Developmental genetics of leaf morphogenesis in dicotyledonous plants

A full understanding of the leaf is essential for a full understanding of plant morphology. However, leaf morphogenesis is still poorly understood, in particular in dicotyledonous plants, because of the complex nature of the development of leaves. Mutational analysis seems to be the most suitable strategy for investigations of such processes, and should allow us to dissect the developmental pathways into genetically programmed unit processes. The techniques of developmental genetics have been applied to the study of leaf morphogenesis in model plants, such asArabidopsis thaliana, and several key processes in leaf morphogenesis have been identified. The fundamental processes in leaf morphogenesis include the identification of leaf organs, determination of leaf primordia (occurrence of marginal meristem), and the polar or non-polar elongation of leaf cells. This review will focus on the genes that are essential for these processes and have been identified in mutational analyses. Mutational analyses of the photomorphogenesis is also briefly summarized from the perspective of the plasticity of leaf morphogenesis.     

Developmental processes of leaf morphogenesis in<Emphasis Type="Italic">arabidopsis</Emphasis>

The leaf is a suitable subject with which to study plant morphogenesis because of its diversity of shape. Although mechanisms for leaf initiation and lateral morphogenesis have been suggested, the exact means for determining shape remain unclear. Many genes involved in those developmental processes have now been identified. Here, we summarize the early events in the genetic regulation ofArabidopsis leaf formation, including initiation, dorsoventrality, and the spatial and temporal control of cell proliferation and enlargement. We focus on recent progress within the model plantArabidopsis, placing special emphasis on our own findings.     

Development states associated with the floral transition.

Floral initiation can be analyzed from a developmental perspective by focusing upon how developmental fates are imprinted, remembered, and expressed. This is not an altogether new perspective, since people studying flowering have been concerned for a long time with the commitment of meristems to form flowers and the morphological, cellular, and molecular changes associated with this commitment. What is novel is the emphasis on developmental states as opposed to physiological processes. This developmental focus indicates that there appear to be at least three major developmental states that are acquired and expressed in the process of a meristem initiating floral morphogenesis. The meristem cells must first become competent to respond to a developmental signal that evokes them into a florally determined state. The leaves are the usual source of this signal and a specific leaf may or may not have the capacity to be inductively active. When a leaf does develop the capacity for inductive activity, this capacity is usually correlated with the ontogeny of the leaf. Inductive activity, however, may be continually expressed as in some day-neutral plants or may be latent as in plants where the photoperiod is the external cue for activity. Competent shoot apical meristems respond to inductive leaf signal by being evoked into a florally determined state. Under permissive conditions this florally determined state is expressed as the initiation of floral morphogenesis. Many mechanisms have evolved to regulate entry into and expression of these developmental states. As we learn more about the developmental states associated with flowering and how they are acquired and expressed, we will understand better how the various patterns of flowering are related to one another as well as which developmental processes are common to all angiosperms.     

Coordination of cell proliferation and cell expansion in the control of leaf size in Arabidopsis thaliana

Size is an important parameter in the characterization of organ morphology and function. To understand the mechanisms that control leaf size, we previously isolated a number of Arabidopsis thaliana mutants with altered leaf size. Because leaf morphogenesis depends on determinate cell proliferation, the size of a mature leaf is controlled by variation in cell size and number. Therefore, leaf-size mutants should be classified according to the effects of the mutations on the cell number and/or size. A group of mutants represented by angustifolia3/grf-interacting factor1 and aintegumenta exhibits an intriguing cellular phenotype termed compensation: when the leaf cell number is decreased due to the mutation, the leaf cell size increases, leading to compensation in leaf area. Several lines of genetic evidence suggest that compensation is probably not a result of the uncoupling of cell division from cell growth. Rather, the evidence suggests an organ-wide mechanism that coordinates cell proliferation with cell expansion during leaf development. Our results provide a key, novel concept that explains how leaf size is controlled at the organ level.     

Growth, water relations and photosynthesis of seedlings and resprouts after fire

Seasonal patterns of growth, water relations, photosynthesis and leaf characteristics were compared between obligate seeders (Cistus monspeliensis and Cistus ladanifer) and resprouters (Arbutus unedo and Pistacia lentiscus) from the first to the second year after fire. We hypothesized that seedlings would be more water-limited than resprouts due to their shallower root systems. Regarding water use strategies, Cistus species are drought semi-deciduous and A. unedo and P. lentiscus are evergreen sclerophylls, therefore, comparisons were based on the relative deviation from mature conspecific plants. Seedlings and resprouts had higher shoot elongation and leaf production than mature plants, and over an extended period. Differences from mature plants were larger in resprouts, with two-fold transpiration, leaf conductance and photosynthesis in late spring/early summer. Seedlings of C. monspeliensis exhibited higher transpiration and leaf conductance than mature plants, while those of C. ladanifer only exhibited higher water potential. Growth increments and ameliorated water relations and photosynthesis after fire were attributed to an increase in water and nutrient availability. The small differences in water relations and photosynthesis between seedlings and mature conspecifics are in accordance with the prediction of seedlings experiencing higher water limitation than resprouts. We attribute these results to differences in root systems: resprouters benefited from an increase in root/shoot ratios and the presence of deep roots whereas Cistus seedlings relied on very shallow roots, which cannot provide assess to deep water during summer. Nevertheless, seedlings did not show evidence of experiencing a more severe water limitation than mature conspecifics, which we attributed to the presence of efficient mechanisms of avoiding and tolerating water stress. The results are discussed in relation to post-fire demography of seeders and resprouters in Mediterranean communities.     

Toward Molecular Understanding of In Vitro and In Planta Shoot Organogenesis

Shoot organogenesis, one of the in vitro plant regeneration processes that occur during in vitro micropropagation, is used in the study of plant development. Morphological, physiological, and molecular aspects of in vitro shoot organogenesis have been extensively studied for over 50 years. Because of the research progress in plant genetics and molecular biology, our understanding of in planta and in vitro shoot meristem development, the cell cycle and cytokinin signal transduction has advanced significantly. These research advances provide useful information as well as molecular tools to study further the genetic and molecular aspects of shoot organogenesis. A number of key molecular markers, genes, and pathways have been shown to play a critical role in the process of in vitro shoot organogenesis. Furthermore, these studies reveal that in vitro shoot organogenesis, as with in planta shoot development, is a complex, well-coordinated developmental process, given that the induction of a single molecular event is likely to be insufficient to induce the entire process. Continued study is required to identify additional molecular events that trigger dedifferentiation and act as developmental switches for de novo shoot development.     

Genetic analysis of leaf form mutants from the Arabidopsis Information Service collection.

Although a vast inventory of morphological mutants of Arabidopsis thaliana is available, only some have been used for genetic studies of leaf development. Such is the case with the Arabidopsis Information Service (AIS) Form Mutants collection, assembled by A. R. Kranz and currently stored at the Nottingham Arabidopsis Stock Centre, which includes a large number of mutant lines, most of which have been little studied. With the aim of contributing to the genetic dissection of leaf ontogeny, we have subjected 57 mutant lines isolated by others to genetic analysis; 47 of which were from the AIS collection. These are characterized by vegetative leaves of abnormal shape or size, and were chosen as candidates for mutations in genes required for leaf morphogenesis. The mutant phenotypes studied were shown to be inherited as single recessive Mendelian traits and were classified into 10 phenotypic classes. These mutant strains were found to fall into 37 complementation groups, 7 of which corresponded to known genes. Results of the phenotypic analysis and data on the genetic interactions of these mutants are presented, and their possible developmental defects discussed. Received: 28 October 1998 / Accepted: 21 February 1999     

Leaf shape variation and herbivore consumption and performance: a case study with Ipomoea hederacea and three generalists

The effect of leaf shape variation on plant-herbivore interactions has primarily been studied from the perspective of host seeking behavior. Yet for leaf shape to affect plant-herbivore coevolution, there must be reciprocal effects of leaf shape variation on herbivore consumption and performance. We investigated whether alternative leaf morphs affected the performance of three generalist insect herbivores by taking advantage of a genetic polymorphism and developmental plasticity in leaf shape in the Ivyleaf morning glory, Ipomoea hederacea. Across four experiments, we found variable support for an effect of leaf shape genotype on insects. For cabbage loopers (Trichoplusia ni) and corn earworms (Helicoverpa zea) we found opposing, non-significant trends: T. ni gained more biomass on lobed genotypes, while H. zea gained more biomass on heart-shaped genotypes. For army beetworms (Spodoptera exigua), the effects of leaf shape genotype differed depending on the age of the plants and photoperiod of growing conditions. Caterpillars feeding on tissue from older plants (95 days) grown under long day photoperiods had significantly greater consumption, dry biomass, and digestive efficiency on lobed genotypes. In contrast, there were no significant differences between heart-shaped and lobed genotypes for caterpillars feeding on tissue from younger plants (50 days) grown under short day photoperiods. For plants grown under short days, we found that S. exigua consumed significantly less leaf area when feeding on mature leaves than juvenile leaves, regardless of leaf shape genotype. Taken together, our results suggest that the effects of leaf shape variation on insect performance are likely to vary between insect species, growth conditions of the plant, and the developmental stage and age of leaves sampled. Handling editor: May Berenbaum.     

Developmental anatomy of cotyledons and leaves in has mutant of Arabidopsis thaliana

Summary. In this work, we analyzed the developmental anatomy of cotyledons and leaves in the has mutant of Arabidopsis thaliana. It is a recessive T-DNA insertion mutation that causes changes in the size, shape, and tissue organization of the cotyledons and leaves of has plants. Analysis of has cotyledons revealed a prominent decrease in the cell number and an increase in the area of cotyledon cells and intercellular spaces of has plants. At early stages of development, has leaves are fingerlike structures, but later they develop small, lobed blades with rare trichomes. An important characteristic of the mutant leaf anatomy is the absence of mesophyll tissue differentiation. In addition, both cotyledons and leaves display a disrupted pattern of vascular bundles. Furthermore, mutant plants are defective in root and shoot morphology, indicating that the has mutation affects a number of aspects in plant development. Correspondence and reprints: Institute of Botany and “Jevremovac” Botanical Garden, Faculty of Biology, Belgrade University, Takovska 43, 11 000 Belgrade, Serbia.     

Adventitious shoot formation in decapitated dicotyledonous seedlings starts with regeneration of abnormal leaves from cells not located in a shoot apical meristem

Regeneration of new shoots in plant tissue culture is often associated with appearance of abnormally shaped leaves. We used the adventitious shoot regeneration response induced by decapitation (removal of all preformed shoot apical meristems, leaving a single cotyledon) of greenhouse-grown cotyledon-stage seedlings to test the hypothesis that such abnormal leaf formation is a normal regeneration progression following wounding and is not conditioned by tissue culture. To understand why shoot regeneration starts with defective organogenesis, the regeneration response was characterized by morphology and scanning electron and light microscopy in decapitated cotyledon-stage Cucurbita pepo seedlings. Several leaf primordia were observed to regenerate prior to differentiation of a de novo shoot apical meristem from dividing cells on the wound surface. Early regenerating primordia have a greatly distorted structure with dramatically altered dorsoventrality. Aberrant leaf morphogenesis in C. pepo gradually disappears as leaves eventually originate from a de novo adventitious shoot apical meristem, recovering normal phyllotaxis. Similarly, following comparable decapitation of seedlings from a number of families (Chenopodiaceae, Compositae, Convolvulaceae, Cucurbitaceae, Cruciferae, Fabaceae, Malvaceae, Papaveraceae, and Solanaceae) of several dicotyledonous clades (Ranunculales, Caryophyllales, Asterids, and Rosids), stems are regenerated bearing abnormal leaves; the normal leaf shape is gradually recovered. Some of the transient leaf developmental defects observed are similar to responses to mutations in leaf shape or shoot apical meristem function. Many species temporarily express this leaf development pathway, which is manifest in exceptional circumstances such as during recovery from excision of all preformed shoot meristems of a seedling.     

The Role of Meristematic Activities in the Formation of Leaf Blades

Arabidopsis thaliana (L.) Heynh. Leaf primordia also have their own meristematic regions and meristematic activity is maintained in part of the leaf blade, in some case, even after maturation. Transgenic plants have been generated that have proved to be useful tools in the analysis of the behavior of meristematic regions in leaf blades of A. thaliana. This review, based on our present understanding of molecular mechanisms for the maintenance and development of shoot apical meristems in A. thaliana, summarizes the variations in patterns and functions of meristematic regions in leaf blades focusing, in particular, on the case of indeterminate leaves. Received 5 April 2000/ Accepted in revised form 12 April 2000     

Gradual Depletion of 2,4-D in the Culture Medium for Indirect Shoot Regeneration from Leaf Explants of Camellia sinensis (L.) O. Kuntze

Adventitious shoot regeneration via callus phase from in vitro leaf explants is reported for the first time in tea. Callus was obtained on Murashige and Skoog medium supplemented with varied concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) (2.5, 5.0, 7.5 and 10.0 mg/l). Rhizogenesis was observed at all concentrations of 2,4-D. Adventitious shoot buds developed indirectly on leaf explants after prolonged culture for 16 weeks on medium supplemented with 10.0 mg/l 2,4-D. GC analysis of the medium and the tissues at different stages of development showed that specific levels of 2,4-D in the tissue were responsible for morphogenesis. Shoot buds developed on rhizogenic calli, only when 2,4-D declined to undetectable or negligible concentrations in the tissue probably due to detoxification and metabolism. Alternatively, shoot buds could also be evoked when rhizogenic calli were transferred to medium supplemented with low concentration of 2,4-D (1.5 mg/l). The adventitious nature of the shoots was confirmed through histological studies.     

Self-shading, carbon gain and leaf dynamics: a test of alternative optimality models

A simple model of shoot-level carbon gain is presented addressing the optimal number and life span of leaves in relation to alternative optimality criteria: (1) maximizing carbon export from the shoot, or (2) maximizing the rate of leaf production at the shoot tip. Additionally, the processes that cause declining assimilation with leaf age are considered in relation to (1) leaf position on the shoot (e.g., self-shading) versus (2) leaf age per se. Using these alternative scenarios, only a model based on position-dependent assimilation and maximization of leaf production rates resulted in quantitative predictions for all aspects of leaf dynamics on the shoot (i.e., leaf number, life span, and birth rate), while other approaches predicted that one or more parameters would be infinite. This formulation of the model also predicted that leaves should be maintained on the shoot until the diurnal carbon balance declines to zero, in contrast with other scenarios which predict that leaves should be shed while maintaining a positive carbon balance. Predictions of the model were supported by the results of a field study of carbon gain and leaf dynamics in saplings of three species of tropical pioneer trees (Carica papaya, Cecropia obtusifolia, and Hampea nutricia) which differ in the number of leaves per shoot. The results illustrate that in these fast-growing plants, leaf production and height growth may be more appropriate measures of performance than net carbon export from the shoot, and suggest that leaf senescence is primarily a function of the position of a leaf within the canopy, rather than its chronological age. Received: 13 October 1998 / Accepted: 27 January 1999     

The<Emphasis Type="Italic"> Arabidopsis thaliana rlp</Emphasis> mutations revert the ectopic leaf blade formation conferred by activation tagging of the<Emphasis Type="Italic"> LEP</Emphasis> gene

Activation tagging of the gene LEAFY PETIOLE ( LEP) with a T-DNA construct induces ectopic leaf blade formation in Arabidopsis, which results in a leafy petiole phenotype. In addition, the number of rosette leaves produced prior to the onset of bolting is reduced, and the rate of leaf initiation is retarded by the activation tagged LEP gene. The ectopic leaf blade results from an invasion of the petiole region by the wild-type leaf blade. In order to isolate mutants that are specifically disturbed in the outgrowth of the leaf blade, second site mutagenesis was performed using ethane methanesulphonate (EMS) on a transgenic line that harbours the activation-tagged LEP gene and exhibits the leafy petiole phenotype. A collection of revertant for leafy petiole ( rlp) lines was isolated that form petiolated rosette leaves in the presence of the activated LEP gene, and could be classified into three groups. The class III rlp lines also display altered leaf development in a wild-type (non-transgenic) background, and are probably mutated in genes that affect shoot or leaf development. The rlp lines of classes I and II, which represent the majority of revertants, do not affect leaf blade outgrowth in a wild-type (non-transgenic) background. This indicates that LEP regulates a subset of the genes involved in the process of leaf blade outgrowth, and that genetic and/or functional redundancy in this process compensates for the loss of RLP function during the formation of the wild-type leaf blade. More detailed genetic and morphological analyses were performed on a selection of the rlp lines. Of these, the dominant rlp lines display complete reversion of (1) the leafy petiole phenotype, (2) the reduction in the number of rosette leaves and (3) the slower leaf initiation rate caused by the activation-tagged LEP gene. Therefore, these lines are potentially mutated in genes for interacting partners of LEP or in downstream regulatory genes. In contrast, the recessive rlp lines exhibit a specific reversion of the leafy petiole phenotype. Thus, these lines are most probably mutated in genes specific for the outgrowth of the leaf blade. Further functional analysis of the rlp mutations will contribute to the dissection of the complex pathways underlying leaf blade outgrowth.Communicated by G. Jürgens     

Molecular Analysis of In Vitro Shoot Organogenesis

Shoot organogenesis is one of the in vitro plant regeneration pathways. It has been widely employed in plant biotechnology for in vitro micropropagation and genetic transformation, as well as in study of plant development. Morphological and physiological aspects of in vitro shoot organogenesis have already been extensively studied in plant tissue culture for more than 50 years. Within the last ten years, given the research progress in plant genetics and molecular biology, our understanding of in vivo plant shoot meristem development, plant cell cycle, and cytokinin signal transduction has advanced significantly. These research advances have provided useful molecular tools and resources for the recent studies on the genetic and molecular aspects of in vitro shoot organogenesis. A few key molecular markers, genes, and probable pathways have been identified from these studies that are shown to be critically involved in in vitro shoot organogenesis. Furthermore, these studies have also indicated that in vitro shoot organogenesis, just as in in vivo shoot development, is a complex, well-coordinated developmental process, and induction of a single molecular event may not be sufficient to induce the occurrence of the entire process. Further study is needed to identify the early molecular event(s) that triggers dedifferentiation of somatic cells and serves as the developmental switch for de novo shoot development.     

Factors Influencing Somatic Embryogenesis Induction and Plant Regeneration with Particular Reference to Arabidopsis thaliana (L.) Heynh

The broad applications of somatic embryogenesis, both in basic and applied research, have stimulated studies on the determination of in vitro conditions for the induction of somatic embryos and their conversion into plants. As a result, efficient protocols on SE induction and plant regeneration have recently become available for many plant species, including Arabidopsis thaliana (L.) Heynh., a model plant in genetics and embryogenesis.Studies on factors controlling in vitro plant morphogenesis are highly desirable not only for the development of improved regeneration systems, but also for the analysis of molecular mechanisms underlying plant embryogenesis. This review focuses on the conditions influencing the induction of embryogenic potential in in vitro cultured plant cells. The roles of explant type, endo- and exogenous plant growth regulators and stress factors in the induction of somatic embryogenesis are especially emphasized. Possible mechanisms by which different factors induce or modify embryogenic competence in cultured plant cells are also discussed. Since the production of genetically solid and true-to-type plants is desired, especially for transformation and micropropagation practice, the problem of the genetic characteristics of regenerants, in terms of their chimerism and somaclonal variation, is discussed in some detail.Special consideration is given to A. thaliana– a major model plant species for classical genetics and genomics. Recent availability of efficient embryogenic cultures in this organism makes it possible to benefit from advanced genomic research of Arabidopsis to study plant embryogenesis on the molecular level.     

Formation, Maintenance and Function of the Shoot Apical Meristem in Rice

In higher plants, the process of embryogenesis establishes the plant body plan (body axes). On the basis of positional information specified by the body axes, the shoot apical meristem (SAM) and root apical meristem (RAM) differentiate at fixed positions early in embryogenesis. After germination, SAM and RAM are responsible for the development of the above-ground and below-ground parts, respectively, of the plant. Because of the importance of SAM function in plant development, the mechanisms of SAM formation during embryogenesis and of SAM maintenance and function in post-embryonic development are priority questions in plant developmental biology. Recent advances in molecular and genetic analysis of morphogenetic mutations in Arabidopsis have revealed several components required for SAM formation, maintenance and function. Although these processes are fundamental to the life cycle of every plant, conservation of the components does not explain the diversity of plant morphologies. Rice is used as a model plant of the grass family and of monocots because of the progress in research infrastructure, especially the collection of unique mutations and genome information. In comparison with the dicot Arabidopsis, rice has many unique organs or processes of development. This review summarizes what is known of the processes of SAM formation, maintenance and function in rice.     

Transformation of plants via the shoot apex

Summary We have transformed petunia byAgrobacterium tumefaciens containing genes for kanamycin resistance and beta-glucuronidase using isolated shoot apices from seedling tissue. Regeneration of transformed plants in this model system was rapid. The technique of shoot apex transformation is an alternative for use inAgrobacterium-mediated transformation of dicotyledonous crop species for which a method of regeneration via protoplasts, leaf disks, or epidermal strips does not exist. This approach offers direct and rapid regeneration of plants and low risk of tissue-culture-induced genetic variation. Texas Agricultural Experiment Station Technical Article No. 23317.