Cytology of beet yellows virus infection inTetragonia

Summary This paper is the second in the series dealing with the ultrastructure ofTetragonia expansa Murr. infected with the beet yellows virus. It considers the relation of the virus to the conducting cells in the phloem and the xylem. Virus particles occurred in mature sieve elements, their amount increasing as the infected leaf became older. In older leaves some sieve elements were completely blocked with virus. Virus particles were seen in pores of sieve plates, in plasmodesmata interconnecting sieve elements and parenchyma cells, and in those between parenchyma cells. Mature and immature tracheary elements also contained virus particles. Presence of inclusions composed of vesicles and virus in some immature tracheary elements may indicate that virus multiplies in these cells. No vesicles and no virus particles were discovered in immature sieve elements.This work was supported in part by National Science Foundation grant GB-5506.     

Specification of Primary Pigment Cell and Outer Photoreceptor Fates byBarH1Homeobox Gene in the DevelopingDrosophilaEye

In the developingDrosophilaeye,BarH1andBarH2, paired homeobox genes expressed in R1/R6 outer photoreceptors and primary pigment cells, are essential for normal eye morphogenesis. Here, we show evidence thatBarH1ectopically expressed under the control of thesevenlessenhancer (sev-BarH1) causes two types of cone cell transformation: transformation of anterior/posterior cone cells into outer photoreceptors and transformation of equatorial/polar cone cells into primary pigment cells.sev-BarH1repressed the endogenous expression of theroughhomeobox gene in R3/R4 photoreceptors, while theBarH2homeobox gene was activated bysev-BarH1in an appreciable fraction of extra outer photoreceptors. In primary pigment cells generated by cone cell transformation, the expression ofcut,a homeobox gene specific to cone cells, was completely replaced with that ofBarhomeobox genes. Extra outer photoreceptor formation was suppressed and enhanced, respectively, by reducing the activity of Ras/MAPK signaling and by dosage reduction ofyan,a negative regulator of the pathway, suggesting interactions betweenBarhomeobox genes (cell fate determinants) and Ras/MAPK signaling in eye development.     

Spatially regulated genes expressed during seed germination and postgerminative development are activated during embryogeny

Summary We investigated the control of genes expressed primarily during seed germination and postgerminative development in Brassica napus L. We identified cloned mRNA sequences which became prevalent within 1 day after the start of imbibition and were at low or undetectable levels in immature embryos, dry seeds, and leaves. Most postgermination-abundant mRNAs accumulated primarily, though not exclusively, in different parts of the seedling. Of the 14 cloned mRNAs, 8 were prevalent in cotyledons, 2 were abundant in seedling axes, and 4 were approximately equally distributed in both parts. We showed that although these mRNAs reached maximal levels in seedlings, the spatially regulated mRNAs were also detected at distinct embryonic stages; mRNAs prevalent in seedling axes accumulated primarily during early embryogenesis while cotyledon-abundant mRNA concentration increased during late embryogeny. We conclude that the temporal and spatial regulation of gene expression in seedlings reflects similarities and differences in the physiological functions of cotyledons and axes. Furthermore, the regulated expression of cotyledon-abundant genes during late embryogeny suggests that the mRNAs and possibly proteins may accumulate in preparation for subsequent seedling growth. Similarities in the accumulation of cotyledon-abundant mRNAs may indicate coordinate regulation of this gene set.Abbreviations DAF days after flowering - DAI days after the start of imbibition - HAI hours after the start of imbibition - kb kilobase(pairs)     

Role of auxin and sucrose in the differentiation of sieve and tracheary elements in plant tissue cultures

The differentiation of sieve and tracheary elements was studied in callus culture of Daucus carota L., Syringa vulgaris L., Glycine max (L.) Merr., Helianthus annuus L., Hibiscus cannabinus L. and Pisum sativum L. By the lacmoid clearing technique it was found that development of the phloem commenced before that of the xylem. In not one of the calluses was differentiation of tracheary elements observed in the absence of sieve elements. The influence of indole-3-acetic acid (IAA) and sucrose was evaluated quantitatively in callus of Syringa, Daucus and Glycine. Low IAA levels resulted in the differentiation of sieve elements with no tracheary cells. High levels resulted in that of both phloem and xylem. IAA thus controlled the number of sieve and tracheary elements, increase in auxin concentration boosting the number of both cell types. Changes in sucrose concentration, while the IAA concentration was kept constant, did not have a specific effect on either sieve element differentiation, or on the ratio between phloem and xylem. Sucrose did, however, affect the quantity of callose deposited on the sieve plates, because increase in the sucrose concentration resulted in an increase in the amount of callose. It is proposed that phloem is formed in response to auxin, while xylem is formed in response to auxin together with some added factor which reaches it from the phloem.     

Comparison of different techniques for gene transfer into mature and immature tobacco pollen

The particle gun, cocultivation withAgrobacterium tumefaciens, and imbibition in DNA solutions were compared as methods to transfer DNA into mature and immature pollen ofNicotiana tabacum. Bombardment of mature pollen with the β-glucuronidase gene cloned behind the pollen-specific PA2 promoter of the chalcone isomerase gene ofPetunia hybrida resulted in the expression of the β-glucuronidase gene in 0.025% of the pollen grains. Bombardment of younger stages followed byin vitro maturation also resulted in the formation of mature pollen that expressed β-glucuronidase, although at a lower frequency. Cocultivation of pollen duringin vitro maturation orin vitro germination withAgrobacterium tumefaciens did not yeild β-glucuronidase-expressing pollen. In these cases, an intron-containing β-glucuronidase gene was used which effectively prevented β-glucuronidase expression in the bacteria. Imbibition of mature, dry pollen in various DNA solutions of the same constructs also did not lead to the formation of β-glucuronidase expressing pollen.     

Wood anatomy ofTrimeniaceae

Four collections of three species ofTrimenia and one collection ofPiptocalyx were studied; early-formed and later-formed wood was analyzed for oneTrimenia. Liquid-preserved material permitted analysis of mucilage and starch storage in wood ofT. neocaledonica andP. moorei. BecausePiptocalyx is scandent whereasTrimenia is arborescent, wood differences relative to evolution of a climbing habit could be examined.Piptocalyx contrasts withTrimenia in having wider vessels, more numerous per mm², resulting in a conductive area five times greater per unit area than that of theTrimenia woods averaged.Piptocalyx has appreciably fewer bars per perforation plate and thus much greater conductive area per perforation plate than have the species ofTrimenia. Rays inPiptocalyx are much taller and wider than those ofTrimenia. Wood ofTrimeniaceae is highly primitive in its scalariform perforation plates, scalariform lateral wall pitting on vessels, relatively long vessels elements, and heterocellular rays. Imperforate tracheary elements are septate nucleate fibertracheids (or even libriform fibers) rather than tracheids, but loss of borders on pits (and thus lowered conductive function of the imperforate tracheary elements) can be explained by the development of these elements into starchstoring cells. Some fiber-tracheids inT. neocaledonica are enlarged mucilagecontaining cells. Details of vessel structure inTrimeniaceae are similar to those ofMonimiaceae (s. s.), but similarity to some other lauralean (annonalean) families may be found: in mucilage presence,Trimeniaceae resembleLauraceae rather thanMonimiaceae. Wood ofTrimeniaceae may be regarded as highly mesomorphic, corresponding to the moist habitats in which all of the species occur.     

Composition and fine structure of minor veins inTetragonia leaf

Summary Mesophyll containing the minor veins from leaves ofTetragonia expansa Murr. was examined in preparation for a study of effects of beet yellows virus on the leaf tissues of this plant. The sieve elements throughout the minor veins exhibit the characteristics commonly found in this type of cell in dicotyledons. The cells are connected with one another by sieve plates and with contiguous parenchyma cells by branched plasmodesmata. Mature sieve elements are enucleate and lack ribosomes. No tonoplast was discerned in these cells. Mitochondria, plastids, and sparse endoplasmic reticulum are retained in mature cells. The plastids, which contain a massive fibrous ring of proteinaceous material, resemble the sieve element plastids ofBeta. The P-protein in the sieve elements is occasionally composed of tubules; more commonly it is represented by loose helices. The tracheary elements have scalariform secondary thickenings. In regions free of these thickenings, the primary wall largely disintegrates; only some loosely arranged fibrils remain. The mesophyll and vascular parenchyma cells contain the various organelles characteristic of living plant cells but vary in degree of vacuolation and in density of cytoplasm. Some vascular parenchyma cells have particularly dense protoplasts. They contain numerous ribosomes and their background matrix consists of a dense population of fine fibrils. Occasional vascular parenchyma cells contain the tubular spiny cell component first recognized inBeta. This work was supported in part by National Science Foundation grant GB-5506.     

Isolation and Activity Analysis of a Seed-Abundant soyAP1 Gene Promoter from Soybean

The soybean aspartic proteinase gene soyAP1 has previously been shown to be expressed specifically in soybean seeds. To investigate the expression pattern and active cis-elements of the soyAP1 promoter, the 1,650-bp 5??-upstream genomic DNA fragment named PS-552 was isolated by PCR walking. Sequence analysis revealed that this fragment contains a series of motifs related to seed-specific promoters and some pollen-expressed elements. Stable expression in transgenic Arabidopsis thaliana showed that the PS-552 promoter can regulate beta-glucuronidase gene accumulation in mature seeds at much higher levels than other tissues, especially vegetative tissues, and exhibits similar activity to the 35S promoter in mature seeds. These results show that the PS-552 promoter is a highly active promoter controlling downstream gene expression, mainly in mature seeds. The 5??-end deletion studies of PS-552 showed that the cis-elements of CAAACAC, AACA, E-box, and CCAA play a role in increasing the seed-specific activity. The proportion of mature seed activity and flower activity was increased as the deletion fragment lengthened, indicating that seed cis-elements possibly lessen or suppress the effect of pollen-expressed elements, increasing the activity of PS-552 in mature seeds.     

Cloning of HumanENC-1and Evaluation of Its Expression and Regulation in Nervous System Tumors

We recently identified and characterized a novel murine gene,ENC-1,that is expressed primarily in the nervous system and encodes an actin-binding protein. To gain insight into a potential role forENC-1gene in the processes of cell differentiation and malignant transformation in the human nervous system, we first cloned and characterized the human homologue ofENC-1.The humanENC-1gene appeared to be highly expressed in adult brain and spinal cord, and in a number of cell lines derived from nervous system tumors we detected low steady-state levels ofENC-1mRNA. We used a neuroblastoma differentiation model, the retinoic acid-induced neuronal differentiation of SMS-KCNR cells, to study the regulation of theENC-1gene during neural crest cell differentiation. We found that the expression ofENC-1increased dramatically in the differentiated SMS-KCNR cells as compared to control undifferentiated cells. These results suggest thatENC-1expression plays a role during differentiation of neural crest cells and may be down regulated in neuroblastoma tumors.     

Nuclear replication activities during imbibition of abscisic acid- and gibberellin-deficient tomato (Lycopersicon esculentum Mill.) seeds

The role of cis-abscisic acid (ABA) and gibberellins (GAs) in the induction of cell-cycle activities has been studied during imbibition and subsequent germination of tomato seeds. Using flow cytometry, nuclear replication activity was investigated in embryo root tips isolated from seeds of the ABA-deficient mutant sit ^w , the GA-deficient mutant gib-1, and the wild-type (MM) tomato (Lycopersicon esculentum Mill. cv. Moneymaker) upon imbibition in water, 10 μM GA₄₊₇, 5 μM ABA or 5 μM ABA+10 μM GA₄₊₇. The nuclei of fully matured dry MM, sit ^w and gib-1 seeds predominantly showed 2C DNA signals, indicating that the cell-cycle activity of most root-tip cells had been arrested at the G₁ phase of nuclear division. However, ABA-deficient sit ^w seeds contained a significantly higher amount of G₂ cells (4C DNA) compared with the other genotypes, suggesting that, during maturation, cell-cycle activity in sit ^w seeds is less efficiently arrested in G₁. Upon imbibition in water, an induction of the 4C signal, indicating nuclear replication, was observed in the root tip cells of both MM and sit ^w embroys. The augmentation in the 4C signal occurred before visible germination. Gib-1 seeds did not show cell-cycle activity and did not germinate in water. Upon imbibition in GA₄₊₇, both cell-cycle activity and subsequent germination were enhanced in MM and sit ^w seeds, and were induced in gib-1. In ABA, the germination of MM and sit ^w seeds was inhibited while nuclear replication of these seeds was not affected. It is concluded that GA influences germination by acting upon processes that precede cell-cycle activation, while ABA affects growth by acting upon processes that follow cell-cycle activation.     

Expression of the phloem lectin is developmentally linked to vascular differentiation in cucurbits

The conducting elements of phloem in angiosperms are a complex of two cell types, sieve elements and companion cells, that form a single developmental and functional unit. During ontogeny of the sieve element/companion cell complex, specific proteins accumulate forming unique structures within sieve elements. Synthesis of these proteins coincides with vascular development and was studied in Cucurbita seedlings by following accumulation of the phloem lectin (PP2) and its mRNA by RNA blot analysis, enzyme-linked immunosorbent assay, immunocytochemistry and in␣situ hybridization. Genes encoding PP2 were developmentally regulated during vascular differentiation in hypocotyls of Cucurbita maxima Duch. Accumulation of PP2 mRNA and protein paralleled one another during hypocotyl elongation, after which mRNA levels decreased, while the protein appeared to be stable. Both PP2 and its mRNA were initially detected during metaphloem differentiation. However, PP2 mRNA was detected in companion cells of both bundle and extrafascicular phloem, but never in differentiating sieve elements. At later stages of development, PP2 mRNA was most often observed in extrafascicular phloem. In developing stems of Cucurbita moschata L., PP2 was immunolocalized in companion cells but not to filamentous phloem protein (P-protein) bodies that characterize immature sieve elements of bundle phloem. In contrast, PP2 was immunolocalized to persistent ␣ P-protein bodies in sieve elements of the extrafascicular phloem. Immunolocalization of PP2 in mature wound sieve elements was similar to that in bundle phloem. It appears that PP2 is synthesized in companion cells, then transported into differentiated sieve elements where it is a component of P-protein filaments in bundle phloem and persistent P-protein bodies in extrafascicular phloem. This differential accumulation in bundle and extrafascicular elements may result from different functional roles of the two types of phloem. Received: 31 July 1996 / Accepted: 27 August 1996     

Phytepsin, a barley vacuolar aspartic proteinase, is highly expressed during autolysis of developing tracheary elements and sieve cells

Vacuolarisation, formation of autophagocytotic vacuoles and tonoplast disruption have been reported in plant cells undergoing developmentally regulated programmed cell death (PCD), but little is known about the vacuolar proteins involved. In HeLa cells, cathepsin D, a lysosomal aspartic proteinase has been shown to mediate PCD. Based on immunohistochemical staining of barley roots, we show here that the previously well characterised barley vacuolar aspartic proteinase (phytepsin), a plant homologue to cathepsin D, is highly expressed both during formation of tracheary elements and during partial autolysis of sieve cells. In serial transverse sections of the vascular cylinder, starting from the root tip, phytepsin is expressed in root cap cells, in the tracheary elements of early and late metaxylem, and in the sieve cells of the protophloem and metaphloem. Aleurain, a barley vacuolar cysteine proteinase, is expressed similarly in root cap cells but differently in the tracheary elements of protoxylem and early metaxylem. This is the first evidence that a vacuolar aspartic proteinase, in analogy to cathepsin D in animals, may play a role in the active autolysis of plant cells.     

<Emphasis Type="Italic">In Vitro</Emphasis> and cellular responses of jack pine embryos to three imbibition parameters

Summary Eighteen imbibition treatments with differing parameters of light conditions, temperature and duration were applied to jack pine seeds. After imbibition, embryos were excised and cultured in a liquid medium for 4 wk under continous agitation. At the end of the culture period, the embryos were classified according to five categories: nodule-forming, callus-forming, nodule plus callus-forming, white (non-responsive), and necrotic. Our results showed variation in the in vitro responsiveness of the embryos for the three parameters of imbibition and interactions among them. Three statistical models were tested to analyze the data on nodule formation, and two of them showed that the interaction between time and temperature, and between time and the combined variable temperature/light, were significant, while the light and duration variables had a significant effect only as single factors. The different morphogenic responses observed might indicate specific metabolic requirements of the embryos for the different developmental pathways. Afterwards, nine imbibition treatments were selected to evaluate the effects of the three parameters on the cell cycle and absolute DNA amount per nucleus. Following imbibition the number of cells in the G0–G1 phase decreased compared to the cells in dry seed, while the number of cells increased in the G2 phase after all the treatments except one. The percentage of cells in the different cell cycle phases varied significantly among the treatments. DNA amount fluctuated from 35.5 to 40.37 pg per nucleus. Compared to dry seeds with 40.19 pg DNA per nucleus, embryos from four imbibition treatments showed a pronounced decrease in the DNA by 5.9–11.8%. This might indicate underreplication of DNA sequences and reflect DNA plasticity with regards to imbibition and environmental factors.     

Molecular cloning,functional expression and characterization of two serine/threonine-specific protein kinases from<Emphasis Type="Italic"> Nicotiana tabacum</Emphasis> pollen

The existence of different kinds of kinases in pollen and pollen tubes suggests that kinase-mediated signaling pathways are likely involved in regulating pollen germination and pollen tube growth during the life cycle of higher plants. We have used RT-PCR and RACE to isolate full-length cDNAs for two pollen-expressed kinases, named NtPK1 and NtPK2, of Nicotiana tabacum. NtPK1 and NtPK2 encode proteins of 365 and 369 amino acids with calculated molecular masses of 39.2 kDa and 39.5 kDa, respectively, and both proteins possess the 12 sub-domains that are conserved among protein kinases. The nucleotide and deduced amino acid sequences of NtPK1 and NtPK2 share 88% and 91% identity, respectively, with the C-terminal region being the most conserved. RT-PCR analysis revealed that NtPK1 was specifically expressed in pollen and pollen tubes, and that NtPK2 was also expressed in pistil and petal. Immunoblot analysis using anti-NtPK1 and anti-NtPK2 antibodies confirmed that both NtPK1 and NtPK2 were produced in pollen and pollen tubes, and that NtPK2 was also produced in developing male gametophytes and other floral tissues. Biochemical fractionation experiments showed that, in all the tissues examined, NtPK1 and NtPK2 were present in the cytosolic fraction and not in the microsomal fraction. NtPK1 and NtPK2 were found to autophosphorylate on threonine and, for NtPK2, on serine as well. All the results taken together suggest that NtPK1 and NtPK2 are novel receptor-like cytosolic serine/threonine kinases, and could mediate signaling pathways required for pollen germination and/or pollen tube growth.The nucleotide sequence data of NtPK1 and NtPK2 reported in this paper will appear in the EMBL/GenBank/DDBJ nucleotide sequence databases under the accession numbers AJ608156 and AJ608157, respectively     

Flow Cytometric Determination of Nuclear Replication Stages in Tomato Seeds during Priming and Germination

Flow cytometric determination of DNA levels in embryos of fullymatured dry tomato (Lycopersicon esculenium) seeds revealedlarge amounts of 2C DNA signals, indicating that most cellshad arrested in the cell cycle at the presynthetic G₁ phaseof nuclear division. After imbibition in water, an augmentationof the 4C signal in the embryonic root tip region was found.This increase could be ascribed to cells entering the syntheticphase of nuclear division leading towards the doubling of chromosomalmaterial. In the root tip cells, 4C:2C ratios increased I dafter imbibition in water though radicle emergence started 2d later. Apparently, DNA synthesis preceded germination. Onlya small increase in the number of cells with 4C DNA levels wasfound in the rest of the embryonic tissues. In whole dry seeds,DNA histograms revealed both a 2C signal and a considerable6C peak, the latter originating from the endoreduplicated endosperm. A priming period of 14 d in PEG-6000 considerably enhanced therate and uniformity of germination. In the ungerminated seeds,the 4C DNA signal of root tip cells started to increase after3 d incubation in PEG. The ratio of 4C:2C steadily increasedduring the 14 d priming period, though did not reach the levelobtained after hydration in water. Upon priming, the 4C:2C ratiowas constant after redrying the seeds towards the original moisturecontent, indicating that the chromosomal material in the rootcells had stably ceased cell cycle activity at the G² phase.The present results indicate that the beneficial effects ofpriming on seedling performance are associated with the actionof replicative DNA synthetic processes prior to germination. Lycopersicon esculeniumMill, tomato, DNA content, flow cytometry, priming, seed, nuclear replication stage, C levels     

Tip growth in xylogenic suspension cultures ofZinnia elegans L.: Implications for the relationship between cell shape and secondary-cell-wall pattern in tracheary elements

Summary The relationship between cell expansion, cortical microtubule orientation, and patterned secondary-cell-wall deposition was investigated in xylogenic cell suspension cultures ofZinnia elegans L. The direction of cell expansion in these cultures is pH dependent; cells elongate at pH 5.5–6.0, but expand isodiametrically at pH 6.5–7.0. Contrary to our expectations, indirect immunofluorescence revealed that cortical microtubules are oriented parallel to the long axis in elongating cells. Pulse labeling of the walls of isolated cells with the fluorochrome Tinopal LPW demonstrated that xylogenic Zinnia mesophyll cells elongate by tip growth in culture. These results confirm that cortical microtubules in developing tracheary elements reorient before bundling to form transverse cortical microtubule bands. This rearrangement may allow the secondary cell wall pattern to conform to cell shape, independent of the direction in which the cell was expanding prior to reorientation.Abbreviations CMT cortical microtubules - Mes 2-[N-morpholino]ethanesulfonic acid - TE tracheary element     

Le phloème deSelaginella willdenowii: histophysiologie comparée

Metaphloem sieve elements ofSelaginella willdenowii are elongated cells with slightly oblique or transverse end walls. Pores are seen on both lateral and end walls, although they are more numerous on the latter. Parenchyma cells exhibiting strong enzyme activities (acid phosphatase, non specific esterase, succinate dehydrogenase, cytochrome oxidase, peroxidase) are present between sieve elements and tracheids in each vascular bundle. A functional association thus appears to exist between these parenchyma cells and the conducting elements.—The occurrence of transverse to slightly oblique end walls in sieve elements seems to characterize the ligulate Lycopsids (as opposed to the aligulateLycopodium where sieve elements possess slanting, very oblique, end walls).

     

Light induction of the clock-controlled geneccg-1 is not transduced through the circadian clock inNeurospora crassa

Ambient light and the circadian clock have been shown to be capable of acting either independently or in an interrelated fashion to regulate the expression of conidiation in the ascomycete fungusNeurospora crassa. Recently several molecular correlates of the circadian clock have been identified in the form of the morning-specific clock-controlled genesccg-1 andccg-2. In this paper we report studies on the regulation ofccg-1, an abundantly expressed gene displaying complex regulation. Consistent with an emerging consensus for clock-controlled genes and conidiation genes inNeurospora, we report thatccg-1 expression is induced by light, and show that this induction is independent of the direct effects of light on the circadian clock. Although circadian regulation of the gene is lost in strains lacking a functional clock, expression ofccg-1 is still not constitutive, but rather fluctuates in concert with changes in developmental potential seen in such strains. Light induction ofccg-1 requires the products of theNeurospora wc-1 andwc-2 genes, but surprisingly the requirement forwc-2 is suppressed in conditional mutants ofcot-1, a gene that encodes a cAMP-dependent protein kinase. These data provide insight into a complex regulatory web, involving at least circadian clock control, light control, metabolic control, and very probably developmental regulation, that governs the expression ofccg-1.     

Constitutive expression of two endochitinases from root nodules ofElaeagnus umbellata confers resistance on transgenicArabidopsis plants against the fungal pathogenBotrytis cinerea

Plant chitinases have been known as pathogenesis-related (PR) proteins, but recent studies suggest that they play functional roles during normal plant growth and development. We previously isolated two cDNA clones encoding endochitinases,EuNOD-CHT1 and -CHT2, from the root nodules ofElaeagnus umbellata. These genes show differential expression patterns, with theEuNOD-CHT1 gene being active in the root nodules and meristems, whileEuNOD-CHT2 is preferentially expressed in the infected cells of those nodules. To elucidate the functional roles of these two endochitinases, we have now constitutively expressed each gene in a heterologous plant system,Arabidopsis thaliana. Stable inheritance and expression of the transgenes were confirmed by genomic Southern hybridization and RT-PCR. Our transgenic plants did not differ morphologically from the wild types. However, constitutive expression ofEuNOD-CHT1 and -CHT2 inArabidopsis resulted in increased resistance against a fungal pathogen,Botrytis cinerea, but not against a bacterial agent,Pseudomonas syringae pv. Tomato DC3000. Expression levels were enhanced by both wounding and jasmonic acid treatments (forEuNOD-CHT1), or by jasmonic acid only (forEuNOD-CHT2). These data suggest thatEuNOD-CHT1 and -CHT2 primarily play defensive roles during root nodule development inE. umbellata.