Extracellular invertase is involved in the regulation of clubroot disease in Arabidopsis thaliana

Clubroot disease of Brassicaceae is caused by an obligate biotrophic protist, Plasmodiophora brassicae. During root gall development, a strong sink for assimilates is developed. Among other genes involved in sucrose and starch synthesis and degradation, the increased expression of invertases has been observed in a microarray experiment, and invertase and invertase inhibitor expression was confirmed using promoter::GUS lines of Arabidopsis thaliana. A functional approach demonstrates that invertases are important for gall development. Different transgenic lines expressing an invertase inhibitor under the control of two root-specific promoters, Pyk10 and CrypticT80, which results in the reduction of invertase activity, showed clearly reduced clubroot symptoms in root tissue with highest promoter expression, whereas hypocotyl galls developed normally. These results present the first evidence that invertases are important factors during gall development, most probably in supplying sugars to the pathogen. In addition, root-specific repression of invertase activity could be used as a tool to reduce clubroot symptoms.     

A hormone and proteome approach to picturing the initial metabolic events during Plasmodiophora brassicae infection on Arabidopsis

We report on the early response of Arabidopsis thaliana to the obligate biotrophic pathogen Plasmodiophora brassicae at the hormone and proteome level. Using a CYCB1;1::GUS construct, the re-initiation of infection-related cell division is shown from 4 days after inoculation on. Sensitivity to cytokinins and auxins as well as the endogenous hormone levels are evaluated. Both an enhanced cytokinin gene response and an accumulation of isopentenyl adenine and adenosine precede this re-initiation of cell division, whereas an enhanced auxin gene response is observed from 6 days after inoculation on. The alhl mutant, impaired in the cross talk between ethylene and auxins, is resistant to P. brassicae. A differential protein analysis of infected versus noninfected roots and hypocotyls was performed using two-dimensional gel electrophoresis and quantitative image analysis, coupled to matrix-assisted laser desorption ionization time of flight-time of flight mass spectrometry-based protein identification. Of the visualized proteins, 12% show altered abundance compared with the noninfected plants, including proteins involved in metabolism, cell defense, cell differentiation, and detoxification. Combining the hormone and proteome data, we postulate that, at the very first stages of Plasmodiophora infection, plasmodial-produced cytokinins trigger a local re-initiation of cell division in the root cortex. Consequently, a de novo meristematic area is established that acts as a sink for host-derived indole-3-acetic acid, carbohydrates, nitrogen, and energy to maintain the pathogen and to trigger gall development.     

A Histopathological Study of Anomalous Growth in the Stem of Coccinia indica W. & A. Infested with Neolasioptera cephalandrae Mani

Gall formation due to attack of the larvae of Neolasiopteracephalandrae is common on the stem of Coccinia indica. Isolatedpatches of secondary meristematic centres develop throughoutthe proliferated tissue and they often differentiate into vasculartissues (either xylem or phloem or both). The larvae bore throughthe stem and live in lysigenous cavities in the ground tissue.Proliferation of cells of the secondary meristematic centresresults in gall formation and the normal vascular pattern isdisturbed and vascular bundles crushed. Development of mechanicaltissues in the affected region is suppressed. Additional cambiallayers with no definite pattern of orientation also developin the ground tissue and these produce patches of xylem tissue.Larval cavities are irregular and are surrounded by a nutritivetissue rich in protein and polysaccharides.     

Myb genes from Hordeum vulgare: tissue-specific expression of chimeric Myb promoter/Gus genes in transgenic tobacco

The structures of the three Myb -related genes Hv1 , Hv5 and Hv33 from barley were determined. They contain a single intron located in the second repeat unit of the Myb -related domain. By analogy to the animal MYB oncoproteins this conserved region of the gene product was shown by filter-binding experiments to exhibit nucleic acid-binding activity. Tobacco plants transgenic for chimeric Myb promoter/ Gus genes express the enzyme in a developmentally controlled and tissue-specific manner. During germination and early stages of plant growth, GUS activity is seen in the root cap and adjacent meristematic tissue. At later stages of plant development, GUS activity is predominantly observed in the shoot apical meristem, the roots and the nodal regions of the stem. Within the stem at stages of secondary growth, Myb promoters are active in defined cell types. In the internode low GUS activity is displayed by the innermost cell layer of the cortex, the starch sheath, that surrounds the vascular cylinder of secondary xylem and phloem tissue, as well as in pith rays originating from vascular cambium initials. In the nodal region Myb promoter-controlled Gus expression is mainly confined to the abaxial starch sheath of the leaf trace, to the branch traces and to internal strands of primary phloem. It is suggested that in addition to their activity in meristematically active plant tissues Myb genes are expressed in conductive tissues that are closely associated with vascular bundles.     

胡萝卜HRGP启动子调控GUS基因的某些特点

HRG(hydroxyproline rich glycoproteins)是高等植物细胞壁中一类富含羟脯氨酸的糖蛋白,是植物细胞壁中主要的结构蛋白。早期研究认为其在细胞壁的形成过程中起作用并称之为伸展蛋白(extensin)。在双子叶植物中,HRGP基因以多基因家族形式存在,具有特定的表达模式。许多条件及处理都能引起HRGP表达量的增加,如伤害、真菌感染、病毒感染、乙烯、细胞培养、红光等,同时也受到发育水平的调节。在单子叶植物中,玉米HRGP的表达是在发育水平上受伤害调节的。HRGP基因还具有组织专一性表达的特点。在大豆种子中,HRGP主要存在于种皮的外两层、表皮栅栏组织和滴漏细胞中,属于起支持作用的厚壁组织。在胡萝卜根韧皮部薄壁细胞中HRGP含量最丰富,而在健康的番茄根中     

The effect of cytokinin- and auxin-like substances on the anatomy and ultrastructure of vascular bundles in wheat

6-benzylaminopurine (BAP), β-indolylbutyric acid (IBA), phenylacetic acid (PAA) and a triazine herbicide (TH) affected the development of vascular bundles both in coleoptiles and the last intemodium below the ear of wheat plants. These substances were applied before the conductive tissues in the organs investigated were fully differentiated, the degree of their effect being dependent not only on the concentration, but also on the developmental stage of the plants and on cultivation conditions. In coleoptiles the area of vascular bundles was increased especially in the dark. In the stalk, BAP, PAA, IBA and IBA + TH increased the area of phloem. BAP also stimulated the activity of parenchymatous cells of xylem. TH had the same effect only on xylem, which became evident by an increase in vascular bundle area, but not in that of phloem. These anatomical aberrations were confirmed by cytological analysis which revealed that the stimulatory effect of the substances investigated consisted in activation both of companion cells of phloem and parenchymatous cells of xylem, whose function is analogous. As a result, both types of cells contained numerous organelles characteristic of meristematic cells, which suggests protein synthesis and an active transport of substances.     

Fine structure,pattern of division,and course of wound phloem in Coleus blumei

The wound phloem bridges which have developed six days after interrupting an internodal vascular bundle contain wound sieve-elements, companion cells, and phloem parenchyma cells. An analysis of the meristematic activity responding to the wounding clearly demonstrates that three consecutive divisions are prerequisite to the formation of phloem mother-cells. Companion cells are obligatory sister cells of wound sieve-elements, connected to the latter by specific plasmatic strands and provided with a dense protoplast. Six days after wounding most of the wound sieve-elements are still at a nucleate state of development, but already have characteristic P-protein bodies and plastids containing sieve-element starch. Their cytoplasmic differentiation corresponds to the changes recorded during maturation of ordinary sieve elements. Sieve-plate pores penetrate through preexisting parenchyma cell walls, only, and develop from primary pitfield-plasmodesmata. Wound sieve-elements do not connect to preexisting bundle sieve-elements, they open a new tier of young sieve elements produced by cambial activity.     

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     

Effects of Infection by Plasmodiophora brassicae on Root Anatomy of Rape

The anatomy of five-week old roots of rape (Brassica napus L.) infected by Plasmodiophora brassicae Wor. was studied and compared with corresponding non-infected roots using light microscopy and scanning electron microscopy techniques. The club-root gall was characterized by atypically initiated vascular strands separated by large, proliferating parenchymatic cells originating from secondary tissues and infected by the parasite. Similarities in the anatomy of the club-root gall and storage roots of Brassica are discussed together with aspcets of the pathogen control of the host anatomy.     

Identification of genes involved in Meloidogyne incognita-induced gall formation processes in Arabidopsis thaliana

Root-knot nematodes (RKN; Meloidogyne incognita) are phytoparasitic nematodes that cause significant damage to crop plants worldwide. Recent studies have revealed that RKNs disrupt various physiological processes in host plant cells to induce gall formation. However, little is known about the molecular mechanisms of gall formation induced by nematodes. We have previously found that RNA expression levels of some of genes related to micro-RNA, cell division, membrane traffic, vascular formation, and meristem maintenance system were modified by nematode infection. Here we evaluated these genes importance during nematode infection by using Arabidopsis mutants and/or β-glucronidase (GUS) marker genes, particularly after inoculation with nematodes, to identify the genes involved in successful nematode infection. Our results provide new insights not only for the basic biology of plant–nematode interactions but also to improve nematode control in an agricultural setting.     

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.     

Immunolocalization of cinnamyl alcohol dehydrogenase 2 (CAD 2) indicates a good correlation with cell-specific activity of CAD 2 promoter in transgenic poplar shoots

Cinnamyl alcohol dehydrogenase 2 (CAD 2) localization and the cell-specific activity of the eucalyptus CAD 2 promoter were investigated by CAD 2 immunogold localization and promoter β-glucuronidase (GUS) histochemistry in apical and mature parts of stable transformed poplar (Populus tremula × P. alba) stems. Both CAD 2 protein and GUS activity were found to be confined in the same types of cells in the shoot apices, particularly in the determined meristematic cells in leaf axils and shell zones, procambium and developing tracheids. Within mature stems, CAD 2 and GUS were also identified in cambium and in fully or partially lignified cells derived from it (young xylem, developing phloem fibres, chambered parenchyma cells around phloem). Additionally, GUS activity was found in the scale leaves of apical shoot buds and in the roots (namely in the procambium, cambium, phellogen, young xylem, pericycle) of transformed plants. By employing immunogold cytochemistry, CAD 2 was shown to be localized in the cytoplasm within cambial, ray and young xylem cells in stems, the gold particles being randomly attached to endoplasmic reticulum and Golgi-derived vesicles. These results support a crucial role for CAD 2 in lignification and indicate a new role for this enzyme in branching events within the shoot apex and during lateral root formation. Received: 24 April 1997 / Accepted: 17 July 1997     

Cell type specific expression of a CaMV 35S-GUS gene in transgenic soybean plants

A number of independently derived transgenic soybean plants expressing a chimeric β-glucuronidase (GUS) gene under the control of the 355 CaMV promoter and a nopaline synthase polyadenylation signal were recovered using direct DNA transfer via electric discharge particle acceleration. Expression of GUS in R, plants was localized using thin tissue sections. Many tissue types expressed GUS at various levels. Pericycle cells in root, parenchyma cells in xylem, and phloem tissues of stem and leaf had high levels of enzyme activity. Procambium, phloem, and cortex cells in root, vascular cambium cells in stem, and the majority of cortex cells in leaf midrib, expressed low or no GUS activity. Intermediate levels of GUS activity were detected in leaf mesophyll cells, certain ground tissue cells in stem and leaf midrib, and in trichome and epidermal guard cells. Thus, we conclude that the 35S CaMV promoter is cell-type specific and is developmentally regulated in soybean.     

The Structure and Development of Vascular Nodules in Apple Bud-unions: With two Plates and two Figures in the Text

An account is given of the development of new meristematic centres(vascular nodules) initiated in callus tissue formed in applestems as a result of budding. Both amphivasal (xylem peripheral)and amphicribral (phloem peripheral) nodules occurred, and attentionis drawn to the relationship between the type of nodule formedand the tissue in which it originated. The similarity of thesestructures to the meristematic nodules arising in tissue culturesis discussed, and their possible practical significance as anaid in the rooting of cuttings pointed out.     

Evidence for a plasma membrane proton pump in phloem cells of higher plants

Metabolic energy is required for the loading of sucrose into the phloem and translocation of sugars throughout the plant. The proton electrochemical gradient generated by a plasma membrane proton pump (H(+)-ATPase) is thought to provide energy for these processes. The plasma membrane H(+)-ATPase is encoded by a multigene family in Arabidopsis thaliana. Here we characterize the expression of isoform AHA3 (Arabidopsis H(+)-ATPase isoform 3). The AHA3 mRNA start site was mapped and 464 bp of the putative upstream regulatory region sequenced. A translational fusion of AHA3 to the beta-glucuronidase (GUS) reporter gene was constructed and used to generate transgenic Nicotiana and Arabidopsis plants. Using a histochemical stain, expression of the AHA3/GUS fusion was found predominantly in phloem cells of leaves, stems, roots, and flowers. Biochemical measurements of GUS activity in pith and vascular explants confirmed the histochemical localization. Our results support the hypothesis that a proton pump is present in phloem cells, possibly providing energy to drive plasma membrane cotransport systems required for phloem loading and translocation of photosynthates. In addition to AHA3/GUS expression in phloem, expression was observed in pollen and regions of the ovule, tissues whose physiological functions correlate with a requirement for high levels of solute transport.