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.

Global analysis of canola genes targeted by SHORT HYPOCOTYL UNDER BLUE 1 during endosperm and embryo development.

Seed development in dicots includes early endosperm proliferation followed by growth of the embryo to replace the endosperm. Endosperm proliferation in dicots not only provides nutrient supplies for subsequent embryo development but also enforces a space limitation, influencing final seed size. Overexpression of Arabidopsis SHORT HYPOCOTYL UNDER BLUE1::uidA (SHB1:uidA) in canola produces large seeds. We performed global analysis of the canola genes that were expressed and influenced by SHB1 during early endosperm proliferation at 8 days after pollination (DAP) and late embryo development at 13 DAP. Overexpression of SHB1 altered the expression of 973 genes at 8 DAP and 1035 genes at 13 DAP. We also surveyed the global SHB1 association sites, and merging of these sites with the RNA sequencing data identified a set of canola genes targeted by SHB1. The 8-DAP list includes positive and negative genes that influence endosperm proliferation and are homologous to Arabidopsis MINI3, IKU2, SHB1, AGL62, FIE and AP2. We revealed a major role for SHB1 in canola endosperm development based on the dynamics of SHB1-altered gene expression, the magnitude of SHB1 chromatin immunoprecipitation enrichment and the over-representation of eight regulatory genes for endosperm development. Our studies focus on an important agronomic trait in a major crop for global agriculture. The datasets on stage-specific and SHB1-induced gene expression and genes targeted by SHB1 also provide a useful resource in the field of endosperm development and seed size engineering. Our practices in an allotetraploid species will impact similar studies in other crop species.

SHORT HYPOCOTYL UNDER BLUE1 or HAIKU2 mixepression alters canola and Arabidopsis seed development.

Canola (Brassica napus) is a widely cultivated species and provides important resources of edible vegetable oil, biodiesel production and animal feed. Seed development in Arabidopsis and canola shares a similar path: an early proliferation of endosperm to form a large seed cavity, followed by a second phase in which the embryo grows to replace the endosperm. In Arabidopsis, the seed reaches almost its final volume before the enlargement of the embryo. SHORT HYPOCOTYL UNDER BLUE1 (SHB1) is a key regulatory gene of seed development with a broad expression beyond endosperm development. By contrast, its two target genes, MINISEED3 (MINI3) and HAIKU2 (IKU2), are narrowly expressed in early developing endosperm and early embryo. We overexpressed SHB1 in canola to explore the possibility of altering seed development. As an alternative strategy, we expressed the canola IKU2 ortholog in Arabidopsis endosperm under the control of a stronger MINI3 promoter. SHB1 targeted canola orthologs of Arabidopsis MINI3 and IKU2 and caused a significantly increased seed mass. Overaccumulation of IKU2 in the early stage of Arabidopsis seed development also significantly increased the final seed mass. Our studies provide a strong case for increasing the final seed mass by manipulating endosperm proliferation at a rather early developmental stage in crops.

OsJAR1 is required for JA-regulated floret opening and anther dehiscence in rice.

Jasmonates are important phytohormones regulating reproductive development. We used two recessive rice Tos17 alleles of OsJAR1, osjar1-2 and osjar1-3, to study the biological function of jasmonates in rice anthesis. The florets of both osjar1 alleles stayed open during anthesis because the lodicules, which control flower opening in rice, were not withering on time. Furthermore, dehiscence of the anthers filled with viable pollen, was impaired, resulting in lower fertility. In situ hybridization and promoter GUS transgenic analysis confirmed OsJAR1 expression in these floral tissues. Flower opening induced by exogenous applied methyl jasmonate was impaired in osjar1 plants and was restored in a complementation experiment with transgenics expressing a wild type copy of OsJAR1 controlled by a rice actin promoter. Biochemical analysis showed that OsJAR1 encoded an enzyme conjugating jasmonic acid (JA) to at least Ile, Leu, Met, Phe, Trp and Val and both osjar1 alleles had substantial reduction in content of JA-Ile, JA-Leu and JA-Val in florets. We conclude that OsJAR1 is a JA-amino acid synthetase that is required for optimal flower opening and closing and anther dehiscence in rice.

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.

Integration of high-density genetic mapping with transcriptome analysis uncovers numerous agronomic QTL and reveals candidate genes for the control of tillering in sorghum.

Phenotypes such as branching, photoperiod sensitivity, and height were modified during plant domestication and crop improvement. Here, we perform quantitative trait locus (QTL) mapping of these and other agronomic traits in a recombinant inbred line (RIL) population derived from an interspecific cross between Sorghum propinquum and Sorghum bicolor inbred Tx7000. Using low-coverage Illumina sequencing and a bin-mapping approach, we generated ∼1920 bin markers spanning ∼875 cM. Phenotyping data were collected and analyzed from two field locations and one greenhouse experiment for six agronomic traits, thereby identifying a total of 30 QTL. Many of these QTL were penetrant across environments and co-mapped with major QTL identified in other studies. Other QTL uncovered new genomic regions associated with these traits, and some of these were environment-specific in their action. To further dissect the genetic underpinnings of tillering, we complemented QTL analysis with transcriptomics, identifying 6189 genes that were differentially expressed during tiller bud elongation. We identified genes such as Dormancy Associated Protein 1 (DRM1) in addition to various transcription factors that are differentially expressed in comparisons of dormant to elongating tiller buds and lie within tillering QTL, suggesting that these genes are key regulators of tiller elongation in sorghum. Our study demonstrates the usefulness of this RIL population in detecting domestication and improvement-associated genes in sorghum, thus providing a valuable resource for genetic investigation and improvement to the sorghum community.

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.

Bulked-Segregant Analysis Coupled to Whole Genome Sequencing (BSA-Seq) for Rapid Gene Cloning in Maize.

Forward genetics remains a powerful method for revealing the genes underpinning organismal form and function, and for revealing how these genes are tied together in gene networks. In maize, forward genetics has been tremendously successful, but the size and complexity of the maize genome made identifying mutant genes an often arduous process with traditional methods. The next generation sequencing revolution has allowed for the gene cloning process to be significantly accelerated in many organisms, even when genomes are large and complex. Here, we describe a bulked-segregant analysis sequencing (BSA-Seq) protocol for cloning mutant genes in maize. Our simple strategy can be used to quickly identify a mapping interval and candidate single nucleotide polymorphisms (SNPs) from whole genome sequencing of pooled F2 individuals. We employed this strategy to identify narrow odd dwarf as an enhancer of teosinte branched1, and to identify a new allele of defective kernel1 Our method provides a quick, simple way to clone genes in maize.

Caffeoyl shikimate esterase (CSE) is an enzyme in the lignin biosynthetic pathway in Arabidopsis.

Lignin is a major component of plant secondary cell walls. Here we describe caffeoyl shikimate esterase (CSE) as an enzyme central to the lignin biosynthetic pathway. Arabidopsis thaliana cse mutants deposit less lignin than do wild-type plants, and the remaining lignin is enriched in p-hydroxyphenyl units. Phenolic metabolite profiling identified accumulation of the lignin pathway intermediate caffeoyl shikimate in cse mutants as compared to caffeoyl shikimate levels in the wild type, suggesting caffeoyl shikimate as a substrate for CSE. Accordingly, recombinant CSE hydrolyzed caffeoyl shikimate into caffeate. Associated with the changes in lignin, the conversion of cellulose to glucose in cse mutants increased up to fourfold as compared to that in the wild type upon saccharification without pretreatment. Collectively, these data necessitate the revision of currently accepted models of the lignin biosynthetic pathway.

Light-regulated stomatal aperture in Arabidopsis.

The stomatal pores of plant leaves, situated in the epidermis and surrounded by a pair of guard cells, allow CO2 uptake for photosynthesis and water loss through transpiration. Blue light is one of the dominant environmental signals that control stomatal movements in leaves of plants in a natural environment. This blue light response is mediated by blue/UV A light-absorbing phototropins (phots) and cryptochromes (crys). Red/far-red light-absorbing phytochromes (phys) also play a role in the control of stomatal aperture. The signaling components that link the perception of light signals to the stomatal opening response are largely unknown. This review discusses a few newly discovered nuclear genes, their function with respect to the phot-, cry-, and phy-mediated signal transduction cascades, and possible involvement of circadian clock.

Comparative transcriptional profiling and preliminary study on heterosis mechanism of super-hybrid rice.

Heterosis is a biological phenomenon whereby the offspring from two parents show improved and superior performance than either inbred parental lines. Hybrid rice is one of the most successful apotheoses in crops utilizing heterosis. Transcriptional profiling of F(1) super-hybrid rice Liangyou-2186 and its parents by serial analysis of gene expression (SAGE) revealed 1183 differentially expressed genes (DGs), among which DGs were found significantly enriched in pathways such as photosynthesis and carbon-fixation, and most of the key genes involved in the carbon-fixation pathway exhibited up-regulated expression in F(1) hybrid rice. Moreover, increased catabolic activity of corresponding enzymes and photosynthetic efficiency were also detected, which combined to indicate that carbon fixation is enhanced in F(1) hybrid, and might probably be associated with the yield vigor and heterosis in super-hybrid rice. By correlating DGs with yield-related quantitative trait loci (QTL), a potential relationship between differential gene expression and phenotypic changes was also found. In addition, a regulatory network involving circadian-rhythms and light signaling pathways was also found, as previously reported in Arabidopsis, which suggest that such a network might also be related with heterosis in hybrid rice. Altogether, the present study provides another view for understanding the molecular mechanism underlying heterosis in rice.

Molecular cloning, functional characterization and expression analysis of a novel monosaccharide transporter gene OsMST6 from rice (Oryza sativa L.).

Monosaccharides transporters play important roles in assimilate supply for sink tissue development. In this study, a new monosaccharide transporter gene OsMST6 was identified from rice (Oryza sativa L.). The predicted OsMST6 protein shows typical features of sugar transporters and shares 79.6% identity with the rice monosaccharide transporter OsMST3. Heterologous expression in yeast (Saccharomyces cerevisiae) demonstrated that OsMST6 is a broad-spectrum monosaccharide transporter, with a K (m) of 266.1 muMu for glucose. OsMST6-green fluorescent protein fusion protein is localized to the plasma membrane in plant. Semi-quantitative RT-PCR analysis exhibited that OsMST6 is expressed in all tested organs/tissues. In developing seeds, OsMST6 expression level is high at the early and middle grain filling stages and gradually declines later. Further analysis detected its expression in both maternal and filial tissues. RNA in situ hybridization analysis indicated that OsMST6 is predominantly expressed in the vascular parenchyma of the chalazal vein, cross-cells, nucellar tissue and endosperm of young seeds, in mesophyll cells of source leaf blades, and in pollens and the connective vein of anthers. In addition, OsMST6 expression is up-regulated by salt stress and sugars. The physiological role of OsMST6 for seed development and its roles in other sink and source tissues are discussed.

Molecular cloning and expression analysis of a monosaccharide transporter gene OsMST4 from rice (Oryza sativa L.).

Monosaccharide transporters mediate the membrane transport of a variable range of monosaccharides, which plays a crucial role in sugar distribution throughout the plant. To investigate the significance of monosaccharide transporters for rice (Oryza sativa L.) seed development, cDNA of a new putative monosaccharide transporter gene OsMST4 was isolated. The deduced OsMST4 protein shows typical features of monosaccharide transporters, and shares high homology with other plant homologues. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that OsMST4 is a functional monosaccharide transporter capable of transporting glucose, fructose, mannose and galactose. Transcriptional analysis revealed that OsMST4 is expressed in all tested organs/tissues. In developing caryopses, its expression is high at the early and middle grain filling stages, and declines gradually to low levels after that. Further analysis revealed that it is expressed in both the maternal tissue and the filial tissue, with its highest expression in embryo. Cellular location in young caryopses through RNA in situ hybridization showed that OsMST4 mRNA mainly accumulates in the vascular parenchyma of the chalazal vein, cross-cells, nucellar tissue and endosperm. The expression pattern of OsMST4 was further confirmed by histochemical analysis of the OsMST4-promoter-beta-glucuronidase (GUS) transgenic rice plants. These data indicate that OsMST4 is actively involved in monosaccharides supply for seed development during the course of grain filling. In addition, the cell type-specific expression patterns of OsMST4 in other sink and source tissues were also investigated, and its corresponding physiological roles were discussed.