<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> as a Model System for Graft Union Development in Homografts and Heterografts

Homografting of Arabidopsis thaliana scions on stocks of A. thaliana and heterografting on other species were used to study the compatibility and the ontogeny of graft union formation. Highly compatible homografting with scions of young leafy inflorescence stems was obtained on stocks of inflorescence stems growing from large 3-month-old A. thaliana plants. Histologic analysis revealed four developmental stages of graft union formation in Arabidopsis homografting: (1) development of a necrotic layer, (2) callus proliferation in the grafted scion, (3) differentiation of new vascular tissues within the scion, and (4) a full vascular graft union formation between the scion and the stock. Vascular connections were formed within the callus bridge between rootstocks and scions 15 days after grafting. Heterografts of Arabidopsis on two members of Brassicaceae, cabbage (Brassica) and radish (Raphanus), showed partial incompatible interaction with a lower level of vascular differentiation. Arabidopsis grafting on tomato (Solanaceae) rootstock showed complete incompatibility and limited noncontinuous differentiation of new vascular tissues that did not cross the scion/stock boundary. Although lacking scion/stock vascular connections, Arabidopsis scions grafted onto tomato rootstock flowered and produced seeds. This may indicate some nonvascular functional connections between the two plants, probably of parenchyma cells, further emphasizing the usefulness of Arabidopsis as a model plant for studying various levels of the complicated scion/stock relationships expressed in grafting biology. Experiments with dye transport in the xylem showed that although in general there was an agreement between the histologic study and dye transport, in Arabidopsis homografts water transport frequency was lower than functional and histologic compatability. We conclude that homografting and heterografting of Arabidopsis inflorescence stems is a convenient and reproducible method for studying the fundamental cellular genetic and molecular aspects of grafting biology.     

Localisation and characterisation of cell wall mannan polysaccharides in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Polysaccharides containing -1,4-mannosyl residues (mannans) are abundant in the lignified secondary cell walls of gymnosperms, and are also found as major seed storage polysaccharides in some plants, such as legume species. Although they have been found in a variety of angiosperm tissues, little is known about their presence and tissue localisation in the model angiosperm, Arabidopsis thaliana (L.) Heynh. In this study, antibodies that specifically recognised mannans in competitive ELISA experiments were raised in rabbits. Using these antibodies, we showed that Golgi-rich vesicles derived from Arabidopsis callus were able to synthesise mannan polysaccharides in vitro. Immunofluorescence light microscopy and immunogold electron microscopy of Arabidopsis inflorescence stem sections revealed that the mannan polysaccharide epitopes were localised in the thickened secondary cell walls of xylem elements, xylem parenchyma and interfascicular fibres. Similarly, mannan epitopes were present in the xylem of the leaf vascular bundles. Surprisingly, the thickened epidermal cell walls of both leaves and stems also contained abundant mannan epitopes. Low levels were observed in most other cell types examined. Thus, mannans are widespread in Arabidopsis tissues, and may be of particular significance in both lignified and non-lignified thickened cell walls. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) of cell wall preparations digested with a specific mannanase showed that there is glucomannan in inflorescence stems. The findings show that Arabidopsis can be used as a model plant in studies of the synthesis and functions of mannans.Abbreviations BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - PACE polysaccharide analysis by carbohydrate gel electrophoresis     

Modulation of eIF5A1 expression alters xylem abundance in Arabidopsis thaliana

Eukaryotic translation initiation factor 5A (eIF5A) is thoughtto facilitate protein synthesis by participating in the nuclearexport of specific mRNAs. In Arabidopsis, there are three isoformsof eIF5A. One of them, AteIF5A1, has been shown to be expressedin vascular tissue, specifically developing vessel members,using GUS as a reporter. In order to determine whether AteIF5A1plays a role in xylem formation, its full-length cDNA was constitutivelyover-expressed in transgenic Arabidopsis plants. Microscopicanalysis revealed that the cross-sectional area of the xylemin the main inflorescence stems of transgenic plants was 1.9-foldhigher than those of corresponding inflorescence stems of wild-typeplants. In wild-type stems, the primary xylem typically comprisedsix cell layers and was 105 µm thick, but increased to9–11 cell layers, 140–155 µm thick, in transgenicstems. Similarly, the secondary xylem increased from six celllayers, 70 µm thick, in control stems to 9 cell layers,95–105 µm thick, in transgenic stems. Moreover,constitutive down-regulation of AteIF5A1 using antisense technologyresulted in the major suppression of xylem formation comparedwith control plants, and the antisense transgenic plants werealso stunted. These data collectively indicate that eIF5A1 playsa role in xylogenesis. Key words: Arabidopsis thaliana, eukaryotic translation initiation factor 5A, inflorescence stem, xylem Received 5 November 2007; Revised 26 December 2007 Accepted 10 January 2008     

Cu2+ inhibition of gel secretion in the xylem and its potential implications for water uptake of cut Acacia holosericea stems

Maintaining a high rate of water uptake is crucial for maximum longevity of cut stems. Physiological gel/tylosis formation decreases water transport efficiency in the xylem. The primary mechanism of action for post‐harvest Cu²⁺ treatments in improving cut flower and foliage longevity has been elusive. The effect of Cu²⁺ on wound‐induced xylem vessel occlusion was investigated for Acacia holosericea A. Cunn. ex G. Don. Experiments were conducted using a Cu²⁺ pulse (5 h, 2.2 mM) and a Cu²⁺ vase solution (0.5 mM) vs a deionized water (DIW) control. Development of xylem blockage in the stem‐end region 10 mm proximal to the wounded stem surface was examined over 21 days by light and transmission electron microscopy. Xylem vessels of stems stood into DIW were occluded with gels secreted into vessel lumens via pits from surrounding axial parenchyma cells. Gel secretion was initiated within 1–2 days post‐wounding and gels were detected in the xylem from day 3. In contrast, Cu²⁺ treatments disrupted the surrounding parenchyma cells, thereby inhibiting gel secretion and maintaining the vessel lumens devoid of occlusions. The Cu²⁺ treatments significantly improved water uptake by the cut stems as compared to the control.     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> as a model for gelatinous fiber formation

Trees and herbaceous plants continuously monitor their position to maintain vertical stem growth and regulate branch orientation. When orientation is altered from the vertical, they form a special type of wood called reaction wood that differs chemically and structurally from normal wood and forces reorientation of the organ or whole plant. The reaction wood of dicotyledons is called tension wood and is characterized by nonlignified gelatinous fibers. The altered chemical and mechanical properties of tension wood reduce wood quality and represent a major problem for the timber and pulping industries. Repeated clipping of the emerging inflorescence stems of Arabidopsis thaliana augments wood formation in organs, including those inflorescence stems that are allowed to develop later. Gravistimulation of such inflorescence stems induces tension wood formation, allowing the use of A. thaliana for a molecular and genetic analysis of the mechanisms of tension wood formation.     

Eucalyptus gunnii CCR and CAD2 promoters are active in lignifying cells during primary and secondary xylem formation in Arabidopsis thaliana

Cell-specific expression patterns of the Eucalyptus gunnii cinnamoyl coenzymeA reductase (EgCCR) and cinnamyl alcohol dehydrogenase (EgCAD2) promoters were analyzed by promoter-GUS histochemistry in the primary and secondary xylem tissues from floral stems and roots of Arabidopsis thaliana. Expression patterns indicated that the EgCCR and EgCAD2 genes were expressed in a coordinated manner in primary and secondary xylem tissues of the Arabidopsis floral stem and root. Both genes were expressed in all lignifying cells (vessel elements, xylem fibers and paratracheal parenchyma cells) of xylem tissues. The capacity for long-term monolignol production appeared to be related to the cell-specific developmental processes and biological roles of different cell types. Our results suggested that lignification of short-lived vessel elements was achieved by a two-step process involving (i) monolignol production by vessel elements prior to vessel programmed cell death and (ii) subsequent monolignol production by vessel-associated living paratracheal parenchyma cells following vessel element cell death. EgCCR and EgCAD2 gene expression patterns suggested that the process of xylem cell lignification was similar in both primary and secondary xylem tissues in Arabidopsis floral stems and roots.     

Wound-induced and naturally occurring regenerative differentiation of xylem in Zea mays L.

The regenerative differentiation of xylem, both around a wound in the stem and at the root junction was studied in seedlings of maize. The regeneration of vessels around a wound was very small, being limited to the very young internodes and sharply declining basipetally. There were more regenerative vessel elements and they differentiated faster above the wound than below it. The regenerative vessel elements around the wound were characterized by helical or annular pattern of secondary wall thickenings. Wounding also resulted in the development of additional vascular anastomoses in the leaf immediately above the wound, and in differentiation of discontinuous vessels in adjacent bundles. Regenerative vessel elements were very common where the adventitious roots connected with the stem internodes, and exhibited pitted or reticulated secondary wall thickenings.     

The Inflorescence Stem Fibers of <Emphasis Type="Italic">Arabidopsis thaliana Revoluta</Emphasis> (<Emphasis Type="Italic">ifl1</Emphasis>) Mutant

Arabidopsis thaliana is gradually gaining significance as a model for wood and fiber formation.revolute/ifl1 is an important mutant in this respect. To better characterize the fiber system of therevolute/ifl1 mutant, we grew plants of two alleles (rev-9 in Israel andrev-1 in the USA) and examined the fiber system of the inflorescence stems using both brightfield and polarized light. Microscopic examination of sections of plants belonging to the two different alleles clearly revealed that, contrary to previous views, in 18 (13 in Israel and 5 in Ohio) out of 30 stems (20 in Israel and 10 in Ohio) the mutant produced the primary wavy fiber system of the inflorescence stems. Our findings are further supported by the fact that fibers are seen in the figures published in other studies of the mutant even when it was stated that there were no fibers. The impression of a total lack of the wavy band of fibers is in many cases just a result of poorly lignified secondary walls. This specific gene that reduces lignification in fibers is of great significance for biotechnological developments for the paper industry and thus for the global economy and ecology. We propose thatrevoluta, the first name given to this mutant (Talbert and others 1995), is more appropriate thanifl1. Online publication: 7 April 2005     

Stem anatomy of Dolichos lablab Linn (Fabaceae): Origin of cambium and reverse orientation of vascular bundles

Secondary growth in the stem of Dolichos lablab is achieved by the formation of eccentric successive rings of vascular bundles. The stem is composed of parenchymatous ground tissue and xylem and phloem confined to portions of small cambial segments. However, development of new cambial segments can be observed from the obliterating ray parenchyma, the outermost phloem parenchyma and the secondary cortical parenchyma. Initially cambium develops as small segments, which latter become joined to form a complete cylinder of vascular cambium. Each cambial ring is functionally divided into two distinct regions. The one segment of cambium produces thick-walled lignified xylem derivatives in centripetal direction and phloem elements centrifugally. The other segment produces only thin-walled parenchyma on both xylem and phloem side. In mature stems, some of the axial parenchyma embedded deep inside the xylem acquires meristematic activity and leads to the formation of thick-walled xylem derivatives centrifugally and phloem elements centripetally. The secondary xylem comprises vessel elements, tracheids, fibres and axial parenchyma. Rays are uni-multiseriate in the region of cambium that produces xylem and phloem derivatives, while in some of the regions of cambium large multiseriate, compound, aggregate and polycentric rays can be noticed.     

The development of phloem anastomoses between vascular bundles and their role in xylem regeneration after wounding in Cucurbita and Dahlia

The differentiation of phloem anastomoses linking the longitudinal vascular bundles has been studied in stem internodes of Cucurbita maxima Duchesne, C. pepo L. and Dahlia pinnata Cav. These anastomoses comprise naturally occurring regenerative sieve tubes which redifferentiate from interfascicular parenchyma cells in the young internodes. In all three species, severing a vascular bundle in a young internode resulted in regeneration of xylem to form a curved by-pass immediately around the wound. The numerous phloem anastomoses in these young internodes were not involved in this process, the regenerated vessels originating from interfascicular parenchyma alone. Conversely, in mature internodes of Dahlia, the regenerated vessels originated from initials of the interfascicular cambia, and their phloem anastomoses did not influence the pattern of xylogenesis. On the other hand, in old internodes of Cucurbita, in which an interfascicular cambium was not yet developed, the parenchyma cells between the bundles had lost the ability to redifferentiate into vessel elements, and instead, regenerated vessels were produced in the phloem anastomoses. Thus, the wounded region of the vascular bundle was not bypassed via the shortest, curved pathway, but by more circuitous routes further away from the wound. Some of the regenerated vessels produced in the phloem anastomoses were extremely wide, and presumably efficient conductors of water. It is proposed that the dense network of phloem anastomoses developed during evolution as a mechanism of adaptation to possible damage in mature internodes by providing flexible alternative pathways for efficient xylem regeneration in plants with limited or no interfascicular cambium.This paper is dedicated to the memory of the late Isaac Blachmann (deceased 19 November 1995), father-in-law of the senior author, for encouragement and advice throughout the yearsThis research was supported by an International Scientific Exchange Award to R.A. from the Israel Academy of Sciences and The Royal Society.     

Stems of the <Emphasis Type="Italic">Arabidopsis pin1-1</Emphasis> mutant are not deficient in free indole-3-acetic acid

The pin1-1 mutant of Arabidopsis thaliana has been pivotal for studies on auxin transport and on the role of auxin in plant development. It was reported previously that when whole shoots were analysed, levels of the major auxin, indole-3-acetic acid (IAA) were dramatically reduced in the mutant, compared with the WT (Okada et al. 1991). The cloning of PIN1, however, provided evidence that this gene encodes a facilitator of auxin efflux, raising the question of how the pin1-1 mutation might reduce overall IAA levels as well as IAA transport. We therefore re-examined IAA levels in individual parts of pin1-1 and WT plants, focusing on inflorescence stems. Our data show that there is in fact no systemic IAA deficiency in the mutant. The previously reported difference between mutant and WT may have been due to the inclusion of reproductive structures in the WT harvest: we show here that the inflorescence itself contains high levels of IAA. We reconcile the normal IAA levels of pin1-1 inflorescence stems with their (previously-reported) reduced ability to transport IAA by presenting evidence that the auxin in mutant stems is not imported from their apical portion. Our data also indicate that levels of another auxin, indole-3-butyric acid (IBA), are very low in stems of the genotypes used in this study.     

The arabidopsis ATHB-8 HD-zip protein acts as a differentiation-promoting transcription factor of the vascular meristems

ATHB-8, -9, -14, -15, and IFL1/REV are members of a small homeodomain-leucine zipper family whose genes are characterized by expression in the vascular tissue. ATHB-8, a gene positively regulated by auxin (Baima et al., 1995), is considered an early marker of the procambial cells and of the cambium during vascular regeneration after wounding. Here, we demonstrate that although the formation of the vascular system is not affected in athb8 mutants, ectopic expression of ATHB-8 in Arabidopsis plants increased the production of xylem tissue. In particular, a careful anatomical analysis of the transgenic plants indicated that the overexpression of ATHB-8 promotes vascular cell differentiation. First, the procambial cells differentiated precociously into primary xylem. In addition, interfascicular cells also differentiated precociously into fibers. Finally, the transition to secondary growth, mainly producing xylem, was anticipated in transgenic inflorescence stems compared with controls. The stimulation of primary and secondary vascular cell differentiation resulted in complex modifications of the growth and development of the ATHB-8 transgenic plants. Taken together, these results are consistent with the hypothesis that ATHB-8 is a positive regulator of proliferation and differentiation, and participates in a positive feedback loop in which auxin signaling induces the expression of ATHB-8, which in turn positively modulates the activity of procambial and cambial cells to differentiate.     

Molecular cloning and functional characterization of Os<Emphasis Type="Italic">JAG</Emphasis> gene based on a complete-deletion mutant in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The evolution of plant vascular tissue is tightly linked to the evolution of specialised cell walls. Mutations in the QUASIMODO2 (QUA2) gene from Arabidopsis thaliana were previously shown to result in cell adhesion defects due to reduced levels of the cell wall component homogalacturonic acid. In this study, we provide additional information about the role of QUA2 and its closest paralogues, QUASIMODO2 LIKE1 (QUL1) and QUL2. Within the extensive QUA2 family, our phylogenetic analysis shows that these three genes form a clade that evolved with vascular plants. Consistent with a possible role of this clade in vasculature development, QUA2 is highly expressed in the vascular tissue of embryos and inflorescence stems and overexpression of QUA2 resulted in temperature-sensitive xylem collapse. Moreover, in-depth characterisation of qua2 qul1 qul2 triple mutant and 35S::QUA2 overexpression plants revealed contrasting temperature-dependent stem development with dramatic effects on stem width. Taken together, our results suggest that the QUA2-specific clade contributed to the evolution of vasculature and illustrate the important role that modification of cell wall composition plays in the adaptation to changing environmental conditions, including changes in temperature.     

Differential anatomical response of Norway spruce stem tissues to sterile and fungus infected inoculations

The anatomical defense responses in stems of Norway spruce (Picea abies) clones of different resistance to pathogenic fungi were characterized over time and distance from small mechanical wounds or wounds inoculated with the root rot fungus Heterobasidion annosum. Common responses for both treatments included division of ray parenchyma and other cells in the cambial zone, accumulation of phenolic inclusions in ray parenchyma cells, activation of phloem parenchyma (PP) cells, and formation of traumatic resin ducts (TDs) in the xylem. TD formation occurred synchronously from a tangential layer of cells, or symplasmic domain, within the zone of xylem mother cells. TD induction is triggered by a signal, which propagates a developmental wave in the axial direction at about 2.5 cm per day. TDs are formed at least 30 cm above single inoculations within 16–36 days after inoculation. The size and number of TDs is attenuated further away from the inoculation site, indicating a dose-dependent activity leading to TD development. Compared to sterile wounding, fungal inoculation gave rise to more and larger TDs in all clones, and multiple rows of TDs in weak clones. Fungal inoculation also induced the formation of more new PP cells, increasing the number of PP cells in the phloem in the year of inoculation up to 100%. TD and PP cell formation was greater in susceptible compared to resistant clones and after fungal versus sterile inoculation. Potential mechanisms responsible for this variable response are discussed.     

Redirection of the phenylpropanoid pathway to feruloyl malate in <Emphasis Type="Italic">Arabidopsis</Emphasis> mutants deficient for cinnamoyl-CoA reductase 1

Cinnamoyl-CoA reductase 1 (CCR1, gene At1g15950) is the main CCR isoform implied in the constitutive lignification of Arabidopsis thaliana. In this work, we have identified and characterized two new knockout mutants for CCR1. Both have a dwarf phenotype and a delayed senescence. At complete maturity, their inflorescence stems display a 25–35% decreased lignin level, some alterations in lignin structure with a higher frequency of resistant interunit bonds and a higher content in cell wall-bound ferulic esters. Ferulic acid-coniferyl alcohol ether dimers were found for the first time in dicot cell walls and in similar levels in wild-type and mutant plants. The expression of CCR2, a CCR gene usually involved in plant defense, was increased in the mutants and could account for the biosynthesis of lignins in the CCR1-knockout plants. Mutant plantlets have three to four-times less sinapoyl malate (SM) than controls and accumulate some feruloyl malate. The same compositional changes occurred in the rosette leaves of greenhouse-grown plants. By contrast and relative to the control, their stems accumulated unusually high levels of both SM and feruloyl malate as well as more kaempferol glycosides. These findings suggest that, in their hypolignified stems, the mutant plants would avoid the feruloyl-CoA accumulation by its redirection to cell wall-bound ferulate esters, to feruloyl malate and to SM. The formation of feruloyl malate to an extent far exceeding the levels reported so far indicates that ferulic acid is a potential substrate for the enzymes involved in SM biosynthesis and emphasizes the remarkable plasticity of Arabidopsis phenylpropanoid metabolism.     

Activities of auxin polar transport in inflorescence axes of flower mutants ofArabidopsis thaliana: Relevance to flower formation and growth

Relationships between the activity of auxin polar transport and flower formation were studied using several flower mutants ofArabidopsis thaliana. The activity of auxin polar transport in the upper portion of inflorescence axis of wildtype plants ofArabidopsis thaliana was significantly lower than that of the basal part. The activities of auxin polar transport in the upper portion of inflorescence axes ofap1 andclv1 mutants were significantly higher than that of wild-type plant. However, those of other flower mutants tested,ap3-1, ag, pi, Fl-40, Fl-54, Fl-89 andpin-formed, were extremely low as compared with that of wild one. We got some evidence that the reduction of the activity of auxin polar transport is concerned with the growth and development of plants. We could mimic it by the removal of all flowers and pods including mature or immature seeds. Moreover, artificial pollination inap3-1 andpi mutants, in which no seeds are found naturally, resulted in the partial recovery of the activity of auxin polar transport in inflorescence axis. Considering these results in this study together with the fact that inhibitors of auxin polar transport generated almost same disruptions ofpin-formed orpinoid mutants which normally had no flowers in inflorescence axis (Okadaet al. 1991, Uedaet al. 1992, Bennettet al. 1995), the systern of auxin polar transport and its activity in inflorescence axis seems to be essential for the development of flower bud in early stage ofArabidopsis thaliana, and the activity of auxin polar transport is also regulated by the formation of flowers and seeds in inflorescence axis.     

Fibres and Fibre-sclereids in Wild-typeArabidopsis thaliana

Examination of the fibre system in roots and shoots ofArabidopsisthaliana(L.) Heynh. revealed three types of fibres, distinguishedaccording to their site of differentiation: (1) short fibre-sclereidsin the secondary phloem of roots and shoots at the rosette level;(2) long fibres in the secondary xylem of the main root; and(3) very long fibres in the xylem of the inflorescence stems.These three types are in addition to the small number of primaryphloem fibres that are formed even in smallA. thalianaindividuals.These findings provide a basis for the use ofArabidopsis thalianaasa model system to study the processes leading to fibre differentiationin dicotyledons. Arabidopsis thaliana; differentiation; fibre-sclereids; inflorescences; phloem fibres; xylem