PEAPOD regulates lamina size and curvature in Arabidopsis
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Edited by Enrico Coen, John Innes Centre, Norwich, United Kingdom, and approved July 5, 2006 (received for review May 25, 2006)
Abstract
Although a complex pattern of interspersed cell proliferation and cell differentiation is known to occur during leaf blade development in eudicot plants, the genetic mechanisms coordinating this growth are unclear. In Arabidopsis, deletion of the PEAPOD (PPD) locus increases leaf lamina size and results in dome-shaped rather than flat leaves. Siliques are also altered in shape because of extra lamina growth. The curvature of a Δppd leaf reflects the difference between excess growth of the lamina and a limitation to the extension capacity of its perimeter. Excess lamina growth in Δppd plants is due to a prolonged phase of dispersed meristematic cell (DMC) proliferation (for example, the meristemoid and procambium cells that form stomatal stem cells and vascular cells, respectively) during blade development. The PPD locus is composed of two homologous genes, PPD1 and PPD2, which encode plant-specific putative DNA-binding proteins. Overexpression of PPD reduces lamina size by promoting the early arrest of DMC proliferation during leaf and silique development. Therefore, by regulating the arrest of DMC proliferation, the PPD genes coordinate tissue growth, modulate lamina size, and limit curvature of the leaf blade. I propose a revised model of leaf development with two cell-cycle arrest fronts progressing from the tip to the base: the known primary front, which determines arrest of general cell proliferation, followed by a secondary front that involves PPD and arrests DMC division.
Despite a life spent in one position, higher plants are able to adapt readily to environmental changes by altering the size of their lateral organs, such as leaves. However, changing the size of the flat, oval-shaped leaf blade of a eudicot plant is not a trivial task; it involves coordinating complex patterns of cellular proliferation, differentiation, and expansion (1, 2), all in the absence of cell migration (3).
The lamina of a eudicot plant leaf is initially formed by general proliferative cell division. Subsequently, during leaf development, a front of cell-cycle arrest moves from the tip to the base (1, 4, 5). Immediately behind this arrest front, there is a gradient of cellular differentiation. Although most of the cells begin to differentiate and enlarge, including those contributing to the lamina margins and trichomes, there are also meristematic cells that undergo division, resulting in the formation of specific cell types within each cell layer (1). For example, in the epidermis, meristemoids (6) are recruited from meristemoid mother cells and undergo a limited number of asymmetric divisions to form stomatal guard and stomatal-lineage ground cells (7). In a similar manner, procambial cells are recruited in the mesophyll tissue layer and divide to form and extend the vascular tissue network of the leaf (1). These cells are defined here as dispersed meristematic cells (DMCs). Each type of DMC proliferates for only a limited duration, and the temporal patterns of DMC cell-cycle arrest differ between tissues. For example, during leaf development, the arrest of epidermal meristemoid cell division occurs before procambium cell-cycle arrest (1).
Recent studies using mutant plants have identified a number of genes that influence the pattern and maintenance of cell proliferation during leaf development. Some of these genes have positive roles in regulating cell proliferation. Included in this category are genes such as AINTEGUMENTA (ANT), ARGOS, ANGUSTIFOLIA (AN3), ERECTA, GROWTH-REGULATING FACTOR 5 (GRF5), JAGGED (JAG), STRUWWELPETER (SWP), and SWELLMAP (SMP1), which act to prolong the proliferative cell division phase during organ development (8–16). There are also general negative regulators of cell proliferation and organ growth, such as the interaction of AUXIN RESPONSE FACTOR 2 (ARF2) with ANT (17), and the BLADE ON PETIOLE (BOP) genes, which repress the transcription of JAG (18). One of the key aspects of lamina development is the coordination of cell proliferation and cell expansion, which are required to promote leaf flatness. The Antirrhinum majus gene CINCINNATA (CIN) determines the shape and progression of the general cell-cycle arrest front during leaf development, and in the cin mutant, excess growth in the margins results in crinkly leaves with negative curvature (19). In Arabidopsis, the microRNA-mediated regulation of class II TCP genes similar to CIN is also required to prevent excess margin growth in leaves (20).
However, despite these advances in our understanding of the genetic elements controlling the proliferative phase during leaf development, genes regulating the arrest of DMC proliferation have yet to be identified. Indeed, it has not been clear what contribution, if any, DMC proliferation makes to plasticity in lamina size. Here, I present information about the genetic control of DMC proliferation in developing plant organs obtained from the characterization of peapod (ppd), an Arabidopsis mutant with altered lamina size and curvature.
Results
The ppd Mutant Phenotype.
The effect of a ppd mutation (Δppd) induced in the Landsberg erecta (Ler) ecotype by fast-neutron treatment was particularly obvious in leaves and siliques. In Δppd plants, leaf and cotyledon laminae were larger, and leaves had a dome-shaped, positive Gaussian curvature, in contrast to flat WT leaves (Fig. 1 A–C). Siliques of the Δppd mutant were shorter, flattened, and wider at the top and had undulations in the seedpod walls, compared with the smooth, narrow, cylindrical shape of WT siliques (Fig. 1 D and E). There was also a reduction in the branching of trichomes on Δppd leaves, to two rather than the three to four branches of WT.
Phenotype of the Δppd mutant. (A) Phenotypes of WT (Left) and Δppd mutant (Right) plants. (B) Side view of WT Ler (Left) and Δppd (Right) seedlings. (C) Abaxial view of WT (Upper) and Δppd (Lower) mature first leaf. (D) Silique shapes: WT Ler (Left), Δppd (Right), and heterozygote (Center). (E) Inflorescences of WT (Left) and Δppd (Right). (Scale bars: A, 10 mm; C and D, 5 mm).
The larger lamina area observed in the leaves and cotyledons of Δppd plants was due to increases in both length and width (Table 1). For cotyledons, this increase in growth occurred without affecting shape or flatness. However, mature leaves of Δppd plants were more oval in shape because of an altered lamina length/width ratio and could not be flattened without introducing cuts in the margin because of their positive curvature. The area of Δppd leaf blades was ≈30–50% greater than that of WT leaves, but perimeter length was similar (Table 1). The positive curvature of Δppd leaves thus appears to be due to additional growth of the lamina exceeding the extension capacity of the perimeter of the blade. The extra leaf growth of Δppd plants appeared to have no detectable effect on mesophyll palisade cell size, spacing, or the general anatomy of tissues in the lamina (Fig. 6, which is published as supporting information on the PNAS web site). However, there was a more extensive vascular network in the cotyledons of Δppd plants than WT plants (4.93 ± 0.53 areoles per cotyledon for Δppd versus 3.97 ± 0.32 for WT) (Fig. 7, which is published as supporting information on the PNAS web site). The number of stomata per area in the abaxial epidermis of leaves was also greater (137%) in Δppd plants than in WT plants.
Influence of PPD gene copy number and expression level on Arabidopsis mature cotyledon, leaf, and silique dimensions
The PPD Locus Has Two Plant-Specific Homologs.
The Δppd mutation was backcrossed five times to Ler before being used for genetic analysis. Although reduced trichome branching of the Δppd allele was recessive, silique width and leaf curvature aspects of the phenotype were weakly semidominant (Fig. 1 D and Table 1). PPD was mapped to the bottom arm of chromosome 4 by using molecular markers (21) and colocation of the trichome branching gene, FRC3 (At4g14750) (22, 23). PCR analysis demonstrated that the Δppd allele is an ≈60-kb deletion encompassing 12 predicted genes, from At4g14700 to At4g14760, including FRC3. Within the deleted region, there is a duplicated genomic segment containing two interspersed homologous gene pairs, At4g14710/At4g14716 and At4g14713/At4g14720 (Fig. 2 A). Although the At4g14710/At4g14716 homologs are members of a wider gene family, there are no genes similar to At4g14713/At4g14720 elsewhere in the Arabidopsis genome. Homozygous transgenic plants with transfer DNA (T-DNA) insertions in either At4g14713 or At4g14720 had the weak semidominant ppd silique width (Table 1) and leaf curvature phenotype but had WT trichome branching, whereas plants with insertions in flanking genes (At4g14700, At4g14716, At4g14730, At4g14740, and At4g14760) were WT. The phenotypes of mutant alleles Δppd, ppd1-1, ppd1-2, and ppd2-1 indicate that PPD is a complex locus made up of two genes, designated here as PPD1 (At4g14713) and PPD2 (At4g14720). Furthermore, the influence of PPD gene copy number on lamina size, illustrated by comparing the silique widths of plants with 0, 1, 2, or 4 copies of a PPD gene (Table 1), suggests that PPD1 and PPD2 are individually haplo-insufficient. The similarity of silique dimensions for both homozygous T-DNA insertion lines (ppd1-1/ppd1-1 and ppd2-1/ppd2-1) and the heterozygote between them indicates that PPD1 and PPD2 are homologs of equivalent function.
Genomic structure of the PPD locus (A) and predicted amino acid sequences of proteins encoded by the Arabidopsis PPD genes (B). (A) Filled triangles indicate the position of T-DNA insertions. (B) Alignment of the predicted amino acid sequences of PPD proteins encoded by At4g14713 (PPD1) and At4g14720 (PPD2). Gray shading indicates similar residues. A protein domain that is highly conserved in PPD homologues is marked by an overline; dotted underlining identifies a putative ZIM DNA-binding motif.
Genomic transgenes of either of the PPD genes rescued the lamina size and curvature mutant phenotype of Δppd (but not the reduced trichome branching component), confirming that PPD1 and PPD2 are redundant. Of the 80-plus transgenic plants obtained from transformation with PPD1 or PPD2, most were WT in appearance. For example, of 26 Δppd/Δppd::PPD1 transgenic plants examined in detail, 20 were WT in leaf and silique dimensions, 2 had siliques similar in width to Δppd/+ heterozygotes, and 4 had smaller cotyledons and leaves and shorter, narrower siliques than WT. Quantitative expression analysis on two of these small genotypes (designated Δppd/Δppd::PPD1-OE) demonstrated that they had elevated levels of PPD mRNA (Fig. 4 E). The cotyledon, leaf, and silique dimensions of representative complemented Δppd/Δppd::PPD1 and Δppd/Δppd::PPD1-OE plants are given in Table 1. Silique dimensions of complemented Δppd/Δppd::PPD1 plants were similar to those of WT plants, as were cotyledon and leaf sizes (data not shown). Leaves of PPD1-OE plants were flat, smaller, and more rounded than those of the WT, and the siliques were reduced in both width and length. There was also a reduced vascular network pattern in the cotyledons of PPD1-OE plants (2.31 ± 0.54 areoles per cotyledon versus 3.97 ± 0.32 for WT) and a reduction in the number of stomata on the abaxial leaf surface (65%) relative to that found in WT plants.
PPD1 and PPD2 have nine exons and encode 314-aa and 316-aa proteins, respectively, with 84% identity. The predicted PPD proteins each have a central putative DNA-binding sequence, termed a ZIM motif (24), and a unique ≈50-aa N-terminal domain spanning amino acids 14–63 (Fig. 2 B). Proteins with homology to this combination of a conserved N-terminal “PPD” domain and a central ZIM motif that were identified by searching databases were found in the lycophyte Selaginella moellendorffii, the conifer Picea sitchensis, and in a wide variety of eudicot genera, including the basal eudicot Aquilegia. However, PPD proteins appear to be absent from rice and other grasses.
PPD Regulates the Arrest of DMC Proliferation During Lamina Development.
To investigate the cause of increased lamina growth in the Δppd mutant, I compared the appearance of epidermal cells in the leaves and siliques of mutant and WT plants at stages of WT leaf and silique development when meristemoids had ceased division (Fig. 3 A and C). WT plants had the expected pattern of pavement and stomata guard cells (7), but in the leaf and silique epidermis of Δppd mutant plants, clusters of small cells, some with the isometric shape characteristic of meristemoids, were interspersed amongst the enlarged pavement cells (Fig. 3 B and D). A transgene marker of cell-cycle progression, a CYCB1;1::GUS fusion (1), was used to compare the extent of DMC proliferation in developing leaves and siliques of Δppd mutant and WT plants (Fig. 3 E and F). In the leaf and carpel epidermal tissues examined, cyclin activity was expressed in foci corresponding to the appearance of meristemoid cells (see Fig. 8, which is published as supporting information on the PNAS web site). In WT plants, meristemoid cell cycling in the abaxial leaf epidermis was arrested 12 days after germination (DAG), whereas in the Δppd mutant, this phase was extended beyond 20 DAG. Similarly, meristemoid cell proliferation in the outer epidermis of the valve of the Δppd mutant was prolonged into more advanced developmental stages, well after cell-cycle arrest had occurred in the silique valves of WT plants (Fig. 3 F). The arrest of cell cycling in the procambium cells of the developing vasculature of Δppd mutant leaves was also delayed (Fig. 8) and continued after division of meristemoid cells in the epidermis had stopped. Therefore, despite a prolonged phase of proliferation in the leaf of Δppd mutant plants, DMC division eventually stops, and the order of this arrest in different tissues is the same as in WT plants.
Variations in meristemoid cell proliferation in the epidermis of developing leaves and siliques. (A–D) Replica SEM of the abaxial epidermal surface of WT (A) and Δppd leaf 1 (B) at 18 DAG. SEM of the outer surface of silique valves of WT (C) and Δppd (D) at developmental stage 17. (Scale bars: 40 μm.) (E and F) Comparison of the frequency of CYCB1;1::GUS-expressing meristemoid cells in WT (gray bars) and Δppd mutant (black bars) plants at different stages of leaf (E) or silique (F) development.
To determine whether prolonged cell division in the Δppd mutant occurred throughout lamina development or was confined to the DMC phase, I compared the position of the general proliferative cell-cycle arrest front in the leaves of WT, Δppd mutant, and transgenic PPD1-OE plants. If the loss of PPD function increases the duration of all cell proliferation during lamina development, then the general cell-cycle arrest front (primary arrest front) should be delayed, and the size of the general proliferation zone should be increased. However, if the effect of PPD gene expression is only on the zone of DMC proliferation, the tip-to-base advance of the primary arrest front should remain unaltered by either PPD loss of function or PPD overexpression. Because the position of this arrest front was not affected by altered expression of the PPD genes (Fig. 9, which is published as supporting information on the PNAS web site), it appears that the additional leaf and silique lamina growth of Δppd mutant plants was solely due to delayed DMC cell-cycle arrest. The CYCB1,1::GUS transgene was also used to determine the effect of PPD1 overexpression on meristemoid cell proliferation in leaves and siliques (Fig. 3 F). At leaf and silique developmental stages where both WT and Δppd mutant plants had a high cyclin index, meristemoid cell cycling in the epidermis of PPD1-OE transgenic plants was reduced by ≈80–90%.
The Expression Pattern of PPD Coincides with DMC Proliferation in Developing Leaves and Siliques.
To establish where PPD genes are expressed relative to the zone of proliferating DMCs, I carried out RNA in situ hybridizations and examined the expression pattern of a PPD1 promoter::GUS (β-glucuronidase) transgene in developing lateral organs (Fig. 4 A–D). During leaf development, PPD expression was first detected at the tip of the developing leaf, distal to the general proliferative cell-cycle arrest front and coincident with the initiation of trichome and margin cell development (Fig. 4 A and D Inset). This expression appeared to exist initially in all cells in the newly formed DMC zone and was not restricted to meristemoid cells. Subsequently, during leaf development, the pattern of PPD expression followed the tip-to-base progression of the general proliferative arrest front before becoming restricted to developing vascular traces and eventually declining in expression in the same pattern as the arrest of procambial cell division. PPD1 gene expression during the development of silique valves also coincided with the occurrence of meristemoid cell proliferation. PPD1::GUS expression occurred throughout the valves during developmental stages 15 and 16 and declined in more mature siliques (Fig. 4 C).
Analysis of PPD1 expression pattern and the influence of altered expression levels on lamina cell division. (A and B) In situ localization of PPD1 RNA in longitudinal sections of WT (A) or Δppd (B) seedlings (at 6 DAG) probed with PPD1. (C and D) Expression pattern of PPD1 promoter::GUS in the inflorescence (C Left), developing stage-15 silique (C Right), vegetative leaves (D Left), and leaf primordia (D Right) of WT plants. Arrow indicates the position of developing trichomes. (E) Relative expression levels of PPD1 in WT Ler, Δppd::PPD1, and Δppd::PPD1-OE plants as monitored by real-time quantitative RT-PCR. C#2, Δppd::PPD1 plant with WT phenotype; C#19 and C#26, independent Δppd::PPD1 transgenic plants with small leaf/silique phenotypes. Each bar represents the mean of three replicates relative to ornithine transfer carboxylase. (F) Comparison of the meristemoid cyclin index of developing leaves and siliques of WT, Δppd, and PPD1-OE plants. Leaves were sampled at 9 DAG (black bars), and siliques were sampled at carpel developmental stage 15 (gray bars).
Discussion
I report the identification of a gene regulating the cell-cycle arrest of a diverse group of stem cell-like meristematic cells during lamina development in Arabidopsis. Alterations solely to the duration of DMC proliferation can account for the effects of PPD gene expression on leaf morphogenesis (Fig. 5). In the absence of the PPD genes, leaf DMC proliferation is prolonged and lamina tissue outgrows the extension capacity of the margin cells, changing the blade from flat to dome shaped. The eventual decline of DMC division in the laminae of Δppd mutant plants suggests that either DMC proliferation is intrinsically limited or that other cell-cycle arrest mechanisms are involved. Conversely, overexpression of PPD reduces the duration of the DMC proliferation zone and thus the number of cells in the lamina, resulting in smaller, flat leaves. The lack of negative curvature in the leaves of PPD-OE plants is consistent with expansion of the margin cells being determined largely by the amount of growth of the lamina. If so, then reduced lamina growth would result in a flat blade, and only increased lamina growth, beyond the expansion capacity of the margin cells, would result in altered leaf curvature. An explanation for the shortened siliques of Δppd mutant plants is less obvious. During the development of WT siliques, cell-cycle arrest occurs before a phase of proximal–distal longitudinal cell expansion, when the length of the septum may be established (25). It is possible that the presence of interspersed small cells in Δppd plants at this phase of silique development may physically limit this expansion. Once the phase of longitudinal expansion has ceased, the shortened length of the septum in Δppd mutant plants may be set. Subsequent prolonged DMC proliferation would then cause the excessive lateral growth and seedpod wall undulations observed in the mutant.
Schematic illustration of the effect of PPD expression levels on DMC proliferation, leaf curvature, and size. Solid black represents the primordium cell proliferation zone. The DMC proliferation zone is shown in speckled gray; regions without cell division are white.
The seeming lack of PPD genes in the grasses may be because the pattern of cell proliferation during the development of grass leaves is quite different from eudicot and other vascular plants. In grasses, cell proliferation is confined to a narrow band of tissue located at the base of the leaf, and specialized cells, such as stomatal guard cells, have a more condensed cell lineage than in eudicots (26, 27). Therefore, PPD genes may only be required to regulate more extensive DMC proliferation during lamina development.
I propose a model in which there are two separate cell-cycle arrest fronts progressing from the tip to the base during leaf development: a primary front that determines arrest of general cell division in the primordium, followed by a secondary front that involves PPD and arrests DMC proliferation. The shape and progression of the primary arrest front is known to be influenced by class II TCP genes, CIN in Antirrhinium (19) or TCP2 and TCP4 in Arabidopsis (20), and it has been suggested that the extent of cell proliferation in the primordium may be regulated by a balance between the antagonistic activities of class I and II TCP genes on the expression of cyclin and ribosomal protein genes (28). Other genes, such as ANT, ARGOS, AN3, GRF5, JAG, SMP, and SWP, which promote cell division during leaf development, also seem to act on proliferation in the primordium (8–16). However, it is has not been established whether any of these genes also influences cell proliferation in the DMC zone. In this model, the arrest of DMC proliferation is regulated independently from the arrest of cell division in the primordium. Overlapping patterns of PPD1 expression and DMC proliferation suggest a possible feedback mechanism whereby DMCs induce PPD gene expression until PPD protein reaches a threshold level that, in turn, restricts DMC recruitment. Hence, a single arrest mechanism, mediated by means of PPD, might act to coordinate the limitation of DMC proliferation. This type of interaction would provide a mechanism for varying the size of a leaf blade distal to the primary arrest front while maintaining lamina anatomy and flatness. It will be interesting to test this model by examining the interactions between the PPD genes and those promoting the maintenance of cell proliferation during leaf development.
Materials and Methods
Plant Materials and Growth Conditions.
Arabidopsis thaliana (L.) Heynh ecotype Ler was used as WT. The mutant designated Δppd was identified during a screen of ≈3,500 M2 plants grown from a fast-neutron-mutagenized population of ecotype Ler obtained from Lehle Seeds (Round Rock, TX). Plants were grown in a temperature-controlled glasshouse at a continuous 21°C or in a controlled environment cabinet at 23°C in 16-h light/8-h dark cycles.
Morphological and Cellular Analyses.
Dimension measurements of fully expanded cotyledons and first and fourth leaves collected at the floral bolt stage were made by flattening the organs between two microscope slides, scanning to produce a digital image, and then calculating length, width, area, and perimeter by using an image analysis program (ImageJ). Because of their curvature, leaves of the Δppd mutant could not be flattened without cutting the margins. Mature silique dimensions were determined by using a digital micrometer. To detect the influence of the ppd mutation on cell proliferation, a CYCB1,1::GUS reporter gene (29) was introgressed into WT and mutant backgrounds. Histochemical detection of GUS activity was carried out as described by Donnelly et al. (1). The frequency of meristemoid cells with GUS activity in the abaxial epidermis of leaves and the outer epidermis of carpel valves at different developmental stages was assessed across the midpoint of the length of the lamina. The total cell number and the number of meristemoid cells with GUS activity in a medial-lateral microscopic (×40) traverse across the leaf were counted and expressed as a cyclin index (number of cells with GUS activity/number of total cells × 100). The cyclin index was determined for at least 15 samples for each leaf or silique developmental stage examined. Silique developmental stages 17.1–17.3 were defined as the progressively older siliques formed before a stage-17 silique (30).
SEM.
To prepare leaf surface replicas for SEM analysis, medial transverse segments dissected from the first leaves of WT and ppd mutant seedlings (at 18 DAG) were coated on the abaxial surface with polyvinylsiloxane dental impression material to make a mold. The leaf tissue was removed and replaced with Spurr’s resin. The positive resin replica was polymerized, detached from the mold, and then sputter-coated with gold for SEM examination.
Genetic Analysis and Mapping of the ppd Locus.
The Δppd mutant was backcrossed five times to WT Ler plants. Inheritance of Δppd was determined in BC5 self-progeny by scoring leaf curvature and trichome branching and measuring silique width. The map-based identification of the PPD locus is described in Supporting Methods, which is published as supporting information on the PNAS web site.
T-DNA Insertion Mutants.
Plant lines containing T-DNA insertions in the coding regions of annotated genes within the ppd deletion region were identified by searching the TAIR Arabidopsis web site (www.arabidopsis.org). Insertion lines for At4g14700 (SALK_140010, SALK_104400), At4g14713 (SALK_149924, SALK_057237), At4g14716 (SALK_119327), At4g14720 (SALK_14698), At4g14730 (SALK_046652), At4g14740 (SALK_013371), and At4g14760 (SALK_003809) were obtained from the Arabidopsis Biological Resource Center (Columbus, OH).
Cloning and Gene Constructs.
For DNA and RNA manipulations, standard molecular biology techniques were used (31). The At4g14713 and At4g14720 genes were amplified by PCR from WT Col-0 ecotype genomic DNA by using Platinum TaqDNA polymerase high fidelity (Invitrogen, Auckland, New Zealand) and gene-specific primers for sequences at the end of the 3′ UTR and ≈1.5 kb upstream of the beginning of the 5′ UTR. Construction of pPPD1, pPPD2, and a PPD1 promoter::GUS reporter gene are described in Supporting Methods. Constructs were transformed into Arabidopsis by the floral dip infiltration method (32). Transgenic plants were confirmed by PCR analysis with a combination of transgene-specific and T-DNA primers. For Δppd complementation experiments, the plants were also tested for the absence of genes in the deleted region and for the mutant reduced trichome branching phenotype.
In Situ Hybridization.
Tissues for in situ hybridization were fixed with 4% (wt/vol) paraformaldehyde/4% (vol/vol) DMSO and passed through a graded butanol series before embedding in Paraplast Plus wax (BioLab, Auckland, New Zealand). Sections (10 μm) were affixed to polylysine-coated slides. Probe preparation is described in Supporting Methods. Slides were taken through prehybridization treatments according to the protocol of Drews and Okamura (33) and hybridized overnight at 45°C. The slides were washed and prepared for immunodetection by following the protocol of Roche (Manheim, Germany).
Real-Time Quantitative RT-PCR.
Total RNA was extracted from inflorescence tips dissected from WT Ler and three individual Δppd::PPD1 complemented transgenic plants (C no. 2, C no. 19, and C no. 26) by using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA). First-strand cDNA was synthesized from total RNA by using an oligo(dT) primer and SuperScript II reverse transcriptase (Invitrogen). Quantitative RT-PCR assays for the relative level of PPD1 (At4g14713) transcript were performed on a MyIQ color real time PCR instrument (Bio-Rad, Auckland, New Zealand) by using ornithine transfer carboxylase as an internal control. Primer sequences and PCR amplification conditions are given in Supporting Methods. The comparative CT method (34) was used to determine relative levels of expression.
Acknowledgments
I thank Roy Meeking, Ruth Cookson (AgResearch), and Douglas Hopcroft (HortResearch, Palmerston North, New Zealand) for technical assistance; David Smyth (Monash, Melbourne, Australia) for the CYCB1,1::GUS transgenic line; and John Bowman, Toshi Foster, and colleagues for comments on the manuscript. This work was funded by Royal Society of New Zealand Marsden Fund Grant AGR304.
Footnotes
- *E-mail: derek.white{at}agresearch.co.nz
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Author contributions: D.W.R.W. designed research, performed research, analyzed data, and wrote the paper.
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Conflict of interest statement: No conflicts declared.
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This paper was submitted directly (Track II) to the PNAS office.
- Abbreviations:
- DMC,
- dispersed meristematic cell;
- Ler,
- Landsberg erecta;
- PPD,
- PEAPOD;
- T-DNA,
- transfer DNA;
- DAG,
- days after germination.
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Freely available online through the PNAS open access option.
- © 2006 by The National Academy of Sciences of the USA
