Comparative mutant analyses reveal a novel mechanism of ARF regulation in land plants.

A major challenge in plant biology is to understand how the plant hormone auxin regulates diverse transcriptional responses throughout development, in different environments, and in different species. The answer may lie in the specific complement of auxin signaling components in each cell. The balance between activators (class-A AUXIN RESPONSE FACTORS) and repressors (class-B ARFs) is particularly important. It is unclear how this balance is achieved. Through comparative analysis of novel, dominant mutants in maize and the moss Physcomitrium patens , we have discovered a ∼500-million-year-old mechanism of class-B ARF protein level regulation, important in determining cell fate decisions across land plants. Thus, our results add a key piece to the puzzle of how auxin regulates plant development.

Terminal ear 1 and phytochromes B1/B2 regulate maize leaf initiation independently.

Higher plants generate new leaves from shoot meristems throughout their vegetative lifespan. The tempo of leaf initiation is dynamically regulated by physiological cues, but little is known about the underlying genetic signaling pathways that coordinate this rate. Two maize (Zea mays) mutants, terminal ear1 (te1) and phytochrome B1;phytochrome B2 (phyB1;phyB2), oppositely affect leaf initiation rates and total leaf number at the flowering time: te1 mutants make leaves faster whereas phyB1;phyB2 mutants make leaves slower than wild-type plants. To test whether PhyB1, PhyB2, and TE1 act in overlapping or distinct pathways to regulate leaf initiation, we crossed te1 and phyB1;phyB2 created an F2 population segregating for these three mutations and quantified various phenotypes among the resulting genotypes, including leaf number, leaf initiation rate, plant height, leaf length, leaf width, number of juvenile leaves, stalk diameter, and dry shoot biomass. Leaf number and initiation rate in phyB1;phyB2;te1 plants fell between the extremes of the two parents, suggesting an additive genetic interaction between te1 and phyB1;phyB2 rather than epistasis. Therefore, we conclude that PhyB1, PhyB2, and TE1 likely control leaf initiation through distinct signaling pathways.

Liguleless narrow and narrow odd dwarf act in overlapping pathways to regulate maize development and metabolism.

Narrow odd dwarf (nod) and Liguleless narrow (Lgn) are pleiotropic maize mutants that both encode plasma membrane proteins, cause similar developmental patterning defects, and constitutively induce stress signaling pathways. To investigate how these mutants coordinate maize development and physiology, we screened for protein interactors of NOD by affinity purification. LGN was identified by this screen as a strong candidate interactor, and we confirmed the NOD-LGN molecular interaction through orthogonal experiments. We further demonstrated that LGN, a receptor-like kinase, can phosphorylate NOD in vitro, hinting that they could act in intersecting signal transduction pathways. To test this hypothesis, we generated Lgn-R;nod mutants in two backgrounds (B73 and A619), and found that these mutations enhance each other, causing more severe developmental defects than either single mutation on its own, with phenotypes including very narrow leaves, increased tillering, and failure of the main shoot. Transcriptomic and metabolomic analyses of the single and double mutants in the two genetic backgrounds revealed widespread induction of pathogen defense genes and a shift in resource allocation away from primary metabolism in favor of specialized metabolism. These effects were similar in each single mutant and heightened in the double mutant, leading us to conclude that NOD and LGN act cumulatively in overlapping signaling pathways to coordinate growth-defense tradeoffs in maize.

The power of classic maize mutants: Driving forward our fundamental understanding of plants.

Since Mendel, maize has been a powerhouse of fundamental genetics research. From testing the Mendelian laws of inheritance, to the first genetic and cytogenetic maps, to the use of whole-genome sequencing data for crop improvement, maize is at the forefront of genetics advances. Underpinning much of this revolutionary work are the classic morphological mutants; the "freaks" that stood out in the field to even the untrained eye. Here we review some of these classic developmental mutants and their importance in the history of genetics, as well as their key role in our fundamental understanding of plant development.

A mixed-linkage (1,3;1,4)-ß-D-glucan specific hydrolase mediates dark-triggered degradation of this plant cell wall polysaccharide.

The presence of mixed-linkage (1,3;1,4)-ß-d-glucan (MLG) in plant cell walls is a key feature of grass species such as cereals, the main source of calorie intake for humans and cattle. Accumulation of this polysaccharide involves the coordinated regulation of biosynthetic and metabolic machineries. While several components of the MLG biosynthesis machinery have been identified in diverse plant species, degradation of MLG is poorly understood. In this study, we performed a large-scale forward genetic screen for maize (Zea mays) mutants with altered cell wall polysaccharide structural properties. As a result, we identified a maize mutant with increased MLG content in several tissues, including adult leaves and senesced organs, where only trace amounts of MLG are usually detected. The causative mutation was found in the GRMZM2G137535 gene, encoding a GH17 licheninase as demonstrated by an in vitro activity assay of the heterologously expressed protein. In addition, maize plants overexpressing GRMZM2G137535 exhibit a 90% reduction in MLG content, indicating that the protein is not only required, but its expression is sufficient to degrade MLG. Accordingly, the mutant was named MLG hydrolase 1 (mlgh1). mlgh1 plants show increased saccharification yields upon enzymatic digestion. Stacking mlgh1 with lignin-deficient mutations results in synergistic increases in saccharification. Time profiling experiments indicate that wall MLG content is modulated during day/night cycles, inversely associated with MLGH1 transcript accumulation. This cycling is absent in the mlgh1 mutant, suggesting that the mechanism involved requires MLG degradation, which may in turn regulate MLGH1 gene expression.

Gene duplication at the Fascicled ear1 locus controls the fate of inflorescence meristem cells in maize.

Plant meristems are self-renewing groups of pluripotent stem cells that produce lateral organs in a stereotypical pattern. Of interest is how the radially symmetrical meristem produces laminar lateral organs. Both the male and female inflorescence meristems of the dominant Fascicled ear (Fas1) mutant fail to grow as a single point and instead show deep branching. Positional cloning of two independent Fas1 alleles identified an ∼160 kb region containing two floral genes, the MADS-box gene, zmm8, and the YABBY gene, drooping leaf2 (drl2). Both genes are duplicated within the Fas1 locus and spatiotemporally misexpressed in the mutant inflorescence meristems. Increased zmm8 expression alone does not affect inflorescence development; however, combined misexpression of zmm8, drl2, and their syntenic paralogs zmm14 and drl1, perturbs meristem organization. We hypothesize that misexpression of the floral genes in the inflorescence and their potential interaction cause ectopic activation of a laminar program, thereby disrupting signaling necessary for maintenance of radially symmetrical inflorescence meristems. Consistent with this hypothesis, RNA sequencing and in situ analysis reveal altered expression patterns of genes that define distinct zones of the meristem and developing leaf. Our findings highlight the importance of strict spatiotemporal patterns of expression for both zmm8 and drl2 and provide an example of phenotypes arising from tandem gene duplications.

Reconstructing the Transcriptional Ontogeny of Maize and Sorghum Supports an Inverse Hourglass Model of Inflorescence Development.

Assembling meaningful comparisons between species is a major limitation in studying the evolution of organismal form. To understand development in maize and sorghum, closely related species with architecturally distinct inflorescences, we collected RNA-seq profiles encompassing inflorescence body-plan specification in both species. We reconstructed molecular ontogenies from 40 B73 maize tassels and 47 BTx623 sorghum panicles and separated them into transcriptional stages. To discover new markers of inflorescence development, we used random forest machine learning to determine stage by RNA-seq. We used two descriptions of transcriptional conservation to identify hourglass-like stages during inflorescence development. Despite a relatively short 12 million years since their last common ancestor, we found maize and sorghum inflorescences are most different during their hourglass-like stages of development, following an inverse-hourglass model of development. We discuss whether agricultural selection may account for the rapid divergence signatures in these species and the observed separation of evolutionary pressure and developmental reprogramming.

The Second Site Modifier, Sympathy for the ligule, Encodes a Homolog of Arabidopsis ENHANCED DISEASE RESISTANCE4 and Rescues the Liguleless narrow Maize Mutant.

Liguleless narrow1 encodes a plasma membrane-localized receptor-like kinase required for normal development of maize (Zea mays) leaves, internodes, and inflorescences. The semidominant Lgn-R mutation lacks kinase activity, and phenotypic severity is dependent on inbred background. We created near isogenic lines and assayed the phenotype in multiple environments. Lgn-R plants that carry the B73 version of Sympathy for the ligule (Sol-B) fail to grow under hot conditions, but those that carry the Mo17 version (Sol-M) survive at hot temperatures and are significantly taller at cool temperatures. To identify Sol, we used recombinant mapping and analyzed the Lgn-R phenotype in additional inbred backgrounds. We identified amino acid sequence variations in GRMZM2G075262 that segregate with severity of the Lgn-R phenotypes. This gene is expressed at high levels in Lgn-R B73, but expression drops to nonmutant levels with one copy of Sol-M An EMS mutation solidified the identity of SOL as a maize homolog of Arabidopsis (Arabidopsis thaliana) ENHANCED DISEASE RESISTANCE4 (EDR4). SOL, like EDR4, is induced in response to pathogen-associated molecular patterns such as flg22. Integrated transcriptomic and phosphoproteomic analyses suggest that Lgn-R plants constitutively activate an immune signaling cascade that induces temperature-sensitive responses in addition to defects in leaf development. We propose that aspects of the severe Lgn-R developmental phenotype result from constitutive defense induction and that SOL potentially functions in repressing this response in Mo17 but not B73. Identification of LGN and its interaction with SOL provides insight into the integration of developmental control and immune responses.

Tasselseed5 overexpresses a wound-inducible enzyme, ZmCYP94B1, that affects jasmonate catabolism, sex determination, and plant architecture in maize.

Maize is monecious, with separate male and female inflorescences. Maize flowers are initially bisexual but achieve separate sexual identities through organ arrest. Loss-of-function mutants in the jasmonic acid (JA) pathway have only female flowers due to failure to abort silks in the tassel. Tasselseed5 (Ts5) shares this phenotype but is dominant. Positional cloning and transcriptomics of tassels identified an ectopically expressed gene in the CYP94B subfamily, Ts5 (ZmCYP94B1). CYP94B enzymes are wound inducible and inactivate bioactive jasmonoyl-L-isoleucine (JA-Ile). Consistent with this result, tassels and wounded leaves of Ts5 mutants displayed lower JA and JA-lle precursors and higher 12OH-JA-lle product than the wild type. Furthermore, many wounding and jasmonate pathway genes were differentially expressed in Ts5 tassels. We propose that the Ts5 phenotype results from the interruption of JA signaling during sexual differentiation via the upregulation of ZmCYP94B1 and that its proper expression maintains maize monoecy.

Drawing a Line: Grasses and Boundaries.

Delineation between distinct populations of cells is essential for organ development. Boundary formation is necessary for the maintenance of pluripotent meristematic cells in the shoot apical meristem (SAM) and differentiation of developing organs. Boundaries form between the meristem and organs, as well as between organs and within organs. Much of the research into the boundary gene regulatory network (GRN) has been carried out in the eudicot model Arabidopsis thaliana. This work has identified a dynamic network of hormone and gene interactions. Comparisons with other eudicot models, like tomato and pea, have shown key conserved nodes in the GRN and species-specific alterations, including the recruitment of the boundary GRN in leaf margin development. How boundaries are defined in monocots, and in particular the grass family which contains many of the world's staple food crops, is not clear. In this study, we review knowledge of the grass boundary GRN during vegetative development. We particularly focus on the development of a grass-specific within-organ boundary, the ligule, which directly impacts leaf architecture. We also consider how genome engineering and the use of natural diversity could be leveraged to influence key agronomic traits relative to leaf and plant architecture in the future, which is guided by knowledge of boundary GRNs.

GRF-interacting factor1 Regulates Shoot Architecture and Meristem Determinacy in Maize.

Plant architecture results from a balance of indeterminate and determinate cell fates. Cells with indeterminate fates are located in meristems, comprising groups of pluripotent cells that produce lateral organs. Meristematic cells are also found in intercalary stem tissue, which provides cells for internodes, and at leaf margins to contribute to leaf width. We identified a maize (Zea mays) mutant that has a defect in balancing determinacy and indeterminacy. The mutant has narrow leaves and short internodes, suggesting a reduction in indeterminate cells in the leaf and stem. In contrast, the mutants fail to control indeterminacy in shoot meristems. Inflorescence meristems are fasciated, and determinate axillary meristems become indeterminate. Positional cloning identified growth regulating factor-interacting factor1 (gif1) as the responsible gene. gif1 mRNA accumulates in distinct domains of shoot meristems, consistent with tissues affected by the mutation. We determined which GROWTH REGULATING FACTORs interact with GIF1 and performed RNA-seq analysis. Many genes known to play roles in inflorescence architecture were differentially expressed in gif1 Chromatin immunoprecipitation identified some differentially expressed genes as direct targets of GIF1. The interactions with these diverse direct and indirect targets help explain the paradoxical phenotypes of maize GIF1. These results provide insights into the biological functions of gif1.

KNOTTED1 Cofactors, BLH12 and BLH14, Regulate Internode Patterning and Vein Anastomosis in Maize.

Monocot stems lack the vascular cambium and instead have characteristic structures in which intercalary meristems generate internodes and veins remain separate and scattered. However, developmental processes of these unique structures have been poorly described. BELL1-like homeobox (BLH) transcription factors (TFs) are known to heterodimerize with KNOTTED1-like homeobox TFs to play crucial roles in shoot meristem maintenance, but their functions are elusive in monocots. We found that maize (Zea mays) BLH12 and BLH14 have redundant but important roles in stem development. BLH12/14 interact with KNOTTED1 (KN1) in vivo and accumulate in overlapping domains in shoot meristems, young stems, and provascular bundles. Similar to kn1 loss-of-function mutants, blh12 blh14 (blh12/14) double mutants fail to maintain axillary meristems. Unique to blh12/14 is an abnormal tassel branching and precocious internode differentiation that results in dwarfism and reduced veins in stems. Micro-computed tomography observation of vascular networks revealed that blh12/14 double mutants had reduced vein number due to fewer intermediate veins in leaves and precocious anastomosis in young stems. Based on these results, we propose two functions of BLH12/14 during stem development: (1) maintaining intercalary meristems that accumulate KN1 and prevent precocious internode differentiation and (2) preventing precocious anastomosis of provascular bundles in young stems to ensure the production of sufficient independent veins.

The Maize MID-COMPLEMENTING ACTIVITY Homolog CELL NUMBER REGULATOR13/NARROW ODD DWARF Coordinates Organ Growth and Tissue Patterning.

Organogenesis occurs through cell division, expansion, and differentiation. How these cellular processes are coordinated remains elusive. The maize (Zea mays) leaf provides a robust system to study cellular differentiation due to its distinct tissues and cell types. The narrow odd dwarf (nod) mutant displays defects at both the cellular and tissue level that increase in severity throughout growth. nod mutant leaves have reduced size due to fewer and smaller cells compared with the wild type. The juvenile-to-adult transition is delayed, and proximal distal-patterning is abnormal in this mutant. Differentiation of specialized cells such as those forming stomata and trichomes is incomplete. Analysis of nod-1 sectors suggests that NOD plays a cell-autonomous function in the leaf. We cloned nod positionally and found that it encodes CELL NUMBER REGULATOR13 (CNR13), the maize MID-COMPLEMENTING ACTIVITY homolog. CNR13/NOD is localized to the membrane and is enriched in dividing tissues. Transcriptome analysis of nod mutants revealed overrepresentation of cell wall, hormone metabolism, and defense gene categories. We propose that NOD coordinates cell activity in response to intrinsic and extrinsic cues.

Keep on growing: building and patterning leaves in the grasses.

Monocot leaves have unique features that arise early in their development. Maturing leaves protectively enclose younger leaves and the meristem, the pool of founder cells from which a leaf emerges. Through the maturation process, proximal sheath and distal blade tissues differentiate and are separated by the ligule and auricle structures. Here we review current research focusing on the contribution of gene regulatory factors and phytohormones on the patterning and differentiation of monocot leaves primarily focusing on research in the grasses (Poaceae). The 10000 members of the grasses include the true grain cereals (wheat, rice, maize, etc.), biofuel crops such as sugarcane, pasture grasses, and bamboo. They are the most studied of the monocots due to their tremendous agricultural and agronomic importance.

Diverse functions of KNOX transcription factors in the diploid body plan of plants.

KNOX genes were initially found as shoot meristem regulators in angiosperms. Recent studies in diverse plant lineages however, have revealed the divergence of KNOX gene function during the evolution of diploid body plans. Using genomic approaches, class I KNOX transcription factors have been shown to regulate multiple hormone pathways including auxin and brassinosteroid as well as many transcription factors that play important roles in plant development. Class I KNOX proteins appear to be activators, whereas class II proteins act as repressors in transcriptional regulation of their target genes.

Organogenesis in plants: initiation and elaboration of leaves.

Plant organs initiate from meristems and grow into diverse forms. After initiation, organs enter a morphological phase where they develop their shape, followed by differentiation into mature tissue. Investigations into these processes have revealed numerous factors necessary for proper development, including transcription factors such as the KNOTTED-LIKE HOMEOBOX (KNOX) genes, the hormone auxin, and miRNAs. Importantly, these factors have been shown to play a role in organogenesis in various diverse model species, revealing both deep conservation of regulatory strategies and evolutionary novelties that led to new plant forms. We review here recent work in understanding the regulation of organogenesis and in particular leaf formation, highlighting how regulatory modules are often redeployed in different organ types and stages of development to achieve diverse forms through the balance of growth and differentiation.

Functionally different PIN proteins control auxin flux during bulbil development in Agave tequilana.

In Agave tequilana, reproductive failure or inadequate flower development stimulates the formation of vegetative bulbils at the bracteoles, ensuring survival in a hostile environment. Little is known about the signals that trigger this probably unique phenomenon in agave species. Here we report that auxin plays a central role in bulbil development and show that the localization of PIN1-related proteins is consistent with altered auxin transport during this process. Analysis of agave transcriptome data led to the identification of the A. tequilana orthologue of PIN1 (denoted AtqPIN1) and a second closely related gene from a distinct clade reported as 'Sister of PIN1' (denoted AtqSoPIN1). Quantitative real-time reverse transcription-PCR (RT-qPCR) analysis showed different patterns of expression for each gene during bulbil formation, and heterologous expression of the A. tequilana PIN1 and SoPIN1 genes in Arabidopsis thaliana confirmed functional differences between these genes. Although no free auxin was detected in induced pedicel samples, changes in the levels of auxin precursors were observed. Taken as a whole, the data support the model that AtqPIN1 and AtqSoPIN1 have co-ordinated but distinct functions in relation to auxin transport during the initial stages of bulbil formation.

Genetic, evolutionary and plant breeding insights from the domestication of maize.

The natural history of maize began nine thousand years ago when Mexican farmers started to collect the seeds of the wild grass, teosinte. Invaluable as a food source, maize permeated Mexican culture and religion. Its domestication eventually led to its adoption as a model organism, aided in large part by its large chromosomes, ease of pollination and growing agricultural importance. Genome comparisons between varieties of maize, teosinte and other grasses are beginning to identify the genes responsible for the domestication of modern maize and are also providing ideas for the breeding of more hardy varieties.