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.

Wuschel2 enables highly efficient CRISPR/Cas-targeted genome editing during rapid de novo shoot regeneration in sorghum.

For many important crops including sorghum, use of CRISPR/Cas technology is limited not only by the delivery of the gene-modification components into a plant cell, but also by the ability to regenerate a fertile plant from the engineered cell through tissue culture. Here, we report that Wuschel2 (Wus2)-enabled transformation increases not only the transformation efficiency, but also the CRISPR/Cas-targeted genome editing frequency in sorghum (Sorghum bicolor L.). Using Agrobacterium-mediated transformation, we have demonstrated Wus2-induced direct somatic embryo formation and regeneration, bypassing genotype-dependent callus formation and significantly shortening the tissue culture cycle time. This method also increased the regeneration capacity that resulted in higher transformation efficiency across different sorghum varieties. Subsequently, advanced excision systems and "altruistic" transformation technology have been developed to generate high-quality morphogenic gene-free and/or selectable marker-free sorghum events. Finally, we demonstrate up to 6.8-fold increase in CRISPR/Cas9-mediated gene dropout frequency using Wus2-enabled transformation, compared to without Wus2, across various targeted loci in different sorghum genotypes.

Developing a rapid and highly efficient cowpea regeneration, transformation and genome editing system using embryonic axis explants.

Cowpea (Vigna unguiculata (L.) Walp.) is one of the most important legume crops planted worldwide, but despite decades of effort, cowpea transformation is still challenging due to inefficient Agrobacterium-mediated transfer DNA delivery, transgenic selection and in vitro shoot regeneration. Here, we report a highly efficient transformation system using embryonic axis explants isolated from imbibed mature seeds. We found that removal of the shoot apical meristem from the explants stimulated direct multiple shoot organogenesis from the cotyledonary node tissue. The application of a previously reported ternary transformation vector system provided efficient Agrobacterium-mediated gene delivery, while the utilization of spcN as selectable marker enabled more robust transgenic selection, plant recovery and transgenic plant generation without escapes and chimera formation. Transgenic cowpea plantlets developed exclusively from the cotyledonary nodes at frequencies of 4% to 37% across a wide range of cowpea genotypes. CRISPR/Cas-mediated gene editing was successfully demonstrated. The transformation principles established here could also be applied to other legumes to increase transformation efficiencies.

Developing a flexible, high-efficiency Agrobacterium-mediated sorghum transformation system with broad application.

Sorghum is the fifth most widely planted cereal crop in the world and is commonly cultivated in arid and semi-arid regions such as Africa. Despite its importance as a food source, sorghum genetic improvement through transgenic approaches has been limited because of an inefficient transformation system. Here, we report a ternary vector (also known as cohabitating vector) system using a recently described pVIR accessory plasmid that facilitates efficient Agrobacterium-mediated transformation of sorghum. We report regeneration frequencies ranging from 6% to 29% in Tx430 using different selectable markers and single copy, backbone free 'quality events' ranging from 45% to 66% of the total events produced. Furthermore, we successfully applied this ternary system to develop transformation protocols for popular but recalcitrant African varieties including Macia, Malisor 84-7 and Tegemeo. In addition, we report the use of this technology to develop the first stable CRISPR/Cas9-mediated gene knockouts in Tx430.

Integument Development in Arabidopsis Depends on Interaction of YABBY Protein INNER NO OUTER with Coactivators and Corepressors.

Arabidopsis thaliana INNER NO OUTER (INO) is a YABBY protein that is essential for the initiation and development of the outer integument of ovules. Other YABBY proteins have been shown to be involved in both negative and positive regulation of expression of putative target genes. YABBY proteins have also been shown to interact with the corepressor LEUNIG (LUG) in several systems. In support of a repressive role for INO, we confirm that INO interacts with LUG and also find that INO directly interacts with SEUSS (SEU), a known corepressive partner of LUG. Further, we find that INO can directly interact with ADA2b/PROPORZ1 (PRZ1), a transcriptional coactivator that is known to interact with the histone acetyltransferase GENERAL CONTROL NONREPRESSIBLE PROTEIN 5 (GCN5, also known as HAG1). Mutations in LUG, SEU, and ADA2b/PRZ1 all lead to pleiotropic effects including a deficiency in the extension of the outer integument. Additive and synergistic effects of ada2b/prz1 and lug mutations on outer integument formation indicate that these two genes function independently to promote outer integument growth. The ino mutation is epistatic to both lug and ada2b/prz1 in the outer integument, and all three proteins are present in the nuclei of a common set of outer integument cells. This is consistent with a model where INO utilizes these coregulator proteins to activate and repress separate sets of target genes. Other Arabidopsis YABBY proteins were shown to also form complexes with ADA2b/PRZ1, and have been previously shown to interact with SEU and LUG. Thus, interaction with these corepressors and coactivator may represent a general mechanism to explain the positive and negative activities of YABBY proteins in transcriptional regulation. The LUG, SEU, and ADA2b/PRZ1 proteins would also separately be recruited to targets of other transcription factors, consistent with their roles as general coregulators, explaining the pleiotropic effects not associated with YABBY function.

Positive- and negative-acting regulatory elements contribute to the tissue-specific expression of INNER NO OUTER, a YABBY-type transcription factor gene in Arabidopsis.

BACKGROUND: The INNER NO OUTER (INO) gene, which encodes a YABBY-type transcription factor, specifies and promotes the growth of the outer integument of the ovule in Arabidopsis. INO expression is limited to the abaxial cell layer of the developing outer integument of the ovule and is regulated by multiple regions of the INO promoter, including POS9, a positive element that when present in quadruplicate can produce low-level expression in the normal INO pattern. RESULTS: Significant redundancy in activity between different regions of the INO promoter is demonstrated. For specific regulatory elements, multimerization or the addition of the cauliflower mosaic virus 35S general enhancer was able to activate expression of reporter gene constructs that were otherwise incapable of expression on their own. A new promoter element, POS6, is defined and is shown to include sufficient positive regulatory information to reproduce the endogenous pattern of expression in ovules, but other promoter regions are necessary to fully suppress expression outside of ovules. The full-length INO promoter, but not any of the INO promoter deletions tested, is able to act as an enhancer-blocking insulator to prevent the ectopic activation of expression by the 35S enhancer. Sequence conservation between the promoter regions of Arabidopsis thaliana, Brassica oleracea and Brassica rapa aligns closely with the functional definition of the POS6 and POS9 regions, and with a defined INO minimal promoter. The B. oleracea INO promoter is sufficient to promote a similar pattern and level of reporter gene expression in Arabidopsis to that observed for the Arabidopsis promoter. CONCLUSIONS: At least two independent regions of the INO promoter contain sufficient regulatory information to direct the specific pattern but not the level of INO gene expression. These regulatory regions act in a partially redundant manner to promote the expression in a specific pattern in the ovule and suppress expression outside of ovules. Establishment of this pattern requires cooperation and competition between multiple positive and negative regulatory elements.

Seed dispersal: same gene, different organs.

A single nucleotide change in a conserved promoter element is responsible for both human-selected retention of rice grains on pedicels and for naturally selected differences in dehiscence-associated fruit structures in mustards.

Overlapping and antagonistic activities of BASIC PENTACYSTEINE genes affect a range of developmental processes in Arabidopsis.

The BASIC PENTACYSTEINE (BPC) proteins are a plant-specific transcription factor family that is present throughout land plants. The Arabidopsis BPC proteins have been categorized into three classes based on sequence similarity, and we demonstrate that there is functional overlap between classes. Single gene mutations produce no visible phenotypic effects, and severe morphological phenotypes occur only in higher order mutants between members of classes I and II, with the most severe phenotype observed in bpc1-1 bpc2 bpc4 bpc6 plants. These quadruple mutants are dwarfed and display small curled leaves, aberrant ovules, altered epidermal cells and reduced numbers of lateral roots. Affected processes include coordinated growth of cell layers, cell shape determination and timing of senescence. Disruption of BPC3 function rescues some aspects of the bpc1-1 bpc2 bpc4 bpc6 phenotype, indicating that BPC3 function may be antagonistic to other members of the family. Ethylene response is diminished in bpc1-1 bpc2 bpc4 bpc6 plants, although not all aspects of the phenotype can be explained by reduced ethylene sensitivity. Our data indicate that the BPC transcription factor family is integral for a wide range of processes that support normal growth and development.