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. 2006 Apr;18(4):852-66.
doi: 10.1105/tpc.105.040568. Epub 2006 Mar 10.

The TORNADO1 and TORNADO2 Genes Function in Several Patterning Processes During Early Leaf Development in Arabidopsis Thaliana

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Free PMC article

The TORNADO1 and TORNADO2 Genes Function in Several Patterning Processes During Early Leaf Development in Arabidopsis Thaliana

Gerda Cnops et al. Plant Cell. .
Free PMC article

Abstract

In multicellular organisms, patterning is a process that generates axes in the primary body plan, creates domains upon organ formation, and finally leads to differentiation into tissues and cell types. We identified the Arabidopsis thaliana TORNADO1 (TRN1) and TRN2 genes and their role in leaf patterning processes such as lamina venation, symmetry, and lateral growth. In trn mutants, the leaf venation network had a severely reduced complexity: incomplete loops, no tertiary or quaternary veins, and vascular islands. The leaf laminas were asymmetric and narrow because of a severely reduced cell number. We postulate that the imbalance between cell proliferation and cell differentiation and the altered auxin distribution in both trn mutants cause asymmetric leaf growth and aberrant venation patterning. TRN1 and TRN2 were epistatic to ASYMMETRIC LEAVES1 with respect to leaf asymmetry, consistent with their expression in the shoot apical meristem and leaf primordia. TRN1 codes for a large plant-specific protein with conserved domains also found in a variety of signaling proteins, whereas TRN2 encodes a transmembrane protein of the tetraspanin family whose phylogenetic tree is presented. Double mutant analysis showed that TRN1 and TRN2 act in the same pathway.

Figures

Figure 1.
Figure 1.
TRN1 Gene Structure and TRN Gene Expression. (A) Genomic and protein structures of TRN1. Top line, gene structure of TRN1 with two boxed exons and a small intron. The positions of the mutations in each of the four trn1 alleles are represented. Bottom line, TRN1 protein with LRR–ribonuclease inhibitor-like repeat domains (gray boxes), a P-loop for ATP/GTP binding (black box), and homology with DAPK (dark gray box). AA, amino acids. (B) Structure of the TRN2/TET1 protein as a representative of the plant TET family based on comparisons of 10 Arabidopsis TET proteins and 51 TET proteins derived from full-length tomato (Lycopersicon esculentum), soybean (Glycine max), daylily (Hemerocallis hybrid), ice plant (Mesembryanthemum crystallinum), maize (Zea mays), wheat (Triticum aestivum), barley (Hordeum vulgare), and rice (Oryza sativa) cDNA and rice genomic sequences (all members of the five conserved plant tetraspanin classes). The most conserved amino acids are positioned along the protein. Residues that are 100% identical among different plants are in red, and those that are 90% identical are in blue. The polar amino acids in the four transmembrane domains are in italics. (C) Expression analysis of TRN1 and TRN2. RT-PCR analysis of TRN2/TET1 with exon 1 and 2 primers. cs, cell suspensions; f, flowers; l, leaves; r, roots; s, stems; sl, seedlings. (D) In situ localization of TRN1 and TRN2 in 7-d-old seedlings. Top two panels are SAMs hybridized with antisense TRN1 (left) and TRN2 (right) probes; bottom two panels are sections through the cotyledon lamina hybridized with the same probes. le, lower epidermis; lp, leaf primordia; p, palisade parenchyma; s, spongy parenchyma; SAM, shoot apical meristem; ue, upper epidermis; vb, vascular bundle. Bar = 200 μm.
Figure 2.
Figure 2.
Phylogenetic Tree of the Plant Tetraspanin Family, Inferred with MrBayes. Posterior probability values are shown at the internodes. The scale measures the evolutionary distance in substitutions per amino acid site. The neighbor-joining analysis yielded similar classes, although deeper nodes were less supported. In addition, the branch containing Arabidopsis TET13 to TET15 branched off at the base of the tree. Because of this incongruence between the two methods, and the low posterior probability values in the Bayesian analysis, the positions of these genes remained uncertain; therefore, they were not assigned to any of the defined classes. The tree was based on 40 plant-deduced protein sequences obtained from the TIGR gene indices database, the 17 Arabidopsis TET proteins, and predictions from rice genomic clones (asterisks). Genes from monocots are indicated in red, from dicots in dark blue, and from Arabidopsis in light blue. EST data are given next to the genes. The Arabidopsis data set was completed with the Gene Atlas data from Genevestigator (https://www.genevestigator.ethz.ch/). C, cold stress; Da, grown in darkness; Dr, drought stress; F, flowers; Fr, fruits; I, inflorescences; L, leaves; N2, nitrogen starvation; Po, pool of conditions; Ps, pathogen response; R, roots; Sa, salt stress; Se, seed; Sl, seedling; Tc, tissue culture. In the analysis, 22K array data were used.
Figure 3.
Figure 3.
Morphology and Anatomy of trn Mutants. (A) Leaf morphology and venation pattern of first leaves of Ws, trn1-1, trn1-2, trn2-1, trn2-4, trn1-2 trn2-4, as1-1, and trn2-1 as1-1. Double-headed arrow, part of the leaf blade missing = asymmetry; asterisk, open top loop. pb, primary bundle; pv, primary vein or midvein; qv, quaternary vein; sv, secondary vein; tv, tertiary vein. Bar = 0.5 cm. (B) and (C) Graphs of the lamina area (B) and total branch points (brp)/lamina area (C) of fully expanded first and second leaves of 30 C24, 58 trn1-1, 30 Col-4, 41 trn2-1, 39 as1-1, 37 trn2-1 as1-1, 23 Ws, 31 trn1-2, 34 trn2-4, and 50 trn1-2 trn2-4 plants. Single, double, and triple asterisks indicate significant differences between mutant and parent by Student's t-test (P < 0.05) and between double mutants and both parental single mutants. (D) Transverse sections through 8-d-old shoot apices of wild-type (top) and trn2-1 (bottom) leaf primordia, respectively. c, cotyledon; g, gap; lp, leaf primordium; mv, midvein; s, stomata. Bar = 300 μm. (E) Transverse sections through fully expanded first leaves of wild-type (top) and two trn2-1 (bottom) laminas. x indicates the position of the midvein, which is bifurcated in the top leaf and eccentric in the bottom trn leaf. Bar = 200 μm. (F) Whole-leaf flow cytometry of the wild type (Ws) and trn mutants. The different stages of leaf growth are indicated with arrows: proliferation period before the yellow arrow, expansion period between the two arrows, and mature stage after the purple arrow. DAG, days after germination.
Figure 4.
Figure 4.
Venation Pattern in Cotyledons of trn Mutants. The venation complexity is given as the percentage of cotyledons displaying a particular venation pattern. The most representative complexity for a wild type or mutant is boxed. The percentages of cotyledons with discontinuities in their venation are circled. The arrow indicates a vascular island. cots, cotyledons; ND, not determined.
Figure 5.
Figure 5.
Venation Patterning in Leaves of trn Mutants. (A) 5-Bromo-4-chloro-3-indolyl-β-d-glucuronide (X-Gluc) staining of the PATHB8:GUS marker gene activity in 5-, 6-, 7-, and 8-d-old first leaves of wild-type and trn2-1 plants. mv, midvein; sv, secondary veins; tv, tertiary veins; vi, vascular island. (B) Activity of PCYCB1-1:GUS in trn1-1. pc, procambium. (C) PDR5:GUS activity in 5- to 8- and 24-d-old first leaves (left) and 10- to 12-d-old third leaves (right) of wild-type (top) and trn2-1 (bottom) plants. df, distal focus; mf, marginal focus. Double-headed arrows indicate ectopic expression. (D) PDR5:GUS activity in excised 10-d-old Col, trn2-1, and trn1-2 leaves after exposure to 0, 1, and 10 mM α-naphthaleneacetic acid (NAA). (E) Auxin transport measurements of Ws and trn1-2/lop1 hypocotyls and stem fragments. The hypocotyls or stem fragments were placed either upside-up (acropetal transport) or upside-down (basipetal = active transport) in [3H]IAA. The pieces were cut in two, and the radioactivity present in the top half was expressed as a percentage of the total amount of radioactive IAA present in the entire explant. For the upside-up direction, 14 (44) WS and 10 (27) trn1-2/lop1 hypocotyls (stems) were used; for the upside-down direction, 36 (28) Ws and 41 (80) trn1-2/lop1 hypocotyls (stems) were used. Error bars indicate se. (F) Top, differential interference contrast images of xylem patterning of primary and secondary veins at the top of the cotyledon or first leaf of 3-week-old seedlings. Bottom, fluorescence microscopy of phloem sieve tubes stained with aniline blue. x indicates the top of the midvein. Bar = 1 mm.

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