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First published online January 23, 2009
doi: 10.1242/10.1242/dev.028365

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The polarly localized D6 PROTEIN KINASE is required for efficient auxin transport in Arabidopsis thaliana

Melina Zourelidou^1,*, Isabel Müller^1,*, Björn C. Willige^1,*, Carola Nill¹, Yusuke Jikumaru², Hanbing Li^1, and Claus Schwechheimer^1,*,

¹ Tübingen University, Center for Plant Molecular Biology, Department of Developmental Genetics, Auf der Morgenstelle 5, 72076 Tübingen, Germany.
² RIKEN, Plant Science Center, Suehiro-cho 1-7-22, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.

View larger version (66K): [in this window] [in a new window]   Fig. 1. Isolation of d6pk mutants and YFP:D6PK overexpression lines. (A) Schematic representation of the four Arabidopsis D6PK genes, names of the mutant alleles, and positions of the respective T-DNA insertions. Black triangles mark the positions of the T-DNA insertions in lines that were used for mutant analyses. Introns are represented by lines, protein-coding exon sequences are presented as black boxes, non-coding exon sequences as gray boxes. (B) The RT-PCR-based gene expression analysis (28 PCR amplification cycles) of D6PK gene expression in the mutant alleles used for this study reveals the absence of full-length transcripts in d6pk-1, d6pkl2-2 and d6pkl3-2, and reduced transcript levels in d6pkl1-1. (C-M) Phenotype of 6-week-old adult plants (top) and vegetative rosettes of 3-week-old plants (bottom) of d6pk single, double, triple and quadruple mutants, as indicated. Scale bars: 6 cm. (N-P) The differentiation defects of apical meristems from lateral shoots of d6pk quadruple mutants (O) are reminiscent of the differentiation defects observed in pin1 mutant alleles (P). Scale bar: 0.5 cm. (Q) The overexpression of YFP:D6PK results in plants with reduced plant height and broader leaves, when compared with wild type. Scale bar: 6 cm. (R) In contrast to wild-type leaves, the leaves of YFP:D6PK plants are shorter and broader, and in some places are uneven. Scale bar: 1 cm.

View larger version (87K): [in this window] [in a new window]   Fig. 2. Arabidopsis d6pk mutants and overexpression lines have defects in lateral root formation and gravitropism. (A-C) The relative number of emerged lateral roots is reduced in 10-day-old light-grown d6pk d6pkl1 d6pkl2 mutants (d6pk012) and YFP:D6PK seedlings when compared with wild type. Scale bar: 0.5 cm. (D-F) Ten-day-old light-grown d6pk d6pkl1 d6pkl2 mutants (d6pk012) grow agravitropically when compared with wild-type and YFP:D6PK seedlings. Scale bar: 0.5 cm. (G) Approximately 10% of the d6pk d6pkl1 d6pkl2 (d6pk012) mutant seedlings (5 days old) have only a single cotyledon or fused cotyledons. Scale bar: 0.5 cm. (H) Light-grown YFP:D6PK seedlings have epinastic cotyledons. Scale bar: 0.5 cm. (I) Dark-grown YFP:D6PK seedlings (7 days old) have a dramatically shortened hypocotyl. The overexpression of YFP:D6PK was confirmed by immunoblots and by fluorescence microscopy (see also Fig. 4). Scale bar: 3 mm. (J) Relative number of lateral roots in 9-day-old d6pk mutant and YFP:D6PK seedlings. Nomenclature: d6pk0 (d6pk-1), d6pk01 (d6pk-1 d6pkl1-1), d6pk012 (d6pk-1 d6pkl1-1 d6pkl2-2), etc. The number of lateral roots decreases with reduced D6PK gene dosage. The graph shows the average root length (and standard deviation) of at least eight seedlings for each genotype. The root length is reduced in YFP:D6PK seedlings but is unaltered in the single d6pk mutants when compared with wild type (compare for example A-F).

View larger version (41K): [in this window] [in a new window]   Fig. 3. Altered auxin transport in d6pk mutants and YFP:D6PK plants. (A-D) DR5:GUS auxin (response) maxima at the sites of lateral root initiation in the primary roots of 7-day-old seedlings grown on standard growth medium (A-C) and on medium supplemented with 1 µM NPA (D). Scale bar: 1 mm. (E-H) The DR5:GUS maxima in primary root tips of 7-day-old seedlings is shifted above the quiescent center (asterisks) in d6pk d6pkl1 d6pkl2 (d6pk012) mutant roots. Scale bar: 0.5 mm. (I,J) Auxin (response) maxima in leaves of wild-type (I) and YFP:D6PK transgenic (J) plants. Maxima in the leaf surface coincide with uneven areas of the leaf. Scale bars: 1 cm. (K,L) Staining for the activity of DR5:GUS reveals the formation of adventitious root primordia in hypocotyls of dark-grown YFP:D6PK seedlings (blue, L), a differentiation event that is never observed in the wild type (K). Scale bar: 3 mm. (M) Relative root elongation of wild type, d6pk d6pkl1 d6pkl2 (d6pk012) mutants, d6pk d6pkl1 d6pkl3 (d6pk013) mutants, and YFP:D6PK transgenic seedlings grown on standard growth medium supplemented with the auxin transport inhibitor NPA. The values displayed in the graph correspond to the average and standard deviation of at least eight replicate measurements. t-test significance: ***P<0.001, **P=0.001-0.01, *P=0.01-0.05. (N) Auxin transport in stem segments of wild type and d6pk d6pkl1 d6pkl2 (d6pk012) mutants, as revealed by measuring the presence of radiolabeled IAA. The measured IAA transport is the result of active auxin transport, as it is blocked in the presence of NPA. In each experiment, four replicate measurements were analyzed, the experiment was repeated four times, the average and the standard deviation of one experiment are shown.

View larger version (97K): [in this window] [in a new window]   Fig. 4. YFP:D6PK colocalizes with PIN proteins at the basal membrane of root cells. (A-D) Immunostaining with anti-PIN1 (red signal) reveals the lower (basal) localization of PIN1 in stele cells of roots of wild-type, d6pk mutant and YFP:D6PK transgenic seedlings (red arrowheads). YFP:D6PK (green signal) and PIN1 (red signal) colocalize at the basal membrane of stele cells (green and red arrowheads). Spearman's correlation coefficient for the PIN1 and YFP:D6PK signal shown in D is 0.586. (E-H) Following treatment with BFA, PIN1 (red) accumulates in endosomal compartments in all three genotypes (red arrowheads). YFP:D6PK (green) looses its association with the membrane and becomes cytoplasmic following BFA treatment (G,H). Spearman's correlation coefficient for the PIN1 and YFP:D6PK signal shown in H is 0.0135. (I,J) Immunostaining with anti-PIN2 (red) reveals the apical localization of PIN2 in root epidermal cells and the basal localization of PIN2 in cortical cells (red arrowheads). YFP:D6PK (green) colocalizes with basal PIN2 in the cortex but not with apical PIN2 in the epidermis (green and red arrowheads; K,L). E, epidermis; C, cortex. Spearman's correlation coefficient for the PIN2 and YFP:D6PK signal in K, in cortical cells is 0.255, in epidermal cells is -0.609. (M-P) Immunostaining with anti-PIN4 (red) reveals the basal localization of PIN4 in cells of the root tips of wild-type, d6pk mutant and YFP:D6PK transgenic seedlings. YFP:D6PK (green) and PIN4 (red) colocalize at the basal membrane of lateral root cap cells (green and red arrowheads; O,P). Spearman's correlation coefficient for the PIN4 and YFP:D6PK signal shown in P is 0.558. (Q-S) Propidium iodide staining of wild-type, d6pk d6pkl1 d6pkl2 mutant and YFP:D6PK roots reveals that the different genotypes have normal root morphology. (S) Although overexpressed, YFP:D6PK (green, live imaging) is not detectable in the central root meristem, but it is expressed in lateral root cap cells, cortical cells, the epidermis and stele cells (see also C,D).

View larger version (61K): [in this window] [in a new window]   Fig. 5. Biochemical and genetic interaction between D6PK and PIN auxin efflux facilitators. (A) Recombinant GST:D6PK but not the kinase-dead variant GST:D6PKin phosphorylates the histidine-tagged cytoplasmic loops of PIN1, PIN2, PIN3, PIN4 and PIN7 in vitro. (B) Following immunoprecipitation from transgenic plants, YFP:D6PK but not the kinase-dead variant YFP:D6PKin phosphorylates a recombinant GST-tagged PIN1 cytoplasmic loop in vitro. (C) Untagged D6PK or YFP:D6PK (but not the kinase-dead variant expressed from YFP:D6PKin) phosphorylate PIN1:GFP when coexpressed in Arabidopsis protoplasts. The phosphorylation is revealed by the appearance of additional low mobility PIN1:GFP-specific bands, whose abundance is diminished following treatment with calf intestinal alkaline phosphatase (CIAP; right panel). (D) Genetic interaction between a pin1 mutant allele and d6pk mutants. The height of pin1 d6pk d6pkl1 d6pkl2 (pin1 d6pk012) and of pin1 d6pk d6pkl2 (pin1 d6pk02) mutants is reduced more strongly than that of the respective pin1 or d6pk mutants (see Table S1 in the supplementary material). Numbers indicate the average plant height and its standard deviation (cm) from at least six plants. The shoot differentiation defects of the pin1 mutants are maintained in the pin1 d6pk mutant combinations (not visible in the case of pin1 d6012), but the leaf shape of pin1 d6pk d6pkl2 mutants is altered in comparison to that of the pin1 and d6pk d6pkl2 mutants, such that the leaves are rounder and have an uneven leaf surface.

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