involving gene integration followed by marker excision, each mediated by a distinct recombination system, was earlier proposed. Experimental validation of this approach is the focus of this work. Using FLPe-FRT system for site-specific gene integration and heat-inducible Cre-lox for marker gene excision, marker-free SSI lines were developed in the first generation itself. More importantly, progeny derived from these lines inherited the marker-free locus, indicating efficient germinal transmission. Finally, as the transgene expression from SSI locus was not altered upon marker excision, this method is suitable for streamlining the production of marker-free SSI lines.

Hydrothermal synthesis of ZnO nanorods in the presence of a surfactant.

Controlling the dimensions, positioning, and shapes of semiconductor nanowires, nanorods, and nanobelts lies in the synthesis and understanding of their growth mechanism. Controlled growth and synthesis is required in the fabrication of nanodevices and nanosensors. Among methods utilized for one-dimensional nanostructure synthesis, the hydrothermal process--a simple and cost-effective technique involving a low process temperature--has emerged as a powerful tool for the fabrication of anisotropic nanomaterials. Under hydrothermal conditions, many starting materials can undergo quite unexpected reactions, which are often accompanied by the formation of nanoscopic morphologies that are not accessible by classical routes. Synthesized ZnO nanostructures from aqueous solutions are usually poor in terms of morphology and size control. To improve the growth conditions and the controllability of the process, the use of surfactants or organic solvents has been attempted. In the present work, ZnO nanorods were grown on templates with a pre-sputtered ZnO seed layer over oxidized Si (100) substrates, and polyvinyl pyrrolidone (PVP) was used as a surfactant. By varying the PVP concentration in the growth solution, we can control the diameter and density of ZnO nanorods. The optical property of ZnO nanorods is highly improved by PVP addition.

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.

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.

Site-specific gene integration in rice genome mediated by the FLP-FRT recombination system.

Plant transformation based on random integration of foreign DNA often generates complex integration structures. Precision in the integration process is necessary to ensure the formation of full-length, single-copy integration. Site-specific recombination systems are versatile tools for precise genomic manipulations such as DNA excision, inversion or integration. The yeast FLP-FRT recombination system has been widely used for DNA excision in higher plants. Here, we report the use of FLP-FRT system for efficient targeting of foreign gene into the engineered genomic site in rice. The transgene vector containing a pair of directly oriented FRT sites was introduced by particle bombardment into the cells containing the target locus. FLP activity generated by the co-bombarded FLP gene efficiently separated the transgene construct from the vector-backbone and integrated the backbone-free construct into the target site. Strong FLP activity, derived from the enhanced FLP protein, FLPe, was important for the successful site-specific integration (SSI). The majority of the transgenic events contained a precise integration and expressed the transgene. Interestingly, each transgenic event lacked the co-bombarded FLPe gene, suggesting reversion of the integration structure in the presence of the constitutive FLPe expression. Progeny of the precise transgenic lines inherited the stable SSI locus and expressed the transgene. This work demonstrates the application of FLP-FRT system for site-specific gene integration in plants using rice as a model.

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.

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.

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.