Fertility restoration of maize CMS-C altered by a single amino acid substitution within the Rf4 bHLH transcription factor.

Type C cytoplasmic male sterility (CMS-C) is the most commonly used form of CMS in maize hybrid seed production. Restorer of fertility 4 (Rf4), the major fertility restorer gene of CMS-C, is located on chromosome 8S. To positionally clone Rf4, a large F3 population derived from a cross between a non-restorer and restorer (n = 5104) was screened for recombinants and then phenotyped for tassel fertility, resulting in a final map-based cloning interval of 12 kb. Within this 12-kb interval, the only likely candidate for Rf4 was GRMZM2G021276, a basic helix-loop-helix (bHLH) transcription factor with tassel-specific expression. The Rf4 gene product contains a nuclear localization signal and is likely to not interact directly with the mitochondria. Sequence analysis of Rf4 revealed four encoded amino acid substitutions between restoring and non-restoring inbreds, however only one substitution, F187Y, was within the highly conserved bHLH domain. The hypothesis that Rf4 restoration is altered by a single amino acid was tested by using clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated protein 9 (Cas9) homology directed repair (HDR) to create isogenic lines that varied for the F187Y substitution. In a population of these CRISPR-Cas9 edited plants (n = 780) that was phenotyped for tassel fertility, plants containing F187 were completely fertile, indicating fertility restoration, and plants containing Y187 were sterile, indicating lack of fertility restoration. Structural modeling shows that this amino acid residue 187 is located within the four helix bundle core, a critical region for stabilizing dimer conformation and affecting interaction partner selection.

Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation.

While transformation of the major monocot crops is currently possible, the process typically remains confined to one or two genotypes per species, often with poor agronomics, and efficiencies that place these methods beyond the reach of most academic laboratories. Here, we report a transformation approach involving overexpression of the maize (Zea mays) Baby boom (Bbm) and maize Wuschel2 (Wus2) genes, which produced high transformation frequencies in numerous previously nontransformable maize inbred lines. For example, the Pioneer inbred PHH5G is recalcitrant to biolistic and Agrobacterium tumefaciens transformation. However, when Bbm and Wus2 were expressed, transgenic calli were recovered from over 40% of the starting explants, with most producing healthy, fertile plants. Another limitation for many monocots is the intensive labor and greenhouse space required to supply immature embryos for transformation. This problem could be alleviated using alternative target tissues that could be supplied consistently with automated preparation. As a major step toward this objective, we transformed Bbm and Wus2 directly into either embryo slices from mature seed or leaf segments from seedlings in a variety of Pioneer inbred lines, routinely recovering healthy, fertile T0 plants. Finally, we demonstrated that the maize Bbm and Wus2 genes stimulate transformation in sorghum (Sorghum bicolor) immature embryos, sugarcane (Saccharum officinarum) callus, and indica rice (Oryza sativa ssp indica) callus.

A truncated FatB resulting from a single nucleotide insertion is responsible for reducing saturated fatty acids in maize seed oil.

KEY MESSAGE: We identified a G-nucleotide insertion in a maize FatB responsible for reducing saturated fatty acids through QTL mapping and map-based cloning and developed an allele-specific DNA marker for molecular breeding. Vegetable oils with reduced saturated fatty acids have signficant health benefits. SRS72NE, a Dow AgroSciences proprietory maize inbred line, was found to contain signficantly reduced levels of palmitic acid and total saturated fatty acids in seed oil when compared to other common inbreds. Using F2 and F3 populations derived from a cross between SRS72NE and a normal inbred SLN74, we have demonstrated that the reduced saturated fatty acid phenotype in SRS72NE is controlled by a single QTL on chromosome 9 that explains 79.1 % of palmitic acid and 79.6 % total saturated fatty acid variations. The QTL was mapped to an interval of 105 kb that contains one single gene, a type B fatty acyl-ACP thioesterase (ZmFatB; GRMZM5G829544). ZmFatB alleles from SRS72NE and common inbreds were cloned and sequenced. SRS72NE fatb allele contains a single nucleotide (G) insertion in the 6th exon, which creates a premature stop codon 22 base pairs down stream. As a result, ZmFatB protein from SRS72NE is predicted to contain eight altered and 90 deleted amino acids at its C-terminus. Because the affected region is part of the conserved acyl-ACP thioesterase catalytic domain, the truncated ZmFatB in SRS72NE is likely non-functional. We also show that fatb RNA level in SRS72NE is reduced by 4.4-fold when compared to the normal allele SNL74. A high throughput DNA assay capable of differentiating the normal and reduced saturate fatty acid alleles has been developed and can be used for accelerated molecular breeding.

Transposon insertion in a cinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize.

Maize brown midrib1 (bm1) mutant plants have reduced lignin content and offer significant advantages when used in silage and biofuel applications. Cinnamyl alcohol dehydrogenase (CAD) catalyzes the conversion of hydroxycinnamyl aldehydes to monolignols, a key step in lignin biosynthesis. Maize CAD2 has been implicated as the underlying gene for bm1 phenotypes since bm1 plants have reduced CAD activity and lower CAD2 transcript level. Here, we describe a Dow AgroSciences maize bm1 mutant (bm1-das1) that contains a 3,444-bp transposon insertion in the first intron of CAD2 gene. As a result of chimeric RNA splicing, cad2 mRNA from bm1-das1 contains a 409-bp insert between its 1st and 2nd exons. This insertion creates a premature stop codon and is predicted to result in a truncated protein of 48 amino acids (AA), compared to 367 AA for the wild type (WT) CAD2. We have also sequenced cad2 from the reference allele bm1-ref in 515D bm1 stock and showed that it contains a two-nucleotide (AC) insertion in the 3rd exon, which is predicted to result in a truncated protein of 147 AA. The levels of cad2 mRNA in the midribs of bm1-das1 and bm1-ref are reduced by 91 and 86 % respectively, leading to reductions in total lignin contents by 24 and 30 %. Taken together, our data show that mutations in maize CAD2 are responsible for maize bm1 phenotypes. Based on specific changes in bm1-das1 and bm1-ref, high throughput TaqMan and KBioscience's allele specific PCR assays capable of differentiating mutant and WT alleles have been developed to accelerate bm1 molecular breeding.

Expression of ZmLEC1 and ZmWRI1 increases seed oil production in maize.

Increasing seed oil production is a major goal for global agriculture to meet the strong demand for oil consumption by humans and for biodiesel production. Previous studies to increase oil synthesis in plants have focused mainly on manipulation of oil pathway genes. As an alternative to single-enzyme approaches, transcription factors provide an attractive solution for altering complex traits, with the caveat that transcription factors may face the challenge of undesirable pleiotropic effects. Here, we report that overexpression of maize (Zea mays) LEAFY COTYLEDON1 (ZmLEC1) increases seed oil by as much as 48% but reduces seed germination and leaf growth in maize. To uncouple oil increase from the undesirable agronomic traits, we identified a LEC1 downstream transcription factor, maize WRINKLED1 (ZmWRI1). Overexpression of ZmWRI1 results in an oil increase similar to overexpression of ZmLEC1 without affecting germination, seedling growth, or grain yield. These results emphasize the importance of field testing for developing a commercial high-oil product and highlight ZmWRI1 as a promising target for increasing oil production in crops.

TREM-2 negatively regulates LPS-mediated inflammatory response in rat bone marrow-derived MSCs.

To the best of our knowledge, our previous study demonstrated the expression of triggering receptor expressed on myeloid cells 2 (TREM­2) in human bone marrow mesenchymal stem cells (MSCs) for the first time. However, the inflammation regulatory role of TREM­2 in MSCs remain elusive. The aim of the present study was to investigate the immune regulation and the underlying mechanism of TREM­2 in rat bone marrow MSCs. MSCs were divided into three groups: NullMSCs, TREM­2MSCs, and NormMSCs. TREM­2 was expressed in MSCs at the mRNA and protein level. Following stimulation by lipopolysaccharide (LPS), the gene transcription levels of TREM­2 and inflammatory cytokines were increased. The expression levels of inflammatory cytokines, including tumor necrosis factor­α (TNF­α) and interleukin­1ß (IL­1ß), in the TREM­2MSCs lentiviral vector group were significantly downregulated, and the expression of IL­10 was significantly upregulated compared with the controls. Western blot analysis revealed that TREM­2 downregulated the LPS­induced inflammatory response in MSCs, which was probably associated with regulating AKT serine/threonine kinase and p38 mitogen­activated protein kinase downstream signaling proteins. The results of the current study demonstrated that TREM­2 negatively regulates the LPS­mediated inflammatory response in MSCs suggesting that TREM­2 is a potential target of immune regulation in rat MSCs.

Regulation of human mesenchymal stem cell differentiation by TREM-2.

Activation of the triggering receptor expressed on myeloid cells 2 (TREM-2) regulates myeloid cell function in vitro. However, the failure to detect TREM-2 protein expression in vivo has hampered studies on immunological and other physiological TREM-2 functions. This study demonstrates that TREM-2 is expressed by human mesenchymal stem cells (h-MSCs) and responds to the toll-like receptor (TLR) ligand lipopolysaccharide (LPS). Knockdown of TREM-2 in h-MSCs using a small interfering RNA (siRNA) reduced the expression levels of TLR2, TLR4, and TLR6, inhibited osteogenic, chondrogenic, and adipogenic differentiation under specific induction conditions, and enhanced LPS-evoked inflammatory cytokine production. Thus, activation of TREM-2 may restrain h-MSC immune activation and promote differentiation for tissue repair.

Whole genome scan detects an allelic variant of fad2 associated with increased oleic acid levels in maize.

We used whole genome scan association mapping to identify loci with major effect on oleic acid content in maize kernels. Single nucleotide polymorphism haplotypes at 8,590 loci were tested for association with oleic acid content in 553 maize inbreds. A single locus with major effect on oleic acid was mapped between 380 and 384 cM in the IBM2 neighbors genetic map on chromosome 4 and confirmed in a biparental population. A fatty acid desaturase, fad2, identified approximately 2 kb from the associated genetic marker, is the most likely candidate gene responsible for the differences in the phenotype. The fad2 alleles with high- and low-oleic acid content were sequenced and allelic differences in fad2 RNA level in developing embryos was investigated. We propose that a non-conservative amino acid polymorphism near the active site of fad2 contributes to the effect on oleic acid content. This is the first report of the use of a high resolution whole genome scan association mapping where a putative gene responsible for a quantitative trait was identified in plants.

A phenylalanine in DGAT is a key determinant of oil content and composition in maize.

Plant oil is an important renewable resource for biodiesel production and for dietary consumption by humans and livestock. Through genetic mapping of the oil trait in plants, studies have reported multiple quantitative trait loci (QTLs) with small effects, but the molecular basis of oil QTLs remains largely unknown. Here we show that a high-oil QTL (qHO6) affecting maize seed oil and oleic-acid contents encodes an acyl-CoA:diacylglycerol acyltransferase (DGAT1-2), which catalyzes the final step of oil synthesis. We further show that a phenylalanine insertion in DGAT1-2 at position 469 (F469) is responsible for the increased oil and oleic-acid contents. The DGAT1-2 allele with F469 is ancestral, whereas the allele without F469 is a more recent mutant selected by domestication or breeding. Ectopic expression of the high-oil DGAT1-2 allele increases oil and oleic-acid contents by up to 41% and 107%, respectively. This work provides insights into the molecular basis of natural variation of oil and oleic-acid contents in plants and highlights DGAT as a promising target for increasing oil and oleic-acid contents in other crops.