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.

Expression, regulation and activity of a B2-type cyclin in mitotic and endoreduplicating maize endosperm.

Cyclin-dependent kinases, the master regulators of the eukaryotic cell cycle, are complexes comprised of a catalytic serine/threonine protein kinase and an essential regulatory cyclin. The maize genome encodes over 50 cyclins grouped in different types, but they have been little investigated. We characterized a type B2 cyclin (CYCB2;2) during maize endosperm development, which comprises a cell proliferation phase based on the standard mitotic cell cycle, followed by an endoreduplication phase in which DNA replication is reiterated in the absence of mitosis or cytokinesis. CYCB2;2 RNA was present throughout the period of endosperm development studied, but its level declined as the endosperm transitioned from a mitotic to an endoreduplication cell cycle. However, the level of CYCB2;2 protein remained relatively constant during both stages of endosperm development. CYCB2;2 was recalcitrant to degradation by the 26S proteasome in endoreduplicating endosperm extracts, which could explain its sustained accumulation during endosperm development. In addition, although CYCB2;2 was generally localized to the nucleus of endosperm cells, a lower molecular weight form of the protein accumulated specifically in the cytosol of endoreduplicating endosperm cells. In dividing cells, CYCB2;2 appeared to be localized to the phragmoplast and may be involved in cytokinesis and cell wall formation. Kinase activity was associated with CYCB2;2 in mitotic endosperm, but was absent or greatly reduced in immature ear and endoreduplicating endosperm. CYCB2;2-associated kinase phosphorylated maize E2F1 and the "pocket" domains of RBR1 and RBR3. CYCB2;2 interacted with both maize CDKA;1 and CDKA;3 in insect cells. These results suggest CYCB2;2 functions primarily during the mitotic cell cycle, and they are discussed in the context of the roles of cyclins, CDKs and proteasome activity in the regulation of the cell cycle during endosperm development.

Cell cycle control and seed development.

Seed development is a complex process that requires coordinated integration of many genetic, metabolic, and physiological pathways and environmental cues. Different cell cycle types, such as asymmetric cell division, acytokinetic mitosis, mitotic cell division, and endoreduplication, frequently occur in sequential yet overlapping manner during the development of the embryo and the endosperm, seed structures that are both products of double fertilization. Asymmetric cell divisions in the embryo generate polarized daughter cells with different cell fates. While nuclear and cell division cycles play a key role in determining final seed cell numbers, endoreduplication is often associated with processes such as cell enlargement and accumulation of storage metabolites that underlie cell differentiation and growth of the different seed compartments. This review focuses on recent advances in our understanding of different cell cycle mechanisms operating during seed development and their impact on the growth, development, and function of seed tissues. Particularly, the roles of core cell cycle regulators, such as cyclin-dependent-kinases and their inhibitors, the Retinoblastoma-Related/E2F pathway and the proteasome-ubiquitin system, are discussed in the contexts of different cell cycle types that characterize seed development. The contributions of nuclear and cellular proliferative cycles and endoreduplication to cereal endosperm development are also discussed.

Cyclin-dependent kinase complexes in developing maize endosperm: evidence for differential expression and functional specialization.

Endosperm development in maize (Zea mays L.) and related cereals comprises a cell proliferation stage followed by a period of rapid growth coupled to endoreduplication. Regulation of the cell cycle in developing endosperm is poorly understood. We have characterized various subunits of cyclin-dependent kinase (CDK) complexes, master cell cycle regulators in all eukaryotes. A-, B-, and D-type cyclins as well as A- and B-type cyclin-dependent kinases were characterized with respect to their RNA and protein expression profiles. Two main patterns were identified: one showing expression throughout endosperm development, and another characterized by a sharp down-regulation with the onset of endoreduplication. Cyclin CYCB1;3 and CYCD2;1 proteins were distributed in the cytoplasm and nucleus of cells throughout the endosperm, while cyclin CYCD5 protein was localized in the cytoplasm of peripheral cells. CDKB1;1 expression was strongly associated with cell proliferation. Expression and cyclin-binding patterns suggested that CDKA;1 and CDKA;3 are at least partially redundant. The kinase activity associated with the cyclin CYCA1 was highest during the mitotic stage of development, while that associated with CYCB1;3, CYCD2;1 and CYCD5 peaked at the mitosis-to-endoreduplication transition. A-, B- and D-type cyclins were more resistant to proteasome-dependent degradation in endoreduplicating than in mitotic endosperm extracts. These results indicated that endosperm development is characterized by differential expression and activity of specific cyclins and CDKs, and suggested that endoreduplication is associated with reduced cyclin proteolysis via the ubiquitin-proteasome pathway.

Control of cell proliferation, endoreduplication, cell size, and cell death by the retinoblastoma-related pathway in maize endosperm.

The endosperm of cereal grains is one of the most valuable products of modern agriculture. Cereal endosperm development comprises different phases characterized by mitotic cell proliferation, endoreduplication, the accumulation of storage compounds, and programmed cell death. Although manipulation of these processes could maximize grain yield, how they are regulated and integrated is poorly understood. We show that the Retinoblastoma-related (RBR) pathway controls key aspects of endosperm development in maize. Down-regulation of RBR1 by RNAi resulted in up-regulation of RBR3-type genes, as well as the MINICHROMOSOME MAINTENANCE 2-7 gene family and PROLIFERATING CELL NUCLEAR ANTIGEN, which encode essential DNA replication factors. Both the mitotic and endoreduplication cell cycles were stimulated. Developing transgenic endosperm contained 42-58% more cells and ∼70% more DNA than wild type, whereas there was a reduction in cell and nuclear sizes. In addition, cell death was enhanced. The DNA content of mature endosperm increased 43% upon RBR1 down-regulation, whereas storage protein content and kernel weight were essentially not affected. Down-regulation of both RBR1 and CYCLIN DEPENDENT KINASE A (CDKA);1 indicated that CDKA;1 is epistatic to RBR1 and controls endoreduplication through an RBR1-dependent pathway. However, the repressive activity of RBR1 on downstream targets was independent from CDKA;1, suggesting diversification of RBR1 activities. Furthermore, RBR1 negatively regulated CDK activity, suggesting the presence of a feedback loop. These results indicate that the RBR1 pathway plays a major role in regulation of different processes during maize endosperm development and suggest the presence of tissue/organ-level regulation of endosperm/seed homeostasis.

A statistical model for estimating maternal-zygotic interactions and parent-of-origin effects of QTLs for seed development.

Proper development of a seed requires coordinated exchanges of signals among the three components that develop side by side in the seed. One of these is the maternal integument that encloses the other two zygotic components, i.e., the diploid embryo and its nurturing annex, the triploid endosperm. Although the formation of the embryo and endosperm contains the contributions of both maternal and paternal parents, maternally and paternally derived alleles may be expressed differently, leading to a so-called parent-of-origin or imprinting effect. Currently, the nature of how genes from the maternal and zygotic genomes interact to affect seed development remains largely unknown. Here, we present a novel statistical model for estimating the main and interaction effects of quantitative trait loci (QTLs) that are derived from different genomes and further testing the imprinting effects of these QTLs on seed development. The experimental design used is based on reciprocal backcrosses toward both parents, so that the inheritance of parent-specific alleles could be traced. The computing model and algorithm were implemented with the maximum likelihood approach. The new strategy presented was applied to study the mode of inheritance for QTLs that control endoreduplication traits in maize endosperm. Monte Carlo simulation studies were performed to investigate the statistical properties of the new model with the data simulated under different imprinting degrees. The false positive rate of imprinting QTL discovery by the model was examined by analyzing the simulated data that contain no imprinting QTL. The reciprocal design and a series of analytical and testing strategies proposed provide a standard procedure for genomic mapping of QTLs involved in the genetic control of complex seed development traits in flowering plants.

Identification of quantitative trait loci that affect endoreduplication in maize endosperm.

Endoreduplication in maize endosperm precedes the onset of starch and storage protein synthesis, and it is generally thought to influence grain filling. We created four backcross populations by reciprocally crossing the F(1) progeny of a cross between Sg18 and Mo17 to the parental inbreds, which differ in endoreduplication by two parameters--mean ploidy and percentage of endoreduplicated nuclei. This four-backcross design allowed us to estimate and test the additive and dominant genetic effects of quantitative trait loci (QTLs) affecting endoreduplication. An analysis of endosperm from the four backcross populations at 16 days after pollination using a modified triploid mapping approach identified three endosperm QTLs influencing mean ploidy and two endosperm QTLs affecting the percentage of endoreduplicated nuclei. Some of these QTLs may manifest their effects on endoreduplication via expression in the embryo. The QTLs detected display strong dominance or over-dominance and interacted epistatically with an embryo-expressed QTL. This helps to explain the genetic basis for transgressive segregation in the backcross progeny. Although the favorable alleles that increase mean ploidy and percentage of endoreduplicated nuclei can be contributed by both parents, the Mo17-derived alleles for endoreduplication were often dominant or over-dominant to the Sg18-derived allele. One QTL on chromosome 7 that may be expressed in both the embryo and endosperm exerted a pleiotropic effect on two different parameters of endoreduplication. The results from this study shed light on the regulation of endoreduplication in maize endosperm and provide a marker-assisted selection strategy for potentially improving grain yield.

RBR3, a member of the retinoblastoma-related family from maize, is regulated by the RBR1/E2F pathway.

Retinoblastoma-related (RBR) proteins regulate cell division in higher eukaryotes by controlling the adenovirus E2 promoter binding factor (E2F)/dimerization partner (DP) family of transcription factors that regulate expression of many genes involved in cell-cycle progression. We identified a previously undescribed member of the maize RBR family, RBR3, which has the characteristic structure and binding activities of pocket proteins, where interaction depends on a LxCxE motif in the partner proteins and a critical cysteine within the B pocket domain. Like other RBR proteins, RBR3 appears to be regulated by phosphorylation mediated by cyclin-dependent kinases. During endosperm development, RBR3 expression is restricted to the mitotic stage preceding the onset of endoreduplication. This finding suggests a role distinct from RBR1, which is constitutively expressed. Two sites in the RBR3 promoter bind to complexes containing maize E2F1 and DP proteins. Expression of wheat dwarf virus RepA protein, which blocks RBR1 activity and stimulates cell proliferation, dramatically up-regulates RBR3, but not RBR1, RNA in embryogenic maize calli. The results indicate that RBR3 expression is controlled by RBR1 through the activity of E2F/DP and that RBR3 is the maize equivalent of mammalian p107. Furthermore, maize and related grasses might have evolved a compensatory mechanism among distinct types of RBR proteins to ensure robust control of pocket protein activity.

Cyclin-dependent kinase inhibitors in maize endosperm and their potential role in endoreduplication.

Two maize (Zea mays) cyclin-dependent kinase (CDK) inhibitors, Zeama;KRP;1 and Zeama;KRP;2, were characterized and shown to be expressed in developing endosperm. Similar to the CDK inhibitors in Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum), the maize proteins contain a carboxy-terminal region related to the inhibitory domain of the mammalian Cip/Kip inhibitors. Zeama;KRP;1 is present in the endosperm between 7 and 21 d after pollination, a period that encompasses the onset of endoreduplication, while the Zeama;KRP;2 protein declines during this time. Nevertheless, Zeama;KRP;1 accounts for only part of the CDK inhibitory activity that peaks coincident with the endoreduplication phase of endosperm development. In vitro assays showed that Zeama;KRP;1 and Zeama;KRP;2 are able to inhibit endosperm Cdc2-related CKD activity that associates with p13(Suc1). They were also shown to specifically inhibit cyclin A1;3- and cyclin D5;1-associated CDK activities, but not cyclin B1;3/CDK. Overexpression of Zeama;KRP;1 in maize embryonic calli that ectopically expressed the wheat dwarf virus RepA protein, which counteracts retinoblastoma-related protein function, led to an additional round of DNA replication without nuclear division.

A dominant negative mutant of cyclin-dependent kinase A reduces endoreduplication but not cell size or gene expression in maize endosperm.

Cells in maize (Zea mays) endosperm undergo multiple cycles of endoreduplication, with some attaining DNA contents as high as 96C and 192C. Genome amplification begins around 10 d after pollination, coincident with cell enlargement and the onset of starch and storage protein accumulation. Although the role of endoreduplication is unclear, it is thought to provide a mechanism that increases cell size and enhances gene expression. To investigate this process, we reduced endoreduplication in transgenic maize endosperm by ectopically expressing a gene encoding a dominant negative mutant form of cyclin-dependent kinase A. This gene was regulated by the 27-kD gamma-zein promoter, which restricted synthesis of the defective enzyme to the endoreduplication rather than the mitotic phase of endosperm development. Overexpression of a wild-type cyclin-dependent kinase A increased enzyme activity but had no effect on endoreduplication. By contrast, ectopic expression of the defective enzyme lowered kinase activity and reduced by half the mean C-value and total DNA content of endosperm nuclei. The lower level of endoreduplication did not affect cell size and only slightly reduced starch and storage protein accumulation. There was little difference in the level of endosperm gene expression with high and low levels of endoreduplication, suggesting that this process may not enhance transcription of genes associated with starch and storage protein synthesis.

Genetic analyses of endoreduplication in Zea mays endosperm: evidence of sporophytic and zygotic maternal control.

Flow cytometry was used to assess the variability of endoreduplication in endosperms of maize inbred lines. Little variation was found between midwestern dent types, and high levels of endoreduplication were observed in popcorns. Endoreduplication is different between inbred lines by 13-18 days after pollination, and flow cytometric analysis of ploidy level was feasible until 20 DAP. To study the genetic regulation of endoreduplication, four inbreds were crossed to B73 and developing endosperms from both parental, reciprocal F(1), and backcross generations were subjected to flow cytometric analysis. Three measurements of endoreduplication were calculated from these data and analyzed as quantitative genetic traits. Multiple models of trait inheritance were considered including triploid, diploid, sporophytic maternal, and maternal and paternal zygotic nuclear inheritance. Maternal zygotic effects, often considered a form of parental imprinting, and maternal sporophytic effects were detected. To test the feasibility of introgressing a high endoreduplication phenotype into a midwestern dent inbred line, a backcross population was generated from B73 x Sg18. Parental and progeny endoreduplication levels were compared and heritabilities assessed. The heritabilities calculated from these data generally agree with the values calculated in the larger crossing experiments.