Peter K. Hepler

Calcium: A Central Regulator of Plant Growth and Development

Today no one questions the assertion that Ca2+ is a crucial regulator of growth and development in plants. The myriad processes in which this ion participates is large and growing and involves nearly all aspects of plant development (recent reviews in [Harper et al., 2004][1]; [Hetherington and

Pollen Tube Growth and the Intracellular Cytosolic Calcium Gradient Oscillate in Phase while Extracellular Calcium Influx Is Delayed.

Ratio images of cytosolic Ca2+ (Ca2+i) in growing, fura-2-dextran-loaded Lilium longiflorum pollen tubes taken at 3- to 5-sec intervals showed that the tip-focused [Ca2+]i gradient oscillates with the same period as growth. Similarly, measurement of the extracellular inward current, using a noninvasive ion-selective vibrating probe, indicated that the tip-directed extracellular Ca2+ (Ca2+o) current also oscillates with the same period as growth. Cross-correlation analysis revealed that whereas the [Ca2+]i gradient oscillates in phase with growth, the influx of Ca2+o lags by ~11 sec. Ion influx thus appears to follow growth, with the effect that the rate of growth at a given point determines the magnitude of the ion influx ~11 sec later. To explain the phase delay in the extracellular inward current, there must be a storage of Ca2+ for which we consider two possibilities: either the inward current represents the refilling of intracellular stores (capacitative calcium entry), or it represents the binding of the ion within the cell wall domain.

Ultrastructure of the cytoskeleton in freeze-substituted pollen tubes ofNicotiana alata

SummaryThe ultrastructure of the cytoskeleton inNicotiana alata pollen tubes grownin vitro has been examined after rapid freeze fixation and freeze substitution (RF-FS). Whereas cytoplasmic microtubules (MTs) and especially microfilaments (MFs) are infrequently observed after conventional chemical fixation, they occur in all samples prepared by RF-FS. Cortical MTs are oriented parallel to the long axis of the pollen tube and usually appear evenly spaced around the circumference of the cell. They are always observed with other components in a structural complex that includes the following: 1. a system of MFs, in which individual elements are aligned along the sides of the MTs and crossbridged to them; 2. a system of cooriented tubular endoplasmic reticulum (ER) lying beneath the MTs, and 3. the plasma membrane (PM) to which the MTs appear to be extensively linked. The cortical cytoskeleton is thus structurally complex, and contains elements such as MFs and ER that must be considered together with the MTs in any attempt to elucidate cytoskeletal function. MTs are also observed within the vegetative cytoplasm either singly or in small groups. Observations reveal that some of these may be closely associated with the envelope of the vegetative nucleus. MTs of the generative cell, in contrast to those of the vegetative cytoplasm, occur tightly clustered in bundles and show extensive cross-bridging. These bundles, especially in the distal tail of the generative cell, are markedly undulated. MFs are observed commonly in the cytoplasm of the vegetative cell. They occur in bundles oriented predominantly parallel to the pollen tube axis. Although proof is not provided, we suggest that they are composed of actin and are responsible for generating the vigorous cytoplasmic streaming characteristic of living pollen tubes.

Pectin Methylesterase, a Regulator of Pollen Tube Growth

The apical wall of growing pollen tubes must be strong enough to withstand the internal turgor pressure, but plastic enough to allow the incorporation of new membrane and cell wall material to support polarized tip growth. These essential rheological properties appear to be controlled by pectins, which constitute the principal component of the apical cell wall. Pectins are secreted as methylesters and subsequently deesterified by the enzyme pectin methylesterase (PME) in a process that exposes acidic residues. These carboxyls can be cross-linked by calcium, which structurally rigidifies the cell wall. Here, we examine the role of PME in cell elongation and the regulation of its secretion and enzymatic activity. Application of an exogenous PME induces thickening of the apical cell wall and inhibits pollen tube growth. Screening a Nicotiana tabacum pollen cDNA library yielded a pollen-specific PME, NtPPME1, containing a pre-region and a pro-region. Expression studies with green fluorescent protein fusion proteins show that the pro-region participates in the correct targeting of the mature PME. Results from in vitro growth analysis and immunolocalization studies using antipectin antibodies (JIM5 and JIM7) provide support for the idea that the pro-region acts as an intracellular inhibitor of PME activity, thereby preventing premature deesterification of pectins. In addition to providing experimental data that help resolve the significance and function of the pro-region, our results give insight into the mechanism by which PME and its pro-region regulate the cell wall dynamics of growing pollen tubes.

Pectin Methylesterases and Pectin Dynamics in Pollen Tubes

Pectic polysaccharides are an essential component of the primary plant cell wall. They are particularly prominent in pollen tubes, where they control the structure and yielding characteristics of the cell wall at the growing apex of these rapidly expanding cells. The properties of pectin meshworks,

The Regulation of Actin Organization by Actin-Depolymerizing Factor in Elongating Pollen Tubes

Pollen tube elongation is a polarized cell growth process that transports the male gametes from the stigma to the ovary for fertilization inside the ovules. Actomyosin-driven intracellular trafficking and active actin remodeling in the apical and subapical regions of pollen tubes are both important aspects of this rapid tip growth process. Actin-depolymerizing factor (ADF) and cofilin are actin binding proteins that enhance the depolymerization of microfilaments at their minus, or slow-growing, ends. A pollen-specific ADF from tobacco, NtADF1, was used to dissect the role of ADF in pollen tube growth. Overexpression of NtADF1 resulted in the reduction of fine, axially oriented actin cables in transformed pollen tubes and in the inhibition of pollen tube growth in a dose-dependent manner. Thus, the proper regulation of actin turnover by NtADF1 is critical for pollen tube growth. When expressed at a moderate level in pollen tubes elongating in in vitro cultures, green fluorescent protein (GFP)–tagged NtADF1 (GFP-NtADF1) associated predominantly with a subapical actin mesh composed of short actin filaments and with long actin cables in the shank. Similar labeling patterns were observed for GFP-NtADF1–expressing pollen tubes elongating within the pistil. A Ser-6-to-Asp conversion abolished the interaction between NtADF1 and F-actin in elongating pollen tubes and reduced its inhibitory effect on pollen tube growth significantly, suggesting that phosphorylation at Ser-6 may be a prominent regulatory mechanism for this pollen ADF. As with some ADF/cofilin, the in vitro actin-depolymerizing activity of recombinant NtADF1 was enhanced by slightly alkaline conditions. Because a pH gradient is known to exist in the apical region of elongating pollen tubes, it seems plausible that the in vivo actin-depolymerizing activity of NtADF1, and thus its contribution to actin dynamics, may be regulated spatially by differential H+ concentrations in the apical region of elongating pollen tubes.

Endoplasmic reticulum in the formation of the cell plate and plasmodesmata

SummaryThe association of endoplasmic reticulum (ER) with the developing cell plate has been analyzed in lettuce roots fixed in glutaraldehyde and post-fixed in a mixture of osmium tetroxide-potassium ferricyanide (OsFeCN). Electron microscopic observations show that elements of ER, which are selectively stained by the OsFeCN reagent, become loosely associated with aggregating dictyosome vesicles at the onset of plate formation. Subsequently the ER, in a tubular reticulate network, surrounds the vesicular aggregates creating a three dimensional membrane matrix. It is suggested that the ER (1) provides a structural framework that holds the vesicles in position and directs their fusion within the plane of the plate and/or (2) regulates the local release of calcium ions required for vesicle fusion.OsFeCN post-fixation also provides new information about the cell plate vesicles themselves. The results demonstrate that vesicles derived from dictyosomes undergo an abrupt increase in staining as they fuse at the plate.Finally the ER associated with developing and mature plasmodesmata has been examined. Electron micrographs reveal that the OsFeCN staining, seen traversing the cell plate in early stages, later becomes restricted from that portion of the ER extending through the plasmodesmatal canal. These structural observations support the idea that during formation of the plasmodesma a tubular element of ER is tightly furled upon itself and that its inner leaflet is compressed into a rod. The ER cisternal space appears occluded and thus it is argued that intercellular transport occurs through the cytoplasmic annulus of the plasmodesmata.

Rab2 GTPase Regulates Vesicle Trafficking between the Endoplasmic Reticulum and the Golgi Bodies and Is Important to Pollen Tube Growth

Pollen tube elongation depends on the secretion of large amounts of membrane and cell wall materials at the pollen tube tip to sustain rapid growth. A large family of RAS-related small GTPases, Rabs or Ypts, is known to regulate both anterograde and retrograde trafficking of transport vesicles between different endomembrane compartments and the plasma membrane in mammalian and yeast cells. Studies on the functional roles of analogous plant proteins are emerging. We report here that a tobacco pollen-predominant Rab2, NtRab2, functions in the secretory pathway between the endoplasmic reticulum and the Golgi in elongating pollen tubes. Green fluorescent protein–NtRab2 fusion protein localized to the Golgi bodies in elongating pollen tubes. Dominant-negative mutations in NtRab2 proteins inhibited their Golgi localization, blocked the delivery of Golgi-resident as well as plasmalemma and secreted proteins to their normal locations, and inhibited pollen tube growth. On the other hand, when green fluorescent protein–NtRab2 was over-expressed in transiently transformed leaf protoplasts and epidermal cells, in which NtRab2 mRNA have not been observed to accumulate to detectable levels, these proteins did not target efficiently to Golgi bodies. Together, these observations indicate that NtRab2 is important for trafficking between the endoplasmic reticulum and the Golgi bodies in pollen tubes and may be specialized to optimally support the high secretory demands in these tip growth cells.

Cellular oscillations and the regulation of growth: the pollen tube paradigm

The occurrence of oscillatory behaviours in living cells can be viewed as a visible consequence of stable, regulatory homeostatic cycles. Therefore, they may be used as experimental windows on the underlying physiological mechanisms. Recent studies show that growing pollen tubes are an excellent biological model for these purposes. They unite experimental simplicity with clear oscillatory patterns of both structural and temporal features, most being measurable during real‐time in live cells. There is evidence that these cellular oscillators involve an integrated input of plasma membrane ion fluxes, and a cytosolic choreography of protons, calcium and, most likely, potassium and chloride. In turn, these can create positive feedback regulation loops that are able to generate and self‐sustain a number of spatial and temporal patterns. Other features, including cell wall assembly and rheology, turgor, and the cytoskeleton, play important roles and are targets or modulators of ion dynamics. Many of these features have similarities with other cell types, notably with apical‐growing cells. Pollen tubes may thus serve as a powerful model for exploring the basis of cell growth and morphogenesis. BioEssays 23:86–94, 2001.

Selective localization of intracellular Ca2+ with potassium antimonate.

Introduction As a result of concerted efforts by scientists from a wide range of disciplines, we are coming to realize the crucial role played by Ca2 in biological functions. In the realm of biochemistry, especially, there has been recently an explosive rise in our knowledge about Ca2 effects on numerous cellular reactions, processes, and structural components. One emerging concept is that Ca2 ‘ serves as regulator of many of these. In such wellexamined phenomena as muscle contraction, a high degree of compartmentalization of Ca2 , coupled with the cell’s ability to mobilize Ca2 among various compartments (and thus to locally alter levels of reactive Ca2 ), is the means by which this is achieved (2). Often, direct measurement of the dynamics of free Ca2 is technologically difficult. Many have chosen instead to cxamine intracellular Ca2 -binding or Ca2 -sequestration sites in the search for clues on regulatory processes. There exist several histochemical techniques for localization of these sites, among them being in situ precipitation ofCa2 with potassium antimonate. The ideal probe should retain the cell’s in vivo Ca2 distribution, maximize its detection, and minimize interferenee from other reacting species. Realistically speaking, few, if any, techniques in science match up to their ideal: limitations and dangers of artifact abound, and histoehemistry holds no exception. Use of antimonate has received its share of criticism, and, indeed, the variety of eations reported to precipitate with antimonate could easily lead one to conclude that specificity for Ca2 is not possible with the reaction. However, a closer look at the literature reveals that “antimonate precipitation” does not specify a unique procedure, but rather encompasses a bewildering array of variations. A survey of results obtained by others using different buffers, pH’s, antimonate concentrations, fixatives, and tissue pretreatments, as well as our own experience in handling the reagent, indicates that reaction parameters strongly influence retention of and precipitation of physiological cations relative to each other. Thus, while originally proposed and used as a means of loealizing Na (40), antimonate’s use recently has been almost exclusively in studies involving Ca2 localization. As elaborated in this review, careful choice of reaction conditions can make the antimonate technique highly selective for Ca2 in comparison to the other cations that are capable of precipitation. Also, other variations on the antimonate reaction, while not so specific for Ca2 , can be used in conjunction with analytical techniques such as X-ray analysis or chelator treatments to ascertain which of the deposits formed contain Ca2 . By means of several different antimonate procedures, coupled thus with deposit analysis, previous studies have localized Ca2 in a wide variety of tissue and cell types, and cumulatively have revealed Ca2 in nearly every type of membranous organelle, as well as in association with some nonmembranous cellular components (Table 1 ). We believe that a discussion of some parameters of antimonate precipitation is instructive for those considering its use, as well as for those trying to understand results obtained with it in the past. When comparing results obtained in various laboratories, it is often difficult to pinpoint the influence exerted by any single parameter of the technique, since even the most similar protocols usually differ from each other in several details. While we have tried to sort these out as much as possible, there are substantial areas ofunavoidable overlap with material discussed in other sections. In these eases, the reader is requested to cross-refer to appropriate sections for a more detailed analysis of other influencing factors. We hope this cxercise provides evidence that it is possible to employ antimonate as a selective electron microscopic histoehemical stain for localization of exchangeable cellular Ca2 and that, in spite of inevitable limitations, it is a useful tool for exploring Ca2 regulation.