Developmental disaster1: A novel mutation causing defects during vegetative and inflorescence development in maize (Zea mays, Poaceae)

Axillary meristems, which give rise to branches and flowers, play a critical role in plant architecture and reproduction. To understand how axillary meristems initiate, we have screened for mutants with defects in axillary meristem initiation to uncover the genes controlling this process. These mutants, called the barren class of mutants in maize (Zea mays), have defects in axillary meristem initiation during both vegetative and reproductive development. Here, we identify and characterize a new member of the barren class of mutants named Developmental disaster1 (Dvd1), due to the pleiotropic effects of the mutation. Similar to the barren mutants, Dvd1 mutants have fewer branches, spikelets, florets, and floral organs in the inflorescence due to defects in the initiation of axillary meristems. Furthermore, double mutant analysis with teosinte branched1 shows that dvd1 also functions in axillary meristems during vegetative development. However, unlike the barren mutants, Dvd1 mutants are semidwarf due to the production of shorter internodes, and they produce leaves in the inflorescence due to the outgrowth of bract leaf primordia. The suite of defects seen in Dvd1 mutants, together with the genetic interaction of Dvd1 with barren inflorescence2, suggests that dvd1 is a novel regulator of axillary meristem and internode development.     

A maize thiamine auxotroph is defective in shoot meristem maintenance

Plant shoots undergo organogenesis throughout their life cycle via the perpetuation of stem cell pools called shoot apical meristems (SAMs). SAM maintenance requires the coordinated equilibrium between stem cell division and differentiation and is regulated by integrated networks of gene expression, hormonal signaling, and metabolite sensing. Here, we show that the maize (Zea mays) mutant bladekiller1-R (blk1-R) is defective in leaf blade development and meristem maintenance and exhibits a progressive reduction in SAM size that results in premature shoot abortion. Molecular markers for stem cell maintenance and organ initiation reveal that both of these meristematic functions are progressively compromised in blk1-R mutants, especially in the inflorescence and floral meristems. Positional cloning of blk1-R identified a predicted missense mutation in a highly conserved amino acid encoded by thiamine biosynthesis2 (thi2). Consistent with chromosome dosage studies suggesting that blk1-R is a null mutation, biochemical analyses confirm that the wild-type THI2 enzyme copurifies with a thiazole precursor to thiamine, whereas the mutant enzyme does not. Heterologous expression studies confirm that THI2 is targeted to chloroplasts. All blk1-R mutant phenotypes are rescued by exogenous thiamine supplementation, suggesting that blk1-R is a thiamine auxotroph. These results provide insight into the role of metabolic cofactors, such as thiamine, during the proliferation of stem and initial cell populations.     

Initiation of axillary and floral meristems in Arabidopsis

Shoot development is reiterative: shoot apical meristems (SAMs) give rise to branches made of repeating leaf and stem units with new SAMs in turn formed in the axils of the leaves. Thus, new axes of growth are established on preexisting axes. Here we describe the formation of axillary meristems and floral meristems in Arabidopsis by monitoring the expression of the SHOOT MERISTEMLESS and AINTEGUMENTA genes. Expression of these genes is associated with SAMs and organ primordia, respectively. Four stages of axillary meristem development and previously undefined substages of floral meristem development are described. We find parallels between the development of axillary meristems and the development of floral meristems. Although Arabidopsis flowers develop in the apparent absence of a subtending leaf, the expression patterns of AINTEGUMENTA and SHOOT MERISTEMLESS RNAs during flower development suggest the presence of a highly reduced, "cryptic" leaf subtending the flower in Arabidopsis. We hypothesize that the STM-negative region that develops on the flanks of the inflorescence meristem is a bract primordium and that the floral meristem proper develops in the "axil" of this bract primordium. The bract primordium, although initially specified, becomes repressed in its growth.     

The bHLH protein ROX acts in concert with RAX1 and LAS to modulate axillary meristem formation in Arabidopsis

During post-embryonic shoot development, new meristems are initiated in the axils of leaves. They produce secondary axes of growth that determine morphological plasticity and reproductive efficiency in higher plants. In this study, we describe the role of the bHLH-protein-encoding Arabidopsis gene REGULATOR OF AXILLARY MERISTEM FORMATION (ROX), which is the ortholog of the branching regulators LAX PANICLE1 (LAX1) in rice and barren stalk1 (ba1) in maize. rox mutants display compromised axillary bud formation during vegetative shoot development, and combination of rox mutants with mutations in RAX1 and LAS, two key regulators of axillary meristem initiation, enhances their branching defects. In contrast to lax1 and ba1, flower development is unaffected in rox mutants. Over-expression of ROX leads to formation of accessory side shoots. ROX mRNA accumulates at the adaxial boundary of leaf and flower primordia. However, in the vegetative phase, axillary meristems initiate after ROX expression has terminated, suggesting an indirect role for ROX in meristem formation. During vegetative development, ROX expression is dependent on RAX1 and LAS activity, and all three genes act in concert to modulate axillary meristem formation.     

barren inflorescence2 regulates axillary meristem development in the maize inflorescence

Organogenesis in plants is controlled by meristems. Shoot apical meristems form at the apex of the plant and produce leaf primordia on their flanks. Axillary meristems, which form in the axils of leaf primordia, give rise to branches and flowers and therefore play a critical role in plant architecture and reproduction. To understand how axillary meristems are initiated and maintained, we characterized the barren inflorescence2 mutant, which affects axillary meristems in the maize inflorescence. Scanning electron microscopy, histology and RNA in situ hybridization using knotted1 as a marker for meristematic tissue show that barren inflorescence2 mutants make fewer branches owing to a defect in branch meristem initiation. The construction of the double mutant between barren inflorescence2 and tasselsheath reveals that the function of barren inflorescence2 is specific to the formation of branch meristems rather than bract leaf primordia. Normal maize inflorescences sequentially produce three types of axillary meristem: branch meristem, spikelet meristem and floral meristem. Introgression of the barren inflorescence2 mutant into genetic backgrounds in which the phenotype was weaker illustrates additional roles of barren inflorescence2 in these axillary meristems. Branch, spikelet and floral meristems that form in these lines are defective, resulting in the production of fewer floral structures. Because the defects involve the number of organs produced at each stage of development, we conclude that barren inflorescence2 is required for maintenance of all types of axillary meristem in the inflorescence. This defect allows us to infer the sequence of events that takes place during maize inflorescence development. Furthermore, the defect in branch meristem formation provides insight into the role of knotted1 and barren inflorescence2 in axillary meristem initiation.     

CORKSCREW1 defines a novel mechanism of domain specification in the maize shoot

In higher plants, determinate leaf primordia arise in regular patterns on the flanks of the indeterminate shoot apical meristem (SAM). The acquisition of leaf form is then a gradual process, involving the specification and growth of distinct domains within the three leaf axes. The recessive corkscrew1 (cks1) mutation of maize (Zea mays) disrupts both leaf initiation patterns in the SAM and domain specification within the mediolateral and proximodistal leaf axes. Specifically, cks1 mutant leaves exhibit multiple midribs and leaf sheath tissue differentiates in the blade domain. Such perturbations are a common feature of maize mutants that ectopically accumulate KNOTTED1-like homeobox (KNOX) proteins in leaf tissue. Consistent with this observation, at least two knox genes are ectopically expressed in cks1 mutant leaves. However, ectopic KNOX proteins cannot be detected. We therefore propose that CKS1 primarily functions within the SAM to establish boundaries between meristematic and leaf zones. Loss of gene function disrupts boundary formation, impacts phyllotactic patterns, and leads to aspects of indeterminate growth within leaf primordia. Because these perturbations arise independently of ectopic KNOX activity, the cks1 mutation defines a novel component of the developmental machinery that facilitates leaf-versus-shoot development in maize.     

The Relationship between auxin transport and maize branching

Maize (Zea mays) plants make different types of vegetative or reproductive branches during development. Branches develop from axillary meristems produced on the flanks of the vegetative or inflorescence shoot apical meristem. Among these branches are the spikelets, short grass-specific structures, produced by determinate axillary spikelet-pair and spikelet meristems. We investigated the mechanism of branching in maize by making transgenic plants expressing a native expressed endogenous auxin efflux transporter (ZmPIN1a) fused to yellow fluorescent protein and a synthetic auxin-responsive promoter (DR5rev) driving red fluorescent protein. By imaging these plants, we found that all maize branching events during vegetative and reproductive development appear to be regulated by the creation of auxin response maxima through the activity of polar auxin transporters. We also found that the auxin transporter ZmPIN1a is functional, as it can rescue the polar auxin transport defects of the Arabidopsis (Arabidopsis thaliana) pin1-3 mutant. Based on this and on the groundbreaking analysis in Arabidopsis and other species, we conclude that branching mechanisms are conserved and can, in addition, explain the formation of axillary meristems (spikelet-pair and spikelet meristems) that are unique to grasses. We also found that BARREN STALK1 is required for the creation of auxin response maxima at the flanks of the inflorescence meristem, suggesting a role in the initiation of polar auxin transport for axillary meristem formation. Based on our results, we propose a general model for branching during maize inflorescence development.     

Transposon tagging of the Defective embryo and meristems gene of tomato.

The shoot and root apical meristems (SAMs and RAMs, respectively) of higher plants are mechanistically and structurally similar. This has led previously to the suggestion that the SAM and RAM represent modifications of a fundamentally homologous plan of organization. Despite recent interest in plant development, especially in the areas of meristem regulation, genes specifically required for the function of both the SAM and RAM have not yet been identified. Here, we report on a novel gene, Defective embryo and meristems (Dem), of tomato. This gene is required for the correct organization of shoot apical tissues of developing embryos, SAM development, and correct cell division patterns and meristem maintenance in roots. Dem was cloned using transposon tagging and shown to encode a novel protein of 72 kD with significant homology to YNV2, a protein of unknown function of Saccharomyces cerevisiae. Dem is expressed in root and shoot meristems and organ primordia but not in callus. The expression pattern of Dem mRNA in combination with the dem mutant phenotype suggests that Dem plays an important role within apical meristems.     

Meristem maintenance and compound-leaf patterning utilize common genetic mechanisms in tomato

Balancing shoot apical meristem (SAM) maintenance and organ formation from its flanks is essential for proper plant growth and development and for the flexibility of organ production in response to internal and external cues. Leaves are formed at the SAM flanks and display a wide variability in size and form. Tomato (Solanum lycopersicum) leaves are compound with lobed margins. We exploited 18 recessive tomato mutants, representing four distinct phenotypic classes and six complementation groups, to track the genetic mechanisms involved in meristem function and compound-leaf patterning in tomato. In goblet (gob) mutants, the SAM terminates following cotyledon production, but occasionally partially recovers and produces simple leaves. expelled shoot (exp) meristems terminate after the production of several leaves, and these leaves show a reduced level of compoundness. short pedicel (spd) mutants are bushy, with impaired meristem structure, compact inflorescences, short pedicels and less compound leaves. In multi drop (mud) mutants, the leaves are more compound and the SAM tends to divide into two active meristems after the production of a few leaves. The range of leaf-compoundness phenotypes observed in these mutants suggests that compound-leaf patterning involves an array of genetic factors, which act successively to elaborate leaf shape. Furthermore, the results indicate that similar mechanisms underlie SAM activity and compound-leaf patterning in tomato.     

Gene expression patterns in seed plant shoot meristems and leaves: homoplasy or homology?

The fossil record reveals that seed plant leaves evolved from ancestral lateral branch systems. Over time, the lateral branch systems evolved to become determinate, planar and eventually laminar. Considering their evolutionary histories, it is instructive to compare the developmental genetics of shoot apical meristems (SAMs) and leaves in extant seed plants. Genetic experiments in model angiosperm species have assigned functions of meristem maintenance, specification of stem cell identity, boundary formation, polarity establishment and primordium initiation to specific genes. Investigation of roles of the same or homologous genes during leaf development has revealed strikingly similar functions in leaves compared to SAMs. Specifically, the marginal blastozone that characterizes many angiosperm leaves appears to function in a manner mechanistically similar to the SAM. We argue here that the similarities may be homologous due to descent from ancestral roles in an ancestral shoot system. Molecular aspects of SAM and leaf development in gymnosperms is largely neglected and could provide insight into seed plant leaf evolution.     

茎顶端分生组织在植物发育过程中的保持、转变和逆转

顶端分生组织(shoot apical meristems,SAM)为产生新的器官和组织而不断提供新的细胞,它的活性依赖于平衡分生组织细胞的增殖和器官发生之间关系的调控基因.来自不具备光合能力的顶端分生组织的细胞可形成具有光合能力的营养器官.在从营养生长到生殖发育的转变过程中,茎顶端分生组织,转变为花序分生组织,最终形成花分生组织.在进入开花决定状态以前,SAM的状态很大程度上受到环境信号和转录调控因子的影响.以模式植物拟南芥为主,对在顶端分生组织的保持和转变中复杂同时又有差异的基因调控网络进行讨论.在花和花序分生组织逆转过程中,SAM中的细胞也受到相关基因的调控,且表达方式存在明显的时空差异.因此,具有决定性的和未决定性双重特性的分生组织之间的转变和相互协调,对于器官发生和形态建成起到至关重要的作用.     

CUC1 gene activates the expression of SAM-related genes to induce adventitious shoot formation

CUP-SHAPED COTYLEDON (CUC)1 encodes members of the NAC family. These are functionally redundant genes that are involved in shoot apical meristem (SAM) formation and cotyledon separation during embryogenesis in Arabidopsis. We analyzed transgenic plants overexpressing CUC1 (35S::CUC1). The cotyledons of these transgenic seedlings regularly had two basal lobes, small and round epidermal cells between the sinuses, and adventitious SAMs on the adaxial surface of this region. This suggests that CUC1 promotes adventitious SAM formation by maintaining epidermal cells in an undifferentiated state. In 35S::CUC1 cotyledons, the class I knotted-like homeobox (KNOX) genes, including SHOOT MERISTEMLESS (STM) and BREVIPEDICELLUS (BP), which are involved in SAM formation and/or maintenance, were ectopically expressed before adventitious SAM formation. In stm mutants, ectopic expression of CUC1 could not induce adventitious SAMs, whereas they continued to be observed in bp mutants. These results suggest that STM, but not BP, is necessary for the formation of adventitious SAMs in 35S::CUC1 cotyledons. Furthermore, we examined the relationship between CUC1 and ASYMMETRIC LEAVES (AS)1 and AS2. The as1 and as2 mutations genetically enhance 35S::CUC1 phenotypes even in the absence of STM function. Interestingly, the as1 mutation can partially rescue the mutant vegetative development phenotypes in the cuc1 cuc2 double mutant. Our results suggest that CUC1 positively regulates SAM formation not only through STM but also through an STM-independent pathway that is negatively regulated by AS1 and AS2.     

Giant shoot apical meristems in cacti have ordinary leaf primordia but altered phyllotaxy and shoot diameter

BACKGROUND AND AIMS: Shoot apical meristems (SAMs) in most seed plants are quite uniform in size and zonation, and molecular genetic studies of Arabidopsis and other model plants are revealing details of SAM morphogenesis. Some cacti have SAMs much larger than those of A. thaliana and other seed plants. This study examined how SAM size affects leaf primordium (LP) size, phyllotaxy and shoot diameter. METHODS: Apices from 183 species of cacti were fixed, microtomed and studied by light microscopy. KEY RESULTS: Cactus SAM diameter varies from 93 to 2565 microm, the latter being 36 times wider than SAMs of A. thaliana and having a volume 45 thousand times larger. Phyllotaxy ranges from distichous to having 56 rows of leaves and is not restricted to Fibonacci numbers. Leaf primordium diameter ranges from 44 to 402 microm, each encompassing many more cells than do LP of other plants. Species with high phyllotaxy have smaller LP, although the correlation is weak. There is almost no correlation between SAM diameter and LP size, but SAM diameter is strongly correlated with shoot diameter, with shoots being about 189.5 times wider than SAMs. CONCLUSIONS: Presumably, genes such as SHOOT-MERISTEMLESS, WUSCHEL and CLAVATA must control much larger volumes of SAM tissue in cacti than they do in A. thaliana, and genes such as PERIANTHIA might establish much more extensive fields of inhibition around LP. These giant SAMs should make it possible to more accurately map gene expression patterns relative to SAM zonation and LP sites.     

Expression of CDC2Zm and KNOTTED1 during in-vitro axillary shoot meristem proliferation and adventitious shoot meristem formation in maize (Zea mays L.) and barley (Hordeum vulgare L.)

Expression of CDC2Zm and KNOTTED1 (KN1) in maize (Zea mays L.) and their cross-reacting proteins in barley (Hordeum vulgare L.) was studied using immunolocalization during in-vitro axillary shoot meristem proliferation and adventitious shoot meristem formation. Expression of CDC2Zm, a protein involved in cell division, roughly correlated with in-vitro cell proliferation and in the meristematic domes CDC2Zm expression was triggered during in-vitro proliferation. Analysis of the expression of KN1, a protein necessary for maintenance of the shoot meristem, showed that KN1 or KN1-homologue(s) expression was retained in meristematic cells during in-vitro proliferation of axillary shoot meristems. Multiple adventitious shoot meristems appeared to form directly from the KN1- or KN1 homologue(s)-expressing meristematic cells in the in-vitro proliferating meristematic domes. However, unlike Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) leaves ectopically expressing KN1 (G. Chuck et al., 1996 Plant Cell 8: 1277–1289; N. Sinha et al., 1993 Genes Dev. 7: 787–797), transgenic maize leaves over-expressing KN1 were unable to initiate adventitious shoot meristems on their surfaces either in planta or in vitro. Therefore, expression of KN1 is not the sole triggering factor responsible for inducing adventitious shoot meristem formation from in-vitro proliferating axillary shoot meristems in maize. Our results show that genes critical to cell division and plant development have utility in defining in-vitro plant morphogenesis at the molecular level and, in combination with transformation technologies, will be powerful tools in identifying the fundamental molecular and-or genetic triggering factor(s) responsible for reprogramming of plant cells during plant morphogenesis in-vitro. Received: 2 June 1997 / Accepted: 21 July 1997