Jörg Fromm

A Molecular Timetable for Apical Bud Formation and Dormancy Induction in Poplar

The growth of perennial plants in the temperate zone alternates with periods of dormancy that are typically initiated during bud development in autumn. In a systems biology approach to unravel the underlying molecular program of apical bud development in poplar (Populus tremula × Populus alba), combined transcript and metabolite profiling were applied to a high-resolution time course from short-day induction to complete dormancy. Metabolite and gene expression dynamics were used to reconstruct the temporal sequence of events during bud development. Importantly, bud development could be dissected into bud formation, acclimation to dehydration and cold, and dormancy. To each of these processes, specific sets of regulatory and marker genes and metabolites are associated and provide a reference frame for future functional studies. Light, ethylene, and abscisic acid signal transduction pathways consecutively control bud development by setting, modifying, or terminating these processes. Ethylene signal transduction is positioned temporally between light and abscisic acid signals and is putatively activated by transiently low hexose pools. The timing and place of cell proliferation arrest (related to dormancy) and of the accumulation of storage compounds (related to acclimation processes) were established within the bud by electron microscopy. Finally, the identification of a large set of genes commonly expressed during the growth-to-dormancy transitions in poplar apical buds, cambium, or Arabidopsis thaliana seeds suggests parallels in the underlying molecular mechanisms in different plant organs.

Characteristics of Electrical Signals in Poplar and Responses in Photosynthesis

To gain an understanding of the role of electrical signaling in trees, poplar (Populus trichocarpa, Populus tremula × P. tremuloides) shoots were stimulated by chilling as well as flaming. Two kinds of signal propagation were detected by microelectrode measurements (aphid technique) in the phloem of leaf veins: (1) basipetal, short-distance signaling that led to rapid membrane hyperpolarization caused by K+-efflux within the leaf lamina; and (2) acropetal, long-distance signaling that triggered depolarization of the membrane potential in the leaf phloem. In the latter, the depolarizing signals travel across the stem from the manipulated leaves to adjacent leaves where the net CO2 uptake rate is temporarily depressed toward compensation. With regard to photosystem II, both heat-induced long-distance and short-distance signaling were investigated using two-dimensional “imaging” analysis of chlorophyll fluorescence. Both types of signaling significantly reduced the quantum yield of electron transport through photosystem II. Imaging analysis revealed that the signal that causes yield reduction spreads through the leaf lamina. Coldblocking of the stem proved that the electrical signal transmission via the phloem becomes disrupted, causing the leaf gas exchange to remain unaffected. Calcium-deficient trees showed a marked contrast inasmuch as the amplitude of the electrical signal was distinctly reduced, concomitant with the absence of a significant response in leaf gas exchange upon flame wounding. In summary, the above results led us to conclude that calcium as well as potassium is involved in the propagation of phloem-transmitted electrical signals that evoke specific responses in the photosynthesis of leaves.

Lignin distribution in wood cell walls determined by TEM and backscattered SEM techniques.

The lignin distribution in cell walls of spruce and beech wood was determined by high-voltage transmission-electron-microscopy (TEM) in sections stained with potassium permanganate as well as by field-emission-scanning-electron-microscopy (FE-SEM) combined with a back-scattered electron detector on mercurized specimens. The latter is a new technique based on the mercurization of lignin and the concomitant visualization of mercury by back-scattered electron microscopy (BSE). Due to this combination it was possible to obtain a visualized overview of the lignin distribution across the different layers of the cell wall. To our knowledge, this combined method was used the first time to analyse the lignin distribution in cell walls. In agreement with previous work the highest lignin levels were found in the compound middle lamella and the cell corners. Back-scattered FE-SEM allows the lignin distribution in the pit membrane of bordered pits as well as in the various cell wall layers to be shown. In addition, by using TEM as well as SEM we observed that lignin closely follows the cellulose microfibril orientation in the secondary cell wall. From these observations, we conclude that the polymerisation of monolignols is affected by the arrangement of the polysaccharides which constitute the cell wall.

The fou2 mutation in the major vacuolar cation channel TPC1 confers tolerance to inhibitory luminal calcium.

The SV channel encoded by the TPC1 gene represents a Ca(2+)- and voltage-dependent vacuolar cation channel. Point mutation D454N within TPC1, named fou2 for fatty acid oxygenation upregulated 2, results in increased synthesis of the stress hormone jasmonate. As wounding causes Ca2+ signals and cytosolic Ca2+ is required for SV channel function, we here studied the Ca(2+)-dependent properties of this major vacuolar cation channel with Arabidopsis thaliana mesophyll vacuoles. In patch clamp measurements, wild-type and fou2 SV channels did not exhibit differences in cytosolic Ca2+ sensitivity and Ca2+ impermeability. K+ fluxes through wild-type TPC1 were reduced or even completely faded away when vacuolar Ca2+ reached the 0.1-mm level. The fou2 protein under these conditions, however, remained active. Thus, D454N seems to be part of a luminal Ca2+ recognition site. Thereby the SV channel mutant gains tolerance towards elevated luminal Ca2+. A three-fold higher vacuolar Ca/K ratio in the fou2 mutant relative to wild-type plants seems to indicate that fou2 can accumulate higher levels of vacuolar Ca(2+) before SV channel activity vanishes and K(+) homeostasis is impaired. In response to wounding fou2 plants might thus elicit strong vacuole-derived cytosolic Ca2+ signals resulting in overproduction of jasmonate.

Heat-induced electrical signals affect cytoplasmic and apoplastic pH as well as photosynthesis during propagation through the maize leaf

Combining measurements of electric potential and pH with such of chlorophyll fluorescence and leaf gas exchange showed heat stimulation to evoke an electrical signal (propagation speed: 3-5 mm s(-1)) that travelled through the leaf while reducing the net CO(2) uptake rate and the photochemical quantum yield of both photosystems (PS). Two-dimensional imaging analysis of the chlorophyll fluorescence signal of PS II revealed that the yield reduction spread basipetally via the veins through the leaf at a speed of 1.6 +/- 0.3 mm s(-1) while the propagation speed in the intervein region was c. 50 times slower. Propagation of the signal through the veins was confirmed because PS I, which is present in the bundle sheath cells around the leaf vessels, was affected first. Hence, spreading of the signal along the veins represents a path with higher travelling speed than within the intervein region of the leaf lamina. Upon the electrical signal, cytoplasmic pH decreased transiently from 7.0 to 6.4, while apoplastic pH increased transiently from 4.5 to 5.2. Moreover, photochemical quantum yield of isolated chloroplasts was strongly affected by pH changes in the surrounding medium, indicating a putative direct influence of electrical signalling via changes of cytosolic pH on leaf photosynthesis.

Wood formation of trees in relation to potassium and calcium nutrition

Potassium and calcium are essential for tree metabolism and various physiological processes related to growth. In recent years, special interest was therefore accorded to the effect of both cations on cambial activity and xylem development. Various studies revealed a distinct correlation between potassium as well as calcium nutrition and wood formation. When poplar trees were grown under low K(+) or Ca²(+) regimes, the cambial activity as well as the seasonal rate of wood increment and the vessel size were significantly reduced. Molecular, biochemical and electrophysiological investigations indicate (i) a strong involvement of specific K(+) channels in the regulation of xylem cell expansion and (ii) a significant influence of Ca²(+) on the onset of cambial reactivation after winter dormancy as well as on wood structure and chemistry. These studies highlight the important role of potassium as well as calcium in xylogenesis. Based on that knowledge, further research will be directed towards a better understanding of the mechanisms governing K(+)- as well as Ca²(+)-dependent wood formation.

Expression of the Arabidopsis Mutant abi1 Gene Alters Abscisic Acid Sensitivity, Stomatal Development, and Growth Morphology in Gray Poplars

The consequences of altered abscisic acid (ABA) sensitivity in gray poplar (Populus × canescens [Ait.] Sm.) development were examined by ectopic expression of the Arabidopsis (Arabidopsis thaliana) mutant abi1 (for abscisic acid insensitive1) gene. The expression resulted in an ABA-insensitive phenotype revealed by a strong tendency of abi1 poplars to wilt, impaired responsiveness of their stomata to ABA, and an ABA-resistant bud outgrowth. These plants therefore required cultivation under very humid conditions to prevent drought stress symptoms. Morphological alterations became evident when comparing abi1 poplars with poplars expressing Arabidopsis nonmutant ABI1 or wild-type plants. abi1 poplars showed increased stomatal size, enhanced shoot growth, and retarded leaf and root development. The increased stomatal size and its reversion to the size of wild-type plants by exogenous ABA indicate a role for ABA in regulating stomatal development. Enhanced shoot growth and retarded leaf and root development support the hypothesis that ABA acts independently from drought stress as a negative regulator of growth in shoots and as a positive regulator of growth in leaves and roots. In shoots, we observed an interaction of ABA with ethylene: abi1 poplars exhibited elevated ethylene production, and the ethylene perception inhibitor Ag+ antagonized the enhanced shoot growth. Thus, we provide evidence that ABA acts as negative regulator of shoot growth in nonstressed poplars by restricting ethylene production. Furthermore, we show that ABA has a role in regulating shoot branching by inhibiting lateral bud outgrowth.

Calcium-dependent physiological processes in trees.

Among the various plant nutrients, calcium appears to occupy a unique position, acting as an important regulator in many processes related to both growth and responses to environmental stresses. This applies to stomatal function, cell division, cell wall synthesis, signalling functions in plant defence, repair of damage from biotic and abiotic stress and to the structural chemistry and function of woody tissues. The calcium content in the cambium of poplar was shown to rise transiently by as much as 40% in spring, indicating the significant role that calcium plays in the onset of cambial reactivation. Moreover, during bud flush and the beginning of cell division, calcium was reported to increase significantly in the apical meristem. A reduction in calcium supplies also proved to strongly affect wood formation, as evidenced in the pronounced reduction in wood increment, vessel size and fibre length, as well as in reduced carbonyl and methoxy groups from S-lignin. Induced wounding revealed that calcium acts as an intracellular signal and, furthermore, proved its involvement in long-distance electrical signalling. Environmental stimuli such as cold shock or wounding showed that poplar grown under calcium-starved conditions was incapable of responding to this type of stress. The above evidence highlights the important role of calcium in tree functions, both as a signal in minute physiologically active pools within the cytoplasm, and in higher concentrations for its impact on the structural integrity of cell walls and woody tissues.

Seasonal Changes of Plasma Membrane H+-ATPase and Endogenous Ion Current during Cambial Growth in Poplar Plants

The plasma membrane H+-ATPase (PM H+-ATPase), potassium ions, and endogenous ion currents might play a fundamental role in the physiology of cambial growth. Seasonal changes of these parameters were studied in twigs ofPopulus nigra and Populus trichocarpa. Monoclonal and polyclonal antibodies against the PM H+-ATPase, x-ray analysis for K+ localization and a vibrating electrode for measurement of endogenous ion currents were used as probes. In dormant plants during autumn and winter, only a slight immunoreactivity against the PM H+-ATPase was found in cross sections and tissue homogenates, K+ was distributed evenly, and the density of endogenous current was low. In spring during cambial growth, strong immunoreactivity against a PM H+-ATPase was observed in cambial cells and expanding xylem cells using the monoclonal antibody 46 E5 B11 F6 for fluorescence microscopy and transmission electron microscopy. At the same time, K+ accumulated in cells of the cambial region, and strong endogenous current was measured in the cambial and immature xylem zone. Addition of auxin to dormant twigs induced the formation of this PM H+-ATPase in the dormant cambial region within a few days and an increase in density of endogenous current in shoot cuttings within a few hours. The increase in PM H+-ATPase abundance and in current density by auxin indicates that auxin mediates a rise in number and activity of an H+-ATPase in the plasma membrane of cambial cells and their derivatives. This PM H+-ATPase generates the necessary H+-gradient (proton-motive force) for the uptake of K+ and nutrients into cambial and expanding xylem cells.

Guard Cell-Specific Calcium Sensitivity of High Density and Activity SV/TPC1 Channels

The slow vacuolar (SV) channel, a Ca2+-regulated vacuolar cation conductance channel, in Arabidopsis thaliana is encoded by the single-copy gene AtTPC1. Although loss-of-function tpc1 mutants were reported to exhibit a stoma phenotype, knowledge about the underlying guard cell-specific features of SV/TPC1 channels is still lacking. Here we demonstrate that TPC1 transcripts and SV current density in guard cells were much more pronounced than in mesophyll cells. Furthermore, the SV channel in motor cells exhibited a higher cytosolic Ca2+ sensitivity than in mesophyll cells. These distinct features of the guard cell SV channel therefore probably account for the published stomatal phenotype of tpc1-2.