Coordinated regulation of photosynthetic and respiratory components is necessary to maintain chloroplast energy balance in varied growth conditions

Abstract

Mitochondria have a non-energy-conserving alternative oxidase (AOX) proposed to support photosynthesis, perhaps by promoting energy balance under varying growth conditions. To investigate this, wild-type (WT) Nicotiana tabacum were compared with AOX knockdown and overexpression lines. In addition, the amount of AOX protein in WT plants was compared with that of chloroplast light-harvesting complex II (LHCB2), whose amount is known to respond to chloroplast energy status. With increased growth irradiance, WT leaves maintained higher rates of respiration in the light (RL), but no differences in RL or photosynthesis were seen between the WT and transgenic lines, suggesting that, under non-stress conditions, AOX was not critical for leaf metabolism, regardless of growth irradiance. However, under drought, the AOX amount became an important determinant of RL, which in turn was an important determinant of chloroplast energy balance (measured as photosystem II excitation pressure, EP), and photosynthetic performance. In the WT, the AOX amount increased and the LHCB2 amount decreased with increased growth irradiance or drought severity. These changes in protein amounts correlated strongly, in opposing ways, with growth EP. This suggests that a signal deriving from the photosynthetic electron transport chain status coordinately controls the amounts of AOX and LHCB2, which then both contribute to maintaining chloroplast energy balance, particularly under stress conditions.

Keywords: Chloroplast energy balance; drought stress; excitation pressure; growth irradiance; light-harvesting complex II protein; mitochondrial alternative oxidase; photosynthesis; respiration.

Fig. 1.

Leaf RWC at different growth irradiances (150, 400, or 700 PPFD) and at different times following the withholding of water. ‘Day’ represents the number of days since water was withheld from well-watered plants. Data are shown for WT plants (gray bars), AOX overexpressors (black bars), and AOX knockdowns (white bars). In each independent experiment, data from two overexpressors (B7 and B8) that acted similarly were averaged, and data from two knockdowns (RI9 and RI29) that acted similarly were averaged. The data shown are the average ±SE of three to five independent experiments.

Fig. 2.

Effect of growth irradiance on photosynthesis during moderate drought in WT tobacco and transgenic lines with altered amounts of AOX protein. (A) A_sat. (B) ETR_sat. (C) EP_sat. (D) NPQ_sat. Plants were grown at 150, 400, or 700 PPFD under well-watered conditions for 19 to 21 days, followed by water being withheld from the plants until they were experiencing a leaf RWC of 63–66%, at which time each of the photosynthetic parameters was measured at saturating irradiance (1600 PPFD). Data are shown for WT plants (gray bars), AOX overexpressors (left to right the two black bars are B8 and B7, respectively), and AOX knockdowns (left to right the two white bars are RI9 and RI29, respectively). Data are the average ±SE of three to five independent experiments. Within an irradiance, values significantly different from the WT are indicated: *P<0.05; **P<0.01; ***P<0.001.

Fig. 3.

Effect of growth irradiance on photosynthesis during moderate drought in WT tobacco and transgenic lines with altered amounts of AOX protein. (A) A_net. (B) ETR_net. (C) EP_net. (D) NPQ_net. Plants were grown at 150, 400, or 700 PPFD under well-watered conditions for 19 to 21 days, followed by water being withheld from the plants until they were experiencing a leaf RWC of 63–66%, at which time each of the photosynthetic parameters was measured at the growth irradiance (150, 400, or 700 PPFD). Data are shown for WT plants (gray bars), AOX overexpressors (left to right the two black bars are B8 and B7, respectively) and AOX knockdowns (left to right the two white bars are RI9 and RI29, respectively). Data are the average ±SE of three to five independent experiments. Within an irradiance, values significantly different from the WT are indicated: *P<0.05; **P<0.01; ***P<0.001.

Fig. 4.

Effect of growth irradiance on respiration during moderate drought in WT tobacco and transgenic lines with altered amounts of AOX protein. (A) R_D. (B) R_L. Plants were grown at 150, 400, or 700 PPFD under well-watered conditions for 19 to 21 days, followed by water being withheld from the plants until they were experiencing a leaf RWC of 63–66%, at which time the measurements were taken. Data are shown for WT plants (gray bars), AOX overexpressors (left to right the two black bars are B8 and B7, respectively) and AOX knockdowns (left to right the two white bars are RI9 and RI29, respectively). Data are the average ±SE of three to five independent experiments. Within an irradiance, values significantly different from the WT are indicated: *P<0.05; **P<0.01; ***P<0.001.

Fig. 5.

Leaf protein amounts. (A) AOX protein amount. (B) LHCB2 protein amount. (C) Plot of LHCB2 protein amount versus AOX protein amount. (D) Representative immunoblots. WT tobacco plants were grown at an irradiance of 150 PPFD (circles), 400 PPFD (squares), or 700 PPFD (triangles) under well-watered conditions for 19 to 21 days. AOX and LHCB2 protein amounts were subsequently determined at different times for up to 8 days following a final watering of the plants on Day 0. Protein amounts in (A) and (B) are relative to that of the well-watered (Day 1) plants grown at 150 PPFD, which was set to 1. Data are the average ±SE of three independent experiments. Data in (C) are derived from that in (A) and (B).

Fig. 6.

Leaf AOX and LHCB2 protein amounts and their relation to EP_net in WT tobacco. (A) The negative relationship between LHCB2 protein amount and EP_net. (B) The positive relationship between AOX protein amount and EP_net. Plants were grown at 150 PPFD (circles), 400 PPFD (squares), or 700 PPFD (triangles) under well-watered conditions for 19 to 21 days, followed by water being withheld from the plants for up to an additional 8 days. At different times following the water being withheld, AOX and LHCB2 protein amounts, as well as the EP_net being experienced by the plant were determined. All protein amounts are relative to that of well-watered plants grown at 150 PPFD, which was set to 1. All data are the average ±SE of three independent experiments.

Fig. 7.

Changes in R_L and EP_sat as a function of growth irradiance and leaf water status in WT tobacco and transgenic tobacco lines with altered amounts of AOX protein. Plants were grown at 150 PPFD (A), 400 PPFD (B), or 700 PPFD (C) under well-watered conditions for 19 to 21 days, followed by water being withheld from the plants for up to an additional 6 days. At different times following the water being withheld, leaf R_L and EP_sat were determined. Data are shown for WT plants (gray circles), AOX overexpressors (black circles; half are B7, half are B8) and AOX knockdowns (white circles; half are RI9, half are RI29). The data points are compiled from three to five independent experiments.

Fig. 8.

A working model for the coordinated regulation of chloroplast and mitochondrial proteins that contribute toward chloroplast energy balance. In response to growth conditions that promote a high steady-state growth excitation pressure (EP_net), plants decrease (red arrow) chloroplast components such as LHCB2 that promote energy absorption, and increase (green arrow) mitochondrial components such as AOX that promote energy dissipation. These coordinated adjustments promote energy balance in the chloroplast by reducing electron flow into the PQ pool and increasing electron flow out of the PQ pool. This figure is highly simplified to highlight just those proteins quantified in this study (LHCB2, AOX). Many other processes also contribute toward chloroplast energy balance in response to growth conditions. PQ_ox, oxidized plastoquinone pool; PQ_red, reduced plastoquinone pool.