Recruitment of an ancient branching program to suppress carpel development in maize flowers.

Carpels in maize undergo programmed cell death in half of the flowers initiated in ears and in all flowers in tassels. The HD-ZIP I transcription factor gene GRASSY TILLERS1 (GT1) is one of only a few genes known to regulate this process. To identify additional regulators of carpel suppression, we performed a gt1 enhancer screen and found a genetic interaction between gt1 and ramosa3 (ra3). RA3 is a classic inflorescence meristem determinacy gene that encodes a trehalose-6-phosphate (T6P) phosphatase (TPP). Dissection of floral development revealed that ra3 single mutants have partially derepressed carpels, whereas gt1;ra3 double mutants have completely derepressed carpels. Surprisingly, gt1 suppresses ra3 inflorescence branching, revealing a role for gt1 in meristem determinacy. Supporting these genetic interactions, GT1 and RA3 proteins colocalize to carpel nuclei in developing flowers. Global expression profiling revealed common genes misregulated in single and double mutant flowers, as well as in derepressed gt1 axillary meristems. Indeed, we found that ra3 enhances gt1 vegetative branching, similar to the roles for the trehalose pathway and GT1 homologs in the eudicots. This functional conservation over ∼160 million years of evolution reveals ancient roles for GT1-like genes and the trehalose pathway in regulating axillary meristem suppression, later recruited to mediate carpel suppression. Our findings expose hidden pleiotropy of classic maize genes and show how an ancient developmental program was redeployed to sculpt floral form.

Necrotic upper tips1 mimics heat and drought stress and encodes a protoxylem-specific transcription factor in maize.

Maintaining sufficient water transport during flowering is essential for proper organ growth, fertilization, and yield. Water deficits that coincide with flowering result in leaf wilting, necrosis, tassel browning, and sterility, a stress condition known as "tassel blasting." We identified a mutant, necrotic upper tips1 (nut1), that mimics tassel blasting and drought stress and reveals the genetic mechanisms underlying these processes. The nut1 phenotype is evident only after the floral transition, and the mutants have difficulty moving water as shown by dye uptake and movement assays. These defects are correlated with reduced protoxylem vessel thickness that indirectly affects metaxylem cell wall integrity and function in the mutant. nut1 is caused by an Ac transposon insertion into the coding region of a unique NAC transcription factor within the VND clade of Arabidopsis NUT1 localizes to the developing protoxylem of root, stem, and leaf sheath, but not metaxylem, and its expression is induced by flowering. NUT1 downstream target genes function in cell wall biosynthesis, apoptosis, and maintenance of xylem cell wall thickness and strength. These results show that maintaining protoxylem vessel integrity during periods of high water movement requires the expression of specialized, dynamically regulated transcription factors within the vasculature.

Understanding Grass Domestication through Maize Mutants.

As an economically important model crop plant, rich in genetic resources, maize has been useful for uncovering the genetic pathways responsible for domestication and plant improvement. However, several of the pathways that have been shown by recent studies to be important for domestication and/or yield in other grasses function differently in maize. In several cases, this unexpectedly wide functional divergence between genes from closely related grasses appears to be due to alternative modes of regulation rather than to simple differences in protein function. This indicates that domestication genes need to be understood within the architecture of the whole genome and the species-specific processes that they influence before they can serve as the foundation to improve plants.

microRNA172 plays a crucial role in wheat spike morphogenesis and grain threshability.

Wheat domestication from wild species involved mutations in the Q gene. The q allele (wild wheats) is associated with elongated spikes and hulled grains, whereas the mutant Q allele (domesticated wheats) confers subcompact spikes and free-threshing grains. Previous studies showed that Q encodes an AP2-like transcription factor, but the causal polymorphism of the domestication traits remained unclear. Here, we show that the interaction between microRNA172 (miR172) and the Q allele is reduced by a single nucleotide polymorphism in the miRNA binding site. Inhibition of miR172 activity by a miRNA target mimic resulted in compact spikes and transition from glumes to florets in apical spikelets. By contrast, overexpression of miR172 was sufficient to induce elongated spikes and non-free-threshing grains, similar to those observed in three Q loss-of-function mutations. These lines showed transitions from florets to glumes in the basal spikelets. These localized homeotic changes were associated with opposing miR172/Q gradients along the spike. We propose that the selection of a nucleotide change at the miR172 binding site of Q contributed to subcompact spikes and free-threshing grains during wheat domestication.

Ideal crop plant architecture is mediated by tassels replace upper ears1, a BTB/POZ ankyrin repeat gene directly targeted by TEOSINTE BRANCHED1.

Axillary branch suppression is a favorable trait bred into many domesticated crop plants including maize compared with its highly branched wild ancestor teosinte. Branch suppression in maize was achieved through selection of a gain of function allele of the teosinte branched1 (tb1) transcription factor that acts as a repressor of axillary bud growth. Previous work indicated that other loci may function epistatically with tb1 and may be responsible for some of its phenotypic effects. Here, we show that tb1 mediates axillary branch suppression through direct activation of the tassels replace upper ears1 (tru1) gene that encodes an ankyrin repeat domain protein containing a BTB/POZ motif necessary for protein-protein interactions. The expression of TRU1 and TB1 overlap in axillary buds, and TB1 binds to two locations in the tru1 gene as shown by chromatin immunoprecipitation and gel shifts. In addition, nucleotide diversity surveys indicate that tru1, like tb1, was a target of selection. In modern maize, TRU1 is highly expressed in the leaf trace vasculature of axillary internodes, while in teosinte, this expression is highly reduced or absent. This increase in TRU1 expression levels in modern maize is supported by comparisons of relative protein levels with teosinte as well as by quantitative measurements of mRNA levels. Hence, a major innovation in creating ideal maize plant architecture originated from ectopic overexpression of tru1 in axillary branches, a critical step in mediating the effects of domestication by tb1.

Maize SBP-box transcription factors unbranched2 and unbranched3 affect yield traits by regulating the rate of lateral primordia initiation.

The separation of male and female flowers in maize provides the potential for independent regulation of traits that affect crop productivity. For example, tassel branch number controls pollen abundance and length of shedding time, whereas ear row number directly affects kernel yield. Mutations in duplicate SBP-box transcription factor genes unbranched2 (ub2) and ub3 affect both of these yield traits. Double mutants display a decrease in tassel branch number and an increase in ear row number, both of which are enhanced by loss of a related gene called tasselsheath4 (tsh4). Furthermore, triple mutants have more tillers and leaves-phenotypes seen in Corngrass1 mutants that result from widespread repression of SBP-box genes. Immunolocalization of UB2 and UB3 proteins revealed accumulation throughout the meristem but absence from the central domain of the meristem where cells regenerate. Thus, ub2, ub3, and tsh4 function as redundant factors that limit the rate of cell differentiation to the lateral domains of meristems. When these genes are mutated, cells are allocated to lateral primordia at a higher rate, causing a net loss of cells from the central domain and premature termination of the inflorescence. The ub3 locus is tightly linked to quantitative trait loci (QTL) for ear row number and tassel branch number in both the nested association mapping (NAM) and intermated B73 by Mo17 (IBM) populations of maize recombinant inbreds, indicating that this gene may be agronomically important. Analysis of ear and tassel QTL across biparental families suggests that multiple mutations in ub3 independently regulate male and female inflorescence development.

The heterochronic maize mutant Corngrass1 results from overexpression of a tandem microRNA.

Retention of juvenile traits in the adult reproductive phase characterizes a process known as neoteny, and speculation exists over whether it has contributed to the evolution of new species. The dominant Corngrass1 (Cg1) mutant of maize is a neotenic mutation that results in phenotypes that may be present in the grass-like ancestors of maize. We cloned Cg1 and found that it encodes two tandem miR156 genes that are overexpressed in the meristem and lateral organs. Furthermore, a target of Cg1 is teosinte glume architecture1 (tga1), a gene known to have had a role in the domestication of maize from teosinte. Cg1 mutant plants overexpressing miR156 have lower levels of mir172, a microRNA that targets genes controlling juvenile development. By altering the relative levels of both microRNAs, it is possible to either prolong or shorten juvenile development in maize, thus providing a mechanism for how species-level heterochronic changes can occur in nature.

Brassinosteroid control of sex determination in maize.

Brassinosteroids (BRs) are plant hormones that regulate growth and development. They share structural similarities with animal steroids, which are decisive factors of sex determination. BRs are known to regulate morphogenesis and environmental stress responses, but their involvement in sex determination in plants has been only speculative. We show that BRs control sex determination in maize revealed through characterization of the classical dwarf mutant nana plant1 (na1), which also feminizes male flowers. na1 plants carry a loss-of-function mutation in a DET2 homolog--a gene in the BR biosynthetic pathway. The mutant accumulates the DET2-specific substrate (24R)-24-methylcholest-4-en-3-one with a concomitant decrease of downstream BR metabolites. Treatment of wild-type maize plants with BR biosynthesis inhibitors completely mimicked both dwarf and tasselseed phenotypes of na1 mutants. Tissue-specific na1 expression in anthers throughout their development supports the hypothesis that BRs promote masculinity of the male inflorescence. These findings suggest that, in the monoecious plant maize, BRs have been coopted to perform a sex determination function not found in plants with bisexual flowers.

Overexpression of the maize Corngrass1 microRNA prevents flowering, improves digestibility, and increases starch content of switchgrass.

Biofuels developed from biomass crops have the potential to supply a significant portion of our transportation fuel needs. To achieve this potential, however, it will be necessary to develop improved plant germplasm specifically tailored to serve as energy crops. Liquid transportation fuel can be created from the sugars locked inside plant cell walls. Unfortunately, these sugars are inherently resistant to hydrolytic release because they are contained in polysaccharides embedded in lignin. Overcoming this obstacle is a major objective toward developing sustainable bioenergy crop plants. The maize Corngrass1 (Cg1) gene encodes a microRNA that promotes juvenile cell wall identities and morphology. To test the hypothesis that juvenile biomass has superior qualities as a potential biofuel feedstock, the Cg1 gene was transferred into several other plants, including the bioenergy crop Panicum virgatum (switchgrass). Such plants were found to have up to 250% more starch, resulting in higher glucose release from saccharification assays with or without biomass pretreatment. In addition, a complete inhibition of flowering was observed in both greenhouse and field grown plants. These results point to the potential utility of this approach, both for the domestication of new biofuel crops, and for the limitation of transgene flow into native plant species.

Transcription factor binding site divergence across maize inbred lines drives transcriptional and phenotypic variation.

Regulatory elements are important constituents of plant genomes that have shaped ancient and modern crops. Their identification, function, and diversity in crop genomes however are poorly characterized, thus limiting our ability to harness their power for further agricultural advances using induced or natural variation. Here, we use DNA affinity purification-sequencing (DAP-seq) to map transcription factor (TF) binding events for 200 maize TFs belonging to 30 distinct families and heterodimer pairs in two distinct inbred lines historically used for maize hybrid plant production, providing empirical binding site annotation for 5.3% of the maize genome. TF binding site comparison in B73 and Mo17 inbreds reveals widespread differences, driven largely by structural variation, that correlate with gene expression changes. TF binding site presence-absence variation helps clarify complex QTL such as vgt1, an important determinant of maize flowering time, and DICE, a distal enhancer involved in herbivore resistance. Modification of TF binding regions via CRISPR-Cas9 mediated editing alters target gene expression and phenotype. Our functional catalog of maize TF binding events enables collective and comparative TF binding analysis, and highlights its value for agricultural improvement.

The maize SBP-box transcription factor encoded by tasselsheath4 regulates bract development and the establishment of meristem boundaries.

Plant architecture consists of repeating units called phytomers, each containing an internode, leaf and axillary meristem. The formation of boundaries within the phytomer is necessary to differentiate and separate these three components, otherwise some will grow at the expense of others. The microRNA-targeted SBP-box transcription factor tasselsheath4 (tsh4) plays an essential role in establishing these boundaries within the inflorescence. tsh4 mutants display altered phyllotaxy, fewer lateral meristems and ectopic leaves that grow at the expense of the meristem. Double-mutant analyses of tsh4 and several highly branched mutants, such as ramosa1-3 and branched silkless1, demonstrated a requirement for tsh4 in branch meristem initiation and maintenance. TSH4 protein, however, was localized throughout the inflorescence stem and at the base of lateral meristems, but not within the meristem itself. Double labeling of TSH4 with the ramosa2, branched silkless1 and knotted1 meristem markers confirmed that TSH4 forms a boundary adjacent to all lateral meristems. Indeed, double labeling of miR156 showed a meristem-specific pattern complementary to that of TSH4, consistent with tsh4 being negatively regulated by this microRNA. Thus, downregulation of TSH4 by a combination of microRNAs and branching pathway genes allows the establishment of lateral meristems and the repression of leaf initiation, thereby playing a major role in defining meristem versus leaf boundaries.

Boundary domain genes were recruited to suppress bract growth and promote branching in maize.

Grass inflorescence development is diverse and complex and involves sophisticated but poorly understood interactions of genes regulating branch determinacy and leaf growth. Here, we use a combination of transcript profiling and genetic and phylogenetic analyses to investigate tasselsheath1 (tsh1) and tsh4, two maize genes that simultaneously suppress inflorescence leaf growth and promote branching. We identify a regulatory network of inflorescence leaf suppression that involves the phase change gene tsh4 upstream of tsh1 and the ligule identity gene liguleless2 (lg2). We also find that a series of duplications in the tsh1 gene lineage facilitated its shift from boundary domain in nongrasses to suppressed inflorescence leaves of grasses. Collectively, these results suggest that the boundary domain genes tsh1 and lg2 were recruited to inflorescence leaves where they suppress growth and regulate a nonautonomous signaling center that promotes inflorescence branching, an important component of yield in cereal grasses.

From organelle to organ: ZRIZI MATE-Type transporter is an organelle transporter that enhances organ initiation.

Plant architecture is a predictable but flexible trait. The timing and position of organ initiation from the shoot apical meristem (SAM) contribute to the final plant form. While much progress has been made recently in understanding how the site of leaf initiation is determined, the mechanism underlying the temporal interval between leaf primordia is still largely unknown. The Arabidopsis ZRIZI (ZRZ) gene belongs to a large gene family encoding multidrug and toxic compound extrusion (MATE) transporters. Unique among plant MATE transporters identified so far, ZRZ is localized to the membrane of a small organelle, possibly the mitochondria. Plants overexpressing ZRZ in initiating leaves are short, produce leaves much faster than wild-type plants and show enhanced growth of axillary buds. These results suggest that ZRZ is involved in communicating a leaf-borne signal that determines the rate of organ initiation.

The regulatory landscape of a core maize domestication module controlling bud dormancy and growth repression.

Many domesticated crop plants have been bred for increased apical dominance, displaying greatly reduced axillary branching compared to their wild ancestors. In maize, this was achieved through selection for a gain-of-function allele of the TCP transcription factor teosinte branched1 (tb1). The mechanism for how a dominant Tb1 allele increased apical dominance, is unknown. Through ChIP seq, RNA seq, hormone and sugar measurements on 1 mm axillary bud tissue, we identify the genetic pathways putatively regulated by TB1. These include pathways regulating phytohormones such as gibberellins, abscisic acid and jasmonic acid, but surprisingly, not auxin. In addition, metabolites involved in sugar sensing such as trehalose 6-phosphate were increased. This suggests that TB1 induces bud suppression through the production of inhibitory phytohormones and by reducing sugar levels and energy balance. Interestingly, TB1 also putatively targets several other domestication loci, including teosinte glume architecture1, prol1.1/grassy tillers1, as well as itself. This places tb1 on top of the domestication hierarchy, demonstrating its critical importance during the domestication of maize from teosinte.

Regulation of developmental transitions.

Plants undergo a series of profound developmental changes throughout their lifetimes in response to both external environmental factors and internal intrinsic ones. When these changes are abrupt and dramatic, the process is referred to as phase change. Recently, several genes have been discovered that play a role in these developmental transitions. Their sequence and expression patterns shed new light on the mechanisms of phase change, and provide a link between the external and internal factors that control them. Examples of these transitions include changes from juvenile to adult leaf formation, vegetative to inflorescence meristem development, and inflorescence to floral meristem initiation.

Small RNAs going the distance during plant development.

Small RNAs are 19-27 nucleotide long RNAs that negatively regulate gene expression and function as important regulators of diverse aspects of plant development. Current models for how they function continue to be modified as new research uncovers additional aspects of their biology. Unexpectedly, several previously characterized small RNAs appear to function non-cell autonomously, some moving a few cells away, others moving throughout the plant. This fact may reflect that small RNAs are an essential component of a larger signaling network that orchestrates plant development.

The unique relationship between tsh4 and ra2 in patterning floral phytomers.

Phytomers are developmental compartments that display stereotypical patterns dependent on whether they are initiated during the vegetative phase or the floral phases. Differences in appearance result from differential partitioning mechanisms responsible for allocation of cells to different components of the phytomer. The tasselsheath loci of maize control cell partitioning within the phytomer, indirectly influencing growth and development of its individual components. The tasselsheath4 (tsh4) gene accomplishes this through regulation of the ramosa2 (ra2) meristem determinacy gene, whereas tasselsheath1 (tsh1) appears to function differently.

Big impacts by small RNAs in plant development.

The identification and study of small RNAs, including microRNAs and trans-acting small interfering RNAs, have added a layer of complexity to the many pathways that regulate plant development. These molecules, which function as negative regulators of gene expression, are now known to have greatly expanded roles in a variety of developmental processes affecting all major plant structures, including meristems, leaves, roots, and inflorescences. Mutants with specific developmental phenotypes have also advanced our knowledge of the biogenesis and mode of action of these diverse small RNAs. In addition, previous models on the cell autonomy of microRNAs may have to be revised as more data accumulate supporting their long distance transport. As many of these small RNAs appear to be conserved across different species, knowledge gained from one species is expected to have general application. However, a few surprising differences in small RNA function seem to exist between monocots and dicots regarding meristem initiation and sex determination. Integrating these unique functions into the overall scheme for plant growth will give a more complete picture of how they have evolved as unique developmental systems.

Floral meristem initiation and meristem cell fate are regulated by the maize AP2 genes ids1 and sid1.

Grass flowers are organized on small branches known as spikelets. In maize, the spikelet meristem is determinate, producing one floral meristem and then converting into a second floral meristem. The APETALA2 (AP2)-like gene indeterminate spikelet1 (ids1) is required for the timely conversion of the spikelet meristem into the floral meristem. Ectopic expression of ids1 in the tassel, resulting from a failure of regulation by the tasselseed4 microRNA, causes feminization and the formation of extra floral meristems. Here we show that ids1 and the related gene, sister of indeterminate spikelet1 (sid1), play multiple roles in inflorescence architecture in maize. Both genes are needed for branching of the inflorescence meristem, to initiate floral meristems and to control spikelet meristem determinacy. We show that reducing the levels of ids1 and sid1 fully suppresses the tasselseed4 phenotype, suggesting that these genes are major targets of this microRNA. Finally, sid1 and ids1 repress AGAMOUS-like MADS-box transcription factors within the lateral organs of the spikelet, similar to the function of AP2 in Arabidopsis, where it is required for floral organ fate. Thus, although the targets of the AP2 genes are conserved between maize and Arabidopsis, the genes themselves have adopted novel meristem functions in monocots.

The maize tasselseed4 microRNA controls sex determination and meristem cell fate by targeting Tasselseed6/indeterminate spikelet1.

In maize (Zea mays), sex determination occurs through abortion of female carpels in the tassel and arrest of male stamens in the ear. The Tasselseed6 (Ts6) and tasselseed4 (ts4) mutations permit carpel development in the tassel while increasing meristem branching, showing that sex determination and acquisition of meristem fate share a common pathway. We show that ts4 encodes a mir172 microRNA that targets APETALA2 floral homeotic transcription factors. Three lines of evidence suggest that indeterminate spikelet1 (ids1), an APETALA2 gene required for spikelet meristem determinacy, is a key target of ts4. First, loss of ids1 suppresses the ts4 sex determination and branching defects. Second, Ts6 mutants phenocopy ts4 and possess mutations in the microRNA binding site of ids1. Finally, IDS1 protein is expressed more broadly in ts4 mutants compared to wild type. Our results demonstrate that sexual identity in maize is acquired by limiting floral growth through negative regulation of the floral homeotic pathway.