A calcium sensor-regulated protein kinase, CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASE19, is required for pollen tube growth and polarity

Abstract

Calcium plays an essential role in pollen tube tip growth. However, little is known concerning the molecular basis of the signaling pathways involved. Here, we identified Arabidopsis (Arabidopsis thaliana) CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASE19 (CIPK19) as an important element to pollen tube growth through a functional survey for CIPK family members. The CIPK19 gene was specifically expressed in pollen grains and pollen tubes, and its overexpression induced severe loss of polarity in pollen tube growth. In the CIPK19 loss-of-function mutant, tube growth and polarity were significantly impaired, as demonstrated by both in vitro and in vivo pollen tube growth assays. Genetic analysis indicated that disruption of CIPK19 resulted in a male-specific transmission defect. Furthermore, loss of polarity induced by CIPK19 overexpression was associated with elevated cytosolic Ca2+ throughout the bulging tip, whereas LaCl3, a Ca2+ influx blocker, rescued CIPK19 overexpression-induced growth inhibition. Our results suggest that CIPK19 may be involved in maintaining Ca2+ homeostasis through its potential function in the modulation of Ca2+ influx.

Figure 1.

Subcellular localization and overexpression phenotype for pollen-expressed CIPKs in tobacco pollen tubes. A, Representative images of pollen tubes transiently expressing CIPK-GFPs. A tube expressing GFP alone was used as a control. Approximately 4 h after bombardment, localization of GFP-tagged CIPKs was analyzed using confocal microscopy. The images were captured in single optical slices. Representative images were selected from 50 to 60 samples. Bar = 5 μm. B, Quantitative analysis of pollen tube phenotypes induced by overexpression of different CIPK-GFPs. Pollen tube length and maximum tip width were measured 3.5 h after bombardment. Asterisks represent significant differences from the GFP control (P < 0.01, Student’s t test), and error bars indicate sd. Data were collected from 100 tubes per experiment.

Figure 2.

Expression patterns of CIPK19 in Arabidopsis. A, RT-PCR analysis of CIPK19 transcripts in different Arabidopsis tissues. Total RNA was isolated from the roots, leaves, stems, flowers, and mature pollen of wild-type Columbia plants. Following complementary DNA (cDNA) synthesis, the relative expression level of CIPK19 in diverse tissues was determined by RT-PCR with gene-specific primers. Amplification of TUBULIN2 served as a loading control. B, CIPK19 promoter-GUS expression in transgenic Arabidopsis plants. Histochemical GUS staining was carried out in various tissues (flower [a], pollen tubes [b], leaf [c], root [d], and stem [e]) of CIPK19 promoter-GUS transgenic plants. Bars = 0.5 mm (a, d, and e), 40 μm (b), and 3 mm (c).

Figure 3.

The tube growth phenotype caused by CA-CIPK19 overexpression in tobacco pollen tubes. A, Diagram of the C-terminal truncation and mutation for CIPK19 used in transient expression. The regulatory C-terminal domain is partitioned into the two interaction domains, the asparagine-alanine-phenylalanine (NAF) domain and the protein phosphatase interaction (PPI) domain. The red arrow indicates the mutation site from Thr to Asp in the activation loop. B, Representative pollen tubes transformed with CA-CIPK19. Images show pollen tubes cultured in vitro for 4 h after bombardment. A tube expressing GFP alone was used as a control. Bar = 30 μm. C, Quantitative analysis of CA-CIPK19-induced growth phenotypes. Pollen tube length and maximum tip width were measured 4 h after bombardment. Asterisks represent significant differences from the GFP control (P < 0.01, Student’s t test). Data are means ± sd; n = 70 to 80.

Figure 4.

CIPK19 is required for normal pollen tube growth. A, Representative in vitro-germinated pollen tubes from wild-type (WT), cipk19, and complemented (COM) lines. Images show pollen tubes grown for approximately 3.5 h. Arrows indicate excess bulging on the tube surface. Bars = 30 μm. B, RT-PCR analysis of a homozygous mutant line shows the absence of CIPK19 transcripts in the T-DNA line. Amplification of Arabidopsis TUBULIN2 was used as a positive internal control. C and D, Statistical analysis of tube length and width from wild-type, cipk19, and complemented lines. Pollen tube length and maximum tip width were measured after 3.5 h of incubation. Data were collected from three individual experiments (80 tubes per experiment). Asterisks indicate significant differences from the wild type (P < 0.01, Student’s t test).

Figure 5.

Growth patterns of wild-type (WT) and cipk19 mutant pollen tubes in female tissues. Wild-type pistils were pollinated with wild-type or cipk19 pollen grains and subsequently stained with Aniline Blue 4, 24, or 48 h after pollination (hap). Bars = 100 μm.

Figure 6.

The distribution of wild-type and heterozygous seeds within the resulting silique harvested from an outcross using pollen grains from cipk19 heterozygous plants and wild-type pistil. The overall percentages of wild-type and heterozygous seeds in the top, middle, and bottom sections of a silique are shown above each distinct area separated by black bars. Bar = 1 mm.

Figure 7.

CIPK19 may regulate Ca²⁺ influx. A, Ratiometric Ca²⁺ imaging of tobacco pollen tubes. Pollen from tobacco was bombarded with CIPK19-GFP or GFP alone (control). Transformed tubes were then microinjected with the fluorescent Ca²⁺ imaging dye indo-1-dextran, and Ca²⁺ distribution was monitored by confocal ratio imaging. Calcium concentrations have been pseudocolor coded according to the scale at bottom. The results are displayed with bright-field images (left), GFP fluorescence images (middle), and ratiometric Ca²⁺ images (right). Bar = 5 μm. B, LaCl₃ rescued CIPK19 overexpression-induced growth inhibition of tobacco pollen tubes. LaCl₃ was added to growth medium at a final concentration of 100 µm. Data were collected from three individual experiments (60 tubes per experiment cultured for 3.5 h). Asterisks indicate significant differences from the GFP control at the same data point (P < 0.01, Student’s t test). C, An Arabidopsis pollen tube from the CIPK19 loss-of-function mutant is less sensitive to high [Ca²⁺]_ex and more sensitive to low [Ca²⁺]_ex. Pollen grains from the wild type (Columbia) and the homozygous cipk19 mutant were incubated for 3.5 h in germination medium with different Ca²⁺ concentrations. Data were collected from three individual experiments (70 tubes per experiment). Asterisks indicate significant differences from the wild-type control at the same [Ca²⁺]_ex level (P < 0.01, Student’s t test). D, A tobacco pollen tube overexpressing CIPK19 (CIPK19 OX) is more sensitive to high [Ca²⁺]_ex and less sensitive to low [Ca²⁺]_ex. LAT52:CIPK19 and LAT52:GFP were transiently co-overexpressed in tobacco pollen tubes, and transformed tubes were cultured for 3.5 h in germination medium with different Ca²⁺ concentrations. Data were collected from three individual experiments (80 tubes per experiment). Asterisks indicate significant differences from the GFP control at the same [Ca²⁺]_ex level (P < 0.01, Student’s t test).