Envelope K+/H+ Antiporters AtKEA1 and AtKEA2Function in Plastid Development1[OPEN]

María Nieves Aranda-Sicilia, Ali Aboukila, Ute Armbruster2, Olivier Cagnac3, Tobias Schumann,Hans-Henning Kunz, Peter Jahns, María Pilar Rodríguez-Rosales, Heven Sze, and Kees Venema*

Departimento de Bioquímica, Biología Celular, y Molecular de Plantas, Estación Experimental del Zaidín,Consejo Superior de Investigaciones Científicas, 18008 Granada, Spain (M.N.A.-S., A.A., O.C., M.P.R.-R., K.V.);Howard Hughes Medical Institute, Department of Plant and Microbial Biology, University of California,Berkeley, California 94720 (U.A.); Institute of Plant Biochemistry, Heinrich-Heine-University Düsseldorf,D–40225 Duesseldorf, Germany (T.S., P.J.); School of Biological Sciences, Washington State University,Pullman, Washington 99164–4236 (H.-H.K.); and Department of Cell Biology and Molecular Genetics,University of Maryland, College Park, Maryland 20742 (H.S.)

ORCID IDs: 0000-0002-0395-9669 (A.A.); 0000-0003-2751-969X (O.C.); 0000-0001-8000-0817 (H.-H.K.); 0000-0002-5340-1153 (P.J.);0000-0001-7711-3239 (M.P.R.-R.); 0000-0003-4118-3758 (H.S.); 0000-0001-5177-6464 (K.V.).

It is well established that thylakoid membranes of chloroplasts convert light energy into chemical energy, yet the development ofchloroplast and thylakoid membranes is poorly understood. Loss of function of the two envelope K+/H+ antiporters AtKEA1and AtKEA2 was shown previously to have negative effects on the efficiency of photosynthesis and plant growth; however, themolecular basis remained unclear. Here, we tested whether the previously described phenotypes of double mutant kea1kea2plants are due in part to defects during early chloroplast development in Arabidopsis (Arabidopsis thaliana). We show thatimpaired growth and pigmentation is particularly evident in young expanding leaves of kea1kea2 mutants. In proliferating leafzones, chloroplasts contain much lower amounts of photosynthetic complexes and chlorophyll. Strikingly, AtKEA1 and AtKEA2proteins accumulate to high amounts in small and dividing plastids, where they are specifically localized to the two caps of theorganelle separated by the fission plane. The unusually long amino-terminal domain of 550 residues that precedes the antiportdomain 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 theinner envelope link local osmotic, ionic, and pH homeostasis to plastid division and thylakoid membrane formation.

Chloroplasts from plants and algae originated froman endosymbiotic event, most likely involving an an-cestral photoautotrophic prokaryote related to today’scyanobacteria. In plants, plastids have become com-plex organelles that are well integrated into the planthost cell, where they differentiate and divide in tunewith plant differentiation and development (Basakand Møller, 2013). Both chloroplasts and cyanobacteriahave thylakoidmembranes, harboring pigment-proteincomplexes that perform the light-dependent reactionsof oxygenic photosynthesis (Pfeil et al., 2014). The thy-lakoid membrane system in chloroplasts has devel-oped a complex structure of grana stacks connected bystroma lamellae. The thylakoid membrane system ap-pears to be separated from the inner envelope mem-brane. This results in the formation of two independentcompartments with differing functions that requirecommunication and transport between bothmembranesduring thylakoid membrane development (Chigri, 2012;Vothknecht et al., 2012).

When seedlings emerge from the soil, light triggersthe transformation of proplastids into chloroplasts andinduces an increase in chloroplast number by fission.This transformation requires the assembly of photo-synthetic complexes into thylakoid membranes and a

massive increase in total volume and solute content ofthe organelle. The photosynthetic pigments and thyla-koid membrane lipids are synthesized at the plastidenvelope and transported to the developing thylakoidmembrane by so-far poorly characterized transportsystems (Jarvis and López-Juez, 2013; Rast et al., 2015).The proplastid-to-chloroplast transition occurs duringthe differentiation of meristematic cells into photosyn-thetic tissue (Sakamoto et al., 2008). Once chloroplastshave been established, they propagate in developing(dividing) tissues. While the signal for the proplastid-to-chloroplast transition is light, little is known aboutthe downstreammechanisms that promote the complexprocesses associated with this transition.

Early studies suggested an important role for K+/H+

exchange fluxes at the chloroplast inner envelope andthylakoid membranes (Wu and Berkowitz, 1992; Fanget al., 1995). The recent discovery of Arabidopsis (Ara-bidopsis thaliana) K+/H+ antiporters, AtKEA1 andAtKEA2, at the inner envelope and AtKEA3 at thethylakoid membrane represented an important break-through in uncovering the molecular basis for chloro-plast K+/H+ homeostasis (Aranda-Sicilia et al., 2012;Armbruster et al., 2014; Kunz et al., 2014). AtKEA3 wasshown to be involved in modulation of the thylakoid

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lumen pH, which is important for photosynthetic per-formance and nonphotochemical quenching underfluctuating light conditions (Armbruster et al., 2014).AtKEA1 and AtKEA2 were suggested to be funda-mental for chloroplast osmotic balance and integrity,while reduced photosynthetic efficiency of the kea1kea2double mutants was attributed mainly to perturbationof ionic (K+ and H+) gradients across envelope andthylakoid membranes (Kunz et al., 2014). The basis forthe chlorotic appearance and reduced photosynthesis inyoung leaves, however, was not addressed. The aim ofthis work was to test if AtKEA1 and AtKEA2 have arole at the early stage of leaf development, beforechloroplasts are fully differentiated, as defective or-ganelle biogenesis could compromise photosyntheticactivities and growth.

RESULTS

Disruption of AtKEA1 and AtKEA2 Causes StuntedGrowth and Reduced Levels of Pigmentation andPhotosynthetic Complexes in Early Growth Stages

Arabidopsis KEA1 and KEA2 are close homologs ofthe KEA Ia family that possess a long N-terminal ex-tension before an antiporter domain (Chanroj et al.,2012). Here, we generated two independent doublekea1kea2 T-DNA insertion lines (Supplemental Fig. S1)and confirmed that kea1kea2 double mutants show re-duced growth and have pale yellowish leaves (Fig. 1, A

and B; Kunz et al., 2014). The pale yellowish color wasespecially evident in young newly formed leaves butdisappeared partially in later growth stages (Fig. 1, Band C). Typically, newly emerging leaves have reducedpigmentation but turn green from top to base, as leafcells exit the cell division cycle and enter the cell ex-pansion phase from tip to base (Fig. 1, C and D). Thestunted growth and pigmentation phenotype wascomplemented by expressing an AtKEA2-GFP fusionprotein. Thus, the phenotype is due to a loss of anti-porter activity (Supplemental Fig. S2).

The reduced pigmentation evident in young leaf tis-sues of kea1kea2 plants and the recovery in later leafstages suggested that chloroplast development wasdelayed when envelope K+/H+ antiporter is impaired.To investigate this idea, we separated the yellowishsectors (close to the petiole and along the middle leafvein) and the green sections (at the tip of the leaf andalong the edges) of mutant and wild-type leaves bycutting semicircles or triangles from the base of the leaf(Supplemental Fig. S3). Pigments in the yellow andgreen sections were analyzed. As expected, the youngerleaf tissue contained significantly less chlorophyll (Chl)on a fresh weight basis than the green mature parts ofkea1kea2 mutant and wild-type leaves (Table I). On aChl basis, however, a significant increase of all xan-thophylls (neoxanthin, lutein, and the xanthophyll cy-cle pigment pool) and a significant reduction ofb-carotene were detectable in young kea1kea2 tissues(Table I). These changes indicate an increased ratio ofantenna proteins to the reaction center, in accordancewith the lower Chl a/b ratio in yellow sections (Table I).This result suggests that the amount of photosystems inyellow sections of the double mutant leaf is reducedand that the existing complexes show an altered an-tenna protein composition.

To verify the abundance of photosynthetic complexesin developing leaf tissues, total proteins from green andyellow leaf sections of kea1kea2mutants were separatedby electrophoresis and immunostained for specificsubunits (Fig. 2). The selected subunits serve as goodindicators for the abundance of the respective com-plexes, because subunits either do not accumulate at allor accumulate only in marginal amounts in the absenceof their functional complexes. Particularly the twosubunits of the PSII reaction center, PsbA and PsbD,were decreased in yellow leaf sections (approximately20% of the wild type), but also subunits of PSI (PsaD),the cytochrome b6f complex, and the light-harvestingcomplex II were strongly reduced compared with thewild type (approximately 40%). The ATP synthasesubunit and Rubisco large subunit accumulated toabout 60% of the wild type, while AtKEA3 and Albino3(involved in protein insertion in the thylakoid mem-brane) were present at wild-type levels. In the differ-entiated mature parts of kea1kea2 leaves, the levels of allanalyzed protein subunits recovered to wild-typelevels. Together, the effect of AtKEA1 and AtKEA2loss of function on pigmentation and photosyntheticprotein complex accumulation, particularly in the

1 This work was supported by ERDF-cofinanced grants from theSpanish Ministry of Economy and Competitiveness (grant nos.BIO2012–33655 and BIO2015–65056-P to K.V. and M.P.R.-R.) and theConsejería de Economía, Innovación, Ciencia, y Empleo, Junta deAndalucía (grant no. CVI–7558 to K.V. andM.P.R.-R.), by the DeutscheForschungsgemeinschaft (grant nos. AR 808/1–1 and AR 808/1–2 toU.A. and grant no. JA 665/10–1 to P.J.), by the U.S. Department ofEnergy, Division of Chemical Sciences, Geosciences, and Biosciences,Office of Basic Energy Sciences (grant no. BES DEFG–0207ER15883 toH.S.), by the National Science Foundation (grant no. IOS–1553506 toH.-H.K.), and by the Agricultural Research for Development Fund,Ministry of Agriculture, Egypt (Ph.D. grant to A.A.).

2 Present address: Max Planck Institute of Molecular Plant Physi-ology, Wissenschaftspark Golm, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany.

3 Present address: Fermentalg, 4 bis rue Rivière, 33500 Libourne,France.

* Address correspondence to kev@eez.csic.es.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Kees Venema (kev@eez.csic.es).

M.N.A.-S., A.A., O.C., U.A., P.J., M.P.R.-R., H.S., and K.V. de-signed experiments; M.N.A.-S., A.A., O.C., U.A., H.-H.K., T.S., M.P.R.-R., and K.V. performed experiments and analyzed data; all authorscontributed to writing draft versions of the article and correcting finalversions; H.S., U.A., M.P.R.-R., and K.V. wrote the article; K.V. con-ceived the project.

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young developing leaf tissues, may suggest a functionof envelope K+/H+ antiport in chloroplast biogenesis.

AtKEA2-GFP Localizes to the Poles inDeveloping Chloroplasts

To visualize the AtKEA2 protein during leaf devel-opment, plants were stably transformed with a custom-designed construct encodingAtKEA2intron1-4-GFP fusionprotein. AtKEA2-GFP fluorescence formed a ring sur-rounding isolated chloroplasts of mesophyll cells(Supplemental Fig. S4). Fluorescencewas not detectablein all chloroplasts, and some plastids showed localpatches of fluorescence. Our results agree with a pre-vious study where AtKEA2 and AtKEA1 localize to theinner envelope membrane of chloroplasts from guardcells (Kunz et al., 2014). In mesophyll cells of intactleaves, AtKEA2-GFP fluorescence was associated withsmall developing chloroplasts, concentrated at two orthree caps or poles, but excluded from the divisionsite (Fig. 3, A–E). In mature chloroplasts, AtKEA2-GFPfluorescence was much less intense and only remainedin discrete spots (Fig. 3F). We observed a very similarlocalization of the AtKEA1-VENUS and AtKEA2-VENUSproteins (Supplemental Fig. S2) using constructs of aprevious study (Kunz et al., 2014). In plastids of etio-lated seedlings, GFP fluorescence was much weakercompared with that in developing chloroplasts of light-grown seedlings (Fig. 3, H and I). Altogether, the resultsshow that AtKEA2 accumulates specifically in youngdeveloping chloroplasts, where it takes on a very dis-tinctive localization at the poles. These images werequantified by scoring AtKEA2-GFP fluorescence at thetwo poles or not as a function of plastid size (measuredalong the longitudinal axis). The analysis clearly dem-onstrated a polar localization in chloroplasts of 2 to3 mm in length (Fig. 3G).

The association of AtKEA2 to the poles of developingchloroplast suggests specific interactions of AtKEA2with proteins or lipids at these locations. We hypothe-sized that the unusual N-terminal domain of more than550 residues (Supplemental Fig. S5) might have a role insuch an interaction. To test its localization, we tried toexpress the N-terminal domain of AtKEA2 in Arabi-dopsis. However, thisDNA sequencewas highly toxic inbacteria. TheN-terminal domain of the rice (Oryza sativa)OsKEA1 homolog could be cloned easily in bacteria, andits GFP fusion protein was transiently expressed inArabidopsis protoplasts. Intriguingly, GFP fluorescencewas localized at distinct spots on the plastid (Fig. 4, Aand B). To test the distribution of the transport domainalone, wild-type plants were stably transformed with aconstruct encoding the N-terminally truncated trans-porter domain fused to GFP. Fluorescence signal wasdetected throughout the plastid periphery (Fig. 4, C andD). After scoring the fluorescence pattern of all chloro-plasts from several views, it is apparent that theN-terminally truncated protein containing the trans-port domain alone is localized predominantly to large

Figure 1. Arabidopsis kea1kea2 double mutant leaves appear chloroticin a developmental gradient. A,Wild-type Columbia-0 (Col-0) and kea1and kea2 mutants were grown on soil for 3 weeks. Two independentkea1kea2 double mutants were stunted in size and had pale yellowishleaves. B, Wild-type plants and kea1-2kea2-1 mutants at 2 weeks (left)and 3 weeks (right). Newly emerging leaves of mutants (insets) are paleyellowish at the base. C, A kea1-1kea2-2 mutant at 4 weeks (left) and5weeks (right). The plants are oriented to show the numbering of visibleleaves 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 thedotted line) ofmutant leaves (right). Representativewild-type (wt) leaveswere taken from 3- and 4-week-old plants (left); mutant leaves are from3-, 4-, and 5-week-old plants. Bars = 0.5 cm.

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patches or distributed uniformly around the plastids(Fig. 4E). These results support the idea that the Nterminus contains the signal or information requiredfor the polar or spotty localization of the full-lengthAtKEA2 protein. While we could readily select dou-ble mutant plants transformed with the full-lengthAtKEA2-GFP protein based on recovery of the wild-type growth phenotype (Supplemental Fig. S2), wecould not identify double mutant plants transformedwith the truncated AtKEA2-GFP protein without theN terminus, as none of the plants recovered normalgrowth and greening (data not shown). This indicatesthat the N-terminal domain is required for the physi-ological function of the antiporter.

Confocal images of mesophyll cells indicated alower chloroplast density in kea1kea2 double mu-tants (Fig. 5A). Careful counting of chloroplasts inglutaraldehyde-fixed cells showed that kea1kea2 dou-ble mutants have only about 60% of the number ofchloroplasts per cell compared with wild-type plants(Fig. 5B). Furthermore, double mutant chloroplastsshowed a heterogenous size distribution, althoughthey were slightly larger at an average size of 45 mm2

relative to 36 mm2 of the wild type (Fig. 5C).

DISCUSSION

pH and K+ homeostasis are fundamental to photo-synthetic reactions in plastids, although the molecularbasis of pH and cation homeostasis is largely unknown.Recently, AtKEA1 and AtKEA2 were shown to act asK+/H+ antiporters (Aranda-Sicilia et al., 2012) and to belocated in the inner envelope membranes (Ferro et al.,2010; Roston et al., 2012; Kunz et al., 2014). Light-induced electrogenic K+ uptake is expected to cause os-motic swelling of the organelle. Photosynthetic reactionsare sensitive to such chloroplast volume changes, and aplausible role for KEA antiporters in the envelopemembrane is thus volume control by catalyzing pHgradient-coupled K+ efflux. In accordance, the increasedsize and fragility of kea1kea2 double mutant chloroplastspointed to osmotic sensitivity (Kunz et al., 2014). Re-duced photosynthetic efficiency by osmotic swellingcould be the result of substrate dilution (Kaiser et al.,

1981). Additionally, kea1kea2 double mutant plantswere reported to have a lower trans-thylakoid pHgradient, which also could explain the observed low-ered PSII quantum efficiency and pH-regulated non-photochemical quenching (Kunz et al., 2014). However,why the lack of envelope K+/H+ antiporter activitycaused a lowered thylakoid pH was not clear. Fur-thermore, Kunz et al. (2014) did not address the ques-tion of why reduced photosynthesis was observed inyoung leaf tissues only. Here, we show that AtKEA1and AtKEA2 are likely critical for proper chloroplastdevelopment and thylakoid membrane biogenesis, es-pecially in developing leaves.

A Role of AtKEA1 and AtKEA2 inChloroplast Development

Intriguingly, kea1kea2 double mutants showed re-ducedChl content primarily in newly developing leaves,which becomes less obvious in later growth stages (Fig.1; Table I). Our results show that all pigment-bindingcomplexes of the thylakoid membrane are reduced inthe developing leaf sections of the double mutant (TableI). Young leaves of kea1kea2 mutants further showstrongly reduced amounts of protein complexes in-volved in photosynthetic electron transport (Fig. 2).The first leaves expand in response to light, andproplastids divide and differentiate to form maturechloroplasts. The zones with reduced pigmentationand protein accumulation of double mutant leavescoincide with the proliferating zones of newly de-veloping leaves. The defect recovers to a great extentin the expansion andmaturation zones (Figs. 1 and 2;Table I; Kalve et al., 2014). These findings point to afunction of AtKEA1 and AtKEA2 in chloroplast de-velopment and thylakoid membrane formation. In-deed, electron microscopy images show that kea1kea2double mutants have fewer stacked and disorga-nized thylakoid membranes (Supplemental Fig. S6;Kunz et al., 2014). The size of mutant leaves issmaller than in wild-type plants at the same age (Fig.1), in agreement with the observation that plastiddivision and cell division are linked (Vanhaerenet al., 2015), although this also can be a consequenceof reduced photosynthesis during development.

Table I. Pigment composition in wild-type and double mutant leaves

Pigment composition is altered in young developing leaves. Mutant leaf was cut to separate green from chlorotic regions (Supplemental Fig. S3).Chl a and b content on a fresh weight basis is reduced in yellow (y) sections of leaves from kea1kea2mutants as compared with green (g) sections andleaves from wild-type Columbia-0 plants. Levels of xanthophylls in relation to Chl are increased, and b-carotene levels are decreased, in yellowsections of leaves from kea1kea2 mutants. Nx, Neoxanthin; Vx, violaxanthin; Ax, antheraxanthin; Lut, lutein; Zx, zeaxanthin; Car, b-carotene; VAZ,sum of Vx, Ax, and Zx. Values are means 6 SE of five independent measurements.

Plant Color Chl a Chl b Chl (a+b) Nx Vx Ax Lut Zx Car VAZ

nmol g21 fresh wt mmol mol21 Chl (a+b)Columbia-0 g 1,181 6 69 403 6 27 1,584 6 97 32 6 1 38 6 2 3 6 0 99 6 4 1 6 0 73 6 2 41 6 2

kea1-1kea2-2 g 1,191 6 72 432 6 28 1,623 6 100 37 6 1 45 6 3 2 6 0 119 6 2 0 6 0 67 6 2 46 6 3y 594 6 26 247 6 9 841 6 34 47 6 1 79 6 2 8 6 1 181 6 3 2 6 0 51 6 2 89 6 2

kea1-2kea2-1 g 1,171 6 179 453 6 70 1,624 6 249 37 6 1 43 6 2 2 6 1 121 6 4 0 6 1 70 6 3 46 6 2y 713 6 82 325 6 35 1,037 6 117 44 6 1 73 6 4 7 6 1 156 6 3 1 6 1 40 6 2 81 6 3

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Furthermore, we observed that AtKEA2 protein ac-cumulation is highest in small developing plastids anddividing plastids of first true leaves (Fig. 3, A–G).AtKEA2 protein accumulation appears to be regulateddevelopmentally and in response to light, because it isreduced significantly or absent in etiolated seedlingsand mature differentiated chloroplasts (Fig. 3, H and I).The AtKEA2 protein is detected in some mature chlo-roplasts, where it is localized in distinct spots (Fig. 3, C–F).The uniform distribution of AtKEA2-GFP proteinlacking the N-terminal domain throughout the enve-lope in all chloroplasts (Fig. 4, C and D) strongly sug-gests that the N terminus is responsible for the specificaccumulation of AtKEA2 in developing chloroplasts

and points to a specific requirement of the protein inthese plastids, possibly by interaction with other de-velopmentally regulated proteins.

The polar distribution of AtKEA1 or AtKEA2 wasnot observed in chloroplasts from guard cells before(Kunz et al., 2014), probably because guard cells are

Figure 2. Levels of essential photosynthetic protein complexes are se-verely decreased in developing kea1kea2 leaves. Total protein from0.5 mg fresh weight of wild-type Columbia-0 (Col-0) or green (g) andyellow (y) sections of kea1kea2 leaves was separated by SDS-gel elec-trophoresis and transferred to polyvinylidene difluoride membranes forimmunostaining. Different amounts (20%–100%) of wild-type proteinwere analyzed to estimate the relative abundance of proteins in kea1-kea2 leaves. PsbA, PSII protein D1; PsbD, PSII subunit D2; PsaD, PSIsubunit D; Cytb6, cytochrome b6f complex subunit; AtpB, b-subunit ofATP synthase; Lhcb2, subunit 2 of light-harvesting complex 2; RbcL,Rubisco large subunit; Alb3, Albino3 thylakoid insertase; KEA3, Ara-bidopsis thylakoid membrane K+/H+ antiporter KEA3; P.R., PonceauRed-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. Imagesare from young leaves of a 2-week-old plant. GFP fluorescence isbrightest in small chloroplasts with weak Chl fluorescence (red). B, GFPfluorescence is localized to the poles in small dividing chloroplasts. C toE, Magnified images of the box in A, GFP (C), Chl (D), and overlay (E),showing a dividing chloroplast with polar AtKEA2-GFP fluorescenceand amature chloroplast with fluorescent spots. F, In amature section ofan expanded leaf, plastids show some isolated GFP spots. The image isfrom a 3-week-old plant. G, Polar distribution of GFP fluorescence inplastids of 2 to 3 mm in length. Fluorescence is nonpolar in 5- to 8-mmchloroplasts (n = 200). H and I, AtKEA2-GFP fluorescence in light-grown (H) versus dark-grown (I) seedlings. Cotyledons of 10-d-oldseedlings were examined. Bars = 10 mm in all images.

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differentiated epidermal cells whose primary role isnot photosynthetic. In contrast, a new emerging leafcontains three overlapping zones: (1) the cell prolif-eration zone near the base; (2) the expansion zone atthe middle; and (3) the differentiation zone at the tipand periphery (Kalve et al., 2014; Vanhaeren et al.,2015; Fig. 1D). The primary function of mesophyll cellsis to harvest light energy by photosynthesis; thus,

mesophyll cells in the different leaf zones wouldcontain varying populations of plastids undergoingfission, elongation, and differentiation. Our resultsare consistent with the current model of leaf devel-opment (Vanhaeren et al., 2015).

Figure 4. The N-terminal domain targets KEA Ia antiporters to distinctfoci. A, N terminus of OsKEA1 (residues Met-1 to Glu-536) expressedtransiently in Arabidopsis protoplasts. Shown are the N-terminal GFPfluorescence (left) and overlay of GFP and Chl fluorescence (right).Bar = 10 mm. B, The N-terminal domain of OsKEA1 shows punctatedistribution in chloroplasts. Overlay of GFP (green) and Chl (red) fluo-rescence of two chloroplasts is shown. Individual chloroplasts wereobtained by breaking a protoplast. Bars = 2.5 mm. C, The transportdomain of AtKEA2-GFP protein (Met-556 to Ile-1174) stably expressedin Arabidopsis distributes evenly around plastids. Overlay of GFP andChl fluorescence is shown. Bar = 10mm.D, GFP (top), Chl (middle), andoverlay of GFP and Chl fluorescence (bottom) images from box 1 in C.Bar = 10 mm. E, Quantitative analysis shows distinct patterns of full-length AtKEA2-GFP (Met-1 to Ile-1174) or the N terminally truncatedAtKEA2-GFP (Met-556 to Ile-1174). Patterns were categorized as fol-lows: 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 threedifferent 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 chlo-roplasts. A, Confocalmicroscopy shows thatmutant leaves have a lowerdensity of chloroplasts compared with wild-type Columbia-0 (Col-0)leaves. B, Quantitative analysis confirms that the double mutant hasfewer chloroplasts per cell. Chloroplasts per cell were counted as de-scribed in Supplemental Methods S1. C, Chloroplasts of doublemutantsare larger. The size of individual chloroplasts was determined by mea-suring the area in Corel Photo Paint. Avalue of 100%= 200 chloroplastseach for mutants and for the wild type. Themean areas of wild-type anddouble mutant chloroplast are 36 6 9 and 45 6 15 mm2, respectively.

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The basis for the partial recovery of pigmentation andphotosynthesis activity in mature leaf tissues of mutantsis less clear. Recovery of pigmentation is typical of vi-rescent mutants, which show defects in photosyntheticpigments in young and emerging tissues but graduallyturn green along the leaf proximal-distal axis. This isoften related to the altered expression of genes involvedin early chloroplast biogenesis, causing delayed chloro-plast development (Archer et al., 1987; Lopez-Juez andPyke, 2005). We suggest that, even though growth isretarded, chloroplasts of kea1kea2 mutants eventuallydevelop to form functioning organelles, resulting in lesssevere phenotypes in older leaves. Our results extendthat of a recent report on a rice AtKEA1/2 homolog(Sheng et al., 2014). Thus, the observed reduction inphotosynthetic activities of kea1kea2 double mutants(Kunz et al., 2014) could be a consequence of impaired ordelayed chloroplast differentiation.

The Long N-Terminal Domain Attaches AtKEA1/2 toSpecific Locations

Our results demonstrate that theN terminus facilitatesthe anchoring of AtKEA2 to specific envelope domains,as the N-terminal domain of the rice OsKEA1 proteinlocalizes to two or three foci on the plastid (Fig. 4, A andB), whereas the N-terminally truncated AtKEA2 pro-tein is distributed homogenously at the periphery (Fig.4, C–E). The N-terminal domain of more than 550 resi-dues, unique to plant and algal KEA Ia sequences, is richin charged residues and is predicted to form coiled-coilstructures (Supplemental Fig. S5; Chanroj et al., 2012).Coiled-coil domains can hold together molecules, sub-cellular structures, and even tissues (Rose et al., 2005). Inaccordance, AtKEA1 and AtKEA2 proteins are presentin large protein complexes of unknown functions at theinner envelope (Roston et al., 2012). Coiled-coil proteinsplay important roles in chloroplast division, thylakoidmembrane development, and chloroplast relocation inresponse to light, by interaction with cytoskeleton-likeelements (Wada, 2013; Karim and Aronsson, 2014;Osteryoung and Pyke, 2014). Furthermore, although theAtKEA2 protein without the N-terminal domain con-stitutes an active antiporter (Aranda-Sicilia et al., 2012),it fails to complement the growth defects of the doublemutant plant, suggesting that the specific localization ofthe protein is crucial for its function. The association ofoligomers of AtKEA2 could depend on membranetension or curvature, which is highest at the poles butmore relaxed at the midcell, as in bacteria (Laloux andJacobs-Wagner, 2014; Strahl et al., 2015).

Relation of AtKEA2 to Chloroplast Fissionand Osmoregulation

kea1kea2 double mutants contain fewer chloroplasts,with a slightly larger average size (Fig. 5), which is afeature of chloroplast division mutants (Osteryoung andPyke, 2014). Curiously,mechanosensitive channelsMSL2

and MSL3 at the chloroplast inner envelope were shownto be important for osmoregulation (Wilson et al., 2014),plastid division, and FtsZ division ring placement, al-though the mechanism is not clear (Wilson and Haswell,2012). MSL2 and MSL3 show a similar patchy localiza-tion in mature chloroplasts (Haswell and Meyerowitz,2006), as observed for AtKEA1 and AtKEA2 proteins.Perhaps both MSL channel and KEA antiporters share afunction in the pathway that links osmoregulation toplastid fission.

A Model: Ion Homeostasis at Biogenesis Centersfor Thylakoids

Although the significance for polar distribution ofcation/H+ antiporters is not understood, we suggest thatthe two poles of dividing and developing plastids mightserve as biogenesis centers, where new thylakoid mem-branes are formed. Biogenesis centers have been shownto exist in cyanobacteria or in Chlamydomonas reinhardtiias so-called translation zones (Rast et al., 2015). It wassuggested that, during the transition of proplastids tochloroplasts, thylakoid membranes originate from in-vaginations of the inner envelope in algal chloroplasts orproplastids of vascular plants, while in mature chloro-plasts, the membrane system is maintained by vesicletransport (Rast et al., 2015). Contact points of thylakoidmembranes with the plasma membrane were seen in 3Dmicrographs of a cyanobacterium (Nierzwicki-Baueret al., 1983). Evidence to support this model in higherplants has been challenging due to the transient nature ofmembrane remodeling in plastids (Shimoni et al., 2005).The formation of thylakoid membranes requires thesynthesis, import, and assembly of lipids, proteins, andpigments to form the complexes for electron transportand ATP synthesis. Distinct biogenesis centers at the en-velope membrane for the formation of thylakoid mem-branes have been proposed for decades (Vothknecht andWesthoff, 2001; Rast et al., 2015). As chloroplasts mature,thylakoids are likely maintained at contact sites betweenthe thylakoid and the inner envelope. Electron micro-graphs show that thylakoid membranes converge at twopoles in wild-type chloroplasts, but in kea1kea2 mutants,thylakoids are disorganized and not alignedwith the axisof the organelle (Supplemental Fig. S6; Kunz et al., 2014).There is no evidence in vascular plants that biogenesiscenters are ultrastructurally distinct. Evidence for bio-chemically distinctmembranemicrodomains is emerging(Rast et al., 2015). Thus, AtKEA1 and AtKEA2 couldserve as candidate markers of a microdomain or centerwhere the local cation and pH homeostasis is importantfor the proper formation and alignment of the thylakoidmembrane in vascular plants.

CONCLUSION

Loss of AtKEA1 and AtKEA2 function at the innerenvelope delayed the formation of pigments and elec-tron transport complexes, thus reducing photosynthetic

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efficiency and leading to reduced growth. The longN-terminal domain of AtKEA1/2 tethers the protein topolar sites in dividing chloroplasts or to specific spots inmature chloroplasts. Furthermore, AtKEA1 and AtKEA2seem to be essential for the proper development andstructural organization of the thylakoid membrane sys-tem. Our results strongly suggest that AtKEA1 andAtKEA2 are localized to inner envelope microdomains orcenters where cation and pHhomeostasis is important forplastid division and thylakoid membrane development.

MATERIALS AND METHODS

Plant Growth

Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 was grown at 23°C,120 mmol photons m22 s21, and a 16-h-light/8-h-dark photoperiod for 2 to4 weeks.

Generation of kea1kea2 Double Mutants

Seeds harboring T-DNA insertions in AtKEA1 (SAIL_1156_H07 [hereafterkea1-2] and SAIL_586_D02 [kea1-1+/2]) and AtKEA2 (SALK_045324.26.15.x[kea2-1] and SALK_009732C [kea2-2]) were obtained from the NottinghamArabidopsis Stock Centre. Double mutants kea1-2kea2-1 and kea1-1kea2-2 wereobtained as described in Supplemental Methods S1.

Cloning of AtKEA2 and OsKEA1 Constructs andPlant Transformation

The complete AtKEA2 gene including the first four introns as well as a shortversionof thegenewithout theN-terminal domain (residuesMet-556 to Ile-1174)were fused to eGFP and used to stably transform wild-type or kea1kea2 doublemutant Arabidopsis plants. The rice (Oryza sativa) OsKEA1 N-terminal domain(Os04g58620.1; residues Met-1 to Glu-536) was used for transient expressionstudies. Details can be found in Supplemental Methods S1.

Other methods are described in detail in Supplemental Methods S1.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under accession numbers At4g00630 (AtKEA2) and XP_015636213(OsKEA1).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Characterization of AtKEA1 and AtKEA2T-DNA mutants.

Supplemental Figure S2. Complementation of the growth defects by ex-pression of AtKEA2-GFP and localization of AtKEA1.

Supplemental Figure S3. Green and yellow sections in young leaves.

Supplemental Figure S4. AtKEA2 is localized to the chloroplast envelope.

Supplemental Figure S5. Analysis of the AtKEA2 N-terminal domain se-quence.

Supplemental Figure S6. Double mutant kea1kea2 chloroplasts show al-tered thylakoid organization and starch grain alignment.

Supplemental Table S1. Primers used in this study.

Supplemental Methods S1.

ACKNOWLEDGMENTS

We thank L.R. Roston and C. Benning (Roston et al., 2012) for the kind gift ofthe kea1kea2 double mutant we used in preliminary studies and María ElenaSánchez Romero for excellent technical assistance.

Received June 21, 2016; accepted July 19, 2016; published July 21, 2016.

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