John A. Webb

Ultrastructure of minor veins inCucurbita pepo leaves

SummaryThe minor veins ofCucurbita pepo leaves were examined as part of a continuing study of leaf development and phloem transport in this species. The minor veins are bicollateral along their entire length. Mature sieve elements are enucleate and lack ribosomes. There is no tonoplast. The sieve elements, which are joined to each other by sieve plates, contain mitochondria, plastids and endoplasmic reticulum as well as fibrillar and tubular (190–195 Ā diameter) P-protein. Fibrillar P-protein is dispersed in mature abaxial sieve elements but remains aggregated as discrete bodies in mature adaxial sieve elements. In both abaxial and adaxial mature sieve elements tubular P-protein remains undispersed. Sieve pores in abaxial sieve elements are narrow, lined with callose and are filled with P-protein. In adaxial sieve elements they are wide, contain little callose and are unobstructed. The intermediary cells (companion cells) of the abaxial phloem are large and dwarf the diminutive sieve elements. Intermediary cells are densely filled with ribosomes and contain numerous small vacuoles and many mitochondria which lie close to the plasmalemma. An unusually large number of plasmodesmata traverse the common wall between intermediary cells and bundle sheath cells suggesting that the pathway for the transport of photosynthate from the mesophyll to the sieve elements is at least partially symplastic. Adaxial companion cells are of approximately the same diameter as the adaxial sieve elements. They are densely packed with ribosomes and have a large central vacuole. They are not conspicuously connected by plasmodesmata to the bundle sheath.

Leaf development and phloem transport in Cucurbita pepo: Transition from import to export.

SummaryThe capacity of a growing leaf blade of Cucurbita pepo L. to import 14C-labelled photoassimilate is lost in a basipetal direction. Import into the lamina tip stops when the blade is 10% expanded. Development of the leaf progresses linearly with time and the lamina base stops importing when the blade is 45% expanded. Export capacity also develops basipetally and follows immediately the loss of import capacity, at least in the lamina base. The small amount of material initially exported from the leaf tip is redistributed to the still-importing leaf base, delaying export from the lamina until the blade is 35% expanded. Loss of import capacity by the petiole is both basipetal and dorsoventral. The proximal, adaxial portion of the petiole is the last region to cease importing 14C. Leaves of Beta vulgaris L. and Nicotiana tabacum L. also lose import capacity in a basipetal direction.

Leaf development and phloem transport in Cucurbita pepo: Carbon economy

SummaryNet photosynthesis, dark respiration and growth for leaf 5 of Cucurbita pepo L. plants grown under controlled conditions were measured and the data used for an assessment of the changes in carbon balance during growth of the leaf through expansion to maturity. The blade is first capable of net CO2 fixation when ca. 8% expanded but the initial rapid growth during this period is sustained almost entirely through imported nutrients. When the growth rate starts to decline rapidly the net photosynthetic capacity of the blade begins to increase. This increase is accompanied by an expansion of the intercellular spaces and by decreasing dark respiration measured at night and in dark periods during the day. The blade becomes completely independent of phloem imported nutrients and begins to export excess photosynthate when the phase of rapid decrease in relative growth rate is almost complete at about 45% expansion. Maximum net photosynthesis of ca. 11 mg CO2 h-1 dm-2 is achieved at 70% expansion. The first detectable synthesis of the transport sugars stachyose and raffinose in the blade coincides with the beginning of intralaminar phloem transport from the tip to the base of the leaf. The synthesis of sucrose, the other major transport sugar, is detectable at all stages of leaf development.

Leaf development and phloem transport in Cucurbita pepo: Maturation of the minor veins.

SummaryYoung leaves of Cucurbita pepo L. were examined by whole-leaf autoradiography and serial paradermal sections were examined by light microscopy to determine whether commencement of sugar export depends upon the minor vein phloem achieving structural maturity. Maturation of these veins develops progressively from the largest toward the smallest elements with the minor veins in the distal region of the leaf maturing before those in the proximal region. Commencement of sugar export is coincident with maturation of the abaxial phloem of the minor veins delimiting the areoles. The abaxial phloem elements of the larger minor veins, which are probably capable of vein loading too but border only relatively few areoles, mature before export starts. The adaxial phloem surrounding the areoles and the xylem elements, mature in advance of the abaxial phloem and well before the beginning of sugar export. It is therefore considered unlikely that structural development alone directly governs the initiation of export. The results suggest that some other rate controlling step is involved.

Development of a simple particle bombardment device for gene transfer into plant cells

A simple particle bombardment device was designed, constructed and shown to be efficient for the delivery of DNA into plant cells. High levels of transient β-glucuronidase expression were observed in alfalfa suspension-cultured cells and embryogenic soybean suspension-cultured cells. Expression of β-glucuronidase in alfalfa suspension-cultured cells was used to optimize the bombardment conditions for the device. Transient gene expression in alfalfa was found to be dependent on the state of the target tissue, the size of particles employed, the helium pressure used to accelerate the particles and the distance travel led by the tungsten particles carrying DNA.

Stachyose synthesis in leaves of Cucurbita pepo

Abstract An enzyme synthesizing stachyose, galactinol-raffinose galactosyltransferase (EC2.4.1.67), has been purified ca 40-fold from mature leaves of Cucurbita pepo using ammonium sulphate precipitation, Sephadex gel filtration and DEAE-Sephadex gel chromatography. The purified enzyme fraction was separated from all but 2 % of the total,α-galactosidase activity extracted from the tissue. The enzyme was optimally active at pH 6.9 and was stable for at least a month at 4° in the presence of 20 mM 2-mercaptoethanol. The enzyme displayed high specificity for the donor galactinol (K m 7.7 mM) and the acceptor raffinose (K m 4.6 mM) and was unable to effect synthesis of any other member of the raffinose series of galactosyl-sucrose oligosaccharides. Co 2+ , Hg 2+ , Mn 2+ and Ni 2+ ions were particularly inhibitory; no metal ion promotion was observed and 5 mM EDTA was ineffective. Myo-inositol was strongly inhibitory (K i 2 mM), melibiose weakly so. Tris buffer (0. 1 M) was also inhibitory. Galactinol hydrolysis occurred in the absence of the acceptor raffinose but there was no hydrolysis of either raffinose or stachyose in the absence of the donor galactinol. The reaction was readily reversible and exchange reactions were detected between substrates and products. It is proposed that the synthesis of stachyose in mature leaves of C. pepo proceeds via this galactosyltransferase and not via α-galactosidase.

In vitro response and pedigree analysis for somatic embryogenesis of long-day photoperiod adapted soybean

Northern-adapted soybean cultivars were screened for their ability to form somatic embryos in vitro in response to exposure to 180 μM 2,4-dichlorophenoxyacetic acid in a modified Murashige & Skoog (1962) medium containing B5 (Gamborg et al. 1968) organics and 43.5 mM sucrose. The 20 cultivars formed between 1 and 7 embryos per cotyledon with between 18% and 98% of the cotyledons responding. The response was genotype dependant. Three ancestral lines, ‘840-7-3’, ‘A.K. Harrow’ and ‘Mandarin’ were prominent in the genetic background of long-photoperiod adapted lines and showed a high degree of somatic embryogenesis.

Multiple forms of α-galactosidase in mature leaves of Cucurbita pepo

Abstract α- D -Galactosidase has been purified from mature leaves of Cucurbita pepo using pH and ammonium sulphate fractionation, Sephadex gel filtration and DEAE Sephadex gel chromatography. Gel filtration produced one peak of α-galactosidase activity from which three distinct enzyme forms were resolved on DEAE Sephadex and designated LI, LII and LIII. Purirications obtained were ca 75, 120 and 30 fold for LI, Lll and LIII respectively. Ll was slightly contaminated with β-galactosidase and LII with β-fructosidase activity. All forms hydrolysed the α-galactosyl linkages of raffinose and stachyose. Differences between each form were found in their pH optima, reactivity toward metal ions, thermal stability and K m values using either p -nitrophenyl-α- D -galactoside (NPG) or raffinose as substrates. All forms were inhibited by NPG at high concentrations and by α- D -galactose. It is proposed that α-galactosidases may be components of a lysosomal system in plant cells.

Alkaline α-galactosidase activity and galactose metabolism in the family Cucurbitaceae

Abstract The distribution of alkaline α-galactosidase in various organs of Cucurbita pepo was studied. Organs such as roots and immature leaves which import stachyose, a major translocated sugar in this species, were found to contain higher levels of this enzyme compared to mature leaves. Immature leaves were also found to contain twice as much galactokinase and UDP-galactose pyrophosphorylase, two key enzymes for galactose incorporation into the hexose phosphate pool, than mature leaf tissue. The presence of an alkaline α-galactosidase is reported for two other cucurbits, Cucumis sativus and C. maxima . The concentrations of the enzyme in these species was similarly correlated with the stage of leaf development, the immature leaves containing 6–8-fold more activity than mature leaves. The alkaline α-galactosidase from C. sativus cotyledons was partially purified and its properties found to be very similar to those of the C. pepo enzyme. The results suggest that in cucurbits immature leaf tissue rapidly metabolizes stachyose through increased levels of alkaline α-galactosidase and other enzymes associated with galactose utilisation.

Physiological and Structural Ontogeny of the Source Leaf

A leaf is transformed during ontogenesis from a heterotrophic, to an autotrophic, organ. Initial dependence on phloem imported nutrients is lost as increasing photosynthetic capacity enables the lamina to satisfy its own carbon requirements, and to export excess photosynthate. This transition is interesting from both a practical and a theoretical viewpoint. The leaf becomes an asset to the carbon economy of the plant only when it is autotrophic and has begun to export. Theoretically, the study of conditions which contribute to the start of assimilate flow should provide insight into the mechanisms of vein loading and long-distance transport.