GRASSY TILLERS1 (GT1) and SIX-ROWED SPIKE1 (VRS1) homologs share conserved roles in growth repression.

Crop engineering and de novo domestication using gene editing are new frontiers in agriculture. However, outside of well-studied crops and model systems, prioritizing engineering targets remains challenging. Evolution can guide us, revealing genes with deeply conserved roles that have repeatedly been selected in the evolution of plant form. Homologs of the transcription factor genes GRASSY TILLERS1 (GT1) and SIX-ROWED SPIKE1 (VRS1) have repeatedly been targets of selection in domestication and evolution, where they repress growth in many developmental contexts. This suggests a conserved role for these genes in regulating growth repression. To test this, we determined the roles of GT1 and VRS1 homologs in maize (Zea mays) and the distantly related grass brachypodium (Brachypodium distachyon) using gene editing and mutant analysis. In maize, gt1; vrs1-like1 (vrl1) mutants have derepressed growth of floral organs. In addition, gt1; vrl1 mutants bore more ears and more branches, indicating broad roles in growth repression. In brachypodium, Bdgt1; Bdvrl1 mutants have more branches, spikelets, and flowers than wild-type plants, indicating conserved roles for GT1 and VRS1 homologs in growth suppression over ca. 59 My of grass evolution. Importantly, many of these traits influence crop productivity. Notably, maize GT1 can suppress growth in arabidopsis (Arabidopsis thaliana) floral organs, despite ca. 160 My of evolution separating the grasses and arabidopsis. Thus, GT1 and VRS1 maintain their potency as growth regulators across vast timescales and in distinct developmental contexts. This work highlights the power of evolution to inform gene editing in crop improvement.

Parallel tuning of semi-dwarfism via differential splicing of Brachytic1 in commercial maize and smallholder sorghum.

In the current genomic era, the search and deployment of new semi-dwarf alleles have continued to develop better plant types in all cereals. We characterized an agronomically optimal semi-dwarf mutation in Zea mays L. and a parallel polymorphism in Sorghum bicolor L. We cloned the maize brachytic1 (br1-Mu) allele by a modified PCR-based Sequence Amplified Insertion Flanking Fragment (SAIFF) approach. Histology and RNA-Seq elucidated the mechanism of semi-dwarfism. GWAS linked a sorghum plant height QTL with the Br1 homolog by resequencing a West African sorghum landraces panel. The semi-dwarf br1-Mu allele encodes an MYB transcription factor78 that positively regulates stalk cell elongation by interacting with the polar auxin pathway. Semi-dwarfism is due to differential splicing and low functional Br1 wild-type transcript expression. The sorghum ortholog, SbBr1, co-segregates with the major plant height QTL qHT7.1 and is alternatively spliced. The high frequency of the Sbbr1 allele in African landraces suggests that African smallholder farmers used the semi-dwarf allele to improve plant height in sorghum long before efforts to introduce Green Revolution-style varieties in the 1960s. Surprisingly, variants for differential splicing of Brachytic1 were found in both commercial maize and smallholder sorghum, suggesting parallel tuning of plant architecture across these systems.

Emb15 encodes a plastid ribosomal assembly factor essential for embryogenesis in maize.

Ribosome assembly factors guide the complex process by which ribosomal proteins and the ribosomal RNAs form a functional ribosome. However, the assembly of plant plastid ribosomes is poorly understood. In the present study, we discovered a maize (Zea mays) plastid ribosome assembly factor based on our characterization of the embryo defective 15 (emb15) mutant. Loss of function of Emb15 retards embryo development at an early stage, but does not substantially affect the endosperm, and causes an albino phenotype in other genetic backgrounds. EMB15 localizes to plastids and possesses a ribosome maturation factor M (RimM) domain in the N-terminus and a predicted UDP-GlcNAc pyrophosphorylase domain in the C-terminus. The EMB15 RimM domain originated in bacteria and the UDP-GlcNAc pyrophosphorylase domain originated in fungi; these two domains came together in the ancestor of land plants during evolution. The N-terminus of EMB15 complemented the growth defect of an Escherichia coli strain with a RimM deletion and rescued the albino phenotype of emb15 homozygous mutants. The RimM domain mediates the interaction between EMB15 and the plastid ribosomal protein PRPS19. Plastid 16S rRNA maturation is also significantly impaired in emb15. These observations suggest that EMB15 functions in maize seed development as a plastid ribosome assembly factor, and the C-terminal domain is not important under normal conditions.

The maize premature senesence2 encodes for PHYTOCHROME-DEPENDENT LATE-FLOWERING and its expression modulation improves agronomic traits under abiotic stresses.

Among the various abiotic stresses, water and nitrogen are major stress factors that limit crop productivity worldwide. Since leaf nutrients remobilization during leaf senescence might impact response to abiotic stress in crops, we undertook a forward screen of the Mutator-active approach to identify premature senescence loci in maize. A mutant line isolated from a cross between a Pioneer Brand elite line and a public Mutator-active material, designated premature senescence2 (pre2), expressed leaf senescence during flower initiation. The Pre2 gene encodes PHYTOCHROME-DEPENDENT LATE-FLOWERING (PHL) protein, a nuclear receptor coactivator. The pre2-1 mutant allele was not a null mutation but produced a functional wild-type transcript along with multiple mRNA species of varying lengths resulting from the alternate splicing of the Pre2 gene. The PHL accelerates flowering by suppressing the inhibitory effect of phyB on flowering in Arabidopsis (Endo et al., 2013). The ZmPRE2 polypeptide is highly conserved in plant species and has two identifiable motifs namely SPT20 and MED15. The Spt20 domain, which is a part of the SAGA (Spt-Ada-Gcn5 acetyltransferase) complex, is involved in histone deacetylation and MED15 proteins have nuclear functions in mediating DNA Pol II transcription. The differential spliced mature transcripts in both the pre2 alleles, as a result of transposon interference, were producing truncated proteins that lacked polyglutamine (Q) tract near the C-terminus and might be causative of the premature senescence phenotype in maize. Endogenous gene suppression of ZmPre2 by RNAi improves maize agronomic performance under both water stress and suboptimal nitrogen conditions. The homozygous T-DNA knockout of the pre2 homolog in Arabidopsis (At1G72390; the same insertional allele used by Endo et al., 2013) results in higher biomass, delayed maturity, enhanced tolerance to drought, and improved nitrogen utilization efficiency. The Arabidopsis mutant also showed hypersensitive response to 1 µM ABA (abscisic acid) concentration. These results indicate that the PHL protein plays a direct or indirect role in ABA-dependent drought and N signaling pathways.

Chitinase-like1 Plays a Role in Stalk Tensile Strength in Maize.

Stalk lodging in maize (Zea mays) causes significant yield losses due to breaking of stalk tissue below the ear node before harvest. Here, we identified the maize brittle stalk4 (bk4) mutant in a Mutator F2 population. This mutant was characterized by highly brittle aerial parts that broke easily from mechanical disturbance or in high-wind conditions. The bk4 plants displayed a reduction in average stalk diameter and mechanical strength, dwarf stature, senescence at leaf tips, and semisterility of pollen. Histological studies demonstrated a reduction in lignin staining of cells in the bk4 mutant leaves and stalk, and deformation of vascular bundles in the stalk resulting in the loss of xylem and phloem tissues. Biochemical characterization showed a significant reduction in p-coumaric acid, Glc, Man, and cellulose contents. The candidate gene responsible for bk4 phenotype is Chitinase-like1 protein (Ctl1), which is expressed at its highest levels in elongated internodes. Expression levels of secondary cell wall cellulose synthase genes (CesA) in the bk4 single mutant, and phenotypic observations in double mutants combining bk4 with bk2 or null alleles for two CesA genes, confirmed interaction of ZmCtl1 with CesA genes. Overexpression of ZmCtl1 enhanced mechanical stalk strength without affecting plant stature, senescence, or fertility. Biochemical characterization of ZmCtl1 overexpressing lines supported a role for ZmCtl1 in tensile strength enhancement. Conserved identity of CTL1 peptides across plant species and analysis of Arabidopsis (Arabidopsis thaliana) ctl1-1 ctl2-1 double mutants indicated that Ctl1 might have a conserved role in plants.

Sucrose transporter2 contributes to maize growth, development, and crop yield.

During daylight, plants produce excess photosynthates, including sucrose, which is temporarily stored in the vacuole. At night, plants remobilize sucrose to sustain metabolism and growth. Based on homology to other sucrose transporter (SUT) proteins, we hypothesized the maize (Zea mays) SUCROSE TRANSPORTER2 (ZmSUT2) protein functions as a sucrose/H+ symporter on the vacuolar membrane to export transiently stored sucrose. To understand the biological role of ZmSut2, we examined its spatial and temporal gene expression, determined the protein subcellular localization, and characterized loss-of-function mutations. ZmSut2 mRNA was ubiquitously expressed and exhibited diurnal cycling in transcript abundance. Expressing a translational fusion of ZmSUT2 fused to a red fluorescent protein in maize mesophyll cell protoplasts revealed that the protein localized to the tonoplast. Under field conditions, zmsut2 mutant plants grew slower, possessed smaller tassels and ears, and produced fewer kernels when compared to wild-type siblings. zmsut2 mutants also accumulated two-fold more sucrose, glucose, and fructose as well as starch in source leaves compared to wild type. These findings suggest (i) ZmSUT2 functions to remobilize sucrose out of the vacuole for subsequent use in growing tissues; and (ii) its function provides an important contribution to maize development and agronomic yield.

Cooperative action of the paralogous maize lateral organ boundaries (LOB) domain proteins RTCS and RTCL in shoot-borne root formation.

The paralogous maize (Zea mays) LBD (Lateral Organ Boundaries Domain) genes rtcs (rootless concerning crown and seminal roots) and rtcl (rtcs-like) emerged from an ancient whole-genome duplication. RTCS is a key regulator of crown root initiation. The diversity of expression, molecular interaction and phenotype of rtcs and rtcl were investigated. The rtcs and rtcl genes display highly correlated spatio-temporal expression patterns in roots, despite the significantly higher expression of rtcs. Both RTCS and RTCL proteins bind to LBD downstream promoters and act as transcription factors. In line with its auxin inducibility and binding to auxin response elements of rtcs and rtcl promoters, ARF34 (AUXIN RESPONSE FACTOR 34) acts as transcriptional activator. Yeast two-hybrid screening combined with bimolecular fluorescence complementation (BiFC) experiments revealed conserved and unique interaction partners of RTCS and RTCL. The rtcl mutation leads to defective shoot-borne root elongation early in development. Cooperative action of RTCS and RTCL during shoot-borne root formation was demonstrated by rtcs-dependent repression of rtcl transcription in coleoptilar nodes. Although RTCS is instrumental in shoot-borne root initiation, RTCL controls shoot-borne root elongation early in development. Their conserved role in auxin signaling, but diverse function in shoot-borne root formation, is underscored by their conserved and unique interaction partners.

A putative plant organelle RNA recognition protein gene is essential for maize kernel development.

Basal endosperm transfer layer (BETL) cells are responsible for transferring apoplastic solutes from the maternal pedicel into the endosperm, supplying the grain with compounds required for embryo development and storage reserve accumulation. Here, we analyze the maize (Zea mays L.) empty pericarp6 (emp6) mutant, which causes early arrest in grain development. The Emp6+gene function is required independently in both the embryo and endosperm. The emp6 mutant causes a notable effect on the differentiation of BETL cells; the extensive cell wall ingrowths that distinguish BETL cells are diminished and BETL marker gene expression is compromised in mutant kernels. Transposon tagging identified the emp6 locus as encoding a putative plant organelle RNA recognition (PORR) protein, 1 of 15 PORR family members in maize. The emp6 transcript is widely detected in plant tissues with highest levels in embryos and developing kernels. EMP6-green fluorescent protein (GFP) fusion proteins transiently expressed in Nicotiana benthamiana leaves were targeted specifically to mitochondria. These results suggest that BETL cell differentiation might be particularly energy intensive, or alternatively, that mitochondria might confer a developmental function.

Genetic perturbation of the maize methylome.

DNA methylation can play important roles in the regulation of transposable elements and genes. A collection of mutant alleles for 11 maize (Zea mays) genes predicted to play roles in controlling DNA methylation were isolated through forward- or reverse-genetic approaches. Low-coverage whole-genome bisulfite sequencing and high-coverage sequence-capture bisulfite sequencing were applied to mutant lines to determine context- and locus-specific effects of these mutations on DNA methylation profiles. Plants containing mutant alleles for components of the RNA-directed DNA methylation pathway exhibit loss of CHH methylation at many loci as well as CG and CHG methylation at a small number of loci. Plants containing loss-of-function alleles for chromomethylase (CMT) genes exhibit strong genome-wide reductions in CHG methylation and some locus-specific loss of CHH methylation. In an attempt to identify stocks with stronger reductions in DNA methylation levels than provided by single gene mutations, we performed crosses to create double mutants for the maize CMT3 orthologs, Zmet2 and Zmet5, and for the maize DDM1 orthologs, Chr101 and Chr106. While loss-of-function alleles are viable as single gene mutants, the double mutants were not recovered, suggesting that severe perturbations of the maize methylome may have stronger deleterious phenotypic effects than in Arabidopsis thaliana.

A pair of homoeolog ClpP5 genes underlies a virescent yellow-like mutant and its modifier in maize.

Gene-background interaction is a commonly observed phenomenon in many species, but the molecular mechanisms of such an interaction is less well understood. Here we report the cloning of a maize mutant gene and its modifier. A recessive mutant with a virescent yellow-like (vyl) phenotype was identified in an ethyl methanesulfonate-mutagenized population derived from the maize inbred line B73. Homozygous mutant maize plants exhibited a yellow leaf phenotype after emergence but gradually recovered and became indistinguishable from wild-type plants after approximately 2 weeks. Taking the positional cloning approach, the Chr.9_ClpP5 gene, one of the proteolytic subunits of the chloroplast Clp protease complex, was identified and validated as the candidate gene for vyl. When introgressed by backcross into the maize inbred line PH09B, the mutant phenotype of vyl lasted much longer in the greenhouse and was lethal in the field, implying the presence of a modifier(s) for vyl. A major modifier locus was identified on chromosome 1, and a paralogous ClpP5 gene was isolated and confirmed as the candidate for the vyl-modifier. Expression of Chr.1_ClpP5 is induced significantly in B73 by the vyl mutation, while the expression of Chr.1_ClpP5 in PH09B is not responsive to the vyl mutation. Moreover, expression and sequence analysis suggests that the PH09B Chr.1_ClpP5 allele is functionally weaker than the B73 allele. We propose that functional redundancy between duplicated paralogous genes is the molecular mechanism for the interaction between vyl and its modifier.

Roothairless5, which functions in maize (Zea mays L.) root hair initiation and elongation encodes a monocot-specific NADPH oxidase.

Root hairs are instrumental for nutrient uptake in monocot cereals. The maize (Zea mays L.) roothairless5 (rth5) mutant displays defects in root hair initiation and elongation manifested by a reduced density and length of root hairs. Map-based cloning revealed that the rth5 gene encodes a monocot-specific NADPH oxidase. RNA-Seq, in situ hybridization and qRT-PCR experiments demonstrated that the rth5 gene displays preferential expression in root hairs but also accumulates to low levels in other tissues. Immunolocalization detected RTH5 proteins in the epidermis of the elongation and differentiation zone of primary roots. Because superoxide and hydrogen peroxide levels are reduced in the tips of growing rth5 mutant root hairs as compared with wild-type, and Reactive oxygen species (ROS) is known to be involved in tip growth, we hypothesize that the RTH5 protein is responsible for establishing the high levels of ROS in the tips of growing root hairs required for elongation. Consistent with this hypothesis, a comparative RNA-Seq analysis of 6-day-old rth5 versus wild-type primary roots revealed significant over-representation of only two gene ontology (GO) classes related to the biological functions (i.e. oxidation/reduction and carbohydrate metabolism) among 893 differentially expressed genes (FDR <5%). Within these two classes the subgroups 'response to oxidative stress' and 'cellulose biosynthesis' were most prominently represented.

Genomic distribution of maize facultative heterochromatin marked by trimethylation of H3K27.

Trimethylation of histone H3 Lys-27 (H3K27me3) plays a critical role in regulating gene expression during plant and animal development. We characterized the genome-wide distribution of H3K27me3 in five developmentally distinct tissues in maize (Zea mays) plants of two genetic backgrounds, B73 and Mo17. There were more substantial differences in the genome-wide profile of H3K27me3 between different tissues than between the two genotypes. The tissue-specific patterns of H3K27me3 were often associated with differences in gene expression among the tissues and most of the imprinted genes that are expressed solely from the paternal allele in endosperm are targets of H3K27me3. A comparison of the H3K27me3 targets in rice (Oryza sativa), maize, and Arabidopsis thaliana provided evidence for conservation of the H3K27me3 targets among plant species. However, there was limited evidence for conserved targeting of H3K27me3 in the two maize subgenomes derived from whole-genome duplication, suggesting the potential for subfunctionalization of chromatin regulation of paralogs. Genomic profiling of H3K27me3 in loss-of-function mutant lines for Maize Enhancer of zeste-like2 (Mez2) and Mez3, two of the three putative H3K27me3 methyltransferases present in the maize genome, suggested partial redundancy of this gene family for maintaining H3K27me3 patterns. Only a portion of the targets of H3K27me3 required Mez2 and/or Mez3, and there was limited evidence for functional consequences of H3K27me3 at these targets.

ROP GTPases act with the receptor-like protein PAN1 to polarize asymmetric cell division in maize.

Plant Rho family GTPases (ROPs) have been investigated primarily for their functions in polarized cell growth. We previously showed that the maize (Zea mays) Leu-rich repeat receptor-like protein PANGLOSS1 (PAN1) promotes the polarization of asymmetric subsidiary mother cell (SMC) divisions during stomatal development. Here, we show that maize Type I ROPs 2 and 9 function together with PAN1 in this process. Partial loss of ROP2/9 function causes a weak SMC division polarity phenotype and strongly enhances this phenotype in pan1 mutants. Like PAN1, ROPs accumulate in an asymmetric manner in SMCs. Overexpression of yellow fluorescent protein-ROP2 is associated with its delocalization in SMCs and with aberrantly oriented SMC divisions. Polarized localization of ROPs depends on PAN1, but PAN1 localization is insensitive to depletion and depolarization of ROP. Membrane-associated Type I ROPs display increased nonionic detergent solubility in pan1 mutants, suggesting a role for PAN1 in membrane partitioning of ROPs. Finally, endogenous PAN1 and ROP proteins are physically associated with each other in maize tissue extracts, as demonstrated by reciprocal coimmunoprecipitation experiments. This study demonstrates that ROPs play a key role in polarization of plant cell division and cell growth and reveals a role for a receptor-like protein in spatial localization of ROPs.

Production of viable gametes without meiosis in maize deficient for an ARGONAUTE protein.

Apomixis is a form of asexual reproduction through seeds in angiosperms. Apomictic plants bypass meiosis and fertilization, developing offspring that are genetically identical to their mother. In a genetic screen for maize (Zea mays) mutants mimicking aspects of apomixis, we identified a dominant mutation resulting in the formation of functional unreduced gametes. The mutant shows defects in chromatin condensation during meiosis and subsequent failure to segregate chromosomes. The mutated locus codes for AGO104, a member of the ARGONAUTE family of proteins. AGO104 accumulates specifically in somatic cells surrounding the female meiocyte, suggesting a mobile signal rather than cell-autonomous control. AGO104 is necessary for non-CG methylation of centromeric and knob-repeat DNA. Digital gene expression tag profiling experiments using high-throughput sequencing show that AGO104 influences the transcription of many targets in the ovaries, with a strong effect on centromeric repeats. AGO104 is related to Arabidopsis thaliana AGO9, but while AGO9 acts to repress germ cell fate in somatic tissues, AGO104 acts to repress somatic fate in germ cells. Our findings show that female germ cell development in maize is dependent upon conserved small RNA pathways acting non-cell-autonomously in the ovule. Interfering with this repression leads to apomixis-like phenotypes in maize.

Identification and characterization of the maize arogenate dehydrogenase gene family.

In plants, the amino acids tyrosine and phenylalanine are synthesized from arogenate by arogenate dehydrogenase and arogenate dehydratase, respectively, with the relative flux to each being tightly controlled. Here the characterization of a maize opaque endosperm mutant (mto140), which also shows retarded vegetative growth, is described The opaque phenotype co-segregates with a Mutator transposon insertion in an arogenate dehydrogenase gene (zmAroDH-1) and this led to the characterization of the four-member family of maize arogenate dehydrogenase genes (zmAroDH-1-zmAroDH-4) which share highly similar sequences. A Mutator insertion at an equivalent position in AroDH-3, the most closely related family member to AroDH-1, is also associated with opaque endosperm and stunted vegetative growth phenotypes. Overlapping but differential expression patterns as well as subtle mutant effects on the accumulation of tyrosine and phenylalanine in endosperm, embryo, and leaf tissues suggest that the functional redundancy of this gene family provides metabolic plasticity for the synthesis of these important amino acids. mto140/arodh-1 seeds shows a general reduction in zein storage protein accumulation and an elevated lysine phenotype typical of other opaque endosperm mutants, but it is distinct because it does not result from quantitative or qualitative defects in the accumulation of specific zeins but rather from a disruption in amino acid biosynthesis.

The HD-ZIP IV transcription factor OCL4 is necessary for trichome patterning and anther development in maize.

Among the genes controlling the differentiation and maintenance of epidermal cell fate are members of the HD-ZIP IV class family of plant-specific transcription factors, most of which are specifically expressed in the epidermis of tissues. Here, we report the functional analysis of the maize HD-ZIP IV gene OCL4 (outer cell layer 4) via the phenotypic analysis of two insertional mutants, and of OCL4-RNAi transgenic plants. In all three materials, the macrohairs, one of the three types of trichomes present on adult maize leaf blades, developed ectopically at the margin of juvenile and adult leaves. Consistent with this phenotype, OCL4 is expressed in the epidermis of the leaf blade, with a maximum at the margin of young leaf primordia. Expression of OCL4 in the model plant Arabidopsis under the control of the GLABRA2 (GL2) promoter, a member of the Arabidopsis HD-ZIP IV family involved in trichome differentiation, did not complement the gl2-1 mutant, but instead aggravated its phenotype. The construct also caused a glabrous appearance of rosette leaves in transformed control plants of the Ler ecotype, suggesting that OCL4 inhibits trichome development both in maize and Arabidopsis. Furthermore, insertional mutants showed a partial male sterility that is likely to result from the presence of an extra subepidermal cell layer with endothecium characteristics in the anther wall. Interestingly, the epidermis-specific OCL4 expression in immature anthers was restricted to the region of the anther locule where the extra cell layer differentiated. Taken together these results suggest that OCL4 inhibits trichome development and influences division and/or differentiation of the anther cell wall.

Disruption of imprinting by mutator transposon insertions in the 5' proximal regions of the Zea mays Mez1 locus.

Imprinting is a form of epigenetic gene regulation in which alleles are differentially regulated according to the parent of origin. The Mez1 gene in maize is imprinted such that the maternal allele is expressed in the endosperm while the paternal allele is not expressed. Three novel Mez1 alleles containing Mutator transposon insertions within the promoter were identified. These mez1-mu alleles do not affect vegetative expression levels or result in morphological phenotypes. However, these alleles can disrupt imprinted expression of Mez1. Maternal inheritance of the mez-m1 or mez1-m4 alleles results in activation of the normally silenced paternal allele of Mez1. Paternal inheritance of the mez1-m2 or mez1-m4 alleles can also result in a loss of silencing of the paternal Mez1 allele. The paternal disruption of imprinting by transposon insertions may reflect a requirement for sequence elements involved in targeting silencing of the paternal allele. The maternal disruption of imprinting by transposon insertions within the Mez1 promoter suggests that maternally produced MEZ1 protein may be involved in silencing of the paternal Mez1 allele. The endosperms with impaired imprinting did not exhibit phenotypic consequences associated with bi-allelic Mez1 expression.

Embryo-specific silencing of a transporter reduces phytic acid content of maize and soybean seeds.

Phytic acid in cereal grains and oilseeds is poorly digested by monogastric animals and negatively affects animal nutrition and the environment. However, breeding programs involving mutants with less phytic acid and more inorganic phosphate (P(i)) have been frustrated by undesirable agronomic characteristics associated with the phytic acid-reducing mutations. We show that maize lpa1 mutants are defective in a multidrug resistance-associated protein (MRP) ATP-binding cassette (ABC) transporter that is expressed most highly in embryos, but also in immature endosperm, germinating seed and vegetative tissues. Silencing expression of this transporter in an embryo-specific manner produced low-phytic-acid, high-Pi transgenic maize seeds that germinate normally and do not show any significant reduction in seed dry weight. This dominant transgenic approach obviates the need for incorporating recessive lpa1 mutations to create maize hybrids with reduced phytic acid. Suppressing the homologous soybean MRP gene also generated low-phytic-acid seed, suggesting that the strategy might be feasible for many crops.

The maize floury1 gene encodes a novel endoplasmic reticulum protein involved in zein protein body formation.

The maize (Zea mays) floury1 (fl1) mutant was first reported almost 100 years ago, but its molecular identity has remained unknown. We report the cloning of Fl1, which encodes a novel zein protein body membrane protein with three predicted transmembrane domains and a C-terminal plant-specific domain of unknown function (DUF593). In wild-type endosperm, the FL1 protein accumulates at a high level during the period of zein synthesis and protein body development and declines to a low level at kernel maturity. Immunogold labeling showed that FL1 resides in the endoplasmic reticulum surrounding the protein body. Zein protein bodies in fl1 mutants are of normal size, shape, and abundance. However, mutant protein bodies ectopically accumulate 22-kD alpha-zeins in the gamma-zein-rich periphery and center of the core, rather than their normal discrete location in a ring at outer edge of the core. The 19-kD alpha-zein is uniformly distributed throughout the core in wild-type protein bodies, and this distribution is unaffected in fl1 mutants. Pairwise yeast two-hybrid experiments showed that FL1 DUF593 interacts with the 22-kD alpha-zein. Results of these studies suggest that FL1 participates in protein body formation by facilitating the localization of 22-kD alpha-zein and that this is essential for the formation of vitreous endosperm.

Functional analysis of maize RAD51 in meiosis and double-strand break repair.

In Saccharomyces cerevisiae, Rad51p plays a central role in homologous recombination and the repair of double-strand breaks (DSBs). Double mutants of the two Zea mays L. (maize) rad51 homologs are viable and develop well under normal conditions, but are male sterile and have substantially reduced seed set. Light microscopic analyses of male meiosis in these plants reveal reduced homologous pairing, synapsis of nonhomologous chromosomes, reduced bivalents at diakinesis, numerous chromosome breaks at anaphase I, and that >33% of quartets carry cells that either lack an organized nucleolus or have two nucleoli. This indicates that RAD51 is required for efficient chromosome pairing and its absence results in nonhomologous pairing and synapsis. These phenotypes differ from those of an Arabidopsis rad51 mutant that exhibits completely disrupted chromosome pairing and synapsis during meiosis. Unexpectedly, surviving female gametes produced by maize rad51 double mutants are euploid and exhibit near-normal rates of meiotic crossovers. The finding that maize rad51 double mutant embryos are extremely susceptible to radiation-induced DSBs demonstrates a conserved role for RAD51 in the repair of mitotic DSBs in plants, vertebrates, and yeast.