Envelope K+/H+ Antiporters AtKEA1 and AtKEA2 Function in Plastid Development

Abstract

It is well established that thylakoid membranes of chloroplasts convert light energy into chemical energy, yet the development of chloroplast and thylakoid membranes is poorly understood. Loss of function of the two envelope K(+)/H(+) antiporters AtKEA1 and AtKEA2 was shown previously to have negative effects on the efficiency of photosynthesis and plant growth; however, the molecular basis remained unclear. Here, we tested whether the previously described phenotypes of double mutant kea1kea2 plants are due in part to defects during early chloroplast development in Arabidopsis (Arabidopsis thaliana). We show that impaired growth and pigmentation is particularly evident in young expanding leaves of kea1kea2 mutants. In proliferating leaf zones, chloroplasts contain much lower amounts of photosynthetic complexes and chlorophyll. Strikingly, AtKEA1 and AtKEA2 proteins accumulate to high amounts in small and dividing plastids, where they are specifically localized to the two caps of the organelle separated by the fission plane. The unusually long amino-terminal domain of 550 residues that precedes the antiport domain appears to tether the full-length AtKEA2 protein to the two caps. Finally, we show that the double mutant contains 30% fewer chloroplasts per cell. Together, these results show that AtKEA1 and AtKEA2 transporters in specific microdomains of the inner envelope link local osmotic, ionic, and pH homeostasis to plastid division and thylakoid membrane formation.

Figure 1.

Arabidopsis kea1kea2 double mutant leaves appear chlorotic in a developmental gradient. A, Wild-type Columbia-0 (Col-0) and kea1 and kea2 mutants were grown on soil for 3 weeks. Two independent kea1kea2 double mutants were stunted in size and had pale yellowish leaves. B, Wild-type plants and kea1-2kea2-1 mutants at 2 weeks (left) and 3 weeks (right). Newly emerging leaves of mutants (insets) are pale yellowish at the base. C, A kea1-1kea2-2 mutant at 4 weeks (left) and 5 weeks (right). The plants are oriented to show the numbering of visible leaves from old to new. Leaves 5 to 8 at 4 weeks show yellowish areas, which turn green progressively from top to base by 5 weeks. D, Greening observed in expansion and maturation zones (above the dotted line) of mutant leaves (right). Representative wild-type (wt) leaves were taken from 3- and 4-week-old plants (left); mutant leaves are from 3-, 4-, and 5-week-old plants. Bars = 0.5 cm.

Figure 2.

Levels of essential photosynthetic protein complexes are severely decreased in developing kea1kea2 leaves. Total protein from 0.5 mg fresh weight of wild-type Columbia-0 (Col-0) or green (g) and yellow (y) sections of kea1kea2 leaves was separated by SDS-gel electrophoresis and transferred to polyvinylidene difluoride membranes for immunostaining. Different amounts (20%–100%) of wild-type protein were analyzed to estimate the relative abundance of proteins in kea1kea2 leaves. PsbA, PSII protein D1; PsbD, PSII subunit D2; PsaD, PSI subunit D; Cytb6, cytochrome b₆f complex subunit; AtpB, β-subunit of ATP synthase; Lhcb2, subunit 2 of light-harvesting complex 2; RbcL, Rubisco large subunit; Alb3, Albino3 thylakoid insertase; KEA3, Arabidopsis thylakoid membrane K⁺/H⁺ antiporter KEA3; P.R., Ponceau Red-stained proteins served as a loading control.

Figure 3.

Polar distribution of AtKEA2-GFP in small chloroplasts. A, AtKEA2-GFP fluorescence (green) varies in a plastid population. Images are from young leaves of a 2-week-old plant. GFP fluorescence is brightest in small chloroplasts with weak Chl fluorescence (red). B, GFP fluorescence is localized to the poles in small dividing chloroplasts. C to E, Magnified images of the box in A, GFP (C), Chl (D), and overlay (E), showing a dividing chloroplast with polar AtKEA2-GFP fluorescence and a mature chloroplast with fluorescent spots. F, In a mature section of an expanded leaf, plastids show some isolated GFP spots. The image is from a 3-week-old plant. G, Polar distribution of GFP fluorescence in plastids of 2 to 3 μm in length. Fluorescence is nonpolar in 5- to 8-μm chloroplasts (n = 200). H and I, AtKEA2-GFP fluorescence in light-grown (H) versus dark-grown (I) seedlings. Cotyledons of 10-d-old seedlings were examined. Bars = 10 μm in all images.

Figure 4.

The N-terminal domain targets KEA Ia antiporters to distinct foci. A, N terminus of OsKEA1 (residues Met-1 to Glu-536) expressed transiently in Arabidopsis protoplasts. Shown are the N-terminal GFP fluorescence (left) and overlay of GFP and Chl fluorescence (right). Bar = 10 μm. B, The N-terminal domain of OsKEA1 shows punctate distribution in chloroplasts. Overlay of GFP (green) and Chl (red) fluorescence of two chloroplasts is shown. Individual chloroplasts were obtained by breaking a protoplast. Bars = 2.5 μm. C, The transport domain of AtKEA2-GFP protein (Met-556 to Ile-1174) stably expressed in Arabidopsis distributes evenly around plastids. Overlay of GFP and Chl fluorescence is shown. Bar = 10 μm. D, GFP (top), Chl (middle), and overlay of GFP and Chl fluorescence (bottom) images from box 1 in C. Bar = 10 μm. E, Quantitative analysis shows distinct patterns of full-length AtKEA2-GFP (Met-1 to Ile-1174) or the N terminally truncated AtKEA2-GFP (Met-556 to Ile-1174). Patterns were categorized as follows: Uniform (as in C, 1), Patchy (as in C, 2), Spots (Fig. 3, C–E), or Polar (Fig. 3B). Total plastids were scored from several images of at least three different leaves. Total plastids scored for truncated (blue) or full-length (red) AtKEA2-GFP are 371 or 171, respectively.

Figure 5.

Mesophyll cells of the kea1kea2 mutant contain fewer chloroplasts. A, Confocal microscopy shows that mutant leaves have a lower density of chloroplasts compared with wild-type Columbia-0 (Col-0) leaves. B, Quantitative analysis confirms that the double mutant has fewer chloroplasts per cell. Chloroplasts per cell were counted as described in Supplemental Methods S1. C, Chloroplasts of double mutants are larger. The size of individual chloroplasts was determined by measuring the area in Corel Photo Paint. A value of 100% = 200 chloroplasts each for mutants and for the wild type. The mean areas of wild-type and double mutant chloroplast are 36 ± 9 and 45 ± 15 µm², respectively.