Plastidial transporters KEA1 and KEA2 at the inner envelope membrane adjust stromal pH in the dark

Abstract

Photosynthesis and carbon fixation depend critically on the regulation of pH in chloroplast compartments in the daylight and at night. While it is established that an alkaline stroma is required for carbon fixation, it is not known how alkaline stromal pH is formed, maintained or regulated. We tested whether two envelope transporters, AtKEA1 and AtKEA2, directly affected stromal pH in isolated Arabidopsis chloroplasts using the fluorescent probe 2ʹ,7ʹ-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). External K +-induced alkalinization of the stroma was observed in chloroplasts from wildtype (WT) plants but not from kea1kea2 mutants, suggesting that KEA1 and KEA2 mediate K + uptake/H + loss to modulate stromal pH. While light-stimulated alkalinization of the stroma was independent of KEA1 and KEA2, the rate of decay to neutral pH in the dark is delayed in kea1kea2 mutants. However, the dark-induced loss of a pH gradient across the thylakoid membrane was similar in WT and mutant chloroplasts. This indicates that proton influx from the cytosol mediated by envelope K + /H + antiporters contributes to adjustment of stromal pH upon light to dark transitions.

Chloroplast pH Homeostasis for the Regulation of Photosynthesis

“…As Ca 2+ accumulation in the chloroplast stroma contributes to the downregulation of CO 2 fixation, because of the inhibition of several enzymes involved in the CBB cycle ( Rocha and Vothknecht, 2012 ), and to the transcription of plastidial genes via the synthesis of the secondary messenger, guanosine tetraphosphate ( Ono et al, 2019 ), BICAT1 is proposed to be deactivated upon the light-to-dark transition (bottom). Both KEA1 and KEA2 antiporters are less active under light conditions (top, middle) than upon light-to-dark transitions (bottom), as reported previously ( Aranda Sicilia et al, 2021 ). The BICAT2 antiporter is active under light conditions (top, middle), as reported by Frank et al (2019) .…”

“…Conversely, stromal pH in the dark was reported to be ~7, which increased to ~7.8–8.0 in the light ( Werdan and Heldt, 1972 ; Heldt et al, 1973 ; Werdan et al, 1975 ; Demmig and Gimmler, 1983 ; Robinson, 1985 ; Wu and Berkowitz, 1992 ). Recently, owing to the use of a pH indicator called BCECF-AM (2′,7′-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester), the stromal pH was reported to increase from 7.32 ± 0.02 in the dark to 7.55 ± 0.09 in the light within less than 1 min upon illumination ( Su and Lai, 2017 ; Aranda Sicilia et al, 2021 ). The proton concentration gradient (ΔpH) across thylakoid membranes under steady light was reported to be ~1.8–2.1 ( Tikhonov et al, 2008 ), indicating that the difference in pH between the stroma and thylakoid lumen upon exposure to light, reported previously, was reliable.…”

“…Knocking out either KEA1 or KEA2 results in no visible effect on plant growth ( Kunz et al, 2014 ); however, knocking out both genes together reduces plant growth and induces leaf yellowing ( Kunz et al, 2014 ), suggesting that K + /H + exchange and pH homeostasis in the stroma are important for chloroplast development and photosynthesis. Recently, the function of KEA1 and KEA2 was shown to be suppressed upon dark-to-light transitions for maintaining alkaline pH levels in the stroma, but their function was fully activated upon light-to-dark transitions to neutralize stromal pH ( Aranda Sicilia et al, 2021 ).…”

Cooperation of chloroplast ascorbate peroxidases and proton gradient regulation 5 is critical for protecting Arabidopsis plants from photo‐oxidative stress

“…Proton gradient regulation 5 (PGR5) is a small chloroplast protein essential for ∆pH formation (Munekage et al ., 2002), and this protein is generally considered to be involved in the major pathway for cyclic electron flow (CEF) around PSI (Munekage et al ., 2002; DalCorso et al ., 2008; Yamori and Shikanai, 2016). There are several chloroplast K + /H + transporters that also regulate ∆pH (Kunz et al ., 2014; Armbruster et al ., 2017; Aranda Sicilia et al ., 2021). The Arabidopsis pgr5 mutant cannot generate a sufficient ∆pH across the thylakoid membrane and therefore fails to fully activate NPQ and photosynthetic control (Suorsa et al ., 2012; Yamamoto and Shikanai, 2019).…”

Two plastid POLLUX ion channel-like proteins are required for stress-triggered stromal Ca2+release

“…Thus far, only two K + /H + exchangers from the K efflux antiporter (KEA) family have been characterized in more detail ( Aranda-Sicilia et al, 2012 ; Kunz et al, 2014 ). Both carriers (KEA1 and KEA2) physiologically function in pH and ion homeostasis which is critical for plastid gene expression and development ( Aranda Sicilia et al, 2021 ; deTar et al, 2021 ). The IE membrane potential of at least −70 mV on the stromal side ( Wu et al, 1991 ) makes K + /H + valves such as KEA1/2 a necessity for osmoregulation to balance K + influx and prevent rupture of plastids ( Bernardi, 1999 ).…”