Distribution Of Callose Research Articles

The different response of Brassica napus genotypes to microspore embryogenesis induced by heat shock and trichostatin A is not determined by changes in cell wall structure and composition but by different stress tolerance

AbstractDuring microspore embryogenesis, microspores are induced to develop into haploid embryos. In Brassica napus, microspore embryogenesis is induced by a heat shock (HS), which initially produces embryogenic structures with different cell wall architectures and compositions, and with different potentials to develop into embryos. The B. napus DH4079 and DH12075 genotypes have high and very low embryo yields, respectively. In DH12075, embryo yield is greatly increased by combining HS and the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). However, we show that HS + TSA inhibits embryogenesis in the highly embryogenic DH4079 line. To ascertain why TSA has such different effects in these lines, we treated DH4079 and DH12075 microspore cultures with TSA and compared the cell wall structure and composition of the different embryogenic structures in both lines, specifically the in situ levels and distribution of callose, cellulose, arabinogalactan proteins and high and low methyl‐esterified pectin. For both lines, HS + TSA led to the formation of cell walls unfavorable for embryogenesis progression, with reduced levels of arabinogalactan proteins, reduced cell adhesion of inner walls and altered pectin composition. Thus, TSA effects on cell walls cannot explain their different embryogenic response to TSA. We also applied TSA to DH4079 cultures at different times and concentrations before HS application, with no negative effects on embryogenic induction. These results indicate that DH4079 microspores are hypersensitive to combined TSA and HS treatments, and open up new hypotheses about the causes of such hypersensitivity.

An efficient method for the extraction and the quantitative determination of callose from HLB-affected and healthy citrus

Callose is a 1,3-β glucan responsible for several processes in planta such as cell division, ripening of pollen mother cells, maintaining the plasmodesmata opening, and providing structure to the sieve plates. Besides the physiological roles, callose is deposited during pathogen attacks, forming papillae to prevent pathogen entrance into the tissue or plugging the sieve pores to limit the spread of phloem pathogens. Candidatus Liberibacter asiaticus (CLas), the putative causal agent of Huanglongbing (HLB), is a phloem-restricted pathogen whose infection leads to the production of callose in the phloem. Characterizing the dynamics of callose plugging during HLB is important for understanding the disease, but no published protocols are available for the extraction and quantification of the callose in citrus trees, and quantitative data are limited. Detection of callose by microscopy is time-consuming and expensive and does not give information about the distribution of callose in the entire plant. Here, we present a short protocol for the efficient extraction and quantification of the total callose from citrus plants. We compared different tissues from healthy and CLas-infected plants and identified an increase in callose levels in the midribs, the stems, and the fruit peduncles of infected plants. Callose levels were highest in leaves, especially midribs, compared to stems, roots, and fruit peduncles. This method could be applied to other woody plant species.

Callose Detection and Quantification at Plasmodesmata in Bryophytes.

In bryophytes (i.e., mosses, liverworts, and hornworts), extant representatives of early land plants, plasmodesmata have been described in a wide range of tissues. Although their contribution to bryophyte morphogenesis remains largely unexplored, several recent studies have suggested that the deposition of callose around plasmodesmata might regulate developmental and physiological responses in mosses. In this chapter, we provide a protocol to image and quantify callose levels in the filamentous body of the model moss Physcomitrium (Physcomitrella) patens and discuss possible alternatives and pitfalls. More generally, this protocol establishes a framework to explore the distribution of callose in other bryophytes.

Treatment with spermidine alleviates the effects of concomitantly applied cold stress by modulating Ca2+, pH and ROS homeostasis, actin filament organization and cell wall deposition in pollen tubes of Camellia sinensis

The aim of the current study was to examine the effect of spermidine treatment concomitant with cold stress on the elongation of Camellia sinensis pollen tube. When exogenous spermidine (0.05 mM) was applied concomitantly with cold stress, pollen germination rate and pollen tube length were significantly increased in comparison with cold stressed pollen tubes. In addition, spermidine treatment concomitantly with cold stress reduced pollen tube abnormalities induced by cold stress. Besides, cold-induced disorganizations of actin filaments were ameliorated after spermidine treatment along with cold stress because anisotropy levels of actin filaments in shank and apex of pollen tubes decreased. Changes in cold-induced callose distribution in the pollen tube cell wall were partially recovered after spermidine/cold stress treatment. Other cold-induced effects (decrease in Ca2+ content, reduction of pH gradient, accumulation of ROS) were reverted to adequate levels after spermidine treatment in conjunction with cold stress, indicating that pollen tubes are able to cope with stress. Thus, spermidine treatment reorganized the growth pattern of pollen tubes by modulating Ca2+ and ROS homeostasis, actin cytoskeleton organization, and cell wall deposition in Camellia sinensis pollen tubes under cold stress.

Effect of methyl jasmonate on in-vitro pollen germination and tube elongation of Pinus nigra.

The purpose of the main research was to investigate the effects of methyl jasmonate (MeJA) (0.05, 0.25, 0.5, and 2.5mM) on the pollen germination and tube elongation of Pinus nigra. Total pollen germination rate increased after MeJA treatments while the most enhancement was observed at 0.05-mM MeJA. No germination was observed at 2.5-mM MeJA. Although the unipolar and bipolar germination were observed in all groups, no significant changes were observed in unipolar and bipolar pollen germination rates after MeJA treatments. Tube length increased only at 0.05-mM MeJA. Although branched tubes were observed in all groups, branched tube rate increased only at 0.05-mM MeJA. Although two branched, three branched, and consecutive branched tubes were observed in all groups, the most common branching type was two branched type in all groups. Although anisotropy of actin filaments in the shank and apex of unbranched tubes decreased after MeJA treatments, the most decrease was observed at 0.05-mM MeJA. Also, anisotropy of actin filaments in the shank and in pre-branching region of branched tubes decreased only at 0.25-mM MeJA. Anisotropy of both two apexes of a branched tube changed only at 0.25- and 0.5-mM MeJA. Callose accumulation in the apex of unbranched and branched tubes increased in parallel with the increase in MeJA concentration. However, more callose is accumulated in one apex than the other apex of a branched tube. In conclusion, MeJA affected the actin organization, changed the callose distribution, and altered the pollen tube growth of Pinus nigra.

Aluminum toxicity-induced pollen tube growth inhibition in apple (Malus domestica) is mediated by interrupting calcium dynamics and modification of cell wall components

Aluminum (Al) is known the most abundant metal in the Earth’s crust which is a major problem limiting crop production and yield throughout the world. Al could inhibit pollen tube elongation, however the mechanism is largely unknown. The aim of this study was to investigate the influence of Al toxicity on Malus domestica pollen tube growth and possible cytological mechanism. The results showed that Al inhibited pollen germination and tube growth in a concentration dependent manner. An non-invasive ion-selective electrode technique together with fluorescent labeling indicated that Al toxicity decreased pollen tube apex calcium influx and disrupted [Ca2+]c gradient. Al toxicity also altered actin filament orientation. Results from immuno-localization and fluorescence labeling indicated that Al toxicity affected the accumulation and distribution of callose, acid pectins, esterified pectins and arabinogalactan proteins in pollen tubes, which were supported by Fourier-transform infrared analysis. In a word, Al induced a series of the cellular toxic changes concerning calcium influx, [Ca2+]c gradient, actin and cell wall components. Based on the information provided in this article, it is concluded that Al toxicity inhibits apical growth of pollen tubes by changing calcium influx and [Ca2+]c and cell wall components.

Callose and homogalacturonan epitope distribution in stomatal complexes of Zea mays and Vigna sinensis.

This article deals with the distribution of callose and of the homogalacturonan (HG) epitopes recognized by LM20, JIM5, and 2F4 antibodies in cell walls of differentiating and functioning stomatal complexes of the monocotyledon Zea mays and the dicotyledon Vigna sinensis. The findings revealed that, during stomatal development, in these plant species, callose appears in an accurately spatially and timely controlled manner in cell walls of the guard cells (GCs). In functioning stomata of both plants, callose constitutes a dominant cell wall matrix material of the polar ventral cell wall ends and of the local GC cell wall thickenings. In Zea mays, the LM20, JIM5, or 2F4 antibody-recognized HG epitopes were mainly located in the expanding cell wall regions of the stomatal complexes, while in Vigna sinensis, they were deposited in the local cell wall thickenings of the GCs as well as at the ledges of the stomatal pore. Consideration of the presented data favors the view that in the stomatal complexes of the monocotyledon Z. mays and the dicotyledon V. sinensis, the esterified HGs contribute to the cell wall expansion taking place during GC morphogenesis and the opening of the stomatal pore. Besides, callose and the highly de-esterified HGs allow to GC cell wall regions to withstand the mechanical stresses exerted during stomatal function.

Cytochalasin B Treatment of Apple (Malus pumila Mill.) Pollen Tubes Alters the Cytoplasmic Calcium Gradient and Causes Major Changes in the Cell Wall Components

It is well established that the actin cytoskeleton is absolutely essential to pollen germination and tube growth. In this study we investigated the effects of cytochalasin B (CB), which affects actin polymerization by binding to the barbed end of actin filaments, on apple (Malus pumila Mill.) pollen tube growth. Results showed that CB altered the morphology of pollen tubes, which had a larger diameter than control tubes beside inhibiting pollen germination and tube growth. Meantime CB also caused an abnormal distribution of actin filaments in the shank of the treated pollen tubes. Fluo-3/AM labeling indicated that the gradient of cytosolic calcium ([Ca2+]c) in the pollen tube tip was abolished by exposure to CB, which induced a much stronger signal in the cytoplasm. Cellulose and callose distribution in the tube apex changed due to the CB treatment. Immunolabeling with different pectin and arabinogalactan protein (AGP) antibodies illustrated that CB induced an accumulation of pectins and AGPs in the tube cytoplasm and apex wall. The above results were further supported by Fourier-transform infrared (FTIR) analysis. The results suggest the disruption of actin can result in abnormal growth by disturbing the [Ca2+]c gradient and the distribution of cell wall components at the pollen tube apex.

The Ceratopteris (fern) developing motile gamete walls contain diversepolysaccharides, but not pectin.

Unlike most plant cell walls, the five consecutive walls laid down during spermatogenesis in the model fern Ceratopteris contain sparse cellulose, lack pectin and are enriched with callose and hemicelluloses. Seed-free plants like bryophytes and pteridophytes produce swimming male gametes for sexual reproduction. During spermatogenesis, unique walls are formed that are essential to the appropriate development and maturation of the motile gametes. Other than the detection of callose and general wall polysaccharides in scattered groups, little is known about the sequence of wall formation and the composition of these walls during sperm cell differentiation in plants that produce swimming sperm.Using histochemistry and immunogold localizations, we examined the distribution of callose, cellulose, mannan and xylan-containing hemicelluloses, and homogalacturonan (HG) pectinsin the special walls deposited during spermatogenesis in Ceratopteris. Five walls are produced in sequenceand each has a unique fate. The first wall (W1) contains callose and sparse xylan-containing hemicelluloses. Wall two (W2) is thin and composed of cellulose crosslinked by xylan-containing hemicelluloses. The third wall (W3) is thick and composed entirely of callose, and the fourth wall (W4) is built of cellulose heavily crosslinked by galactoxyloglucan hemicelluloses. Wall five (W5) is an arabinogalactan protein (AGP)-rich matrix in which the gamete changes shape and multiple flagella elongate. We detected no esterified or unesterified HG pectins in any of thewalls laid down during spermatogenesis.To consider evolutionary modifications in cell walls associated with motile gametes, comparisons are presented with male gametophyte and spermatogenous cell walls across plant groups.

Pattern of callose deposition during the course of meiotic diplospory in Chondrilla juncea (Asteraceae, Cichorioideae)

Total absence of callose in the ovules of diplosporous species has been previously suggested. This paper is the first description of callose events in the ovules of Chondrilla juncea, which exhibits meiotic diplospory of the Taraxacum type. We found the presence of callose in the megasporocyte wall and stated that the pattern of callose deposition is dynamically changing during megasporogenesis. At the premeiotic stage, no callose was observed in the ovules. Callose appeared at the micropylar pole of the cell entering prophase of the first meioticdivision restitution but did not surround the megasporocyte. After the formation of a restitution nucleus, a conspicuous callose micropylar cap and dispersed deposits of callose were detected in the megasporocyte wall. During the formation of a diplodyad, the micropylar callose cap decreased and the walls of a newly formed megaspores showed scattered distribution of callose. Within the older diplodyad, callose was mainly accumulated in the wall between megaspores, as well as in the wall of the micropylar cell; however, a dotted fluorescence of callose was also visible in the wall of the chalazal megaspore. Gradual degradation of callose in the wall of the chalazal cell and intense callose accumulation in the wall of the micropylar cell were related to the selection of the functional megaspore. Thus, our findings may suggest that callose fulfills a similar role both during megasporogenesis in sexual angiosperms and in the course of meiotic diplospory in apomicts and seems to form a regulatory interface between reproductive and somatic cells.

Boron Toxicity Causes Multiple Effects on Malus domestica Pollen Tube Growth.

Boron is an important micronutrient for plants. However, boron is also toxic to cells at high concentrations, although the mechanism of this toxicity is not known. This study aimed to evaluate the effect of boron toxicity on Malus domestica pollen tube growth and its possible regulatory pathway. Our results showed that a high concentration of boron inhibited pollen germination and tube growth and led to the morphological abnormality of pollen tubes. Fluorescent labeling coupled with a scanning ion-selective electrode technique detected that boron toxicity could decrease [Ca2+]c and induce the disappearance of the [Ca2+]c gradient, which are critical for pollen tube polar growth. Actin filaments were therefore altered by boron toxicity. Immuno-localization and fluorescence labeling, together with fourier-transform infrared analysis, suggested that boron toxicity influenced the accumulation and distribution of callose, de-esterified pectins, esterified pectins, and arabinogalactan proteins in pollen tubes. All of the above results provide new insights into the regulatory role of boron in pollen tube development. In summary, boron likely plays a structural and regulatory role in relation to [Ca2+]c, actin cytoskeleton and cell wall components and thus regulates Malus domestica pollen germination and tube polar growth.

Callose localization in the walls of megasporocytes and megaspores in the course of development of monospore embryo sacs

The localization of callose in the cell walls of megasporocytes and megaspores was studied by means of fluorescence microscopy. It was found that the part of the meiocyte cell wall in which callose begins to appear corresponds to the region of active megaspore formation. During the whole megasporogensis more of callose was found in walls of such cells which differentiate afterwards. For different species the pattern of callose distribution in walls during megasporogenesis is a constant specific trait.

Cell Wall Assembly and Intracellular Trafficking in Plant Cells Are Directly Affected by Changes in the Magnitude of Gravitational Acceleration

Plants are able to sense the magnitude and direction of gravity. This capacity is thought to reside in selected cell types within the plant body that are equipped with specialized organelles called statoliths. However, most plant cells do not possess statoliths, yet they respond to changes in gravitational acceleration. To understand the effect of gravity on the metabolism and cellular functioning of non-specialized plant cells, we investigated a rapidly growing plant cell devoid of known statoliths and without gravitropic behavior, the pollen tube. The effects of hyper-gravity and omnidirectional exposure to gravity on intracellular trafficking and on cell wall assembly were assessed in Camellia pollen tubes, a model system with highly reproducible growth behavior in vitro. Using an epi-fluorescence microscope mounted on the Large Diameter Centrifuge at the European Space Agency, we were able to demonstrate that vesicular trafficking is reduced under hyper-gravity conditions. Immuno-cytochemistry confirmed that both in hyper and omnidirectional gravity conditions, the characteristic spatial profiles of cellulose and callose distribution in the pollen tube wall were altered, in accordance with a dose-dependent effect on pollen tube diameter. Our findings suggest that in response to gravity induced stress, the pollen tube responds by modifying cell wall assembly to compensate for the altered mechanical load. The effect was reversible within few minutes demonstrating that the pollen tube is able to quickly adapt to changing stress conditions.

A clearing protocol for whole tissues: An example using haustoria of Orobanchaceae.

• Premise of the study: Due to lack of success in clearing whole tissues using only classical clearing techniques (e.g., Herr’s 4½ solution, KOH, NaOH, lactic acid saturated with chloral hydrate), and because tissue degradation is often a result of harsh clearing agents (e.g., KOH, NaOH), a novel combined treatment was sought to improve the removal of obscuring tannins from intact haustoria.• Methods and Results: Stockwell’s bleach proved to be useful in removing tannins from haustoria, usually within 3 d (up to 10 d), rendering them opaque to (rarely) translucent. After bleaching, haustoria were successfully cleared in 1–3 d in a solution of lactic acid saturated with chloral hydrate at 42°C.• Conclusions: The two-step clearing protocol reported here will now facilitate structural studies on haustoria, such as those examining the presence and distribution of callose, and three-dimensional reconstruction using confocal microscopy. Tissues in this study did not suffer from the degradation in quality observed using harsher treatments. This protocol should be useful for other difficult-to-clear tissues that are unable to be cleared using classical protocols alone.

Zonal responses of sensitive vs. tolerant wheat roots during Al exposure and recovery

Aluminium (Al) irreversibly inhibits root growth in sensitive, but not in some tolerant genotypes. To better understand tolerance mechanisms, seedlings from tolerant (‘Barbela 7/72’ line) and sensitive (‘Anahuac’) Triticum aestivum L. genotypes were exposed to AlCl3 185μM for: (a) 24h followed by 48h without Al (recovery); (b) 72h of continuous exposure. Three root zones were analyzed (meristematic (MZ), elongation (EZ) and hairy (HZ)) for callose deposition, reserves (starch and lipids) accumulation, endodermis differentiation and tissue architecture. Putative Al-induced genotoxic or cytostatic/mytogenic effects were assessed by flow cytometry in root apices.Tolerant plants accumulated less Al, presented less root damage and a less generalized callose distribution than sensitive ones. Starch and lipid reserves remained constant in tolerant roots but drastically decreased in sensitive ones. Al induced different profiles of endodermis differentiation: differentiation was promoted in EZ and HZ, respectively, in sensitive and tolerant genotypes. No ploidy changes or clastogenicity were observed. However, differences in cell cycle blockage profiles were detected, being less severe in tolerant roots. After Al removal, only the ‘Barbela 7/72’ line reversed Al-induced effects to values closer to the control, mostly with respect to callose deposition and cell cycle progression. We demonstrate for the first time that: (a) cell cycle progression is differently regulated by Al-tolerant and Al-sensitive genotypes; (b) Al induces callose deposition >3cm above root apex (in HZ); (c) callose deposition is a transient Al-induced effect in tolerant plants; and (d) in HZ, endodermis differentiation is also stimulated only in tolerant plants, probably functioning in tolerant genotypes as a protective mechanism in addition to callose.

Contrasting reactions of roots of two coniferous tree species to aluminum stress

The formation of cell-wall polysaccharide callose in tree roots has been suggested to detect aluminum (Al) stress at an early stage in forest trees. The objective of this study was to clarify whether the Japanese plantation tree species Cryptomeria japonica, which grows in acid soils, and Pinus thunbergii, distributed in coastal regions, have sensitive or tolerant root reactions against Al stress. We examined the accumulation and distribution of callose and Al, and the exudation of organic acid anion (OA) of the roots of C. japonica seedlings, and compared them with those of P. thunbergii after one day exposure to Al in hydroponic systems. Callose in the root apices of P. thunbergii was significantly induced at 0.5 mM Al or higher. However, no induction of callose was recorded in C. japonica supplied with 1.0 mM Al. Experiments with the callose elicitor digitonin confirmed that both species could have a capacity of callose induction. Aluminum was found to accumulate mainly in the epidermal cell walls of C. japonica. In contrast, in P. thunbergii, Al penetrated into more inner root cells, such as cortical cell walls and lumen, and callose deposition was also evident in these compartments. Exudations of OA, such as oxalate, malate and citrate, were observed in both tree species after the treatment of Al, but the exudation patterns of the species differed. We conclude that roots of C. japonica with lower callose productions are tolerant to Al due to not allowing Al to penetrate readily into its inner cells of the roots, partly via OA exudation, but the detailed mechanism still remains unclear.

Is callose a barrier for lead ions entering Lemna minor L. root cells?

Plants have developed a range of strategies for resisting environmental stresses. One of the most common is the synthesis and deposition of callose, which functions as a barrier against stress factor penetration. The aim of our study was to examine whether callose forms an efficient barrier against Pb penetration in the roots of Lemna minor L. exposed to this metal. The obtained results showed that Pb induced callose synthesis in L. minor roots, but it was not deposited regularly in all tissues and cells. Callose occurred mainly in the protoderm and in the centre of the root tip (procambial central cylinder). Moreover, continuous callose bands, which could form an efficient barrier for Pb penetration, were formed only in the newly formed and anticlinal cell walls (CWs); while in other CWs, callose formed only small clusters or incomplete bands. Such an arrangement of callose within root CWs inefficiently protected the protoplast from Pb penetration. As a result, Pb was commonly present inside the root cells. In the light of the results, the barrier role of callose against metal ion penetration appears to be less obvious than previously believed. It was indicated that induction of callose synthesis is not enough for a successful blockade of the stress factor penetration. Furthermore, it would appear that the pattern of callose distribution has an important role in this defence strategy.

Russian wheat aphid biotype RWASA2 causes more vascular disruption than RWASA1 on resistant barley lines

We investigated the comparative effects of the feeding damage caused by two Russian wheat aphid (RWA, Diuraphis noxia Kurdjumov) biotypes, RWASA1 and RWASA2, on leaves of three RWA-resistant barley ( Hordeum vulgare L.) lines from the USDA-ARS, and used a South African non-resistant cultivar as control. The relationship between aphid breeding capacity and the structural damage inflicted by the aphids was studied, using wide-field fluorescence and transmission electron microscopy (TEM). Colonies of the two biotypes grew rapidly on all four barley lines during a 10 day feeding exposure but as expected, population size and density were generally lower on the resistant lines than on the non-resistant cultivar. The new South African biotype, RWASA2, bred significantly faster than the original RWASA1 biotype. The feeding and water uptake-related damage sustained by phloem and xylem tissues of the resistant lines suggest that RWASA2 was a more aggressive feeder and caused substantially more cell damage than RWASA1. Examination of wound callose distribution after aphid feeding revealed that high levels of wound callose occurred in non-resistant and in resistant lines. Reduction in aphid population size, as well as ultrastructural damage during feeding by RWA biotypes on resistant lines, signals potential antibiotic and tolerant responses of the barley lines to aphid feeding. We infer from callose distribution and ultrastructural studies, that phloem transport would be substantially reduced in the non-resistant PUMA and to a lesser extent in the resistant STARS lines, which suggests that the STARS lines may be a potential source of RWASA1 and RWASA2-resistance.

Dynamics of callose deposition and beta-1,3-glucanase expression during reproductive events in sexual and apomictic Hieracium.

Callose accumulates in the walls of cells undergoing megasporogenesis during embryo sac formation in angiosperm ovules. Deficiencies in callose deposition have been observed in apomictic plants and causal linkages between altered callose deposition and apomictic initiation proposed. In apomictic Hieracium, embryo sacs initiate by sexual and apomictic processes within an ovule, but sexual development terminates in successful apomicts. Callose deposition and the events that lead to sexual termination were examined in different Hieracium apomicts that form initials pre- and post-meiosis. In apomictic plants, callose was not detected in initial cell walls and deficiencies in callose deposition were not observed in cells undergoing megasporogenesis. Multiple initial formation pre-meiosis resulted in physical distortion of cells undergoing megasporogenesis, persistence of callose and termination of the sexual pathway. In apomictic plants, callose persistence did not correlate with altered spatial or temporal expression of a beta-1,3-glucanase gene (HpGluc) encoding a putative callose-degrading enzyme. Expression analysis indicated HpGluc might function during ovule growth and embryo sac expansion in addition to callose dissolution in sexual and apomictic plants. Initial formation pre-meiosis might therefore limit the access of HpGluc protein to callose substrate while the expansion of aposporous embryo sacs is promoted. Callose deposition and dissolution during megasporogenesis were unaffected when initials formed post-meiosis, indicating other events cause sexual termination. Apomixis in Hieracium is not caused by changes in callose distribution but by events that lead to initial cell formation. The timing of initial formation can in turn influence callose dissolution.

The mac1 mutation alters the developmental fate of the hypodermal cells and their cellular progeny in the maize anther.

In angiosperm ovules and anthers, the hypodermal cell layer provides the progenitors of meiocytes. We have previously reported that the multiple archesporial cells1 (mac1) mutation identifies a gene that plays an important role in the switch of the hypodermal cells from the vegetative pathway to the meiotic (sporogenous) pathway in maize ovules. Here we report that the mac1 mutation alters the developmental fate of the hypodermal cells of the maize anther. In a normal anther a hypodermal cell divides periclinally with the inner cell giving rise to the sporogenous archesporial cells while the outer cell, together with adjacent cells, forms the primary parietal layer. The cells of the parietal layer then undergo two cycles of periclinal divisions to give rise to three wall layers. In mac1 anthers the primary parietal layer usually fails to divide periclinally so that the three wall layers do not form, while the archesporial cells divide excessively and most fail to form microsporocytes. The centrally located mutant microsporocytes are abnormal in appearance and in callose distribution and they fail to proceed through meiosis. These failures in development and function appear to reflect the failure of mac1 gene function in the hypodermal cells and their cellular progeny.