Plant Developmental Biology

Originally coined to emphasize morphological differences, ‘microphyll’ and ‘megaphyll’ became synonymous with the idea that vascular plant leaves are not homologous. Although it is now accepted that leaves evolved independently in several euphyllophyte lineages, ‘megaphyll’ has grown to reflect another type of homology, that of euphyllophyte leaf precursor structures. However, evidence from the fossil record and developmental pathways fails to indicate homology and suggests homoplasy of precursor structures. Thus, as I discuss here, ‘megaphyll’ should be abandoned because it perpetuates an unsupported idea of homology, leading to misconceptions that pervade plant biology thinking and can bias hypothesis and inference in developmental and phylogenetic studies. Alternative definitions are needed that are based on development and phylogeny for different independently evolved leaf types.

The microsporangia (anther) and male gametophyte development were observed in Canna generalis Bailey, an economically important plant from the Order Zingiberales, which also includes bananas (Musa spp.). This was done by using the following techniques: light microscopy, scanning electron microscopy, acetocarmine squash and smear method and pollen tube growth induction by sucrose solution. It was observed that there are distinct changes in the morphology of the anther as it matures as evident with the maturation of sporogenous cells into pollen grains and the disintegration of the septa and tapetum. It was also observed that the male gametophyte or pollen grain development in C. generalis follows the basic or typical monocot type of development with microsporogenesis (formation of spores) preceding microgametogenesis (formation of gametes). It was also observed that pollen grain development is similar with other members of Zingiberales most especially with regards to the tetrad formation: isobilateral or T-shape. The mature pollen grain is around 87.98 μm in size, spherical in shape and has two distinct layers: a thick outer exine with spinules and a smooth inner intine. Pollen tube growth can occur at any site and was induced by soaking the pollen grain under 30% sucrose solution for an hour.

Keywords: microsporangia, microsporogenesis, microgametogenesis, pollen grain, pollen tube,
Canna generalis

Global food security is vulnerable due to massive growth of the human population, changes in global climate, the emergence of novel/more virulent pathogens, and demands from increasingly discerning consumers for chemical-free, sustainably produced food products. Bacterium- based biological control agents (BCAs), if used as part of an integrated management system, may satisfy the above demands. We focus on the advantages, limitations, problems, and challenges involved in such strategies.

Ustilago maydis is a biotrophic pathogen and well-established genetic model to understand the molecular basis of biotrophic interactions. U. maydis suppresses plant defense and induces tumors on all aerial parts of its host plant maize. In a previous study we found that U. maydis induced leaf tumor formation builds on two major processes: the induction of hypertrophy in the mesophyll and the induction of cell
division (hyperplasia) in the bundle sheath. In this study we analyzed the cell-type specific transcriptome of maize leaves 4 days post infection. This analysis allowed identification of key features underlying the hypertrophic and hyperplasic cell identities derived from mesophyll and bundle sheath cells, respectively. We examined the differentially expressed (DE) genes with particular focus on maize cell cycle genes and found that three A-type cyclins, one B-, D- and T-type are upregulated in the hyperplasic tumorous cells, in which the U. maydis effector protein See1 promotes cell division. Additionally, most of the proteins involved in the formation of the pre-replication complex (pre-RC, that assure that each daughter cell receives identic DNA copies), the transcription factors E2Fand DPa as well as several D-type cyclins are deregulated in the hypertrophic cells.

The adaptive success of living organisms depends on the maintenance of a dynamic equilibrium between creating new genetic combinations and fixing those which are more adapted to the present environment. Sexual reproduction is universally the main route to recombine genes in the short term; on the other hand, different strategies have been adopted to fix the genetic composition of individuals demonstrating high fitness. In plants, genotypes may be “immortalized” via vegetative propagation or “photocopied” by selfing of highly homozygous individuals. A third, more technically sophisticated pathway is represented by the implementation of apomixis, where a functional sexual machine is short-circuited to asexually produce embryos with the fixed genotype of the mother plant. This developmental sophistication represents a challenging research field for the reproduction biologist and a desirable trait for the plant breeder to be used in seed production schemes of elite varieties.

Leaf shape formation is a fascinating process involving the coordinated regulation of cell division and cell expansion, which ultimately relies on the control of cell wall expansion. Expansins comprise a large family of cell wall proteins which can non-enzymatically trigger the relaxation of the cell wall for expansion. They have been implicated in organ initiation, cell differentiation and organ growth. However, the exact mechanism of expansin action and functional role of different members of the expansin gene family is unclear.
Here, I focus on the largest subgroup of expansins (EXPA) to identify which genes are expressed during different stages of leaf development in Arabidopsis. This information was used to design an inducible artificial microRNA construct to target the knock-down of multiple expansin genes expressed during early leaf development. Through a series of experiments knocking down expansin gene expression at different stages of leaf development, I show that the suppression of expansin gene expression at later stages of leaf development is sufficient to repress leaf growth and alter leaf shape. This molecular genetic approach is combined with an atomic force microscopy (AFM) approach to investigate the role of cell wall mechanics in leaf growth. I describe the use of AFM for probing the leaf primordium mechanics and outline consideration for future work.

• Premise of the Study: Secondary xylem (wood) produced by a vascular cambium supports increased plant size and underpins
the most successful model of arborescence among tracheophytes. Woody plants established the extensive forest ecosystems that dramatically changed the Earth’s biosphere. Secondary growth evolved in several lineages in the Devonian, but only two occurrences have been reported previously from the Early Devonian. The evolutionary history and phylogeny of wood production are poorly understood, and Early Devonian plants are key to illuminating them.
• Methods: A fossil plant preserved anatomically by cellular permineralization in the Lower Devonian (Emsian, ca. 400–395
million years old) Battery Point Formation of Gaspé Bay (Quebec, Canada) is described using the cellulose acetate peel
technique.
• Key Results: The plant, Franhueberia gerriennei Hoffman et Tomescu gen. et sp. nov., is a basal euphyllophyte with a centrarch protostele and metaxylem tracheids with circular and oval to scalariform bordered multiaperturate pits (P-type tracheids). The outer layers of xylem, consisting of larger-diameter P-type tracheids, exhibit the features diagnostic of secondary xylem: radial fi les of tracheids, multiplicative divisions, and a combination of axial and radial components.
• Conclusions: Franhueberia is one of the three oldest euphyllophytes exhibiting secondary growth documented in the Early Devonian. Within the euphyllophyte clade, these plants represent basal lineages that predate the evolution of stem-leaf-root organography and indicate that underlying mechanisms for secondary growth became part of the euphyllophyte developmental toolkit very early in the clade’s evolution.

Expansins are cell wall proteins implicated in the control of plant growth via loosening of the extracellular matrix. They are encoded by a large gene family, and data linked to loss of single gene function to support a role of expansins in leaf growth remain limited. Here, we provide a quantitative growth analysis of transgenics containing an inducible artificial microRNA construct designed to down-regulate the expression of a number of expansin genes that an expression analysis indicated are expressed during the development of Arabidopsis (Arabidopsis thaliana) leaf 6. The results support the hypothesis that expansins are required for leaf growth and show that decreased expansin gene expression leads to a more marked repression of growth during the later stage of leaf development. In addition, a histological analysis of leaves in which expansin gene expression was suppressed indicates that, despite smaller leaves, mean cell size was increased. These data provide functional evidence for a role of expansins in leaf growth, indicate the importance of tissue/organ developmental context for the outcome of altered expansin gene expression, and highlight the separation of the outcome of expansin gene expression at the cellular and organ levels.

In multicellular organisms, the coordination of cell behaviors largely relies on biochemical and biophysical signals. Understanding how such signals control development is often challenging, because their distribution relies on the activity of individual cells and, in a feedback loop, on tissue behavior and geometry. This review focuses on one of the best-studied structures in biology, the shoot apical meristem (SAM). This tissue is responsible for the production of all the aerial parts of a plant. In the SAM, a population of stem cells continuously produces new cells that are incorporated in lateral organs, such as leaves, branches, and flowers. Organogenesis from stem cells involves a tight regulation of cell identity and patterning as well as large-scale morphogenetic events. The gene regulatory network controlling these processes is highly coordinated in space by various signals, such as plant hormones, peptides, intracellular mobile factors, and mechanical stresses. Many crosstalks and feedback loops interconnecting these pathways have emerged in the past 10 years. The plant hormone auxin and mechanical forces have received more attention recently and their role is more particularly detailed here. An integrated view of these signaling networks is also presented in order to help understanding how robust shape and patterning can emerge from these networks.

Germination of spores, growth and development of gametophytes in Pteris Vittata were investigated mainly to study sexual development and morphology of the gametophyte. Fifty spores of Pteris Vittata were sown in four sterile Petri dishes containing agar solidified Parker's medium and one Petri dish containing water which serves as the control. Germination of the spores, sexual development and gametophytes development were monitored daily using dissecting microscope. The mean percentage germination of the spores ranges from 90% to 100%. The results also showed that maximum germination of the spores took place by the end of the 3 rd week in all the Petri dishes except the control where no growth was observed. Germination is vittaria type while the prothallial development is Ceratopteris type. Gametophyte has slow growth rate and it is bisexual. Gametangia developments are of the common type for leptosporangiate ferns and the adult gametophytes were hairy. Archegonia were multiserate while antheridia were uniserate. There was high rate of germination of freshly harvested spores of P. Vittata in Parker's medium and this reveals the viability and suitability of this plant to be raised from spores whenever the need arises. These gametophyte morphological features are highly useful in the taxonomy of this plant.

Food security (a pressing issue for all nations) faces a threat due to population growth, land availability for growing crops, a changing climate (leading to increases in both abiotic and biotic stresses), heightened consumer awareness of the risks related to the use of agrichemicals, and also the reliance on depleting fossil fuel reserves for their production. Legislative changes in Europe mean that fewer agrichemicals will be available in the future for the control of crop pests and pathogens. The need for the implementation of a more sustainable
agricultural system globally, incorporating an integrated approach to disease management, has never been more urgent. To that end, the Valorizing Andean Microbial Diversity (VALORAM) project (http://valoram.ucc.ie), funded under FP7, examined the role of microbial
communities in crop production and protection to improve the sustainability, food security, environmental protection, and productivity for rural Andean farmers. During this work, microbial volatile organic compounds (mVOCs) of 27 rhizobacterial isolates were identified using gas chromatography/mass spectrometry (GC/MS), and their antifungal
activity against Rhizoctonia solani was determined in vitro and compared to the activity of a selection of pure volatile compounds. Five of these isolates, Pseudomonas palleroniana R43631, Bacillus sp. R47065, R47131, Paenibacillus sp. B3a R49541, and Bacillus simplex M3-4 R49538 trialled in the field in their respective countries of origin, i.e., Bolivia, Peru, and Ecuador, showed significant increase in the yield of potato. The strategy followed in the VALORAM project may offer a template for the future isolation and determination of putative biocontrol and plant growth-promoting agents, useful as part of a low-input integrated pest management system.

Expansins are cell wall proteins implicated in the control of plant growth via loosening of the extracellular matrix. They are encoded by a large gene family, and data linked to loss of single gene function to support a role of expansins in leaf growth remain limited. Here, we provide a quantitative growth analysis of transgenics containing an inducible artificial microRNA construct designed to down-regulate the expression of a number of expansin genes that an expression analysis indicated are expressed during the development of Arabidopsis (Arabidopsis thaliana) leaf 6. The results support the hypothesis that expansins are required for leaf growth and show that decreased expansin gene expression leads to a more marked repression of growth during the later stage of leaf development. In addition, a histological analysis of leaves in which expansin gene expression was suppressed indicates that, despite smaller leaves, mean cell size was increased. These data provide functional evidence for a role of expansins in leaf growth, indicate the importance of tissue/organ developmental context for the outcome of altered expansin gene expression, and highlight the separation of the outcome of expansin gene expression at the cellular and organ levels.

The concept of the discrete ontogenesis of plants, by the first, was made by T. А. Rabotnov (1950). Detailed ontogenetic  researches of tree plants  had  begun  since  1970th. At present,  the most  tree species  ontogeny have been described in the East-European forests.  Development of all plants has the universal periods that we divide into  the  structural-functional  stages.  The  last  ones  were  named  «ontogenetic  stages».  The  ontogenetic  stages mark  biological  age  of  plants.  Individuals  of different  species  show  quite  similar  enlistment  of  ontogenetic stages.  Each  stage  in  marked  by  specific  growth  and  morphological  characteristics.  The  plants  of  the  each ontogenetic  stage  can  be  distinguished  by  vitality  level.  Each  vitality  level  is  characterized  by specific  signs. The vitality of plants may be changed many times during ontogeny. The plants of different life forms and type of propagation have specific traits of ontogeny. The species show high individuality of ontogenetic behavior; it is clear especially in the heterogeneous natural forest community. These results we take into account as a basis for forecast of behavior of species and their groups in permanent changing mosaic environment of natural community.
Key  w ords:  ontogeny,  biological age,  ontogenetic  stages, vitality  level,  life forms.

The Schizosaccharomyces pombe Mei2 gene encodes an RNA recognition motif (RRM) protein that stimulates meiosis upon binding a specific non-coding RNA and subsequent accumulation in a "mei2-dot" in the nucleus. We present here the first systematic characterization of the family of proteins with characteristic Mei2-like amino acid sequences. Mei2-like proteins are an ancient eukaryotic protein family with three identifiable RRMs. The C-terminal RRM (RRM3) is unique to Mei2-like proteins and is the most highly conserved of the three RRMs. RRM3 also contains conserved sequence elements at its C-terminus not found in other RRM domains. Single copy Mei2-like genes are present in some fungi, in alveolates such as Paramecium and in the early branching eukaryote Entamoeba histolytica, while plants contain small families of Mei2-like genes. While the C-terminal RRM is highly conserved between plants and fungi, indicating conservation of molecular mechanisms, plant Mei2-like genes have changed biological context to regulate various aspects of developmental pattern formation.

This study reveals that anatomical development of anther wall and pollen in Averrhoa carambola L. flower. The developments, in different stages, were observed through a preparation by paraffin method, LR White resin method, and SEM. The preparation was observed using a light microscope. Results showed that anther derived from archesporial cells surrounded by epidermal cells. The archesporial cells divided into primary sporogenous cell and primary parietal cells. The former was then to form microspore mother cells that meiotically divided into dyad and tetrad, respectively. Pollen with one nucleus was released from the tetrad present in sporangia of the anther. The nucleus of the pollen was mitotically divided into pollen with two nuclei named vegetative and generative nuclei. The latter divided and differentiated into anther wall consisted of the epidermis, endothecium, two middle layers, and tapetum.

Tolerance to salinity varies throughout plant development. Stable phenophases with a relatively-defined level of tolerance are interspaced with transition periods during which, in some species, an increase in tolerance may be achieved. This adaptive response involves changes at many levels of biological organization, from gene expression to inter-organ relationships. They include overexpression of protective proteins, control of Na+ uptake, changes in photosynthetic metabolism, restructuring of the hormonal balance and modification of the cellular sensitivity to growth factors. Individuality is endemic to this adaptive response, and results from an extreme sensitivity to environmental and developmental factors during its maturation. For this reason, the adaptive response is interpreted as an expression of the nonlinear dynamics characterizing the transitory period between two stable developmental phases. Accordingly, plant response to salinity is reconsidered as the mutual expression of a pre-existing (genetic) component and the self-organized dynamics of its rearrangement. 14.1 Towards a non-linear approach of plant response to salinity Exposure to salinity induces modifications at all levels of biological organization, from gene expression to whole plant physiology and development. Hundreds of reports are focused on a given change, comparing tile response of salt-tolerant and salt-sensitive species or genotypes. However, it remains difficult to integrate all these modifications into a general framework of plant response to stress. Concerning salt tolerance, a cellular basis is observed in some species but not in others, and growth inhibition is not always proportional to tile accumulation of Na+ and cr ions in the shoot (see below). It seems, therefore, that there are many ways to cope with presence of sublethal concentrations of NaCI in the root medium, disproving any unifying scheme for tolerance. Nevertheless, plant tolerance is assumed to be a stable function of the genetic 291 A. Lduchli and U. Liittge (eds.), Salinity: Environment-Plants> Molecules, 291-312.

Phosphate (Pi) availability is a significant limiting factor for plant growth and productivity in both natural and agricultural systems. To cope with such limiting conditions, plants have evolved a myriad of developmental and biochemical strategies to enhance the efficiency of Pi acquisition and assimilation to avoid nutrient starvation. In the past decade, these responses have been studied in detail at the level of gene expression; however, the possible epigenetic components mod- ulating plant Pi starvation responses have not been thoroughly investigated. Here, we report that an extensive remodeling of global DNA methylation occurs in Arabidopsis plants exposed to low Pi avail- ability, and in many instances, this effect is related to changes in gene expression. Modifications in methylation patterns within genic regions were often associated with transcriptional activation or repression, revealing the important role of dynamic methylation changes in mod- ulating the expression of genes in response to Pi starvation. Moreover, Arabidopsis mutants affected in DNA methylation showed that changes in DNA methylation patterns are required for the accurate regulation of a number of Pi-starvation–responsive genes and that DNA methylation is necessary to establish proper morphological and physiological phosphate starvation responses.

Proteas are woody perennial plants that develop large, complex inflorescences which are popular as cut flowers.
Four paralogous geneswith sequence similarity to the meristemidentity gene LEAFY (LFY) of Arabidopsis thaliana
were identified in the Protea cultivar ‘Carnival’ (Protea compacta x Protea neriifolia). The deduced amino acid
sequences of Protea LFY (ProLFY) paralogues displayed highly conserved N- and C-terminal regions comparable
to that of other LFY proteins. Paralogous sequences shared 97–99% similarity at nucleotide and deduced amino
acid levels. To determine if these ProLFY paralogues are involved in floral transition and development, three
paralogues were heterologously expressed in A. thaliana Col-0 wild-type and lfy-2 mutant plants. These three
paralogueswere chosen as they had the greatest sequence dissimilarities which may suggest a different function.
Expression of the ProLFY-WL4 paralogue rescued the low fertility of the lfy-2 mutant by restoring seed yield to
wild-type levels, and resulted in the conversion of ‘leaf-like’ inflorescences to flowers with a wild-type appearance.
ProLFY-WL4 expression levels during ‘Carnival’ meristem development confirmed a role for this paralogue
in inflorescence development. The other two paralogues used in heterologous A. thaliana expression studies,
ProLFY-L5 and ProLFY-D1, did not rescue fertility or the abnormal floral phenotype of lfy-2. However, transgenic
plants developed either smaller leaves when expressing 35S::ProLFY-L5, or larger leaves when expressing
35S::ProLFY-D1, compared to the non-transgenic lfy-2 plants. Together these results suggest that ProLFY
paralogues may be involved in both floral and vegetative development of Protea.

A predominantly plant-based family of genes encoding RNA binding proteins is defined by the presence of a highly conserved RNA binding motif first described in the mei2 gene of the fission yeast Schizosaccharomyces pombe. In silico analyses reveal nine mei2 -like genes in Arabidopsis thaliana and six in Oryza sativa. These predicted genes group into four distinct clades, based on overall sequence similarity and subfamily-specific sequence elements. In situ analysis show that Arabidopsis genes from one of these clades, TEL1 and TEL2, are specifically expressed in central zone of the shoot apical meristem and the quiescent center of the root apical meristem, suggesting that they may somehow function to maintain indeterminacy in these tissues. By contrast, members of two sister clades, AML1 through AML5, are expressed more broadly, a trend that was confirmed by Q-PCR analysis. mei2 -like transcripts with similar sequences showed similar expression patterns, suggesting functional redundancy within the four clades. Phenotypic analyses of lines that contain T-DNA insertions to individual mei2 -like genes reveal no obvious phenotypes, further suggesting redundant activities for these gene products.

The negative gravitropic response in the grass leaf-sheath pulvinus is a consequence ofcell elongation involving c// cells except those of the uppermost region of the upper flank of an horizontally orientfi pulvinus. Thi lowermost layer of cells elongate maximally, and the regions in between elongate to intermediate extents. The resulting curvatures ofa responding_pulvinus can be exprissed mathematicalyly relating the angle ofcurvature (d) to the original length (zo).and the maximal length of the lower surface (Lri and thJdiametir of the organ (D)' using the equation, 0 : (Lr-L)lD, where d ii in radians. The elongation response (^9) of any individial cell within the pulvinus can be expressed by the equation, S : 0.5-r cos d, where r is the radius ofihe pulvinus and_d is in degrees. Microscopic measurement of ceil lengths in ditrerent iegions of the pulvinus supiorts the mathematical predictions. Indirect support is also obtained from the use Jfcolchicine, coumarin, dichloro-benzonitrile (DCBN) and iso_propyl N-chlorophenyl carbamate which exaggerate the inherent asymmetry during gravitropic response. Coumarin and DCBN also induce thickenings ii the radial walls whicir appeai first in the statenchyma, and later, in cells located towards the outer periphJry ofthe pulvinus. The distribution patterns.of these thickenings-suggest that the asymmetric growth iespbnse-of the pulvinus may be due to a differential and radial, centrifugal transport ofgrowth pro;otors from the central statenchyma region. rNinooucrloN When young shoots and roots are reoriented from their normal vertical positions, perception of the now altered position in relation to the gravitational vectoi elicits a response of differential growth that causes the organs to return to their original positions. Sachs (1887) was the first to make a lucid analysis of the geometry of ihe giavitropic ql_o-wth r€sponse. Recently, Firn and Digby (1980) have pointed oui the imp.-ortance of differential growth response on the upper and lower flanks of a horizontaily oriented organ' We intend to show that the response of att the cells in the upper and lower flanks of horizontally placed organs is of significance for the understanding of the perception phase and the geometry of growth during the response phase. Our cuirent study is based on the grass leaf-sheath pulvinus (referred to aJpulvinus hereafter). The struiture and gravitropic response of the pulvinus in different grasses have been previously reported (Bridges and wilkins, !973; Dayanandan, Hebard and Kaufman, i976; Dayanandan, 1977; osborne and Wright, 1977). rn this paper, we present a detailed quantitative analysis of the growth response in the pulvini ofbats andbarley, and report on inhibitors that exaggerate the asymmetric growth response in these and ofher grasses. It is suggested th^at the_observed growth patterns and effects of inhibitors may beiestablished b/ireans of a radial transport of growth promoters in these pulvini. 29 030s-736418410r0029 + 16 $03.00/0

African nightshades (Solanum nigrum-related species) are important traditional
green leafy vegetables whose yields are limited by prolific early flowering and
excessive berry set in competition with leaf production. As a step towards elimination
of this competition, male-sterile lines were developed through ion-beam irradiation.
Locally collected S. villosum seeds from Kenyan farmers were subjected to 220 MeV
12C5+ ion beam irradiation at the Japan Atomic Energy Research Institute (JAERI),
Gunma, Japan. At the M2 progeny, four male-sterile mutant types (T-1, T-2, T-3, T-4)
similar to those reported from a previous 􀈖-ray mutation were isolated. In addition, a
novel abnormal floral organ mutant (T-5) was isolated and its flower structure further
observed throughout the year. The mutant flowers were sepaloid in mid-spring,
stamenless in late-spring, indeterminate in summer, partially restored with seeded
berries in autumn, but the florets aborted at the bud stage in winter. The M3 progeny
also showed a similar pattern. These phenotypes suggest a loss or impairment of
function of one or more of B, C or E genes, the activation or repression of which seems
to be under environmental influence. Partial fertility restoration and the resulting
berry and seed set observed in autumn is advantageous for maintenance and seed
propagation while the leaf-like flowers in spring, summer and winter form part of the
vegetative yield. In a field trial, the T-5 had higher shoot-tip fresh and dry weights
compared to wild-type (W-T) and other male-sterile mutants.

"Background and Aims Leaf venation in many C4 species is characterized by high vein density, essential in facilitating rapid intercellular diffusion of C4 photosynthetic metabolites between different tissues (mesophyll, bundle sheath). Greater vein density has been hypothesized to be an early step in C4 photosynthesis evolution. Development of C4 vein patterning is thought to occur from either accelerated or prolonged procambium formation, relative to ground tissue development.

Methods Cleared and sectioned tissues of phylogenetically basal C3 Flaveria robusta and more derived C4 Flaveria bidentis were compared for vein pattern in mature leaves and vein pattern formation in developing leaves.

Key Results In mature leaves, major vein density did not differ between C3 and C4 Flaveria species, whereas minor veins were denser in C4 species than in C3 species. The developmental study showed that both major and minor vein patterning in leaves of C3 and C4 species were initiated at comparable stages (based on leaf length). An additional vein order in the C4 species was observed during initiation of the higher order minor veins compared with the C3 species. In the two species, expansion of bundle sheath and mesophyll cells occurred after vein pattern was complete and xylem differentiation was continuous in minor veins. In addition, mesophyll cells ceased dividing sooner and enlarged less in C4 species than in C3 species.

Conclusions Leaf vein pattern characteristic to C4 Flaveria was achieved primarily through accelerated and earlier offset of higher order vein formation, rather than other modifications in the timing of vein pattern formation, as compared with C3 species. Earlier cessation of mesophyll cell division and reduced expansion also contributed to greater vein density in the C4 species. The relatively late expansion of bundle sheath and mesophyll cells shows that vein patterning precedes ground tissue development in C4 species."