Sieve Element Differentiation Research Articles

SYNAPTOTAGMIN 4 is expressed mainly in the phloem and participates in abiotic stress tolerance in Arabidopsis.

Plant synaptotagmins structurally resemble animal synaptotagmins and extended-synaptotagmins. Animal synaptotagmins are well-characterized calcium sensors in membrane trafficking, and extended-synaptotagmins mediate lipid transfer at the endoplasmic reticulum-plasma membrane contact sites. Here, we characterize SYNAPTOTAGMIN 4 (SYT4), which belongs to the six-member family in Arabidopsis. Fluorometric GUS assay showed that the SYT4 promoter was strongest in roots and the least active in rosettes and cauline leaves, which was confirmed by qPCR. In seedlings, promoter activity was influenced by several factors, such as plant growth regulators, mannitol, sucrose, polyethylene glycol and cold. GUS histochemistry revealed SYT4 promoter activity in the phloem of all organs and even almost exclusively in sieve element precursors and differentiating sieve elements. Accordingly, the SYT-GFP fusion protein also accumulated in these cells with maximal abundance in sieve element precursors. The protein formed a network in the cytoplasm, but during sieve tube differentiation, it deposited at the cell periphery and disappeared from mature tubes. Using photoconvertible fluorescence technology, we showed that a high abundance of SYT4 protein in meristematic protophloem cells was due to its extensive synthesis. SYT4 protein synthesis was interrupted in differentiating sieve elements, but protein degradation was also reduced. In addition to phloem, the fusion protein was detected in shoot and root stem cell niche as early as the late heart stage of the embryo. We isolated and molecularly and biologically characterized five syt4 T-DNA insertion alleles and subjected them to phenotype analysis. The allele with the C2B domain interrupted by an T-DNA insertion exhibits increased sensitivity to factors such as auxins, osmotics, salicylic acid, sodium chloride, and the absence of sucrose in the root growth test.

Phloem development.

The evolution of the plant vascular system is a key process in Earth history because it enabled plants to conquer land and transform the terrestrial surface. Among the vascular tissues, the phloem is particularly intriguing because of its complex functionality. In angiosperms, its principal components are the sieve elements, which transport phloem sap, and their neighboring companion cells. Together, they form a functional unit that sustains sap loading, transport, and unloading. The developmental trajectory of sieve elements is unique among plant cell types because it entails selective organelle degradation including enucleation. Meticulous analyses of primary, so-called protophloem in the Arabidopsis thaliana root meristem have revealed key steps in protophloem sieve element formation at single-cell resolution. A transcription factor cascade connects specification with differentiation and also orchestrates phloem pole patterning via noncell-autonomous action of sieve element-derived effectors. Reminiscent of vascular tissue patterning in secondary growth, these involve receptor kinase pathways, whose antagonists guide the progression of sieve element differentiation. Receptor kinase pathways may also safeguard phloem formation by maintaining the developmental plasticity of neighboring cell files. Our current understanding of protophloem development in the A. thaliana root has reached sufficient detail to instruct molecular-level investigation of phloem formation in other organs.

Putative pectate lyase PLL12 and callose deposition through polar CALS7 are necessary for long-distance phloem transport in Arabidopsis

In plants, the phloem distributes photosynthetic products for metabolism and storage over long distances. It relies on specialized cells, the sieve elements, which are enucleated and interconnected through large so-called sieve pores in their adjoining cell walls. Reverse genetics identified PECTATE LYASE-LIKE 12 (PLL12) as critical for plant growth and development. Using genetic complementations, we established that PLL12 is required exclusively late during sieve element differentiation. Structural homology modeling, enzyme inactivation, and overexpression suggest a vital role for PLL12 in sieve-element-specific pectin remodeling. While short distance symplastic diffusion is unaffected, the pll12 mutant is unable to accommodate sustained plant development due to an incapacity to accommodate increasing hydraulic demands on phloem long-distance transport as the plant grows-a defect that is aggravated when combined with another sieve-element-specific mutant callose synthase 7 (cals7). Establishing CALS7 as a specific sieve pore marker, we investigated the subcellular dynamics of callose deposition in the developing sieve plate. Using fluorescent CALS7 then allowed identifying structural defects in pll12 sieve pores that are moderate at the cellular level but become physiologically relevant due to the serial arrangement of sieve elements in the sieve tube. Overall, pectin degradation through PLL12 appears subtle in quantitative terms. We therefore speculate that PLL12 may act as a regulator to locally remove homogalacturonan, thus potentially enabling further extracellular enzymes to access and modify the cell wall during sieve pore maturation.

Local auxin competition explains fragmented differentiation patterns

Trajectories of cellular ontogeny are tightly controlled and often involve feedback-regulated molecular antagonism. For example, sieve element differentiation along developing protophloem cell files of Arabidopsis roots requires two antagonistic regulators of auxin efflux. Paradoxically, loss-of-function in either regulator triggers similar, seemingly stochastic differentiation failures of individual sieve element precursors. Here we show that these patterning defects are distinct and non-random. They can be explained by auxin-dependent bistability that emerges from competition for auxin between neighboring cells. This bistability depends on the presence of an auxin influx facilitator, and can be triggered by either flux enhancement or repression. Our results uncover a hitherto overlooked aspect of auxin uptake, and highlight the contributions of local auxin influx, efflux and biosynthesis to protophloem formation. Moreover, the combined experimental-modeling approach suggests that without auxin efflux homeostasis, auxin influx interferes with coordinated differentiation.

Local and Systemic Effects of Brassinosteroid Perception in Developing Phloem.

The plant vasculature is an essential adaptation to terrestrial growth. Its phloem component permits efficient transfer of photosynthates between source and sink organs but also transports signals that systemically coordinate physiology and development. Here, we provide evidence that developing phloem orchestrates cellular behavior of adjacent tissues in the growth apices of plants, the meristems. Arabidopsis thaliana plants that lack the three receptor kinases BRASSINOSTEROID INSENSITIVE 1 (BRI1), BRI1-LIKE 1 (BRL1), and BRL3 ("bri3" mutants) can no longer sense brassinosteroid phytohormones and display severe dwarfism as well as patterning and differentiation defects, including disturbed phloem development. We found that, despite the ubiquitous expression of brassinosteroid receptors in growing plant tissues, exclusive expression of the BRI1 receptor in developing phloem is sufficient to systemically correct cellular growth and patterning defects that underlie the bri3 phenotype. Although this effect is brassinosteroid-dependent, it cannot be reproduced with dominant versions of known downstream effectors of BRI1 signaling and therefore possibly involves a non-canonical signaling output. Interestingly, the rescue of bri3 by phloem-specific BRI1 expression is associated with antagonism toward phloem-specific CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 45 (CLE45) peptide signaling in roots. Hyperactive CLE45 signaling causes phloem sieve element differentiation defects, and consistently, knockout of CLE45 perception in bri3 background restores proper phloem development. However, bri3 dwarfism is retained in such lines. Our results thus reveal local and systemic effects of brassinosteroid perception in the phloem: whereas it locally antagonizes CLE45 signaling to permit phloem differentiation, it systemically instructs plant organ formation via a phloem-derived, non-cell-autonomous signal.

Plasma Membrane Domain Patterning and Self-Reinforcing Polarity in Arabidopsis.

Cell polarity is a key feature in the development of multicellular organisms. For instance, asymmetrically localized plasma-membrane-integral PIN-FORMED (PIN) proteins direct transcellular fluxes of the phytohormone auxin that govern plant development. Fine-tuned auxin flux is important for root protophloem sieve element differentiation and requires the interacting plasma-membrane-associated BREVIS RADIX (BRX) and PROTEIN KINASE ASSOCIATED WITH BRX (PAX) proteins. We observed "donut-like" polar PIN localization in developing sieve elements that depends on complementary, "muffin-like" polar localization of BRX and PAX. Plasma membrane association and polarity of PAX, and indirectly BRX, largely depends on phosphatidylinositol-4,5-bisphosphate. Consistently, mutants in phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) display protophloem differentiation defects similar to brx mutants. The same PIP5Ks are in complex with BRX and display "muffin-like" polar localization. Our data suggest that the BRX-PAX module recruits PIP5Ks to reinforce PAX polarity and thereby the polarity of all three proteins, which is required to maintain a local PIN minimum.

What Can Cell Culture Systems Reveal About Sieve Element Differentiation?

Tissue culture systems can be powerful tools for studying the process of cell differentiation in detail. Although a large number of cultures for xylem differentiation have been developed and utilized, there are only few reports on culture systems for ectopic phloem differentiation. Recently, a novel tissue culture system named Vascular cell Induction culture System Using Arabidopsis Leaves (VISUAL) was established, in which both xylem and phloem cells can be rapidly and efficiently induced in the model plant Arabidopsis thaliana. This chapter discusses the principle of VISUAL and how it can be used to investigate phloem differentiation, for example in combination with genetic experiments or transcriptome analysis. In addition, the protocol for establishing a phloem cell culture is provided.

Sieve Elements: The Favourite Habitat of Phytoplasmas.

The sieve elements are the only plant compartments, where phytoplasmas can survive and propagate. Therefore, this chapter is focussed on the specific molecular and cell-biological properties of the sieve element. Sieve element-companion cell complexes arise from (pro)cambial mother cells induced by key genes known to be decisive for sieve-element differentiation. The special anatomy, cell biology, and plasma-membrane outfit of sieve elements allows them to act collectively as a tube system that is able to drive a mass flow against the flow induced by transpiration. Plasmodesmal corridors are vital for the translocation of photoassimilates and systemic signals and for survival of the enucleate sieve elements. Of paramount importance is the Ca2+-dependent gating of plasmodesmata by callose and proteins. Hence, some of the complex, regulatory mechanisms to maintain Ca2+ homoeostasis in sieve elements are presented. Finally, the peculiarities of the chemical and physical sieve-element environment offered to phytoplasmas are discussed.

Non-dispersive phloem-protein bodies (NPBs) of Populus trichocarpa consist of a SEOR protein and do not respond to cell wounding and Ca2.

Differentiating sieve elements in the phloem of angiosperms produce abundant phloem-specific proteins before their protein synthesis machinery is degraded. These P-proteins initially form dense bodies, which disperse into individual filaments when the sieve element matures. In some cases, however, the dense protein agglomerations remain intact and are visible in functional sieve tubes as non-dispersive P-protein bodies, or NPBs. Species exhibiting NPBs are distributed across the entire angiosperm clade. We found that NPBs in the model tree, Populus trichocarpa, resemble the protein bodies described from other species of the order Malpighiales as they all consist of coaligned tubular fibrils bundled in hexagonal symmetry. NPBs of all Malpighiales tested proved unresponsive to sieve tube wounding and Ca2+. The P. trichocarpa NPBs consisted of a protein encoded by a gene that in the genome database of this species had been annotated as a homolog of SEOR1 (sieve element occlusion-related 1) in Arabidopsis. Sequencing of the gene in our plants corroborated this interpretation, and we named the gene PtSEOR1. Previously characterized SEOR proteins form irregular masses of P-protein slime in functional sieve tubes. We conclude that a subgroup of these proteins is involved in the formation of NPBs at least in the Malpighiales, and that these protein bodies have no role in rapid wound responses of the sieve tube network.

Brassinosteroid signaling directs formative cell divisions and protophloem differentiation in Arabidopsis root meristems.

ABSTRACTBrassinosteroids (BRs) trigger an intracellular signaling cascade through its receptors BR INSENSITIVE 1 (BRI1), BRI1-LIKE 1 (BRL1) and BRL3. Recent studies suggest that BR-independent inputs related to vascular differentiation, for instance root protophloem development, modulate downstream BR signaling components. Here, we report that protophloem sieve element differentiation is indeed impaired in bri1 brl1 brl3 mutants, although this effect might not be mediated by canonical downstream BR signaling components. We also found that their small meristem size is entirely explained by reduced cell elongation, which is, however, accompanied by supernumerary formative cell divisions in the radial dimension. Thus, reduced cell expansion in conjunction with growth retardation, because of the need to accommodate supernumerary formative divisions, can account for the overall short root phenotype of BR signaling mutants. Tissue-specific re-addition of BRI1 activity partially rescued subsets of these defects through partly cell-autonomous, partly non-cell-autonomous effects. However, protophloem-specific BRI1 expression essentially rescued all major bri1 brl1 brl3 root meristem phenotypes. Our data suggest that BR perception in the protophloem is sufficient to systemically convey BR action in the root meristem context.

Evolution of plant conducting cells: perspectives from key regulators of vascular cell differentiation.

One crucial problem that plants faced during their evolution, particularly during the transition to growth on land, was how to transport water, nutrients, metabolites, and small signaling molecules within a large, multicellular body. As a solution to this problem, land plants developed specific tissues for conducting molecules, called water-conducting cells (WCCs) and food-conducting cells (FCCs). The well-developed WCCs and FCCs in extant plants are the tracheary elements and sieve elements, respectively, which are found in vascular plants. Recent molecular genetic studies revealed that transcriptional networks regulate the differentiation of tracheary and sieve elements, and that the networks governing WCC differentiation are largely conserved among land plant species. In this review, we discuss the molecular evolution of plant conducting cells. By focusing on the evolution of the key transcription factors that regulate vascular cell differentiation, the NAC transcription factor VASCULAR-RELATED NAC-DOMAIN for WCCs and the MYB-coiled-coil (CC)-type transcription factor ALTERED PHLOEM DEVELOPMENT for sieve elements, we describe how land plants evolved molecular systems to produce the specialized cells that function as WCCs and FCCs.

Microautophagy involves programmed cell semi-death of sieve elements in developing caryopsis of Triticum aestivum L.

Differentiation of sieve elements (SEs) involves programmed cell semi-death, in which a small amount of organelles is retained. However, the mechanisms by which a large amount of SE cytoplasm is degraded and the specific proteases involved are not clear. In this study, we confirmed that the degradation of cytoplasm outside of the vacuole was mediated by microautophagy of the vacuole, and that the tonoplast selectively fused with the plasma membrane after most of the cytoplasm in the vacuoles was degraded. The integration of space enclosed a small amount of cytoplasm. Therefore, that fraction of the cytoplasm was preserved. At the same time, the cytosol was weakly acidic during membrane fusion because part of the tonoplast was ruptured. We also demonstrated that wheat aspartic protease (WAP1) and proteases including cathepsin B activity (PICA) were involved in programmed cell semi-death of SEs. PICA showed strongest activity before mass of the cytoplasm was degraded, which might contribute toward SE stability. We found that WAP1 mainly degraded the cytoplasm. Therefore, programmed cell semi-death of SEs might result from the joint action of vacuoles and multiple proteases.

Definitive Callose Deposition in Tapping Panel Dryness Affected Bark of Hevea brasiliensis

The structural and functional aspects of sieve elements in Tapping Panel Dryness (TPD) affected bark of Hevea brasiliensis, with special reference to definitive callose deposition, have been studied using histochemical methods. The effect of ethephon in definitive callose deposition in TPD affected bark also has been investigated, as the ethylene is reported to have a major role in TPD. The phloem of Hevea is characterized by sieve elements with compound sieve plates. The deposition of definitive callose on the sieve areas following TPD limits the translocation of sap through sieve elements. The amount of functional phloem was narrow and the accumulation of definitive callose was more in the sieve elements of TPD affected bark compared to that of unaffected bark of the same tree and of healthy trees. The affected area showed an increment in deposition of definitive callose in both vertical and horizontal extent of sieve areas; and there was a reduction in length of sieve plate, density of sieve pores, length of inter-sieve area wall, and number of sieve element rows in the inner bark. The lateral walls of non-functional sieve elements became thick and lignified. The anatomical changes in the sieve elements of TPD affected bark suggest that the translocation of phloem sap is regulated by deposition of definitive callose and less differentiation of sieve elements from vascular cambium. The external application of ethephon on bark enhanced the definitive callose deposition on sieve elements. The findings from the study reveal that ethylene plays a major role in the senescence of sieve elements.

Molecular and phylogenetic characterization of the sieve element occlusion gene family in Fabaceae and non-Fabaceaeplants

BackgroundThe phloem of dicotyledonous plants contains specialized P-proteins (phloem proteins) that accumulate during sieve element differentiation and remain parietally associated with the cisternae of the endoplasmic reticulum in mature sieve elements. Wounding causes P-protein filaments to accumulate at the sieve plates and block the translocation of photosynthate. Specialized, spindle-shaped P-proteins known as forisomes that undergo reversible calcium-dependent conformational changes have evolved exclusively in the Fabaceae. Recently, the molecular characterization of three genes encoding forisome components in the model legume Medicago truncatula (MtSEO1, MtSEO2 and MtSEO3; SEO = sieve element occlusion) was reported, but little is known about the molecular characteristics of P-proteins in non-Fabaceae.ResultsWe performed a comprehensive genome-wide comparative analysis by screening the M. truncatula, Glycine max, Arabidopsis thaliana, Vitis vinifera and Solanum phureja genomes, and a Malus domestica EST library for homologs of MtSEO1, MtSEO2 and MtSEO3 and identified numerous novel SEO genes in Fabaceae and even non-Fabaceae plants, which do not possess forisomes. Even in Fabaceae some SEO genes appear to not encode forisome components. All SEO genes have a similar exon-intron structure and are expressed predominantly in the phloem. Phylogenetic analysis revealed the presence of several subgroups with Fabaceae-specific subgroups containing all of the known as well as newly identified forisome component proteins. We constructed Hidden Markov Models that identified three conserved protein domains, which characterize SEO proteins when present in combination. In addition, one common and three subgroup specific protein motifs were found in the amino acid sequences of SEO proteins. SEO genes are organized in genomic clusters and the conserved synteny allowed us to identify several M. truncatula vs G. max orthologs as well as paralogs within the G. max genome.ConclusionsThe unexpected occurrence of forisome-like genes in non-Fabaceae plants may indicate that these proteins encode species-specific P-proteins, which is backed up by the phloem-specific expression profiles. The conservation of gene structure, the presence of specific motifs and domains and the genomic synteny argue for a common phylogenetic origin of forisomes and other P-proteins.

An early nodulin-like protein accumulates in the sieve element plasma membrane of Arabidopsis.

Membrane proteins within the sieve element-companion cell complex have essential roles in the physiological functioning of the phloem. The monoclonal antibody line RS6, selected from hybridomas raised against sieve elements isolated from California shield leaf (Streptanthus tortuosus; Brassicaceae) tissue cultures, recognizes an antigen in the Arabidopsis (Arabidopsis thaliana) ecotype Columbia that is associated specifically with the plasma membrane of sieve elements, but not companion cells, and accumulates at the earliest stages of sieve element differentiation. The identity of the RS6 antigen was revealed by reverse transcription-PCR of Arabidopsis leaf RNA using degenerate primers to be an early nodulin (ENOD)-like protein that is encoded by the expressed gene At3g20570. Arabidopsis ENOD-like proteins are encoded by a multigene family composed of several types of structurally related phytocyanins that have a similar overall domain structure of an amino-terminal signal peptide, plastocyanin-like copper-binding domain, proline/serine-rich domain, and carboxy-terminal hydrophobic domain. The amino- and carboxy-terminal domains of the 21.5-kD sieve element-specific ENOD are posttranslationally cleaved from the precursor protein, resulting in a mature peptide of approximately 15 kD that is attached to the sieve element plasma membrane via a carboxy-terminal glycosylphosphatidylinositol membrane anchor. Many of the Arabidopsis ENOD-like proteins accumulate in gametophytic tissues, whereas in both floral and vegetative tissues, the sieve element-specific ENOD is expressed only within the phloem. Members of the ENOD subfamily of the cupredoxin superfamily do not appear to bind copper and have unknown functions. Phenotypic analysis of homozygous T-DNA insertion mutants for the gene At3g20570 shows minimal alteration in vegetative growth but a significant reduction in the overall reproductive potential.

Effect of nickel on growth, proliferation, and differentiation of root cells in Triticum aestivum seedlings

The effect of 10−6 and 10−4 M NiSO4 on cell growth, proliferation, and differentiation was studied over 48 h in seminal and lateral roots of five-day-old Triticum aestivum seedlings. 10−6 M NiSO4 did not significantly affect the root system, whereas 10−4 M NiSO4 inhibited its development. However, 10−6 M NiSO4 disturbed the contacts between the groups of closely related cells of the rhizodermis in the meristem. In the exodermis, an additional layer of cells was formed. At the nickel concentration of 10−4 M, cell divisions in the outer layers of the root cells and metaxylem ceased earlier than in other root tissues positioned both centripetally and acropetally. Differentiation of protophloem sieve elements was completed in the meristem but at a greater distance from the root tip. Cell elongation started at the same distance from the root tip as in control plants. The rate of elongation decreased, and acropetally it stopped. Therefore, the cells of the xylem and metaphloem started to differentiate, and primordia of lateral roots were initiated and formed closer to the root tip. At a lethal concentration (10−4 M), nickel induced necroses of elongating cells of the endodermis and pericycle. Nickel is supposed to enter the tissues of the central cylinder predominantly via the protoxylem and rapidly translocate along the xylem. As a result, the incubation of the roots at this concentration for 48 h almost did not affect the development of the phloem and probably sugar unloading, that makes possible to maintain the growth of meristematic cells and the cell division of the most important tissues for longer time.

The Phloem Sieve Element: A River Runs through It.

31 9-335-3620. the sieve tube, which is formed by a series of connected phloem sieve elements. This fundamental difference imposes requirements on the development of the sieve element that are unique to these cells, and the differentiation of sieve elements follows a course that is unparalleled in the biological world. Essentially, a series of differentiating sieve elements form a syncytium, a single compartment bound by a plasma membrane (Murphy and Aikman, 1989). Within the developing syncytium, the larger cellular organelles, which could impede assimilate flow, are degraded and removed, and the mature sieve tube lacks nuclei, vacuoles, Golgi bodies, or ribosomes. The plasmodesmata in the walls between the individual sieve elements are converted into large openings (sieve pores) that facilitate flow from cell to cell through the sieve tube. Significantly, this differentiation process, including the formation of sieve pores, is accomplished while maintaining the continuity of the plasma membrane around the individual cells and through the pores formed between the cells. The result of this unique differentiation process is the formation of a continuous, membrane-lined compartment, the sieve tube, that provides a pathway for nutrient flow and for signaling activities (Ryals et al., 1996) throughout the plant body.

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.

Differentiation of abnormal sieve elements in roots of wheat (Triticum aestivum L.) affected by colchicine.

Protophloem sieve elements (PSEs) of seminal roots of wheat (Triticum aestivum L.) treated with 2 MM colchicine solution differentiate into an extraordinary cell type not found in normal roots. Colchicine-treated PSEs stop elongating and increase in diameter considerably. Abnormal ultrastructural features appear gradually with increasing time of exposure to the drug. Microtubules disorganize and disappear first. Cell divisions are blocked and nuclei become polyploid, with multiple lobes and deep imaginations. Profuse paracrystalline material, presumably consisting of tubulin or, more likely, of tubulin-colchicine polymers, accumulates in the cytoplasm of the polyploid cells. Endoplasmic reticulum (ER) cisternae do not aggregate in stacks and mitochondria are not enveloped by ER cisternae, as normal. Golgi bodies appear active and distinct, while unusual vesicles with dense contents occur in the cytoplasm. However, development of plastids and degeneration of nuclei do not seem to be significantly affected by colchicine. The rate of development of affected PSEs is retarded considerably, as judged from ultrastructural features (plastid inclusions, wall thickenings), which appear much later in the vertical files of cells. Due to the retardation and to the overall stoppage of Toot elongation, differentiation of PSEs is caught up by other vascular elements such as protoxylem elements and metaphloem sieve elements. The aberrant PSEs undergo autolysis and reach maturity, but plastids, mitochondria, nuclear remnants and ER configurations characterizing normal PSEs are retained. Transverse walls differentiate into abnormal sieve plates, in which sieve pores are not formed. Lateral walls develop unusual thickenings projecting in the cytoplasm. The observations show that: (a) Differentiation of the affected PSEs proceeds in the absence of divisions; (b) the number and degree of aberrations depend largely on the timing of exposure to the drug, and (c) strands of paracrystalline material and vacuoles are formed in microtubule-free PSEs after long and continuous colchicine treatment.

Aberrant sieve element differentiation in primary leaves of Vigna sinensis Endl. affected by colchicine

summaryIn primary leaves of Vigna sinensis Endl. seedlings growing in the presence of colchicine, some giant polyploid cells at the phloem pole of the vascular bundles differentiate into an extraordinary cell type acquiring most of the prominent features of the sieve elements. These aberrant sieve elements (a‐SEs) show some exceptional features not found in normal sieve‐tube elements. A heavy secondary thickening is deposited on their walls, which in surface view exhibits a reticulate or ‘pitted’ appearance. The thickenings reach the same depth into the cytoplasm and show a diffuse orientation of cellulose microfibrils. Amorphous callose is deposited around some fine cytoplasmic strands penetrating the secondary wall up to the primary one along the longitudinal and particularly the transverse walls. In the latter walls sieve pores are not formed. At first, the a‐SEs undergo a partial protoplasmic degradation like normal sieve‐tube elements but finally autolyse completely. During this process, the major portion of the unmasked primary and secondary wall is dissolved.The observations show that (a) a vascular bundle initiation and development proceeds in the affected primary leaves, in the absence of cell divisions.‘Vascular bundles' are formed consisting of a single row of cells, in which a‐SE differentiation dominates. (b) The ‘sieve element differentiation’ is initiated without differential divisions. (c) Although the microtubules arc absent, the cells determined to become a‐SEs activate another morphogenetic mechanism to control a more or less patterned deposition of a secondary wall layer.