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First published online 31 August 2004
doi: 10.1242/jcs.01360

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Arabidopsis VARIEGATED 3 encodes a chloroplast-targeted, zinc-finger protein required for chloroplast and palisade cell development

¹ Institute of Molecular Biology, Copenhagen University, Øster Farimagsgade 2A, 1353 Copenhagen K, Denmark
² BioCentrum, Technical University of Denmark, Building 208, 2800 Lyngby, Denmark
³ Long Ashton Research Station, University of Bristol, Weston Road, Bristol, BS41 9AF, UK
⁴ Research Center Foulum, PO Box 50, 8839 Tjele, Denmark
⁵ Institute of Plant Biology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark
⁶ CNRS, Institut de Biologie Moleculaire, Rue de General Zimmer 12, 67084 Strasbourg, France

View larger version (34K): [in a new window]   Fig. 2. Gene expression in wild type and var3. (A) RNA blot of 20 µg total RNA from wild type (wt) and var3 probed with labeled cDNAs of CAB (X56062), RbcS (X13611; Lhcb1) and EF-1 (16260) control. (B) RT-PCR detection of Lhcb1 in wt and var3 under nonphoto-oxidative conditions (green, wtg and var3g) and photo-oxidative conditions (5 µM norflurazon, wtNf and var3Nf) compared with EF-1 control. (C) Scatter plot of z-score of slide 1 versus z-score of slide 2 (z-score=(intvar3-intwt)/std14wt). Dots in first and third quadrants represent consistent measurements of clones from both arrays from which the var3 sample was measured higher or lower relative to wt, respectively. Dots in second and fourth quadrants represent inconsistent measurements. The line represents a geometric mean of 4 std., equal to the most extreme, inconsistent measurement (geometric mean of absolute values). (D) The table shows the top genes ranked after the geometric mean of z-scores. C, chloroplast encoded; M, mitochondrion encoded; N, nuclear encoded; NC, nuclear encoded chloroplast targeted. *The protein was formerly designated HSP70-7 but is now known as Hsc70-2 (Sung et al., 2001).

View larger version (113K): [in a new window]   Fig. 1. The var3 mutant. (A-C) Leaves of 20-day-old, wild type (Ler), var3 grown under high light and immutans (im) under low light. (D-F) Second or third true leaves at 28 days. (G-H) Light micrographs of cross sections of 12-day-old Ler and var3 leaves. Scale bars, 100 µm. (I-J) Scanning electron micrographs of first true leaves of 12-day-old Ler and var3. Scale bars, 100 µm. (K-P) Confocal images with merged red and Nomarsky channels. Scale bars, 50 µm for K,O; 5 µm for L,N,P; 20 µm for M. K and N are of live material, other images are of formaldehyde fixated tissue. (K) Leaf initial before emergence at the shoot tip. (L) Detail from K showing cells with normal, reduced and undetectable chloroplast development. (M) Variegation in pre-emergent leaf. (N) Detail from M of chloroplasts with normal and arrested development. (O) Light patch with reduced or no chloroplast development, surrounded by normal mesophyll cells in a 16-day-old third leaf. (P) Plastids from the leaf in O with reduced development.

View larger version (48K): [in a new window]   Fig. 3. VAR3 locus and mRNA. (A) VAR3 wild type, sequence of exon one (Var3), sequence of var3 allele with Ds left border (in gray) (var3 left) and var3 with Ds right border (gray) and the 8-bp repeat underlined (var3 right). Numbers give nucleotide position from the same sequence of wild type Col-O (complement of P1 clone MVA3, AB006706). (B) RT-PCR detection of VAR3 mRNA in wild type (wt) and var3 (lanes 2 and 4) compared to control EF-1 mRNA (lanes 1 and 3). Molecular markers are shown on both sides of the gel.

View larger version (91K): [in a new window]   Fig. 4. VAR3 protein. (A) Alignment of VAR3 (accession number ATG517790) with three related Arabidopsis proteins (AAG51125, AAG52346 and AAF79711). Conserved residues are in black and similar residues in gray. Arrows indicate intron sites and the double arrow the Ds insertion site. (B) Top: similarity between VAR3 zinc-finger domains (ZnF1 and 2) with those from yeast (ScARP, P32770) and human nucleoporins (HsLUCA15, P52756; HsNUP358, P49792). Bottom: similarities defining the novel repeated regions of VAR3.

View larger version (88K): [in a new window]   Fig. 5. VAR3 sub-cellular localization. Micrographs of stomata from wild type (top), var3 expressing the 35S-GUS:GFP reporter from pCAMBIA 1303 (middle) and var3 complemented to wild type by expression of the 35S-VAR3:GUS:GFP reporter (bottom). Panels 1, 5 and 9: confocal micrographs of chlorophyll red fluorescence. Panels 2, 6 and 10: fluorescence from GFP. Panels 3, 7 and 11: merged red and green images. Panels 4, 8 and 12: corresponding reference scanning Nomarsky micrographs.

View larger version (32K): [in a new window]   Fig. 6. VAR3 protein interaction and targeting. (A) Yeast two-hybrid interactions. Left panel shows growth of yeast strains carrying different plasmids on minimal medium lacking Trp, Leu, His and adenine. Right panel identifies fusion proteins expressed from the DNA-binding (BD bait) and activation domain (AD prey) plasmids in the yeast strains shown on the left. LAM is human lamin C, TD1 and VA3 (B) Co-immunoprecipitation (Co-IP) of VAR3 and NCED4. (Left to right) Lane 1, IP of cMyc-tagged VAR3; lane 2, IP of HA-tagged NCED4 (partial length open reading frame isolated from the cDNA library screen); lane 3, IP of HA-tagged LAM; lane 4, Co-IP of NCED4 with cMyc-tagged VAR3; lane 5, lack of Co-IP of control LAM with cMyc-tagged VAR3. Proteins and antibodies are noted above. (C) In vitro chloroplast import assay. Isolated, intact chloroplasts were incubated with in vitro translated, radiolabeled precursor proteins, and chloroplasts were then fractionated and samples analyzed by SDS-PAGE and fluorography. Gel lanes are: Tr, in vitro translation mixture; Tr+, translation mixture digested with thermolysin; C, total chloroplasts; C+, thermolysin-treated chloroplasts; S, stroma; T, thylakoid membranes; T+, thermolysin-treated thylakoids. Pre- and m- denote precursor and mature protein, respectively. SSU is a RbcS stromal marker and 33 K is the 33 kDa subunit of the oxygen-evolving complex, thylakoid lumen marker. The sizes (kDa) of precursor and mature forms of VAR3 and NCED4 are indicated.