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, 128 (3), 911-23

The Expression of an Extensin-Like Protein Correlates With Cellular Tip Growth in Tomato

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The Expression of an Extensin-Like Protein Correlates With Cellular Tip Growth in Tomato

Marcel Bucher et al. Plant Physiol.

Erratum in

  • Plant Physiol 2002 Jul;129(3):1417

Abstract

Extensins are abundant proteins presumed to determine physical characteristics of the plant cell wall. We have cloned a cDNA encoding LeExt1 from a tomato (Lycopersicon esculentum Mill.) root hair cDNA library. The deduced sequence of the LeExt1 polypeptide defined a novel type of extensin-like proteins in tomato. Patterns of mRNA distribution indicated that expression of the LeExt1 gene was initiated in the root hair differentiation zone of the tomato rhizodermis. Cloning of the corresponding promoter and fusion to the -glucuronidase (GUS) reporter gene allowed detailed examination of LeExt1 expression in transgenic tomato plants. Evidence is presented for a direct correlation between LeExt1 expression and cellular tip growth. LeExt1/GUS expression was detectable in trichoblasts (=root hair-bearing cells), but not in atrichoblasts of the tomato rhizodermis. Both hair formation and LeExt1 expression was inducible by the plant hormone ethylene. Comparative analysis of the LeExt1/GUS expression was performed in transgenic tomato, potato (Solanum tuberosum), tobacco (Nicotiana tabacum), and Arabidopsis plants. In the apical/basal dimension, GUS staining was absent from the root cap and undifferentiated cells at the root tip in all species investigated. It was induced at the distal end of the differentiation zone and remained high proximally to the root/hypocotyl boundary. In the radial dimension, GUS expression was root hair specific in the solanaceous species. Whereas LeExt1 mRNA was exclusively detectable in the rhizodermis, root hair-specific expression correlated with GUS expression in germinating pollen tubes. This is correlative evidence for a role of LeExt1 in root hair tip growth [corrected].

Figures

Figure 1
Figure 1
Peptide structure of LeExt1 and genomic DNA gel-blot analysis. A, Deduced LeExt1 amino acid sequence. Repetitive amino acid units (indicated in bold) are arranged to emphasize various amino acid repeat units and their periodicity. The signal peptide is underlined and the putative cleavage site is marked with an arrow. B, Hydropathy plot of LeExt1 polypeptide. Hydrophilicity and hydrophobicity values are indicated at left. C, Genomic DNA was digested with the designated restriction enzymes. The LeExt1 cDNA was used as a radioactive probe. The positions of DNA marker fragments and their lengths in kb are indicated at left.
Figure 2
Figure 2
Alignment of the deduced amino acid sequence of LeExt1 with that of Dif10 and Dif54. Identical amino acids are shaded in black, similar amino acids are shaded in gray. The conserved pentapeptides YxKxP and SPPPP are underlined.
Figure 3
Figure 3
LeExt1 transcript abundance in seedlings and flowering plants. A, RNA gel-blot analysis was performed with 5 μg of total RNA from seedling root hairs, stripped roots, hypocotyls, cotyledons, and mature leaves and hybridized with randomly labeled cDNA probes, as indicated at left. A cDNA encoding 25S rDNA from tomato served as a control for equal loading of total RNA on the gel and equal transfer to the membrane. Transcript sizes are indicated at right. B, RNA gel-blot analysis was performed with 5 μg of total RNA from stripped seedling roots and root hairs, and with 10 μg of total RNA from the organs indicated and hybridized with randomly labeled cDNA probes, as indicated at left. Transcript sizes are indicated at right.
Figure 4
Figure 4
Localization of LeExt1 transcripts in tomato seedling roots. A through D, Bright-field microscopy of root sections. Shown in A and B are sections hybridized with the LeExt1 antisense probe. The purple dye reflects LeExt1 mRNA. C, Section hybridized with the LeExt1 sense probe as a negative control. D, Section hybridized with an antisense Rpl2 probe as a positive control. Bar = 0.25 mm in A through C; bar = 0.125 mm in D.
Figure 5
Figure 5
Upstream sequence of the LeExt1 gene. Bold letters underlined with dashed arrows indicate putative binding sites of transcription factors P, MYB.Ph3, SBF-1, and Athb-1 as determined using TFSEARCH. Bold arrows on top of a base indicate the start of the different promoter fragments as indicated to the right. The TATA box is underlined and indicated in bold. An asterisk indicates a stop codon upstream of the open reading frame (ORF) in the genomic sequence. Amino acids encoded by the ORF are given in the one letter code. Bold letters designate the N terminus of the LeExt1 protein.
Figure 6
Figure 6
Qualitative GUS assay in Δgen/GUS tobacco plants. A, Schematic drawing of the constructs used. The drawing is to scale. Negative numbers indicate the length of the promoter fragments. NcoI designates the fusion site at the ATG of the GUS gene. NOS is the nopaline synthase transcriptional terminator. Bold lines indicate vector sequence. B, Visual evaluation of GUS staining in roots of T0 lines, containing the promoter constructs as indicated in A. The number of independently transformed lines exhibiting strong GUS staining is listed. The total number of lines analyzed is given in parentheses.
Figure 7
Figure 7
Histochemical localization of GUS activity in germinating tomato seeds and pollen. Tomato plants were transformed with the Δgen1.1/GUS construct. A through C, F1 seeds at various stages of germination assayed for GUS activity. D and E, Pollen harvested from dehisced anthers and germinated in vitro. Bar in A through C = 1 mm; bar in D = 0.1 mm; bar in E = 0.01 mm.
Figure 8
Figure 8
Comparison of LeExt1 and Rpl2 transcript levels in tomato tissues by RT-PCR. RT-PCR was performed with total RNA from roots, leaves, and anthers and hybridized with randomly labeled cDNA probes, as indicated at left. RNA from leaves of transgenic plants constitutively expressing LeExt1 served as a control for primer specificity (control). RNA samples that were not subjected to reverse transcription (−rt) served as a control to determine residual amounts of genomic DNA in the samples (absent in −rt treatments). Black columns indicate relative abundance of transcripts as determined by phosphorimager analysis. The signal for root RNA was arbitrarily set to 100%.
Figure 9
Figure 9
Histochemical localization of GUS activity in transgenic tomato, potato, and Arabidopsis. A, D, and G, Expression of the Δgen1.1/GUS chimeric gene in seedlings of tomato; B, E, and H, potato; C, F, and I, Arabidopsis. D through F, Stereomicroscopy images of GUS-stained root tips. G through I, Bright-field microscopy images of resin-imbedded cross sections of stained roots using Nomarski optics. Bar in A and B = 2 mm; bar in C = 1 mm; bar in D and E = 0.5 mm; bar in F = 0.2 mm; bar in G through I = 0.05 mm.
Figure 10
Figure 10
Modulation of ethylene biosynthesis and root hair development. A, Δgen1.1/GUS tomato seeds were germinated on control medium. At a primary root length of 3 to 5 mm, control seedlings were transferred to fresh medium for 2 d before GUS activity was assayed. B, Seedling root grown in presence of 20 μm AVG and stained for GUS activity after pretreatment as described in A. C, Ectopic root hairs 2 d after transfer to medium containing AVG and 1 mm ethephon. Bar in A = 1 mm; bar in B = 0.5 mm; bar in C = 0.2 mm.

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