Null Segregants Research Articles

ZmWRKY30 modulates drought tolerance in maize by influencing myo-inositol and reactive oxygen species homeostasis.

Maize (Zea mays L.) is an important food crop with a wide range of uses in both industry and agriculture. Drought stress during its growth cycle can greatly reduce maize crop yield and quality. However, the molecular mechanisms underlying maize responses to drought stress remain unclear. In this work, a WRKY transcription factor-encoding gene, ZmWRKY30, from drought-treated maize leaves was screened out and characterized. ZmWRKY30 gene expression was induced by dehydration treatments. The ZmWRKY30 protein localized to the nucleus and displayed transactivation activity in yeast. Compared with wild-type (WT) plants, Arabidopsis lines overexpressing ZmWRKY30 exhibited a significantly enhanced drought stress tolerance, as evidenced by the improved survival rate, increased antioxidant enzyme activity by superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), elevated proline content, and reduced lipid peroxidation recorded after drought stress treatment. In contrast, the mutator (Mu)-interrupted ZmWRKY30 homozygous mutant (zmwrky30) was more sensitive to drought stress than its null segregant (NS), characterized by the decreased survival rate, reduced antioxidant enzyme activity (SOD, POD, and CAT) and proline content, as well as increased malondialdehyde accumulation. RNA-Seq analysis further revealed that, under drought conditions, the knockout of the ZmWRKY30 gene in maize affected the expression of genes involved in reactive oxygen species (ROS), proline, and myo-inositol metabolism. Meanwhile, the zmwrky30 mutant exhibited significant downregulation of myo-inositol content in leaves under drought stress. Combined, our results suggest that ZmWRKY30 positively regulates maize responses to water scarcity. This work provides potential target genes for the breeding of drought-tolerant maize.

Generation of major glutelin-deficient (GluA, GluB, and GluC) semi-dwarf Koshihikari rice line.

We generated a new Koshihikari rice line with a drastically reduced content of glutelin proteins and higher lodging resistance by using new and conventional plant breeding techniques. Using CRISPR/Cas9-mediated genome editing, we generated mutant rice with drastically decreased contents of major glutelins. A Koshihikari rice mutant line, a123, lacking four glutelins (GluA1, GluA2, GluB4, and GluB5) was used as a host, and another five major glutelin genes (GluA3, GluB1a, GluB1b, GluB2, and GluC) were knocked out through two iterations of Agrobacterium-mediated transformation. Mutant seeds were deficient in the GluA family, GluB family, and GluC, and the line obtained was named GluABC KO. Glutelin content was much lower in GluABC KO than in the existing low-glutelin rice mutant LGC-1. A null segregant of GluABC KO was selected using new-generation sequencing and backcrossing, and the sd-1 allele for the semi-dwarf trait was introduced to increase lodging resistance.

Storage protein activator controls grain protein accumulation in bread wheat in a nitrogen dependent manner

The expression of cereal grain storage protein (GSP) genes is controlled by a complex network of transcription factors (TFs). Storage protein activator (SPA) is a major TF acting in this network but its specific function in wheat (Triticum aestivum L.) remains to be determined. Here we generated an RNAi line in which expression of the three SPA homoeologs was reduced. In this line and its null segregant we analyzed GSP accumulation and expression of GSP and regulatory TF genes under two regimes of nitrogen availability. We show that down regulation of SPA decreases grain protein concentration at maturity under low but not high nitrogen supply. Under low nitrogen supply, the decrease in SPA expression also caused a reduction in the total quantity of GSP per grain and in the ratio of GSP to albumin-globulins, without significantly affecting GSP composition. The slight reduction in GSP gene expression measured in the SPA RNAi line under low nitrogen supply did not entirely account for the more significant decrease in GSP accumulation, suggesting that SPA regulates additional levels of GSP synthesis. Our results demonstrate a clear role of SPA in the regulation of grain nitrogen metabolism when nitrogen is a limiting resource.

Efficient double-flowered gentian plant production using the CRISPR/Cas9 system.

Japanese cultivated gentians are highly valued ornamental flowers in Japan, but the flower shape is mostly limited to the single-flower type, unlike other flowers such as roses and carnations. To overcome this limitation, we used the CRISPR/Cas9 genome editing system to increase double-flowered genetic resources in gentians. Our approach targeted an AGAMOUS (AG) floral homeotic gene (AG1), which is responsible for the natural mutation that causes double flowers in gentians. We designed two targets in exon 1 of AG1 for genome editing and found that 9 of 12 herbicide-resistant shoots had biallelic mutations in the target regions of AG1. These nine lines all produced double flowers, with stamens converted into petaloid organs, similar to the natural mutant. We also analyzed the off-target effects of AG2, which is homologous to AG1, and found that such effects occurred in gentian genome editing but with low frequency. Furthermore, we successfully produced transgene-free genome-edited plants (null segregants) by crossing with wild-type pollen. F1 seedlings were subjected to PCR analysis to determine whether foreign DNA sequences, two partial regions of the CaMV35S promoter and Cas9 gene, were present in the genome. As a result, foreign genes were segregated at a 1 : 1 ratio, indicating successful null segregant production. Using PCR analysis, we confirmed that four representative null segregants did not contain transfer DNA. In summary, our study demonstrates that the CRISPR/Cas9 system can efficiently produce double-flowered gentians, and null segregants can also be obtained. These genome-edited plants are valuable genetic resources for future gentian breeding programs.

Are null segregants new combinations of heritable material and should they be regulated?

Through genome editing and other techniques of gene technology, it is possible to create a class of organism called null segregants. These genetically modified organisms (GMOs) are products of gene technology but are argued to have no lingering vestige of the technology after the segregation of chromosomes or deletion of insertions. From that viewpoint regulations are redundant because any unique potential for the use of gene technology to cause harm has also been removed. We tackle this question of international interest by reviewing the early history of the purpose of gene technology regulation. The active ingredients of techniques used for guided mutagenesis, e.g., site-directed nucleases, such as CRISPR/Cas, are promoted for having a lower potential per reaction to create a hazard. However, others see this as a desirable industrial property of the reagents that will lead to genome editing being used more and nullifying the promised hazard mitigation. The contest between views revolves around whether regulations could alter the risks in the responsible use of gene technology. We conclude that gene technology, even when used to make null segregants, has characteristics that make regulation a reasonable option for mitigating potential harm. Those characteristics are that it allows people to create more harm faster, even if it creates benefits as well; the potential for harm increases with increased use of the technique, but safety does not; and regulations can control harm scaling.

Trait stacking simultaneously enhances provitamin A carotenoid and mineral bioaccessibility in biofortified Sorghum bicolor.

Vitamin A, iron, and zinc deficiencies are major nutritional inadequacies in sub-Saharan Africa and disproportionately affect women and children. Biotechnology strategies have been tested to individually improve provitamin A carotenoid or mineral content and/or bioaccessibility in staple crops including sorghum (Sorghum bicolor). However, concurrent carotenoid and mineral enhancement has not been thoroughly assessed and antagonism between these chemical classes has been reported. This work evaluated two genetically engineered constructs containing a suite of heterologous genes to increase carotenoid stability and pathway flux, as well as phytase to catabolize phytate and increase mineral bioaccessibility. Model porridges made from transgenic events were evaluated for carotenoid and mineral content as well as bioaccessibility. Transgenic events produced markedly higher amounts of carotenoids (26.4 μg g-1 DW) compared to null segregants (4.2 μg g-1 DW) and wild-type control (Tx430; 3.7 μg g-1 DW). Phytase activation by pre-steeping flour resulted in significant phytate reduction (9.4 to 4.2 mg g-1 DW), altered the profile of inositol phosphate catabolites, and reduced molar ratios of phytate to iron (16.0 to 4.1), and zinc (19.0 to 4.9) in engineered material, suggesting improved mineral bioaccessibility. Improved phytate : mineral ratios did not significantly affect micellarization and bioaccessible provitamin A carotenoids were over 23 times greater in transgenic events compared to corresponding null segregants and wild-type controls. A 200 g serving of porridge made with these transgenic events provide an estimated 53.7% of a 4-8-year-old child's vitamin A estimated average requirement. These data suggest that combinatorial approaches to enhance micronutrient content and bioaccessibility are feasible and warrant further assessment in human studies.

Co-overexpression of AtSAT1 and EcPAPR improves seed nutritional value in maize.

Maize seeds synthesize insufficient levels of the essential amino acid methionine (Met) to support animal and livestock growth. Serine acetyltransferase1 (SAT1) and 3′-phosphoadenosine-5′-phosphosulfate reductase (PAPR) are key control points for sulfur assimilation into Cys and Met biosynthesis. Two high-MET maize lines pRbcS:AtSAT1 and pRbcS:EcPAPR were obtained through metabolic engineering recently, and their total Met was increased by 1.4- and 1.57-fold, respectively, compared to the wild type. The highest Met maize line, pRbcS:AtSAT1-pRbcS:EcPAPR, was created by stacking the two transgenes, causing total Met to increase 2.24-fold. However, the pRbcS:AtSAT1-pRbcS:EcPAPR plants displayed progressively severe defects in plant growth, including early senescence, stunting, and dwarfing, indicating that excessive sulfur assimilation has an adverse effect on plant development. To explore the mechanism of correlation between Met biosynthesis in maize leaves and storage proteins in developing endosperm, the transcriptomes of the sixth leaf at stage V9 and 18 DAP endosperm of pRbcS:AtSAT1, pRbcS:AtSAT1-pRbcS:EcPAPR, and the null segregants were quantified and analyzed. In pRbcS:AtSAT1-pRbcS:EcPAPR, 3274 genes in leaves (1505 up- and 1769 downregulated) and 679 genes in the endosperm (327 up- and 352 downregulated) were differentially expressed. Gene ontology (GO) and KEGG (Kyoto encyclopedia of genes and genomes) analyses revealed that many genes were associated with Met homeostasis, including transcription factors and genes involved in cysteine and Met metabolism, glutathione metabolism, plant hormone signal transduction, and oxidation–reduction. The data from gene network analysis demonstrated that two genes, serine/threonine-protein kinase (CCR3) and heat shock 70 kDa protein (HSP), were localized in the core of the leaves and endosperm regulation networks, respectively. The results of this study provide insights into the diverse mechanisms that underlie the ideal establishment of enhanced Met levels in maize seeds.

Trait Stacking Simultaneously Enhances Mineral and Provitamin a Carotenoid Bioaccessibility in Biofortified Sorghum bicolor

ObjectivesVitamin A, iron, and zinc deficiencies represent major dietary inadequacies in Sub-Saharan Africa and disproportionately affect women and children. Biotechnology strategies have been tested to individually improve carotenoid or mineral content and/or bioaccessibility in relevant cereal crops such as sorghum (Sorghum bicolor). However, approaches combining carotenoid and mineral enhancement in the same event have not been thoroughly evaluated. This work evaluated genetically engineered sorghum events that simultaneously enhanced provitamin A carotenoid accumulation and reduced the mineral limiting antinutrient phytate.MethodsTwo sorghum transformation constructs containing HGGT, to increase vitamin E accumulation and stabilize provitamin A carotenoids during grain storage, CRTI, to increase provitamin A biosynthesis, PSY1 or CRTB, to increase flux through the carotenoid pathway, and PhyA, to decrease phytate, were engineered to produce transgenic events. These sorghum events were processed into model porridges and evaluated for carotenoid and mineral content as well as bioaccessibility.ResultsAll transgenic events produced markedly higher amounts of carotenoids compared to corresponding null segregants and wild-type control (Tx430). A steeping step prior to porridge production to pre-activate phytase drastically reduced phytate content, altered the profile of inositol phosphate conversion products, and reduced molar ratios of phytate to iron and zinc; preventing the chelation of minerals by phytate and enhancing their bioaccessibility. The subsequent release of minerals did not affect micellarization efficiency and the bioaccessible fraction of provitamin A carotenoids were over 2300% greater in transgenic events compared to corresponding null segregants and wild-type controls; providing 53.7% of a 4–8-year-old child's vitamin A estimated average requirement in a standard 200 g serving of porridge.ConclusionsThese data suggest that a combination of strategies to enhance micronutrient content and bioaccessibility are feasible and warrant further assessment in human studies.Funding SourcesThis work was supported in part by the U.S. Department of Agriculture, Agricultural Research Service as well as the Bill and Melinda Gates Foundation.

TALEN-mediated depletion of the mitochondrial gene orf312 proves that it is a Tadukan-type cytoplasmic male sterility-causative gene in rice.

Cytoplasmic male sterility (CMS) is a trait that causes pollen or anther dysfunctions, resulting in the lack of seed setting. CMS is considered to be caused by the expression of a unique mitochondrial open reading frame referred to as CMS-associated gene. orf312 has been reported as a CMS-associated gene of Tadukan-type CMS (TAA) in rice (Oryza sativa L.), which exhibits impaired anther dehiscence; however, evidence thereof has not yet been reported. Here, we took a loss-of-function approach, using a mitochondria-targeted transcription activator-like effector nuclease (mitoTALEN) designed to knock out orf312 in TAA, to prove that orf312 indeed is a CMS-causative gene. Out of 28 transgenic TAA plants harboring the mitoTALEN expression vector, deletion of orf312 was detected in 24 plants by PCR, Southern blot, and sequencing analyses. The 24 plants were grouped into three groups based on the deleted regions. All orf312-depleted TAA plants exhibited recovery of anther dehiscence and seed setting. The depletion of orf312 and fertility restoration was maintained in the next generation, even in mitoTALEN expression cassette null segregants. In contrast, orf312-retaining plants were sterile. These results provide robust evidence that orf312 is a Tadukan-type CMS-causative gene.

Mini-Review: Transgenerational CRISPR/Cas9 Gene Editing in Plants.

CRISPR/Cas9 genome editing has been used extensively in a wide variety of plant species. Creation of loss-of-function alleles, promoter variants and mutant collections are a few of the many uses of genome editing. In a typical workflow for sexually reproducing species, plants are generated that contain an integrated CRISPR/Cas9 transgene. After editing of the gene of interest, T-DNA null segregants can be identified in the next generation that contain only the desired edit. However, maintained presence of the CRISPR/Cas9 transgene and continued editing in the subsequent generations offer a range of applications for model plants and crops. In this review, we define transgenerational gene editing (TGE) as the continued editing of CRISPR/Cas9 after a genetic cross. We discuss the concept of TGE, summarize the current main applications, and highlight special cases to illustrate the importance of TGE for plant genome editing research and breeding.

Multi-year field evaluation of nicotianamine biofortified bread wheat.

Conventional breeding efforts for iron (Fe) and zinc (Zn) biofortification of bread wheat (Triticum aestivum L.) have been hindered by a lack of genetic variation for these traits and a negative correlation between grain Fe and Zn concentrations and yield. We have employed genetic engineering to constitutively express (CE) the rice (Oryza sativa) nicotianamine synthase 2 (OsNAS2) gene and upregulate biosynthesis of two metal chelators - nicotianamine (NA) and 2'-deoxymugineic acid (DMA) - in bread wheat, resulting in increased Fe and Zn concentrations in wholemeal and white flour. Here we describe multi-location confined field trial (CFT) evaluation of a low-copy transgenic CE-OsNAS2 wheat event (CE-1) over 3 years and demonstrate higher concentrations of NA, DMA, Fe, and Zn in CE-1 wholemeal flour, white flour, and white bread and higher Fe bioavailability in CE-1 white flour relative to a null segregant (NS) control. Multi-environment models of agronomic and grain nutrition traits revealed a negative correlation between grain yield and grain Fe, Zn, and total protein concentrations, yet no correlation between grain yield and grain NA and DMA concentrations. White flour Fe bioavailability was positively correlated with white flour NA concentration, suggesting that NA-chelated Fe should be targeted in wheat Fe biofortification efforts.

Utilizing MIKC-type MADS-box protein SOC1 for yield potential enhancement in maize

Key messageOverexpression of Zea mays SOC gene promotes flowering, reduces plant height, and leads to no reduction in grain production per plant, suggesting enhanced yield potential, at least, through increasing planting density.MIKC-type MADS-box gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) is an integrator conserved in the plant flowering pathway. In this study, the maize SOC1 (ZmSOC1) gene was cloned and overexpressed in transgenic maize Hi-II genotype. The T0 plants were backcrossed with nontransgenic inbred B73 to produce first generation backcross (BC1) seeds. Phenotyping of both transgenic and null segregant (NT) BC1 plants was conducted in three independent experiments. The BC1 transgenic plants showed new attributes such as increased vegetative growth, accelerated flowering time, reduced overall plant height, and increased grain weight. Second generation backcross (BC2) plants were evaluated in the field using two planting densities. Compared to BC2 NT plants, BC2 transgenic plants, were 12–18% shorter, flowered 5 days earlier, and showed no reduction in grain production per plant and an increase in fat, starch, and simple sugars in the grain. Transcriptome comparison in young leaves of 56-day-old BC1 plants revealed that the overexpressed ZmSOC1 resulted in 107 differentially expressed genes. The upregulated transcription factor DNA BINDING WITH ONE FINGER 5.4 (DOF5.4) was among the genes responsible for the reduced plant height. Modulating expression of SOC1 opens a new and effective approach to promote flowering and reduce plant height, which may have potential to enhance crop yield and improve grain quality.

Small "Nested" Introgressions from Wild Thinopyrum Species, Conferring Effective Resistance to Fusarium Diseases, Positively Impact Durum Wheat Yield Potential.

Today wheat cultivation is facing rapidly changing climate scenarios and yield instability, aggravated by the spreading of severe diseases such as Fusarium head blight (FHB) and Fusarium crown rot (FCR). To obtain productive genotypes resilient to stress pressure, smart breeding approaches must be envisaged, including the exploitation of wild relatives. Here we report on the assessment of the breeding potential of six durum wheat-Thinopyrum spp. recombinant lines (RLs) obtained through chromosome engineering. They are characterized by having 23% or 28% of their 7AL chromosome arm replaced by a “nested” alien segment, composed of homoeologous group 7 chromosome fractions from Th. ponticum and Th. elongatum (=7el1L + 7EL) or from different Th. ponticum accessions (=7el1L + 7el2L). In addition to the 7el1L genes Lr19 + Yp (leaf rust resistance, and yellow pigment content, respectively), these recombinant lines (RLs) possess a highly effective QTL for resistance to FHB and FCR within their 7el2L or 7EL portion. The RLs, their null segregants and well-adapted and productive durum wheat cultivars were evaluated for 16 yield-related traits over two seasons under rainfed and irrigated conditions. The absence of yield penalties and excellent genetic stability of RLs was revealed in the presence of all the alien segment combinations. Both 7el2L and 7EL stacked introgressions had positive impacts on source and sink yield traits, as well as on the overall performance of RLs in conditions of reduced water availability. The four “nested” RLs tested in 2020 were among the top five yielders, overall representing good candidates to be employed in breeding programs to enhance crop security and safety.

TALEN-mediated bar-knockout Rice Production and Transcriptome Profiling

Gene editing technologies such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) systems have been developed to create targeted DNA mutagenesis in many crop plants. This report describes application of the TALEN system to generate bialaphos resistance (bar)-knockout null segregants in herbicide-tolerant rice (Ba15) and microarray analysis on transcriptome changes of mutated lines, to identify unexpected effects resulting from off-targets. We generated 41 T0 plants and identified TALEN-mediated bar sequence mutations in 14 of them. Non-target site single nucleotide polymorphisms (SNPs) and small insertion/deletions (InDels) accounted for a large proportion of the mutations. Segregations of phosphinothricin acetyltransferase (PAT) protein expression levels were observed in T1 generations of two lines, R6 and R9. In addition, most T1 offspring harbored the TALE-R expression cassette and acquired some de novo mutations that were not inherited from their T0 parents. Three bar-knockout T1 lines were tested for PAT protein expression in progeny seedlings, and their T2 plants possessed inactive bar. We selected three bar-knockout T2 plants that were TALE-DNA-free for microarray analysis, aiming to understand the transcriptome differences between mutated null segregants and their recipient line. Only 31 differentially expressed genes (DEGs) were identified in the bar-knockout rice lines, possibly resulting from somaclonal variations from the in vitro cell culture process. Taken together, TALEN-mediated bar mutations have little effect on the whole transcriptome profile of rice. We believe our results will be helpful to study unexpected consequences in gene-edited crops.

CRISPR/Cas9-Mediated Knockout of Galactinol Synthase-Encoding Genes Reduces Raffinose Family Oligosaccharide Levels in Soybean Seeds.

Raffinose family oligosaccharides (RFOs) are major soluble carbohydrates in soybean seeds that cannot be digested by human and other monogastric animals. Hence, a major goal is to reduce RFO levels to improve the nutritional quality of soybean. In this study, we utilized a dual gRNAs CRISPR/Cas9 system to induce knockouts in two soybean galactinol synthase (GOLS) genes, GmGOLS1A and its homeolog GmGOLS1B. Genotyping of T0 plants showed that the construct design was efficient in inducing various deletions in the target sites or sequences spanning the two target sites of both GmGOLS1A and GmGOLS1B genes. A subset of induced alleles was successfully transferred to progeny and, at the T2 generation, we identified null segregants of single and double mutant genotypes without off-target induced mutations. The seed carbohydrate analysis of double mutant lines showed a reduction in the total RFO content of soybean seed from 64.7 mg/g dry weight to 41.95 mg/g dry weight, a 35.2% decrease. On average, the stachyose content, the most predominant RFO in soybean seeds, decreased by 35.4% in double mutant soybean, while the raffinose content increased by 41.7%. A slight decrease in verbascose content was also observed in mutant lines. Aside from changes in soluble carbohydrate content, some mutant lines also exhibited increased protein and fat contents. Otherwise, no difference in seed weight, seed germination, plant development and morphology was observed in the mutants. Our findings indicate that GmGOLS1A and GmGOLS1B contribute to the soybean oligosaccharide profile through RFO biosynthesis pathways, and are promising targets for future investigation, as well as crop improvement efforts. Our results also demonstrate the potential in using elite soybean cultivars for transformation and targeted genome editing.

Effect of Rice GDP-L-Galactose Phosphorylase Constitutive Overexpression on Ascorbate Concentration, Stress Tolerance, and Iron Bioavailability in Rice.

Ascorbate (vitamin C) is an essential multifunctional molecule for both plants and mammals. In plants, ascorbate is the most abundant water-soluble antioxidant that supports stress tolerance. In humans, ascorbate is an essential micronutrient and promotes iron (Fe) absorption in the gut. Engineering crops with increased ascorbate levels have the potential to improve both crop stress tolerance and human health. Here, rice (Oryza sativa L.) plants were engineered to constitutively overexpress the rice GDP-L-galactose phosphorylase coding sequence (35S-OsGGP), which encodes the rate-limiting enzymatic step of the L-galactose pathway. Ascorbate concentrations were negligible in both null segregant (NS) and 35S-OsGGP brown rice (BR, unpolished grain), but significantly increased in 35S-OsGGP germinated brown rice (GBR) relative to NS. Foliar ascorbate concentrations were significantly increased in 35S-OsGGP plants in the vegetative growth phase relative to NS, but significantly reduced at the reproductive growth phase and were associated with reduced OsGGP transcript levels. The 35S-OsGGP plants did not display altered salt tolerance at the vegetative growth phase despite having elevated ascorbate concentrations. Ascorbate concentrations were positively correlated with ferritin concentrations in Caco-2 cells – an accurate predictor of Fe bioavailability in human digestion – exposed to in vitro digests of NS and 35S-OsGGP BR and GBR samples.

ZmDREB2A regulates ZmGH3.2 and ZmRAFS, shifting metabolism towards seed aging tolerance over seedling growth.

Seed aging tolerance and rapid seedling growth are important agronomic traits for crop production; however, how these traits are controlled at the molecular level remains largely unknown. The unaged seeds of two independent maize DEHYDRATION-RESPONSIVE ELEMENT-BINDING2A mutant (zmdreb2a) lines, with decreased expression of GRETCHEN HAGEN3.2 (ZmGH3.2, encoding indole-3-acetic acid [IAA] deactivating enzyme), and increased IAA in their embryo, produced longer seedling shoots and roots, than the null segregant (NS) controls. However, the zmdreb2a seeds, with decreased expression of RAFFINOSE SYNTHASE (ZmRAFS) and less raffinose in their embryo, exhibit decreased seed aging tolerance, than the NS controls. Overexpression of ZmDREB2A in maize protoplasts increased the expression of ZmGH3.2, ZmRAFS genes and that of a Rennila LUCIFERASE reporter (Rluc) gene, which was controlled by either the ZmGH3.2- or ZmRAFS-promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation assay quantitative polymerase chain reaction showed that ZmDREB2A directly binds to the DRE motif of the promoters of both ZmGH3.2 and ZmRAFS. Exogenous supplementation of IAA to the unaged, germinating NS seeds increased subsequent seedling growth making them similar to the zmdreb2a seedlings from unaged seeds. These findings provide evidence that ZmDREB2A regulates the longevity of maize seed by stimulating the production of raffinose while simultaneously acting to limit auxin-mediated cell expansion.

Targeted genome editing in tetraploid potato through transient TALEN expression by Agrobacterium infection.

Genome editing using site-specific nucleases, such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9), is a powerful technology for crop breeding. For plant genome editing, the genome-editing reagents are usually expressed in plant cells from stably integrated transgenes within the genome. This requires crossing processes to remove foreign nucleotides from the genome to generate null segregants. However, in highly heterozygous plants such as potato, the progeny lines have different agronomic traits from the parent cultivar and do not necessarily become elite lines. Agrobacteria can transfer exogenous genes on T-DNA into plant cells. This has been used both to transform plants stably and to express the genes transiently in plant cells. Here, we infected potato, with Agrobacterium tumefaciens harboring TALEN-expression vector targeting sterol side chain reductase 2 (SSR2) gene and regenerated shoots without selection. We obtained regenerated lines with disrupted-SSR2 gene and without transgene of the TALEN gene, revealing that their disruption should be caused by transient gene expression. The strategy using transient gene expression by Agrobacterium that we call Agrobacterial mutagenesis, developed here should accelerate the use of genome-editing technology to modify heterozygous plant genomes.

Raffinose synthase enhances drought tolerance through raffinose synthesis or galactinol hydrolysis in maize and Arabidopsis plants

Raffinose and its precursor galactinol accumulate in plant leaves during abiotic stress. RAFFINOSE SYNTHASE (RAFS) catalyzes raffinose formation by transferring a galactosyl group of galactinol to sucrose. However, whether RAFS contributes to plant drought tolerance and, if so, by what mechanism remains unclear. In this study, we report that expression of RAFS from maize (or corn, Zea mays) (ZmRAFS) is induced by drought, heat, cold, and salinity stresses. We found that zmrafs mutant maize plants completely lack raffinose and hyper-accumulate galactinol and are more sensitive to drought stress than the corresponding null-segregant (NS) plants. This indicated that ZmRAFS and its product raffinose contribute to plant drought tolerance. ZmRAFS overexpression in Arabidopsis enhanced drought stress tolerance by increasing myo-inositol levels via ZmRAFS-mediated galactinol hydrolysis in the leaves due to sucrose insufficiency in leaf cells and also enhanced raffinose synthesis in the seeds. Supplementation of sucrose to detached leaves converted ZmRAFS from hydrolyzing galactinol to synthesizing raffinose. Taken together, we demonstrate that ZmRAFS enhances plant drought tolerance through either raffinose synthesis or galactinol hydrolysis, depending on sucrose availability in plant cells. These results provide new avenues to improve plant drought stress tolerance through manipulation of the raffinose anabolic pathway.

Identification and Characterization of Carotenoids, Vitamin E and Minerals of Biofortified Sorghum

Abstract Objectives Sorghum is a critical staple crop in Sub-Saharan Africa and has been included in biofortification efforts to enhance shortfall micronutrient content including provitamin A carotenoids (pVA), zinc and iron. The purpose of this study was to evaluate progress in enhancement of pVA, tocochromanol (vitamin E), iron and zinc content as well as improvements in stability from a new generation of transgenic biofortified sorghum events in order to prioritize selection for translation to food systems. Methods Transgenic sorghum events (n = 16) from three different genotypic backgrounds developed to increase provitamin A biosynthesis (PSY1 + CRTI genes, +/− CRTB gene) and increase tocochromanol accumulation (HGGT gene) for vitamin A stability were characterized for carotenoid and tocochromanol profile using liquid chromatography coupled with diode array detection. Events were compared against their respective null segregants and wild type (WT) sorghum. Mineral content of sorghum lines and those developed to increase iron and zinc accumulation (YSL2 + NAS2 genes) was assessed by inductively coupled plasma - optical emission spectrometry. Storage stability of carotenoids and tocochromanols was assessed under controlled conditions (25°C; 57%RH) over 3 months. Results Total pVA content from transgenic sorghum (5.9 ± 0.1 – 28.6 ± 0.6 mg/gFW) was significantly different (P < 0.05) from null (0.4 ± 0 – 1.2 ± 0 mg/gFW) or WT (0.73 ± 0.2 mg/gFW). A general increase in total tocochromanol accumulation was observed in transgenic sorghum events (14.9 ± 0.8 – 36.2 ± 1.3 mg/gFW) compared to null/WT (14.2 ± 0.5 – 32.4 ± 4.4 mg/gFW) with significant differences observed among transgenic events across different sorghum genotypes (P < 0.05). Mineral accumulation varied among sorghum events with levels ranging from 28.44 ± 1.9 – 48.85 ± 4.9 mg/g and 27.65 ± 4.5 – 63.59 ± 13.4 mg/g FW for zinc and iron respectively. Highest level of zinc and iron was observed in Tx430 sorghum event. Conclusions Findings from this study highlight progress in pVA levels of transgenic biofortified sorghum and the potential for increased vitamin E levels to improve pVA stability over storage. Studies are underway to characterize the bioaccessibility of pVA carotenoids, Fe and Zn from these events. Funding Sources Pioneer Foundation.