Luke Hendrickson

The pentatricopeptide repeat gene OTP51 with two LAGLIDADG motifs is required for the cis-splicing of plastid ycf3 intron 2 in Arabidopsis thaliana

Summary The Arabidopsis thaliana chloroplast contains 20 group-II introns in its genome, and seven known splicing factors are required for the splicing of overlapping subsets of 19 of them. We describe an additional protein (OTP51) that specifically promotes the splicing of the only group-II intron for which no splicing factor has been described previously. This protein is a pentatricopeptide repeat (PPR) protein containing two LAGLIDADG motifs found in group-I intron maturases in other organisms. Amino acids thought to be important for the homing endonuclease activity of other LAGLIDADG proteins are missing in this protein, but the amino acids described to be important for maturase activity are conserved. OTP51 is absolutely required for the splicing of ycf3 intron 2, and also influences the splicing of several other group-IIa introns. Loss of OTP51 has far-reaching consequences for photosystem-I and photosystem-II assembly, and for the photosynthetic fluorescence characteristics of mutant plants.

A simple chlorophyll fluorescence parameter that correlates with the rate coefficient of photoinactivation of photosystem II.

A method of partitioning the energy in a mixed population of active and photoinactivated Photosystem II (PS II) complexes based on chlorophyll fluorescence measurements is presented. There are four energy fluxes, each with its quantum efficiency: a flux associated with photochemical electron flow in active PS II reaction centres (JPSII), thermal dissipation in photoinactivated, non-functional PS IIs (JNF), light-regulated thermal dissipation in active PS IIs (JNPQ) and a combined flux of fluorescence and constitutive, light-independent thermal dissipation (Jf,D). The four quantum efficiencies add up to 1.0, without the need to introduce an ‘excess’ term E, which in other studies has been claimed to be linearly correlated with the rate coefficient of photoinactivation of PS II (kpi). We examined the correlation of kpi with various fluxes, and found that the combined flux (JNPQ + Jf,D= Jpi) is as well correlated with kpi as is E. This combined flux arises from Fs/Fm′, the ratio of steady-state to maximum fluorescence during illumination, which represents the quantum efficiency of combined non-photochemical dissipation pathways in active PS IIs. Since Fs/Fm′ or its equivalent, Jpi, is a likely source of events leading to photoinactivation of PS II, we conclude that Fs/Fm′ is a simple predictor of kpi.

Photoinactivation of Photosystem II in leaves

Photoinactivation of Photosystem II (PS II), the light-induced loss of ability to evolve oxygen, inevitably occurs under any light environment in nature, counteracted by repair. Under certain conditions, the extent of photoinactivation of PS II depends on the photon exposure (light dosage, x), rather than the irradiance or duration of illumination per se, thus obeying the law of reciprocity of irradiance and duration of illumination, namely, that equal photon exposure produces an equal effect. If the probability of photoinactivation (p) of PS II is directly proportional to an increment in photon exposure (p = kΔx, where k is the probability per unit photon exposure), it can be deduced that the number of active PS II complexes decreases exponentially as a function of photon exposure: N = Noexp(−kx). Further, since a photon exposure is usually achieved by varying the illumination time (t) at constant irradiance (I), N = Noexp(−kI t), i.e., N decreases exponentially with time, with a rate coefficient of photoinactivation kI, where the product kI is obviously directly proportional to I. Given that N = Noexp(−kx), the quantum yield of photoinactivation of PS II can be defined as −dN/dx = kN, which varies with the number of active PS II complexes remaining. Typically, the quantum yield of photoinactivation of PS II is ca. 0.1μmol PS II per mol photons at low photon exposure when repair is inhibited. That is, when about 107 photons have been received by leaf tissue, one PS II complex is inactivated. Some species such as grapevine have a much lower quantum yield of photoinactivation of PS II, even at a chilling temperature. Examination of the longer-term time course of photoinactivation of PS II in capsicum leaves reveals that the decrease in N deviates from a single-exponential decay when the majority of the PS II complexes are inactivated in the absence of repair. This can be attributed to the formation of strong quenchers in severely-photoinactivated PS II complexes, able to dissipate excitation energy efficiently and to protect the remaining active neighbours against damage by light.

Reductions of Rubisco Activase by Antisense RNA in the C4 Plant Flaveria bidentis Reduces Rubisco Carbamylation and Leaf Photosynthesis

To function, the catalytic sites of Rubisco (EC 4.1.1.39) need to be activated by the reversible carbamylation of a lysine residue within the sites followed by rapid binding of magnesium. The activation of Rubisco in vivo requires the presence of the regulatory protein Rubisco activase. This enzyme is thought to aid the release of sugar phosphate inhibitors from Rubiscos catalytic sites, thereby influencing carbamylation. In C3 species, Rubisco operates in a low CO2 environment, which is suboptimal for both catalysis and carbamylation. In C4 plants, Rubisco is located in the bundle sheath cells and operates in a high CO2 atmosphere close to saturation. To explore the role of Rubisco activase in C4 photosynthesis, activase levels were reduced in Flaveria bidentis, a C4 dicot, by transformation with an antisense gene directed against the mRNA for Rubisco activase. Four primary transformants with very low activase levels were recovered. These plants and several of their segregating T1 progeny required high CO2 (>1 kPa) for growth. They had very low CO2 assimilation rates at high light and ambient CO2, and only 10% to 15% of Rubisco sites were carbamylated at both ambient and very high CO2. The amount of Rubisco was similar to that of wild-type plants. Experiments with the T1 progeny of these four primary transformants showed that CO2 assimilation rate and Rubisco carbamylation were severely reduced in plants with less than 30% of wild-type levels of activase. We conclude that activase activity is essential for the operation of the C4 photosynthetic pathway.

Energy partitioning in photosystem II complexes subjected to photoinhibitory treatment

Chlorophyll a fluorescence measured in vivo is frequently used to study the role of different processes influencing the distribution of excitation energy in PSII complexes. Such studies are important for understanding the regulation of photosynthetic electron transport. However, at the present time, there is no unified methodology to analyse the energy partitioning in PSII. In this article, we critically assess several approaches recently developed in this area of research and propose new simple equations, which can be used for de-convolution of non-photochemical energy quenching in PSII complexes.

Photoinactivation of photosystem II in high light-acclimated grapevines

Grapevines are considered well adapted to high irradiance during growth. It is still controversial, however, whether photoinactivation of photosystem II is completely avoided in high light-acclimated grapevines growing in the field. This study examines the functional stability of PSII in leaf discs (floated on water) of field-grown, high light-acclimated grapevines as a function of photon exposure. Measuring functional PSII units by flash-induced oxygen evolution, it was found that the susceptibility of PSII to photoinactivation was less in sun-exposed leaves than shade leaves of Vitis riparia Michaux, and enhanced by lincomycin, an inhibitor of chloroplast-encoded protein synthesis. Vitis vinifera L. cv. Chardonnay, grown in a glasshouse with slightly lower irradiance, exhibited an intermediate susceptibility. Significantly, the dark-relaxed quantum efficiency of PSII, measured as (Fm – Fo)/Fm, where Fm and Fo are the chlorophyll (Chl) fluorescence yields for closed and open reaction centres, respectively, declined much more slowly than did the number of functional PSII units in V. riparia. Thus, measurements of (Fm – Fo)/Fm may give an impression of little photoinactivation of PSII, even when nearly half of functional PSII units may be lost. By contrast, the parameter 1/Fo – 1/Fm is a more linear indicator of functional PSII units. The results indicate that grapevines may suffer photoinactivation of PSII, at least when leaf discs are floated on water.

A rapid, whole-tissue determination of the functional fraction of PSII after photoinhibition of leaves based on flash-induced P700 redox kinetics.

Assaying the number of functional PSII complexes by the oxygen yield from leaf tissue per saturating, single-turnover flash, assuming that each functional PSII evolves one oxygen molecule after four flashes, is one of the most direct methods but time-consuming. The ratio of variable to maximum Chl fluorescence yield (F(v)/F(m)) in leaves can be correlated with the oxygen yield per flash during a progressive loss of PSII activity associated with high-light stress and is rapid and non-intrusive, but suffers from being representative of chloroplasts near the measured leaf surface; consequently, the exact correlation depends on the internal leaf structure and on which leaf surface is being measured. Our results show that the average F(v)/F(m) of the adaxial and abaxial surfaces has a reasonable linear correlation with the oxygen yield per flash after varied extents of photoinactivation of PSII. However, we obtained an even better linear correlation between (1) the integrated, transient electron flow (Sigma) to P700+, the dimeric Chl cation in PSI, after superimposing a single-turnover flash on steady background far-red light and (2) the relative oxygen yield per flash. Leaves of C3 and C4 plants, woody and herbaceous species, wild-type and a Chl-b-less mutant, and monocot and dicot plants gave a single straight line, which seems to be a universal relation for predicting the relative oxygen yield per flash from Sigma. Measurement of Sigma is non-intrusive, representative of the whole leaf tissue, rapid and applicable to attached leaves; it may even be applicable in the field.

Assessment of photoprotection mechanisms of grapevines at low temperature

The photosynthetic response of grapevine leaves (Vitis vinifera L. cv. Riesling) to low temperature was studied in the field and laboratory. Light-saturated rates of photosynthetic electron transport were lower and non-photochemical energy dissipation was higher when leaves were subject to low morning temperatures than to high afternoon temperatures under field conditions. These responses to low temperatures occurred without sustained reduction of quantum efficiency of PSII as measured by the variable to maximum chlorophyll fluorescence yield ratio, Fv/Fm, after dark adaptation. The temperature dependence of light-saturated apparent electron transport rate, gas exchange and non-photochemical quenching (NPQ) was also examined in laboratory experiments with glasshouse-grown material. NPQ reached saturation at lower light intensity with decreasing temperature. The relationship between the quantum efficiency of PSII and CO2 fixation at 25°C (2-21% O2) and 10°C (2-21% O2) indicated a decreased dependence of electron transport on both photorespiration and the Mehler reaction at the lower temperature. The calculated percentage of electron flow to the Mehler reaction declined faster than photorespiration at low temperature. Warm- and cold-treated leaf discs under saturating light showed very little photoinhibition as measured by sustained reduction in Fv/Fm, which was linearly related to the percentage of functional PSII reaction centres. However, the addition of dithiothreitol greatly enhanced the rate of photoinhibition, indicating a potentially strong dependence on xanthophyll de-epoxidation for photoprotection at low temperature.

Low temperature effects on grapevine photosynthesis: the role of inorganic phosphate

The photosynthetic response of grapevine leaves (Vitis vinifera L. cv. Riesling) to low temperature was studied to determine the role of end-product limitation and orthophosphate (Pi) recycling to the chloroplast under these conditions. As reported previously, the response of photosynthesis in air to stomatal conductance declined at temperatures below 15°C, suggesting that at low temperatures inhibition of photosynthesis in grapevine has a strong non-stomatal component. Stimulation of carbon assimilation at ambient CO2 by reducing O2 from 21 to 2 kPa, O2 declined to zero below 15°C, a phenomenon often associated with a restriction in photosynthesis due to end-product-synthesis limitation. This stimulation could be restored by feeding Pi. Photosynthesis in leaf disks at both high and low irradiances in non-photorespiratory conditions (1% CO2) was highly sensitive to reductions in temperature. Below 15°C, feeding Pi caused a large stimulation of photosynthetic O2 evolution. Metabolite measurements indicated that despite a decline in Rubisco carbamylation state, ribulose 1,5-bisphosphate (RuBP) levels dropped at low temperature and the ratio of 3-phosphoglycerate (3-PGA) to triose phosphate (TP) remained largely unchanged. These results suggest that grapevine-leaf photosynthesis is severely restricted at low temperature by non-stomatal mechanisms. The return of Pi to the chloroplast plays an important role in this limitation but a coordinated set of regulatory processes maintain a homeostasis of phosphorylated sugar levels.

The effects of Rubisco activase on C4 photosynthesis and metabolism at high temperature

The activation of Rubisco in vivo requires the presence of the regulatory protein Rubisco activase. This enzyme facilitates the release of sugar phosphate inhibitors from Rubisco catalytic sites thereby influencing carbamylation. T(1) progeny of transgenic Flaveria bidentis (a C(4) dicot) containing genetically reduced levels of Rubisco activase were used to explore the role of the enzyme in C(4) photosynthesis at high temperature. A range of T(1) progeny was screened at 25 degrees C and 40 degrees C for Rubisco activase content, photosynthetic rate, Rubisco carbamylation, and photosynthetic metabolite pools. The small isoform of F. bidentis activase was expressed and purified from E. coli and used to quantify leaf activase content. In wild-type F. bidentis, the activase monomer content was 10.6+/-0.8 micromol m(-2) (447+/-36 mg m(-2)) compared to a Rubisco site content of 14.2+/-0.8 micromol m(-2). CO(2) assimilation rates and Rubisco carbamylation declined at both 25 degrees C and 40 degrees C when the Rubisco activase content dropped below 3 mumol m(-2) (125 mg m(-2)), with the status of Rubisco carbamylation at an activase content greater than this threshold value being 44+/-5% at 40 degrees C compared to 81+/-2% at 25 degrees C. When the CO(2) assimilation rate was reduced, ribulose-1,5-bisphosphate and aspartate pools increased whereas 3-phosphoglycerate and phosphoenol pyruvate levels decreased, demonstrating an interconnectivity of the C(3) and C(4) metabolites pools. It is concluded that during short-term treatment at 40 degrees C, Rubisco activase content is not the only factor modulating Rubisco carbamylation during C(4) photosynthesis.