Cytokinin Promotes Jasmonic Acid Accumulation in the Control of Maize Leaf Growth.

Plant organ growth results from the combined activity of cell division and cell expansion. The co-ordination of these two processes depends on the interplay between multiple hormones that determine the final organ size. Using the semidominant Hairy Sheath Frayed1 (Hsf1) maize mutant that hypersignals the perception of cytokinin (CK), we show that CK can reduce leaf size and growth rate by decreasing cell division. Linked to CK hypersignaling, the Hsf1 mutant has an increased jasmonic acid (JA) content, a hormone that can inhibit cell division. The treatment of wild-type seedlings with exogenous JA reduces maize leaf size and growth rate, while JA-deficient maize mutants have increased leaf size and growth rate. Expression analysis revealed the increased transcript accumulation of several JA pathway genes in the Hsf1 leaf growth zone. A transient treatment of growing wild-type maize shoots with exogenous CK also induced the expression of JA biosynthetic genes, although this effect was blocked by the co-treatment with cycloheximide. Together, our results suggest that CK can promote JA accumulation, possibly through the increased expression of specific JA pathway genes.

Utilizing CRISPR-Cas in Tropical Crop Improvement: A Decision Process for Fitting Genome Engineering to Your Species.

Adopting modern gene-editing technologies for trait improvement in agriculture requires important workflow developments, yet these developments are not often discussed. Using tropical crop systems as a case study, we describe a workflow broken down into discrete processes with specific steps and decision points that allow for the practical application of the CRISPR-Cas gene editing platform in a crop of interest. While we present the steps of developing genome-edited plants as sequential, in practice parts can be done in parallel, which are discussed in this perspective. The main processes include 1) understanding the genetic basis of the trait along with having the crop's genome sequence, 2) testing and optimization of the editing reagents, development of efficient 3) tissue culture and 4) transformation methods, and 5) screening methods to identify edited events with commercial potential. Our goal in this perspective is to help any lab that wishes to implement this powerful, easy-to-use tool in their pipeline, thus aiming to democratize the technology.

The Maize Hairy Sheath Frayed1 (Hsf1) Mutation Alters Leaf Patterning through Increased Cytokinin Signaling.

Leaf morphogenesis requires growth polarized along three axes-proximal-distal (P-D) axis, medial-lateral axis, and abaxial-adaxial axis. Grass leaves display a prominent P-D polarity consisting of a proximal sheath separated from the distal blade by the auricle and ligule. Although proper specification of the four segments is essential for normal morphology, our knowledge is incomplete regarding the mechanisms that influence P-D specification in monocots such as maize (Zea mays). Here, we report the identification of the gene underlying the semidominant, leaf patterning maize mutant Hairy Sheath Frayed1 (Hsf1). Hsf1 plants produce leaves with outgrowths consisting of proximal segments-sheath, auricle, and ligule-emanating from the distal blade margin. Analysis of three independent Hsf1 alleles revealed gain-of-function missense mutations in the ligand binding domain of the maize cytokinin (CK) receptor Z. mays Histidine Kinase1 (ZmHK1) gene. Biochemical analysis and structural modeling suggest the mutated residues near the CK binding pocket affect CK binding affinity. Treatment of the wild-type seedlings with exogenous CK phenocopied the Hsf1 leaf phenotypes. Results from expression and epistatic analyses indicated the Hsf1 mutant receptor appears to be hypersignaling. Our results demonstrate that hypersignaling of CK in incipient leaf primordia can reprogram developmental patterns in maize.

Over-expression of the photoperiod response regulator ZmCCT10 modifies plant architecture, flowering time and inflorescence morphology in maize.

Maize originated as a tropical plant that required short days to transition from vegetative to reproductive development. ZmCCT10 [CO, CONSTANS, CO-LIKE and TIMING OF CAB1 (CCT) transcription factor family] is a regulator of photoperiod response and was identified as a major QTL controlling photoperiod sensitivity in maize. We modulated expression of ZmCCT10 in transgenic maize using two constitutive promoters with different expression levels. Transgenic plants over expressing ZmCCT10 with either promoter were delayed in their transition from vegetative to reproductive development but were not affected in their switch from juvenile-to-adult vegetative growth. Strikingly, transgenic plants containing the stronger expressing construct had a prolonged period of vegetative growth accompanied with dramatic modifications to plant architecture that impacted both vegetative and reproductive traits. These plants did not produce ears, but tassels were heavily branched. In more than half of the transgenic plants, tassels were converted into a branched leafy structure resembling phyllody, often composed of vegetative plantlets. Analysis of expression modules controlling the floral transition and meristem identity linked these networks to photoperiod dependent regulation, whereas phase change modules appeared to be photoperiod independent. Results from this study clarified the influence of the photoperiod pathway on vegetative and reproductive development and allowed for the fine-tuning of the maize flowering time model.

A Pectin Methylesterase ZmPme3 Is Expressed in Gametophyte factor1-s (Ga1-s) Silks and Maps to that Locus in Maize (Zea mays L.).

The ga1 locus of maize confers unilateral cross incompatibility, preventing cross pollination between females carrying the incompatible Ga1-s allele and males not carrying a corresponding compatible allele. To characterize this system at the molecular level, we carried out a transcript profiling experiment in which silks from near isogenic lines carrying the Ga1-s and ga1 alleles were compared. While several differentially expressed genes were identified, only one mapped to the known location of ga1. This gene is a pectin methylesterase (PME), which we designated as ZmPme3, and is present and expressed only in Ga1-s genotypes. While a functional ZmPME3 is not present in the ga1 genotypes examined, a pectin methylesterase gene cluster is found in ga1 genotypes. The gene cluster in W22 contains 58 tandem full-length or partial PME pseudo genes. These data combined with a wealth of previously published data on the involvement of PMEs in pollen tube growth suggest a role for cell wall modification enzymes in the pollen exclusion component of Ga1-s gametophytic incompatibility. Consistent with this role, a third allele which lacks the female function of Ga1-s, Ga1-m, has a mutationally inactivated version of ZmPme3.

Three FLOWERING LOCUS T-like genes function as potential florigens and mediate photoperiod response in sorghum.

Sorghum is a typical short-day (SD) plant and its use in grain or biomass production in temperate regions depends on its flowering time control, but the underlying molecular mechanism of floral transition in sorghum is poorly understood. Here we characterized sorghum FLOWERING LOCUS T (SbFT) genes to establish a molecular road map for mechanistic understanding. Out of 19 PEBP genes, SbFT1, SbFT8 and SbFT10 were identified as potential candidates for encoding florigens using multiple approaches. Phylogenetic analysis revealed that SbFT1 clusters with the rice Hd3a subclade, while SbFT8 and SbFT10 cluster with the maize ZCN8 subclade. These three genes are expressed in the leaf at the floral transition initiation stage, expressed early in grain sorghum genotypes but late in sweet and forage sorghum genotypes, induced by SD treatment in photoperiod-sensitive genotypes, cooperatively repressed by the classical sorghum maturity loci, interact with sorghum 14-3-3 proteins and activate flowering in transgenic Arabidopsis plants, suggesting florigenic potential in sorghum. SD induction of these three genes in sensitive genotypes is fully reversed by 1 wk of long-day treatment, and yet, some aspects of the SD treatment may still make a small contribution to flowering in long days, indicating a complex photoperiod response mediated by SbFT genes.

Beyond flowering time: pleiotropic function of the maize flowering hormone florigen.

The transition from vegetative to reproductive development is a critical turning point in a plant's life cycle. It is now widely accepted that a leaf-borne signal, florigen, moves via the phloem from leaves to the shoot apical meristem to trigger its reprogramming to produce flowers. In part, the florigenic signal comprises a protein that belongs to the phosphatidylethanolamine-binding protein (PEBP) family that is present in all living organisms but displays diverse functions. The founding floral-promoting PEBP gene in Arabidopsis is FLOWERING LOCUS T (FT) whose functional homologs have been indentified in many flowering plants. We recently accumulated sufficient evidence to demonstrate the maize FT homolog ZCN8 has florigenic function. This task was particularly challenging due to the large number of FT-homologous genes in the maize genome. Here we show that ZCN8 function is more complex than simply regulating the floral transition. ZCN8 appears to play a pleiotropic role in the regulation of generalized growth of vegetative and reproductive tissues.

The FT-like ZCN8 Gene Functions as a Floral Activator and Is Involved in Photoperiod Sensitivity in Maize.

The mobile floral-promoting signal, florigen, is thought to consist of, in part, the FT protein named after the Arabidopsis thaliana gene FLOWERING LOCUS T. FT is transcribed and translated in leaves and its protein moves via the phloem to the shoot apical meristem where it promotes the transition from vegetative to reproductive development. In our search for a maize FT-like floral activator(s), seven Zea mays CENTRORADIALIS (ZCN) genes encoding FT homologous proteins were studied. ZCN8 stood out as the only ZCN having the requisite characteristics for possessing florigenic activity. In photoperiod sensitive tropical lines, ZCN8 transcripts were strongly upregulated in a diurnal manner under floral-inductive short days. In day-neutral temperate lines, ZCN8 mRNA level was independent of daylength and displayed only a weak cycling pattern. ZCN8 is normally expressed in leaf phloem, but ectopic expression of ZCN8 in vegetative stage shoot apices induced early flowering in transgenic plants. Silencing of ZCN8 by artificial microRNA resulted in late flowering. ZCN8 was placed downstream of indeterminate1 and upstream of delayed flowering1, two other floral activator genes. We propose a flowering model linking photoperiod sensitivity of tropical maize to diurnal regulation of ZCN8.

Involvement of the MADS-box gene ZMM4 in floral induction and inflorescence development in maize.

The switch from vegetative to reproductive growth is marked by the termination of vegetative development and the adoption of floral identity by the shoot apical meristem (SAM). This process is called the floral transition. To elucidate the molecular determinants involved in this process, we performed genome-wide RNA expression profiling on maize (Zea mays) shoot apices at vegetative and early reproductive stages using massively parallel signature sequencing technology. Profiling revealed significant up-regulation of two maize MADS-box (ZMM) genes, ZMM4 and ZMM15, after the floral transition. ZMM4 and ZMM15 map to duplicated regions on chromosomes 1 and 5 and are linked to neighboring MADS-box genes ZMM24 and ZMM31, respectively. This gene order is syntenic with the vernalization1 locus responsible for floral induction in winter wheat (Triticum monococcum) and similar loci in other cereals. Analyses of temporal and spatial expression patterns indicated that the duplicated pairs ZMM4-ZMM24 and ZMM15-ZMM31 are coordinately activated after the floral transition in early developing inflorescences. More detailed analyses revealed ZMM4 expression initiates in leaf primordia of vegetative shoot apices and later increases within elongating meristems acquiring inflorescence identity. Expression analysis in late flowering mutants positioned all four genes downstream of the floral activators indeterminate1 (id1) and delayed flowering1 (dlf1). Overexpression of ZMM4 leads to early flowering in transgenic maize and suppresses the late flowering phenotype of both the id1 and dlf1 mutations. Our results suggest ZMM4 may play roles in both floral induction and inflorescence development.

tie-dyed1 Regulates carbohydrate accumulation in maize leaves.

Acquisition of cell identity requires communication among neighboring cells. To dissect the genetic pathways regulating cell signaling in later leaf development, a screen was performed to identify mutants with chloroplast pigmentation sectors that violate cell lineage boundaries in maize (Zea mays) leaves. We have characterized a recessive mutant, tie-dyed1 (tdy1), which develops stable, nonclonal variegated yellow and green leaf sectors. Sector formation requires high light, occurs during a limited developmental time, and is restricted to leaf blade tissue. Yellow tdy1 sectors accumulate excessive soluble sugars and starch, whereas green sectors appear unaffected. Significantly, starch accumulation precedes chlorosis in cells that will become a yellow sector. Retention of carbohydrates in tdy1 leaves is associated with a delay in reproductive maturity, decreased stature, and reduced yield. To explain the tdy1 sectoring pattern, we propose a threshold model that incorporates the light requirement and the hyperaccumulation of photoassimilates. A possible function consistent with this model is that TDY1 acts as a sugar sensor to regulate an inducible sugar export pathway as leaves develop under high light conditions.

delayed flowering1 Encodes a basic leucine zipper protein that mediates floral inductive signals at the shoot apex in maize.

Separation of the life cycle of flowering plants into two distinct growth phases, vegetative and reproductive, is marked by the floral transition. The initial floral inductive signals are perceived in the leaves and transmitted to the shoot apex, where the vegetative shoot apical meristem is restructured into a reproductive meristem. In this study, we report cloning and characterization of the maize (Zea mays) flowering time gene delayed flowering1 (dlf1). Loss of dlf1 function results in late flowering, indicating dlf1 is required for timely promotion of the floral transition. dlf1 encodes a protein with a basic leucine zipper domain belonging to an evolutionarily conserved family. Three-dimensional protein modeling of a missense mutation within the basic domain suggests DLF1 protein functions through DNA binding. The spatial and temporal expression pattern of dlf1 indicates a threshold level of dlf1 is required in the shoot apex for proper timing of the floral transition. Double mutant analysis of dlf1 and indeterminate1 (id1), another late flowering mutation, places dlf1 downstream of id1 function and suggests dlf1 mediates floral inductive signals transmitted from leaves to the shoot apex. This study establishes an emergent framework for the genetic control of floral induction in maize and highlights the conserved topology of the floral transition network in flowering plants.

Duplicated fie genes in maize: expression pattern and imprinting suggest distinct functions.

Two maize genes with predicted translational similarity to the Arabidopsis FIE (Fertilization-Independent Endosperm) protein, a repressor of endosperm development in the absence of fertilization, were cloned and analyzed. Genomic sequences of fie1 and fie2 show significant homology within coding regions but none within introns or 5' upstream. The fie1 gene is expressed exclusively in the endosperm of developing kernels starting at approximately 6 days after pollination. fie1 is an imprinted gene showing no detectable expression of the paternally derived fie1 allele during kernel development. Conversely, fie2 is expressed in the embryo sac before pollination. After pollination, its expression persists, predominantly in the embryo and at lower levels in the endosperm. The paternal fie2 allele is not expressed early in kernel development, but its transcription is activated at 5 days after pollination. fie2 is likely to be a functional ortholog of the Arabidopsis FIE gene, whereas fie1 has evolved a distinct function. The maize FIE2 and sorghum FIE proteins form a monophyletic group, sharing a closer relationship to each other than to the FIE1 protein, suggesting that maize fie genes originated from two different ancestral genomes.

Maximum likelihood methods reveal conservation of function among closely related kinesin families.

We have reconstructed the evolution of the anciently derived kinesin superfamily using various alignment and tree-building methods. In addition to classifying previously described kinesins from protists, fungi, and animals, we analyzed a variety of kinesin sequences from the plant kingdom including 12 from Zea mays and 29 from Arabidopsis thaliana. Also included in our data set were four sequences from the anciently diverged amitochondriate protist Giardia lamblia. The overall topology of the best tree we found is more likely than previously reported topologies and allows us to make the following new observations: (1) kinesins involved in chromosome movement including MCAK, chromokinesin, and CENP-E may be descended from a single ancestor; (2) kinesins that form complex oligomers are limited to a monophyletic group of families; (3) kinesins that crosslink antiparallel microtubules at the spindle midzone including BIMC, MKLP, and CENP-E are closely related; (4) Drosophila NOD and human KID group with other characterized chromokinesins; and (5) Saccharomyces SMY1 groups with kinesin-I sequences, forming a family of kinesins capable of class V myosin interactions. In addition, we found that one monophyletic clade composed exclusively of sequences with a C-terminal motor domain contains all known minus end-directed kinesins.