Proteins in Intercellular Washing Fluids from Leaves of Barley (<i>Hordeum vulgare</i>L.)

Rohringer, R.; Ebrahim‐Nesbat, F.; Wolf, Gerhard

doi:10.1093/jxb/34.12.1589

Cited by 86 publications

(44 citation statements)

References 31 publications

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“…Rohringer et al (21) used a centrifugation technique to obtain infiltrated washing fluid from barley leaves and our results with centrifugation (Table I) were similar to theirs (21). Less soluble protein and less peroxidase activity, but higher G6P-D activity, was obtained with the centrifugation technique than with our simple peeling and vacuum infiltration technique, suggesting that out technique is more efficient and less damaging for these tissues.…”

Section: Discussionsupporting

confidence: 85%

“…The malate dehydrogenase (MDH, EC 1.1.1.37) assay mix (3) contained 0.16 ,umol NADH, 1 umol oxaloacetic acid, 45 ,umol k-phosphate (pH 7.5), and enzyme, and the oxidation of NADH was followed at 340 nm. Peroxidase (EC Rohringer et al (21) extracted an intercellular washing fluid from intact barley leaves by vacuum infiltration with buffer followed by gentle centrifugation. We directly compared our technique of washing peeled leaves with theirs in an experiment in which NaCl was omitted from the buffered washing solution.…”

Section: Enzyme Assaysmentioning

confidence: 99%

“…By criteria developed in several laboratories (6,9,11,15,17,18,21,25): (a) enzymes washed from tissues by solutions not damaging the plasmalemma are soluble in the apoplast or weakly bound to the cell wall; (b) soluble cytoplasmic enzymes can be obtained by disrupting cells and pelleting organelles and broken cell walls; (c) ionically bound enzymes may be extracted from washed wall pellets by salt solutions of high ionic strength; and (d) enzyme activity remaining in the salt-washed wall pellet is covalently bound or entrapped into the wall. We have applied these criteria in our attempt to localize activity for four enzymes which may have roles in regulating apoplastic phenolic metabolism in young primary leaves of oats and barley.…”

mentioning

confidence: 99%

“…Soluble or weakly bound enzymes have been extracted from the apoplast by vacuum infiltration followed by gentle centrifugation (21,(23)(24)(25). This technique works well with stems which require minimal support during centrifugation, but when used with cereals which have thin and relatively fragile leaves it yields small volumes of solution with low protein content (21).…”

mentioning

confidence: 99%

“…This technique works well with stems which require minimal support during centrifugation, but when used with cereals which have thin and relatively fragile leaves it yields small volumes of solution with low protein content (21). In 1984, Smith et al (22) reported that buffered salt solutions rapidly (60% within 10 s) eluted peroxidase and hydroxyproline-rich glycoproteins from the surface of intact tomato suspension culture cells without loss of cytoplasmic constituents.…”

mentioning

confidence: 99%

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Soluble and Bound Apoplastic Activity for Peroxidase, β-d-Glucosidase, Malate Dehydrogenase, and Nonspecific Arylesterase, in Barley (Hordeum vulgare L.) and Oat (Avena sativa L.) Primary Leaves

Li¹,

McClure²,

Hagerman³

1989

Plant Physiol.

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An intercellular washing solution containing about 1% of the soluble protein, 0.3% or less of the glucose-6-phosphate dehydrogenase activity, but up to 20% of the peroxidase and 6-Dglucosidase activity of barley (Hordeum vulgare L.) or oat (Avena sativa L.) primary leaves was obtained by vacuum infiltrating peeled leaves with pH 6.9 buffered 200 millimolar NaCI. After this wash, segments were homogenized in buffer, centrifuged, and the supernatant was assayed for soluble cytoplasmic enzymes. The pellet was washed and resuspended in 1 molar NaCI to solubilize enzymes strongly ionically bound to the cell wall. The final pellet was assayed for enzyme activity covalently bound in the cell wall. Apoplastic (intercellular washing solution, ionically bound, and covalently bound) fractions contained up to 76% of the ,6-D-glucosidase activity, 36% of the peroxidase activity, 11% of the nonspecific arylesterase activity, 4% of the malate dehydrogenase activity, but less than 2% of the glucose-6-phosphate dehydrogenase activity of peeled leaf segments. The partitioning and salt-solubility of the enzymes between the apoplast and symplast differed considerably between these two species. Intercellular washing fluid prepared by centrifuging unpeeled leaves had higher activity for glucose-6-phosphate dehydrogenase, less soluble protein, and less peroxidase activity per leaf than intercellular washing solution obtained by our peeling-infiltration-washing technique. The results are discussed in relation to the roles of these enzymes in phenolic metabolism in the cell wall.

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Section: Discussionsupporting

confidence: 85%

Section: Enzyme Assaysmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Soluble and Bound Apoplastic Activity for Peroxidase, β-d-Glucosidase, Malate Dehydrogenase, and Nonspecific Arylesterase, in Barley (Hordeum vulgare L.) and Oat (Avena sativa L.) Primary Leaves

Li¹,

McClure²,

Hagerman³

1989

Plant Physiol.

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Zur enzymatischen Differenzierung der pflanzlichen Zellwand

Mäder

1985

Biologie in unserer Zeit

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Protein extraction for proteome analysis from cacao leaves and meristems, organs infected by Moniliophthora perniciosa, the causal agent of the witches' broom disease

Pirovani

Carvalho

Machado³

et al. 2008

Electrophoresis

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Preparation of high-quality proteins from cacao vegetative organs is difficult due to very high endogenous levels of polysaccharides and polyphenols. In order to establish a routine procedure for the application of proteomic and biochemical analysis to cacao tissues, three new protocols were developed; one for apoplastic washing fluid (AWF) extraction, and two for protein extraction--under denaturing and nondenaturing conditions. The first described method allows a quick and easy collection of AWF--using infiltration-centrifugation procedure--that is representative of its composition in intact leaves according to the smaller symplastic contamination detected by the use of the hexose phosphate isomerase marker. Protein extraction under denaturing conditions for 2-DE was remarkably improved by the combination of chemically and physically modified processes including phenol, SDS dense buffer and sonication steps. With this protocol, high-quality proteins from cacao leaves and meristems were isolated, and for the first time well-resolved 1-DE and 2-DE protein patterns of cacao vegetative organs are shown. It also appears that sonication associated with polysaccharide precipitation using tert-butanol was a crucial step for the nondenaturing protein extraction and subsequent enzymatic activity detection. It is expected that the protocols described here could help to develop high-level proteomic and biochemical studies in cacao also being applicable to other recalcitrant plant tissues.

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Proteins in Intercellular Washing Fluids from Leaves of Barley (Hordeum vulgareL.)

Cited by 86 publications

References 31 publications

Soluble and Bound Apoplastic Activity for Peroxidase, β-d-Glucosidase, Malate Dehydrogenase, and Nonspecific Arylesterase, in Barley (Hordeum vulgare L.) and Oat (Avena sativa L.) Primary Leaves

Soluble and Bound Apoplastic Activity for Peroxidase, β-d-Glucosidase, Malate Dehydrogenase, and Nonspecific Arylesterase, in Barley (Hordeum vulgare L.) and Oat (Avena sativa L.) Primary Leaves

Zur enzymatischen Differenzierung der pflanzlichen Zellwand

Protein extraction for proteome analysis from cacao leaves and meristems, organs infected by Moniliophthora perniciosa, the causal agent of the witches' broom disease

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