Plant memory: a tentative model.

All memory functions have molecular bases, namely in signal reception and transduction, and in storage and recall of information. Thus, at all levels of organisation living organisms have some kind of memory. In plants one may distinguish two types. There are linear pathways from reception of signals and propagation of effectors to a type of memory that may be described by terms such as learning, habituation or priming. There is a storage and recall memory based on a complex network of elements with a high degree of integration and feedback. The most important elements envisaged are calcium waves, epigenetic modifications of DNA and histones, and regulation of timing via a biological clock. Experiments are described that document the occurrence of the two sorts of memory and which show how they can be distinguished. A schematic model of plant memory is derived as emergent from integration of the various modules. Possessing the two forms of memory supports the fitness of plants in response to environmental stimuli and stress.

Modularity and emergence: biology's challenge in understanding life.

This essay juxtaposes modularity and emergence in the consideration of biological systems at various scalar levels of spatio-temporal organisation. It is noted that reductionism, specialisation and modularity are basic prerequisites for understanding life. It is realised that increased progress of scientific biology in elucidating mechanisms at the level of modular components supports the accusation that the more it advances in materialistic description of details, the more it diverts from understanding the innate properties of life. It is clear that modularity, by taking the whole as the sum of its parts, is insufficient for understanding living systems. At the same time, however, there is emergence, as advocated by Robert Laughlin. Emergence after the integration of modules leads to completely new properties of individual organisms as unique unitary entities, and also of systems of organisms with synergistic and antagonistic interactions of the integrated species. The discussion is predominantly based on examples from plant biology. At hierarchically higher scalar levels emergent biological systems are networks integrating species, biotopes, ecosystems and the entire biosphere of Earth, also named Gaia by James Lovelock, in a natural scientific respect. While investigating modules remains essential, biology as a nature science needs to merge and integrate such information to be able to unfold emergence. Through efforts towards visualising and understanding emergent diversity and complexity, the research discipline of biology will provide invaluable contributions to understanding life, and thus refute the accusation that it diverts from embracing the innate properties of life.

Photosynthetic efficiency of Clusia arrudae leaf tissue with and without Cecidomyiidae galls.

Leaf galls induced by a still undescribed new species of Cecidomyiidae (Diptera) are frequent on leaves of Clusia arrudae Planchon & Tirana (Clusiaceae) in the rupestrian fields at 1400 m a.s.l. in Serra do Cipó, Minas Gerais, Brazil. Galls were 7.1 ± 0.7 mm in diameter, one chambered with only one larva inside. Gall tissue is green and soft. Assessments of photosynthetic capacity using chlorophyll-a fluorescence measurements revealed that photosynthetic performance of gall tissue and healthy leaf tissue were rather similar. Hence, the morphological changes due to gall development were not associated with significant changes in the photosynthetic capacity of the tissue.

Photosynthetic efficiency of Clusia arrudae leaf tissue with and without Cecidomyiidae galls

Leaf galls induced by a still undescribed new species of Cecidomyiidae (Diptera) are frequent on leaves of Clusia arrudae Planchon & Tirana (Clusiaceae) in the rupestrian fields at 1400 m a.s.l. in Serra do Cipó, Minas Gerais, Brazil. Galls were 7.1 ± 0.7 mm in diameter, one chambered with only one larva inside. Gall tissue is green and soft. Assessments of photosynthetic capacity using chlorophyll-a fluorescence measurements revealed that photosynthetic performance of gall tissue and healthy leaf tissue were rather similar. Hence, the morphological changes due to gall development were not associated with significant changes in the photosynthetic capacity of the tissue.

Galhas induzidas por uma espécie nova ainda não descrita de Cecidomyiidae (Diptera) são frequentes nas folhas de Clusia arrudae Planchon & Tirana (Clusiaceae) em áreas de campos rupestres a 1400 metros de altitude na Serra do Cipó, Minas Gerais, Brasil. As galhas apresentaram um diâmetro médio de 7,1 ± 0,7 mm e uma loja com uma larva dentro. As galhas são verdes e de tecido macio. Análises da capacidade fotossintética usando medidas de fluorescência da clorofila a revelaram que o desempenho fotossintético dos tecidos da galha e sadios foi similar. Assim, as mudanças induzidas pelo desenvolvimento das galhas não foram associadas a mudanças significativas da capacidade fotossintética do tecido.

Resurrection kinetics of photosynthesis in desiccation-tolerant terrestrial green algae (Chlorophyta) on tree bark.

The rough bark of orchard trees (Malus) around Darmstadt is predominantly covered in red to purple-brown layers (biofilms) of epiphytic terrestrial alga of Trentepohlia umbrina. The smooth bark of forest trees (Fagus sylvatica L. and Acer sp.) in the same area is covered by bright green biofilms composed of the green algae Desmococcus, Apatococcus and Trebouxia, with a few cells of Coccomyxa and 'Chlorella' trebouxioides between them. These algae are desiccation tolerant. After samples of bark with the biofilms were kept in dry air in darkness for various periods of time, potential quantum yield of PSII, F(v)/F(m), recovered during rehydration upon rewetting. The kinetics and degree of recovery depended on the length of time that the algae were kept in dry air in the desiccated state. Recovery was better for green biofilm samples, i.e. quite good even after 80 days of desiccation (F(v)/F(m) = ca. 50% of initial value), than the red samples, where recovery was only adequate up to ca. 30-40 days of desiccation (F(v)/F(m) = ca. 20-55% of initial value). It is concluded that the different bark types constitute different ecophysiological niches that can be occupied by the algae and that can be distinguished by their capacity to recover from desiccation after different times in the dry state.

Redundancy of stomatal control for the circadian photosynthetic rhythm in Kalanchoë daigremontiana Hamet et Perrier.

In continuous light, the Crassulacean acid metabolism plant Kalanchoe daigremontiana Hamet et Perrier has a circadian rhythm of gas exchange with peaks occurring during the subjective night. The rhythm of gas exchange is coupled to a weak, reverse phased rhythm of quantum yield of photosystem II (Phi (PSII)). To test if the rhythm of Phi (PSII) persists in the absence of stomatal control, leaves were coated with a thin layer of translucent silicone grease which prevented CO2 and H2O exchange. In spite of this treatment, the rhythm of Phi (PSII) occurred with close to normal phase timing and with a much larger amplitude than in uncoated leaves. The mechanism underlying the Phi (PSII) rhythm in coated leaves can be explained by a circadian activity of phosphoenolpyruvate carboxylase (PEPC). At peaks of PEPC activity, the small amount of CO2 contained in the coated leaf could have become depleted, preventing the carboxylase activity of Rubisco and causing decreases in electron transport rates (observed as deep troughs of Phi (PSII) at 23-h in LL and at ca. 24-h intervals afterwards). Peaks of Phi (PSII) would be caused by a downregulation of PEPC leading to improved supply of CO2 to Rubisco. Substrate limitation of photochemistry at 23 h (trough of Phi (PSII)) was also suggested by the weak response of ETR in coated leaves to stepwise light enhancement. These results show that photosynthetic rhythmicity in K. daigremontiana is independent of stomatal regulation and may originate in the mesophyll.

Period-2 cycles and 2:1 phase locking in a biological clock driven by temperature pulses.

Crassulacean acid metabolism (CAM) serves as a botanical model system for the investigation of circadian rhythmicity. In a new set of experiments with the obligatory CAM plant Kalanchoë daigremontiana the response to periodic stimulations with temperature pulses has been studied. On the basis of an experimental phase-response curve of net CO(2)-gas exchange the effect of periodic stimulation has been simulated using a finite-difference equation. These simulations revealed the locations of two period-2 cycles in the CO(2) uptake of the CAM plant. In subsequent experiments based upon the simulated bifurcation diagram the position and amplitude of one of these cycles were confirmed, while experimental evidence for the second cycle could be found. Possible roles of such dynamics for the functioning of the biological clock are discussed.

Midday depression in savanna trees: coordinated adjustments in photochemical efficiency, photorespiration, CO2 assimilation and water use efficiency.

High irradiances, high air temperatures and low relative humidities characterize the seasonal savannas of central Brazil, locally known as cerrados. In the present study, we investigated the implications to photoprotection of diurnal adjustments in photochemical and non-photochemical processes in five cerrado woody species that differed in photosynthetic capacity and in the duration and extension of the midday depression of photosynthesis. We also evaluated the contribution of photorespiration to minimize the danger of photoinhibition and the potential carbon costs of the operation of this mechanism in response to changes in irradiance levels. Notwithstanding the large differences in diurnal patterns of photosynthesis and in stomatal conductance, four out of the five species showed a tight common linear relationship between net CO2 assimilation rates and transpiration over the large range of environmental conditions that prevailed during typical sunny days at the end of the rainy season. Large reversible decreases in photochemical efficiency were compensated by proportional increases in non-photochemical processes related to photoprotection, irrespective of the prevailing irradiance levels and degree of stomata closure. Light response curves were used to evaluate the relative contribution of photorespiratory CO2 production to electron flow in response to changes in irradiance levels. A large percentage of the electron flow was used to drive photorespiration in light-saturated leaves. In conclusion, an efficient control of photochemical and non-photochemical quenching and adjustments in the partition of electron flow between assimilative and non-assimilative processes alleviated the danger of photoinhibition. However, the results also suggest that losses in potential carbon gain because of high photorespiratory costs could impose strong limitations on leaf carbon balance of cerrado woody species.

Day-to-night variations of cytoplasmic pH in a crassulacean acid metabolism plant.

In crassulacean acid metabolism (CAM) large amounts of malic acid are redistributed between vacuole and cytoplasm in the course of night-to-day transitions. The corresponding changes of the cytoplasmic pH (pHcyt) were monitored in mesophyll protoplasts from the CAM plant Kalanchoe daigremontiana Hamet et Perrier by ratiometric fluorimetry with the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein as a pHcyt indicator. At the beginning of the light phase, pHcyt was slightly alkaline (about 7.5). It dropped during midday by about 0.3 pH units before recovering again in the late-day-to-early-dark phase. In the physiological context the variation in pHcyt may be a component of CAM regulation. Due to its pH sensitivity, phosphoenolpyruvate carboxylase appears as a likely target enzyme. From monitoring delta pHcyt in response to loading the cytoplasm with the weak acid salt K-acetate a cytoplasmic H(+)-buffer capacity in the order of 65 mM H+ per pH unit was estimated at a pHcyt of about 7.5. With this value, an acid load of the cytoplasm by about 10 mM malic acid can be estimated as the cause of the observed drop in pHcyt. A diurnal oscillation in pHcyt and a quantitatively similar cytoplasmic malic acid is predicted from an established mathematical model which allows simulation of the CAM dynamics. The similarity of model predictions and experimental data supports the view put forward in this model that a phase transition of the tonoplast is an essential functional element in CAM dynamics.

Effects of salt treatment and osmotic stress on V-ATPase and V-PPase in leaves of the halophyte Suaeda salsa.

The Chenopodiaceae Suaeda salsa L. was grown under different salt concentrations and under osmotic stress. The fresh weight was markedly stimulated by 0.1 M NaCl, 0.4 M NaCl and 0.1 M KCl and reduced by osmotic stress (PEG iso-osmotic to 0.1 M NaCl). Treatment with 0.4 M KCl severely damaged the plants. Membrane vesicle fractions containing tonoplast vesicles were isolated by sucrose gradient from leaves of the S. salsa plants and modulations of V-ATPase and V-PPase depending on the growth conditions were determined. Western blot analysis revealed that V-ATPase of S. salsa consists of at least nine subunits (apparent molecular masses 66, 55, 52, 48, 36, 35, 29, 18, and 16 kDa). This polypeptide pattern did not depend on culture conditions. V-PPase is composed of a single polypeptide (69 kDa). An additional polypeptide (54 kDa) was detected in the fractions of NaCl-, KCl- and PEG-treated plants. It turned out that the main strategy of salt-tolerance of S. salsa seems to be an up-regulation of V-ATPase activity, which is required to energize the tonoplast for ion uptake into the vacuole, while V-PPase plays only a minor role. The increase in V-ATPase activity is not obtained by structural changes of the enzyme, but by an increase in V-ATPase protein amount.

Spatiotemporal variation of metabolism in a plant circadian rhythm: the biological clock as an assembly of coupled individual oscillators.

The complex dynamic properties of biological timing in organisms remain a central enigma in biology despite the increasingly precise genetic characterization of oscillating units and their components. Although attempts to obtain the time constants from oscillations of gene activity and biochemical units have led to substantial progress, we are still far from a full molecular understanding of endogenous rhythmicity and the physiological manifestations of biological clocks. Applications of nonlinear dynamics have revolutionized thinking in physics and in biomedical and life sciences research, and spatiotemporal considerations are now advancing our understanding of development and rhythmicity. Here we show that the well known circadian rhythm of a metabolic cycle in a higher plant, namely the crassulacean acid metabolism mode of photosynthesis, is expressed as dynamic patterns of independently initiated variations in photosynthetic efficiency (phi(PSII)) over a single leaf. Noninvasive highly sensitive chlorophyll fluorescence imaging reveals randomly initiated patches of varying phi(PSII) that are propagated within minutes to hours in wave fronts, forming dynamically expanding and contracting clusters and clearly dephased regions of phi(PSII). Thus, this biological clock is a spatiotemporal product of many weakly coupled individual oscillators, defined by the metabolic constraints of crassulacean acid metabolism. The oscillators operate independently in space and time as a consequence of the dynamics of metabolic pools and limitations of CO(2) diffusion between tightly packed cells.

Stochastic noise interferes coherently with a model biological clock and produces specific dynamic behaviour.

The influence of noise is unavoidable in all living systems. Its impact on a model of a biological clock, normally running in regular oscillating modes, is examined. It is shown that in a specific system in which endogenous rhythmicity is produced by a beat oscillator acting on a feedback coupled metabolic pool system, noise can act coherently to produce unexpected dynamic behaviour, running from regular over pseudo-regular to irregular time-structures. If the biological system consists of a set of identical weakly coupled cells, stochasticity may lead to phase decoupling producing irregular spatio-temporal patterns. Synchronization via phase resetting can be achieved by external short-time temperature pulses. Explicit results are obtained for the well-studied circadian photosynthesis oscillations in plants performing crassulacean acid metabolism. Because of the generic structure of the underlying nonlinear dynamics they can, however, be regarded as a general property of the influence of noise on nonlinear excitable systems with fixed points occuring close to limit cycles.

The role of vacuolar malate-transport capacity in crassulacean acid metabolism and nitrate nutrition. Higher malate-transport capacity in ice plant after crassulacean acid metabolism-induction and in tobacco under nitrate nutrition.

Anion uptake by isolated tonoplast vesicles was recorded indirectly via increased H(+)-transport by H(+)-pumping of the V-ATPase due to dissipation of the electrical component of the electrochemical proton gradient, Deltamu(H+), across the membrane. ATP hydrolysis by the V-ATPase was measured simultaneously after the Palmgren test. Normalizing for ATP-hydrolysis and effects of chloride, which was added to the assays as a stimulating effector of the V-ATPase, a parameter, J(mal)(rel), of apparent ATP-dependent malate-stimulated H(+)-transport was worked out as an indirect measure of malate transport capacity. This allowed comparison of various species and physiological conditions. J(mal)(rel) was high in the obligate crassulacean acid metabolism (CAM) species Kalanchoë daigremontiana Hamet et Perrier, it increased substantially after CAM induction in ice plant (Mesembryanthemum crystallinum), and it was positively correlated with NO(3)(-) nutrition in tobacco (Nicotiana tabacum). For tobacco this was confirmed by measurements of malate transport energized via the V-PPase. In ice plant a new polypeptide of 32-kD apparent molecular mass appeared, and a 33-kD polypeptide showed higher levels after CAM induction under conditions of higher J(mal)(rel). It is concluded that tonoplast malate transport capacity plays an important role in physiological regulation in CAM and NO(3)(-) nutrition and that a putative malate transporter must be within the 32- to 33-kD polypeptide fraction of tonoplast proteins.

The tonoplast functioning as the master switch for circadian regulation of crassulacean acid metabolism.

From the initial discovery of free-running endogenous circadian oscillations of Crassulacean acid metabolism (CAM) under constant conditions in the light and in air, it has been disputed whether the underlying oscillator is enzymic or biophysical. The hypothesis of a biophysical hysteresis switch or beat oscillator started from osmotic considerations of malate accumulation and remobilisation, indicating a tonoplast tension/relaxation mechanism. It then advanced to application of non-linear dynamics theory for the analysis of rhythmic and arrhythmic time series of CO2 exchange under the regime of external control parameters, mainly temperature, and the implementation of models for computer simulations of CAM rhythms. This provided strong evidence for the tonoplast functioning as a master switch for circadian regulation of CAM. Conversely, the hypothesis of an enzymic beat oscillator strongly developed on the experimental basis of phosphorylation/dephosphorylation of phosphoenolpyruvate carboxylase (PEPC) regulating the enzyme activity, and hence CO2 fixation and malate synthesis via this enzyme. It was much supported by the discovery that PEPC-kinase gene-transcription was under circadian control. However, biochemical and molecular analysis, as well as model simulation, strongly suggests that this is a secondary and not the primary oscillator. The synchronisation/desynchronisation of leaf patches has revealed spatiotemporal characteristics of circadian rhythmicity that may open new ways for understanding biological clocks.

Diurnal changes in chlorophyll a fluorescence, CO(2)-exchange and organic acid decarboxylation in the tropical CAM tree Clusia hilariana.

Crassulacean acid metabolism (CAM) plants are dependent on the organic acids that accumulate overnight in the vacuoles as a source of CO(2) during the daylight deacidification period, when stomata are closed and high irradiances generally prevail. We performed an integrative analysis of diurnal changes in gas exchange, chlorophyll fluorescence parameters and organic acid decarboxylation to understand the adjustments in photochemical and non-photochemical processes during the different CAM phases in Clusia hilariana Schlecht., a dominant tree species in the sandy coastal plains of southeastern Brazil. A linear relationship was obtained between the quantum yields of photochemical and non-photochemical quenching, irrespective of the CAM phase and prevailing irradiance. Degradation of malic and citric acids during the midday stomatal closure period could lead to potential CO(2) fixation rates of 23 &mgr;mol m(-2) s(-1), whereas CO(2) losses, measured as CO(2) evolution, corresponded to about 3% of this value. Thus, decarboxylation of malate and citrate provided high internal CO(2) concentrations during phase III of CAM, even though the stomata were closed, allowing optimal utilization of light energy, as indicated by the non-saturating electron transport rates (ETR) in the light response curves, with highest rates of ETR occurring at midday in the diurnal curves. At the transition from phase III to IV of CAM, depletion of internal CO(2) sources and low stomatal conductances, which restricted the supply of exogenous CO(2), reduced the demand for photochemical energy to drive carbon assimilation. This was compensated by increases in thermal energy dissipation as indicated by higher rates of non-photochemical quenching, while high irradiances still prevailed. Shifts in the CAM phases and changes in protective thermal dissipation potential allowed C. hilariana to match changes in PPFD patterns for leaves of different orientations. Evidence that most of the decline in photochemical efficiency was probably related to the fast-relaxing component of non-photochemical quenching is provided by the high values of the quantum yield of photosystem II after 20 min of relaxation in darkness, and an almost complete recovery after sunset. These adjustments in photosynthetic machinery minimized the danger of photo-inhibition in C. hilariana, which is commonly found in fully exposed habitats.

Thermodynamics and energetics of the tonoplast membrane operating as a hysteresis switch in an oscillatory model of Crassulacean acid metabolism.

The observed endogenous circadian rhythm in plants performing Crassulacean acid metabolism is effected by malate transport at the tonoplast membrane. Experimental and theoretical work asks for a hysteresis switch, regulating this transport via the ordering state of the membrane. We apply a schematic molecular model to calculate the thermally averaged order parameter of the membrane lipid structure in its dependence on external parameters temperature and area per molecule. The model shows a first order structural phase transition in a biologically relevant temperature range. Osmotic consequences of malate accumulation can trigger a transition between the two phases by changing the surface area of the cell vacuole. Estimation of the energy needed to expand the vacuole under turgor pressure because of osmotic changes while acidifying shows that energy needed as latent heat for the calculated change between phases can easily be afforded by the cell. Thus, malate content and the coexisting two phases of lipid order, showing hysteretic behavior, can serve as a feedback system in an oscillatory model of Crassulacean acid metabolism, establishing the circadian clock needed for endogenous rhythmicity.

On the Mechanism of Reinitiation of Endogenous Crassulacean Acid Metabolism Rhythm by Temperature Changes.

Under continuous light the endogenous Crassulacean acid metabolism (CAM) rhythm of Kalanchoe daigremontiana Hamet et Perrier de la Bathie disappears at high (>29.0[deg]C) or low (<8.0[deg]C) temperatures. We investigated the reinitiation of rhythmicity when temperature was reduced from above the upper and increased from below the lower threshold level via measurements of (a) short-term changes in carbon-isotope discrimination to illustrate shifts between C3 and C4 carboxylation in vivo, and (b) the malate sensitivity of phosphoenolpyruvate carboxylase (PEPC) in vitro. When the net CO2-exchange rhythm disappears at both temperatures, the instantaneous discrimination indicates low PEPC activity. Leaf malate concentration and osmolarity attain high and low values at low and high temperatures, respectively. After small temperature increases or reductions from the low and high temperatures, respectively, the rhythm is reinitiated, with phases shifted by 180[deg] relative to each other. This can be related to the contrasting low and high leaf malate concentrations due to direct inhibition of PEPC and possibly also of the phosphorylation of PEPC by malate. The experimental results were satisfactorily simulated by a mathematical CAM-cycle model, with temperature acting only on the passive efflux of malate from the vacuole. We stress the important role of the tonoplast in malate compartmentation and of malate itself for the reinitiation and generation of endogenous CAM rhythmicity.

A Model for Photosynthetic Oscillations in Crassulacean Acid Metabolism (CAM).

We propose a model of crassulacean acid metabolism (CAM) describing the varying concentrations of pools of major metabolites by a system of coupled nonlinear differential equations. The model shows regular oscillations in normal day night and free-running endogeneous oscillations in continuous light. The effect of temperature is incorporated in a realistic way. It leads to the correct dependence of the oscillatory period length on temperature as compared to experimental observations.

Photosynthesis and photoinhibition in a tropical alpine giant rosette plant, Lobelia rhynchopetalum.

Carbodioxide uptake, oxygen evolution and chlorophyll fluorescence of leaves of Lobelia Lobelia rhynchopetalum Hemsl., a giant rosette plant of the tropical alpine regions of Ethiopia, were studied under field conditions at 4000 m above sea level. Our objective was to investigate the photosynthetic adaptation to the combination of wide fluctuation in diurnal temperature, high photon flux densities (PFD) and low CO2 partial pressure encountered in these regions. At an ambient CO2 partial pressure of c. 17 Pa, maximal rates of CO2 uptake were low, ranging between 4 and 6 µmol m-2 s-1 . Such rates, however, required high PFDs and were observed only at levels of 1500 µmol photons m-2 s-2 . Carbon dioxide uptake was significantly inhibited when PFD was ≤ 2000 µmol photons m-2 s-1 . On the other hand, at saturating CO2 levels, maximal photosynthetic oxygen evolution was higher (30 µmol C2 m-2 s-1 ). saturating at the same PFD as CO2 uptake. Quantum efficiency of CO2 uptake (0.006 mol CO2 mol photons-1 , at high altitude and a low CO, partial pressure of 17 Pa) and even of oxygen evolution under CO2 -saturating conditions in the leaf O2 electrode (0.05 mol O2 mo) photons-1 ) indicated reduced photosynthetic efficiency. Electron transport rate (ETR) was strongly correlated with the leaf temperature. Non-photochemical quenching (NPQ) responded inversely to leaf temperature and stomatal conductance. The results indicated that in the morning, when the sun irradiates the partly frozen leaves with closed stomata, NPQ is the principal mechanism by which Lobelia leaves protect their photosynthetic apparatus. However, during the day, the predominant upright inclination of the leaves significantly contributes to protecting the leaves from excess light absorption. A comparison of the chlorophyll fluorescence of young and old leaves revealed that the former had high ETR and quantum efficiency of photosynthetic electron transport but a lower capacity for NPQ. Extremely high NPQ values but low ETR and low quantum efficiency were recorded for the old leaves. Thus, in the course of maturation the leaves apparently lose photosynthetic efficiency but increase their capability for protective non-photochemical quenching.

The 32 kDa tonoplast polypeptide Di associated with the V-type H+-ATPase of Mesembryanthemum crystallinum L. in the CAM state: A proteolytically processed subunit B?

In the facultative halophyte Mesembryanthemum crystallinum, the salt- or age-induced transition to crassulacean acid metabolism (CAM) leads to the occurrence of a tonoplast-bound 32 kDa polypeptide (Di). The alignment of its N-terminal protein sequence with protein sequences of recently cloned higher plant V-ATPase B-subunits indicates that Di may be derived from subunit B by proteolytic removal of a protein fragment of about 20 kDa from its N-terminus. Furthermore, an antiserum directed against Di cross-reacts with subunit B from Nicotiana tabacum. It inhibits both proton pumping and ATP hydrolysis of the holoenzyme in M. crystallinum. As Di remains firmly attached to the holoenzyme the proteolytic processing may have functional implications.