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. 2009 Mar;57(6):1000-14.
doi: 10.1111/j.1365-313X.2008.03742.x. Epub 2008 Nov 3.

VH1/BRL2 Receptor-Like Kinase Interacts With Vascular-Specific Adaptor Proteins VIT and VIK to Influence Leaf Venation

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Free PMC article

VH1/BRL2 Receptor-Like Kinase Interacts With Vascular-Specific Adaptor Proteins VIT and VIK to Influence Leaf Venation

Teresa Ceserani et al. Plant J. .
Free PMC article

Abstract

VH1/BRL2 is a receptor-like kinase of the BRI1 family with a role in vascular development. In developing Arabidopsis leaves it is expressed first in ground cells and then becomes restricted to provascular and procambial cells as venation forms. We isolated proteins interacting with the activated (phosphorylated) cytoplasmic domain of VH1/BRL2, and found that most belong to three processes: proteasome activity, vesicle traffic and intracellular signal transduction. Two adaptor proteins are included that we named VIT [VH1-interacting tetratricopeptide repeat (TPR)-containing protein] and VIK (VH1-interacting kinase), which are co-expressed in the same cells as VH1/BRL2 at two distinct time points in vein differentiation. Mutation of either adaptor or of VH1 results in vein pattern defects and in alterations in response to auxin and brassinosteroids. We propose that these two adaptors facilitate the diversification and amplification of a ligand signal perceived by VH1/BRL2 in multiple downstream pathways affecting venation.

Figures

Figure 1
Figure 1
Cotyledon vein patterns are altered in insertion mutants of 11 of the isolated candidates for interaction with the VH1/BRL2 catalytic domain. Columns 3-8: percentage of cotyledons displaying the venation pattern depicted at top; “gap” = percentage of cotyledons presenting one or more loops unconnected apically to the midvein or interrupted by a gap; n = total number of scored cotyledons. Patterns of wild-type siblings of each mutant were indistinguishable from the Col-0 wild-type depicted.
Figure 2
Figure 2
VH1/BRL2 direct interactions imply three signaling branches. Listed below each branch are the interaction partners supporting that process.
Figure 3
Figure 3
Thioredoxin-like domain of VIT is inactive. Standard insulin reduction assay of VIT and thioredoxin (Trx), as described in Materials and Methods. VIT is inactive. Standard error bars from three independent experiments were smaller than the point symbols plotted.
Figure 4
Figure 4
Vein pattern is altered in cotyledons of vh1 and vit mutants. A: Col-0 wild-type. B-E: Pattern defects exhibited by vh1 mutants, including gap (E), mis-positioning of top (B) or bottom (C) secondary loop and the formation of extra veins (D). F-I: Defects in vit mutants, including gaps (F-I), reduced pattern (G,I), islands (H) and alteration in the overall cotyledon shape associated with a simplified and discontinuous pattern (F,I). J-K: Higher magnification of vit mutant cotyledons reveal discontinuities in midvein (J) as well as in the top loops (K). A-I: dark field; J-K: DIC images. Note that Col-0 wild-type cotyledons exhibit some variation from the 4-loop pattern in (A), but at far lower frequencies and without the occurrence of gaps (see Figure 1). Patterns of wild-type siblings of each mutant were indistinguishable from the Col-0 wild-type depicted
Figure 5
Figure 5
Vein pattern is altered in primary leaves of vh1, vit and vik mutants. A. Total number of veins per leaf in Col-0 (col) and the three mutants. B. Ratio of free ending veins to closed loops in Col-0 (col) and the three mutant lines. C. Percentage of branches emanating from the primary (1°), secondary (2°) or tertiary (3°) veins in Col-0 (col) and the three mutant lines. D. Dark field and E. DIC images of the top secondary loop of a vit primary leaf exhibiting a gap (D) and absence of cell elongation (E), indicating lack of procambial differentiation.
Figure 6
Figure 6
Responses of root and hypocotyl elongation to four major hormones and to NPA are altered in vh1, vit and vik mutants. The graphs represent the mean of three independent experiments in which 3 day old seedlings were transferred to media containing increasing concentrations of 2,4D, NPA, eBL, ABA or GA3 as indicated. Inhibition of root growth or induction of hypocotyls growth is expressed as percentage of the mean growth without hormone. Error bars are standard errors.
Figure 7
Figure 7
Transcript levels for VH1, VIT and VIK are hormone-responsive in Col-0 wild-type plants. Plant were treated with IAA (A) (20 μM), eBL (B) (1 μM), GA3 (C) (10 μM), or ABA (D) (100 μM), for 1 or 3 hours, and the indicated mRNAs measured by semiquantitative RT-PCR. Amount of each transcript after hormone treatment is expressed as percentage of the mean level without treatment (100%). Error bars are standard errors of three independent experiments on two biological replicates.
Figure 8
Figure 8
Levels of a subset of BR-responsive and auxin-responsive transcripts are altered in vit mutants. 7 day old Col-0 (Col) wild-type or vit mutant seedlings were treated with 20 μM IAA (top panel) or 1 μM eBL (bottom panel). Three independent RTPCR measurements on two biological replicas were made of the indicated transcripts with similar results. – and + indicate untreated and treated seedlings, respectively.
Figure 9
Figure 9
pVIT::GUS expression is highly dynamic and specific. A-S: GUS-reporter activity from expression directed by VIT promoter. A-C: embryo globular (A), late heart (B), and bent cotyledon stage (C). D-F: seedling 75 hours (D), 84 hours (E) and 120 hours (F) post-imbibition. Note high expression in the shoot apical meristem, in the border between radicle and hypocotyl and in the transition zone after. G-I: leaf primordia. J-L: developing leaves. Note expression in stipule (J), hydathodes and fragmented pattern along the venation (K-L), in the petiole and stomata (K-L). M: cauline leaves; N: petals. O-P: flower. Note expression from early stages in the carpel and stamens (O) that becomes restricted to pollen grains, ovules and funiculi (P). Q-S: secondary root. Note expression throughout from early stages (Q-R), subsequent restriction to more internal tissue (S).
Figure 10
Figure 10
Vascular cell-specific expression of VIK. VIK transcripts were assayed by RTPCR in RNA obtained by laser microdissection of leaf epidermal (E), guard (G), mesophyll (M) and vascular bundle (V) cells. VIK RNA is present only in the V lane (upper panel). Lower panel: actin control.

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