Enabling genome editing in tropical maize lines through an improved, morphogenic regulator-assisted transformation protocol.

The recalcitrance exhibited by many maize (Zea mays) genotypes to traditional genetic transformation protocols poses a significant challenge to the large-scale application of genome editing (GE) in this major crop species. Although a few maize genotypes are widely used for genetic transformation, they prove unsuitable for agronomic tests in field trials or commercial applications. This challenge is exacerbated by the predominance of transformable maize lines adapted to temperate geographies, despite a considerable proportion of maize production occurring in the tropics. Ectopic expression of morphogenic regulators (MRs) stands out as a promising approach to overcome low efficiency and genotype dependency, aiming to achieve 'universal' transformation and GE capabilities in maize. Here, we report the successful GE of agronomically relevant tropical maize lines using a MR-based, Agrobacterium-mediated transformation protocol previously optimized for the B104 temperate inbred line. To this end, we used a CRISPR/Cas9-based construct aiming at the knockout of the VIRESCENT YELLOW-LIKE (VYL) gene, which results in an easily recognizable phenotype. Mutations at VYL were verified in protoplasts prepared from B104 and three tropical lines, regardless of the presence of a single nucleotide polymorphism (SNP) at the seed region of the VYL target site in two of the tropical lines. Three out of five tropical lines were amenable to transformation, with efficiencies reaching up to 6.63%. Remarkably, 97% of the recovered events presented indels at the target site, which were inherited by the next generation. We observed off-target activity of the CRISPR/Cas9-based construct towards the VYL paralog VYL-MODIFIER, which could be partly due to the expression of the WUSCHEL (WUS) MR. Our results demonstrate efficient GE of relevant tropical maize lines, expanding the current availability of GE-amenable genotypes of this major crop.

Plant microbiomes harbor potential to promote nutrient turnover in impoverished substrates of a Brazilian biodiversity hotspot.

The substrates of the Brazilian campos rupestres, a grassland ecosystem, have extremely low concentrations of phosphorus and nitrogen, imposing restrictions to plant growth. Despite that, this ecosystem harbors almost 15% of the Brazilian plant diversity, raising the question of how plants acquire nutrients in such a harsh environment. Here, we set out to uncover the taxonomic profile, the compositional and functional differences and similarities, and the nutrient turnover potential of microbial communities associated with two plant species of the campos rupestres-dominant family Velloziaceae that grow over distinct substrates (soil and rock). Using amplicon sequencing data, we show that, despite the pronounced composition differentiation, the plant-associated soil and rock communities share a core of highly efficient colonizers that tend to be highly abundant and is enriched in 21 bacterial families. Functional investigation of metagenomes and 522 metagenome-assembled genomes revealed that the microorganisms found associated to plant roots are enriched in genes involved in organic compound intake, and phosphorus and nitrogen turnover. We show that potential for phosphorus transport, mineralization, and solubilization are mostly found within bacterial families of the shared microbiome, such as Xanthobacteraceae and Bryobacteraceae. We also detected the full repertoire of nitrogen cycle-related genes and discovered a lineage of Isosphaeraceae that acquired nitrogen-fixing potential via horizontal gene transfer and might be also involved in nitrification via a metabolic handoff association with Binataceae. We highlight that plant-associated microbial populations in the campos rupestres harbor a genetic repertoire with potential to increase nutrient availability and that the microbiomes of biodiversity hotspots can reveal novel mechanisms of nutrient turnover.

Purification and characterization of a small (7.3 kDa) putative lipid transfer protein from maize seeds.

The present study reports, for the first time in literature, the purification and biochemical characterization of a small basic protein from maize seeds similar to plant lipid transfer proteins-2, named mLTP2. The mLTP2 consists of 70 amino acid residues and has an M(r) of 7303.83, determined by electrospray ionization mass spectrometry. The primary structure of mLTP2 was determined by automated Edman degradation of the intact protein and peptides obtained from digestions with trypsin and by C-terminal sequencing using carboxypeptidase Y. The mLTP2 exhibits high sequence similarity (51-44% identical positions) with other plant LTP2s previously described.