George Chuck

KNAT1 induces lobed leaves with ectopic meristems when overexpressed in Arabidopsis.

Plant development depends on the activity of apical meristems, which are groups of indeterminate cells whose derivatives elaborate the organs of the mature plant. Studies of knotted1 (kn1) and related gene family members have determined potential roles for homeobox genes in the function of shoot meristems. The Arabidopsis kn1-like gene, KNAT1, is expressed in the shoot apical meristem and not in determinate organs. Here, we show that ectopic expression of KNAT1 in Arabidopsis transforms simple leaves into lobed leaves. The lobes initiate in the position of serrations yet have features of leaves, such as stipules, which form in the sinus, the region at the base of two lobes. Ectopic meristems also arise in the sinus region close to veins. Identity of the meristem, that is, vegetative or floral, depends on whether the meristem develops on a rosette or cauline leaf, respectively. Using in situ hybridization, we analyzed the expression of KNAT1 and another kn1-like homeobox gene, SHOOT MERISTEMLESS, in cauliflower mosaic virus 35S::KNAT1 transformants. KNAT1 expression is strong in vasculature, possibly explaining the proximity of the ectopic meristems to veins. After leaf cells have formed a layered meristem, SHOOT MERISTEMLESS expression begins in only a subset of these cells, demonstrating that KNAT1 is sufficient to induce meristems in the leaf. The shootlike features of the lobed leaves are consistent with the normal domain of KNAT1s expression and further suggest that kn1-related genes may have played a role in the evolution of leaf diversity.

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.

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.

ramosa2 Encodes a LATERAL ORGAN BOUNDARY Domain Protein That Determines the Fate of Stem Cells in Branch Meristems of Maize

Genetic control of grass inflorescence architecture is critical given that cereal seeds provide most of the worlds food. Seeds are borne on axillary branches, which arise from groups of stem cells in axils of leaves and whose branching patterns dictate most of the variation in plant form. Normal maize (Zea mays) ears are unbranched, and tassels have long branches only at their base. The ramosa2 (ra2) mutant of maize has increased branching with short branches replaced by long, indeterminate ones. ra2 was cloned by chromosome walking and shown to encode a LATERAL ORGAN BOUNDARY domain transcription factor. ra2 is transiently expressed in a group of cells that predicts the position of axillary meristem formation in inflorescences. Expression in different mutant backgrounds places ra2 upstream of other genes that regulate branch formation. The early expression of ra2 suggests that it functions in the patterning of stem cells in axillary meristems. Alignment of ra2-like sequences reveals a grass-specific domain in the C terminus that is not found in Arabidopsis thaliana. The ra2-dm allele suggests this domain is required for transcriptional activation of ra1. The ra2 expression pattern is conserved in rice (Oryza sativa), barley (Hordeum vulgare), sorghum (Sorghum bicolor), and maize, suggesting that ra2 is critical for shaping the initial steps of grass inflorescence architecture.

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.

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.

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.

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.

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.

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

Significance Crop yields are dependent on the number of lateral primordia made by the inflorescence. In maize unbranched mutants, excess lateral primordia are made at the expense of the stem cells located in the center of the meristem. Ultimately, the unbranched mutant meristem lacks enough cells to regenerate and thus, terminates prematurely. This study shows that the duplicate transcription factors unbranched2 and unbranched3 function together to decrease the rate of lateral primordia initiation, thus giving the stem cells of the meristem enough time to regenerate. Variants of the unbranched3 gene affect different aspects of lateral primordia initiation that control crop yield. 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.