JXB Advance Access originally published online on July 26, 2006
Journal of Experimental Botany 2006 57(12):3019-3031; doi:10.1093/jxb/erl063
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© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Mutations in the RETICULATA gene dramatically alter internal architecture but have little effect on overall organ shape in Arabidopsis leaves
1División de Genética and Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, E-03202 Elche, Spain
2Plant Science Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK
3School of Human and Life Sciences, Roehampton University, Holybourne Avenue, London SW15 4J, UK
4División de Genética, Universidad Miguel Hernández, Campus de San Juan, E-03550 Alicante, Spain
To whom correspondence should be addressed. E-mail: jlmicol{at}umh.es
Received 29 March 2006; Accepted 24 May 2006
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Abstract |
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A number of mutants have been described in Arabidopsis, whose leaf vascular network can be clearly distinguished as a green reticulation on a paler lamina. One of these reticulate mutants was named reticulata (re) by Rédei in 1964 and has been used for years as a classical genetic marker for linkage analysis. Seven recessive alleles of the RE gene were studied, at least four of which seem to be null. Contrary to many other leaf mutants studied in Arabidopsis, very little pleiotropy was observed in the external morphology of the re mutants, whose only aberration obvious at first sight is the reticulation exhibited by cotyledons and leaves. The re alleles caused a marked reduction in the density of mesophyll cells in interveinal regions of the leaf, which does not result from perturbed plastid development in specific cells, but rather from a dramatic change in internal leaf architecture. Loss of function of the RE gene seems to specifically perturb mesophyll cell division in the early stages of leaf organogenesis. The leaves of re mutants were nearly normal in shape in spite of their extremely reduced mesophyll cell density, suggesting that the epidermis plays a major role in regulating leaf shape in Arabidopsis. The RE gene was positionally cloned and found to be expressed in all the major organs studied. RE encodes a protein of unknown function and is identical to the LCD1 gene, which was identified based on the increased sensitivity to ozone caused by its mutant allele lcd1-1. Double mutant analyses suggest that RE acts in a developmental pathway that involves CUE1 but does not include DOV1.
Key words: Arabidopsis, leaf organogenesis, mesophyll development, reticulata
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Introduction |
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The correct development of plant leaf internal tissues is crucial to leaf function in order to optimize gaseous exchange and light capture. However, the nature of the developmental mechanisms that underlie the construction of the internal leaf architecture from different leaf cell types is unclear (reviewed in Fleming, 2002; Tsukaya, 2002, 2003, 2005; Tsiantis and Hay, 2003; Byrne, 2005; Canales et al., 2005). A detailed genetic dissection of the development of the internal anatomy of leaves is required, and although specific epidermal cell types (Larkin et al., 1997) and vascular cell differentiation (Carland and McHale, 1996; Nelson and Dengler, 1997; Candela et al., 1999; Ye et al., 2002) have been studied in detail, an understanding of the interplay of epidermal, palisade mesophyll, spongy mesophyll, and vascular development in constructing internal leaf structure and whole leaf shape is lacking (Pyke and López-Juez, 1999).
A problem with screening approaches that require the isolation of mutants specifically perturbed in leaf internal architecture is the prediction of a clear morphological leaf phenotype which will reveal an underlying change in the anatomical structure of the organ. Multiple sectioning as a screening method is likely to be both arduous and problematic, especially in comparing tissues of equivalent developmental status. In addition, pleiotropic effects on leaf morphogenesis may result from altered cell physiology and consequently blur the distinction between leaf-specific mutants and general developmental mutants (Poethig, 1997; Bohmert et al., 1998; Berná et al., 1999).
Morphological mutants of Arabidopsis thaliana have been examined, in which the vascular network stands out as a colour difference on the lamina. Three broad phenotypic classes exist: a dark green vascular pattern on a green lamina, a green vascular pattern on a pale lamina, and a pale vascular pattern on a green lamina (EA Kinsman, KA Pyke, unpublished data). Although many reticulate mutants are present in stock centres and have been reported in different mutant collections (Li et al., 1995; Berná et al., 1999; Serrano-Cartagena et al., 1999; Streatfield et al., 1999), the relationship of this phenotype to the underlying internal leaf structure has been characterized in only a very few cases.
reticulata (re) was the first mutant with reticulate leaves isolated in Arabidopsis (Rédei and Hirono, 1964). It has been used for decades as a classical morphological mapping line because of the ease of recognition and low degree of pleiotropy of its phenotype. The characterization of re and another six mutant alleles of the RE gene is reported here. The results indicate that re mutations dramatically affect mesophyll cell proliferation, but only slightly change overall leaf shape, which suggests that the correct development of the mesophyll tissues contributes little to leaf shape.
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Materials and methods |
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Plant material and growth conditions
Seeds of the Arabidopsis thaliana (L.) Heynh. wild-type accessions Landsberg erecta (Ler), Columbia-0 (Col-0), Wassilewskija-2 (Ws-2), and Enkheim-2 (En-2), as well as the N129 (which carries the re-1 and glabrous1 mutations) and N734 (re-2) mutant lines, were obtained from the Nottingham Arabidopsis Stock Centre (NASC). They were grown for the morphological and anatomical analyses illustrated in Figs 2, 5, 6, and Table 2, as described in Kinsman and Pyke (1998). All the remaining cultures were performed as described in Ponce et al. (1998). The re-3 and re-4 mutants were isolated as described in Berná et al. (1999), and re-5 was identified in a collection of alleles kindly provided by Javier Paz-Ares. Lines carrying the re-6 and re-7 alleles were supplied by the NASC and are described at http://signal.salk.edu. Crosses were performed as described by Berná et al. (1999).
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The genetic backgrounds of the mutants are shown in Table 1. Although the genetic background of re-1 was assumed to be Col, based on the information available at the NASC, it was found to be a hybrid, as determined by genotyping microsatellite markers, as described in Ponce et al. (1999). Among the 20 microsatellites genotyped in re-1, 11 were homozygous for the Col-0 allele, 5 for the Ler allele, and 4 for alleles unequivocally different from those of Ler and Col-0. These four alleles could not be assigned to any of the 27 wild-type accessions that were genotyped in previous works (Quesada et al., 2002; Pérez-Pérez et al., 2002).
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Morphological and ultrastructural analyses
Methods for leaf clearing and fixation, embedding, quantification of leaf and cellular anatomical features, and observation of leaf tissue by confocal microscopy are as described previously (Kinsman and Pyke, 1998; Candela et al., 1999). Venation pattern diagrams were obtained by drawing leaf veins on the screen of a Wacom Cintiq 18SX Interactive Pen Display and using the Adobe Photoshop 5.0 program.
Leaf cell fixation and separation were performed as described previously (Pyke and Leech, 1992; Kinsman and Pyke, 1998). Suspensions of separated cells were tapped out from pieces of processed leaf tissue mounted in 0.1 M Na2EDTA on microscope slides. Numbers of chloroplasts per cell were counted by eye by viewing with Nomarski microscope optics, and areas of individual cells and plastids were measured using the Lucia image analysis system.
For scanning electron microscopy, plant material was prepared as described in Serrano-Cartagena et al. (2000). Micrographs were taken in a JSM-840 JEOL scanning electron microscope.
Positional cloning and molecular characterization of the re mutations
High-resolution mapping of the re-3 mutation was performed using SSLP markers (Bell and Ecker, 1994) as described in Ponce et al. (1999). For allele sequencing, PCR products covering the At2g37860 transcription unit were obtained using as a template DNA extracted from Col-0, Ler, En-2, and re-2, re-3, re-4, and re-5 homozygous plants. Sequencing reactions were carried out with ABI PRISM BigDye Terminator Cycle Sequencing kits in 5 µl reaction volumes. Sequencing electrophoreses were performed on an ABI PRISM 3100 Genetic Analyser.
To confirm the presence and position of T-DNA inserts, DNA was extracted from the N584529 and N537745 lines and PCR amplified using the primers shown in supplementary Table 1 at JXB online, and both strands of the amplification products obtained were sequenced. Southern blot analyses were performed as previously described (Pérez-Pérez et al., 2004), after ScaI or BamHI restriction of genomic DNA extracted from Col-0, Ler and re-1 and re-3 homozygous plants. Sequences of the primers used to PCR synthesize the probe are shown in supplementary Table 1 at JXB online.
Gene expression analyses
For semi-quantitative reverse-transcriptase PCR (RT-PCR) analysis, RNA was extracted from plant material (50–100 mg), reverse transcribed using random hexanucleotide primers, and PCR amplified as described in Quesada et al. (1999). Sequences of the gene-specific primers used are detailed in supplementary Table 1 at JXB online. Control reactions using primers for the ORNITHINE TRANSCARBAMILASE (OTC) gene (Quesada et al., 1999) were performed on the same cDNA samples.
To produce the PRE:GUS construct, the complete genomic region (0.5 kb) located upstream of the RE transcription unit and downstream of the preceding gene (At2g37840) was PCR amplified using Col-0 genomic DNA as a template and the pGusAt2g37860F and pGusAt2g37860R primers, cloned into the pGEM-3Zf(+) vector and transferred to the pDW137 binary vector, which contains the ß-Glucuronidase (GUS) reporter gene (Blázquez et al., 1997). The PRE:GUS construct was mobilized into Agrobacterium tumefaciens LBA4404 cells, and fully sequenced to confirm its structural integrity prior to being transferred into Col-0 plants by the floral dip method (Clough and Bent, 1998). Transgenic plants were selected on 50 µg ml–1 kanamycin-supplemented medium. GUS staining was performed as described in Blázquez et al. (1997).
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Results |
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Leaf morphology of the re mutants
The mutants studied here are listed in Table 1. Crosses of the N129 line (carrying the re recessive mutation, here named re-1) to several other reticulate mutants from stock centres revealed a second recessive allele of RE, which was named re-2 (N734). Another three reticulate allelic mutants were obtained (Berná et al., 1999; this work) and initially named ven2-1 (venosa2-1), ven2-2, and ven2-3. Complementation tests demonstrated that these lines carried recessive alleles of the RE gene, for which reason they were renamed re-3, re-4, and re-5. After the positional cloning of the RE gene, two additional recessive alleles, re-6 and re-7, were obtained from a collection of knockouts.
The only abnormality obvious in the re mutants at the level of the overall plant form is the fact that their cotyledons, vegetative leaves, and cauline leaves display a conspicuous vein pattern, which is normally seen as a dark-green venation on a paler green background (Fig. 1). No obvious mutant phenotypic traits were found in the roots, the inflorescence, the floral organs, or the fruits of the re mutants (data not shown).
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All the re mutants displayed very similar leaf phenotypes, irrespective of their genetic background. A few extensions of the vasculature to the leaf margins in the re/re leaves resulted in teeth that correspond to the presence of the apical and lateral hydathodes. Lateral teeth are visible only during the early stages of leaf expansion in the wild types (Candela et al., 1999), but remain evident in re/re fully expanded leaves.
Overall leaf shape is not very different between the re mutants and their wild types, the only exceptions being the above-mentioned marginal irregularities and organ size, which is slightly, but clearly, reduced (Fig. 1). Measurements of the 1st to 9th node leaf laminae showed no consistent differences between the wild type and the re-1 mutant in the width/length and perimeter/area ratios during most of the stages of leaf development (Fig. 2A, B). Consistent with the small reduction observed for the third leaf (Fig. 1), the width, length, perimeter, and area were slightly reduced in all the leaves of the re-1 mutant compared with the wild type (Fig. 2A, B).
Measurement of the vascular length in relation to leaf expansion revealed that re-1/re-1 and wild-type first leaves are very similar in the early stages of leaf development (Fig. 2C). As the leaf reaches full expansion, however, vascular development is decreased in the mutant, resulting in a reduction in the vascular length per unit area in re-1/re-1 mature first leaves. The leaf venation pattern of the re mutants was also analysed and it was found in all cases that it is simpler than that of their corresponding wild types (Fig. 3).
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Internal architecture of re/re leaves
In the re mutants, the leaf lamina was ridged rather than smooth, with the contours of the vascular pattern evident on the leaf surface (Fig. 4A, B). This suggested that there may be changes in the internal leaf anatomy, which correlate with the venation pattern. Examination of intact first leaves by confocal microscopy showed that their interveinal regions were extremely reduced in cell density and appeared to have holes within the matrix of mesophyll cells (Fig. 4C–K). This was confirmed by transverse sections of re/re leaves, which revealed an internal leaf structure dramatically different from the wild type and which directly reflects the reticulation pattern observed at the whole leaf level (Fig. 5). Areas which lie between the vascular strands had a greatly reduced number of both palisade and spongy mesophyll cells compared with the wild type (Fig. 5B, C). By contrast, the vascular bundles consisting of vascular cells and bundle sheath cells were normal and complete. Extensive sectioning indicated that a cellular bridge between the adaxial and abaxial epidermis often occurs in the proximity of vascular strands while in the interveinal regions, mesophyll cell density is much lower and contiguous cells linking the two epidermises in the vertical plane are much rarer (Fig. 5). Consequently, the density of chloroplast-containing cells per unit leaf area is greatly reduced in the interveinal regions, compared with the vascular regions of the lamina (Table 2).
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Quantification of the internal leaf architecture of the re-1 mutant (Table 2) showed that for transverse sections across half leaves, i.e. from the midvein to leaf margin, the proportion of transect occupied by mesophyll cells is reduced and the fraction occupied by airspace is concomitantly greater than in wild-type leaves. This difference is also manifested as a reduction in the number of palisade and spongy mesophyll cells as a proportion of the cell transects (data not shown). There appears to be a preferential loss of palisade mesophyll cells over spongy mesophyll cells since the ratio of palisade:spongy cell transects is only 0.37 in re-1/re-1 leaves compared with 0.48 in the wild type. It is clear from the transverse sections of these leaves (Fig. 5) that, in the interveinal regions, the loss of mesophyll cells and the resultant increase in airspace is even greater than the average data reveal for entire half-leaf transects. The palisade and spongy mesophyll cells which are present in re-1/re-1 leaves appeared normal (data not shown) and contained chloroplasts similar in number and morphology to those of the wild type (Table 2, and data not shown). The content of chloroplasts per bundle sheath cell, on the other hand, was significantly higher in the re-1 mutant. Thus, its reticulate phenotype does not result from perturbed plastid development, but rather from a dramatic change in internal leaf architecture. The paleness of the interveinal regions seems to be mainly due to reduced mesophyll cell density and increased air space, which, furthermore, makes the vasculature more apparent. Nevertheless, the increased number of chloroplasts shown in the bundle sheath cells would also contribute to the dark green colour of the vasculature.
Generally, re-1/re-1 leaves were thinner than wild-type leaves (Table 2), but increased leaf thickness was often observed close to a vascular strand, which is consistent with the observation that the vascular pattern has a raised profile on the adaxial surface of the mutant leaves (Fig. 4B). Transverse sections revealed lobes of epidermal tissue, particularly on the abaxial leaf surface, which are not observed in wild-type leaves (Fig. 5). Scanning electron microscopy indicated that epidermal cell size and morphology are not modified in the leaves of re mutants (see supplementary Fig. 1 at JXB online).
Changes in the development of the internal mesophyll tissue in the re-1 mutant were evident at the early stages of leaf primordial development (Fig. 6), although the shoot apical meristem was unchanged (data not shown). It appears that reduced cell division in the re-1/re-1 palisade cell layer results in looser cell packing even at this early stage. The mutant palisade cells also appeared to be more vacuolated than the wild type in sections of equivalent developmental stage (Fig. 6).
Positional cloning of the RE gene and characterization of re alleles
The re-1 mutation has been known for a long time to map at chromosome 2 (Rédei and Hirono, 1964). An F2 mapping population derived from a Col-0xre-3/re-3 cross was genotyped for molecular markers (see Materials and methods), which allowed us to find linkage to the nga361 and nga168 markers. For one of the genes within the candidate region, At2g37860, recently named LCD1 (LOW CELL DENSITY1), a mutant allele had been described, lcd1-1, which causes a reticulate leaf phenotype (Barth and Conklin, 2003). The At2g37860 gene was sequenced in all the re mutants (Fig. 7), finding the mutations shown in Table 1. This demonstrated that lcd1-1 and the re mutations are alleles of the same gene, which is named RE in this work. The RE gene symbol has been used for four decades in significant publications and is widely known to the Arabidopsis community, criteria that have been in many cases followed to decide which gene symbol should be given precedence, as indicated in the guidelines of Meinke and Koornneef (1997).
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symbol is used to indicate a deletion. The positions and directions of the oligonucleotide primers used for the structural characterization of re alleles are represented by horizontal arrows below the RE gene map. Oligonucleotide sequences are detailed in the supplementary Table 1 at JXB online.A search of the Salk collection of T-DNA insertion lines revealed two with the At2g37860 gene disrupted, which were confirmed by complementation tests to carry re alleles and named re-6 and re-7. The sequence of the RE gene in the wild-type accessions studied was identical, the only exception being nucleotide position 2060 of its transcription unit, which was C in Col-0, but G in Ler and En-2. This means that the RE protein has a phenylalanine residue in Col-0 and a leucine in Ler and En-2 at position 399.
As regards the mutants, the re-3 point mutation converts a glycine into arginine at position 328, and re-4 causes the substitution of proline by leucine at position 295. These two last changes affect a conserved region of the RE protein (Fig. 7; see supplementary Fig. 2 at JXB online). The re-2 mutation removes 189 amino acids from the C-terminal region of the RE protein, eliminating most of their conserved domains (see supplementary Fig. 2 at JXB online). The re-5 mutant carries a rearrangement joining nucleotide -45 of RE (At2g37860) to nucleotide 6508 of At2g41790. This mutant carries, in addition, a T-DNA insertion between the At2g41770 and At2g41780 genes, which, together with At2g41790, are located at a distance of 1.6 Mb from RE in the wild type. In the re-5 mutant, the promoter of At2g41790 drives the expression of an At2g41790-RE chimeric transcript, which is predicted to be translated only into the protein product of At2g41790, which in turn is partially aberrant because its C-terminus includes amino acids translated from the promoter of RE. The distance between the 5' cap of the chimeric At2g41790-RE mRNA and the initiation codon of its RE portion is about 3 kb, which would not allow ribosome assembly at the RE translation initiation signal (Kawaguchi and Bailey-Serres, 2002). As regards re-1, it carries a deletion of 24 kb, encompassing RE and another 10 genes (At2g37790–At2g37880). Consistent with this, no signal was detected in Southern blots of restricted genomic DNA of the re-1 mutant probed with a RE-specific probe (see Materials and methods and supplementary Table 1 at JXB online).
The expression of the RE gene in the third leaves of the re mutants and their wild types was analysed using several primer sets (Fig. 7; see supplementary Table 1 at JXB online). Transcripts of the expected size were detected in the re-2, re-3, and re-4 mutants, which carry point mutations, but not in re-1, as a consequence of its deletion affecting the whole RE gene. Amplification and sequencing of the full-length re-5 cDNA confirmed its predicted chimeric nature. Two RE truncated mRNAs were identified in the re-6 mutant, one of them transcribed upstream and the other downstream of the T-DNA insert, as occurs in other insertional mutants. The upstream mRNA is predicted to be translated into only 37 amino acids of the RE wild-type protein. The downstream mRNA lacks 217 nucleotides in its 5' region, includes some T-DNA nucleotides, and cannot be translated because it does not include a translation initiation signal.
The RE gene encodes a predicted protein of 432 amino acids with a molecular weight of 46.6 kDa (see supplementary Fig. 2A at JXB online). Searches in the databases made it possible to identify two full length cDNAs, At2g37860.1 and At2g37860.2, the first one 96 nucleotides longer than the other. Translation of At2g37860.1 would produce a protein of 348 amino acids, 84 residues shorter than the predicted full length protein. The attempts to identify the At2g37860.1 transcript were not successful, suggesting that it is either artefactual, unstable, produced in developmental stages other than those studied here, or strictly restricted to a reduced number of cells during development.
In silico analyses (http://crombec.botanik.uni-koeln.de/seq_view.ep) detected a putative transit peptide to chloroplasts in the N-terminal part of the protein (amino acids 1–47) and two probable transmembrane regions (amino acids 247–269 and 321–343). RE is closely related to other plant proteins of unknown function in Arabidopsis (the closest homologue to RE in the genome of Arabidopsis being the At5g22790 gene, which maps at chromosome 5), Euphorbia esula, and Oryza sativa. The C-terminal regions of all these proteins are highly homologous, whereas the N-terminal region of all of them, except CAE05717 [GenBank] of rice, is rich in glycine residues. No homology was found with proteins of biological systems other than plants, suggesting that RE is a plant-specific protein.
RE expression analyses
To determine the spatial pattern of expression of RE in the wild types, total RNA was isolated from roots, rosette leaves, stems, flower buds, and open flowers of Col-0, En-2 and Ler. RT-PCR amplifications indicated that expression of RE is not restricted to leaves, as shown by the single band of 288 bp (the expected size for the amplification product corresponding to the last three exons of At2g37860.2) detected in all the organs studied (see supplementary Fig. 2B at JXB online). Similar amounts of RE transcripts were obtained from the different organs studied, except in stems, where fewer transcripts were detected. The same conclusions can also be reached by examining the expression profiles of RE obtained from Genevestigator (http://www.genevestigator.ethz.ch; Zimmermann et al., 2004).
RE expression was further analysed by transforming Col-0 plants with a RE promoter: ß-glucuronidase (GUS) construct (see Materials and methods). GUS activity was high in the vasculature of developing leaf primordia and decreased as leaves aged, becoming restricted in fully expanded leaves to the margins, hydathodes, and to the basal region of the lamina (Fig. 8). GUS activity was also detected in stipules, root tips, hydathodes of cauline leaves, stamens, and in the abscission zone of the funiculus. This pattern of GUS activity is consistent with that of RE expression detected by RT-PCR.
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Double mutant analysis
To gain insight into the genetic interactions between mutations causing reticulation, re-3/re-3 plants were crossed to homozygotes for the loss-of-function mutations cue1-5 (chlorophyll a/b binding protein underexpressed; Li et al., 1995) and dov1 (differential development of vascular associated cells; Kinsman and Pyke, 1998), which exhibit a dark-green leaf vasculature on a pale-green lamina (Fig. 9). The cue1-5 mutant is smaller than the re mutants and its reticulation does not become less apparent with ageing. These two traits were shared by the RE/-;cue1-5/cue1-5 and re-3/re-3;cue1-5/cue1-5 siblings found in the F2 progeny of a re-3/re-3xcue1-5/cue1-5 cross, whose genotypes were confirmed by sequencing the CUE1 and RE genes. This indicates that cue1-5 is epistatic to re-3 and suggests that RE and CUE1 act in the same developmental pathway affecting leaf development. The phenotype of the dov1/dov1;re-3/re-3 double mutants was merely additive, as expected if the DOV1 and RE genes act in an independent manner.
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The re mutants seem to be leaf-specific
The anatomical development of the wild-type leaf has been characterized in some detail (Pyke et al., 1991) and a plethora of mutants with altered leaf shape is available (Tsuge et al., 1996; Berná et al., 1999; Serrano-Cartagena et al., 1999) for the model species Arabidopsis thaliana. However, many leaf mutants are not exclusively affected in leaf organogenesis. For example, in a large-scale screening for mutants affected in their leaf development 153 mutants were isolated, 78 of which were also visibly affected in their flower development (Berná et al., 1999; Robles and Micol, 2001). Since the lateral organs of plants are assumed to have evolved from leaves, it is not surprising that a number of leaf mutants display, in addition, defects in floral organs and fruits. On the other hand, pleiotropy appears in some cases because the leaf phenotype is due to the ectopic derepression in the leaves of the shoot apical meristem (Lincoln et al., 1994) or floral organ identity genes (Goodrich et al., 1997; Serrano-Cartagena et al., 1999).
The final shape and size of all lateral organs is achieved through the genetic control of cell expansion, proliferation, and identity along the dorsoventral, mediolateral, and proximodistal axes. Mutants affected in leaf dorsoventrality, such as kanadi (kan, Kerstetter et al., 2000), phabulosa (phb; McConnell and Barton, 1998; McConnell et al., 2001), and phavoluta (phv; McConnell et al., 2001), display radialization in both their leaves and floral organs, as happens in the phantastica (phan) mutants of Antirrhinum majus (Waites and Hudson, 1995; Waites et al., 1998) and lam1 of Nicotiana tabacum (McHale and Marcotrigiano, 1998). Furthermore, the angustifolia mutants of Arabidopsis are affected in cell expansion (an; Tsuge et al., 1996) and cell proliferation (an3; Horiguchi et al., 2005) along the mediolateral axis, and display narrow leaves and flower organs. The same can be said with regard to the proximodistal axis, as shown by the rotundifolia mutants of Arabidopsis, which display short leaves and floral organs because of defective cell expansion (rot3; Tsuge et al., 1996) and cell proliferation (rot4; Narita et al., 2004). The CINCINNATA (CIN) gene of Antirrhinum majus (Nath et al., 2003; Crawford et al., 2004) controls the growth of both leaves and petals. The asymmetric leaves 1 (as1; Byrne et al., 2000) and as2 (Semiarti et al., 2001) Arabidopsis mutants are affected in both the proximodistal and mediolateral patterning of leaves, and their flowers are also abnormal.
Although many mutants isolated based on their leaf phenotypes are pleiotropic, this is not the case for the re mutants, whose only abnormality as regards external morphology is the reticulation displayed by the cotyledons, vegetative leaves, and cauline leaves. Only a few other leaf mutants are known to be leaf-specific, such as liguleless1 and liguleless2, which are uniquely necessary for ligule and auricle development in the leaves of maize (Harper and Freeling, 1996).
The re alleles strongly disrupt internal leaf architecture but not plastid development
Mutation at the RE locus causes reticulation, due to a marked reduction in the density of mesophyll cells in interveinal regions of the leaf, which does not result from perturbed plastid development in specific cells but rather from a dramatic change in internal leaf architecture. Contrary to what has been concluded from previous studies on other mutants, this characterization of the RE gene revealed that the cellular basis for the reticulate phenotype of leaves does not necessarily result from aberrations in plastid development. It has been shown, in addition, that this phenotypic class may be usefully exploited as a base for mutant screens which may reveal altered internal leaf architecture.
It has been suggested that altered internal leaf architecture results from the absence of a putative chloroplast-derived signal which controls palisade mesophyll cell differentiation and expansion (Pyke et al., 2000). In fact, a number of mutants with reticulate, pale or albino phenotypes show aberrant mesophyll development. Unlike re, all these mutants show defective chloroplast development, which suggests a role for the RE gene in a mesophyll-specific cell proliferation regulatory system that is not dependent on plastid development.
Mutations that disrupt internal leaf architecture and cause pale or albino phenotypes include pac (pale cress; Reiter et al., 1994; Grevelding et al., 1996), dal1 (DAG-like1; Babiychuk et al., 1997), var1 and var2 (yellow variegated1 and 2; Chen et al., 2000; Takechi et al., 2000; Sakamoto et al., 2002), im (immutans; Wetzel et al., 1994; Carol et al., 1999; Wu et al., 1999) and sca3 (scabra3; Hricová et al., 2006), all of them in Arabidopsis, and the dcl (defective chloroplast and leaves-mutable; Keddie et al., 1996) and dag (Dof affecting germination; Sommer et al., 1985; Chatterjee et al., 1996) mutants in tomato and Antirrhinum majus, respectively.
The reticulate mutants reported in Arabidopsis include cue1 and dov1. In the leaves of cue1, palisade parenchyma cells show a spheric rather than a columellar shape with larger air-spaces and smaller chloroplasts than the wild type (Li et al., 1995). CUE1 encodes the plastid phosphoenolpyruvate (PEP)/phosphate translocator (PPT), which is localized on the inner envelope and imports PEP, the first substrate of the shikimate pathway which produces aromatic amino acids and a variety of secondary metabolites (Streatfield et al., 1999). The normal-looking bundle sheath cells and plastids found in paraveinal regions of cue1/cue1 leaves, but abnormal mesophyll cells and plastids, indicate that mesophyll cells specifically require PPT or that it is not required by bundle sheath cells. The epistasis of cue1-5 to re-3 that was observed indicates a functional relationship between CUE1 and RE and suggests a role for RE in the shikimate pathway.
The dov1 mutant has normal bundle sheath cells and chloroplasts, whereas its mesophyll cells are normal in size and number, but contain plastids that are reduced in size and number, are vacuolated, and lack grana. The DOV1 gene remains to be cloned. Our double mutant analyses suggest that RE and DOV1 are involved in different processes affecting leaf development.
The re alleles have little effect on leaf shape
Whilst many studies have focused on roles for differential planes of cell division and leaf expansion in controlling leaf shape (Poethig, 1997), the relative roles of the epidermis and the mesophyll are far from clear (Dale, 1988). The an and rot3 mutations in Arabidopsis result in changes in leaf allometry, to which a major contributory factor is abnormality in the polarity of epidermal cell expansion (Tsukaya et al., 1994; Tsuge et al., 1996). In leaves of cotton, the Okra mutation dramatically affects leaf shape, yet can produce an altered leaf phenotype when present solely in the L1-derived epidermis, as found by studies of chimeras (Dolan and Poethig, 1998).
Impaired mesophyll cell development resulting from mutation at the RE locus dramatically alters internal leaf architecture, but has little effect on leaf shape. Only a slight decrease in the final leaf area of the re mutants was found, suggesting that RE might also contribute to some extent to leaf expansion. Leaf thickness was reduced in re/re interveinal regions, resulting in ridges of venation on the leaf surface. In addition, it was found that the size and morphology of the epidermal cells was not different in the re mutants and their wild types. Taken together, these observations suggest that the epidermis plays a major role in controlling leaf shape in Arabidopsis, whereas the division and expansion of the internal mesophyll parenchyma tissue would appear to be secondary in this process. This is not totally surprising, since Avery (1933) already claimed that the epidermis controls leaf expansion, based on observations performed in tobacco. Since the epidermis, which is genetically distinct and lineage-related, is the only contiguous cellular structure which covers the entire leaf, it would seem logical that the epidermal layer may contain pre-determined information concerning leaf shape, which can be modulated to varying extents by the underlying mesophyll tissue. In an analogous developmental system, the Arabidopsis petal, the epidermis is the primary tissue since the mesophyll is rudimentary and poorly developed (Pyke and Page, 1998). As a result, the expansion of epidermal cells is a major factor in controlling overall petal expansion and petal shape.
The evidence derived from this characterization of the leaf phenotype of re mutants is also compatible with the hypothesis that the control of leaf shape is non-cell autonomous, as suggested by the tangled mutant of maize (Smith et al., 1996). If leaf shape were controlled at tissue or organ level rather than at cell level, reduced mesophyll cell packing would not presumably change leaf shape at all, but merely produce a leaf with more internal airspace, as was shown in the re mutants.
The fact that the leaves of re mutants are thinner in interveinal areas because the mesophyll tissue is incomplete and the observation that the abaxial epidermis appears to be lobed in places suggest that the development of the mesophyll parenchyma plays a more important role in the control of leaf thickness. Indeed, the extent of palisade mesophyll cell elongation in a plane perpendicular to the lamina surface is closely correlated with leaf thickness throughout the process of Arabidopsis leaf development and in mature leaves (Pyke et al., 1991). Despite the reduction in mesophyll cell number in the re lines, the palisade and spongy mesophyll cells which are present maintain their position-specific characteristics, with similar size and shape and chloroplast complement as the wild type. Nevertheless, an increased number of chloroplasts per bundle sheath cell was observed in the re-1 mutant, suggesting that RE might also restrict the number of chloroplasts in these cells. It is interesting that reduced internal mesophyll proliferation affects vascular development, which might indicate that recruitment of cells along the vascular cell differentiation pathway from mesophyll parenchyma is limiting in this mutant.
Nature and action of the RE gene
The RE gene was positionally cloned and it was found to correspond to LCD1, which was identified based on the increased sensitivity to ozone caused by its mutant allele lcd1-1 (Barth and Conklin, 2003). The lcd1-1 mutation does not affect the photosynthetic apparatus or chloroplast-protective mechanisms (Barth and Conklin, 2003), which is consistent with the observation here that the reticulate phenotype was due to defects in mesophyll cells but not in chloroplasts.
Mutants that display both developmental and defence defects have been reported, in which some defence components affect cell proliferation or vice versa (see, among others, Vanacker et al., 2000; Holt et al., 2002; Jin et al., 2002; Song et al., 2004). This was also the case for lcd1-1, whose impaired defence responses have been interpreted as secondary effects of the reduction in the number of leaf parenchyma cells, since O3 degradation would yield a higher number of radical oxygen species per cell, and pronounced tissue damage, in a mesophyll extremely reduced in cell density (Barth and Conklin, 2003). Further research will be required, however, to exclude completely that the perturbations in defence associated with mutations at RE (LCD) cause their developmental aberrations.
The lcd1-1 mutant carries a nonsense mutation that eliminates only the last C-terminal amino acid of the RE protein. No other allele of a plant gene has been described in which the absence of a single, C-terminal amino acid results in a mutant phenotype (Barth and Conklin, 2003). The results presented here demonstrate for re-1 and predict for re-2, re-5, and re-6 that they are null mutations, all of which damage the RE gene much more severely than lcd1-1. Therefore, the isolation and characterization of a large series of loss-of-function re alleles allowed the involvement of the RE gene in the control of internal leaf architecture in Arabidopsis to be confirmed.
RE encodes a plant-specific protein, to which no function could be assigned on the basis of its similarity to other proteins found in the databases. In silico analysis detected the presence of two transmembrane regions, and a putative transit peptide to the chloroplast in the N-terminal part of the RE protein. Therefore, the involvement of RE in some plastid function not required for plastid development cannot be ruled out, given that its absence has no visible effects on plastid biogenesis. On the other hand, RE was not identified as being included in the chloroplast proteome in a recent attempt to isolate all chloroplast proteins, by means of tandem mass spectrometry (Kleffman et al., 2004). Therefore, a function for RE outside this organelle cannot be excluded.
Using a reporter transgene, RE activity was detected not only in leaves but also in roots and floral organs. Interestingly, RE activity was especially high in leaf primordia, as expected from the role assumed for this gene in the early stages of leaf development. In fact, the leaf phenotype of the re mutants suggests that RE functions in early primordia development to control the division of mesophyll parenchyma cells as the lamina expands. Subsequent differentiation pathways of those cells are normal in all features other than the final ratio of the major cell types of the leaf.
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Supplementary data can be found at JXB online.
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Acknowledgements |
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We wish to thank the NASC and the ABRC for seeds, Javier Paz-Ares for providing the T-DNA seed collection from which re-5 was isolated, and Anton Page, Sarah Case, José Manuel Serrano, and Verónica García-Sempere for excellent technical assistance. This work was funded by the Biotechnology and Biological Sciences Research Council (grant no. GO4843 to KAP), and the Ministerio de Educación y Ciencia of Spain (grant nos BMC2002-02840 and BFU2005-01031 to JLM). RGB was fellow of the Ministerio de Educación y Ciencia of Spain.
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* These authors contributed equally to this work.
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
|---|
Avery GS. (1933) Structure and development of the tobacco leaf. American Journal of Botany 20:565–592.[CrossRef][Web of Science]
Babiychuk E, Fuangthong M, Van Montagu M, Inzé D, Kushnir S. (1997) Efficient gene tagging in Arabidopsis thaliana using a gene trap approach. Proceedings of the National Academy of Sciences, USA 94:12722–12727.
Barth C and Conklin PL. (2003) The lower cell density of leaf parenchyma in the Arabidopsis thaliana mutant lcd1-1 is associated with increased sensitivity to ozone and virulent Pseudomonas syringae. The Plant Journal 35:206–218.[CrossRef][Web of Science][Medline]
Bell DJ and Ecker JR. (1994) Assignment of 30 microsatellite loci to the linkage map of Arabidopsis. Genomics 19:137–144.[CrossRef][Web of Science][Medline]
Berná G, Robles P, Micol JL. (1999) A mutational analysis of leaf morphogenesis in Arabidopsis thaliana. Genetics 152:729–742.
Blazquez MA, Soowal LN, Lee I, Weigel D. (1997) LEAFY expression and flower initiation in Arabidopsis. Development 124:3835–3844.[Abstract]
Bohmert K, Camus I, Bellini C, Bouchez D, Caboche M, Benning C. (1998) AGO1 defines a novel locus of Arabidopsis controlling leaf development. EMBO Journal 17:170–180.[CrossRef][Web of Science][Medline]
Byrne ME. (2005) Networks in leaf development. Current Opinion in Plant Biology 8:59–66.[CrossRef][Web of Science][Medline]
Byrne ME, Barley R, Curtis M, Arroyo JM, Dunham M, Hudson A, Martienssen RA. (2000) Asymmetric leaves1 mediates leaf patterning and stem cell function in Arabidopsis. Nature 408:967–971.[CrossRef][Medline]
Canales C, Grigg S, Tsiantis M. (2005) The formation and patterning of leaves: recent advances. Planta 221:752–756.[CrossRef][Web of Science][Medline]
Candela H, Martínez-Laborda A, Micol JL. (1999) Venation pattern formation in Arabidopsis thaliana vegetative leaves. Developmental Biology 205:205–216.[CrossRef][Web of Science][Medline]
Carland FM and McHale NA. (1996) LOP1: a gene involved in auxin transport and vascular patterning in Arabidopsis. Development 122:1811–1819.[Abstract]
Carol P, Stevenson D, Bisanz C, Breitenbach J, Sandmann G, Mache R, Coupland G, Kuntz M. (1999) Mutations in the Arabidopsis gene IMMUTANS cause a variegated phenotype by inactivating a chloroplast terminal oxidase associated with phytoene desaturation. The Plant Cell 11:57–68.
Chatterjee M, Sparvoli S, Edmunds C, Garosi P, Findlay K, Martin C. (1996) DAG, a gene required for chloroplast differentiation and palisade development in Antirrhinum majus. EMBO Journal 15:4194–4207.[Web of Science][Medline]
Chen M, Choi Y, Voytas DF, Rodermel S. (2000) Mutations in the Arabidopsis VAR2 locus cause leaf variegation due to the loss of a chloroplast FtsH protease. The Plant Journal 22:303–313.[CrossRef][Web of Science][Medline]
Clough SJ and Bent AF. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis. The Plant Journal 16:735–743.[CrossRef][Web of Science][Medline]
Crawford BC, Nath U, Carpenter R, Coen ES. (2004) CINCINNATA controls both cell differentiation and growth in petal lobes and leaves of Antirrhinum. Plant Physiology 135:244–253.
Dale JE. (1988) The control of leaf expansion. Annual Review of Plant Physiology and Plant Molecular Biology 39:267–295.[CrossRef][Web of Science]
Dolan L and Poethig RS. (1998) The Okra leaf shape mutation in cotton is active in all cell layers of the leaf. American Journal of Botany 85:322–327.[Abstract]
Fleming AJ. (2002) The mechanism of leaf morphogenesis. Planta 216:17–22.[CrossRef][Web of Science][Medline]
Goodrich J, Puangsomlee P, Martin M, Long D, Meyerowitz EM, Coupland G. (1997) A Polycomb-group gene regulates homeotic gene expression in Arabidopsis. Nature 386:44–51.[CrossRef][Medline]
Grevelding C, Suter-Crazzolara C, von Menges A, Kemper E, Masterson R, Schell J, Reiss B. (1996) Characterisation of a new allele of pale cress and its role in greening in Arabidopsis thaliana. Molecular and General Genetics 251:532–541.
Harper L and Freeling M. (1996) Interactions of liguleless1 and liguleless2 function during ligule induction in maize. Genetics 144:1871–1882.[Abstract]
Holt BF III, Boyes DC, Ellerstrom M, Siefers N, Wiig A, Kauffman S, Grant MR, Dangl J. (2002) An evolutionarily conserved mediator of plant disease resistance gene function is required for normal Arabidopsis development. Developmental Cell 2:807–817.[CrossRef][Web of Science][Medline]
Horiguchi G, Kim GT, Tsukaya H. (2005) The transcription factor AtGRF5 and the transcription coactivator AN3 regulate cell proliferation in leaf primordia of Arabidopsis. The Plant Journal 43:68–78.[CrossRef][Web of Science][Medline]
Hricová A, Quesada V, Micol JL. (2006) The SCABRA3 nuclear gene encodes the plastid Rpo Tp RNA polymerase, which is required for chloroplast biogenesis and mesophyll cell proliferation in Arabidopsis. Plant Physiology 141:942–958.
Jin H, Axtell MJ, Dahlbeck D, Ekwenna O, Zhang S, Staskawicz B, Baker B. (2002) NPK1, an MEKK1-like mitogen-activated protein kinase, regulates innate immunity and development in plants. Developmental Cell 3:291–297.[CrossRef][Web of Science][Medline]
Kawaguchi R and Bailey-Serres J. (2002) Regulation of translational initiation in plants. Current Opinion in Plant Biology 5:460–465.[CrossRef][Web of Science][Medline]
Keddie J, Carroll B, Jones J, Gruissem W. (1996) The DCL gene of tomato is required for chloroplast development and palisade cell morphogenesis in leaves. EMBO Journal 15:4208–4217.[Web of Science][Medline]
Kerstetter RA, Bollman K, Taylor RA, Bomblies K, Poethig RS. (2001) KANADI regulates organ polarity in Arabidopsis. Nature 411:706–709.[CrossRef][Medline]
Kinsman EA and Pyke KA. (1998) Bundle sheath cells and cell-specific plastid development in Arabidopsis leaves. Development 125:1815–1822.[Abstract]
Kleffmann T, Russenberger D, Von Zychlinski A, Christopher W, Sjolander K, Gruissem W, Baginsky S. (2004) The Arabidopsis thaliana chloroplast proteome reveals pathway abundance and novel protein functions. Current Biology 14:354–362.[CrossRef][Web of Science][Medline]
Larkin JC, Marks MD, Nadeau J, Sack F. (1997) Epidermal cell fate and patterning in leaves. The Plant Cell 9:1109–1120.[CrossRef][Web of Science][Medline]
Li H, Culligan K, Dixon RA, Chory J. (1995) CUE1: a mesophyll cell-specific positive regulator of light-controlled gene expression in Arabidopsis. The Plant Cell 7:1599–1610.[Abstract]
Lincoln C, Long J, Yamaguchi J, Serikawa K, Hake S. (1994) A knotted 1-like homeobox gene in Arabidopsis is expressed in the vegetative meristem and dramatically alters leaf morphology when overexpressed in transgenic plants. The Plant Cell 6:1859–1876.
McConnell JR and Barton MK. (1998) Leaf polarity and meristem formation in Arabidopsis thaliana. Development 125:2935–2942.[Abstract]
McConnell JR, Emery J, Eshed Y, Bao N, Bowman J, Barton MK. (2001) Role of PHABULOSA and PHAVOLUTA in radial patterning in shoots. Nature 411:709–713.[CrossRef][Medline]
McHale NA and Marcotrigiano M. (1998) LAM1 is required for dorsoventrality and lateral growth of the leaf blade in Nicotiana. Development 125:4235–4243.[Abstract]
Meinke DW and Koornneef M. (1997) Community standards for Arabidopsis genetics. The Plant Journal 12:247–253.[CrossRef][Web of Science]
Narita NN, Moore S, Horiguchi G, Kubo M, Demura T, Fukuda H, Goodrich J, Tsukaya H. (2004) Overexpression of a novel small peptide ROTUNDIFOLIA4 decreases cell proliferation and alters leaf shape in Arabidopsis. The Plant Journal 38:699–713.[CrossRef][Web of Science][Medline]
Nath U, Crawford BC, Carpenter R, Coen E. (2003) Genetic control of surface curvature. Science 299:1404–1407.
Nelson T and Dengler N. (1997) Leaf vascular pattern formation. The Plant Cell 9:1121–1135.[CrossRef][Web of Science][Medline]
Pérez-Pérez JM, Ponce MR, Micol JL. (2004) The ULTRACURVATA2 gene of Arabidopsis thaliana encodes an FK506-binding protein involved in auxin and brassinosteroid signaling. Plant Physiology 134:101–117.
Pérez-Pérez JM, Serrano-Cartagena J, Micol JL. (2002) Genetic analysis of natural variations in the architecture of vegetative leaves in Arabidopsis thaliana. Genetics 162:893–915.
Poethig RS. (1997) Leaf morphogenesis in flowering plants. The Plant Cell 9:1077–1087.[CrossRef][Web of Science][Medline]
Ponce MR, Quesada V, Micol JL. (1998) Rapid discrimination of sequences flanking and within T-DNA insertions in the Arabidopsis genome. The Plant Journal 14:497–501.[CrossRef][Web of Science][Medline]
Ponce MR, Robles P, Micol JL. (1999) High-throughput genetic mapping in Arabidopsis. Molecular and General Genetics 261:408–415.
Pyke KA and Leech RM. (1992) Nuclear mutations radically alter chloroplast division and expansion in Arabidopsis thaliana. Plant Physiology 99:1005–1008.
Pyke KA and Lopez-Juez E. (1999) Cell differentiation and leaf morphogenesis in Arabidopsis. Critical Reviews of Plant Science 18:527–546.[CrossRef]
Pyke KA, Marrison JL, Leech RM. (1991) Temporal and spatial development of the cells of the expanding first leaf of Arabidopsis thaliana (L.) Heynh. Journal of Experimental Botany 42:1407–1416.
Pyke KA and Page AM. (1998) Plastid ontogeny during petal development in Arabidopsis. Plant Physiology 116:797–803.
Pyke KA, Zwobka M, Day A. (2000) Marking cell layers in Brassica napus with spectinomycin provides a new tool for studying cell fate and demonstrates a requirement for chloroplasts in palisade cell differentiation. Journal of Experimental Botany 51:1713–1720.
Quesada V, García-Martínez S, Piqueras P, Ponce MR, Micol JL. (2002) Genetic architecture of NaCl tolerance in Arabidopsis thaliana. Plant Physiology 130:951–963.
Quesada V, Ponce MR, Micol JL. (1999) OTC and AUL1, two convergent and overlapping genes in the nuclear genome of Arabidopsis thaliana. FEBS Letters 461:101–106.[CrossRef][Web of Science][Medline]
Rédei GP and Hirono Y. (1964) Linkage studies. Arabidopsis Information Service 1:9–10.
Reiter RS, Coomber SA, Bouret TM, Bartley GE, Scolnik PA. (1994) Control of leaf and chloroplast development by the Arabidopsis gene pale cress. The Plant Cell 6:1253–1264.[Abstract]
Robles P and Micol JL. (2001) Genome-wide linkage analysis of Arabidopsis genes required for leaf development. Molecular and General Genetics 266:12–19.
Sakamoto W, Tamura T, Hanba-Tomita Y, Sodmergen Y, Murata M. (2002) The VAR1 locus of Arabidopsis encodes a chloroplastic FtsH and is responsible for leaf variegation in the mutant alleles. Genes and Cells 7:769–780.
Semiarti E, Ueno Y, Tsukaya H, Iwakawa H, Machida C, Machida Y. (2001) The ASYMMETRIC LEAVES2 gene of Arabidopsis thaliana regulates formation of a symmetric lamina, establishment of venation and repression of meristem-related homeobox genes in leaves. Development 128:1771–1783.[Abstract]
Serrano-Cartagena J, Candela H, Robles P, Ponce MR, Pérez-Pérez JM, Piqueras P, Micol JL. (2000) Genetic analysis of incurvata mutants reveals three independent genetic operations at work in Arabidopsis leaf morphogenesis. Genetics 156:1363–1377.
Serrano-Cartagena J, Robles P, Ponce MR, Micol JL. (1999) Genetic analysis of leaf form mutants from the Arabidopsis Information Service collection. Molecular and General Genetics 261:725–739.
Smith LG, Hake S, Sylvester AW. (1996) The tangled-1 mutation alters cell division orientations throughout maize leaf development without altering leaf shape. Development 122:481–489.[Abstract]
Sommer H, Carpenter R, Harrison BJ, Saedler H. (1985) The transposable element Tam3 of Antirrhinum majus generates a novel type of sequence alteration upon excision. Molecular and General Genetics 199:225–231.[CrossRef]
Song JT, Lu H, Greenberg JT. (2004) Divergent roles in Arabidopsis thaliana development and defense of two homologous genes, ABERRANT GROWTH AND DEATH2 and AGD2-LIKE DEFENSE RESPONSE PROTEIN1, encoding novel aminotransferases. The Plant Cell 16:353–366.
Streatfield SJ, Weber A, Kinsman EA, Hausler RE, Li J, Post-Beittenmiller D, Kaiser WM, Pyke KA, Flugge UI, Chory J. (1999) The phosphoenolpyruvate/phosphate translocator is requiredfor phenolic metabolism, palisade cell development, and plastid-dependent nuclear gene expression. The Plant Cell 11:1609–1622.
Takechi K, Sodmergen K, Murata M, Motoyoshi F, Sakamoto W. (2000) The YELLOW VARIEGATED (VAR2) locus encodes a homologue of FtsH, an ATP-dependent protease in Arabidopsis. Plant Cell Physiology 41:1334–1346.
Tsiantis M and Hay A. (2003) Comparative plant development: the time of the leaf? . Nature Reviews Genetics 4:169–180.[Web of Science][Medline]
Tsuge T, Tsukaya H, Uchimiya H. (1996) Two independent and polarized processes of cell elongation regulate leaf blade expansion in Arabidopsis thaliana (L.) Heynh. Development 122:1589–1600.[Abstract]
Tsukaya H. (2002) Leaf development. In Somerville CR and Meyerowitz EM (Eds.). The Arabidopsis Book (American Society of Plant Biologists, Rockville, FL) pp. 1–23.
Tsukaya H. (2003) Organ shape and size: a lesson from studies of leaf morphogenesis. Current Opinion in Plant Biology 6:57–62.[CrossRef][Web of Science][Medline]
Tsukaya H. (2005) Leaf shape: genetic controls and environmental factors. International Journal of Developmenta Biology 49:547–555.
Tsukaya H, Tsuge T, Uchimiya H. (1994) The cotyledon: a superior system for studies of leaf development. Planta 195:309–312.[Web of Science]
Vanacker H, Lu H, Rate DN, Greenberg JT. (2001) A role for salicylic acid and NPR1 in regulating cell growth in Arabidopsis. The Plant Journal 28:209–216.[CrossRef][Web of Science][Medline]
Waites R and Hudson A. (1995) phantastica: a gene required for dorsoventrality of leaves in Antirrhinum majus. Development 121:2143–2154.[Abstract]
Waites R, Selvadurai HR, Oliver IR, Hudson A. (1998) The PHANTASTICA gene encodes a MYB transcription factor involved in growth and dorsoventrality of lateral organs in Antirrhinum. Cell 93:779–789.[CrossRef][Web of Science][Medline]
Wetzel CM, Jiang CZ, Meehan LJ, Voytas DF, Rodermel SR. (1994) Nuclear-organelle interactions: the immutans variegation mutant of Arabidopsis is plastid autonomous and impaired in carotenoid biosynthesis. The Plant Journal 6:161–175.[CrossRef][Web of Science][Medline]
Wu D, Wright DA, Wetzel C, Voytas DF, Rodermel S. (1999) The IMMUTANS variegation locus of Arabidopsis defines a mitochondrial alternative oxidase homolog that functions during early chloroplast biogenesis. The Plant Cell 11:43–55.
Ye ZH, Freshour G, Hahn MG, Burk DH, Zhong R. (2002) Vascular development in Arabidopsis. International Review of Cytology 220:225–256.[Web of Science][Medline]
Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W. (2004) Genevestigator. Arabidopsis microarray database and analysis toolbox. Plant Physiology 136:2621–2632.
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