Inhibition of phenylalanine ammonia-lyase activity causes the depression of lignin accumulation of secondary wall thickening in isolatedZinnia mesophyll cells

Summary Isolated mesophyll cells ofZinnia elegans L. cv. Canary Bird differentiate into tracheary elements in differentiation (D) medium. These elements develop lignified secondary wall thickenings. The influence of 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase (PAL), on lignification ofZinnia tracheary elements was examined. The mesophyll cells were cultured in D and AIP media. The latter medium, in which 100 M AIP was added to the D medium, inhibited PAL activity, though the differentiation proceeded. Morphological differences of secondary wall thickenings cultured in these two types of media were investigated under an UV microscope and a transmission electron microscope. The secondary wall thickenings at 96 h in the D medium showed strong UV absorption. The fibrillar structure of the thickenings observed clearly at 72 h was covered with electron opaque materials by 96 h. The secondary wall thickenings at 96 h in the AIP medium showed weak UV absorption. The thickenings at 96 h had a cracked appearance. Furthermore, the thickenings showed a little irregular or wavy arrangement of cellulose microfibrils and had many pores and spaces between microfibrils. From these results, the role of lignin accumulation in the formation of secondary wall thickenings was discussed.Abbreviations AIP 2-aminoindan-2-phosphonic acid - PAL phenylalanine ammonia-lyase     

Immunocytochemical Localization of Phenylalanine Ammonia-Lyase and Cinnamyl Alcohol Dehydrogenase in Differentiating Tracheary Elements Derived from Zinnia Mesophyll Cells

Secondary wall thickening is the most characteristic morphologicalfeature of the differentiation of tracheary elements. Isolatedmesophyll cells of Zinnia elegans L. cv. Canary Bird in differentiationmedium are converted to tracheary elements, which develop lignifiedsecondary wall thickenings. Using this system, we investigatedthe distribution of two enzymes, phenylalanine ammonia-Iyase(PAL) (EC 4.3.1.5 [EC] ) and cinnamyl alcohol dehydrogenase (CAD)(EC 1.1.1.195 [EC] ), by both biochemical and immunological methods.Both PAL and CAD appear to be key enzymes in the biosynthesisof lignin precursors, and they have been shown to be associatedwith the differentiation of tracheary elements. Cultured cellswere collected after various times in culture. The culture mediumwas separated from cells by centrifugation and designated fraction(1), the extracellular fraction. The collected cells were homogenizedand separated into four fractions: (2) cytosol; (3) microsomes;(4) cell walls (loosely bound material); and (5) cell walls(tightly bound material). PAL activity was detected in eachfraction. The extracellular fraction consistently had the greatestPAL activity. Moreover, PAL activity in the cytosolic fractionincreased rapidly prior to lignification, as it did in boththe microsomal and the cell wall (tightly bound) fractions duringlignification. Antisera against PAL and against CAD detectedthe proteins with molecular masses that corresponded to thoseof PAL and CAD in Zinnia. Immuno-electron microscopy revealedthat, in differentiating tracheary elements, PAL was dispersedin the cytoplasmic matrix and was located on Golgi-derived vesiclesand on the secondary wall thickenings. "Cell-free" immuno-lightmicroscopy supported the putative distribution of PAL on lignifyingsecondary walls. The pattern of distribution of CAD was similarto that of PAL. Thus, both PAL and CAD seemed to be localizedin secondary wall thickenings. From the results of both biochemicalassays and immunocytochemical staining, it appeared that atleast two types of PAL and CAD are present in differentiatingcells. One type of each enzyme is distributed in the cytosol,while the other is secreted from the Golgi apparatus and transportedby Golgi-derived vesicles to the secondary wall thickenings. (Received April 19, 1996; Accepted November 18, 1996)     

Production of Salicylic Acid Precursors Is a Major Function of Phenylalanine Ammonia-Lyase in the Resistance of Arabidopsis to Peronospora parasitica

Arabidopsis ecotype Columbia (Col-0) seedlings, transformed with a phenylalanine ammonia-lyase 1 promoter (PAL1)-[beta]-glucuronidase (GUS) reporter construct, were inoculated with virulent and avirulent isolates of Peronospora parasitica. The PAL1 promoter was constitutively active in the light in vascular tissue but was induced only in the vicinity of fungal structures in the incompatible interaction. A double-staining procedure was developed to distinguish between GUS activity and fungal structures. The PAL1 promoter was activated in cells undergoing lignification in the incompatible interaction in response to the pathogen. Pretreatment of the seedlings with 2-aminoindan-2-phosphonic acid (AIP), a highly specific PAL inhibitor, made the plants completely susceptible. Lignification was suppressed after AIP treatment, and surprisingly, pathogen-induced PAL1 promoter activity could not be detected. Treatment of the seedlings with 2-hydroxyphenylaminosulphinyl acetic acid (1,1-dimethyl ester) (OH-PAS), a cinnamyl alcohol dehydrogenase inhibitor specific for the lignification pathway, also caused a shift toward susceptibility, but the effect was not as pronounced as it was with AIP. Significantly, although OH-PAS suppressed pathogen-induced lignification, it did not suppress pathogen-induced PAL1 promoter activation. Salicylic acid (SA), supplied to AIP-treated plants, restored resistance and both pathogen-induced lignification and activation of the PAL1 promoter. Endogenous SA levels increased significantly in the incompatible but not in the compatible combination, and this increase was suppressed by AIP but not by OH-PAS. These results provide evidence of the central role of SA in genetically determined plant disease resistance and show that lignification per se, although providing a component of the resistance mechanism, is not the deciding factor between resistance and susceptibility.     

Deposition of glycine-rich structural protein in xylem cell walls of french bean seedlings is independent of lignification

Lignin biosynthesis was inhibited in young bean seedlings by 2-aminoindan-2-phosphonic acid (AIP). AIP is a specific and potent inhibitor of phenylalanine ammonialyase, an enzyme involved in lignin biosynthesis. At a concentration of 100 μM AIP in the growth medium, no lignin could be detected in roots and hypocotyls of 7- or 9-day-old seedlings when stained with phloroglucinol/HCl. At an AIP concentration of 70 μM only a very weak lignification was observed, whereas at 30 μM, no inhibition of lignification was detectable. Glycine-rich protein GRP 1.8, a cell wall protein present in protoxylem of beans, was studied by immunocytochemistry in hypocotyls grown in the presence of 100 μM AIP. No difference of the GRP deposition pattern at sites of normally lignified secondary cell wall thickenings, as well as along the protoxylem vessels, was found in unlignified tissue when compared to controls. The cell-type specific synthesis of GRP 1.8 was not affected by AIP. Thus, deposition of the GRP 1.8 structural cell wall protein is independent of lignification, and lignin does not act as an essential scaffold for correct GRP 1.8 deposition in the complex wall structure of xylem.     

Tracheary element differentiation in Zinnia mesophyll cell cultures

Mechanically isolated mesophyll cells of Zinnia elegans differentiate into tracheary elements (TEs) when cultured in a medium containing adequate auxin and cytokinin. Differentiation in this culture system is relatively synchronous, rapid (occuring within 3 days of cell isolation) and efficient (with up to 65% of the mesophyll cells differentiating into TEs), and does not require prior mitosis. The Zinnia system has been used to investigate (a) cytological and ultrastructural changes occurring during TE differentiation, such as the reorganization of microtubules controlling secondary wall deposition, (b) the influences of calcium and of various plant hormones and antihormones on TE differentiation, and (c) biochemical changes during differentiation, including those occurring during secondary wall deposition, lignification and autolysis. This review summarizes experiments in which the Zinnia system has served as a model for the study of TE differentiation.     

Regulation of secondary cell wall development by cortical microtubules during tracheary element differentiation in Arabidopsis cell suspensions

Cortical microtubules participate in the deposition of patterned secondary walls in tracheary element differentiation. In this study, we established a system to induce the differentiation of tracheary elements using a transgenic Arabidopsis (Arabidopsis thaliana) cell suspension stably expressing a green fluorescent protein-tubulin fusion protein. Approximately 30% of the cells differentiated into tracheary elements 96 h after culture in auxin-free media containing 1 mum brassinolide. With this differentiation system, we have been able to time-sequentially elucidate microtubule arrangement during secondary wall thickening. The development of secondary walls could be followed in living cells by staining with fluorescein-conjugated wheat germ agglutinin, and the three-dimensional structures of the secondary walls could be simultaneously analyzed. A single microtubule bundle first appeared beneath the narrow secondary wall and then developed into two separate bundles locating along both sides of the developing secondary wall. Microtubule inhibitors affected secondary wall thickening, suggesting that the pair of microtubule bundles adjacent to the secondary wall played a crucial role in the regulation of secondary wall development.     

L-α-Aminooxy-β-phenylpropionic acid inhibits lignification but not the differentiation to tracheary elements of isolated mesophyll cells of Zinnia elegans

The influence of L-α-aminooxy-β-phenylpropionic acid (AOPP), an inhibitor of L-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), and thus of lignin formation, on the differentiation of tracheary elements from isolated mesophyll cells of Zinnia elegans L. cv. Canary Bird was investigated. At low concentrations of AOPP (5–25 μ,M) lignification of differentiating cells was almost completely prevented whereas number of differentiating cells, viability of the cells, fresh and dry weights, and the packed cell volume were higher than corresponding parameters in control cultures. At higher concentrations of AOPP (50–75 μ M ) the formation of tracheary elements was inhibited but division, elongation and expansion of cells were still observed. Cells cultured for 96 h in the presence of 100 μ M AOPP were morphologically similar to cells at 12 h of culture, the time at which AOPP was added. At concentrations of AOPP that did not inhibit differentiation, AOPP caused an increase in the amounts of uronic acid and total carbohydrate (per unit volume of cell suspension) in the extracellular polysaccharide fraction and in the total cell wall fraction, although these parameters were not significantly different from control values when expressed on a dry weight basis. AOPP caused the release of polysaccharides which contained xylose into the medium when added before the onset of visible differentiation and the release of polysaccharides which contained glucose when added at the time when the formation of the secondary cell wall thickenings took place. The results indicate that AOPP at low concentrations specifically inhibits the formation of lignin without adversely affecting the synthesis of cell wall polysaccharides, although the proper integration of these compounds into the wall may be disturbed. O-Benzylhydroxyla-mine, on the other hand, did not prove to be a useful agent to affect lignin synthesis in differentiating Zinnia cells.     

Involvement of extracellular dilignols in lignification during tracheary element differentiation of isolated Zinnia mesophyll cells

During differentiation of isolated Zinnia mesophyll cells into tracheary elements (TEs), lignification on TEs progresses by supply of monolignols not only from TEs themselves but also from surrounding xylem parenchyma-like cells through the culture medium. However, how lignin polymerizes from the secreted monolignols has not been resolved. In this study, we analyzed phenol compounds in culture medium with reversed-phase HPLC, gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry, and found 12 phenolic compounds including coniferyl alcohol and four dilignols, i.e. erythro-guaiacylglycerol-beta-coniferyl ether, threo-guaiacylglycerol-beta-coniferyl ether, dehydrodiconiferyl alcohol and pinoresinol, in the medium in which TEs were developing. Coniferyl alcohol applied to TE-inductive cultures during TE formation rapidly disappeared from the medium, and caused a sudden increase in dilignols. Addition of the dilignols promoted lignification of TEs in which monolignol biosynthesis was blocked by an inhibitor of phenylalanine anmmonia-lyase (PAL), L-alpha-aminooxy-beta-phenylpropionic acid (AOPP). These results suggested that dilignols can act as intermediates of lignin polymerization.     

Effect of high salinity on tracheary element differentiation in light-grown callus of Mesembryanthemum crystallinum

This study investigated the inhibitory effects of NaCl on tracheary element (TE) differentiation in light-grown callus of ice plant Mesembryanthemum crystallinum L., a halophyte which adaptes well to saline environments. When ice plant callus was grown in a modified Linsmaier-Bednar and Skoog culture medium containing no NaCl (control medium), up to 20% of ice plant cells differentiated into tracheary elements during in vitro culture. Close examination of callus tissues stained with potassium permanganate revealed that tracheary elements were aggregated as discrete nodules. Some strikingly elongated tracheary elements were found in the macerated tissues. Experimental results indicated that adding 200 mM NaCl to the control medium reversibly inhibited the formation of tracheary element in the halophytic cells. The rate of tracheary element formation increased accordingly as the rate of cell growth in control medium. In the presence of high salt, the degree of tracheary element differentation remained low through the growth cycle. The inhibitory effect of salt on tracheary element differentiation was overcome by adding 10 mg l⁻¹ salicylic acid, a known signaling compound that induces a diverse group of defense-related genes, including genes involved in reinforcing the host cell wall. Furthermore, microscopic examination revealed that most tracheary elements formed under this treatment (200 mM NaCl plus 10 mg l⁻¹ salicylic acid) were round shaped. The results suggest that high salt inhibits both the biosynthesis of secondary wall components and cell elongation ice plant in vitro culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Inhibition of xylogenesis by morphactin in pith parenchyma explants of Lactuca

Pith parenchyma explants of Romaine lettuce (Lactuca salivaLinn. var. Roman?) incubated in the dark for 7 days at 25?Con a nutrient medium containing sucrose, IAA. and kinetin exhibitedextensive differentiation of tracheary elements. The additionof CFL to the medium strongly inhibited tracheary element formation.The lack of tracheary strand formation in the CFL-treated explantssuggests the inhibition of auxin transport. Conclusive evidencethat CFL influences the anatomy of differentiating xylem elementswas lacking. The addition of CFL to various combinations ofxylogenic media was not stimulatory to xylem element formationbeyond the differentiation response observed in the absenceof CFL. Unique patterns of tracheary element formation producedby cytokinin media containing IAA, 2,4-D, and NAA, respectively,were abolished by CFL. As indicated by counts of total trachearyelements formed per explant, the addition of cysteine to a CFL-containingmedium reversed the inhibitory effect of CFL. Tracheary strandformation was not re-established in the explants cultured onthe cysteine+CFL medium. Tracheary element formation was completelysuppressed by TIBA. Cysteine had a slight effect on the inhibitionof differentiation by TIBA. These observations suggest thatCFL inhibits some sulfhydryl- containing system involved eitherin the process of xylem differentiation or in some prerequisiterole necessary for the induction of tracheary element formation. (Received December 27, 1972; )     

Touch- and Methyl Jasmonate-induced Lignification in Tendrils of Bryonia dioica Jacq.

The touch-induced free coiling response of Bryonia dioica tendrils is accompanied by the differentiation of supporting tissue at the ventral side of the organ, becoming the inner (concave) side of the coiled tendril. As part of this process, the Bianconi plate, a continuous sclerenchyma sheath stretching along the ventral face of the five bicollateral vascular bundles, becomes strongly lignified. During this reaction, the extractable activity of phenylalanine ammonia-lyase in the tendrils increases four- to five-fold, while the amount of PAL, as demonstrated by immunoblotting, remains unchanged. This touch-induced process can also be elicited by airborne application of methyl jasmonate. The PAL inhibitor, 2-aminoindan-2-phosphonic acid (AIP) completely inhibits both the touch- and methyl jasmonate-induced deposition of lignin-like material in the Bianconi plate, but has no effect on coiling. From these results, it can be concluded that the cessation of growth at the ventral side of a free-coiling tendril is not due to induced lignification but rather, lignification seems to serve the function of increasing the mechanical strength of the coiled tendril.     

Zinnia elegans uses the same peroxidase isoenzyme complement for cell wall lignification in both single-cell tracheary elements and xylem vessels

The nature of the peroxidase isoenzyme complement responsible for cell wall lignification in both Zinnia elegans seedlings and Z. elegans tracheary single-cell cultures have been studied. Results showed that both hypocotyls and stems from lignifying Z. elegans seedlings express a cell wall-located basic peroxidase of pI approximately 10.2, which was purified to homogeneity. Molecular mass determination under non-denaturing conditions showed an M(r) of about 43 000, similar to that of other plant peroxidases. The purified Z. elegans peroxidase showed absorption maxima at 403 (Soret band), and at 496-501 and 632-635 (alpha and beta absorption bands), indicating that this enzyme is a high spin ferric haem protein, belonging to the plant peroxidase superfamily, the prosthetic group being ferric protoporphyrin IX. The N-terminal amino acid sequence of this Z. elegans basic peroxidase was KVAVSPLS (peptide motif in bold), which shows strong homologies with the N-amino acid terminus of other strongly basic plant peroxidases. Isoenzyme and western blot analyses showed that this peroxidase isoenzyme is also expressed in trans-differentiating Z. elegans tracheary single-cell cultures. The results also showed that Z. elegans tracheary single-cell cultures not only express the same peroxidase isoenzyme as the Z. elegans lignifying xylem, but that this peroxidase isoenzyme acts as a marker of tracheary element differentiation in Z. elegans mesophyll single-cell cultures. From these results, it may be concluded that Z. elegans uses a single programme, i.e. an identical peroxidase isoenzyme complement, for lignification of the xylem, regardless of the existence of different ontogenesis pathways from either mesophyll cells (in the case of tracheary elements) or cambial derivatives (in the case of xylem vessels).     

Effects of auxin on the spatial distribution of cell division and xylogenesis in lettuce pith explants

Summary Cylinders of pith parenchyma were tissue-cultured with their opposite ends on media which differed only in content of the morphogens auxin (IAA), sucrose, or zeatin. A range of concentrations of each of these morphogens applied at one end (none at the other end) resulted in distribution patterns of cell division and xylogenesis that were attributable to interaction between inductive levels and morphogen mobility. Auxin was crucial for tracheary patterns: large tracheary elements formed by direct differentiation of pith cells near the auxin source, smaller but still roughly isodiametric tracheary elements formed after cell division, and tracheary strands developed where, presumably, auxin transport had become polarized and then canalized. Xylogenesis was confined to regions within millimeters of the auxin source, and [¹⁴C]IAA studies showed a steep logarithmic concentration gradient along the cylinder. Patterns of tracheary strands and rings revealed that the pith explants retained some polarity from the stem from which they had been excised. However, the direction of flow of applied auxin was more effective than original polarity in controlling the orientation of tracheary strands and their constituent tracheary elements. It seems that, in tissues with little or no polarity, diffusive flow of auxin gradually induces polar flow in the same direction, together with an associated bioelectric current, and that this orients the cortical microtubules that in turn determine the orientations of cell elongation and of the secondary wall banding in tracheary elements.Abbreviations IAA indoleacetic acid - NAA naphthaleneacetic acid - TIBA triiodobenzoic acid Dedicated to the memory of Professor John G. Torrey     

Restoration of secondary metabolism in birch seedlings relieved from PAL-inhibitor

Phenylalanine ammonia lyase (PAL) plays a key role in phenylpropanoid metabolism, catalyzing the deamination of phenylalanine (Phe) to form trans-cinnamic acid. Inhibitors of PAL have been used to study the physiological role of the different compounds derived from trans-cinnamic acid, and to test theories about a trade-off between growth and defence in plants. In a previous study with birch (Betula pubescens Ehrh.) seedlings, the PAL inhibitor 2-aminoindane-2-phosphonic acid monohydrate (AIP) caused an accumulation of Phe and a strong decrease in the quantity of simple phenolics, soluble condensed tannins and growth, whereas flavonol glycosides were generally not affected. The present study demonstrates restoration of secondary metabolism in the previously AIP treated birch seedlings. Our results indicate that Phe accumulated during PAL inhibition could be partly used to increase the content of the phenolic acids, flavan-3-ols and to some extent the soluble condensed tannins. Seedling growth also increased when the supply of PAL inhibitor ceased. We thereby show that the inhibition of PAL by AIP in vivo is reversible, at least for moderate AIP concentrations and the rate of restoration is dependent on the inhibitor concentration.     

Xylogenesis in tissue culture: Taxol effects on microtubule reorientation and lateral association in differentiating cells

Summary InZinnia elegans tissue cultures, cortical microtubules reorient from longitudinal to transverse arrays as the culture age increases and before differentiation of tracheary elements is visible. The orientation of microtubules, in the period just before visible differentiation, determines the direction of the secondary wall bands in forming tracheary elements. Taxol, applied early in culture, stabilizes the microtubules of most cells in the longitudinal direction. Tracheary elements differentiating in these taxol treated cultures show secondary wall bands parallel to the long axis of the cell while those differentiating in control cultures always have wall bands transverse to the long axis of the cell.It is proposed that, in untreatedZinnia cultures, microtubules are reoriented by a gradual shift from longitudinal to transverse and this reorientation normally occurs before differentiation becomes visible. Once initiated, tracheary element differentiation involves lateral association of microtubules to form the discrete bands typical of secondary wall patterns.     

Cytoskeletal organization during xylem cell differentiation

The water and mineral conductive tube, the xylem vessel and tracheid, is a highly conspicuous tissue due to its elaborately patterned secondary-wall deposition. One constituent of the xylem vessel and tracheid, the tracheary element, is an empty dead cell that develops secondary walls in the elaborate patterns. The wall pattern is appropriately regulated according to the developmental stage of the plant. The cytoskeleton is an essential component of this regulation. In fact, the cortical microtubule is well known to participate in patterned secondary cell wall formation. The dynamic rearrangement of the microtubules and actin filaments have also been recognized in the cultured cells differentiating into tracheary elements in vitro. There has recently been considerable progress in our understanding of the dynamics and regulation of cortical microtubules, and several plant microtubule associated proteins have been identified and characterized. The microtubules have been observed during tracheary element differentiation in living Arabidopsis thaliana cells. Based on this recently acquired information on the plant cytoskeleton and tracheary element differentiation, this review discusses the role of the cytoskeleton in secondary cell wall formation.     

Developmental changes in the secondary xylem ofAcer rubrum induced by various auxins and 2,3,5-tri-iodobenzoic acid

Summary TIBA has been applied laterally to actively growing stems of uprightAcer rubrum seedlings. The frequency of initiation of tracheary elements is reduced and a complete ring of tension wood is developed in the stem locally below the TIBA application site. Rings of tension wood were never formed above the TIBA treatment site. In regard to anatomy, lignin distribution and peroxidase activity, the tension wood fibers formed as a result of TIBA treatment are identical to those which can be induced by bending.In the region of the stem above the site of TIBA application there is a particularly strong alteration in the development of tracheary elements.Application of IAA, NAA, or 2,4-D to the TIBA treatment site suppresses the capacity of TIBA to induce the development of tension wood and at the same time generally increases the frequency of initiation of tracheary elements.The effect of auxin alone on theAcer rubrum system has been studied. The secondary xylem formed during treatment with auxins (especially 2,4-D and NAA) at the stated concentrations is generally characterized by large groups of tracheary elements with a conspicuous angular outline in transverse section.The evidence suggests that auxins are involved in the regulatory systems which bring about the orderly development of the secondary xylem in arborescent angiosperms.This material was included in a doctoral thesis submitted by P. R.Morey to the graduate school of Yale University, New Haven.     

Neighboring Parenchyma Cells Contribute to Arabidopsis Xylem Lignification,while Lignification of Interfascicular Fibers Is Cell Autonomous

Lignin is a critical structural component of plants, providing vascular integrity and mechanical strength. Lignin precursors (monolignols) must be exported to the extracellular matrix where random oxidative coupling produces a complex lignin polymer. The objectives of this study were twofold: to determine the timing of lignification with respect to programmed cell death and to test if nonlignifying xylary parenchyma cells can contribute to the lignification of tracheary elements and fibers. This study demonstrates that lignin deposition is not exclusively a postmortem event, but also occurs prior to programmed cell death. Radiolabeled monolignols were not detected in the cytoplasm or vacuoles of tracheary elements or neighbors. To experimentally define which cells in lignifying tissues contribute to lignification in intact plants, a microRNA against CINNAMOYL CoA-REDUCTASE1 driven by the promoter from CELLULOSE SYNTHASE7 (ProCESA7:miRNA CCR1) was used to silence monolignol biosynthesis specifically in cells developing lignified secondary cell walls. When monolignol biosynthesis in ProCESA7:miRNA CCR1 lines was silenced in the lignifying cells themselves, but not in the neighboring cells, lignin was still deposited in the xylem secondary cell walls. Surprisingly, a dramatic reduction in cell wall lignification of extraxylary fiber cells demonstrates that extraxylary fibers undergo cell autonomous lignification.     

Hydrogen peroxide and expression of hipI-superoxide dismutase are associated with the development of secondary cell walls in Zinnia elegans

A special form of a CuZn-superoxide dismutase with a high isoelectric point (hipI-SOD; EC 1.15.1.1) and hydrogen peroxide (H2O2) production were studied during the secondary cell wall formation of the inducible tracheary element cell-culture system of Zinnia elegans L. Confocal microscopy after labelling with 2',7'-dichlorofluorescin diacetate showed H2O2 to be located largely in the secondary cell walls in developing tracheary elements. Fluorescence-activated cell sorting analysis showed there were lower levels of H2O2 in the population containing tracheary elements when H2O2 scavengers such as ascorbate, catalase, and reduced glutathione were applied to the cell culture. Inhibitors of NADPH oxidase and SOD also reduced the amount of H2O2 in the tracheary elements. Furthermore, addition of these compounds to cell cultures at the time of tracheary element initiation reduced the amount of lignin and the development of the secondary cell walls. Analysis of UV excitation under a confocal laser scanning microscope confirmed these results. The expression of hipI-SOD increased as the number of tracheary elements in the cell culture increased and developed. Additionally, immunolocalization of a hipI-SOD isoform during the tracheary element differentiation showed a developmental build-up of the protein in the Golgi apparatus and the secondary cell wall. These findings suggest a novel hipI-SOD could be involved in the regulation of H2O2 required for the development of the secondary cell walls of tracheary elements.     

TERE; a novel cis-element responsible for a coordinated expression of genes related to programmed cell death and secondary wall formation during differentiation of tracheary elements

The differentiation of water-conducting tracheary elements (TEs) is the result of the orchestrated construction of secondary wall structure, including lignification, and programmed cell death (PCD), including cellular autolysis. To understand the orchestrated regulation of differentiation of TEs, we investigated the regulatory mechanism of gene expression directing TE differentiation. Detailed loss-of-function and gain-of-function analyses of the ZCP4 (Zinniacysteine protease 4) promoter, which confers TE-specific expression, demonstrated that a novel 11-bp cis-element is necessary and sufficient for the immature TE-specific promoter activity. The 11-bp cis-element-like sequences were found in promoters of many Arabidopsis TE differentiation-related genes. A gain-of-function analysis with similar putative cis-elements from secondary wall formation or modification-related genes as well as PCD-related genes indicated that the cis-elements are also sufficient for TE-specific expression of genes. These results demonstrate that a common sequence, designated as the tracheary-element-regulating cis-element, confers TE-specific expression to both genes related to secondary wall formation or modification and PCD.