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.     

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.     

ZCN8 encodes a potential orthologue of Arabidopsis FT florigen that integrates both endogenous and photoperiod flowering signals in maize

Higher plants use multiple perceptive measures to coordinate flowering time with environmental and endogenous cues. Physiological studies show that florigen is a mobile factor that transmits floral inductive signals from the leaf to the shoot apex. Arabidopsis FT protein is widely regarded as the archetype florigen found in diverse plant species, particularly in plants that use inductive photoperiods to flower. Recently, a large family of FT homologues in maize, the Zea CENTRORADIALIS (ZCN) genes, was described, suggesting that maize also contains FT-related proteins that act as a florigen. The product of one member of this large family, ZCN8, has several attributes that make it a good candidate as a maize florigen. Mechanisms underlying the floral transition in maize are less well understood than those of other species, partly because flowering in temperate maize is dependent largely on endogenous signals. The maize indeterminate1 (id1) gene is an important regulator of maize autonomous flowering that acts in leaves to mediate the transmission or production of florigenic signals. This study finds that id1 acts upstream of ZCN8 to control its expression, suggesting a possible new link to flowering in day-neutral maize. Moreover, in teosinte, a tropical progenitor of maize that requires short-day photoperiods to induce flowering, ZCN8 is highly up-regulated in leaves under inductive photoperiods. Finally, vascular-specific expression of ZCN8 in Arabidopsis complements the ft-1 mutation, demonstrating that leaf-specific expression of ZCN8 can induce flowering. These results suggest that ZCN8 may encode a florigen that integrates both endogenous and environmental signals in maize.     

Tie-dyed2 functions with tie-dyed1 to promote carbohydrate export from maize leaves

Regulation of carbon partitioning is essential for plant growth and development. To gain insight into genes controlling carbon allocation in leaves, we identified mutants that hyperaccumulate carbohydrates. tie-dyed2 (tdy2) is a recessive mutant of maize (Zea mays) with variegated, nonclonal, chlorotic leaf sectors containing excess starch and soluble sugars. Consistent with a defect in carbon export, we found that a by-product of functional chloroplasts, likely a sugar, induces tdy2 phenotypic expression. Based on the phenotypic similarities between tdy2 and two other maize mutants with leaf carbon accumulation defects, tdy1 and sucrose export defective1 (sxd1), we investigated whether Tdy2 functioned in the same pathway as Tdy1 or Sxd1. Cytological and genetic studies demonstrate that Tdy2 and Sxd1 function independently. However, in tdy1/+; tdy2/+ F(1) plants, we observed a moderate chlorotic sectored phenotype, suggesting that the two genes are dosage sensitive and have a related function. This type of genetic interaction is referred to as second site noncomplementation and has often, though not exclusively, been found in cases where the two encoded proteins physically interact. Moreover, tdy1; tdy2 double mutants display a synergistic interaction supporting this hypothesis. Additionally, we determined that cell walls of chlorotic leaf tissues in tdy mutants contain increased cellulose; thus, tdy mutants potentially represent enhanced feedstocks for biofuels production. From our phenotypic and genetic characterizations, we propose a model whereby TDY1 and TDY2 function together in a single genetic pathway, possibly in homo- and heteromeric complexes, to promote carbon export from leaves.     

Differential expression of alternative oxidase genes in maize mitochondrial mutants

We have examined the expression of three alternative oxidase (aox) genes in two types of maize mitochondrial mutants. Nonchromosomal stripe (NCS) mutants carry mitochondrial DNA deletions that affect subunits of respiratory complexes and show constitutively defective growth. Cytoplasmic male-sterile (CMS) mutants have mitochondrial DNA rearrangements, but they are impaired for mitochondrial function only during anther development. In contrast to normal plants, which have very low levels of AOX, NCS mutants exhibit high expression of aox genes in all nonphotosynthetic tissues tested. The expression pattern is specific for each type of mitochondrial lesion: the NADH dehydrogenase-defective NCS2 mutant has high expression of aox2, whereas the cytochrome oxidase-defective NCS6 mutant predominantly expresses aox3. Similarly, aox2 and aox3 can be induced differentially in normal maize seedlings by specific inhibitors of these two respiratory complexes. Translation-defective NCS4 plants show induction of both aox2 and aox3. AOX2 and AOX3 proteins differ in their ability to be regulated by reversible dimerization. CMS mutants show relatively high levels of aox2 mRNAs in young tassels but none in ear shoots. Significant expression of aox1 is detected only in NCS and CMS tassels. The induction pattern of maize aox genes could serve as a selective marker for diverse mitochondrial defects.     

The indeterminate floral apex1 gene regulates meristem determinacy and identity in the maize inflorescence

Meristems may be determinate or indeterminate. In maize, the indeterminate inflorescence meristem produces three types of determinate meristems: spikelet pair, spikelet and floral meristems. These meristems are defined by their position and their products. We have discovered a gene in maize, indeterminate floral apex1 (ifa1) that regulates meristem determinacy. The defect found in ifa1 mutants is specific to meristems and does not affect lateral organs. In ifa1 mutants, the determinate meristems become less determinate. The spikelet pair meristem initiates more than a pair of spikelets and the spikelet meristem initiates more than the normal two flowers. The floral meristem initiates all organs correctly, but the ovule primordium, the terminal product of the floral meristem, enlarges and proliferates, expressing both meristem and ovule marker genes. A role for ifa1 in meristem identity in addition to meristem determinacy was revealed by double mutant analysis. In zea agamous1 (zag1) ifa1 double mutants, the female floral meristem converts to a branch meristem whereas the male floral meristem converts to a spikelet meristem. In indeterminate spikelet1 (ids1) ifa1 double mutants, female spikelet meristems convert to branch meristems and male spikelet meristems convert to spikelet pair meristems. The double mutant phenotypes suggest that the specification of meristems in the maize inflorescence involves distinct steps in an integrated process.     

The circadian clock‐associated gene gigantea1 affects maize developmental transitions

The circadian clock is an internal timing mechanism that allows plants to make developmental decisions in accordance with environmental conditions. In model plants, circadian clock‐associated gigantea (gi) genes are directly involved in control of growth and developmental transitions. The maize gigantea1 (gi1) gene is the more highly expressed of the two gi homeologs, and its function is uncharacterized. To understand the role of gi1 in the regulatory networks of the maize circadian clock system, gi1 mutants were evaluated for changes in flowering time, phase change and growth control. When grown in long‐day (LD) photoperiods, gi1 mutants flowered earlier than non‐mutant plants, but this difference was not apparent in short‐day (SD) photoperiods. Therefore, gi1 participates in a pathway that suppresses flowering in LD photoperiods, but not in SD. Part of the underlying cause of early flowering was up‐regulated expression of the FT‐like floral activator gene zea mays centroradialis8 (zcn8) and the CONSTANS‐like flowering regulatory gene constans of zea mays1 (conz1). gi1 mutants also underwent vegetative phase change earlier and grew taller than non‐mutant plants. These findings indicate gi1 has a repressive function in multiple regulatory pathways that govern maize growth and development.     

Interactions among APETALA1, LEAFY, and TERMINAL FLOWER1 specify meristem fate.

Upon floral induction, the primary shoot meristem of an Arabidopsis plant begins to produce flower meristems rather than leaf primordia on its flanks. Assignment of floral fate to lateral meristems is primarily due to the cooperative activity of the flower meristem identity genes LEAFY (LFY), APETALA1 (AP1), and CAULIFLOWER. We present evidence here that AP1 expression in lateral meristems is activated by at least two independent pathways, one of which is regulated by LFY. In lfy mutants, the onset of AP1 expression is delayed, indicating that LFY is formally a positive regulator of AP1. We have found that AP1, in turn, can positively regulate LFY, because LFY is expressed prematurely in the converted floral meristems of plants constitutively expressing AP1. Shoot meristems maintain an identity distinct from that of flower meristems, in part through the action of genes such as TERMINAL FLOWER1 (TFL1), which bars AP1 and LFY expression from the influorescence shoot meristem. We show here that this negative regulation can be mutual because TFL1 expression is downregulated in plants constitutively expressing AP1. Therefore, the normally sharp phase transition between the production of leaves with associated shoots and formation of the flowers, which occurs upon floral induction, is promoted by positive feedback interactions between LFY and AP1, together with negative interactions of these two genes with TFL1.     

Expression and Functional Analysis of the ZCN1(ZmTFL1) Gene, a TERMINAL FLOWER 1 Homologue that Regulates the Vegetative to Reproductive Transition in Maize

TERMINAL FLOWER 1 (TFL1) homologs play critical roles in regulating flowering time and/or maintaining flowering of meristems. In this study, the gene of maize TFL1 ortholog ZmTFL1 (ZCN1) was cloned from both the tropical inbred line CML288 and temperate inbred line Huangzao 4, and the function of ZmTFL1 (ZCN1) was determined during different periods of floral development. Spatial and temporal expression patterns revealed that ZCN1 was predominantly localized in shoot apical meristems that develop into flowers, and only at low levels in leaves. To further identify the role of ZCN1 in floral development of maize, the morphology of shoot apices in maize during floral development was investigated using laser scanning confocal microscopy. Moreover, the relative levels of expression of ZCN1, ZCN8, DLF1, and ZAP1 genes were determined. Over-expression of ZCN1 partially complemented the late flowering phenotype in the tfl1-14 Arabidopsis mutant. Moreover, transgenic Arabidopsis plants exhibited indeterminate inflorescence with increased shoot length and higher numbers of trichomes on leaves. In addition, expression levels of AP1 were significantly down-regulated in 35S::ZCN1 transgenic Arabidopsis plants. These results indicated that ZCN1 as well as its homolog TFL1 in Arabidopsis are involved in the regulation of floral transition in maize.     

A MADS domain gene involved in the transition to flowering in Arabidopsis

Flowering time in many plants is triggered by environmental factors that lead to uniform flowering in plant populations, ensuring higher reproductive success. So far, several genes have been identified that are involved in flowering time control. AGL20 (AGAMOUS LIKE 20) is a MADS domain gene from Arabidopsis that is activated in shoot apical meristems during the transition to flowering. By transposon tagging we have identified late flowering agl20 mutants, showing that AGL20 is involved in flowering time control. In previously described late flowering mutants of the long-day and constitutive pathways of floral induction the expression of AGL20 is down-regulated, demonstrating that AGL20 acts downstream to the mutated genes. Moreover, we can show that AGL20 is also regulated by the gibberellin (GA) pathway, indicating that AGL20 integrates signals of different pathways of floral induction and might be a central component for the induction of flowering. In addition, the constitutive expression of AGL20 in Arabidopsis is sufficient for photoperiod independent flowering and the over-expression of the orthologous gene from mustard, MADSA, in the classical short-day tobacco Maryland Mammoth bypasses the strict photoperiodic control of flowering.     

The Medicago FLOWERING LOCUS T homolog, MtFTa1, is a key regulator of flowering time

FLOWERING LOCUS T (FT) genes encode proteins that function as the mobile floral signal, florigen. In this study, we characterized five FT-like genes from the model legume, Medicago (Medicago truncatula). The different FT genes showed distinct patterns of expression and responses to environmental cues. Three of the FT genes (MtFTa1, MtFTb1, and MtFTc) were able to complement the Arabidopsis (Arabidopsis thaliana) ft-1 mutant, suggesting that they are capable of functioning as florigen. MtFTa1 is the only one of the FT genes that is up-regulated by both long days (LDs) and vernalization, conditions that promote Medicago flowering, and transgenic Medicago plants overexpressing the MtFTa1 gene flowered very rapidly. The key role MtFTa1 plays in regulating flowering was demonstrated by the identification of fta1 mutants that flowered significantly later in all conditions examined. fta1 mutants do not respond to vernalization but are still responsive to LDs, indicating that the induction of flowering by prolonged cold acts solely through MtFTa1, whereas photoperiodic induction of flowering involves other genes, possibly MtFTb1, which is only expressed in leaves under LD conditions and therefore might contribute to the photoperiodic regulation of flowering. The role of the MtFTc gene is unclear, as the ftc mutants did not have any obvious flowering-time or other phenotypes. Overall, this work reveals the diversity of the regulation and function of the Medicago FT family.     

Effects of sugar on vegetative development and floral transition in Arabidopsis

Although sugar has been suggested to promote floral transition in many plant species, growth on high concentrations (5% [w/v]) of sucrose (Suc) significantly delayed flowering time, causing an increase in the number of leaves at the time of flowering in Arabidopsis. The effect of high concentrations of Suc seemed to be metabolic rather than osmotic. The delay of floral transition was due to extension of the late vegetative phase, which resulted in a delayed activation of LFY expression. In addition, growth on low concentrations (1% [w/v]) of Suc slightly inhibited flowering in wild-type plants. This delay resulted from effects on the early vegetative phase. This inhibition was more pronounced in tfl1, an early flowering mutant, than in the wild type. Although 1% (w/v) Suc was reported to promote floral transition of late-flowering mutants such as co, fca, and gi, floral transition in these mutants was delayed by a further increase in Suc concentration. These results suggest that sugar may affect floral transition by activating or inhibiting genes that act to control floral transition, depending on the concentration of sugars, the genetic background of the plants, and when the sugar is introduced. Growth on 1% (w/v) Suc did not restore the reduced expression levels of FT and SOC1/AGL20 in co or fca mutants. Rather, expression of FT and SOC1/AGL20 was repressed by 1% (w/v) Suc in wild-type background. The possible effects of sugar on gene expression to promote floral transition are discussed.     

A petunia MADS box gene involved in the transition from vegetative to reproductive development.

We have identified a novel petunia MADS box gene, PETUNIA FLOWERING GENE (PFG), which is involved in the transition from vegetative to reproductive development. PFG is expressed in the entire plant except stamens, roots and seedlings. Highest expression levels of PFG are found in vegetative and inflorescence meristems. Inhibition of PFG expression in transgenic plants, using a cosuppression strategy, resulted in a unique nonflowering phenotype. Homozygous pfg cosuppression plants are blocked in the formation of inflorescences and maintain vegetative growth. In these mutants, the expression of both PFG and the MADS box gene FLORAL BINDING PROTEIN26 (FBP26), the putative petunia homolog of SQUAMOSA from Antirrhinum, are down-regulated. In hemizygous pfg cosuppression plants initially a few flowers are formed, after which the meristem reverts to the vegetative phase. This reverted phenotype suggests that PFG, besides being required for floral transition, is also required to maintain the reproductive identity after this transition. The position of PFG in the hierarchy of genes controlling floral meristem development was investigated using a double mutant of the floral meristem identity mutant aberrant leaf and flower (alf) and the pfg cosuppression mutant. This analysis revealed that the pfg cosuppression phenotype is epistatic to the alf mutant phenotype, indicating that PFG acts early in the transition to flowering. These results suggest that the petunia MADS box gene, PFG, functions as an inflorescence meristem identity gene required for the transition of the vegetative shoot apex to the reproductive phase and the maintenance of reproductive identity.